Ranges of Normal Values in Human Whole Blood (B), Plasma (P), or Serum (S)a Normal Value (Varies with Procedure Used)

Determination Traditional Units SI Units

Normal Value (Varies with Procedure Used) Determination

Acetoacetate plus acetone (S) Aldosterone (supine) (P)

Alpha-amino nitrogen (P) Aminotransferases

Alanine aminotransferase Aspartate aminotransferase Ammonia (B)

Amylase (S)

Ascorbic acid (B)

Bilirubin (S)

Calcium (S)

Carbon dioxide content (S) Carotenoids (S)

Ceruloplasmin (S)

Chloride (S)

Cholesterol (S)

Cholesteryl esters (S)

Copper (total) (S)

Cortisol (P) (AM, fasting) Creatinine (P)

Glucose, fasting (P)

Iron (S)

Lactic acid (B)

Lipase (S)

Lipids, total (S)

Magnesium (S)

Osmolality (S)

PCO2 (arterial) (B)

Pepsinogen (P)

Phenylalanine (S)

Phosphatase, acid (S)

Phosphatase, alkaline (S) Phospholipids (S)

Phosphorus, inorganic (S) PO2 (arterial) (B)

Potassium (S)

Protein

Total (S)

Albumin (S)

Globulin (S)

Pyruvic acid (P)

Sodium (S)

Urea nitrogen (S)

Uric acid (S)

Women

Men

Traditional Units

0.3–2.0 mg/dL

3.0–10 ng/dL

3.0–5.5 mg/dL

SI Units

3–20 mg/L

83–227 pmol/L 2.1–3.9 mmol/L

3–48 units/L

0–55 units/L

12–55 μmol/L

53–123 units/L

0.4–1.5 mg/dL (fasting)

Conjugated (direct): up to 0.4 mg/dL

Total (conjugated plus free): up to 1.0 mg/dL

8.5–10.5 mg/dL; 4.3–5.3 meq/L

24–30 meq/L

0.8–4.0 μg/mL

23–43 mg/dL

100–108 meq/L

< 200 mg/dL

60–70% of total cholesterol

70–155 μg/dL

5–25 μg/dL

0.6–1.5 mg/dL

70–110 mg/dL

50–150 μg/dL

0.5–2.2 meq/L

3–19 units/L

450–1000 mg/dL

1.4–2.0 meq/L

280–296 mosm/kg H

35–45 mm Hg

200–425 units/mL

0–2 mg/dL

Males: 0–0.8 sigma unit/mL

Females: 0.01–0.56 sigma unit/mL

13–39 units/L (adults)

9–16 mg/dL as lipid phosphorus

2.6–4.5 mg/dL (infants in first year: up to 6.0 mg/dL)

75–100 mm Hg

3.5–5.0 meq/L

12–55 μmol/L

884–2050 nmol s

–1

/L 23–85 μmol/L

Up to 7 μmol/L

Up to 17 μmol/L

2.1–2.6 mmol/L

24–30 mmol/L

1.5–7.4 μmol/L

240–430 mg/L

100–108 mmol/L < 5.17 mmol/L

11.0–24.4 μmol/L

0.14–0.69 μmol/L

53–133 μmol/L

3.9–6.1 mmol/L

9.0–26.9 μmol/L

0.5–2.2 mmol/L

4.5–10 g/L

0.7–1.0 mmol/L

280–296 mmol/kg H

4.7–6.0 kPa

pH (B) 7.35–7.45

0–120 μmol/L

0.22–0.65 μmol s

–1

2.9–5.2 mmol/L

0.84–1.45 mmol/L

10.0–13.3 kPa

3.5–5.0 mmol/L

6.0–8.0 g/dL

3.1–4.3 g/dL

2.6–4.1 g/dL

0–0.11 meq/L

135–145 meq/L

8–25 mg/dL

60–80 g/L

31–43 g/L

26–41 g/L

0–110 μmol/L 135–145 mmol/L 2.9–8.9 mmol/L

2.3–6.6 mg/dL 3.6–8.5 mg/dL 137–393 μmol/L 214–506 μmol/L

Based in part on Kratz A, et al. Laboratory reference values. N Engl J Med 2004;351:1548. Ranges vary somewhat from one laboratory to another depending on the

details of the methods used, and specific values should be considered in the context of the range of values for th e laboratory that made the determination. a LANGE

medical book

Ganong’s

Review of

Medical Physiology

Twenty-Third Edition

Kim E. Barrett, PhD

Professor

Department of Medicine

Dean of Graduate Studies

University of California, San Diego La Jolla, California

Scott Boitano, PhD

Associate Professor, Physiology Arizona Respiratory Center

Bio5 Collaborative Research Institute University of Arizona

Tucson, Arizona

Susan M. Barman, PhD

Professor

Department of Pharmacology/Toxicology Michigan State University East Lansing, Mich igan

Heddwen L. Brooks, PhD

Associate Professor

Department of Physiology

College of Medicine

University of Arizona

Tucson, Arizona

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Dedication to

WILLIAM FRANCIS GANONG

William Francis (“Fran”) Ganong was an outstanding scientist, educator, and writer. He was completely dedicated to the field of

physiology and medical education in general. Chairman of the Department of Physiolog y at the University of California, San Francisco,

for many years, he received numerous teaching awards and loved working with medic al students.

Over the course of 40 years and some 22 editions, he was the sole author of the best se lling Review of Medical Physiology, and a co-

author of 5 editions of Pathophysiology of Disease: An Introduction to Clinical Medicin e. He was one of the “deans” of the Lange group

of authors who produced concise medical text and review books that to this day remain extraordinarily popular in print and now in digital

formats. Dr. Ganong made a gigantic impact on the education of countless medical stud ents and clinicians.

A general physiologist par excellence and a neuroendocrine physiologist by subspecial ty, Fran developed and maintained a rare

understanding of the entire field of physiology. This allowed him to write each new edi tion (every 2 years!) of the Review of Medical

Physiology as a sole author, a feat remarked on and admired whenever the book came up for discussion among physiologists. He was an

excellent writer and far ahead of his time with his objective of distilling a complex subjec t into a concise presentation. Like his good

friend, Dr. Jack Lange, founder of the Lange series of books, Fran took great pride in the many different translations of the Review of

Medical Physiology and was always delighted to receive a copy of the new edition in an y language.

He was a model author, organized, dedicated, and enthusiastic. His book was his pride and joy and like other best-selling authors, he

would work on the next edition seemingly every day, updating references, rewriting as needed, and always ready and on time when the

next edition was due to the publisher. He did the same with his other book, Pathophysio logy of Disease: An Introduction to Clinical

Medicine, a book that he worked on meticulously in the years following his formal retir ement and appointment as an emeritus professor

at UCSF.

Fran Ganong will always have a seat at the head table of the greats of the art of medical science education and communication. He died

on December 23, 2007. All of us who knew him and worked with him miss him greatl y.

iii

Key Features of the 23rd Edition of

Ganong’s Review of Medical Physiology

• Thoroughly updated to reflect the latest research and developments in imp ortant areas such as the cellular basis of neurophysiology

• Incorporates examples from clinical medicine throughout the chapters to illustrate important physiologic concepts

• Delivers more detailed, clinically-relevant, high-yield information per page than any similar text or review

• NEW full-color illustrations—the authors have worked with an outstanding team of medical illustrators, photographers, educators,

and students to provide an unmatched collection of 600 illustrations and tables

• NEW boxed clinical cases—featuring examples of diseases that illus trate important physiologic principles

• NEW high-yield board review questions at the end of each chapter

• NEW larger 8½ X 11” trim-size enhances the rich visual content

• NEW companion online learning center (LangeTextbooks.com) offers a wealth of innovative learning tools and illustrations

NEW iPod-compatible

review—Medical PodClass

offers audio and text for

study on the go

Full-color illustrations

enrich the text

KEY FEATURES v

Clinical Cases illustrate essential

physiologic principles

Summary tables and charts

encapsulate important information

Chapters conclude with Chapter Summaries and review questions

About the Authors

KIM E. BARRETT

Kim Barrett received her PhD in biological chemistry from University College London in 1982. Following postdoctoral training at the

National Institutes of Health, she joined the faculty at the University of California, San D iego, School of Medicine in 1985, rising to her

current rank of Professor of Medicine in 1996. Since 2006, she has also served the U niversity as Dean of Graduate Studies. Her

American Physiological Society (APS) and recently served on its council. She has also s erved as Chair of the Central Nervous System

Section of APS as well as Chair of both the Women in Physiology and Section Advisory Committees of APS. In her spare time, she

enjoys daily walks, aerobic exercising, and mind-challenging activities like puzzles of va rious sorts.

SCOTT BOITANO

research interests focus on the physiology and pathophysiology of the intestinal epitheliu m, and how its function is altered by

commensal, probiotics, and pathogenic bacteria as well as in specific disease states, such as inflammatory bowel diseases. She has

published almost 200 articles, chapters, and reviews, and has received several honors f or her research accomplishments including the

Bowditch and Davenport Lectureships from the American Physiological Society and the degree of Doctor of Medical Sciences, honoris

causa, from Queens University, Belfast. She is also a dedicated and award-winning inst ructor of medical, pharmacy, and graduate

students, and has taught various topics in medical and systems physiology to these group s for more than 20 years. Her teaching

experiences led her to author a prior volume (Gastrointestinal Physiology, McGraw-Hi ll, 2005) and she is honored to have been invited to

take over the helm of Ganong.

Scott Boitano received his PhD in genetics and cell biology from Washington State Univ ersity in Pullman, Washington, where he
acquired an interest in cellular signaling. He fostered this interest at University of Califor nia, Los Angeles, where he focused his research
on second
messengers and cellular physiology of the lung epithelium. He continued to foster these  research interests at the University of Wyoming
and at his current positions with the Department of Physiology and the Arizona Respira tory Center, both at the University of Arizona.
HEDDWEN L. BROOKS
SUSAN M. BARMAN
Susan Barman received her PhD in physiology from Loyola University School of Med icine in Maywood, Illinois. Afterward she went to
Michigan State University (MSU) where she is currently a Professor in the Department  of Pharmacology/ Toxicology and the
Neuroscience Program. Dr Barman has had a career-long interest in neural control of  cardiorespiratory function with an emphasis on the
characterization
Heddwen Brooks received her PhD from Imperial College, University of London and  is an Associate Professor in the Department of
Physiology at the University of Arizona (UA). Dr Brooks is a renal physiologist and is  best known for her development of microarray
technology to address in vivo signaling pathways involved in the hormonal regulation o f
and origin of the naturally occurring discharges of sympathetic and phrenic nerves. Sh e was a recipient of a prestigious National
Institutes of Health MERIT (Method to Extend Research in Time) Award. She is also a  recipient of an Outstanding University Woman
Faculty Award from the MSU Faculty Professional Women's Association and an MSU C ollege of Human Medicine Distinguished
Faculty Award. She has been very active in the renal function. Dr Brooks’ many awar ds include the American Physiological Society
(APS) Lazaro J. Mandel Young Investigator Award, which is for an individual demon strating outstanding promise in epithelial or renal
physiology. She will receive the APS Renal Young Investigator Award at the 20 09 annual meeting of the Federation of American
Societies for Experimental Biology. Dr Brooks is a member of the APS Renal Stee ring Section and the APS Committee of Committees.
She is on the Editorial Board of the American Journal of Physiology-Renal Physiology  (since 2001), and she has also served on study
sections of the National Institutes of Health and the American Heart Association.
Contents
Preface ix
SECTION I
CELLULAR & MOLECULAR BASIS FOR MEDICAL PHYSIOLOGY 1
1. General Principles & Energy 
Production in Medical Physiology 1 2. Overview of Cellular Physiology 
in Medical Physiology 31
3. Immunity, Infection, & Inflammation 63
SECTION II
PHYSIOLOGY OF NERVE 
& MUSCLE CELLS 79
4. Excitable Tissue: Nerve 79 
5. Excitable Tissue: Muscle 93 
6. Synaptic & Junctional Transmission 115
7. Neurotransmitters & Neuromodulators 129
8. Properties of Sensory Receptors 149
9. Reflexes 157
SECTION III
CENTRAL & PERIPHERAL 
NEUROPHYSIOLOGY 167
10. Pain & Temperature 167
11. Somatosensory Pathways 173 
12. Vision 181
13. Hearing & Equilibrium 203 
14. Smell & Taste 219
15. Electrical Activity of the Brain, Sleep–Wake States, & Circadian Rhythms 229
16. Control of Posture & Movement 241
17. The Autonomic Nervous System 261
18. Hypothalamic Regulation of 

Hormonal Functions 273
19. Learning, Memory, Language, 
& Speech 289
SECTION IV
ENDOCRINE & REPRODUCTIVE PHYSIOLOGY 301
20. The Thyroid Gland 301
21. Endocrine Functions of the 
Pancreas & Regulation of 
Carbohydrate Metabolism 315
22. The Adrenal Medulla & 
Adrenal Cortex 337
23. Hormonal Control of Calcium 
and Phosphate Metabolism & 
the Physiology of Bone 363
24. The Pituitary Gland 377
25. The Gonads: Development & Function of the Reproductive System 391
SECTION V
GASTROINTESTINAL 
PHYSIOLOGY 429
26. Overview of Gastrointestinal 
Function & Regulation 429 
vii viii CONTENTS
27. Digestion, Absorption, & 
Nutritional Principles 451
28. Gastrointestinal Motility 469 
29. Transport & Metabolic 
Functions of the Liver 479 
SECTION VI
CARDIOVASCULAR 
PHYSIOLOGY 489
30. Origin of the Heartbeat & the 
Electrical Activity of the Heart 489 31. The Heart as a Pump 507 
32. Blood as a Circulatory Fluid & the 
Dynamics of Blood & Lymph Flow 521 33. Cardiovascular Regulatory Mec hanisms 555 34. Circulation Through Special Regions
569 
SECTION VII
RESPIRATORY PHYSIOLOGY 587
35. Pulmonary Function 587 
36. Gas Transport & pH in the Lung 609 
37. Regulation of Respiration 625
SECTION VIII
RENAL PHYSIOLOGY 639
38. Renal Function & Micturition 639
39. Regulation of Extracellular Fluid Composition & Volume 665 
40. Acidification of the Urine & 
Bicarbonate Excretion 679 
Answers to Multiple Choice Questions 687 Index 689
Preface
From the Authors
We are very pleased to launch the 23rd edition of Ganong's Review of Medical P hysiology. The current authors have attempted to
maintain the highest standards of excellence, accuracy, and pedagogy developed by Fr an Ganong over the 46 years in which he educated
countless students worldwide with this textbook.
At the same time, we have been attuned to the evolving needs of both students and prof essors in medical physiology. Thus, in addition to
usual updates on the latest research and developments in areas such as the cellular basis  of physiology and neurophysiology, this edition
has added both outstanding pedagogy and learning aids for students.
We are truly grateful for the many helpful insights, suggestions, and reviews from arou nd the world that we received from colleagues
and students. We hope you enjoy the new features and the 23rd edition!

This edition is a revision of the original works of Dr. Francis Ganong.

New 4 Color Illustrations

• We have worked with a large team of medical illustrators, photographers, educators, and students to build an accurate, up-to-date, and

visually appealing new illustration program. Full-color illustrations and tables are provide d throughout, which also include detailed

figure legends that tell a short story or describes the key point of the illustration.

New 8

x 11 Format

• Based on student and instructor focus groups, we have increased the trim size, which will provide additional white space and allow our

new art program to really show!

New Boxed Clinical Cases

• Highlighted in a shaded background, so students can recognize the boxed clinical case s, examples of diseases illustrating important

physiological principles are provided.

New End of Chapter Board

Review Questions

• New to this edition, chapters now conclude with board review questions.

New Media

• This new edition has focused on creating new student content that is built upon learnin g outcomes and assessing student performance.

Free with every student copy is an iPod Review Tutorial Product. Questions and art bas ed from each chapter tests students

comprehension and is easy to navigate with a simple click of the scroll bar!

• Online Learning Center will provide students and faculty with cases and art and board review questions on a dedicated website.

This page intentionally left blank

SECTION I CELLULAR & MOLECULAR

BASIS OF MEDICAL PHYSIOLOGY

General Principles & Energy Production in Medical Physiology

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Name the different fluid compartments in the human body.

■

Define moles, equivalents, and osmoles.

■

Define pH and buffering.

■

Understand electrolytes and define diffusion, osmosis, and tonicity.

■

Define and explain the resting membrane potential.

■

Understand in general terms the basic building blocks of the cell: nucleotides, amino acids, car bohydrates, and fatty acids.

■

Understand higher-order structures of the basic building blocks: DNA, RNA, proteins, and lipi ds.

■

Understand the basic contributions of these building blocks to cell structure, function, and energy balance.

INTRODUCTION

In unicellular organisms, all vital processes occur in a single cell. As the evolution of mu lticellular organisms has progressed, various cell

groups organized into tissues and organs have taken over particular functions. In hum ans and other vertebrate animals, the specialized

cell groups include a gastrointestinal system to digest and absorb food; a respiratory sys tem to take up O

and eliminate CO

; a urinary

system to remove wastes; a cardiovascular system to distribute nutrients, O

, and the products of metabolism; a reproductive system to

perpetuate the species; and nervous and endocrine systems to coordinate and integrate t he functions of the other systems. This book is

concerned with the way these systems function and the way each contributes to the fun ctions of the body as a whole.

In this section, general concepts and biophysical and biochemical principles that are basi c to the function of all the systems are presented.

In the first chapter, the focus is on review of basic biophysical and biochemical princip les and the introduction of the molecular building

blocks that contribute to cellular physiology. In the second chapter, a review of basic ce llular morphology and physiology is presented. In

the third chapter, the process of immunity and inflammation, and their link to physiology , are considered.

GENERAL PRINCIPLES

THE BODY AS AN

ORGANIZED “SOLUTION”

The cells that make up the bodies of all but the simplest multicellular animals, both aquatic and terrestrial, exist in an “internal sea” of

extracellular fluid (ECF) enclosed within the integument of the animal. From th is fluid, the cells take up O

and nutrients; into it, they

discharge metabolic waste products. The ECF is more dilute than present-day seawater, but its composition closely resembles that of the

primordial oceans in which, presumably, all life originated.

In animals with a closed vascular system, the ECF is divided into two components: the interstitial fluid and the circulating blood

plasma. The plasma and the cellular elements of the blood, principally red blood cells , fill the vascular system, and together they

constitute the total blood volume. The interstitial fluid is that part of the ECF that is o utside the vascular system, bathing the cells. The

special fluids considered together as transcellular fluids are discussed in the following te xt. About a third of the total body water is

extracellular; the remaining two thirds is intracellular (intracellular fluid). In the aver age young adult male, 18% of the body weight is

protein and related substances, 7% is mineral, and 15% is fat. The remaining 60% is w ater. The distribution of this water is shown in

Figure 1–1A.

The intracellular component of the body water accounts for about 40% of body weigh t and the extracellular component for about 20%.

Approximately 25% of the extracellular component is in the vascular system (plasma = 5% of body weight) and 75% outside the blood

vessels (interstitial fluid = 15% of body weight). The total blood volume is about 8% of body weight. Flow between these compartments

is tightly regulated.

UNITS FOR MEASURING

CONCENTRATION OF SOLUTES

In considering the effects of various physiologically important substances and the intera ctions between them, the number of molecules,

electric charges, or particles of a substance per unit volume of a particular body fluid a re often more meaningful than simply the weight

of the substance per unit volume. For this reason, physiological concentrations are freq uently expressed in moles, equivalents, or

osmoles.

Moles

A mole is the gram-molecular weight of a substance, ie, the molecular weight of the sub stance in grams. Each mole (mol) consists of 6 ×

molecules. The millimole (mmol) is 1/1000 of a mole, and the micromole (μmol) is 1/1,0 00,000 of a mole. Thus, 1 mol of NaCl =

23 g + 35.5 g = 58.5 g, and 1 mmol = 58.5 mg. The mole is the standard unit for expr essing the amount of substances in the SI unit

system.

The molecular weight of a substance is the ratio of the mass of one molecule of the subs tance to the mass of one twelfth the mass of an

atom of carbon-12. Because molecular weight is a ratio, it is dimensionless. The dalton ( Da) is a unit of mass equal to one twelfth the

mass of an atom of carbon-12. The kilodalton (kDa = 1000 Da) is a useful unit for ex pressing the molecular mass of proteins. Thus, for

example, one can speak of a 64-kDa protein or state that the molecular mass of the pro tein is 64,000 Da. However, because molecular

weight is a dimensionless ratio, it is incorrect to say that the molecular weight of the prote in is 64 kDa.

Equivalents

The concept of electrical equivalence is important in physiology because many of the so lutes in the body are in the form of charged

particles. One equivalent (eq) is 1 mol of an ionized substance divided by its valence. O ne mole of NaCl dissociates into 1 eq of Na

and

1 eq of Cl

–

. One equivalent of Na

= 23 g, but 1 eq of Ca

= 40 g/2 = 20 g. The milliequivalent (meq) is 1/1000 of 1 eq.

Electrical equivalence is not necessarily the same as chemical equivalence. A gram equiv alent is the weight of a substance that is

chemically equivalent to 8.000 g of oxygen. The normality (N) of a solution is the num ber of gram equivalents in 1 liter. A 1 N solution

of hydrochloric acid contains both H

(1 g) and Cl

–

(35.5 g) equivalents, = (1 g + 35.5 g)/L = 36.5 g/L.

WATER, ELECTROLYTES, & ACID/BASE

The water molecule (H

O) is an ideal solvent for physiological reactions. H

O has a dipole moment where oxygen slightly pulls away

electrons from the hydrogen atoms and creates a charge separation that makes the mole cule polar. This allows water to dissolve a variety

of charged atoms and molecules. It also allows the H

O molecule to interact with other H

O molecules via hydrogen bonding. The

resultant hydrogen bond network in water allows for several key properties in physiolo gy: (1) water has a high surface tension, (2) water

has a high heat of vaporization and heat capacity, and (3) water has a high dielectric co nstant. In layman’s terms, H

O is an excellent

biological fluid that serves as a solute; it provides optimal heat transfer and conduction o f current.

Electrolytes (eg, NaCl) are molecules that dissociate in water to their cation (Na

) and anion (Cl

–

) equivalents. Because of the net

charge on water molecules, these electrolytes tend not to reassociate in water. There are many important electrolytes in physiology,

notably Na

, K

, Ca

, Mg

, Cl

–

, and HCO3–. It is important to note that electrolytes and other charged compounds (eg , proteins) are

unevenly distributed in the body fluids (Figure 1–1B). These separations play an impor tant role in physiology.

Stomach

Intestines

Lungs

Blood plasma:

Skin

5% body weight

Kidneys

Extra

cellular

fluid: Interstitial fluid:20% body

15% body weight

weight

Intracellular fluid: 40% body weight

200

Extracellular fluid Intracellular fluid

Plasma

Interstitial fluid Misc. 150 phosphates K

100

−

+ 50

HCO

− Prot− K+ K+

HCO

−

HCO

−

Prot−

−

FIGURE 1–1 Organization of body fluids and electrolytes into compartments. A) Body fluids are divided into Intracellular and

extracellular fluid compartments (ICF and ECF, respectively). Their contribution to perc entage body weight (based on a healthy young

adult male; slight variations exist with age and gender) emphasizes the dominance of flui d makeup of the body. Transcellular fluids,

which constitute a very small percentage of total body fluids, are not shown. Arrows represent fluid movement between compartments.

B) Electrolytes and proteins are unequally distributed among the body fluids. This unev en distribution is crucial to physiology. Prot

–

protein, which tends to have a negative charge at physiologic pH.

pH AND BUFFERING

The maintenance of a stable hydrogen ion concentration ([H

]) in body fluids is essential to life. The pH of a solution is defined as the

logarithm to the base 10 of the reciprocal of the H

concentration ([H

]), ie, the negative logarithm of the [H

]. The pH of water at 25

°C, in which H

and OH

–

ions are present in equal numbers, is 7.0 (Figure 1–2). For each pH unit less than 7.0, the [H

] is increased

tenfold; for each pH unit above 7.0, it is decreased tenfold. In the plasma of healthy ind ividuals, pH is slightly alkaline, maintained in the

narrow range of 7.35 to 7.45. Conversely, gastric fluid pH can be quite acidic (on the order of 2.0) and pancreatic secretions can be quite

alkaline (on the order of 8.0). Enzymatic activity and protein structure are frequently se nsitive to pH; in any given body or cellular

compartment, pH is maintained to allow for maximal enzyme/protein efficiency.

Molecules that act as H

donors in solution are considered acids, while those that tend to remove H

from solutions are considered

bases. Strong acids (eg, HCl) or bases (eg, NaOH) dissociate completely in water and t hus can most change the [H

] in solution. In

physiological compounds, most acids or bases are considered “weak,” that is, they cont ribute relatively few H

or take away relatively

few H

from solution. Body pH is stabilized by the bufferi ng capacity of the body fluids. A buffer is a substance that has the ability to

bind or release H

in solution, thus keeping the pH of the solution relatively constant despite the addition of considerable quantities of

acid or base. Of course there are a number of buffers at work in biological fluids at an y given time. All buffer pairs in a homogenous

solution are in equilibrium with the same [H

]; this is known as the isohydric principle. One outcome of this principle is that by

assaying a single buffer system, we can understand a great deal about all of the biologic al buffers in that system.

concentration

(mol/L) pH

10 −

10−

For pure water,

10−6 6

] = 10

−

mol/L

10 −

10−

−9

−10

−11

−12

−13

−14

FIGURE 1–2 Proton concentration and pH. Relative proton (H

) concentrations for solutions on a pH scale are shown. (Redraw n from

Alberts B et al: Molecular Biology of the Cell, 4th ed. Garland Science, 2002.)

When acids are placed into solution, there is a dissociation of some of the component ac id (HA) into its proton (H

) and free acid (A

–

This is frequently written as an equation:

←

+ A

–

→ .

According to the laws of mass action, a relationship for the dissociation can be defined mathematically as:

= [H

] [A

–

] / [HA]

where K

is a constant, and the brackets represent concentrations of the individual species. In lay man’s terms, the product of the proton

concentration ([H

]) times the free acid concentration ([A

–

]) divided by the bound acid concentration ([HA]) is a defined constant (K).

This can be rearranged to read:

] = K

[HA]/[A

–

]

If the logarithm of each side is taken:

log [H

] = logK

+ log[HA]/[A

–

]

Both sides can be multiplied by –1 to yield:

–log [H

] = –logK

+ log[A

–

]/[HA]

This can be written in a more conventional form known as the Henderson Hasselbach equation:

pH = pK

+ log [A

–

]/[HA]

This relatively simple equation is quite powerful. One thing that we can discern right aw ay is that the buffering capacity of a particular

weak acid is best when the pK

of that acid is equal to the pH of the solution, or when:

[A–] = [HA], pH = pK

Similar equations can be set up for weak bases. An important buffer in the body is carb onic acid. Carbonic acid is a weak acid, and thus

is only partly dissociated into H

and bicarbonate:

←

3 →

+ HCO3–

If H

is added to a solution of carbonic acid, the equilibrium shifts to the left and most of the a dded H

is removed from solution. If OH

–

is added, H

and OH

–

combine, taking H

out of solution. However, the decrease is countered by more dissociation of H

, and the

decline in H

concentration is minimized. A unique feature of bicarbonate is the linkage between its b uffering ability and the ability for

the lungs to remove carbon dioxide from the body. Other important biological buffers include phosphates and proteins.

DIFFUSION

Diffusion is the process by which a gas or a substance in a solution expands, because o f the motion of its particles, to fill all the available

volume. The particles (molecules or atoms) of a substance dissolved in a solvent are in c ontinuous random movement. A given particle is

equally likely to move into or out of an area in which it is present in high concentration. However, because there are more particles in the

area of high concentration, the total number of particles moving to areas of lower conc entration is greater; that is, there is a net flux of

solute particles from areas of high to areas of low concentration. The time required for equilibrium by diffusion is proportionate to the

square of the diffusion distance. The magnitude of the diffusing tendency from one re gion to another is directly proportionate to the

cross-sectional area across which diffusion is taking place and the concentration gradient, or chemical gradient, which is the

difference in concentration of the diffusing substance divided by the thickness of the b oundary (Fick’s law of diffusion). Thus,

J = –DA Δc

Δx

where J is the net rate of diffusion, D is the diffusion coefficient, A is the area, and Δc/Δ x is the concentration gradient. The minus sign

indicates the direction of diffusion. When considering movement of molecules from a h igher to a lower concentration, Δc/Δx is negative,

so multiplying by –DA gives a positive value. The permeabilities of the boundaries acro ss which diffusion occurs in the body vary, but

diffusion is still a major force affecting the distribution of water and solutes.

OSMOSIS

When a substance is dissolved in water, the concentration of water molecules in the solu tion is less than that in pure water, because the

addition of solute to water results in a solution that occupies a greater volume than does the water alone. If the solution is placed on one

side of a membrane that is permeable to water but not to the solute, and an equal volume of water is placed on the other, water molecules

diffuse down their concentration (chemical) gradient into the solution (Figure 1–3). Thi s process—the diffusion of solvent molecules

into a region in which there is a higher concentration of a solute to which the membrane is imp ermeable—is called osmosis. It is an

important factor in physiologic processes. The tendency for movement of solvent molec ules to a region of greater solute concentration

can be prevented by applying pressure to the more concentrated solution. The pressure necessary to prevent solvent migration is the

osmotic pressure of the solution.

Osmotic pressure—like vapor pressure lowering, freezingpoint depression, and boiling -point elevation—depends on the number rather

than the type of particles in a solution; that is, it is a fundamental colligative property of so lutions. In an ideal solution, osmotic pressure

(P) is related to temperature and volume in the same way as the pressure of a gas:

nRT

P = ---------

where n is the number of particles, R is the gas constant, T is the absolute temperature, a nd V is the volume. If T is held constant, it is

clear that the osmotic pressure is proportional to the number of particles in solution per u nit volume of solution.

Semipermeable

membrane

Pressure

FIGURE 1–3 Diagrammatic representation of osmosis. Water molecules are represented by sma ll open circles, solute molecules by

large solid circles. In the diagram on the left, water is placed on one side of a memb rane permeable to water but not to solute, and an

equal volume of a solution of the solute is placed on the other. Water molecules move down the ir concentration (chemical) gradient into

the solution, and, as shown in the diagram on the right, the volume of the solution increases . As indicated by the arrow on the right, the

osmotic pressure is the pressure that would have to be applied to prevent the movement of the water molecules.

For this reason, the concentration of osmotically active particles is usually expressed in osm oles. One osmole (Osm) equals the gram-

molecular weight of a substance divided by the number of freely moving particles that e ach molecule liberates in solution. For biological

solutions, the milliosmole (mOsm; 1/1000 of 1 Osm) is more commonly used.

If a solute is a nonionizing compound such as glucose, the osmotic pressure is a functio n of the number of glucose molecules present. If

the solute ionizes and forms an ideal solution, each ion is an osmotically active particle. F or example, NaCl would dissociate into Na

and Cl

–

ions, so that each mole in solution would supply 2 Osm. One mole of Na

would dissociate into Na

, Na

, and SO42–

supplying 3 Osm. However, the body fluids are not ideal solutions, and although the di ssociation of strong electrolytes is complete, the

number of particles free to exert an osmotic effect is reduced owing to interactions betw een the ions. Thus, it is actually the effective

concentration (activity) in the body fluids rather than the number of equivalents of an electro lyte in solution that determines its osmotic

capacity. This is why, for example, 1 mmol of NaCl per liter in the body fluids contribu tes somewhat less than 2 mOsm of osmotically

active particles per liter. The more concentrated the solution, the greater the deviation fro m an ideal solution.

The osmolal concentration of a substance in a fluid is measured by the degree to which it depresses the freezing point, with 1 mol of an

ideal solution depressing the freezing point 1.86 °C. The number of milliosmoles per lite r in a solution equals the freezing point

depression divided by 0.00186. The osmolarity is the number of osmoles per liter of so lution (eg, plasma), whereas the osmolality is the

number of osmoles per kilogram of solvent. Therefore, osmolarity is affected by the vo lume of the various solutes in the solution and the

temperature, while the osmolality is not. Osmotically active substances in the body are dis solved in water, and the density of water is 1,

so osmolal concentrations can be expressed as osmoles per liter (Osm/L) of water. In th is book, osmolal (rather than osmolar)

concentrations are considered, and osmolality is expressed in milliosmoles per liter (of w ater).

Note that although a homogeneous solution contains osmotically active particles and can be said to have an osmotic pressure, it can exert

an osmotic pressure only when it is in contact with another solution across a membrane permeable to the solvent but not to the solute.

OSMOLAL CONCENTRATION

OF PLASMA: TONICITY

The freezing point of normal human plasma averages –0.54 °C, which corresponds to an osmolal concentration in plasma of 290

mOsm/L. This is equivalent to an osmotic pressure against pure water of 7.3 atm. The os molality might be expected to be higher than

this, because the sum of all the cation and anion equivalents in plasma is over 300. It is n ot this high because plasma is not an ideal

solution and ionic interactions reduce the number of particles free to exert an osmotic ef fect. Except when there has been insufficient

time after a sudden change in composition for equilibrium to occur, all fluid compartme nts of the body are in (or nearly in) osmotic

equilibrium. The term tonicity is used to describe the osmolality of a solu tion relative to plasma. Solutions that have the same osmolality

as plasma are said to be isotonic; those with greater osmolality are hypertonic; an d those with lesser osmolality are hypotonic. All

solutions that are initially isosmotic with plasma (ie, that have the same actual osmotic pres sure or freezing-point depression as plasma)

would remain isotonic if it were not for the fact that some solutes diffuse into cells and o thers are metabolized. Thus, a 0.9% saline

solution remains isotonic because there is no net movement of the osmotically active part icles in the solution into cells and the particles

are not metabolized. On the other hand, a 5% glucose solution is isotonic when initially i nfused intravenously, but glucose is

metabolized, so the net effect is that of infusing a hypotonic solution.

It is important to note the relative contributions of the various plasma components to the total osmolal concentration of plasma. All but

about 20 of the 290 mOsm in each liter of normal plasma are contributed by Na

and its accompanying anions, principally Cl

–

and

HCO3–. Other cations and anions make a relatively small contribution. Although the co ncentration of the plasma proteins is large when

expressed in grams per liter, they normally contribute less than 2 mOsm/L because of th eir very high molecular weights. The major

nonelectrolytes of plasma are glucose and urea, which in the steady state are in equilibri um with cells. Their contributions to osmolality

are normally about 5 mOsm/L each but can become quite large in hyperglycemia or ure mia. The total plasma osmolality is important in

assessing dehydration, overhydration, and other fluid and electrolyte abnormalities (Clin ical Box 1–1).

CLINICAL BOX 1–1 Plasma Osmolality & Disease

Unlike plant cells, which have rigid walls, animal cell membranes are flexible. Therefore , animal cells swell when exposed to

extracellular hypotonicity and shrink when exposed to extracellular hypertonicity. Cells contain ion channels and pumps that can be

activated to offset moderate changes in osmolality; however, these can be overwhelmed under certain pathologies. Hyperosmolality can

cause coma (hyperosmolar coma). Because of the predominant role of the major solute s and the deviation of plasma from an ideal

solution, one can ordinarily approximate the plasma osmolality within a few mosm/liter b y using the following formula, in which the

constants convert the clinical units to millimoles of solute per liter:

Osmolality (mOsm/L) = 2[Na

] (mEq/L) + 0.055[Glucose] (mg/dL) + 0.36[BUN] (mg/dL) BUN is the blood urea nitrogen. T he formula

is also useful in calling attention to abnormally high concentrations of other solutes. An o bserved plasma osmolality (measured by

freezing-point depression) that greatly exceeds the value predicted by this formula prob ably indicates the presence of a foreign substance

such as ethanol, mannitol (sometimes injected to shrink swollen cells osmotically), or pois ons such as

ethylene glycol or methanol (components of antifreeze).

NONIONIC DIFFUSION

Some weak acids and bases are quite soluble in cell membranes in the undissociated form , whereas they cannot cross membranes in the

charged (ie, dissociated) form. Consequently, if molecules of the undissociated substanc e diffuse from one side of the membrane to the

other and then dissociate, there is appreciable net movement of the undissociated substan ce from one side of the membrane to the other.

This phenomenon is called nonionic diffusion.

DONNAN EFFECT

When an ion on one side of a membrane cannot diffuse through the membrane, the di stribution of other ions to which the membrane is

permeable is affected in a predictable way. For example, the negative charge of a nond iffusible anion hinders diffusion of the diffusible

cations and favors diffusion of the diffusible anions. Consider the following situation,

X Y

–

Prot

–

in which the membrane (m) between compartments X and Y is impermeable to charged proteins (Prot

–

) but freely permeable to K

and

–

. Assume that the concentrations of the anions and of the cations on the two sides are in itially equal. Cl

–

diffuses down its

concentration gradient from Y to X, and some K

moves with the negatively charged Cl

–

because of its opposite charge. Therefore

[K+x] > [K+y]

Furthermore,

[K+x] + [Cl–x] + [Prot–x] > [K+y] + [Cl–y] that is, more osmotically active particles a re on side X than on side Y.

Donnan and Gibbs showed that in the presence of a nondiffusible ion, the diffusible io ns distribute themselves so that at equilibrium their

concentration ratios are equal:

[K+x

] = [Cl

–y]

[K+y] [Cl–x]

Cross-multiplying,

[K+x] + [Cl–x] = [K+y] + [Cl–y]

This is the Gibbs–Donnan equation. It holds for any pair of cations and anions of the same valence.

The Donnan effect on the distribution of ions has three effects in the body introduced h ere and discussed below. First, because of

charged proteins (Prot

–

) in cells, there are more osmotically active particles in cells than in interstitial fluid, and be cause animal cells

have flexible walls, osmosis would make them swell and eventually rupture if it were no t for Na, K ATPase pumping ions back out of

cells. Thus, normal cell volume and pressure depend on Na, K ATPase. Second, becau se at equilibrium the distribution of permeant ions

across the membrane (m in the example used here) is asymmetric, an electrical differenc e exists across the membrane whose magnitude

can be determined by the Nernst equation. In the example used here, side X will be negative relative to side Y. The charges line up

along the membrane, with the concentration gradient for Cl

–

exactly balanced by the oppositely directed electrical gradient, and the same

holds true for K

. Third, because there are more proteins in plasma than in interstitial fluid, there is a Don nan effect on ion movement

across the capillary wall.

FORCES ACTING ON IONS

The forces acting across the cell membrane on each ion can be analyzed mathematically . Chloride ions (Cl

–

) are present in higher

concentration in the ECF than in the cell interior, and they tend to diffuse along this concentration gradient into the cell. The interior of

the cell is negative relative to the exterior, and chloride ions are pushed out of the cell alo ng this electrical gradient. An equilibrium is

reached between Cl

–

influx and Cl

–

efflux. The membrane potential at which this equilibrium exists is the equilib rium potential. Its

magnitude can be calculated from the Nernst equation, as follows:

RT ln

[Clo–]

ECl =

FZ i–]

[Cl

where

= equilibrium potential for Cl

–

R = gas constant

T = absolute temperature

F = the faraday (number of coulombs per mole of charge) Z

= valence of Cl

–

(–1)

[Clo–] = Cl

–

concentration outside the cell

[Cli–] = Cl

–

concentration inside the cell

Converting from the natural log to the base 10 log and replacing some of the constants with numerical values, the equation becomes:

ECl

= 61.5 log [Cl

i–

] at 37 °C

[Clo–]

Note that in converting to the simplified expression the concentration ratio is reversed be cause the –1 valence of Cl

–

has been removed

from the expression.

The equilibrium potential for Cl

–

), calculated from the standard values listed in Table 1–1, is –70 mV, a value identical to the

measured resting membrane potential of –70 mV. Therefore, no forces other than thos e represented by the chemical and electrical

gradients need be invoked to explain

the distribution of Cl– across the membrane. +

A similar equilibrium potential can be calculated for K (E

= RT ln [K

] = 61.5log [K

] at 37 °C

[Ki+] [Ki+]

where

= equilibrium potential for K

(+1)

= valence of K

[Ko+] = K

concentration outside the cell

[Ki+] = K

concentration inside the cell

R, T, and F as above

In this case, the concentration gradient is outward and the electrical gradient inward. In mammalian spinal motor neurons, E

is –90 mV

(Table 1–1). Because the resting membrane potential is –70 mV, there is somewhat mor e K

in the neurons than can be accounted for by

the electrical and chemical gradients.

The situation for Na

is quite different from that for K

and Cl

–

. The direction of the chemical gradient for Na

is inward, to the area

where it is in lesser concentration, and the electrical gradient is in the same direction. E

is +60 mV (Table 1–1). Because neither E

nor E

is equal to the membrane potential,

TABLE 1–1 Concentration of some ions inside and outside mammalian spinal motor neu rons.

Concentration (mmol/L of H

Ion Inside Cell Outside Cell Equilibrium Potential (mV)

15.0 150.0

150.0 5.5 +60

–90

–

9.0 125.0 –70 NH

Adenine N

RiboseO− O− O−

H H

−

O PO PO P

HO OH

O O O

Resting membrane potential = –70 mV Adenosine 5'-monophosphate (AMP)

Adenosine 5'-diphosphate (ADP)

one would expect the cell to gradually gain Na

and lose K

if only passive electrical and chemical forces were acting across the

membrane. However, the intracellular concentration of Na

and K

remain constant because of the action of the Na, K ATPase that

actively transports Na

out of the cell and K

into the cell (against their respective electrochemical gradients).

Adenosine 5'-triphosphate (ATP)

FIGURE 1–4 Energy-rich adenosine derivatives. Ade nosine triphosphate is broken down into its backbone purine base and sugar (at

right) as well as its high energy phosphate derivatives (across bottom). (Reproduc ed, with permission, from Murray RK et al: Harper’s Biochemistry,

26th ed. McGraw-Hill, 2003.)

GENESIS OF THE MEMBRANE POTENTIAL

The distribution of ions across the cell membrane and the nature of this membrane prov ide the explanation for the membrane potential.

The concentration gradient for K

facilitates its movement out of the cell via K

channels, but its electrical gradient is in the opposite

(inward) direction. Consequently, an equilibrium is reached in which the tendency of K

to move out of the cell is balanced by its

tendency to move into the cell, and at that equilibrium there is a slight excess of cations o n the outside and anions on the inside. This

condition is maintained by Na, K ATPase, which uses the energy of ATP to pump K

back into the cell and keeps the intracellular

concentration of Na

low. Because the Na, K ATPase moves three Na

out of the cell for every two K

moved in, it also contributes to

the membrane potential, and thus is termed an electrogenic pump. It should b e emphasized that the number of ions responsible for the

membrane potential is a minute fraction of the total number present and that the total con centrations of positive and negative ions are

equal everywhere except along the membrane.

ENERGY PRODUCTION

ENERGY TRANSFER

Energy is stored in bonds between phosphoric acid residues and certain organic compo unds. Because the energy of bond formation in

some of these phosphates is particularly high, relatively large amounts of energy (10–1 2 kcal/mol) are released when the bond is

hydrolyzed. Compounds containing such bonds are called high-energy phosphate com pounds. Not all organic phosphates are of the

high-energy type. Many, like glucose 6-phosphate, are low-energy phosphates that on hydrolysis liberate 2–3 kcal/mol. Some of the

intermediates formed in carbohydrate metabolism are high-energy phosphates, but the m ost important high-energy phosphate compound

is adenosine triphosphate (ATP). This ubiquitous molecule (Fig ure 1–4) is the energy storehouse of the body. On hydrolysis to

adenosine diphosphate (ADP), it liberates energy directly to such processes as muscle c ontraction, active transport, and the synthesis of

many chemical compounds. Loss of another phosphate to form adenosine monophosp hate (AMP) releases more energy.

Another group of high-energy compounds are the thioesters, the acyl derivatives of m ercaptans. Coenzyme A (CoA) is a widely

distributed mercaptan-containing adenine, ribose, pantothenic acid, and thioethanolamin e (Figure 1–5). Reduced CoA (usually

abbreviated HS–CoA) reacts with acyl groups (R–CO–) to form R–CO–S–CoA deriv atives. A prime example is the reaction of HS-CoA

with acetic acid to form acetylcoenzyme A (acetyl-CoA), a compound of pivotal impor tance in intermediary metabolism. Because acetyl-

CoA has a much higher energy content than acetic acid, it combines readily with substan ces in reactions that would otherwise require

outside energy. Acetyl-CoA is therefore often called “active acetate.” From the point of view of energetics, formation of 1 mol of any

acyl-CoA compound is equivalent to the formation of 1 mol of ATP.

BIOLOGIC OXIDATIONS

Oxidation is the combination of a substance with O

, or loss of hydrogen, or loss of electrons. The corresponding reverse processes are

called reduction. Biologic oxidations are catalyzed by specific enzymes. Cofactors (s imple ions) or coenzymes (organic, nonprotein

substances) are accessory substances that

Pantothenic acid β-Alanine Thioethanolamine

C OH O

C CH C N CH

C N CH

NH 2

O P O

−

Pyrophosphate Adenine O

Coenzyme A

−

H H

Ribose 3-phosphate

O OH

PO O O O

−

R C OH + HS CoA R CS CoA + HOH FIGURE 1–5 Coenzym e A (CoA) and its derivatives. Left: Formula of reduced

coenzyme A (HS-CoA) with its components highlighted. Right: Form ula for reaction of CoA with biologically important compounds to

form thioesters. R, remainder of molecule.

usually act as carriers for products of the reaction. Unlike the enzymes, the coenzymes may catalyze a variety of reactions.

A number of coenzymes serve as hydrogen acceptors. One common form of biologic oxidation is removal of hydrogen from an R–OH

group, forming R=O. In such dehydrogenation reactions, nicotinamide adenine dinucl eotide (NAD

) and dihydronicotinamide adenine

dinucleotide phosphate (NADP

) pick up hydrogen, forming dihydronicotinamide adenine dinucleotide (NADH) and

dihydronicotinamide adenine dinucleotide phosphate (NADPH) (Figure 1–6). The hyd rogen is then transferred to the flavoprotein–

cytochrome system, reoxidizing the NAD

and NADP

. Flavin adenine dinucleotide (FAD) is formed when riboflavin is

phosphorylated, forming flavin mononucleotide (FMN). FMN then combines with AM P, forming the dinucleotide. FAD can accept

hydrogens in a similar fashion, forming its hydro (FADH) and dihydro (FADH

) derivatives.

The flavoprotein–cytochrome system is a chain of enzymes that transfers hydrogen to oxygen, forming water. This process occurs in the

mitochondria. Each enzyme in the chain is reduced

N OH

POCH2O

OH* OH

CONH

−

OCH

N O

HH OH OH

Adenine Ribose Diphosphate Ribose Nicotinamide

CONH

+ R' N

+ R'H

+ H N

R R

Oxidized coenzyme Reduced coenzyme

FIGURE 1–6 Structures of molecules important in oxidation reduction reactio ns to produce energy. Top: Formula of the oxidized

form of nicotinamide adenine dinucleotide (NAD

). Nicotinamide adenine dinucleotide phosphate (NADP

) has an additional phosphate

group at the location marked by the asterisk. Bottom: Reaction by which NAD

and NADP

become reduced to form NADH and

NADPH. R, remainder of molecule; R’, hydrogen donor.

+ Outer lamella

nner lamella

ATP ADP

FIGURE 1–7 Simplified diagram of transport of protons across the inner a nd outer lamellas of the inner mitochondrial

membrane. The electron transport system (flavoprotein-cytochrome system) h elps create H

movement from the inner to the outer

lamella. Return movement of protons down the proton gradient generates ATP.

and then reoxidized as the hydrogen is passed down the line. Each of the enzymes is a protein with an attached nonprotein prosthetic

group. The final enzyme in the chain is cytochrome c oxidase, which transfers hydrog ens to O

, forming H

O. It contains two atoms of

Fe and three of Cu and has 13 subunits.

The principal process by which ATP is formed in the body is oxidative ph osphorylation. This process harnesses the energy from a

proton gradient across the mitochondrial membrane to produce the high-energy bond of ATP and is briefly outlined in Figure 1–7.

Ninety percent of the O

consumption in the basal state is mitochondrial, and 80% of this is coupled to ATP synth esis. About 27% of the

ATP is used for protein synthesis, and about 24% is used by Na, K ATPase, 9% by glu coneogenesis, 6% by Ca

ATPase, 5% by

myosin ATPase, and 3% by ureagenesis.

MOLECULAR BUILDING BLOCKS

NUCLEOSIDES, NUCLEOTIDES,

& NUCLEIC ACIDS

Nucleosides contain a sugar linked to a nitrogen-containing base. The physiologically important bases, purines and pyrimidines, have

ring structures (Figure 1–8). These structures are bound to ribose or 2-deoxyribose to complete the nucleoside. When inorganic

phosphate is added to the nucleoside, a nucleotide is formed. Nucleosides and nucleotides for m the backbone for RNA and DNA, as

well as a variety of coenzymes and regulatory molecules (eg, NAD

, NADP

, and ATP) of physiological importance (Table 1–2).

Nucleic acids in the diet are digested and their constituent purines and pyrimidines absor bed, but most of the purines and pyrimidines are

synthesized from amino acids, principally in the liver. The nucleotides and RNA and DN A are then synthesized. RNA is in dynamic

equilibrium with the amino acid pool, but DNA, once formed, is metabolically stable thro ughout life. The purines and pyrimidines

released by the breakdown of nucleotides may be reused or catabolized. Minor amoun ts are excreted unchanged in the urine.

The pyrimidines are catabolized to the β-amino acids, β- alanine and β-a minoisobutyrate. These amino acids have their amino group on

β-carbon, rather than the α-carbon typical to physiologically active amino acids. Becaus e β-aminoisobutyrate is a product of thymine

degradation, it can serve as a measure of DNA turnover. The β-amino acids are furthe r degraded to CO

and NH

Uric acid is formed by the breakdown of purines and by

Uric acid is formed by the breakdown of purines and by PRPP) and glutamine (Figur e 1–9). In humans, uric acid is excreted in the urine,

but in other mammals, uric acid is further oxidized to allantoin before excretion. The no rmal blood uric acid level in humans is

approximately 4 mg/dL (0.24 mmol/L). In the kidney, uric acid is filtered, reabsorbed, and secreted. Normally, 98% of the filtered uric

acid is reabsorbed and the remaining 2% makes up approximately 20% of the amount excreted. The remaining 80% comes from the

tubular secretion. The uric acid excretion on a purine-free diet is about 0.5 g/24 h and on a regular diet about 1 g/24 h. Excess uric acid in

the blood or urine is a characteristic of gout (Clinical Box 1–2).

Adenine:

C 7

Guanine: 8CH

H C2 3 4 C 9

Hypoxanthine:

N N

6-Aminopurine

1-Amino

6-oxypurine

6-Oxypurine

2,6-Dioxypurine

Xanthine: Purine nucleus

Cytosine: C

N 3 4 5C H

Uracil: C

CHH Thymine: N

Pyrimidine nucleus

TABLE 1–2 Purine- and pyrimidinecontaining compounds.

Type of

Compound Components

Nucleoside Purine or pyrimidine plus ribose or 2-deoxyribose

4-Amino

2-oxypyrimidine 2,4-Dioxypyrimidine

5-Methyl

2,4-dioxypyrimidine Nucleotide

(mononucleotide) Nucleoside plus phosphoric acid residue

Nucleic acid Many nucleotides forming double-helical structures of two polynucleotide chains

Nucleoprotein

Contain ribose Nucleic acid plus one or more simple basic proteins

Ribonucleic acids (RNA)

FIGURE 1–8 Principal physiologically important purines and pyrimidines. Purine and pyrimidine structures are shown next to

representative molecules from each group. Oxypurines and oxypyrimidines may form enol d erivatives (hydroxypurines and

hydroxypyrimidines) by migration of hydrogen to the oxygen substituents.

Contain

2-deoxyribose Deoxyribonucleic acids (DNA) Adenosine Guanosine

CLINICAL BOX 1–2

Hypoxanthine

Xanthine oxidase

5-PRPP + Glutamine

Xanthine

Xanthine oxidase

C NH HN C

C O O C C

Uric acid (excreted in humans)

O NH H

N C

C O O C C

Allantoin (excreted in other mammals)

FIGURE 1–9 Synthesis and breakdown of uric acid. Adenosine is converted to hypoxa nthine, which is then converted to xanthine, and

xanthine is converted to uric acid. The latter two reactions are both catalyzed by xanthin e oxidase. Guanosine is converted directly to

xanthine, while 5-PRPP and glutamine can be converted to uric acid. An additional oxida tion of uric acid to allantoin occurs in some

mammals.

Gout

Gout is a disease characterized by recurrent attacks of arthritis; urate deposits in the joint s, kidneys, and other tissues; and elevated blood

and urine uric acid levels. The joint most commonly affected initially is the metatarsophal angeal joint of the great toe. There are two

forms of “primary” gout. In one, uric acid production is increased because of various enzyme abnormalities. In the other, there is a

selective deficit in renal tubular transport of uric acid. In “secondary” gout, the uric acid levels in the body fluids are elevated as a result

of decreased excretion or increased production secondary to some other disease proce ss. For example, excretion is decreased in patients

treated with thiazide diuretics and those with renal disease. Production is increased in leu kemia and pneumonia because of increased

breakdown of uric acid-rich white blood cells.

The treatment of gout is aimed at relieving the acute arthritis with drugs such as colchicin e or nonsteroidal anti-inflammatory agents and

decreasing the uric acid level in the blood. Colchicine does not affect uric acid metabolis m, and it apparently relieves gouty attacks by

inhibiting the phagocytosis of uric acid crystals by leukocytes, a process that in some wa y produces the joint symptoms. Phenylbutazone

and probenecid inhibit uric acid reabsorption in the renal tubules. Allopurinol, which di rectly inhibits xanthine oxidase in the purine

degradation pathway, is one of the drugs used to decrease uric acid production.

DNA

Deoxyribonucleic acid (DNA) is found in bacteria, in the nuclei of eukaryotic cells, an d in mitochondria. It is made up of two extremely

long nucleotide chains containing the bases adenine (A), guanine (G), thymine (T), and cytosine (C) (Figure 1–10). The chains are bound

together by hydrogen bonding between the bases, with adenine bonding to thymine an d guanine to cytosine. This stable association forms

a double-helical structure (Figure 1–11). The double helical structure of DNA is compa cted in the cell by association with histones, and

further compacted into chromosomes. A diploid human cell contains 46 chromosom es.

A fundamental unit of DNA, or a gene, can be defined as the sequence of DNA n ucleotides that contain the information for the

production of an ordered amino acid sequence for a single polypeptide chain. Interesti ngly, the protein encoded by a single gene may be

subsequently divided into several different physiologically active proteins. Information i s accumulating at an accelerating rate about the

structure of genes and their regulation. The basic structure of a typical eukaryotic gene is shown in diagrammatic form in Figure 1–12. It

is made up of a strand of DNA that includes coding and noncoding regions. In eukary otes, unlike prokaryotes, the portions of the genes

that dictate the formation of proteins are usually broken into several segments (exo ns) separated by segments that are not translated

(introns). Near the transcription start site of the gene is a promoter, which is the site at which RNA polymerase and its cofactors bind .

It often includes a thymidine–adenine–thymidine–adenine (TATA) sequence (TATA b ox), which ensures that transcription starts at the

proper point. Farther out in the 5' region are regulatory elements, which include enhancer and silencer sequences. It has been estimated

that each gene has an average of five regulatory sites. Regulatory sequences are somet imes found in the 3'-flanking region as well.

Gene mutations occur when the base sequence in the DNA is altered from its origina l sequence. Such alterations can affect protein

structure and be passed on to daughter cells after cell division. Point mutations are single base substitutions. A variety of chemical

modifications (eg, alkylating or intercalating agents, or ionizing radiation) can lead to ch anges in DNA sequences and mutations. The

collection of genes within the full expression of DNA from an organism is termed its genome. An indication of the complexity of DNA

in the human haploid genome (the total genetic message) is its size; it is made up of 3 × 1 0

base pairs that can code for approximately

30,000 genes. This genetic message is the blueprint for

Phosphate

Base (cytosine)

O Base (cytosine)

–

O P O CH

O –

O P O CH2 N

O–

H H

Sugar (deoxyribose)

H H

Sugar (ribose)

H C C H H C C H

OH H

OH OH

A Typical deoxyribonucleotide Typical ribonucleotide

Phosphate

NH2

O N N

Adenine (DNA and RNA) OP O CH

– O

Sugar N HNO

Guanine (DNA and RNA) OP O CH

N NH2Nucleotide – OO

N Cytosine (DNA and RNA) OP O CH

N O

–

O NH

Thymine (DNA only) OP O CH

N O

– O

Uracil (RNA only) NH

OP O CH

N O

–

FIGURE 1–10 Basic structure of nucleotides and nucleic acids. A) At left, the nucleotide cytosine is shown with deoxyribose and at

right with ribose as the principal sugar. B) Purine bases adenine and guanine are bound to each other or to pyrimidine bases, cytosine,

thymine, or uracil via a phosphodiester backbone between 2'-deoxyribosyl moieties attach ed to the nucleobases by an N-glycosidic bond.

Note that the backbone has a polarity (ie, a 5' and a 3' direction). Thymine is only fo und in DNA, while the uracil is only found in RNA.

T A

C G A T

Minor groove

G C

3.4 nm C G

A T Major groove

T A

2.0 nm

FIGURE 1–11 Double-helical structure of DNA. The compact structure has an approximatel y 2.0 nm thickness and 3.4 nm between full

turns of the helix that contains both major and minor grooves. The structure is maintained in the double helix by hydrogen bonding

between purines and pyrimidines across individual strands of DNA. Adenine (A) is bound to thymine (T) and cytosine (C) to guanine

(G). (Reproduced with permission from Murray RK et al: Harper’s Biochemistry, 26th ed. McGraw-Hill, 2003.)

REPLICATION: MITOSIS & MEIOSIS

At the time of each somatic cell division (mitosis), the two DNA chains separ ate, each serving as a template for the synthesis of a new

complementary chain. DNA polymerase catalyzes this reaction. One of the double helic es thus formed goes to one daughter cell and one

goes to the other, so the amount of DNA in each daughter cell is the same as that in the parent cell. The life cycle of the cell that begins

after mitosis is highly regulated and is termed the cell cycle (Figure 1–13). The G

(or Gap 1) phase represents a period of cell growth

and divides the end of mitosis from the DNA synthesis (or S) phase. Following DNA sy nthesis, the cell enters another period of cell

growth, the G

(Gap 2) phase. The ending of this stage is marked by chromosome condensation and the beginning of mitosis (M stage).

In germ cells, reduction division (meiosis) takes place during maturation. The net result is that one of ea ch pair of chromosomes ends up

in each mature germ cell; consequently, each mature germ cell contains half the amount of chromosomal material found in somatic cells.

Therefore, when a sperm unites with an ovum, the resulting zygote has the full complem ent of DNA, half of which came from the father

and half from the mother. The term “ploidy” is sometimes used to refer to the number o f chromosomes in cells. Normal resting diploid

cells are euploid and become tetraploid just before division. Aneuploidy is the condition in which a cell contains other than the haploid

number of chromosomes or an exact multiple of it, and this condition is common in canc erous cells.

the heritable characteristics of the cell and its descendants. The proteins formed from the DNA blueprint include all the enzymes, and

these in turn control the metabolism of the cell.

Each nucleated somatic cell in the body contains the full genetic message, yet there is gre at differentiation and specialization in the

functions of the various types of adult cells. Only small parts of the message are normal ly transcribed. Thus, the genetic message is

normally maintained in a repressed state. However, genes are controlled both spatially a nd temporally. First, under physiological

conditions, the double helix requires highly regulated interaction by proteins to unravel for replication, transcription, or both.

RNA

The strands of the DNA double helix not only replicate themselves, but also serve as tem plates by lining up complementary bases for the

formation in the nucleus of ribonucleic acids (RNA). RNA differs from DNA in that it is single-st randed, has uracil in place of

thymine, and its sugar moiety is ribose rather than 2'-deoxyribose (Figure 1–13). The p roduction of RNA from DNA is called

transcription. Transcription can lead to several types of RNA including: messenger RN A (mRNA), transfer RNA (tRNA), ribosomal

RNA (rRNA), and other RNAs. Transcription is catalyzed by various forms of RNA polymerase.

Basal Regulatory promoter region region Transcription Poly(A)

start site addition site Exon Exon

DNA 5' CAAT TATA AATAAA 3'

5' Intron 3' Noncoding Noncoding region region

FIGURE 1–12 Diagram of the components of a typical eukaryotic gene. The region that produces intro ns and exons is flanked by

noncoding regions. The 5'-flanking region contains stretches of DNA that interact with proteins to facilitate or inhibit transcription. The

3'-flanking region contains the poly(A) addition site. (Modified from Murray RK et al: Harper’s Biochem istry, 26th ed. McGraw-Hill, 2003.)

Mitotic phase

elophase

Mitosis

Final growth and activity before mitosis

Cytokinesis

Centrioles replicate

DNA replication

Interphase FIGURE 1–13 Sequence of events during the cell cycle. Immediate ly following mitosis (M) the cell enters a gap phase

(G1) before a DNA synthesis phase (S) a second gap phase (G2) and back to mi tosis. Collectively G1, S, and G2 phases are referred to as

interphase (I).

Typical transcription of an mRNA is shown in Figure 1–14. When suitably activated, tra nscription of the gene into a premRNA starts at

the cap site and ends about 20 bases beyond the AATAAA sequence. The RN A transcript is capped in the nucleus by addition of 7-

methylguanosine triphosphate to the 5' end; this cap is necessary for proper binding to t he ribosome. A poly(A) tail of about 100 bases is

added to the untranslated segment at the 3' end to help maintain the stability of the mRNA . The pre-mRNA formed by capping and

addition of the poly(A) tail is then processed by elimination of the introns, and once this posttranscriptional modification is complete, the

mature mRNA moves to the cytoplasm. Posttranscriptional modification of the pre-mRN A is a regulated process where differential

splicing can occur to form more than one mRNA from a single pre-mRNA. The intron s of some genes are eliminated by spliceosomes,

complex units that are made up of small RNAs and proteins. Other introns are eliminated by selfsplicing by the RNA they contain.

Because of introns and splicing, more than one mRNA can be formed from the same g ene.

Most forms of RNA in the cell are involved in translation, or protein synthesis. A b rief outline of the transition from transcription to

translation is shown in Figure 1–15. In the cytoplasm, ribosomes provide a template for tRNA to deliver specific amino acids to a

growing polypeptide chain based on specific sequences in mRNA. The mRNA molecu les are smaller than the DNA molecules, and each

represents a

Flanking DNA Introns

Gene Exons

Transcription Cap

Pre

mRNA

Flanking DNA

Poly(A)

RNA processing Poly(A)

mRNA Poly(A) Translation

FIGURE 1–14 Transcription of a typical mRNA. Steps in transcription from a typical gene to a processed mRNA are shown. Cap, cap

site. (Modified from Baxter JD: Principles of endocrinology. In: Cecil Textbook of Medicine, 16th ed. Wyng aarden JB, Smith LH Jr (editors). Saunders, 1982.)

transcript of a small segment of the DNA chain. For comparison, the molecules of tRNA contain only 70–80 nitrogenous bases,

compared with hundreds in mRNA and 3 billion in DNA.

AMINO ACIDS & PROTEINS

AMINO ACIDS

Amino acids that form the basic building blocks for proteins are identified in Table 1–3. These amino acids are often referred to by their

corresponding three-letter, or single-letter abbreviations. Various other important amino acids such as ornithine, 5-hydroxytryptophan, L-

dopa, taurine, and thyroxine (T

) occur in the body but are not found in proteins. In higher animals, the L isomers of th e amino acids are

the only naturally occurring forms in proteins. The L isomers of hormones such as thyr oxine are much more active than the D isomers.

The amino acids are acidic, neutral, or basic in reaction, depending on the relative prop ortions of free acidic (–COOH) or basic (–NH

)

groups in the molecule. Some of the amino acids are nutritionally essential amino acids, that is, they must be obtained in the diet,

because they cannot be made in the body. Arginine and histidine must be provided thro ugh diet during times of rapid growth or recovery

from illness and are termed conditionally essential. All others are nonessentia l amino acids in the sense that they can be synthesized in

vivo in amounts sufficient to meet metabolic needs.

DNA

RNA strand formed on DNA strand

(transcription)

tRNA

Amino acid

adenylate

Chain separation

Posttranscriptional modification

Activating enzyme Messenger RNA Coding triplets for

Translation Posttranslational modification

Ribosome

tRNA-amino acid-adenylate A

complex

Peptide chain

FIGURE 1–15 Diagrammatic outline of transcription to translation. Fr om the DNA molecule, a messenger RNA is produced and

presented to the ribosome. It is at the ribosome where charged tRNA match up with their com plementary codons of mRNA to position

the amino acid for growth of the polypeptide chain. DNA and RNA are represented as lines with multiple short projections representing

the individual bases . Small boxes labeled A represent individual amino acids.

TABLE 1–3 Amino acids found in proteins.*

Amino acids with aliphatic side chains Amino acids with acidic side chains, or their amide s Alanine (Ala, A) Aspartic acid (Asp, D)

Valine (Val, V) Asparagine (Asn, N) Leucine (Leu, L) Glutamine (Gln, Q) Isoleucine (IIe, I) Glutamic acid (Glu, E) Hydroxy l-

substituted amino acids γ-Carboxyglutamic acid

(Gla) Serine (Ser, S) Threonine (Thr, T)

Amino acids with side chains containing basic groups Arginine

(Arg, R)

Sulfur-containing amino acids Cysteine (Cys, C) Methionine (Met, M)

Selenocysteine

Lysine (Lys, K)

Hydroxylysine

(Hyl) Histidine

(His, H)

Imino acids (contain imino group but no amino group) Amino acids with aromatic ring side chains Proline (Pro, P)

Phenylalanine (Phe, F) Tyrosine (Tyr, Y) 4-Hydroxyproline

(Hyp)

3-Hydroxyproline

Tryptophan (Trp, W)

*Those in bold type are the nutritionally essential amino acids. The generally accepted three-letter and on e-letter abbreviations for the amino acids are shown in

parentheses.

Selenocysteine is a rare amino acid in which the sulfur of cysteine is replaced by selenium. The codon UG A is usually a stop codon, but in certain

situations it codes for selenocysteine.

There are no tRNAs for these four amino acids; they are formed by post-translational modification of the corresponding

unmodified amino acid in peptide linkage. There are tRNAs for selenocysteine and the remaining 20 amino acids, and they are incorporated into peptides and proteins

under direct genetic control.

Arginine and histidine are sometimes called “conditionally essential”—they are not necessary for maintena nce of nitrogen balance, but are

needed for normal growth.

THE AMINO ACID POOL

Although small amounts of proteins are absorbed from the gastrointestinal tract and som e peptides are also absorbed, most ingested

proteins are digested and their constituent amino acids absorbed. The body’s own prote ins are being continuously hydrolyzed to amino

acids and resynthesized. The turnover rate of endogenous proteins averages 80–100 g /d, being highest in the intestinal mucosa and

practically nil in the extracellular structural protein, collagen. The amino acids formed by endogenous protein breakdown are identical to

those derived from ingested protein. Together they form a common amino acid po ol that supplies the needs of the body (Figure 1–16).

this text, amino acid chains containing 2–10 amino acid residues are called peptides, cha ins containing more than 10 but fewer than 100

amino acid residues are called polypeptides, and chains containing 100 or more amino a cid residues are called proteins.

Diet Body Inert protein protein (hair, etc)

Urinary excretion Amino acid pool

Transamination

CommonAmination metabolicDeamination pool

PROTEINS

Proteins are made up of large numbers of amino acids linked into chains by pepti de bonds joining the amino group of one amino acid to

the carboxyl group of the next (Figure 1–17). In addition, some proteins contain carbo hydrates (glycoproteins) and lipids (lipoproteins).

Smaller chains of amino acids are called peptides or polypeptides. The boundaries between peptides, p olypeptides, and proteins are not

well defined. For

Creatine Purines, Hormones, NH

pyrimidines neurotransmitters

Urea

FIGURE 1–16 Amino acids in the body. There is an extensive network of amino acid turnover in the body. Boxes represent large pools

of amino acids and some of the common interchanges are represented by arrows. Note that most amino acids come from the diet and end

up in protein, however, a large portion of amino acids are interconverted and can feed into and out of a common metabolic pool through

amination reactions.

H OR H O

HH H H NC C N C C OH H–N C C H

R O R

Amino acid Polypeptide chain

FIGURE 1–17 Amino acid structure and formation of peptide bonds. The dashed line shows where peptide bonds are formed

between two amino acids. The highlighted area is released as H

O. R, remainder of the amino acid. For example, in glycine, R = H; in

glutamate, R = —(CH

–

)

—COO

The order of the amino acids in the peptide chains is called the primary structure of a protein. The chains are twisted and folded in

complex ways, and the term secondary structure of a protein refers to the spatial arrange ment produced by the twisting and folding. A

common secondary structure is a regular coil with 3.7 amino acid residues per turn (α- helix). Another common secondary structure is a

β-sheet. An antiparallel β-sheet is formed when extended polypeptide chains fold back and forth on one another and hydrogen bonding

occurs between the peptide bonds on neighboring chains. Parallel β-sheets between pol ypeptide chains also occur. The tertiary

structure of a protein is the arrangement of the twisted chains into layers, crystals, or fib ers. Many protein molecules are made of several

proteins, or subunits (eg, hemoglobin), and the term quaternary structure is used to refer to the a rrangement of the subunits into a

functional structure.

PROTEIN SYNTHESIS

The process of protein synthesis, translation, is the conversion of information en coded in mRNA to a protein (Figure 1–15). As

described previously, when a definitive mRNA reaches a ribosome in the cytoplasm, it dictates the formation of a polypeptide chain.

Amino acids in the cytoplasm are activated by combination with an enzyme and adenosi ne monophosphate (adenylate), and each

activated amino acid then combines with a specific molecule of tRNA. There is at least one tRNA for each of the 20 unmodified amino

acids found in large quantities in the body proteins of animals, but some amino acids hav e more than one tRNA. The tRNA–amino acid–

adenylate complex is next attached to the mRNA template, a process that occurs in the ri bosomes. The tRNA “recognizes” the proper

spot to attach on the mRNA template because it has on its active end a set of three bases that are complementary to a set of three bases in

a particular spot on the mRNA chain. The genetic code is made up of such triplets (c odons), sequences of three purine, pyrimidine, or

purine and pyrimidine bases; each codon stands for a particular amino acid.

Translation typically starts in the ribosomes with an AUG (transcribed from ATG in the gene), which codes for methionine. The amino

terminal amino acid is then added, and the chain is lengthened one amino acid at a time. The mRNA attaches to the 40S subunit of the

ribosome during protein synthesis, the polypeptide chain being formed attaches to the 6 0S subunit, and the tRNA attaches to both. As the

amino acids are added in the order dictated by the codon, the ribosome moves along th e mRNA molecule like a bead on a string.

Translation stops at one of three stop, or nonsense, codons (UGA, UAA, or UAG), an d the polypeptide chain is released. The tRNA

molecules are used again. The mRNA molecules are typically reused approximately 10 times before being replaced. It is common to

have more than one ribosome on a given mRNA chain at a time. The mRNA chain plus its collection of ribosomes is visible under the

electron microscope as an aggregation of ribosomes called a polyribosome.

POSTTRANSLATIONAL MODIFICATION

After the polypeptide chain is formed, it “folds” into its biological form and can be furth er modified to the final protein by one or more

of a combination of reactions that include hydroxylation, carboxylation, glycosylation, o r phosphorylation of amino acid residues;

cleavage of peptide bonds that converts a larger polypeptide to a smaller form; and the further folding, packaging, or folding and

packaging of the protein into its ultimate, often complex configuration. Protein folding is a complex process that is dictated primarily by

the sequence of the amino acids in the polypeptide chain. In some instances, however, n ascent proteins associate with other proteins

called chaperones, which prevent inappropriate contacts with other proteins and ensure that the final “proper” conformation of the

nascent protein is reached.

Proteins also contain information that helps to direct them to individual cell compartments . Many proteins that are going to be secreted or

stored in organelles and most transmembrane proteins have at their amino terminal a signal peptide (leader sequence) that guides them

into the endoplasmic reticulum. The sequence is made up of 15 to 30 predominantly hy drophobic amino acid residues. The signal

peptide, once synthesized, binds to a signal recognition particle (SRP), a complex molecule made up of six polypeptides and 7S RNA,

one of the small RNAs. The SRP stops translation until it binds to a translocon, a pore in the endoplasmic reticulum that is a

heterotrimeric structure made up of Sec 61 proteins. The ribosome also binds, and the s ignal peptide leads the growing peptide chain

into the cavity of the endoplasmic reticulum (Figure 1–18). The signal

5' 3'

UAA

SRP

NN NN

C C C C N

N N

FIGURE 1–18 Translation of protein into endoplasmic reticulum according to the signal hypothesis. The ribosomes synthesizing a

protein move along the mRNA from the 5' to the 3' end. When the signal pep tide of a protein destined for secretion, the cell membrane,

or lysosomes emerges from the large unit of the ribosome, it binds to a signal recognition particle (SRP), and this arrests further

translation until it binds to the translocon on the endoplasmic reticulum. N, amino end of protein; C, carboxyl end of protein. (Reproduced,

with permission, from Perara E, Lingappa VR: Transport of proteins into and across the endoplasm ic reticulum membrane. In: Protein Transfer and Organelle

Biogenesis. Das RC, Robbins PW (editors). Academic Press, 1988.)

peptide is next cleaved from the rest of the peptide by a signal peptidase while the rest o f the peptide chain is still being synthesized.

SRPs are not the only signals that help to direct proteins to their proper place in or out o f the cell; other signal sequences,

posttranslational modifications, or both (eg, glycosylation) can serve this function.

PROTEIN DEGRADATION

Like protein synthesis, protein degradation is a carefully regulated, complex process. It has been estimated that overall, up to 30% of

newly produced proteins are abnormal, such as can occur during improper folding. A ged normal proteins also need to be removed as

they are replaced. Conjugation of proteins to the 74-amino-acid polypeptide ubiquit in marks them for degradation. This polypeptide is

highly conserved and is present in species ranging from bacteria to humans. The proce ss of binding ubiquitin is called ubiquitination,

and in some instances, multiple ubiquitin molecules bind (polyubiquitination). Ubiquitin ation of cytoplasmic proteins, including

integral proteins of the endoplasmic reticulum, marks the proteins for degradation in mu ltisubunit proteolytic particles, or proteasomes.

Ubiquitination of membrane proteins, such as the growth hormone receptors, also mark s them for degradation, however these can be

degraded in lysosomes as well as via the proteasomes.

There is an obvious balance between the rate of production of a protein and its destruc tion, so ubiquitin conjugation is of major

importance in cellular physiology. The rates at which individual proteins are metabolized vary, and the body has mechanisms by which

abnormal proteins are recognized and degraded more rapidly than normal body consti tuents. For example, abnormal hemoglobins are

metabolized rapidly in individuals with congenital hemoglobinopathies.

CATABOLISM OF AMINO ACIDS

The short-chain fragments produced by amino acid, carbohydrate, and fat catabolism a re very similar (see below). From this common

metabolic pool of intermediates, carbohydrates, proteins, and fats can be synthesized. These fragments can enter the citric acid cycle, a

final common pathway of catabolism, in which they are broken down to hydrogen ato ms and CO

. Interconversion of amino acids

involve transfer, removal, or formation of amino groups. Transamination reactions, conversion of one amino acid to the corresponding

keto acid with simultaneous conversion of another keto acid to an amino acid, occur in many tissues:

Alanine +

-Ketoglutarate

←

→

Pyruvate + Glutamate

The transaminases involved are also present in the circulation. When damage to many active cells occurs as a result of a pathologic

process, serum transaminase levels rise. An example is the rise in plasma aspartate aminotr ansferase (AST) following myocardial

infarction.

Oxidative deamination of amino acids occurs in the liver. An imino acid is formed by dehydrogenation, and this compound is

hydrolyzed to the corresponding keto acid, with production of NH4+:

Amino acid + NAD

→ Imino acid + NADH + H

Imino acid + H

O → Keto acid + NH4+

Interconversions between the amino acid pool and the common metabolic pool are sum marized in Figure 1–19. Leucine, isoleucine,

phenylalanine, and tyrosine are said to be ketogenic because they are converted to th e ketone body acetoacetate (see below). Alanine

and many other amino acids are glucogenic or gluconeogenic; that is, they give rise to compou nds that can readily be converted to

glucose.

UREA FORMATION

Most of the NH 4+ formed by deamination of amino acids in the liver is converted to u rea, and the urea is excreted in the urine. The

NH4+ forms carbamoyl phosphate, and in the mitochondria it is transferred to ornithine , forming citrulline. The enzyme involved is

ornithine carbamoyltransferase. Citrulline is converted to arginine, after which urea is s plit off and ornithine is regenerated (urea cycle;

Figure 1–20). The overall reaction in the urea cycle consumes 3 ATP (not shown) and thus requires significant energy. Most of the urea

is formed in the liver, and in severe liver disease the blood urea nitrogen (BUN) falls a nd blood NH

rises (see Chapter 29). Congenital

deficiency of ornithine carbamoyltransferase can also lead to NH

intoxication, even in individuals who are heterozygous for this

deficiency.

Hydroxyproline Serine

Cysteine

Threonine

Glycine

Tryptophan Lactate

Transaminase Alanine

Pyruvate Acetyl-CoA

Glucose Phosphoenolpyruvate

carboxykinase

Phosphoenolpyruvate Oxaloacetate

Tyrosine

Phenylalanine Fumarate Transaminase

Aspartate Citrate

Isoleucine Methionine Valine

Succinyl-CoA

Propionate α-Ketoglutarate

Histidine Proline

Glutamine Arginine

Transaminase

Glutamate

FIGURE 1–19 Involvement of the citric acid cycle in transamination and gluco neogenesis. The bold arrows indicate the main

pathway of gluconeogenesis. Note the many entry positions for groups of amino acids into the citric acid cycle. (Reproduced with permission

from Murray RK et al: Harper’s Biochemistry, 26th ed. McGraw-Hill, 2003.)

METABOLIC FUNCTIONS

OF AMINO ACIDS

In addition to providing the basic building blocks for proteins, amino acids also have me tabolic functions. Thyroid hormones,

catecholamines, histamine, serotonin, melatonin, and intermediates in the urea cycle are f ormed from specific amino acids. Methionine

and cysteine provide the sulfur contained in proteins, CoA, taurine, and other biologica lly important compounds. Methionine is

converted into S-adenosylmethionine, which is the active methylating agent in the synthe sis of compounds such as epinephrine.

CARBOHYDRATES

Carbohydrates are organic molecules made of equal amounts of carbon and H

O. The simple sugars, or monosaccharides, including

pentoses (5 carbons; eg, ribose) and hexoses (6 carbons; eg, glucose) perform both structura l (eg, as part of nucleotides discussed

previously) and functional roles (eg, inositol 1,4,5 trisphosphate acts as a cellular signali ng molecules) in the body. Monosaccharides can

be linked together to form disaccharides (eg, sucrose), or polysaccharides (eg, glycog en). The placement of sugar moieties onto proteins

(glycoproteins) aids in cellular targeting, and in the case of some

Argininosuccinate

Aspartate Fumarate

Cyto

N H

—

O C

—

HN HN Citrulline

NO Arginine (CH

)

(CH

)

HC NH

Ornithine

−COO− COOH3N+

Mito (CH

)

3 Urea

HC NH3+ NH2

Carbamoyl

phosphate

COO− C

—

FIGURE 1–20 Urea cycle. The processing of NH

to urea for excretion contains several coordinative steps in both the cytoplasm (Cyto)

and the mitochondria (Mito). The production of carbamoyl phosphate and its c onversion to citrulline occurs in the mitochondria, whereas

other processes are in the cytoplasm.

H CO H CO CH

OH H COH H COH CO

the body because, except for the quantitatively unimportant production from glycerol, t here is no pathway for conversion.

HO CH HO CH HO CH H COH HO CH H COH H COH H COH H COH

OH CH

D-Glucose D-Galactose D-Fructose

FIGURE 1–21 Structures of principal dietary hexoses. Glucose, galactose, a nd fructose are shown in their naturally occurring D

isomers.

receptors, recognition of signaling molecules. In this section we will discuss a major role for carbohydrates in physiology, the production

and storage of energy.

Dietary carbohydrates are for the most part polymers of hexoses, of which the most im portant are glucose, galactose, and fructose

(Figure 1–21). Most of the monosaccharides occurring in the body are the D isomers. The principal product of carbohydrate digestion

and the principal circulating sugar is glucose. The normal fasting level of plasma glucos e in peripheral venous blood is 70 to 110 mg/dL

(3.9–6.1 mmol/ L). In arterial blood, the plasma glucose level is 15 to 30 mg/ dL higher than in venous blood.

Once it enters the cells, glucose is normally phosphorylated to form glucose 6-phosphat e. The enzyme that catalyzes this reaction is

hexokinase. In the liver, there is an additional enzyme called glucokina se, which has greater specificity for glucose and which, unlike

hexokinase, is increased by insulin and decreased in starvation and diabetes. The glucos e 6-phosphate is either polymerized into

glycogen or catabolized. The process of glycogen formation is called glycogenesis, and glycogen breakd own is called glycogenolysis.

Glycogen, the storage form of glucose, is present in most body tissues, but the major su pplies are in the liver and skeletal muscle. The

breakdown of glucose to pyruvate or lactate (or both) is called glycolysis. Glucos e catabolism proceeds via cleavage through fructose to

trioses or via oxidation and decarboxylation to pentoses. The pathway to pyruvate throu gh the trioses is the Embden–Meyerhof

pathway, and that through 6-phosphogluconate and the pentoses is the direct oxidative pathway (hexose monophosphate shunt).

Pyruvate is converted to acetyl-CoA. Interconversions between carbohydrate, fat, and protein include conversion of the glycerol from fats

to dihydroxyacetone phosphate and conversion of a number of amino acids with carbo n skeletons resembling intermediates in the

Embden–Meyerhof pathway and citric acid cycle to these intermediates by deamination. In this way, and by conversion of lactate to

glucose, nonglucose molecules can be converted to glucose (gluconeogenesis). Glucose c an be converted to fats through acetyl-CoA, but

because the conversion of pyruvate to acetyl-CoA, unlike most reactions in glycolysis, is irreversible, fats are not converted to glucose

via this pathway. There is therefore very little net conversion of fats to carbohydrates in

CITRIC ACID CYCLE

The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is a sequence of reactions in which ac etyl-CoA is metabolized to CO

and H

atoms. Acetyl-CoA is first condensed with the anion of a four-carbon acid, oxaloacetat e, to form citrate and HS-CoA. In a series of seven

subsequent reactions, 2CO

molecules are split off, regenerating oxaloacetate (Figure 1–22). Four pairs of H atom s are transferred to the

flavoprotein– cytochrome chain, producing 12ATP and 4H

O, of which 2H

O is used in the cycle. The citric acid cycle is the common

pathway for oxidation to CO

and H

O of carbohydrate, fat, and some amino acids. The major entry into it is through acetylC oA, but a

number of amino acids can be converted to citric acid cycle intermediates by deaminatio n. The citric acid cycle requires O

and does not

function under anaerobic conditions.

ENERGY PRODUCTION

The net production of energy-rich phosphate compounds during the metabolism of glu cose and glycogen to pyruvate depends on

whether metabolism occurs via the Embden– Meyerhof pathway or the hexose monop hosphate shunt. By oxidation at the substrate level,

the conversion of 1 mol of phosphoglyceraldehyde to phosphoglycerate generates 1 m ol of ATP, and the conversion of 1 mol of

phosphoenolpyruvate of ATP, and the conversion of 1 mol of phosphoenolpyruvate phosp hate produces, via the Embden–Meyerhof

pathway, 2 mol of phosphoglyceraldehyde, 4 mol of ATP is generated per mole of glu cose metabolized to pyruvate. All these reactions

occur in the absence of O

and consequently represent anaerobic production of energy. However, 1 mol of ATP is used in forming

fructose 1,6-diphosphate from fructose 6-phosphate and 1 mol in phosphorylating glu cose when it enters the cell. Consequently, when

pyruvate is formed anaerobically from glycogen, there is a net production of 3 mol of ATP per mole of glucose 6-phosphate; however,

when pyruvate is formed from 1 mol of blood glucose, the net gain is only 2 mol of A TP.

A supply of NAD

is necessary for the conversion of phosphoglyceraldehyde to phosphoglycerate. Und er anaerobic conditions

(anaerobic glycolysis), a block of glycolysis at the phosphoglyceraldehyde conversion step might be expected to develop as soon as the

available NAD

is converted to NADH. However, pyruvate can accept hydrogen from NADH, form ing NAD

and lactate:

Pyruvate + NADH

←

Lactate + NAD

→

In this way, glucose metabolism and energy production can continue for a while withou t O

. The lactate that accumulates is converted

back to pyruvate when the O

supply is restored, with NADH transferring its hydrogen to the flavoprotein– cytochro me chain.

Pyruvate 3C

NAD

NADH + H

+ CO2

Acetyl-CoA 2C

Oxaloacetate 4C

NADH + H+

Citrate 6C NAD

Malate 4C

Isocitrate 6C

Fumarate 4C

NAD+

FADH

2 CO2

NADH + H

FAD

-Ketoglutarate 5C Succinate 4C

P CO

2 GTP

NAD

Succinyl-CoA 4C NADH

H+

GDP

FIGURE 1–22 Citric acid cycle. The numbers (6C, 5C, etc) indicate the number o f carbon atoms in each of the intermediates. The

conversion of pyruvate to acetyl-CoA and each turn of the cycle provide four NADH an d one FADH

for oxidation via the flavoprotein-

cytochrome chain plus formation of one GTP that is readily converted to ATP.

During aerobic glycolysis, the net production of ATP is 19 times greater than the two A TPs formed under anaerobic conditions. Six

ATPs are formed by oxidation via the flavoprotein–cytochrome chain of the two NAD Hs produced when 2 mol of

phosphoglyceraldehyde is converted to phosphoglycerate (Figure 1–22), six ATPs are formed from the two NADHs produced when 2

mol of pyruvate is converted to acetyl-CoA, and 24 ATPs are formed during the subse quent two turns of the citric acid cycle. Of these,

18 are formed by oxidation of six NADHs, 4 by oxidation of two FADH

s, and 2 by oxidation at the substrate level when succinyl-CoA

is converted to succinate. This reaction actually produces GTP, but the GTP is converted to ATP. Thus, the net production of ATP per

mol of blood glucose metabolized aerobically via the Embden–Meyerhof pathway and citric acid cycle is 2 + [2 × 3] + [2 × 3] + [2 × 12]

= 38.

Glucose oxidation via the hexose monophosphate shunt generates large amounts of NA DPH. A supply of this reduced coenzyme is

essential for many metabolic processes. The pentoses formed in the process are building blocks for nucleotides (see below). The amount

of ATP generated depends on the amount of NADPH converted to NADH and then o xidized.

“DIRECTIONAL-FLOW VALVES”

Metabolism is regulated by a variety of hormones and other factors. To bring about an y net change in a particular metabolic process,

regulatory factors obviously must drive a chemical reaction in one direction. Most of th e reactions in intermediary metabolism are freely

reversible, but there are a number of “directional-flow valves,” ie, reactions that procee d in one direction under the influence of one

enzyme or transport mechanism and in the opposite direction under the influence of an other. Five examples in the intermediary

metabolism of carbohydrate are shown in Figure 1–23. The different pathways for fat ty acid synthesis and catabolism (see below) are

another example. Regulatory factors exert their influence on metabolism by acting direc tly or indirectly at these directional-flow valves.

GLYCOGEN SYNTHESIS & BREAKDOWN

Glycogen is a branched glucose polymer with two types of glycoside linkages: 1:4α and 1:6α (Figure 1–24). It is synthesized on

glycogenin, a protein primer, from glucose 1-phosphate via uridine diphosphogluco se (UDPG). The enzyme glycogen synthase

catalyses the final synthetic step. The availability of

1. Glucose entry into cells and glucose exit from cells

Hexokinase

2. Glucose Glucose 6-phosphate Glucose 6-phosphatase

glycogenin is one of the factors determining the amount of glycogen synthesized. The b reakdown of glycogen in 1:4α linkage is

catalyzed by phosphorylase, whereas another enzyme catalyzes the breakdown of gly cogen in 1:6α linkage.

Glycogen synthase 3. Glucose 1-phosphate Glycogen Phosphorylase

Phospho

fructokinase

4. Fructose 6-phosphate

5. Phosphoenolpyruvate 5. Phosphoenolpyruvate 5. Phosphoenolpyr uvate

biphosphate

biphosphatase

ADP ATP

Pyruvate kinase

Pyruvate

Phosphoenolpyruvate carboxykinase Oxaloacetate

Pyruvate

Oxaloacetate

Malate Malate

FIGURE 1–23 Directional flow valves in energy production reactions. In carbohydrate met abolism there are several reactions that

proceed in one direction by one mechanism and in the other direction by a diff erent mechanism, termed “directional-flow valves.” Five

examples of these reactions are illustrated (numbered at left). The double line in example 5 repr esents the mitochondrial membrane.

Pyruvate is converted to malate in mitochondria, and the malate diffuses out of the mitochondria to the cytosol, where it is converted to

phosphoenolpyruvate.

FACTORS DETERMINING THE

PLASMA GLUCOSE LEVEL

The plasma glucose level at any given time is determined by the balance between the am ount of glucose entering the bloodstream and

the amount leaving it. The principal determinants are therefore the dietary intake; the rate of entry into the cells of muscle, adipose

tissue, and other organs; and the glucostatic activity of the liver (Figure 1–25). Five perc ent of ingested glucose is promptly converted

into glycogen in the liver, and 30–40% is converted into fat. The remainder is metaboliz ed in muscle and other tissues. During fasting,

liver glycogen is broken down and the liver adds glucose to the bloodstream. With mor e prolonged fasting, glycogen is depleted and

there is increased gluconeogenesis from amino acids and glycerol in the liver. Plasma gl ucose declines modestly to about 60 mg/dL

during prolonged starvation in normal individuals, but symptoms of hypoglycemia do n ot occur because gluconeogenesis prevents any

further fall.

OH CH

OH O O

1:6α linkage

OH CH

OH Glycogen

O O O

OH CH

O O

O O O O O Glycogen synthase 1:4α linkage

Uridine

Phosphorylase a

diphospho

glucose

O−

OH CH

O PO O

O−

O PO

−

Glucose Glucose

1-phosphate 6-phosphate

FIGURE 1–24 Glycogen formation and breakdown. Glycogen is the main storage for glu cose in the cell. It is cycled: built up from

glucose

6-phosphate when energy is stored and broken down to glucose 6-phosphate when e nergy is required. Note the intermediate glucose

1-phosphate and enzymatic control by phosphorylase a and glycogen kinase.

Diet

Amino Glycerol acids

Intestine Liver Lactate

Plasma glucose 70 mg/dL

(3.9 mmol/L)

Kidney Brain Fat Muscle and other tissues

Urine (when plasma glucose > 180 mg/dL)

the intestines and liver, so its value in replenishing carbohydrate elsewhere in the body i s limited.

Fructose 6-phosphate can also be phosphorylated in the 2 position, forming fructose 2, 6-diphosphate. This compound is an important

regulator of hepatic gluconeogenesis. When the fructose 2,6-diphosphate level is high, conversion of fructose 6-phosphate to fructose

1,6-diphosphate is facilitated, and thus breakdown of glucose to pyruvate is increased. A decreased level of fructose 2,6-diphosphate

facilitates the reverse reaction and consequently aids gluconeogenesis.

FIGURE 1–25 Plasma glucose homeostasis. Notice the glucostat ic function of the liver, as well as the loss of glucose in the urine when

the renal threshold is exceeded (dashed arrows).

METABOLISM OF HEXOSES

OTHER THAN GLUCOSE

Other hexoses that are absorbed from the intestine include galactose, which is liberated b y the digestion of lactose and converted to

glucose in the body; and fructose, part of which is ingested and part produced by hydr olysis of sucrose. After phosphorylation, galactose

reacts with uridine diphosphoglucose (UDPG) to form uridine diphosphogalactose. Th e uridine diphosphogalactose is converted back to

UDPG, and the UDPG functions in glycogen synthesis. This reaction is reversible, and conversion of UDPG to uridine

diphosphogalactose provides the galactose necessary for formation of glycolipids and m ucoproteins when dietary galactose intake is

inadequate. The utilization of galactose, like that of glucose, depends on insulin. In the in born error of metabolism known as

galactosemia, there is a congenital deficiency of galactose 1-phosphate uridyl transfe rase, the enzyme responsible for the reaction

between galactose 1-phosphate and UDPG, so that ingested galactose accumulates in the circulation. Serious disturbances of growth and

development result. Treatment with galactose-free diets improves this condition without l eading to galactose deficiency, because the

enzyme necessary for the formation of uridine diphosphogalactose from UDPG is pre sent.

Fructose is converted in part to fructose 6-phosphate and then metabolized via fructose 1,6-diphosphate. The enzyme catalyzing the

formation of fructose 6-phosphate is hexokinase, the same enzyme that catalyzes the co nversion of glucose to glucose 6-phosphate.

However, much more fructose is converted to fructose 1-phosphate in a reaction cataly zed by fructokinase. Most of the fructose 1-

phosphate is then split into dihydroxyacetone phosphate and glyceraldehyde. The glyce raldehyde is phosphorylated, and it and the

dihydroxyacetone phosphate enter the pathways for glucose metabolism. Because the r eactions proceeding through phosphorylation of

fructose in the 1 position can occur at a normal rate in the absence of insulin, it has been recommended that fructose be given to

diabetics to replenish their carbohydrate stores. However, most of the fructose is metab olized in

FATTY ACIDS & LIPIDS

The biologically important lipids are the fatty acids and their derivatives, the neutral fats ( triglycerides), the phospholipids and related

compounds, and the sterols. The triglycerides are made up of three fatty acids bound to glycerol (Table 1–4). Naturally occurring fatty

acids contain an even number of carbon atoms. They may be saturated (no double bon ds) or unsaturated (dehydrogenated, with various

numbers of double bonds). The phospholipids are constituents of cell membranes and p rovide structural components of the cell

membrane, as well as an important source of intra- and intercellular signaling molecules . Fatty acids also are an important source of

energy in the body.

FATTY ACID OXIDATION & SYNTHESIS

In the body, fatty acids are broken down to acetyl-CoA, which enters the citric acid cy cle. The main breakdown occurs in the

mitochondria by β-oxidation. Fatty acid oxidation begins with activation (formation of th e CoA derivative) of the fatty acid, a reaction

that occurs both inside and outside the mitochondria. Medium- and short-chain fatty aci ds can enter the mitochondria without difficulty,

but long-chain fatty acids must be bound to carnitine in ester linkage before they can cross the inner mitochondrial membrane. Carnitine

is β-hydroxyγ-trimethylammonium butyrate, and it is synthesized in the body from lysin e and methionine. A translocase moves the fatty

acid–carnitine ester into the matrix space. The ester is hydrolyzed, and the carnitine recy cles. β-oxidation proceeds by serial removal of

two carbon fragments from the fatty acid (Figure 1–26). The energy yield of this proc ess is large. For example, catabolism of 1 mol of a

six-carbon fatty acid through the citric acid cycle to CO

and H

O generates 44 mol of ATP, compared with the 38 mol generated by

catabolism of 1 mol of the six-carbon carbohydrate glucose.

KETONE BODIES

In many tissues, acetyl-CoA units condense to form acetoacetylCoA (Figure 1–27). In the liver, which (unlike other tissues) contains a

deacylase, free acetoacetate is formed. This β-keto acid is converted to β-hydroxybutyr ate and acetone, and because these compounds are

metabolized with difficulty in

TABLE 1–4. Lipids.

Typical fatty acids:

Palmitic acid: CH

(CH

)

—C—OH O

Stearic acid: CH

(CH

)

—C—OH O

Oleic acid: CH

(CH

)

CH=CH(CH

)

—C—OH (Unsaturated)

Triglycerides (triacylglycerols): Esters of glycerol and three fatty acids.

O CH

—O—C—R CH

O O

—O—C—R + 3H

O CHOH + 3HO—C—R O

—O—C—R CH

OH Triglyceride Glycerol

R = Aliphatic chain of various lengths and degrees of saturation.

Phospholipids:

A. Esters of glycerol, two fatty acids, and

1. Phosphate = phosphatidic acid

2. Phosphate plus inositol = phosphatidylinositol

3. Phosphate plus choline = phosphatidylcholine (lecithin)

4. Phosphate plus ethanolamine = phosphatidyl-ethanolamine (cephalin)

5. Phosphate plus serine = phosphatidylserine

CELLULAR LIPIDS

The lipids in cells are of two main types: structural lipids, which are an inherent part of the membranes and other parts of cells; and

neutral fat, stored in the adipose cells of the fat depots. Neutral fat is mobi lized during starvation, but structural lipid is preserved. The

fat depots obviously vary in size, but in nonobese individuals they make up about 15% of body weight in men and 21% in women. They

are not the inert structures they were once thought to be but, rather, active dynamic tissu es undergoing continuous breakdown and

resynthesis. In the depots, glucose is metabolized to fatty acids, and neutral fats are synth esized. Neutral fat is also broken down, and

free fatty acids are released into the circulation.

A third, special type of lipid is brown fat, which makes up a small percentage of total bo dy fat. Brown fat, which is somewhat more

abundant in infants but is present in adults as well, is located between the scapulas, at the nape of the neck, along the great vessels in the

thorax and abdomen, and in other scattered locations in the body. In brown fat depots, the fat cells as well as the blood vessels have an

extensive sympathetic innervation. This is in contrast to white fat depots, in which some f at cells may be innervated but the principal

sympathetic innervation is solely on blood vessels. In addition, ordinary lipocytes have o nly a single large droplet of white fat, whereas

brown fat cells contain several small droplets of fat. Brown fat cells also contain many m itochondria. In these mitochondria, an inward

proton conductance that generates ATP takes places as usual, but in addition there is a se cond proton conductance that does not generate

ATP. This “shortcircuit” conductance depends on a 32-kDa uncoupling protein (UCP1 ). It causes uncoupling of metabolism and

generation of ATP, so that more heat is produced.

B. Other phosphate-containing derivatives of glycerol

C. Sphingomyelins: Esters of fatty acid, phosphate, choline, and the amino alcohol sph ingosine.

Cerebrosides: Compounds containing galactose, fatty acid, and sphingosine.

Sterols: Cholesterol and its derivatives, including steroid hormones, bile acids, and various vitam ins.

the liver, they diffuse into the circulation. Acetoacetate is also formed in the liver via the formation of 3-hydroxy-3-methylglutaryl-CoA,

and this pathway is quantitatively more important than deacylation. Acetoacetate, β-hydr oxybutyrate, and acetone are called ketone

bodies. Tissues other than liver transfer CoA from succinyl-CoA to acetoaceta te and metabolize the “active” acetoacetate to CO

and

O via the citric acid cycle. Ketone bodies are also metabolized via other pathways. Aceto ne is discharged in the urine and expired air.

An imbalance of ketone bodies can lead to serious health problems (Clinical Box 1–3).

PLASMA LIPIDS & LIPID TRANSPORT

The major lipids are relatively insoluble in aqueous solutions and do not circulate in the f ree form. Free fatty acids (FFAs) are bound to

albumin, whereas cholesterol, triglycerides, and phospholipids are transported in the fo rm of lipoprotein complexes. The complexes

greatly increase the solubility of the lipids. The six families of lipoproteins (Table 1–5) ar e graded in size and lipid content. The density

of these lipoproteins is inversely proportionate to their lipid content. In general, the lipop roteins consist of a hydrophobic core of

triglycerides and cholesteryl esters surrounded by phospholipids and protein. These lipo proteins can be transported from the intestine to

the liver via an exogenous pathway, and between other tissues via an endogenous pathway.

Dietary lipids are processed by several pancreatic lipases in the intestine to form mixed m icelles of predominantly FFA, 2-

monoglycerols, and cholesterol derivatives (see Chapter 27). These micelles ad ditionally can contain important water-insoluble

molecules such as vitamins A, D, E, and K. These mixed micelles a re taken up into cells of the intestinal

Fatty acid "Active" fatty acid

O O Mg

R CH

C OH + HS-CoA

ATP ADP

O + R CH

C S CoA

Oxidized

flavoprotein

Reduced

flavoprotein

OH O O R C CH

—

CH C S CoA

C S CoA H

O + R CH

H β-Hydroxy fatty acid–CoA α,β-Unsaturated fatty acid–CoA NAD

NADH + H

O O O O R C CH

CS CoA + HS-CoA R C S CoA + CH

CS CoA

β-Keto fatty acid–CoA "Active" fatty acid + Acetyl–CoA

R = Rest of fatty acid chain. FIGURE 1–26 Fatty acid oxidation. This process, splitting off two ca rbon fragments at a time, is repeated

to the end of the chain.

O O

β-Ketothiolase

O O

C S CoA + CH

C S CoA CH

C CH

C S CoA + HS-CoA 2 Acetyl-CoA Acetoacetyl-CoA

CH O O O O

Deacylase

C CH

C O

−

+ H

+ HS-CoA

C CH

C S CoA + H

(liver only)

Acetoacetyl-CoA Acetoacetate

OH O

Acetyl-CoA + Acetoacetyl-CoA CH

C CH

C S CoA + H

COO

−

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)

HMG-CoA Acetoacetate + H

+ Acetyl-CoA

Acetoacetate

O O

−

+ H

Tissues except liver CO

+ ATP

–CO

+2H –2H

C CH

3 O

Acetone

CHOH CH

C O

−

+ H

β-Hydroxybutyrate FIGURE 1–27 Formation and metabolism of ketone bodies. No te the two pathways

for the formation of acetoacetate.

CLINICAL BOX 1–3 Diseases Associated with Imbalance of β-oxidation of F atty Acids

Ketoacidosis

The normal blood ketone level in humans is low (about 1 mg/dL) and less than 1 mg is excreted per 24 h, because the ketones are

normally metabolized as rapidly as they are formed. However, if the entry of acetyl-Co A into the citric acid cycle is depressed because

of a decreased supply of the products of glucose metabolism, or if the entry does not in crease when the supply of acetyl-CoA increases,

acetyl-CoA accumulates, the rate of condensation to acetoacetyl-CoA increases, and mo re acetoacetate is formed in the liver. The ability

of the tissues to oxidize the ketones is soon exceeded, and they accumulate in the bloods tream (ketosis). Two of the three ketone bodies,

acetoacetate and β-hydroxybutyrate, are anions of the moderately strong acids acetoace tic acid and β-hydroxybutyric acid. Many of their

protons are buffered, reducing the decline in pH that would otherwise occur. Howeve r, the buffering capacity can be exceeded, and the

metabolic acidosis that develops in conditions such as diabetic ketosis can be severe

mucosa where large lipoprotein complexes, chylomicrons, are formed. The chylomicrons and their remn ants constitute a transport

system for ingested exogenous lipids (exogenous pathway). Chylomicrons can enter th e circulation via the lymphatic ducts. The

chylomicrons are cleared from the circulation by the action of lipoprotein lipase , which is located on the surface of the endothelium of

the capillaries. The enzyme catalyzes the breakdown of the triglyceride in the chylomicr ons to FFA and glycerol, which then enter

adipose

and even fatal. Three conditions lead to deficient intracellular glucose supplies, and henc e to ketoacidosis: starvation; diabetes mellitus;

and a high-fat, low-carbohydrate diet. The acetone odor on the breath of children who have been vomiting is due to the ketosis of

starvation. Parenteral administration of relatively small amounts of glucose abolishes the k etosis, and it is for this reason that

carbohydrate is said to be antiketogenic.

Carnitine Deficiency

Deficient β-oxidation of fatty acids can be produced by carnitine deficiency or genetic defects in the translocase or other enzymes

involved in the transfer of long-chain fatty acids into the mitochondria. This causes card iomyopathy. In addition, it causes

hypoketonemic hypoglycemia with coma, a serious and often fatal condition triggered by fasting, in which glucose stores are used up

because of the lack of fatty acid oxidation to provide energy. Ketone bodies are not fo rmed in normal amounts because of the lack of

adequate CoA in the liver.

cells and are reesterified. Alternatively, the FFA can remain in the circulation bound to a lbumin. Lipoprotein lipase, which requires

heparin as a cofactor, also removes triglycerides from circulating very low density lipop roteins (VLDL). Chylomicrons depleted of

their triglyceride remain in the circulation as cholesterol-rich lipoproteins called chylom icron remnants, which are 30 to 80 nm in

diameter. The remnants are carried to the liver, where they are internalized and degrad ed.

TABLE 1–5 The principal lipoproteins.*

Composition (%)

Lipoprotein Size (nm) Protein Free Cholesterol

Cholesteryl Esters Triglyceride Phospholipid Origin

Chylomicrons 75–1000 2 2 3 90 3 Intestine

Chylomicron remnants 30–80 … … … … … Capillaries

Very low density lipoproteins 30–80 (VLDL)

17 Liver and intestine 8 4 16 55

Intermediate-density lipo25–40 proteins (IDL)

10 5 25 40 20 VLDL

Low-density lipoproteins 20 (LDL)

20 7 46 6 21 IDL

High-density lipoproteins 7.5–10 (HDL)

50 4 16 5 25 Liver and intestine

*The plasma lipids include these components plus free fatty acids from adipose tissue, which circulate bou nd to albumin.

The endogenous system, made up of VLDL, intermediate-density lipoproteins (IDL), low -density lipoproteins (LDL), and high-

density lipoproteins (HDL), also transports triglycerides and cholesterol throughout t he body. VLDL are formed in the liver and

transport triglycerides formed from fatty acids and carbohydrates in the liver to extrahe patic tissues. After their triglyceride is largely

removed by the action of lipoprotein lipase, they become IDL. The IDL give up phosp holipids and, through the action of the plasma

enzyme lecithin-cholesterol acyltransferase (LCAT), pick up cholester yl esters formed from cholesterol in the HDL. Some IDL are

taken up by the liver. The remaining IDL then lose more triglyceride and protein, prob ably in the sinusoids of the liver, and become

LDL. LDL provide cholesterol to the tissues. The cholesterol is an essential constituent in cell membranes and is used by gland cells to

make steroid hormones.

FREE FATTY ACID METABOLISM

In addition to the exogenous and endogenous pathways described above, FFA are also synthesized in the fat depots in which they are

stored. They can circulate as lipoproteins bound to albumin and are a major source of e nergy for many organs. They are used extensively

in the heart, but probably all tissues can oxidize FFA to CO

and H

The supply of FFA to the tissues is regulated by two lipases. As noted above, lipoprotein lipase on the surface of the endothelium of the

capillaries hydrolyzes the triglycerides in chylomicrons and VLDL, providing FFA and glycerol, which are reassembled into new

triglycerides in the fat cells. The intracellular hormone-sensitive lipase of adip ose tissue catalyzes the breakdown of stored triglycerides

into glycerol and fatty acids, with the latter entering the circulation. Hormone-sensitive lip ase is increased by fasting and stress and

decreased by feeding and insulin. Conversely, feeding increases and fasting and stress decrease the activity of lipoprotein lipase.

CHOLESTEROL METABOLISM

Cholesterol is the precursor of the steroid hormones and bile acids and is an essential cons tituent of cell membranes. It is found only in

animals. Related sterols occur in plants, but plant sterols are not normally absorbed from the gastrointestinal tract. Most of the dietary

cholesterol is contained in egg yolks and animal fat.

Cholesterol is absorbed from the intestine and incorporated into the chylomicrons forme d in the intestinal mucosa. After the

chylomicrons discharge their triglyceride in adipose tissue, the chylomicron remnants br ing cholesterol to the liver. The liver and other

tissues also synthesize cholesterol. Some of the cholesterol in the liver is excreted in the b ile, both in the free form and as bile acids.

Some of the biliary cholesterol is reabsorbed from the intestine. Most of the cholesterol i n the liver is incorporated into VLDL and

circulates in lipoprotein complexes.

The biosynthesis of cholesterol from acetate is summarized in Figure 1–28. Cholesterol feeds back to inhibit its own synthesis by

inhibiting HMG-CoA reductase, the enzyme that converts 3-hydroxy-3-methylglutaryl-coen zyme A (HMG-CoA) to mevalonic acid.

Thus, when dietary cholesterol intake is high, hepatic cholesterol synthesis is decreased, and vice versa. However, the feedback

compensation is incomplete, because a diet that is low in cholesterol and saturated fat lead s to only a modest decline in circulating

plasma cholesterol. The most effective and most commonly used cholesterol-lowering d rugs are lovastatin and other statins, which

reduce cholesterol synthesis by inhibiting HMG-CoA. The relationship between choleste rol and vascular disease is discussed in Clinical

Box 1–4.

ESSENTIAL FATTY ACIDS

Animals fed a fat-free diet fail to grow, develop skin and kidney lesions, and become in fertile. Adding linolenic, linoleic, and

arachidonic acids to the diet cures all the deficiency symptoms. These three acids are po lyunsaturated fatty acids and because of their

action are called essential fatty acids. Similar deficiency symptoms h ave not been unequivocally demonstrated in humans, but there is

reason to believe that some unsaturated fats are essential dietary constituents, especially f or children.

Acetyl-CoA

Acetoacetyl-CoA

Acetoacetyl-CoA methylglutaryl-CoA HMG-CoA reductase

Mevalonic acid

Acetoacetate Acetoacetate Squalene

Cholesterol

HOOC CH

COH Squalene

) HO Mevalonic acid Cholesterol (C

FIGURE 1–28 Biosynthesis of cholesterol. Six mevalonic acid molecules condense to form squal ene, which is then hydroxylated to

cholesterol. The dashed arrow indicates feedback inhibition by cholesterol of HMG-CoA reductase, the e nzyme that catalyzes mevalonic

acid formation.

CLINICAL BOX 1–4 CLINICAL BOX 1–5 Cholesterol & Athero sclerosis

The interest in cholesterol-lowering drugs stems from the role of cholesterol in the etiolo gy and course of atherosclerosis. This

extremely widespread disease predisposes to myocardial infarction, cerebral thrombosis , ischemic gangrene of the extremities, and other

serious illnesses. It is characterized by infiltration of cholesterol and oxidized cholesterol into macrophages, converting them into foam

cells in lesions of the arterial walls. This is followed by a complex sequence of changes involving platelets, macrophages, smooth muscle

cells, growth factors, and inflammatory mediators that produces proliferative lesions wh ich eventually ulcerate and may calcify. The

lesions distort the vessels and make them rigid. In individuals with elevated plasma choles terol levels, the incidence of atherosclerosis

and its complications is increased. The normal range for plasma cholesterol is said to be 120 to 200 mg/dL, but in men, there is a clear,

tight, positive correlation between the death rate from ischemic heart disease and plasma cholesterol levels above 180 mg/dL.

Furthermore, it is now clear that lowering plasma cholesterol by diet and drugs slows an d may even reverse the progression of

atherosclerotic lesions and the complications they cause.

In evaluating plasma cholesterol levels in relation to atherosclerosis, it is important to anal yze the LDL and HDL levels as well. LDL

delivers cholesterol to peripheral tissues, including atheromatous lesions, and the LDL pl asma concentration correlates positively with

myocardial infarctions and ischemic strokes. On the other hand, HDL picks up choleste rol from peripheral tissues and transports it to the

liver, thus lowering plasma cholesterol. It is interesting that women, who have a lower in cidence of myocardial infarction than men, have

higher HDL levels. In addition, HDL levels are increased in individuals who exercise an d those who drink one or two alcoholic drinks

per day, whereas they are decreased in individuals who smoke, are obese, or live sede ntary lives. Moderate drinking decreases the

incidence of myocardial infarction, and obesity and smoking are risk factors that increa se it. Plasma cholesterol and the incidence of

cardiovascular diseases are increased in familial hypercholesterolemia, due to vari ous loss-of-function mutations in the genes for LDL

receptors.

Dehydrogenation of fats is known to occur in the body, but there does not appear to b e any synthesis of carbon chains with the

arrangement of double bonds found in the essential fatty acids.

EICOSANOIDS

One of the reasons that essential fatty acids are necessary for health is that they are the p recursors of prostaglandins, prostacyclin,

thromboxanes, lipoxins, leukotrienes, and related compounds. These substances are cal led eicosanoids, reflecting

Pharmacology of Prostaglandins

Because prostaglandins play a prominent role in the genesis of pain, inflammation, and fever, pharmacologists have long sought drugs to

inhibit their synthesis. Glucocorticoids inhibit phospholipase A

and thus inhibit the formation of all eicosanoids. A variety of

nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both cyclooxygenases, inhibiting the production of PGH

and its derivatives.

Aspirin is the bestknown of these, but ibuprofen, indomethacin, and others are also use d. However, there is evidence that prostaglandins

synthesized by COX2 are more involved in the production of pain and inflammation, a nd prostaglandins synthesized by COX1 are more

involved in protecting the gastrointestinal mucosa from ulceration. Drugs such as celeco xib and rofecoxib that selectively inhibit COX2

have been developed, and in clinical use they relieve pain and inflammation, possibly w ith a significantly lower incidence of

gastrointestinal ulceration and its complications than is seen with nonspecific NSAIDs. H owever, rofecoxib has been withdrawn from the

market in the United States because of a reported increase of strokes and heart attacks in individuals using it. More research is underway

to better understand all the effects of the COX enzymes, their products, and their inhibi tors.

their origin from the 20-carbon (eicosa-) polyunsaturated fatty acid arachidonic acid (arachidonate) and the 20-carbon derivatives of

linoleic and linolenic acids.

The prostaglandins are a series of 20-carbon unsaturated fatty acids containing a cyclopen tane ring. They were first isolated from semen

but are now known to be synthesized in most and possibly in all organs in the body. Pr ostaglandin H

(PGH

) is the precursor for

various other prostaglandins, thromboxanes, and prostacyclin. Arachidonic acid is form ed from tissue phospholipids by phospholipase

. It is converted to prostaglandin H

(PGH

) by prostaglandin G/H synthases 1 and 2. These are bifunctional enzymes that have both

cyclooxygenase and peroxidase activity, but they are more commonly known by the n ames cyclooxygenase 1 (COX1) and

cyclooxygenase 2 (COX2). Their structures are very similar, but COX1 is constitutive where as COX2 is induced by growth factors,

cytokines, and tumor promoters. PGH

is converted to prostacyclin, thromboxanes, and prostaglandins by various tissue isome rases. The

effects of prostaglandins are multitudinous and varied. They are particularly important i n the female reproductive cycle, in parturition, in

the cardiovascular system, in inflammatory responses, and in the causation of pain. Dru gs that target production of prostaglandins are

among the most common over the counter drugs available (Clinical Box 1–5).

Arachidonic acid also serves as a substrate for the production of several physiologically important leukotrienes and lipoxins. The

leukotrienes, thromboxanes, lipoxins, and prostaglandins have been called local hormon es. They have short half-lives and are inactivated

in many different tissues. They undoubtedly act mainly in the tissues at sites in which the y are produced. The leukotrienes are mediators

of allergic responses and inflammation. Their release is provoked when specific allerge ns combine with IgE antibodies on the surfaces of

mast cells (see Chapter 3). They produce bronchoconstriction, constrict arterioles, incre ase vascular permeability, and attract neutrophils

and eosinophils to inflammatory sites. Diseases in which they may be involved include a sthma, psoriasis, adult respiratory distress

syndrome, allergic rhinitis, rheumatoid arthritis, Crohn’s disease, and ulcerative colitis.

CHAPTER SUMMARY

■ Cells contain approximately one third of the body fluids, while the remaining extracellular fluid is found between cells (interstitial

fluid) or in the circulating blood plasma.

■ The number of molecules, electrical charges, and particles of substances in sol ution are important in physiology.

■ The high surface tension, high heat capacity, and high electrical capacity allow H

O to function as an ideal solvent in physiology.

■ Biological buffers including bicarbonate, proteins, and phosphates can bind or rel ease protons in solution to help maintain pH.

Biological buffering capacity of a weak acid or base is greatest when pK

= pH.

■ Fluid and electrolyte balance in the body is related to plasma osmolality. Isotonic sol utions have the same osmolality as blood plasma,

hypertonic have higher osmolality, while hypotonic have lower osmolality.

■ Although the osmolality of solutions can be similar across a plasma membrane, the distrib ution of individual molecules and

distribution of charge across the plasma membrane can be quite different. These are affect ed by the Gibbs-Donnan equilibrium and can

be calculated using the Nernst potential equation.

■ There is a distinct difference in concentration of ions in the extracellular and intracellular fluids (concentration gradient). The

separation of concentrations of charged species sets up an electrical gradient at the plas ma membrane (inside negative). The

electrochemical gradient is in large part maintained by the Na, K ATPase.

■ Cellular energy can be stored in high-energy phosphate compounds, including ad enosine triphosphate (ATP). Coordinated oxidation-

reduction reactions allow for production of a proton gradient at the inner mitocho ndrial membrane that ultimately yields to the

production of ATP in the cell.

■ Nucleotides made from purine or pyrimidine bases linked to ribose or 2-deoxyrib ose sugars with inorganic phosphates are the basic

building blocks for nucleic acids, DNA, and RNA.

■ DNA is a double-stranded structure that contains the fundamental information for an organism. During cell division, DNA is faithfully

replicated and a full copy of DNA is in every cell. The fundamental unit of DNA is the ge ne, which encodes information to make

proteins in the cell. Genes are transcribed into messenger RNA, and with the help of r ibosomal RNA and transfer RNAs, translated into

proteins.

■ Amino acids are the basic building blocks for proteins in the cell and can also serve as sour ces for several biologically active

molecules. They exist in an “amino acid pool” that is derived from the diet, protein degradat ion, and de novo and resynthesis.

■ Translation is the process of protein synthesis. After synthesis, proteins ca n undergo a variety of posttranslational modifications prior

to obtaining their fully functional cell state.

■ Carbohydrates are organic molecules that contain equal amounts of C and H

O. Carbohydrates can be attached to proteins

(glycoproteins) or fatty acids (glycolipids) and are critically important for the production and storage of cellular and body energy, with

major supplies in the form of glycogen in the liver and skeletal muscle. The breakdown of glucose to generate energy, or glycolysis, can

occur in the presence or absence of O

(aerobic or anaerobically). The net production of ATP during aerobic glycolysis is 19 tim es

higher than anaerobic glycolysis.

■ Fatty acids are carboxylic acids with extended hydrocarbon chains. They are an important energy source for cells and their derivatives,

including triglycerides, phospholipids and sterols, and have additional important cellula r applications. Free fatty acids can be bound to

albumin and transported throughout the body. Triglycerides, phospholipids, and cholestero l are transported as lipoprotein complexes.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. The membra ne potential of a particular cell is at the K

equilibrium. The intracellular concentration for K

is at 150 mmol/L

and the extracellular concentration for K

is at 5.5 mmol/L. What is the resting potential?

A) –70 mv

B) –90 mv

C) +70 mv

D) +90 mv

2. The difference in concentration of H

in a solution of pH 2.0 compared with one of pH 7.0 is

A) 5-fold.

B) 1/5 as much.

C) 10

fold.

D) 10

–5

as much.

3. Transcription refers to

A) the process where an mRNA is used as a template for protein production.

B) the process where a DNA sequence is copied into RNA for the purpose of gene expression .

C) the process where DNA wraps around histones to form a nucleosome.

D) the process of replication of DNA prior to cell division. 4. The primary structure of a protein refers to

A) the twist, folds, or twist and folds of the amino acid sequence into stabilized structure s within the protein (ie, α-helices and β-sheets).

B) the arrangement of subunits to form a functional structure.

C) the amino acid sequence of the protein.

D) the arrangement of twisted chains and folds within a protein into a stable structure.

5. Fill in the blanks: Glycogen is a storage form of glucose. _______ refers to the process of making glycogen and _______ refers to the

process of breakdown of glycogen.

A) Glycogenolysis, glycogenesis

B) Glycolysis, glycogenolysis

C) Glycogenesis, glycogenolysis

D) Glycogenolysis, glycolysis

6. The major lipoprotein source of the cholesterol used in cells is A) chylomicrons.

B) intermediate-density lipoproteins (IDLs).

C) albumin-bound free fatty acids.

D) LDL.

E) HDL.

7. Which of the following produces the most high-energy phosphate compounds?

A) aerobic metabolism of 1 mol of glucose

B) anaerobic metabolism of 1 mol of glucose

C) metabolism of 1 mol of galactose

D) metabolism of 1 mol of amino acid

E) metabolism of 1 mol of long-chain fatty acid

8. When LDL enters cells by receptor-mediated endocytosis, which of the follo wing does not occur?

A) Decrease in the formation of cholesterol from mevalonic acid.

B) Increase in the intracellular concentration of cholesteryl esters.

C) Increase in the transfer of cholesterol from the cell to HDL. D) Decrease in the rate of synthesis of LDL receptors. E) Decrease in the

cholesterol in endosomes.

CHAPTER RESOURCES

Alberts B, et al: Molecular Biology of the Cell, 5th ed. Garland Science, 2007.

Hille B: Ionic Channels of Excitable Membranes, 3rd ed. Sinauer Associates, 2001.

Kandel ER, Schwartz JH, Jessell TM: Principles of Neural Science, 4th ed. McGraw- Hill, 2000.

Macdonald RG, Chaney WG: USMLE Road Map, Biochemistry. McGraw- Hill, 2007 .

Murray RK, et al: Harper’s Biochemistry, 26th ed. McGraw-Hill, 2003.

Pollard TD, Earnshaw WC: Cell Biology, 2nd ed. Saunders, Elsevier, 2008.

Sack GH, Jr. USMLE Road Map, Genetics. McGraw Hill, 2008.

Scriver CR, et al (editors): The Metabolic and Molecular Bases of Inherited Disease, 8th ed. McGraw-Hill, 2001.

Sperelakis N (editor): Cell Physiology Sourcebook, 3rd ed. Academic Press, 2001 .

Overview of Cellular Physiology in Medical Physiology

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Name the prominent cellular organelles and state their functions in cells.

■

Name the building blocks of the cellular cytoskeleton and state their contributions to cell structure and function.

■

Name the intercellular and cellular to extracellular connections.

■

Define the processes of exocytosis and endocytosis, and describe the contribution of each to normal cell function.

■

Define proteins that contribute to membrane permeability and transport.

■

Describe specialized transport and filtration across the capillary wall.

■

Recognize various forms of intercellular communication and describe ways in which chemical messengers (including second

messengers) affect cellular physiology.

■

Define cellular homeostasis.

INTRODUCTION

The cell is the fundamental working unit of all organisms. In humans, cells can be highl y specialized in both structure and function;

alternatively, cells from different organs can share features and function. In the previo us chapter, we examined some basic principles of

biophysics and the catabolism and metabolism of building blocks found in the cell. In so me of those discussions, we examined how the

building blocks could contribute to basic cellular physiology (eg, DNA replication, trans cription, and translation). In this chapter, we will

briefly review more of the fundamental aspects of cellular and molecular physiology. A dditional aspects that concern specialization of

cellular and molecular physiology are considered in the next chapter concerning immun e function and in the relevant chapters on the

various organs.

FUNCTIONAL MORPHOLOGY OF THE CELL

A basic knowledge of cell biology is essential to an understanding of the organ systems in the body and the way they function. A key

tool for examining cellular constituents is the microscope. A light microscope can resolv e structures as close as 0.2 μm, while an electron

microscope can resolve structures as close as 0.002 μm. Although cell dimensions are q uite variable, this resolution can give us a good

look at the inner workings of the cell. The advent of common access to fluorescent, con focal, and other microscopy along with

specialized probes for both static and dynamic cellular structures further expanded the examination of cell structure and function.

Equally revolutionary advances in the modern biophysical, biochemical, and molecular biology techniques have also greatly contributed

to our knowledge of the cell.

The specialization of the cells in the various organs is considerable, and no cell can be c alled “typical” of all cells in the body. However,

a number of structures (organelles) are common to most cells. These structures are shown in F igure 2–1. Many of them can be isolated

by ultracentrifugation combined with

Secretory granules

Golgi

apparatus

Centrioles

Rough endoplasmic reticulum Smooth

endoplasmic

reticulum

Lysosomes Nuclear envelope

Lipid

droplets Mitochondrion

Nucleolus Globular heads

FIGURE 2–1 Diagram showing a hypothetical cell in the center as seen with th e light microscope. Individual organelles are

expanded for closer examination. (Adapted from Bloom and Fawcett. Reproduced with permission fro m Junqueira LC, Carneiro J, Kelley RO: Basic Histology,

9th ed. McGraw-Hill, 1998.)

other techniques. When cells are homogenized and the resulting suspension is centrifug ed, the nuclei sediment first, followed by the

mitochondria. High-speed centrifugation that generates forces of 100,000 times gravity or more causes a fraction made up of granules

called the microsomes to sediment. This fraction includes organelles such as the ribosomes and peroxisomes.

CELL MEMBRANES

The membrane that surrounds the cell is a remarkable structure. It is made up of lipids a nd proteins and is semipermeable, allowing

some substances to pass through it and excluding others. However, its permeability can also be varied because it contains numerous

regulated ion channels and other transport proteins that can change the amounts of sub stances moving across it. It is generally referred to

as the plasma membrane. The nucleus and other organelles in the cell are bound by similar membranous structures.

Although the chemical structures of membranes and their properties vary considerably from one location to another, they have certain

common features. They are generally about 7.5 nm (75 Å) thick. The major lipids are phospholipids such as phosphatidylcholine and

phosphatidylethanolamine. The shape of the phospholipid molecule reflects its solubility properties: the head end of the molecule

contains the phosphate portion and is relatively soluble in water (polar, hydrophilic) and the tail s are relatively insoluble (nonpolar,

hydrophobic). The possession of both hydrophilic and hydrophobic properties make the lipid an amphipathic molecule. In the

membrane, the hydrophilic ends of the molecules are exposed to the aqueous environm ent that bathes the exterior of the cells and the

aqueous cytoplasm; the hydrophobic ends meet in the water-poor interior of the membr ane (Figure 2–2). In prokaryotes (ie, bacteria in

which there is no nucleus), the membranes are relatively simple, but in eukaryotes (cells containing nuclei), cell membranes contain

various glycosphingolipids, sphingomyelin, and cholesterol in addition to phospholipids and phosphatidylcholine.

Many different proteins are embedded in the membrane. They exist as separate globula r units and many pass through the membrane

(integral proteins), whereas others (peripheral proteins) stud the inside and outside of the membrane (Fi gure 2–2). The amount of

protein varies significantly with the function of the membrane but makes up on average 50% of the mass of the membrane; that is, there

is about one protein molecule per 50 of the much smaller phospholipid molecules. The p roteins in the membranes carry out many

functions. Some are cell adhesion molecules that anchor cells to their neighbors or to basal laminas. Some proteins function as pumps,

actively

Extracellular fluid

Carbohydrate portion of

glycoprotein Transmembrane

Phospholipidsproteins

Channel

Intregral

proteins

Peripheral protein

Polar regions

Nonpolar regions

Intracellular fluid

FIGURE 2–2 Organization of the phospholipid bilayer and associated protein s in a biological membrane. The phospholipid

molecules each have two fatty acid chains (wavy lines) attached to a phosphate head (o pen circle). Proteins are shown as irregular

colored globules. Many are integral proteins, which extend into the membrane, but periph eral proteins are attached to the inside or

outside (not shown) of the membrane. Specific protein attachments and cholesterol co mmonly found in the bilayer are omitted for clarity.

(Reproduced with permission from Widmaier EP, Raff H, Strang K: Vander’s Human Physiology: The Mechanisms of Body Function, 11th ed. McGraw-Hill, 2008.)

transporting ions across the membrane. Other proteins function as carriers, transporting substances down electrochemical gradients by

facilitated diffusion. Still others are ion channels, which, when activated, permit the passage of ions into or out of the cell. The role of

the pumps, carriers, and ion channels in transport across the cell membrane is discussed below. Proteins in another group function as

receptors that bind ligands or messenger molecules, initiating physiologic changes inside the cell. Proteins also function as enzymes,

catalyzing reactions at the surfaces of the membrane. Examples from each of these gro ups are discussed later in this chapter.

The uncharged, hydrophobic portions of the proteins are usually located in the interior of the membrane, whereas the charged,

hydrophilic portions are located on the surfaces. Peripheral proteins are attached to the surfaces of the membrane in various ways. One

common way is attachment to glycosylated forms of phosphatidylinositol. Proteins held b y these glycosylphosphatidylinositol (GPI)

anchors (Figure 2–3) include enzymes such as alkaline phosphatase, various antigens, a nu mber of cell adhesion molecules, and three

proteins that combat cell lysis by complement. Over 45 GPIlinked cell surface proteins h ave now been described in humans. Other

proteins are lipidated, that is, they have specific lipids attached to them (Figure 2–3). Proteins may be myristolated, palmitoylated, or

prenylated (ie, attached to geranylgeranyl or farnesyl groups).

The protein structure—and particularly the enzyme content—of biologic membranes va ries not only from cell to cell, but also within the

same cell. For example, some of the enzymes embedded in cell membranes are differen t from those in mitochondrial membranes. In

epithelial cells, the enzymes in the cell membrane on the mucosal surface differ from tho se in the

Lipid membrane

N-Myristoyl

Cytoplasmic or external face of membrane O

N Gly Protein COOH H

S-Cys Protein NH

S-Palmitoyl O

S-Cys Protein NH

Geranylgeranyl

S-Cys Protein NH

Farnesyl O C CCH

C C CH O O GPI anchor O C OO OC Protein (Glycosylphosphatidylinositol)

H2 O

Hydrophobic domain Hydrophilic domain

FIGURE 2–3 Protein linkages to membrane lipids. Some are linked by their am ino terminals, others by their carboxyl terminals. Many

are attached via glycosylated forms of phosphatidylinositol (GPI anchors). (R eproduced with permission from Fuller GM, Shields D: Molecular Basis

of Medical Cell Biology. McGraw-Hill, 1998.)

Intramemb space

H+ H+ H+ H+

Inner mito

CoQ Cyt c

membrane

Matrix space ADP

ATP

Complex I II III IV V

Subunits from 70 1 3 2

mDNA

Subunits from 39 4 10 10 14

nDNA

FIGURE 2–4 Components involved in oxidative phosphorylation in mitochondr ia and their origins. As enzyme complexes I

through IV convert 2-carbon metabolic fragments to CO

and H

O, protons (H

) are pumped into the intermembrane space. The

proteins diffuse back to the matrix space via complex V, ATP synthase (AS), in which ADP is converted to ATP. The enzyme

complexes are made up of subunits coded by mitochondrial DNA (mDNA) and nuclear DNA (nDNA), and the figures document the

contribution of each DNA to the complexes.

cell membrane on the basal and lateral margins of the cells; that is, the cells are polarized. Such polarization makes transport acro ss

epithelia possible. The membranes are dynamic structures, and their constituents are bein g constantly renewed at different rates. Some

proteins are anchored to the cytoskeleton, but others move laterally in the membrane.

Underlying most cells is a thin, “fuzzy” layer plus some fibrils that collectively make up t he basement membrane o r, more properly,

the basal lamina. The basal lamina and, more generally, the extracellu lar matrix are made up of many proteins that hold cells together,

regulate their development, and determine their growth. These include collagens, laminin s, fibronectin, tenascin, and various

proteoglycans.

MITOCHONDRIA

Over a billion years ago, aerobic bacteria were engulfed by eukaryotic cells and evolv ed into mitochondria, providing the eukaryotic

cells with the ability to form the energy-rich compound ATP by oxidative pho sphorylation. Mitochondria perform other functions,

including a role in the regulation of apoptosis (programmed cell death), but oxidative phosphory lation is the most crucial. Each

eukaryotic cell can have hundreds to thousands of mitochondria. In mammals, they are generally depicted as sausage-shaped organelles

(Figure 2–1), but their shape can be quite dynamic. Each has an outer membrane, an in termembrane space, an inner membrane, which is

folded to form shelves (cristae), and a central matrix space. The enzyme complexes responsib le for oxidative phosphorylation are lined

up on the cristae (Figure 2–4).

Consistent with their origin from aerobic bacteria, the mitochondria have their own gen ome. There is much less DNA in the

mitochondrial genome than in the nuclear genome, and 99% of the proteins in the mitoc hondria are the products of nuclear genes, but

mitochondrial DNA is responsible for certain key components of the pathway for oxid ative phosphorylation. Specifically, human

mitochondrial DNA is a double-stranded circular molecule containing approximately 16 ,500 base pairs (compared with over a billion in

nuclear DNA). It codes for 13 protein subunits that are associated with proteins encode d by nuclear genes to form four enzyme

complexes plus two ribosomal and 22 transfer RNAs that are needed for protein produ ction by the intramitochondrial ribosomes.

The enzyme complexes responsible for oxidative phosphorylation illustrate the interactio ns between the products of the mitochondrial

genome and the nuclear genome. For example, complex I, reduced nicotinamide adeni ne dinucleotide dehydrogenase (NADH), is made

up of 7 protein subunits coded by mitochondrial DNA and 39 subunits coded by nucle ar DNA. The origin of the subunits in the other

complexes is shown in Figure 2–4. Complex II, succinate dehydrogenase-ubiquinone oxidoreductase; complex III,

ubiquinonecytochrome c oxidoreductase; and complex IV, cytochrome c oxidase, act w ith complex I, coenzyme Q, and cytochrome c to

convert metabolites to CO

and water. Complexes I, III, and IV pump protons (H

) into the intermembrane space during this electron

transfer. The protons then flow down their electrochemical gradient through complex V , ATP synthase, which harnesses this energy to

generate ATP.

As zygote mitochondria are derived from the ovum, their inheritance is maternal. This m aternal inheritance has been used as a tool to

track evolutionary descent. Mitochondria have an ineffective DNA repair system, and the mutation rate for mitochondrial DNA is over

10 times the rate for nuclear DNA. A large number of relatively rare diseases have now been traced to mutations in mitochondrial DNA.

These include for the most part disorders of tissues with high metabolic rates in which en ergy production is defective as a result of

abnormalities in the production of ATP.

LYSOSOMES

In the cytoplasm of the cell there are large, somewhat irregular structures surrounded b y membrane. The interior of these structures,

which are called lysosomes, is more acidic than the

TABLE 2–1 Some of the enzymes found in lysosomes and the cell component s that are their substrates.

CLINICAL BOX 2–1

Enzyme Substrate

Ribonuclease RNA

Deoxyribonuclease DNA

Phosphatase Phosphate esters

Glycosidases Complex carbohydrates; glycosides and polysaccharides

Arylsulfatases Sulfate esters

Collagenase Collagens

Cathepsins Proteins

Lysosomal Diseases

When a lysosomal enzyme is congenitally absent, the lysosomes become engorged with the material the enzyme normally degrades.

This eventually leads to one of the lysosomal storage diseases. For example, α-gala ctosidase A deficiency causes Fabry disease, and β-

galactocerebrosidase deficiency causes Gaucher disease. These diseases are rare, but th ey are serious and can be fatal. Another example

is the lysosomal storage disease called Tay–Sachs disease, which causes mental retardatio n and blindness. Tay–Sachs is caused by the

loss of hexosaminidase A, a lysosomal enzyme that catalyzes the biodegradation of gang liosides (fatty acid derivatives).

rest of the cytoplasm, and external material such as endocytosed bacteria, as well as wor n-out cell components, are digested in them. The

interior is kept acidic by the action of a proton pump, or H

, ATPase. This integral membrane protein uses the energy of ATP to move

protons from the cytosol up their electrochemical gradient and keep the lysosome relativ ely acidic, near pH 5.0. Lysosomes can contain

over 40 types of hydrolytic enzymes, some of which are listed in Table 2–1. Not surpr isingly, these enzymes are all acid hydrolases, in

that they function best at the acidic pH of the lysosomal compartment. This can be a safe ty feature for the cell; if the lysosome were to

break open and release its contents, the enzymes would not be efficient at the near neut ral cytosolic pH (7.2), and thus would be unable

to digest cytosolic enzymes they may encounter. Diseases associated with lysosomal dysf unction are discussed in Clinical Box 2–1.

PEROXISOMES

Peroxisomes are 0.5 μm in diameter, are surrounded by a membrane, and contain enzymes that can either produce H

(oxidases) or

break it down (catalases). Proteins are directed to the peroxisome by a unique signal s equence with the help of protein chaperones,

peroxins. The peroxisome membrane contains a number of peroxisome-specifi c proteins that are concerned with transport of substances

into and out of the matrix of the peroxisome. The matrix contains more than 40 enzyme s, which operate in concert with enzymes outside

the peroxisome to catalyze a variety of anabolic and catabolic reactions (eg, breakdown of lipids). Peroxisomes can form by budding of

endoplasmic reticulum, or by division. A number of synthetic compounds were found to cause proliferation of peroxisomes by acting on

receptors in the nuclei of cells. These peroxisome proliferation activated receptors (PPARs) are members of the nuclear receptor

superfamily. When activated, they bind to DNA, producing changes in the production of mRNAs. The known effects for PPARs are

extensive and can affect most tissues and organs.

CYTOSKELETON

All cells have a cytoskeleton, a system of fibers that not only maintains the structure of the cell b ut also permits it to change shape and

move. The cytoskeleton is made up primarily of microtubules, intermediate filaments, a nd microfilaments (Figure 2–5), along with

proteins that anchor them and tie them together. In addition, proteins and organelles mo ve along microtubules and microfilaments from

one part of the cell to another, propelled by molecular motors.

Microtubules (Figures 2–5 and 2–6) are long, hollow structures with 5-nm walls surrounding a cavity 15 nm in diameter. They are made

up of two globular protein subunits: α- and β-tubulin. A third subunit, γ-tubulin, is assoc iated with the production of microtubules by the

centrosomes. The α and β subunits form heterodimers, which aggregate to form long tu bes made up of stacked rings, with each ring

usually containing 13 subunits. The tubules interact with GTP to facilitate their formation. Although microtubule subunits can be added

to either end, microtubules are polar with assembly predominating at the “+” end and di sassembly predominating at the “–” end. Both

processes occur simultaneously in vitro. The growth of microtubules is temperature sen sitive (disassembly is favored under cold

conditions) as well as under the control of a variety of cellular factors that can directly i nteract with microtubules in the cell.

Because of their constant assembly and disassembly, microtubules are a dynamic portion of the cell skeleton. They provide the tracks

along which several different molecular motors move transport vesicles, organelles such as secretory granules, and mitochondria, from

one part of the cell to another. They also form the spindle, which moves the chromosom es in mitosis. Cargo can be transported in either

direction on microtubules.

There are several drugs available that disrupt cellular function through interaction with m icrotubules. Microtubule assembly is prevented

by colchicine and vinblastine. The anticancer drug paclitaxel (Taxol) bi nds to microtubules and

Cytoskeletal filaments Diameter (nm) Protein subunit

Microfilament 7 Actin

Intermediate filament 10 Several proteins

Microtubule 25 Tubulin

FIGURE 2–5 Cytoskeletal elements of the cell. Artistic impressions that depict the major cytoskeletal elements are shown on the left,

with basic properties of these elements on the right. (Reproduced with permission from Widm aier EP, Raff H, Strang KT: Vander’s Human Physiology: The

Mechanisms of Body Function, 11th ed. McGraw-Hill, 2008.)

makes them so stable that organelles cannot move. Mitotic spindles cannot form, and the cells die.

Intermediate filaments (Figures 2–5 and 2–6) are 8 to 14 nm in diameter and are made up of various subunits. Some of these filaments

connect the nuclear membrane to the cell membrane. They form a flexible scaffolding f or the cell and help it resist external pressure. In

their absence, cells rupture more easily, and when they are abnormal in humans, blister ing of the skin is common. The proteins that make

up intermediate filaments are celltype specific, and are thus frequently used as cellular m arkers. For example, vimentin is a major

intermediate filament in fibroblasts, whereas cytokeratin is expressed in epithelial cells.

Microfilaments (Figures 2–5 and 2–6) are long solid fibers with a 4 to 6 nm diam eter that are made up of actin. Although actin is most

often associated with muscle contraction, it is present in all types of cells. It is the most ab undant protein in mammalian cells, sometimes

accounting for as much as 15% of the total protein in the cell. Its structure is highly con served; for example, 88% of the amino acid

sequences in yeast and rabbit actin are identical. Actin filaments polymerize and depolym erize in vivo, and it is not uncommon to find

polymerization occurring at one end of the filament while depolymerization is occurring at the other end. Filamentous (F) actin refers to

intact microfilaments and globular (G) actin refers to the unpolymerized protein actin s ubunits. F-actin fibers attach to various parts of

the cytoskeleton and can interact directly or indirectly with membrane-bound proteins. T hey reach to the tips of the microvilli on the

epithelial cells of the intestinal mucosa. They are also abundant in the lamellipodia that cel ls put out when they crawl along surfaces. The

actin filaments interact with integrin

FIGURE 2–6 Microfilaments and microtubules. Electron microg raph (Left) of the cytoplasm of a fibroblast, displaying actin

microfilaments (MF) and microtubules (MT). (Reproduced, with permission, from Junque ira LC, Carneiro J: Basic Histology, 10th ed. McGraw-Hill, 2003.)

Fluorescent micrographs of airway epithelial cells displaying actin microfilaments stained with phalloidin (Middle) and microtubules

visualized with an antibody to β-tubulin (Right). Both fluorescent microgr aphs are counterstained with Hoechst dye (blue) to visualize

nuclei. Note the distinct differences in cytoskeletal structure.

receptors and form focal adhesion complexes, which serve as points of traction with the su rface over which the cell pulls itself. In

addition, some molecular motors use microfilaments as tracks. they perform functions a s diverse as contraction of muscle and cell

migration.

MOLECULAR MOTORS

The molecular motors that move proteins, organelles, and other cell parts (collectively re ferred to as “cargo”) to all parts of the cell are

100 to 500 kDa ATPases. They attach to their cargo at one end of the molecule and to microtubules or actin polymers with the other end,

sometimes referred to as the “head.” They convert the energy of ATP into movement a long the cytoskeleton, taking their cargo with

them. There are three super families of molecular motors: kinesin, dynein, and myosin. Examples of individual proteins from each

superfamily are shown in Figure 2–7. It is important to note that there is extensive variat ion among superfamily members, allowing for

specialization of function (eg, choice of cargo, cytoskeletal filament type, and/or directio n of movement).

The conventional form of kinesin is a doubleheaded molecule that tends to move it s cargo toward the “+” ends of microtubules. One

head binds to the microtubule and then bends its neck while the other head swings forw ard and binds, producing almost continuous

movement. Some kinesins are associated with mitosis and meiosis. Other kinesins perform different functions, including, in some

instances, moving cargo to the “–” end of microtubules. Dyneins have two heads, with the ir neck pieces embedded in a complex of

proteins. Cytoplasmic dyneins have a function like that of conventional kinesin, except they tend to move particles and membranes to

the “–” end of the microtubules. The multiple forms of myosin in the body are divid ed into 18 classes. The heads of myosin molecules

bind to actin and produce motion by bending their neck regions (myosin II) or walking along microfilaments, one head after the other

(myosin V). In these ways,

CENTROSOMES

Near the nucleus in the cytoplasm of eukaryotic animal cells is a centrosome . The centrosome is made up of two centrioles and

surrounding amorphous pericentriolar material. The centrioles are short cylinders arra nged so that they are at right angles to each

other. Microtubules in groups of three run longitudinally in the walls of each centriole ( Figure 2–1). Nine of these triplets are spaced at

regular intervals around the circumference.

The centrosomes are microtubule-organizing centers (MTOCs) that con tain γ-tubulin. The microtubules grow out of this γ-tubulin in

the pericentriolar material. When a cell divides, the centrosomes duplicate themselves, an d the pairs move apart to the poles of the

mitotic spindle, where they monitor the steps in cell division. In multinucleate cells, a centr osome is near each nucleus.

CILIA

Cilia are specialized cellular projections that are used by unicellular organisms to prop el themselves through liquid and by multicellular

organisms to propel mucus and other substances over the surface of various epithelia. C ilia are functionally indistinct from the eukaryotic

flagella of sperm cells. Within the cilium there is an axoneme that comprises a unique arrangemen t of nine outer microtubule doublets

and two inner microtubules (“9+2” arrangement). Along this cytoskeleton is axon emal dynein. Coordinated dynein-microtubule

interactions within the axoneme are the basis of ciliary and sperm movement. At the bas e of the axoneme and just inside lies the basal

body. It has nine circumferential triplet microtubules, like a centriole, and there is ev idence that basal bodies and centrioles are

interconvertible.

Cargo

Conventional kinesin Light

chains

4 nm

80 nm

Cytoplasmic dynein

Cargo-binding domain

Head 1 Head 2 Head 2 Head 1 ADP ADP ATP

Actin Myosin V

FIGURE 2–7 Three examples of molecular motors. Conventional kinesin is shown attached to cargo, in this case a membrane-bound

organelle. The way that myosin V “walks” along a microtubule is also shown. Note that the heads of the motors hydrolyze ATP and use

the energy to produce motion.

CELL ADHESION MOLECULES

Cells are attached to the basal lamina and to each other by cell adhesion molecu les (CAMs) that are prominent parts of the intercellular

connections described below. These adhesion proteins have attracted great attention in r ecent years because of their unique structural and

signaling functions found to be important in embryonic development and formation of the nervous system and other tissues, in holding

tissues together in adults, in inflammation and wound healing, and in the metastasis of tum ors. Many CAMs pass through the cell

membrane and are anchored to the cytoskeleton inside the cell. Some bind to like molecu les on other cells (homophilic binding), whereas

others bind to nonself molecules (heterophilic binding). Many bind to laminins, a family of large crossshaped molecules with multiple

receptor domains in the extracellular matrix.

Nomenclature in the CAM field is somewhat chaotic, partly because the field is growing so rapidly and partly because of the extensive

use of acronyms, as in other areas of modern biology. However, the CAMs can be div ided into four broad families: (1) integrins,

heterodimers that bind to various receptors; (2) adhesion molecules of the IgG superfamily of immunoglobulins; (3) cadherins, Ca

dependent molecules that mediate cell-to-cell adhesion by homophilic reactions; and (4) selectins, which have lectin-like domains that

bind carbohydrates. Specific functions of some of these molecules are addressed in late r chapters.

The CAMs not only fasten cells to their neighbors, but they also transmit signals into and out of the cell. For example, cells that lose their

contact with the extracellular matrix via integrins have a higher rate of apoptosis than an chored cells, and interactions between integrins

and the cytoskeleton are involved in cell movement.

INTERCELLULAR CONNECTIONS

Intercellular junctions that form between the cells in tissues can be broadly split into two groups: junctions that fasten the cells to one

another and to surrounding tissues, and junctions that permit transfer of ions and other molecules from one cell to another. The types of

junctions that tie cells together and endow tissues with strength and stability include tight jun ctions, which are also known as the

zonula occludens (Figure 2–8). The desmosome and zonula adherens also help to hold cells together, and the hemidesmosome and

focal adhesions attach cells to their basal laminas. The gap junction forms a cytoplas mic “tunnel” for diffusion of small molecules (<

1000 Da) between two neighboring cells.

Tight junctions characteristically surround the apical margins of the cells in epithelia such as the intestinal mucosa, the walls of the renal

tubules, and the choroid plexus. They are also important to endothelial barrier function. They are made up of ridges—half from one cell

and half from the other—which adhere so strongly at cell junctions that they

Tight

junction

(zonula

occludens)

Zonula

adherens

Desmosomes

Gap

junctions

Hemidesmosome

FIGURE 2–8 Intercellular junctions in the mucosa of the small intestine. Tight jun ctions (zonula occludens), adherens junctions

(zonula adherens), desmosomes, gap junctions, and hemidesmosomes are all shown in relative positions in a polarized epithelial cell.

almost obliterate the space between the cells. There are three main families of transmemb rane proteins that contribute to tight junctions:

occludin, junctional adhesion molecules (JAMs), and claudins; and several more p roteins that interact from the cytosolic side. Tight

junctions permit the passage of some ions and solute in between adjacent cells (parac ellular pathway) and the degree of this

“leakiness” varies, depending in part on the protein makeup of the tight junction. Extrac ellular fluxes of ions and solute across epithelia

at these junctions are a significant part of overall ion and solute flux. In addition, tight ju nctions prevent the movement of proteins in the

plane of the membrane, helping to maintain the different distribution of transporters and channels in the apical and basolateral cell

membranes that make transport across epithelia possible.

In epithelial cells, each zonula adherens is usually a continuous structure on the basal sid e of the zonula occludens, and it is a major site

of attachment for intracellular microfilaments. It contains cadherins.

Desmosomes are patches characterized by apposed thickenings of the membranes of tw o adjacent cells. Attached to the thickened area in

each cell are intermediate filaments, some running parallel to the membrane and others r adiating away from it. Between the two

membrane thickenings the intercellular space contains filamentous material that includes c adherins and the extracellular portions of

several other transmembrane proteins.

Hemidesmosomes look like half-desmosomes that attach cells to the underlying basal lam ina and are connected

Presynaptic cytoplasm

3.5 nm 20 nm

Postsynaptic cytoplasm Normal

extracellular

space

Channel formed by pores in

each membrane

B 6 connexin subunits = 1 connexon (hemichannel) Each of the 6 connexins has 4 membrane-s panning regions

Presynaptic cytoplasm Cytoplasmic loops for regulation

Extracellular space

Extracellular loops for

homophilic interactions FIGURE 2–9 Gap junction connecting the cytoplasm of two cells. A) A gap junction plaque, or collection of

individual gap junctions, is shown to form multiple pores between cells that allow for the tran sfer of small molecules. Inset is electron

micrograph from rat liver (N. Gilula). B) Topographical depiction of individual connexon and corresponding 6 connexin proteins that

traverse the membrane. Note that each connexin traverses the membrane four times. (Reproduced with permission from Kandel ER, Schwartz JH,

Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill, 2000.)

intracellularly to intermediate filaments. However, they contain integrins rather than cadh erins. Focal adhesions also attach cells to their

basal laminas. As noted previously, they are labile structures associated with actin filamen ts inside the cell, and they play an important

role in cell movement.

GAP JUNCTIONS

At gap junctions, the intercellular space narrows from 25 nm to 3 nm, and units called conne xons in the membrane of each cell are lined

up with one another (Figure 2–9). Each connexon is made up of six protein subunits c alled connexins. They surround a channel that,

when lined up with the channel in the corresponding connexon in the adjacent cell, per mits substances to pass between the cells without

entering the ECF. The diameter of the channel is normally about 2 nm, which permits th e passage of ions, sugars, amino acids, and other

solutes with molecular weights up to about 1000. Gap junctions thus permit the rapid pro pagation of electrical activity from cell to cell,

as well as the exchange of various chemical messengers. However, the gap junction ch annels are not simply passive, nonspecific

conduits. At least 20 different genes code for connexins in humans, and mutations in th ese genes can lead to diseases that are highly

selective in terms of the tissues involved and the type of communication between cells pr oduced. For instance, X-linked Charcot–

Marie–Tooth disease is a peripheral neuropathy associated with mutation of o ne particular connexin gene. Experiments in mice in

which particular connexins are deleted by gene manipulation or replaced with different connexins confirm that the particular connexin

subunits that make up connexons determine their permeability and selectivity. Recently it has been shown that connexons can be used as

channels to release small molecules from the cytosol into the ECF.

NUCLEUS & RELATED STRUCTURES

A nucleus is present in all eukaryotic cells that divide. If a cell is cut in half, the anucleate portion eventually dies without dividing. The

nucleus is made up in large part of the chromosomes, the structures in the nucleus that carry a co mplete blueprint for all the heritable

species and individual characteristics of the animal. Except in germ cells, the chromosom es occur in pairs, one originally from each

parent. Each chromosome is made up of a giant molecule of DNA. The DNA stra nd is about 2 m long, but it can fit in the nucleus

because at intervals it is wrapped around a core of histone proteins to form a nucleosome. There are about 25 million nucleosomes in

each nucleus. Thus, the structure of the chromosomes has been likened to a string of be ads. The beads are the nucleosomes, and the

linker DNA between them is the string. The whole complex of DNA and proteins is cal led chromatin. During cell division, the coiling

around histones is loosened, probably by acetylation of the histones, and pairs of chrom osomes become visible, but between cell

divisions only clumps of chromatin can be discerned in the nucleus. The ultimate units o f heredity are the genes on the chromosomes).

As discussed in Chapter 1, each gene is a portion of the DNA molecule.

The nucleus of most cells contains a nucleolus (Figure 2–1), a patchwork of gran ules rich in RNA. In some cells, the nucleus contains

several of these structures. Nucleoli are most prominent and numerous in growing cells . They are the site of synthesis of ribosomes, the

structures in the cytoplasm in which proteins are synthesized.

The interior of the nucleus has a skeleton of fine filaments that are attached to the nuclear membrane , or envelope (Figure 2–1), which

surrounds the nucleus. This membrane is a double membrane, and spaces between the two folds are called perinuclear cisterns. The

membrane is permeable only to small molecules. However, it contains nuclear pore complexes. Each complex has eightfold symmetry

and is made up of about 100 proteins organized to form a tunnel through which transp ort of proteins and mRNA occurs. There are many

transport pathways, and proteins called importins and exportins have been isola ted and characterized. Much current research is focused

on transport into and out of the nucleus, and a more detailed understanding of these pr ocesses should emerge in the near future.

ENDOPLASMIC RETICULUM

The endoplasmic reticulum is a complex series of tubules in the cytoplasm of the cell (Figure 2–1). The inner limb of its membrane is

continuous with a segment of the nuclear membrane, so in effect this part of the nuclear membrane is a cistern of the endoplasmic

reticulum. The tubule walls are made up of membrane. In rough, or granular, endoplasmic r eticulum, ribosomes are attached to the

cytoplasmic side of the membrane, whereas in smooth, or agranular, endoplasmic reticulum , ribosomes are absent. Free ribosomes are

also found in the cytoplasm. The granular endoplasmic reticulum is concerned with pro tein synthesis and the initial folding of

polypeptide chains with the formation of disulfide bonds. The agranular endoplasmic re ticulum is the site of steroid synthesis in steroid-

secreting cells and the site of detoxification processes in other cells. A modified endopla smic reticulum, the sarcoplasmic reticulum,

plays an important role in skeletal and cardiac muscle. In particular, the endoplasmic or sarcoplasmic reticulum can sequester Ca

ions

and allow for their release as signaling molecules in the cytosol.

RIBOSOMES

The ribosomes in eukaryotes measure approximately 22 × 32 nm. Each is made up of a large and a small subunit called, on the basis of

their rates of sedimentation in the ultracentrifuge, the 60S and 40S subunits. The ribosom es are complex structures, containing many

different proteins and at least three ribosomal RNAs. They are the sites of protein synth esis. The ribosomes that become attached to the

endoplasmic reticulum synthesize all transmembrane proteins, most secreted proteins, an d most proteins that are stored in the Golgi

apparatus, lysosomes, and endosomes. These proteins typically have a hydrophobic signal peptide at one end (Figure 2–10). The

polypeptide chains that form these proteins are extruded into the endoplasmic reticulum. The free ribosomes synthesize cytoplasmic

proteins such as hemoglobin and the proteins found in peroxisomes and mitochondria.

GOLGI APPARATUS

& VESICULAR TRAFFIC

The Golgi apparatus is a collection of membrane-enclosed sacs (cisterns) that are stacke d like dinner plates (Figure 2–1). There are

usually about six sacs in each apparatus, but there may be more. One or more Golgi ap parati are present in all eukaryotic cells, usually

near the nucleus. Much of the organization of the Golgi is directed at proper glycosylat ion of proteins and lipids. There are more than

200 enzymes that function to add, remove, or modify sugars from proteins and lipids in the Golgi apparatus.

mRNA from Gene A

Cytoplasm mRNA from Gene B

Free ribosome

Signal sequence

Rough

endoplasmic reticulum

Carbohydrate group

Growing

polypeptide chain

Cleaved signal sequences

Vesicle

Secretory vesicle

Golgi apparatus

Lysosome

Exocytosis

Secreted protein

from Gene A Extracellular fluid

Digestive protein

from

Gene B

Plasma membrane

FIGURE 2–10 Rough endoplasmic reticulum and protein translation. Messenger RNA and ribosomes meet up in the cytosol for

translation. Proteins that have appropriate signal peptides begin translation, then associate with the endoplasmic reticulum (ER) to

complete translation. The association of ribosomes is what gives the ER its “rough” appea rance. (Reproduced with permission from Widmaier EP,

Raff H, Strang KT: Vander’s Human Physiology: The Mechanisms of Body Function, 11th ed. McGraw -Hill, 2008.)

ER Golgi apparatus Secretory granules Regulated secretion

Constitutive secretion

Recycling

Endocytosis

Nucleus Lysosome Late endosome Early endosome FIGURE 2–11 Cellular st ructures involved in protein processing. See text for

details.

The Golgi apparatus is a polarized structure, with cis and trans sides (Figure 2–11). Me mbranous vesicles containing newly synthesized

proteins bud off from the granular endoplasmic reticulum and fuse with the cistern on t he cis side of the apparatus. The proteins are then

passed via other vesicles to the middle cisterns and finally to the cistern on the trans side, from which vesicles branch off into the

cytoplasm. From the trans Golgi, vesicles shuttle to the lysosomes and to the cell exterior via constitutive and nonconstitutive pathways,

both involving exocytosis. Conversely, vesicles are pinched off from the cell membrane b y endocytosis and pass to endosomes. From

there, they are recycled.

Vesicular traffic in the Golgi, and between other membranous compartments in the cell, is regulated by a combination of common

mechanisms along with special mechanisms that determine where inside the cell they will go. One prominent feature is the involvement

of a series of regulatory proteins controlled by GTP or GDP binding (small G proteins) associated with vesicle assembly and delivery. A

second prominent feature is the presence of proteins called SNAREs (for soluble N-eth ylmaleimide-sensitive factor attachment receptor).

The v- (for vesicle) SNAREs on vesicle membranes interact in a lock-and-key fashion with t- (for target) SNAREs. Individual vesicles

also contain structural protein or lipids in their membrane that help to target them for spe cific membrane compartments (eg, Golgi sacs,

cell membranes).

QUALITY CONTROL

The processes involved in protein synthesis, folding, and migration to the various parts of the cell are so complex that it is remarkable

that more errors and abnormalities do not occur. The fact that these processes work as well as they do is because of mechanisms at each

level that are responsible for “quality control.” Damaged DNA is detected and repaired or bypassed. The various RNAs are also checked

during the translation process. Finally, when the protein chains are in the endoplasmic re ticulum and Golgi apparatus, defective

structures are detected and the abnormal proteins are degraded in lysosomes and protea somes. The net result is a remarkable accuracy in

the production of the proteins needed for normal body function.

APOPTOSIS

In addition to dividing and growing under genetic control, cells can die and be absorbe d under genetic control. This process is called

programmed cell death, or apoptosis (Gr. apo “away” + ptosis “fall”). It can be called “cell suicide” in the sense that the cell’s own

genes play an active role in its demise. It should be distinguished from necrosis (“cell mu rder”), in which healthy cells are destroyed by

external processes such as inflammation.

Apoptosis is a very common process during development and in adulthood. In the cent ral nervous system, large numbers of neurons are

produced and then die during the remodeling that occurs during development and syna pse formation. In the immune system, apoptosis

gets rid of inappropriate clones of immunocytes and is responsible for the lytic effects o f glucocorticoids on lymphocytes. Apoptosis is

also an important factor in processes such as removal of the webs between the fingers i n fetal life and regression of duct systems in the

course of sexual development in the fetus. In adults, it participates in the cyclic breakdow n of the endometrium that leads to

menstruation. In epithelia, cells that lose their connections to the basal lamina and neighbo ring cells undergo apoptosis. This is

responsible for the death of the enterocytes sloughed off the tips of intestinal villi. Abno rmal apoptosis probably occurs in autoimmune

diseases, neurodegenerative diseases, and cancer. It is interesting that apoptosis occurs i n invertebrates, including nematodes and insects.

However, its molecular mechanism is much more complex than that in vertebrates.

One final common pathway bringing about apoptosis is activation of caspases, a group of cysteine proteases. Many of these have been

characterized to date in mammals; 11 have been found in humans. They exist in cells as inactive proenzymes until activated by the

cellular machinery. The net result is DNA fragmentation, cytoplasmic and chromatin con densation, and eventually membrane bleb

formation, with cell breakup and removal of the debris by phagocytes (see Clinical Box 2–2).

TRANSPORT ACROSS

CELL MEMBRANES

There are several mechanisms of transport across cellular membranes. Primary pathway s include exocytosis, endocytosis, movement

through ion channels, and primary and secondary active transport. Each of these are d iscussed below.

EXOCYTOSIS

Vesicles containing material for export are targeted to the cell membrane (Figure 2–11) , where they bond in a similar manner to that

discussed in vesicular traffic between Golgi stacks, via the v-SNARE/t-SNARE arrange ment. The area of fusion then breaks down,

leaving the contents of the vesicle outside and the cell membrane intact. This is the Ca

-dependent process of exocytosis (Figure 2–12).

Note that secretion from the cell occurs via two pathways (Figure 2–11). In the nonconstitutive pathway, proteins from the Golgi

apparatus initially enter secretory granules, where processing of prohormones to the m ature hormones occurs before exocytosis. The

other pathway, the constitutive pathway, involves the prompt transpor t of proteins to the cell membrane in vesicles, with little or no

processing or storage. The nonconstitutive pathway is sometimes called the regulated pathw ay, but this term is misleading because the

output of proteins by the constitutive pathway is also regulated.

ENDOCYTOSIS

Endocytosis is the reverse of exocytosis. There are various types of endocytosis named for the size of particles being ingested as well as

the regulatory requirements for the particular process. These include phagocytosis, pinocytos is, clathrinmediated endocytosis,

caveolae-dependent uptake, and nonclathrin/noncaveolae endocytosis.

Phagocytosis (“cell eating”) is the process by which bacteria, dead tissue, or other bits of microscopic material are engulfed by cells such

as the polymorphonuclear leukocytes of the blood. The material makes contact with the cell membrane, which then invaginates. The

invagination is pinched off, leaving the engulfed material in the membrane-enclosed vac uole and the cell membrane intact. Pinocytosis

(“cell drinking”) is a similar process with the vesicles much smaller in size and the substa nces ingested are in solution. The small size

membrane that is ingested should not be misconstrued; cells undergoing active pinocytos is (eg, macrophages) can ingest the equivalent

of their entire cell membrane in just 1 hour.

CLINICAL BOX 2–2 Molecular Medicine

Fundamental research on molecular aspects of genetics, regulation of gene expression, and protein synthesis has been paying off in

clinical medicine at a rapidly accelerating rate.

One early dividend was an understanding of the mechanisms by which antibiotics exert their effects. Almost all act by inhibiting protein

synthesis at one or another of the steps described previously. Antiviral drugs act in a sim ilar way; for example, acyclovir and ganciclovir

act by inhibiting DNA polymerase. Some of these drugs have this effect primarily in ba cteria, but others inhibit protein synthesis in the

cells of other animals, including mammals. This fact makes antibiotics of great value for research as well as for treatment of infections.

Single genetic abnormalities that cause over 600 human diseases have now been identifi ed. Many of the diseases are rare, but others are

more common and some cause conditions that are severe and eventually fatal. Example s include the defectively regulated Cl

–

channel in

cystic fibrosis and the unstable trinucleotide repeats in various parts of the genome th at cause Huntington’s disease, the fragile X

syndrome, and several other neurologic diseases. Abnormalities in mitochondrial DNA can also cause human diseases such as Leber’s

hereditary optic neuropathy and some forms of cardiomyopathy. Not surprisingly, gen etic aspects of cancer are probably receiving the

greatest current attention. Some cancers are caused by oncogenes, genes that are carried in the gen omes of cancer cells and are

responsible for producing their malignant properties. These genes are derived by soma tic mutation from closely related proto-

oncogenes, which are normal genes that control growth. Over 100 oncogenes have been de scribed. Another group of genes produce

proteins that suppress tumors, and more than 10 of these tumor suppressor gen es have been described. The most studied of these is the

p53 gene on human chromosome 17. The p53 protein produced by this gene triggers apoptosis. It is also a nuclear transcription factor

that appears to increase production of a 21-kDa protein that blocks two cell cycle enzym es, slowing the cycle and permitting repair of

mutations and other defects in DNA. The p53 gene is mutated in up to 50% of human cancers, with the production of p53 proteins that

fail to slow the cell cycle and permit other mutations in DNA to persist. The accumulated mutations eventually cause cancer.

Clathrin-mediated endocytosis occurs at membrane indentations where the protein cla thrin accumulates. Clathrin molecules have the

shape of triskelions, with three “legs” radiating from a central hub (Figure 2–13). As e ndocytosis progresses,

Exocytosis

Cytoplasm

Endocytosis

FIGURE 2–12 Exocytosis and endocytosis. Note that in exocytosis the cytoplasm ic sides of two membranes fuse, whereas in

endocytosis two noncytoplasmic sides fuse. (Reproduced with permission from A lberts B et al: Molecular Biology of the Cell, 4th ed. Garland Science, 2002.)

the clathrin molecules form a geometric array that surrounds the endocytotic vesicle. At the neck of the vesicle, the GTP binding protein

dynamin is involved, either directly or indirectly, in pinching off the vesicle. Once the complete v esicle is formed, the clathrin falls off

and the three-legged proteins recycle to form another vesicle. The vesicle fuses with an d dumps its contents into an early endosome

(Figure 2–11). From the early endosome, a new vesicle can bud off and return to the cell membrane. Alternatively, the early endosome

can become a late endosome and fuse with a lysosome (Figure 2–11) in w hich the contents are digested by the lysosomal proteases.

Clathrin-mediated endocytosis is responsible for the internal

FIGURE 2–13 Clathrin molecule on the surface of an endocytotic vesicle. Note the characteristic triskelion shape and the fact that

with other clathrin molecules it forms a net supporting the vesicle.

ization of many receptors and the ligands bound to them— including, for example, ner ve growth factor and low-density lipoproteins. It

also plays a major role in synaptic function.

It is apparent that exocytosis adds to the total amount of membrane surrounding the cell, and if membrane were not removed elsewhere at

an equivalent rate, the cell would enlarge. However, removal of cell membrane occurs by endocytosis, and such exocytosis–endocytosis

coupling maintains the surface area of the cell at its normal size.

RAFTS & CAVEOLAE

Some areas of the cell membrane are especially rich in cholesterol and sphingolipids and have been called rafts. These rafts are probably

the precursors of flask-shaped membrane depressions called caveolae (little caves) when their walls become infiltrated with a protein

called caveolin that resembles clathrin. There is considerable debate about the functions of rafts and caveolae, with evidence that they

are involved in cholesterol regulation and transcytosis. It is clear, however, that choleste rol can interact directly with caveolin, effectively

limiting the protein’s ability to move around in the membrane. Internalization via caveola e involves binding of cargo to caveolin and

regulation by dynamin. Caveolae are prominent in endothelial cells, where they help in the uptake of nutrients from the blood.

COATS & VESICLE TRANSPORT

It now appears that all vesicles involved in transport have protein coats. In humans, 53 coat complex subunits have been identified.

Vesicles that transport proteins from the trans Golgi to lysosomes have assembly pro tein 1 (AP-1) clathrin coats, and endocytotic

vesicles that transport to endosomes have AP-2 clathrin coats. Vesicles that transport bet ween the endoplasmic reticulum and the Golgi

have coat proteins I and II (COPI and COPII). Certain amino acid sequences or attach ed groups on the transported proteins target the

proteins for particular locations. For example, the amino acid sequence Asn–Pro–any a mino acid–Tyr targets transport from the cell

surface to the endosomes, and mannose-6-phosphate groups target transfer from the G olgi to mannose-6-phosphate receptors (MPR) on

the lysosomes.

Various small G proteins of the Rab family are especially important in vesicular traffic. T hey appear to guide and facilitate orderly

attachments of these vesicles. To illustrate the complexity of directing vesicular traffic, hu mans have 60 Rab proteins and 35 SNARE

proteins.

MEMBRANE PERMEABILITY &

MEMBRANE TRANSPORT PROTEINS

An important technique that has permitted major advances in our knowledge about tran sport proteins is patch clamping. A micropipette

is placed on the membrane of a cell and forms a tight seal to the membrane. The patch o f membrane under the pipette tip usually contains

only a few transport proteins, allowing for their detailed biophysical study (Figure 2–14 ). The cell can be left intact (cell-attached patch

clamp). Alternatively, the patch can be pulled loose from the cell, forming an inside-out patch. A third alternative is to suck out the

patch with the micropipette still attached to the rest of the cell membrane, providing direc t access to the interior of the cell (whole cell

recording).

Small, nonpolar molecules (including O

and N

) and small uncharged polar molecules such as CO

diffuse across the lipid membranes

of cells. However, the membranes have very limited permeability to other substances. In stead, they cross the membranes by endocytosis

and exocytosis and by passage through highly specific transport proteins, tr ansmembrane proteins that form channels for ions or

transport substances such as glucose, urea, and amino acids. The limited permeability ap plies even to water, with simple diffusion being

supplemented throughout the body with various water channels (aquaporins). For reference, the sizes of ions and other biologically

important substances are summarized in Table 2–2.

Some transport proteins are simple aqueous ion channels, though many of these have speci al features that make them selective for a

given substance such as Ca

or, in the case of aquaporins, for water. These membrane-spanning proteins (or colle ctions of proteins)

have tightly regulated pores that can be gated opened or closed in response to local changes

Inside-out patch

Electrode

Pipette

Cell

membrane

Closed

ms Open

FIGURE 2–14 Patch clamp to investigate transport. In a patch clamp exper iment, a small pipette is carefully maneuvered to seal off a

portion of a cell membrane. The pipette has an electrode bathed in an appropriat e solution that allows for recording of electrical changes

through any pore in the membrane (shown below). The illustrated setup is termed an “inside-out patch” because of the orientation of the

membrane with reference to the electrode. Other configurations include cell attache d, whole cell, and outside-out patches. (Modified from

Ackerman MJ, Clapham DE: Ion channels: Basic science and clinical disease. N Engl J Med 1997;336:1575.)

(Figure 2–15). Some are gated by alterations in membrane potential (voltage-gated), whereas others are opened or closed in response to

a ligand (ligand-gated). The ligand is

TABLE 2–2 Size of hydrated ions and other substances of biologic interest.

Substance Atomic or Molecular Weight Radius (nm)

–

35 0.12

+ 39 0.12

O 18 0.12

40 0.15

23 0.18

Urea 60 0.23

7 0.24

Glucose 180 0.38

Sucrose 342 0.48

Inulin 5000 0.75

Albumin 69,000 7.50 Data from Moore EW: Physiology of Intestinal Water and Electrolyte Abso rption. American Gastroenterological Association, 1976.

Closed Open A Ligand-gated

Bind ligand

B Phosphorylation-gated

Phosphorylate

Dephosphorylate Pi P C Voltage-gated

Change

membrane

potential

+++ + – – – –

– – – – +++ +

D Stretch or pressure-gated

the bound molecule from one side of the cell membrane to the other. Molecules move f rom areas of high concentration to areas of low

concentration (down their chemical gradient), and cations move to negatively charged areas whereas anions move to positively charged

areas (down their electrical gradient). When carrier proteins move substances in th e direction of their chemical or electrical gradients,

no energy input is required and the process is called facilitated diffusion. A typical example is glu cose transport by the glucose

transporter, which moves glucose down its concentration gradient from the ECF to the cytoplasm of the cell. Other carriers transport

substances against their electrical and chemical gradients. This form of transport require s energy and is called active transport. In

animal cells, the energy is provided almost exclusively by hydrolysis of ATP. Not surpr isingly, therefore, many carrier molecules are

ATPases, enzymes that catalyze the hydrolysis of ATP. One of these ATPases is sodium– potassium adenosine triphosphatase (Na, K

ATPase), which is also known as the Na, K pump. Th ere are also H, K ATPases in the gastric mucosa and the renal tubules.

ATPase pumps Ca

out of cells. Proton ATPases acidify many intracellular organelles, including parts of the Golgi complex and

lysosomes.

Some of the transport proteins are called uniports because they transport only one substa nce. Others are called symports because

transport requires the binding of more than one substance to the transport protein and t he substances are transported across the

membrane together. An example is the symport in the intestinal mucosa that is responsibl e for the cotransport by facilitated diffusion of

and glucose from the intestinal lumen into mucosal cells. Other transporters are called anti ports because they exchange one

substance for another.

Stretch

Cytoskeleton

FIGURE 2–15 Regulation of gating in ion channels. Several types of gating are shown for ion channels. A) Ligand-gated channels

open in response to ligand binding. B) Protein phosphorylation or dephosphoryla tion regulate opening and closing of some ion channels.

C) Changes in membrane potential alter channel openings. D) Mechanical stretch of the membrane results in channel opening.

(Reproduced with permission from Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural S cience, 4th ed. McGraw-Hill, 2000.)

often external (eg, a neurotransmitter or a hormone). However, it can also be internal; intracellular Ca

, cAMP, lipids, or one of the G

proteins produced in cells can bind directly to channels and activate them. Some channe ls are also opened by mechanical stretch, and

these mechanosensitive channels play an important role in cell movement.

Other transport proteins are carriers that bind ions and other molecules and then change their c onfiguration, moving

ION CHANNELS

There are ion channels specific for K

, Na

, Ca

, and Cl

–

, as well as channels that are nonselective for cations or anions. Each type of

channel exists in multiple forms with diverse properties. Most are made up of identical o r very similar subunits. Figure 2–16 shows the

multiunit structure of various channels in diagrammatic cross-section.

Most K

channels are tetramers, with each of the four subunits forming part of the pore throug h which K

ions pass. Structural analysis

of a bacterial voltage-gated K

channel indicates that each of the four subunits have a paddle-like extension containing four charges.

When the channel is closed, these extensions are near the negatively charged interior of the cell. When the membrane potential is

reduced, the paddles containing the charges bend through the membrane to its exterior surface, causing the channel to open. The

bacterial K

channel is very similar to the voltage-gated K

channels in a wide variety of species, including mammals. In the

acetylcholine ion channel and other ligand-gated cation or anion channels, five subunits make up the pore. Members of the ClC family of

–

channels are dimers, but they have two pores, one in each subunit. Finally, aquaporins are tetramers

AB C D

FIGURE 2–16 Different ways in which ion channels form pores. Many K

channels are tetramers (A), with each protein subunit

forming part of the channel. In ligand-gated cation and anion channels (B) such as the ace tylcholine receptor, five identical or very

similar subunits form the channel. Cl

–

channels from the ClC family are dimers (C), with an intracellular pore in each subunit.

Aquaporin water channels (D) are tetramers with an intracellular channel in each subun it. (Reproduced with permission from Jentsch TJ: Chloride

channels are different. Nature 2002;415:276.)

with a water pore in each of the subunits. Recently, a number of ion channels with intri nsic enzyme activity have been cloned. More than

30 different voltage-gated or cyclic nucleotide-gated Na

and Ca

channels of this type have been described. Representative Na

, Ca

and K

channels are shown in extended diagrammatic form in Figure 2–17.

Another family of Na

channels with a different structure has been found in the apical membranes of epithelial cells in the kidneys, colon,

lungs, and brain. The epithelial sodium channels (ENaCs) are made up of three subunits encoded by three different genes. Each of the

subunits probably spans the membrane twice, and the amino terminal and carboxyl term inal are located inside the cell. The α subunit

transports Na

, whereas the β and γ subunits do not. However, the addition of the β and γ subunits in creases Na

transport through the α

subunit. ENaCs are inhibited by the diuretic amiloride, which binds to the α subunit, and they used to be called amilorideinhibitable

channels. The ENaCs in the kidney play an important role in the regulation of ECF volume by aldosterone. ENaC knockout mice

are born alive but promptly die because they cannot move Na

, and hence water, out of their lungs.

Humans have several types of Cl

–

channels. The ClC dimeric channels are found in plants, bacteria, and animals, and ther e are nine

different ClC genes in humans. Other Cl

–

channels have the same pentameric form as the acetylcholine receptor; examples includ e the γ-

aminobutyric acid A (GABA

) and glycine receptors in the central nervous system (CNS). The cystic fibrosis transme mbrane

conductance regulator (CFTR) that is mutated in cystic fibrosis is also a Cl

–

channel. Ion channel mutations cause a variety of

channelopathies—diseases that mostly affect muscle and brain tissue and produce episodic paralyses or convulsions.

Na, K ATPase

As noted previously, Na, K ATPase catalyzes the hydrolysis of ATP to adenosine dipho sphate (ADP) and uses the energy to extrude

three Na

from the cell and take two K

into the cell for each molecule of ATP hydrolyzed. It is an electro genic pump in that it moves

three positive charges out of the cell for each two that it moves in, and it is therefore said to have a coupling ratio of 3:2. It is found in

all parts of the body. Its activity is inhibited by ouabain and related digitalis glycosides us ed in the treatment of heart failure. It is a

heterodimer made up of an α subunit with a molecular weight of approximately 100,00 0 and a β subunit with a molecular weight of

approximately 55,000. Both extend through the cell membrane (Figure 2–18). Separat ion of the subunits eliminates activity. The β

subunit is a glycoprotein, whereas Na

and K

transport occur through the α subunit. The β subunit has a single membrane-spanning

domain and three extracellular glycosylation sites, all of which appear to have attached c arbohydrate residues. These residues account for

one third of its molecular weight. The α subunit probably spans the cell membrane 10 tim es, with the amino and carboxyl terminals both

located intracellularly. This subunit has intracellular Na

- and ATP-binding sites and a phosphorylation site; it also has extracellular

binding sites for K

and ouabain. The endogenous ligand of the ouabain-binding site is unsettled. When N a

binds to the α subunit, ATP

also binds and is converted to ADP, with a phosphate being transferred to Asp 376, the phosphorylation site. This causes a change in the

configuration of the protein, extruding Na

into the ECF. K

then binds extracellularly, dephosphorylating the α subunit, which returns to

its previous conformation, releasing K

into the cytoplasm.

The α and β subunits are heterogeneous, with α

, α

, and α

subunits and β

, β

, and β

subunits described so far. The α

isoform is found in the membranes of most cells, whereas α

is present in muscle, heart, adipose tissue, and brain, and α

is present in

heart and brain. The β

subunit is widely distributed but is absent in certain astrocytes, vestibular cells of the inne r ear, and glycolytic

fast-twitch muscles. The fast-twitch muscles contain only β

subunits. The different α and β subunit structures of Na, K ATPase in

various tissues probably represent specialization for specific tissue functions.

REGULATION OF Na, K ATPase ACTIVITY

The amount of Na

normally found in cells is not enough to saturate the pump, so if the Na

increases, more is pumped out. Pump

The amount of Na

normally found in cells is not enough to saturate the pump, so if the Na

increases, more is pumped out. Pump

activity is affected by second messenger molecules (eg, cAMP and diacylglycerol [DAG ]). The magnitude and direction of the altered

pump effects vary with the experimental conditions. Thyroid hormones increase pump activity by a genomic action to increase the

formation of Na, K ATPase molecules. Aldosterone also increases the number of pump s, although this effect is probably secondary.

Dopamine in the kidney inhibits the pump by phosphorylating it, causing a natriuresis. I nsulin increases pump activity, probably by a

variety of different mechanisms.

channel I II III IV Extracellular side

123 5

6 123 5

Cytoplasmic side

2 COOH

channel

123 5

6 123 5

COOH NH

channel

123 5

COOH NH

FIGURE 2–17 Diagrammatic representation of the pore-forming subunits of three ion channels. The

subunit of the Na

and Ca

channels traverse the membrane 24 times in four repeats of six membrane-spanning un its. Each repeat has a “P” loop between membrane

spans 5 and 6 that does not traverse the membrane. These P loops are though t to form the pore. Note that span 4 of each repeat is colored

in red, representing its net “+” charge. The K

channel has only a single repeat of the six spanning regions and P loop. Four K

subunits

are assembled for a functional K

channel. (Reproduced with permission from Kandel ER, Schwartz JH, Jessell TM [editors]: Princi ples of Neural Science,

4th ed. McGraw-Hill, 2000.)

SECONDARY ACTIVE TRANSPORT

In many situations, the active transport of Na

is coupled to the transport of other substances (secondary active transport). For

example, the luminal membranes of mucosal cells in the small intestine contain a symport that transports glucose into the cell only if Na

binds to the protein and is transported into the cell at the same time. From the cells, the glu cose enters the blood. The electrochemical

gradient for Na

is maintained by the active transport of Na

out of the mucosal cell into ECF. Other examples are shown in Figure 2–

19. In the heart, Na,K ATPase indirectly affects Ca

transport. An antiport in the membranes of cardiac muscle cells normally

exchanges intracellular Ca

for extracellular Na

Active transport of Na

and K

is one of the major energyusing processes in the body. On the average, it accounts for

Ouabain β

ECF

Active transport 2K

Ouabain

3Na

+ ATP

ADP + Pi

Cl− Na

+ Ca

Cytoplasm

1 α

3Na

FIGURE 2–18 Na

–K

ATPase. The intracellular portion of the

subunit has a Na

-binding site (1), a phosphorylation site (4), and an

ATP-binding site (5). The extracellular portion has a K

-binding site (2) and an ouabain-binding site (3). (From Horisberger J-D et al: Structure–

by Annual Reviews)

about 24% of the energy utilized by cells, and in neurons it accounts for 70%. Thus, it accounts for a large part of the basal metabolism.

A major payoff for this energy use is the establishment of the electrochemical gradient i n cells.

15 meq/L

K+, 2Cl−

150 − Na

H+ Cl− 7 − SugarsK+

or amino

acids Cl−

−− − − Vm = −70 mV + + + +

140 meq/L K

4 −

Cl− 105 −

FIGURE 2–19 Composite diagram of main secondary effects of active transport of Na

and K

. Na,K ATPase converts the

chemical energy of ATP hydrolysis into maintenance of an inward gradient for Na

and an outward gradient for K

. The energy of the

gradients is used for countertransport, cotransport, and maintenance of the membrane poten tial. Some examples of cotransport and

countertransport that use these gradients are shown. (Reproduced with permission from Sk ou JC: The Na–K pump. News Physiol Sci 1992;7:95.)

TRANSPORT ACROSS EPITHELIA

In the gastrointestinal tract, the pulmonary airways, the renal tubules, and other structure s, substances enter one side of a cell and exit

another, producing movement of the substance from one side of the epithelium to the o ther. For transepithelial transport to occur, the

cells need to be bound by tight junctions and, obviously, have different ion channels an d transport proteins in different parts of their

membranes. Most of the instances of secondary active transport cited in the preceding p aragraph involve transepithelial movement of

ions and other molecules.

THE CAPILLARY WALL

FILTRATION

The capillary wall separating plasma from interstitial fluid is different from the cell memb ranes separating interstitial fluid from

intracellular fluid because the pressure difference across it makes filtration a significant factor in pr oducing movement of water and

solute. By definition, filtration is the process by which fluid is forced through a membra ne or other barrier because of a difference in

pressure on the two sides.

ONCOTIC PRESSURE

The structure of the capillary wall varies from one vascular bed to another. However, in skeletal muscle and many other organs, water

and relatively small solutes are the only substances that cross the wall with ease. The aper tures in the junctions between the endothelial

cells are too small to permit plasma proteins and other colloids to pass through in signific ant quantities. The colloids have a high

molecular weight but are present in large amounts. Small amounts cross the capillary wa ll by vesicular transport, but their effect is slight.

Therefore, the capillary wall behaves like a membrane impermeable to colloids, and thes e exert an osmotic pressure of about 25 mm Hg.

The colloid osmotic pressure due to the plasma colloids is called the oncotic pressure. F iltration across the capillary membrane as a

result of the hydrostatic pressure head in the vascular system is opposed by the oncotic pressure. The way the balance between the

hydrostatic and oncotic pressures controls exchanges across the capillary wall is conside red in detail in Chapter 32.

TRANSCYTOSIS

Vesicles are present in the cytoplasm of endothelial cells, and tagged protein molecules in jected into the bloodstream have been found in

the vesicles and in the interstitium. This indicates that small amounts of protein are transpo rted out of capillaries across endothelial cells

by endocytosis on the capillary side followed by exocytosis on the interstitial side of the cells. The transport mechanism makes use of

coated vesicles that appear to be coated with caveolin and is called transcytosis, vesicular transport, or cytopempsis.

GAP JUNCTIONS SYNAPTIC PARACRINE AND AUTOCRINE ENDOCRINE

Message transmission Directly from cell

to cell

Across synaptic cleft

By diffusion in interstitial fluid By circulating body fluids

Local or general Local Local Locally diffuse General

Specificity depends on Anatomic location Anatomic location and receptors

Receptors

FIGURE 2–20 Intercellular communication by chemical mediators. A, autocrine; P, para crine.

INTERCELLULAR COMMUNICATION

Cells communicate with one another via chemical messengers. Within a given tissue, som e messengers move from cell to cell via gap

junctions without entering the ECF. In addition, cells are affected by chemical messenge rs secreted into the ECF, or by direct cell–cell

contacts. Chemical messengers typically bind to protein receptors on the surface of the c ell or, in some instances, in the cytoplasm or the

nucleus, triggering sequences of intracellular changes that produce their physiologic eff ects. Three general types of intercellular

communication are mediated by messengers in the ECF: (1) neural communicat ion, in which neurotransmitters are released at synaptic

junctions from nerve cells and act across a narrow synaptic cleft on a postsynaptic cell; (2) endocrine communication, in which

hormones and growth factors reach cells via the circulating blood or the lymph; and (3 ) paracrine communication, in which the

products of cells diffuse in the ECF to affect neighboring cells that may be some distanc e away (Figure 2–20). In addition, cells secrete

chemical messengers that in some situations bind to receptors on the same cell, that is, the cell that secreted the messenger (autocrine

communication). The chemical messengers include amines, amino acids, steroids, polyp eptides, and in some instances, lipids, purine

nucleotides, and pyrimidine nucleotides. It is worth noting that in various parts of the bo dy, the same chemical messenger can function as

a neurotransmitter, a paracrine mediator, a hormone secreted by neurons into the blood (neural hormone), and a hormone secreted by

gland cells into the blood.

An additional form of intercellular communication is called juxtacrine communication. Some cells express multiple repeats of growth

factors such as transforming growth factor alpha (TGFα) extracellularly on transmembrane pro teins that provide an anchor to the cell.

Other cells have TGFα receptors. Consequently, TGFα anchored to a cell can bind to a TGFα receptor on another cell, linking the two.

This could be important in producing local foci of growth in tissues.

RECEPTORS FOR CHEMICAL MESSENGERS

The recognition of chemical messengers by cells typically begins by interaction with a re ceptor at that cell. There have been over 20

families of receptors for chemical messengers characterized. These proteins are not stati c components of the cell, but their numbers

increase and decrease in response to various stimuli, and their properties change with ch anges in physiological conditions. When a

hormone or neurotransmitter is present in excess, the number of active receptors gener ally decreases (down-regulation), whereas in the

presence of a deficiency of the chemical messenger, there is an increase in the number of active receptors (up-regulation). In its actions

on the adrenal cortex, angiotensin II is an exception; it increases rather than decreases th e number of its receptors in the adrenal. In the

case of receptors in the membrane, receptor-mediated endocytosis is responsible for do wn-regulation in some instances; ligands bind to

their receptors, and the ligand– receptor complexes move laterally in the membrane to c oated pits, where they are taken into the cell by

endocytosis (internalization). This decreases the number of receptors in the membrane. Some receptors are recycled after

internalization, whereas others are replaced by de novo synthesis in the cell. Another ty pe of down-regulation is desensitization, in

which receptors are chemically modified in ways that make them less responsive.

Receptors

MECHANISMS BY WHICH

CHEMICAL MESSENGERS ACT

Receptor–ligand interaction is usually just the beginning of the cell response. This event is transduced into secondary responses within

the cell that can be divided into four broad categories: (1) ion channel activation, (2) G-protein activation, (3) activation of enzyme

activity within the cell, or (4) direct activation of transcription. Within each of these grou ps, responses can be quite varied. Some of the

common mechanisms by which

TABLE 2–3 Common mechanisms by which chemical messengers in the ECF bring about changes in cell function.

Mechanism Examples

Open or close ion channels in cell membrane

Acetylcholine on nicotinic cholinergic receptor; norepinephrine on K

channel in the heart

Act via cytoplasmic or nuclear receptors to increase transcription of selected mRNAs

Thyroid hormones, retinoic acid, steroid hormones

Activate phospholipase C with intracellular production of DAG, IP

, and other inositol phosphates Angiotensin II, norepinephrine via

-adrenergic receptor, vasopressin via V

receptor

Activate or inhibit adenylyl cyclase, causing increased or decreased intracellular

production of cAMP

Norepinephrine via

β1

-adrenergic receptor (increased cAMP); norepinephrine via

α2

-adrenergic receptor (decreased cAMP)

Increase cGMP in cell Atrial natriuretic peptide; nitric oxide

Increase tyrosine kinase activity of cytoplasmic portions of transmembrane rec eptors

Insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), monocyte colo nystimulating factor (M-CSF)

pathways. Cellular phosphorylation is under the control of two groups of proteins: k inases, enzymes that catalyze the phosphorylation of

tyrosine or serine and threonine residues in proteins (or in some cases, in lipids); and phosph atases, proteins that remove phosphates

from proteins (or lipids). Some of the larger receptor families are themselves kinases. T yrosine kinase receptors initiate phosphorylation

on tyrosine residues on complementary receptors following ligand binding. Serine/threo nine kinase receptors initiate phosphorylation on

serines or threonines in complementary receptors following ligand binding. Cytokine re ceptors are directly associated with a group of

protein kinases that are activated following cytokine binding. Alternatively, second mess engers changes can lead to phosphorylation

further downstream in the signaling pathway. More than 300 protein kinases have been described. Some of the principal ones that are

important in mammalian cell signaling are summarized in Table 2–4. In general, addition of phosphate groups changes the conformation

of the proteins, altering their functions and consequently the functions of the cell. The c lose relationship between phosphorylation and

dephosphorylation of cellular proteins allows for a temporal control of activation of cel l signaling pathways. This is sometimes referred

to as a “phosphate timer.”

Increase serine or threonine kinase activity

TGFβ, activin, inhibin

STIMULATION OF TRANSCRIPTION

The activation of transcription, and subsequent translation, is a common outcome of cell ular signaling. There are three chemical

messengers exert their intracellular effects are summarized in Table 2–3. Ligands such a s acetylcholine bind directly to ion channels in

the cell membrane, changing their

conductance. Thyroid and steroid hormones, 1,25-dihydroxycholecalciferol, and retin oids enter cells and act on one or another member

of a family of structurally related cytoplasmic or

nuclear receptors. The activated receptor binds to DNA and increases transcription of s elected mRNAs. Many other ligands in

the ECF bind to receptors on the surface of cells and trigger the

release of intracellular mediators such as cAMP, IP

, and DAG

that initiate changes in cell function. Consequently, the extracellular ligands are called “first messengers” and the intracellular mediators

are called “second messengers.” Second

messengers bring about many short-term changes in cell function by altering enzyme fu nction, triggering exocytosis, and so

on, but they also can lead to the alteration of transcription of

various genes. A variety of enzymatic changes, protein–protein

interactions or second messenger changes can be activated

within a cell in an orderly fashion following receptor recognition of the primary messen ger. The resulting cell signaling

pathway provides amplification of the primary signal and distribution of the sign al to appropriate targets within the cell. Extensive cell

signaling pathways also provide opportunities for

feedback and regulation that can fine tune the signal for the correct physiological respo nse by the cell.

The most predominant posttranslation modification of proteins, phosphorylation, is a com mon theme in cell signaling

TABLE 2–4 Sample protein kinases.

Phosphorylate serine or threonine residues, or both

Calmodulin-dependent

Myosin light-chain kinase

Phosphorylase kinase

/calmodulin kinase I

/calmodulin kinase II

/calmodulin kinase III

Calcium-phospholipid-dependent

Protein kinase C (seven subspecies)

Cyclic nucleotide-dependent

cAMP-dependent kinase (protein kinase A; two subspecies)

cGMP-dependent kinase

Phosphorylate tyrosine residues

Insulin receptor, EGF receptor, PDGF receptor, and

M-CSF receptor

distinct pathways for primary messengers to alter transcription of cells. First, as is the cas e with steroid or thyroid hormones, the primary

messenger is able to cross the cell membrane and bind to a nuclear receptor, which then can directly interact with DNA to alter gene

expression. A second pathway to gene transcription is the activation of cytoplasmic pro tein kinases that can move to the nucleus to

phosphorylate a latent transcription factor for activation. This pathway is a common end point of signals that go through the mitogen

activated protein (MAP) kinase cascade. MAP kinases can be activated foll owing a variety of receptor ligand interactions through

second messenger signaling. They comprise a series of three kinases that coordinate a s tepwise phosphorylation to activate each protein

in series in the cytosol. Phosphorylation of the last MAP kinase in series allows it to migra te to the nucleus where it phosphorylates a

latent transcription factor. A third common pathway is the activation of a latent transcript ion factor in the cytosol, which then migrates to

the nucleus and alters transcription. This pathway is shared by a diverse set of transcrip tion factors that include nuclear factor kappa B

(NFκB; activated following tumor necrosis family receptor binding and others), and signal transducers of activated trans cription

(STATs; activated following cytokine receptor binding). In all cases the binding of the activated tran scription factor to DNA increases

(or in some cases, decreases) the transcription of mRNAs encoded by the gene to whic h it binds. The mRNAs are translated in the

ribosomes, with the production of increased quantities of proteins that alter cell function .

INTRACELLULAR Ca

AS A SECOND MESSENGER

regulates a very large number of physiological processes that are as diverse as prolifer ation, neural signaling, learning, contraction,

secretion, and fertilization, so regulation of intracellular Ca

is of great importance. The free Ca

concentration in the cytoplasm at rest

is maintained at about 100 nmol/ L. The Ca

concentration in the interstitial fluid is about 12,000 times the cytoplasmic concentration

(ie, 1,200,000 nmol/L), so there is a marked inwardly directed concentration gradient a s well as an inwardly directed electrical gradient.

Much of the intracellular Ca

is stored at relatively high concentrations in the endoplasmic reticulum and other organe lles (Figure 2–

21), and these organelles provide a store from which Ca

can be mobilized via ligand-gated channels to increase the concentration of

free Ca

in the cytoplasm. Increased cytoplasmic Ca

binds to and activates calcium-binding proteins. These proteins can have direct

effects in cellular physiology, or can activate other proteins, commonly protein kinases, to further cell signaling pathways.

can enter the cell from the extracellular fluid, down its electrochemical gradient, throug h many different Ca

channels. Some of

these are ligand-gated and others are voltagegated. Stretch-activated channels exist in so me cells as well.

Ca 2+ CaBP Effects

(volt)

2H+

ATP

Ca2+ Ca2+(lig) Ca2+

Ca2+

3Na

(SOCC)

2+Ca

Mitochondrion Endoplasmic reticulum

handling in mammalian cells. Ca

is FIGURE 2–21 Ca

stored in the endoplasmic reticulum and, to a lesser extent, mitochondria and can be released from them to replenish cytoplasmic Ca

Calciumbinding proteins (CaBP) bind cytoplasmic Ca

and, when activated in this fashion, bring about a variety of physiologic effects.

enters the cells via voltage-gated (volt) and ligand-gated (lig) Ca

channels and store-operated calcium channels ( SOCCs). It is

transported out of the cell by Ca, Mg ATPases (not shown), Ca, H ATPase and an Na, Ca an tiport. It is also transported into the ER by

Ca ATPases.

Many second messengers act by increasing the cytoplasmic Ca

concentration. The increase is produced by releasing Ca

from

intracellular stores—primarily the endoplasmic reticulum—or by increasing the entry of Ca

into cells, or by both mechanisms. IP

the major second messenger that causes Ca

release from the endoplasmic reticulum through the direct activation of a ligand-gated

channel, the IP

receptor. In effect, the generation of one second messenger (IP

) can lead to the release of another second messenger

(Ca

). In many tissues, transient release of Ca

from internal stores into the cytoplasm triggers opening of a population of Ca

channels in the cell membrane (store-operated Ca

channels; SOCCs). The resulting Ca

influx replenishes the total intracellular

supply and refills the endoplasmic reticulum. The exact identity of the SOCCs is still unk nown, and there is debate about the signal

from the endoplasmic reticulum that opens them.

As with other second messenger molecules, the increase in Ca

within the cytosol is rapid, and is followed by a rapid decrease. Because

the movement of Ca

outside of the cytosol (ie, across the plasma membrane or the membrane of the internal store) requires that it

move up its electrochemical gradient, it requires energy. Ca

movement out of the cell is facilitated by the plasma membrane Ca

ATPase. Alternatively, it can be transported by an antiport that exchanges three Na

for each Ca

driven by the energy stored in the Na

electrochemical gradient. Ca

movement into the internal stores is through the action of the sarcoplasmic or endoplasmic reticulum

ATPase, also known as the SERCA pump.

CALCIUM-BINDING PROTEINS

Many different Ca

-binding proteins have been described, including troponin, calmodulin, and calbindin. Troponin is the

70 90

L F

R V

F A

K D E

M M A R K

R E D

GD M I N Ca E LCa I K E G R

V D

T D E

N G

S E

I S

A A H

D G

N 60 80

100

M I 50 A E N E G L N

(Me)3

110M D Q L E KT

G Q N T D

120

L E

E V D E

M E

G K

30 G K

V G D G IT T T I T

COOH

V D 130 R

Ca N F E A

Ca I

G K

10 A

T Y N A E

E E

140

20 E K

F A M Q

NH Ac

FIGURE 2–22 Structure of calmodulin from bovine brain. Single-letter abbreviations are used fo r the amino acid residues. Note the

four calcium domains (purple residues) flanked on either side by stretches of α helix. (R eproduced with permission from Cheung WY: Calmodulin:

An overview. Fed Proc 1982;41:2253.)

-binding protein involved in contraction of skeletal muscle (Chapter 5). Calmodulin con tains 148 amino acid residues (Figure 2–22)

and has four Ca

-binding domains. It is unique in that amino acid residue 115 is trimethylated, and it is ex tensively conserved, being

found in plants as well as animals. When calmodulin binds Ca

, it is capable of activating five different calmodulin-dependent kinases

(CaMKs; Table 2–4), among other proteins. One of the kinases is myosin light-chain kinase, whi ch phosphorylates myosin. This brings

about contraction in smooth muscle. CaMKI and CaMKII are concerned with synaptic function, and CaMKIII is concerned with protein

synthesis. Another calmodulin-activated protein is calcineurin, a phosphatase that inactivates Ca

channels by dephosphorylating them.

It also plays a prominent role in activating T cells and is inhibited by some immunosuppr essants.

MECHANISMS OF DIVERSITY

OF Ca

ACTIONS

It may seem difficult to understand how intracellular Ca

can have so many varied effects as a second messenger. Part of the

explanation is that Ca

may have different effects at low and at high concentrations. The ion may be at high c oncentration at the site of

its release from an organelle or a channel (Ca

sparks) and at a subsequent lower concentration after it diffuses throughout the cell.

Some of the changes it produces can outlast the rise in intracellular Ca

concentration because of the way it binds to some of the Ca

binding proteins. In addition, once released, intracellular Ca

concentrations frequently oscillate at regular intervals, and there is

evidence that the frequency and, to a lesser extent, the amplitude of those oscillations co des information for effector mechanisms.

Finally, increases in intracellular Ca

concentration can spread from cell to cell in waves, producing coordinated events such as the

rhythmic beating of cilia in airway epithelial cells.

G PROTEINS

A common way to translate a signal to a biologic effect inside cells is by way of nucleoti de regulatory proteins that are activated after

binding GTP (G proteins). When an activating signal reaches a G protein, the protein exc hanges GDP for GTP. The GTP–protein

complex brings about the activating effect of the G protein. The inherent GTPase activit y of the protein then converts GTP to GDP,

restoring the G protein to an inactive resting state. G proteins can be divided into two pr incipal groups involved in cell signaling: small

G proteins and heterotrimeric G proteins. Other groups that have similar regulation and are also important to cel l physiology include

elongation factors, dynamin, and translocation GTPases.

There are six different families of small G proteins (or small GTPases) that are all highly regulate d. GTPase activating proteins

(GAPs) tend to inactivate small G proteins by encouraging hydrolysis of GTP to GDP in the central binding site. Guanine exchange

factors (GEFs) tend to activate small G proteins by encouraging exchange of GDP for GTP in the active site. Some of the small G

proteins contain lipid modifications that help to anchor them to membranes, while others are free to diffuse throughout the cytosol. Small

G proteins are involved in many cellular functions. Members of the Rab family regulate the rate of vesicle traffic between the

endoplasmic reticulum, the Golgi apparatus, lysosomes, endosomes, and the cell membra ne. Another family of small GTPbinding

proteins, the Rho/Rac family, mediates interactions between the cytoskeleton and cell me mbrane; and a third family, the Ras family,

regulates growth by transmitting signals from the cell membrane to the nucleus.

Another family of G proteins, the larger heterotrimeric G proteins, couple cell surface receptors to catalytic u nits that catalyze the

intracellular formation of second messengers or couple the receptors directly to ion cha nnels. Despite the knowledge of the small G

proteins described above, the heteromeric G proteins are frequently referred to in the s hortened “G protein” form because they were the

first to be identified. Heterotrimeric G proteins are made up of three subunits designated α, β, and γ (Figure 2–23). Both the α and the γ

subunits have lipid modifications that anchor these proteins to plasma membrane. The α subunit is bound to GDP. When a ligand binds to

a G protein-coupled receptor (GPCR), this GDP is exchanged for GTP and the α subu nit separates from the combined β and γ subunits.

The separated α subunit brings about many biologic effects. The β and γ subunits are tig htly bound in the cell and together form a

signaling molecule that can also activate a variety of effectors. The intrinsic

Nucleotide exchange

TABLE 2–5 Some of the ligands for receptors coupled to heterotrimeric G proteins.

Input

GDP

GTP Output Class Ligand GTPase activity Neurotransmitters Epinephrine

Norepinephrine

Dopamine

5-Hydroxytryptamine

β γ

Histamine

Acetylcholine Effectors Adenosine

FIGURE 2–23 Heterotrimeric G proteins. Top Summary of overall reaction that occurs in the Gα subunit. Bottom: When the ligand

(square) binds to the G protein-coupled receptor in the cell membrane, GTP replaces GDP on the α subunit. GTPα separates from the βγ

subunit and GTPα and βγ both activate various effectors, producing physiologic effects . The intrinsic GTPase activity of GTPα then

converts GTP to GDP, and the α, β, and γ subunits reassociate. Opioids

Tachykinins Substance P

Neurokinin A

Neuropeptide K

Other peptides Angiotensin II

GTPase activity of the α subunit then converts GTP to GDP, and this leads to reassociation of the α with the βγ subunit and termination

of effector activation. The GTPase activity of the α subunit can be accelerated by a fam ily of regulators of G protein signaling (RGS).

Heterotrimeric G proteins relay signals from over 1000 GPCRs, and their effectors in t he cells include ion channels and enzymes (Table

2–5). There are 20 α, 6 β, and 12 γ genes, which allow for over 1400 α, β, and γ com binations. Not all combinations occur in the cell, but

over 20 different heterotrimeric G proteins have been well documented in cell signaling . They can be divided into five families, each

with a relatively characteristic set of effectors.

Arginine vasopressin

Oxytocin

VIP, GRP, TRH, PTH

Glycoprotein hormones TSH, FSH, LH, hCG

Arachidonic acid derivatives Thromboxane A

Other Odorants

Tastants

Endothelins

Platelet-activating factor

Cannabinoids

G PROTEIN-COUPLED RECEPTORS

All the heterotrimeric G protein-coupled receptors (GPCRs) that have been characterized to d ate are proteins that span the cell

membrane seven times. Because of this structure they are alternatively referred to as seven-helix receptors or serpentine receptors. A

very large number have been cloned, and their functions are multiple and diverse. The topological structures of two of them are shown in

Figure 2–24. These receptors further assemble into a barrel-like structure. Upon ligand binding, a conformational change activates a

resting heterotrimeric G protein associated with the cytoplasmic leaf of the plasma memb rane. Activation of a single receptor can result

in 1, 10, or more active heterotrimeric G proteins, providing amplification as well as tran sduction of the first messenger. Bound receptors

can be inactivated to limit the amount of cellular signaling. This frequently occurs throug h phosphorylation of the cytoplasmic side of the

receptor.

Light

INOSITOL TRISPHOSPHATE

& DIACYLGLYCEROL AS

SECOND MESSENGERS

The link between membrane binding of a ligand that acts via Ca

and the prompt increase in the cytoplasmic Ca

concentration is

often inositol trisphosphate (inositol 1,4,5-trisphosphate; IP

). When one of these ligands binds to its receptor, activation of the

receptor produces activation of phospholipase C (PLC) on the inner surface of the me mbrane. Ligands bound to G protein-coupled

receptor can do this through the G

heterotrimeric G proteins, while ligands bound to tyrosine kinase receptors can do this through other

cell signaling pathways. PLC has at least eight isoforms; PLC

H D

P V H S G N T T L L F D S D N G P P G M NH

2-Adrenergic receptor

C Y H K E TC

surface

Extracellular

MW N F G N

F H

W T F

D N L

VVG M A

K W

M C R

N Q

L I N E F W Y W H Q A Y I V H

P K

E V Y

I L M S B A G F T S I D M Q I P A I A S V I N V I L L N V I V P V V V L C L F S S I V I F F W L G L A I V A L G M V T A S T L Q S S F Y V P L W C Y V N S F G N V L D I E T V I W P L V L T F A F N

V L V I A C A L L C V I V M L I V M V F T G M I P L I Y

T A I S T I A V D V M R V YSL C R S P D F R

I A F Q F N I K V KK Y

R Y A R

A F

N F A K H E K L C

E R L Q T V T

A K

I T

Q V A K R Q

L Q K K L

Cytoplasmic

T L I S C

S P F K Y Q S L D

H F R G E S K S L

surface

R R

N L G Q V E Q D G R S G H G

S S

K S

A E G M Y D T K G N G N S S Y G N G Y A G

Q L G Q E K E S E R L C E D P P G T E S F V N C

Q HOOC L P S D N T S C N R G Q S D L S L S P V T

P S

R V V C T K N S F P V Y F N P G E T G N M NH

A Intradiskal S C G I D Y

Rhodopsin

surface

G P

F C E

E G L I E

G S

DH E Y T QP

WQ F

L S

N H F

F S N T

S M T Y L F A T W G V E S F F I Y

G P

I F M

L A A Y T T T F L G G E L P P A V I Y M F A V Q T I P A M F L Q G F I A L A C A F V V A Y P F F A L I M L V M F L W S L A L A M V H F I I L W C I K T S A G F P D A V V L A W T F P L I L F A V

Y N I N F L A L N L

E R Y A V G M V I F F I V M I

P V I

Y T L Y L I Y V V V I A H C Y G I V M I M M

N C

N Q

T N K Q

F R N C M

PM S N F R FG

E K

E A

K Q V

Q H K K

L R T

F T V

T T

K E

A A A Q QQ E S

Cytoplasmic surface

T T

C C G

HOOC A P A V Q S T E T K S V T T S A E D D G L P N K

FIGURE 2–24 Structures of two G protein-coupled receptors. The individual amino acid residue s are identified by their single-letter

codes, and the orange residues are sites of phosphorylation. The Y-shaped symbols identify gly cosylation sites. Note the extracellular

amino terminal, the intracellular carboxyl terminal, and the seven membrane-spann ing portions of each protein. (Reproduced with permission

from Benovic JL et al: Lightdependent phosphorylation of rhodopsin by

1986 by Macmillan Magazines)

is activated by heterotrimeric G proteins, while PLC γ forms are activated through tyros ine kinase receptors. PLC isoforms can catalyze

the hydrolysis of the membrane lipid phosphatidylinositol 4,5-diphosphate (PIP

) to form IP

and diacylglycerol (DAG) (Figure 2–25).

The IP

diffuses to the endoplasmic reticulum, where it triggers the release of Ca

into the cytoplasm by binding the IP

receptor, a

ligand-gated Ca

channel (Figure 2–26). DAG is also a second messenger; it stays in the cell membrane, where it activates one of

several isoforms of protein kinase C.

Phosphatidylinositol PIP (PI)

PIP

Diacylglycerol

Phospholipase C

P P 1 1

4 4

Inositol IP +

CDP-diacylglycerol

1 P

54 PP

Phosphatidic acid

FIGURE 2–25 Metabolism of phosphatidylinositol in cell membranes. Phosphatidy linositol is successively phosphorylated to form

phosphatidylinositol 4-phosphate (PIP), then phosphatidylinositol 4,5-bisphosphate (PIP

). Phospholipase C

and phospholipase Cγ

catalyze the breakdown of PIP

to inositol 1,4,5-trisphosphate (IP

) and diacylglycerol. Other inositol phosphates and

phosphatidylinositol derivatives can also be formed. IP

is dephosphorylated to inositol, and diacylglycerol is metabolized to cytosine

diphosphate (CDP)-diacylglycerol. CDP-diacylglycerol and inositol then combine to form phosphatidylinositol, completing the cycle.

(Modified from Berridge MJ: Inositol triphosphate and diacylglycerol as second messengers. Biochem J 198 4;220:345.)

Stimulatory receptor ISF

PIP2

PLC

β γ

Tyrosine Gq, etc

kinase

Cytoplasm DAG

O O O HO P OO CH

Adenine

O O O HO P OO CH

Adenine

OH OH OH

PKC ATP

Phosphoproteins OH OH

Adenylyl cyclase

CaBP Ca

Physiologic effects ER

Physiologic effects PP

FIGURE 2–26 Diagrammatic representation of release of inositol tripho sphate (IP

) and diacylglycerol (DAG) as second

messengers. Binding of ligand to G protein-coupled receptor activates phospholipase C (PL C)

. Alternatively, activation of receptors

with intracellular tyrosine kinase domains can activate PLCγ. The resulting hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP

)

produces IP

, which releases Ca

from the endoplasmic reticulum (ER), and DAG, which activates protein kinase C (PKC). CaBP,

-binding proteins. ISF, interstitial fluid.

O CH

Adenine

cAMP

H2O

O P O OH OH

Phosphodiesterase

CYCLIC AMP

Another important second messenger is cyclic adenosine 3',5'monophosphate (cyclic AMP or cAMP; Figure 2–27). Cyclic AM P is

formed from ATP by the action of the enzyme adenylyl cyclase and conv erted to physiologically inactive 5'AMP by the action of the

enzyme phosphodiesterase. Some of the phosphodiesterase isoforms that break down cAMP are inhibited by methylxanthines such as

caffeine and theophylline. Consequently, these compounds can augment hormonal and transmitter effects mediated via cAMP. Cyclic

AMP activates one of the cyclic nucleotide-dependent protein kinases (protein k inase A, PKA) that, like protein kinase C, catalyzes the

phosphorylation of proteins, changing their conformation and altering their activity. In addition, the active catalytic subunit of PKA

moves to the nucleus and phosphorylates the cAMP-responsive element-binding prote in (CREB). This transcription factor then binds

to DNA and alters transcription of a number of genes.

PRODUCTION OF cAMP

BY ADENLYL CYCLASE

Adenylyl cyclase is a transmembrane protein, and it crosses the membrane 12 times. Ten isoforms of this enzyme have been described

and each can have distinct regulatory properties, permitting the cAMP pathway to be cu stomized to specific tissue needs. Notably,

stimulatory heterotrimeric G proteins (G

) activate, while inhibitory heterotrimeric G proteins (G

) inactivate adenylyl cyclase (Figure 2–

28). When the appropriate ligand binds to a stimulatory receptor, a G

α subunit activates one of the adenylyl cyclases. Conversely, when

the

AMP

HO P O CH

Adenine

OH OH

FIGURE 2–27 Formation and metabolism of cAMP. The second me ssenger cAMP is a made from ATP by adenylyl cyclase and

broken down into cAMP by phosphodiesterase.

appropriate ligand binds to an inhibitory receptor, a G

α subunit inhibits adenylyl cyclase. The receptors are specific, responding at low

threshold to only one or a select group of related ligands. However, heterotrimeric G p roteins mediate the stimulatory and inhibitory

effects produced by many different ligands. In addition, cross-talk occurs between the phospholipase C system and the adenylyl cyclase

system, as several of the isoforms of adenylyl cyclase are stimulated by calmodulin. Fina lly, the effects of protein kinase A and protein

kinase C are very widespread and can also affect directly, or indirectly, the activity at ad enylyl cyclase. The close relationship between

activation of G proteins and adenylyl cyclases also allows for spatial regulation of cAM P production. All of these events, and others,

allow for fine-tuning the cAMP response for a particular physiological outcome in the c ell.

Two bacterial toxins have important effects on adenylyl cyclase that are mediated by G proteins. The A subunit of cholera toxin

catalyzes the transfer of ADP ribose to an arginine residue in the middle of the α subun it of G

. This inhibits

Stimulatory

receptor

ISF

Adenylyl cyclase Inhibitory receptor ANP

β γ

Cytoplasm

ATP

CAMP

β γ

PDE

ISF

PTK

NH2

5' AMP

Protein kinase A

Phosphoproteins

cyc cyc cyc COOH

COOH COOH

EGF

PDGF

PTK PTP

PTK

PTPPTP

COOH COOH

COOHCOOH

Physiologic effects Guanylyl cyclases Tyrosine kinases Tyrosine

phosphatases FIGURE 2–28 The cAMP system. Activation of adenylyl cy clase catalyzes the conversion of ATP to cAMP. Cyclic AMP

activates protein kinase A, which phosphorylates proteins, producing physiologic effects. St imulatory ligands bind to stimulatory

receptors and activate adenylyl cyclase via G

. Inhibitory ligands inhibit adenylyl cyclase via inhibitory receptors and G

. ISF, interstitial

fluid.

its GTPase activity, producing prolonged stimulation of adenylyl cyclase. Pertussis toxin catalyzes ADP-ribosylation of a cysteine

residue near the carboxyl terminal of the α subunit of G

. This inhibits the function of G

. In addition to the implications of these

alterations in disease, both toxins are used for fundamental research on G protein funct ion. The drug forskolin also stimulates adenylyl

cyclase activity by a direct action on the enzyme.

GUANYLYL CYCLASE

Another cyclic nucleotide of physiologic importance is cyclic guanosine monoph osphate (cyclic GMP or cGMP). Cyclic GMP is

important in vision in both rod and cone cells. In addition, there are cGMP-regulated io n channels, and cGMP activates cGMP-dependent

kinase, producing a number of physiologic effects.

Guanylyl cyclases are a family of enzymes that catalyze the formation of cGMP. They e xist in two forms (Figure 2–29). One form has an

extracellular amino terminal domain that is a receptor, a single transmembrane domain, a nd a cytoplasmic portion with guanylyl cyclase

catalytic activity. Three such guanylyl cyclases have been characterized. Two are recep tors for atrial natriuretic peptide (ANP; also

known as atrial natriuretic factor), and a third binds an Escherichia coli enterotoxin and the gastrointestinal polypeptide guanylin. The

other form of guanylyl cyclase is soluble, contains heme, and is not bound to the memb rane. There appear to be several isoforms of the

intracellular enzyme. They are activated by nitric oxide (NO) and NO-containing comp ounds.

FIGURE 2–29 Diagrammatic representation of guanylyl cyclases, tyrosine kin ases, and tyrosine phosphatases. ANP, atrial

natriuretic peptide; C, cytoplasm; cyc, guanylyl cyclase domain; EGF, epidermal gro wth factor; ISF, interstitial fluid; M, cell membrane;

PDGF, platelet-derived growth factor; PTK, tyrosine kinase domain; PTP, tyrosine ph osphatase domain; ST, E. coli enterotoxin. (Modified

from Koesling D, Böhme E, Schultz G: Guanylyl cyclases, a growing family of signal transducing e nzymes. FASEB J 1991;5:2785.)

GROWTH FACTORS

Growth factors have become increasingly important in many different aspects of physi ology. They are polypeptides and proteins that are

conveniently divided into three groups. One group is made up of agents that foster the multiplication or development of various types of

cells; nerve growth factor (NGF), insulin-like growth factor I (IGF-I), activins and inh ibins, and epidermal growth factor (EGF) are

examples. More than 20 have been described. The cytokines are a second group. The se factors are produced by macrophages and

lymphocytes, as well as other cells, and are important in regulation of the immune system (see Chapter 3). Again, more than 20 have

been described. The third group is made up of the colony-stimulating factors that regula te proliferation and maturation of red and white

blood cells.

Receptors for EGF, platelet-derived growth factor (PDGF), and many of the other fac tors that foster cell multiplication and growth have

a single membrane-spanning domain with an intracellular tyrosine kinase domain (Figur e 2–29). When ligand binds to a tyrosine kinase

receptor, it first causes a dimerization of two similar receptors. The dimerization results in partial activation of the intracellular tyrosine

kinase domains and a cross-phosphorylation to fully activate each other. One of the pa thways activated by phosphorylation leads,

through the small G protein Ras, to MAP kinases, and eventually to the production of tr anscription factors in the nucleus that alter gene

expression (Figure 2–30).

Receptors for cytokines and colony-stimulating factors differ from the other growth fa ctors in that most of them do not have tyrosine

kinase domains in their cytoplasmic portions and

Growth factor Ligand Receptor

Cell membrane

Receptor ISF

Inactive

Ras Ras

Active Ras Ras Cytoplasm

GDP GTP

Grb2

SOS Raf

STAT STAT MAP KK

MAP K

B Ligand

TF P P P P Nucleus STAT STAT

Altered gene activity

FIGURE 2–30 One of the direct pathways by which growth factors alter gene activity. TK, tyrosine kinase domain; Grb2, Ras

activator controller; Sos, Ras activator; Ras, product of the ras gene; MAP K, mitogen-activ ated protein kinase; MAP KK, MAP kinase

kinase; TF, transcription factors. There is cross-talk between this pathway and the cAMP pathway, as well as cross-talk with the IP

–

DAG pathway.

Ligand

P P P P

STAT

P P

STAT

some have little or no cytoplasmic tail. However, they initiate tyrosine kinase activity in th e cytoplasm. In particular, they activate the

so-called Janus tyrosine kinases (JAKs) in the cytoplasm (Figure 2–31). These i n turn phosphorylate STAT proteins. The

phosphorylated STATs form homo- and heterodimers and move to the nucleus, where they act as transcription factors. There are four

known mammalian JAKs and seven known STATs. Interestingly, the JAK–STAT path way can also be activated by growth hormone and

is another important direct path from the cell surface to the nucleus. However, it should be emphasized that both the Ras and the JAK–

STAT pathways are complex and there is cross-talk between them and other signaling p athways discussed previously.

Finally, note that the whole subject of second messengers and intracellular signaling has become immensely complex, with multiple

pathways and interactions. It is only possible in a book such as this to list highlights and p resent general themes that will aid the reader in

understanding the rest of physiology (see Clinical Box 2–3).

Ligand

P P P P

STAT Nucleus

DNA

FIGURE 2–31 Signal transduction via the JAK–STAT pathway. A) Ligan d binding leads to dimerization of receptor. B) Activation

and tyrosine phosphorylation of JAKs. C) JAKs phosphorylate STATs. D) STATs dimerize and move to nucleus, where they bind to

response elements on DNA. (Modified from Takeda K, Kishimoto T, Akira S: STAT6: Its role in interleukin 4-m ediated biological functions. J Mol Med

1997;75:317.)

HOMEOSTASIS

The actual environment of the cells of the body is the interstitial component of the ECF. Because normal cell function

CLINICAL BOX 2–3 Receptor & G Protein Diseases

Many diseases are being traced to mutations in the genes for receptors. For example, lo ss-of-function receptor mutafor receptors. For

example, loss-of-function receptor muta dihydroxycholecalciferol receptor and the ins ulin receptor. Certain other diseases are caused by

production of antibodies against receptors. Thus, antibodies against thyroidstimulating ho rmone (TSH) receptors cause Graves’ disease,

and antibodies against nicotinic acetylcholine receptors cause myasthenia gravis.

An example of loss of function of a receptor is the type of nephrogenic diabetes insipidus that is due to loss o f the ability of mutated V

vasopressin receptors to mediate concentration of the urine. Mutant receptors can gain as well as lose function. A gain-of-function

mutation of the Ca

receptor causes excess inhibition of parathyroid hormone secretion and familial hypercalciuric hypocalcemia. G

proteins can also undergo loss-of-function or gain-of-function mutations that cause dis ease (Table 2–6), In one form of

pseudohypoparathyroidism, a mutated G

α fails to respond to parathyroid hormone, producing the symptoms of hypoparathyro idism

without any decline in circulating parathyroid hormone. Testotoxicosis is an interesting disease tha t combines gain and loss of function.

In this condition, an activating mutation of G

α causes excess testosterone secretion and prepubertal sexual maturation. However, this

mutation is temperature-sensitive and is active only at the relatively low temperature of th e testes (33 °C). At 37 °C, the normal

temperature of the rest of the body, it is replaced by loss of function, with the productio n of hypoparathyroidism and decreased

responsiveness to TSH. A different activating mutation in G

α is associated with the rough-bordered areas of skin pigmentation and

hypercortisolism of the McCune–Albright syndrome. This mutation occurs during fetal development, creating a mosaic of normal and

abnormal cells. A third mutation in G

α reduces its intrinsic GTPase activity. As a result, it is much more active than normal, an d excess

cAMP is produced. This causes hyperplasia and eventually neoplasia in somatotrope ce lls of the anterior pituitary. Forty percent of

somatotrope tumors causing acromegaly have cells containing a somatic mutation of this type.

depends on the constancy of this fluid, it is not surprising that in multicellular animals, an immense number of regulatory mechanisms

have evolved to maintain it. To describe “the various physiologic arrangements which s erve to restore the normal state, once it has been

disturbed,” W.B. Cannon coined the term homeostasis. The buffering p roperties of the body fluids and the renal and respiratory

adjustments to the presence of excess acid or alkali are examples of homeostatic

TABLE 2–6 Examples of abnormalities caused by loss- or gain-of-function mutations of he terotrimeric G protein-coupled

receptors and G proteins.

Site Type of

Mutation Disease

Receptor

Cone opsins Loss Color blindness

Rhodopsin Loss Congenital night blindness; two forms of retinitis pigmentosa

vasopressin Loss X-linked nephrogenic diabetes insipidus

ACTH Loss Familial glucocorticoid deficiency

LH Gain Familial male precocious puberty

TSH Gain Familial nonautoimmune hyperthyroidism

TSH Loss Familial hypothyroidism

Gain Familial hypercalciuric hypocalcemia

Thromboxane A

Loss Congenital bleeding

Endothelin B Loss Hirschsprung disease

G protein

α Loss Pseudohypothyroidism type 1a

α Gain/loss Testotoxicosis

α Gain

(mosaic) McCune–Albright syndrome

s α Gain Somatotroph adenomas with acromegaly

α Gain Ovarian and adrenocortical tumors

Modified from Lem J: Diseases of G-protein-coupled signal transduction pathways: Th e mammalian visual system as a model. Semin

Neurosci 1998;9:232.

mechanisms. There are countless other examples, and a large part of physiology is con cerned with regulatory mechanisms that act to

maintain the constancy of the internal environment. Many of these regulatory mechanis ms operate on the principle of negative feedback;

deviations from a given normal set point are detected by a sensor, and signals from the sensor trigger compensatory changes that

continue until the set point is again reached.

CHAPTER SUMMARY

■ The cell and the intracellular organelles are surrounded by a semipermeable membr ane. Biological membranes have a lipid bilayer with

a hydrophobic core and hydrophilic outer regions that provide a barrier between inside and outside compartments as well as a template

for biochemical reactions. The membrane is populated by structural and functional p roteins that can be integrated into the membrane or

be associated with one side of the lipid bilayer. These proteins contribute greatly to the semipermeable properties of biological

membrane.

■ Mitochondria are organelles that allow for oxidative phosphorylation in eukaryotic cells. They contain their own DNA, however,

proteins in the mitochondria are encoded by both mitochondrial and cellular DNA. Mito chondria also are important in specialized

cellular signaling.

■ Lysosomes and peroxisomes are membrane-bound organelles that contribute to protein and lipid processing. They do this in part by

creating acidic (lysosomes) or oxidative (peroxisomes) contents relative to the cell cytosol.

■ The cytoskeleton is a network of three types of filaments that provide struc tural integrity to the cell as well as a means for trafficking of

organelles and other structures. Actin is the fundamental building block for thin filamen ts and represents as much as 15% of cellular

protein. Actin filaments are important in cellular contraction, migration, and signaling. A ctin filaments also provide the backbone for

muscle contraction. Intermediate filaments are primarily structural. Proteins that make up intermediate filaments are cell-type specific.

Microtubules are made up of tubulin subunits. Microtubules provide a dynamic structure in cells tha t allows for movement of cellular

components around the cell.

■ There are three superfamilies of molecular motor proteins in the cell that use th e energy of ATP to generate force, movement, or both.

Myosin is the force generator for muscle cell contraction. There are also cellular myosins th at interact with the cytoskeleton (primarily

thin filaments) to participate in contraction as well as movement of cell contents. Kinesins and cellular dyneins are motor proteins that

primarily interact with microtubules to move cargo around the cells.

■ Cellular adhesion molecules aid in tethering cells to each other or to the extrace llular matrix as well as providing for initiation of

cellular signaling. There are four main families of these proteins: integrins, immunoglobu lins, cadherins, and selectins.

■ Cells contain distinct protein complexes that serve as cellular connections to other cells or the extracellular matrix. Tight junctions

provide intercellular connections that link cells into a regulated tissue barrier. T ight junctions also provide a barrier to movement of

proteins in the cell membrane and thus, are important to cellular polarization. Gap junctio ns provide contacts between cells that allow for

direct passage of small molecules between two cells. Desmosomes and adherens junction s are specialized structures that hold cells

together. Hemidesmosomes and focal adhesions attach cells to their basal lamina.

■ The nucleus is an organelle that contains the cellular DNA and is the site of transcription. T here are several organelles that emanate

from the nucleus, including the endoplasmic reticulum and the Golgi apparatus. These two organe lles are important in protein processing

and the targeting of proteins to correct compartments within the cell.

■ Exocytosis and endocytosis are vesicular fusion events that allow for movement of proteins and lipids between the cell interior, the

plasma membrane, and the cell exterior. Exocytosis can be constitutive or non constitutive; both are regulated processes that require

specialized proteins for vesicular fusion. Endocytosis is the formation of vesicles at the p lasma membrane to take material from the

extracellular space into the cell interior. Some endocytoses are defined in part by the size of the vesicles formed whereas others are

defined by membrane structures that contribute to the endocytosis. All are tightly regula ted processes.

■ Membranes contain a variety of proteins and protein complexes that allow for transport of small molecules. Aqueous ion channels are

membrane-spanning proteins that can be gated open to allow for selective diffusion of ions acros s membranes and down their

electrochemical gradient. Carrier proteins bind to small molecules and undergo confo rmational changes to deliver small molecules across

the membrane. This facilitated transport can be passive or active. Active transport requires ener gy for transport and is typically provided

by ATP hydrolysis.

■ Cells can communicate with one another via chemical messengers. Individual mess engers (or ligands) typically bind to a plasma

membrane receptor to initiate intracellular changes that lead to physiologic changes. Plas ma membrane receptor families include ion

channels, G protein-coupled receptors, or a variety of enzyme-linked receptors (eg, tyrosine kin ase receptors). There are additional

cytosolic receptors (eg, steroid receptors) that can bind membrane-permeant compounds. Act ivation of receptors lead to cellular changes

that include changes in membrane potential, activation of heterotrimeric G proteins, incre ase in second messenger molecules, or initiation

of transcription.

■ Second messengers are molecules that undergo a rapid concentration changes in th e cell following primary messenger recognition.

Common second messenger molecules include Ca

, cyclic adenosine monophosphate (cAMP), cyclic guanine monophosphate (c GMP),

inositol trisphosphate (IP

) and nitric oxide (NO).

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. The electrog enic Na, K ATPase plays a critical role in

cellular physiology by

A) using the energy in ATP to extrude 3 Na

out of the cell in

exchange for taking two K

into the cell.

B) using the energy in ATP to extrude 3 K

out of the cell in exchange for taking two Na

into the cell.

C) using the energy in moving Na

into the cell or K

outside

the cell to make ATP. +

outside of the cell or K

D) using the energy in moving Na

inside the cell to make ATP.

2. Cell membranes

A) contain relatively few protein molecules.

B) contain many carbohydrate molecules.

C) are freely permeable to electrolytes but not to proteins. D) have variable protein and lipid contents depending on their

location in the cell.

E) have a stable composition throughout the life of the cell. 3. Second messengers

A) are substances that interact with first messengers outside cells.

B) are substances that bind to first messengers in the cell membrane.

C) are hormones secreted by cells in response to stimulation by another hormone.

D) mediate the intracellular responses to many different hormones and neurotransmitter s.

E) are not formed in the brain.

4. The Golgi complex

A) is an organelle that participates in the breakdown of proteins and lipids.

B) is an organelle that participates in posttranslational processing of proteins.

C) is an organelle that participates in energy production.

D) is an organelle that participates in transcription and translation.

E) is a subcellular compartment that stores proteins for trafficking to the nucleus.

5. Endocytosis

A) includes phagocytosis and pinocytosis, but not clathrinmediated or caveolae-depend ent uptake of extracellular contents.

B) refers to the merging of an intracellular vesicle with the plasma membrane to deliver intracellular contents to the extracellular milieu.

C) refers to the invagination of the plasma membrane to uptake extracellular contents in to the cell.

D) refers to vesicular trafficking between Golgi stacks. 6. G protein-coupled receptors

A) are intracellular membrane proteins that help to regulate movement within the cell.

B) are plasma membrane proteins that couple the extracellular binding of primary s ignaling molecules to activation of small G proteins.

C) are plasma membrane proteins that couple the extracellular binding of primary s ignaling molecules to the activation of heterotrimeric

G proteins.

D) are intracellular proteins that couple the binding of primary messenger molecules with transcription.

7. Gap junctions are intercellular connections that

A) primarily serve to keep cells separated and allow for transport across a tissue barrier .

B) serve as a regulated cytoplasmic bridge for sharing of small molecules between cells.

C) serve as a barrier to prevent protein movement within the cellular membrane.

D) are cellular components for constitutive exocytosis that occurs between adjacent cells .

CHAPTER RESOURCES

Alberts B et al: Molecular Biology of the Cell, 5th ed. Garland Science, 2007.

Cannon WB: The Wisdom of the Body. Norton, 1932.

Junqueira LC, Carneiro J, Kelley RO: Basic Histology, 9th ed. McGraw-Hill, 1998.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural Science, 4th ed. Mc Graw-Hill, 2000.

Pollard TD, Earnshaw WC: Cell Biology, 2nd ed. Saunders, Elsevier, 2008.

Sperelakis N (editor): Cell Physiology Sourcebook, 3rd ed. Academic Press, 2001 .

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Immunity, Infection, & Inflammation

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Understand the significance of immunity, particularly with respect to defending

■

the body against microbial invaders.

■

Define the circulating and tissue cell types that contribute to immune and inflammatory r esponses.

■

Describe how phagocytes are able to kill internalized bacteria.

■

Identify the functions of hematopoietic growth factors, cytokines, and chemokines.

■

Delineate the roles and mechanisms of innate, acquired, humoral, and cellular immunity .

■

Understand the basis of inflammatory responses and wound healing.

INTRODUCTION

As an open system, the body is continuously called upon to defend itself from potentiall y harmful invaders such as bacteria, viruses, and

other microbes. This is accomplished by the immune system, which is subdivided into inn ate and adaptive (or acquired) branches. The

immune system is composed of specialized effector cells that sense and respond to forei gn antigens and other molecular patterns not

found in human tissues. Likewise, the immune system clears the body’s own cells that ha ve become senescent or abnormal, such as

cancer cells. Finally, occasionally, normal host tissues become the subject of inappropria te immune attack, such as in autoimmune

diseases or in settings where normal cells are harmed as innocent bystanders when the i mmune system mounts an inflammatory response

to an invader. It is beyond the scope of this volume to provide a full treatment of all asp ects of modern immunology. Nevertheless, the

student of physiology should have a working knowledge of immune functions and the ir regulation, due to a growing appreciation for the

ways in which the immune system can contribute to normal physiological regulation in a variety of tissues, as well as contributions of

immune effectors to pathophysiology.

IMMUNE EFFECTOR CELLS

Many immune effector cells circulate in the blood as the white blood cells. In addition, th e blood is the conduit for the precursor cells

that eventually develop into the immune cells of the tissues. The circulating immunologic cells include granulocytes

(polymorphonuclear leukocytes, PMNs), comprising neutrophils, eosinophils, and basop hils; lymphocytes; and

monocytes. Immune responses in the tissues are further amplified by these cells follow ing their extravascular migration, as well as tissue

macrophages (derived from monocytes) and mast cells (related to basophils). Act ing together, these cells provide the body with

powerful defenses against tumors and viral, bacterial, and parasitic infections.

GRANULOCYTES

All granulocytes have cytoplasmic granules that contain biologically active substances inv olved in inflammatory and allergic reactions.

The average half-life of a neutrophil in the circulation is 6 hours. To maintain the norm al circulating blood level, it is therefore necessary

to produce over 100 billion neutrophils per day. Many neutrophils enter the tissues, par ticularly if triggered to do so by an infection or by

inflammatory cytokines. They are attracted to the endothelial surface by cell adhesion m olecules known as selectins, and they roll along

it. They then bind firmly to neutrophil adhesion molecules of the integrin family. They n ext insinuate themselves through the walls of

the capillaries between endothelial cells by a process called diapedesis. Many of those that le ave the circulation enter the gastrointestinal

tract and are eventually lost from the body.

Invasion of the body by bacteria triggers the inflammatory response. The bo ne marrow is stimulated to produce and release large

numbers of neutrophils. Bacterial products interact with plasma factors and cells to prod uce agents that attract neutrophils to the infected

area (chemotaxis). The chemotactic agents, which are part of a large and expa nding family of chemokines (see following text), include

a component of the complement system (C5a); leukotrienes; and polypeptides from lym phocytes, mast cells, and basophils. Other plasma

factors act on the bacteria to make them “tasty” to the phagocytes (opsonizatio n). The principal opsonins that coat the bacteria are

immunoglobulins of a particular class (IgG) and complement proteins (see following tex t). The coated bacteria then bind to receptors on

the neutrophil cell membrane. This triggers, via heterotrimeric G protein-mediated respo nses, increased motor activity of the cell,

exocytosis, and the socalled respiratory burst. The increased motor activity leads to prom pt ingestion of the bacteria by endocytosis

(phagocytosis). By exocytosis, neutrophil granules discharge their contents into th e phagocytic vacuoles containing the bacteria and also

into the interstitial space (degranulation). The granules contain various pr oteases plus antimicrobial proteins called defensins. In

addition, the cell membrane-bound enzyme NADPH oxidase is act ivated, with the production of toxic oxygen metabolites. The

combination of the toxic oxygen metabolites and the proteolytic enzymes from the gran ules makes the neutrophil a very effective killing

machine.

Activation of NADPH oxidase is associated with a sharp increase in O

uptake and metabolism in the neutrophil (the respiratory burst)

and generation of O2– by the following reaction:

NADPH + H

+ 2O

+ → NADP

+ 2H

+ 2O2– O2– is a free radical formed by the addition of one electron to O

. Two O2– react

with two H

to form H

in a reaction catalyzed by the cytoplasmic form of superoxide dismutase (SOD-1):

O 2–+ O2– + H

+ H

+ SOD-1

→ H

+ O

O2– and H

are both oxidants that are effective bactericidal agents, but H

converted to H

O and O

by the enzyme catalase. The cytoplasmic form of SOD contains both Zn a nd Cu. It is found in many parts of

the body. It is defective as a result of genetic mutation in a familial form of amyotrophic lateral sclerosis (ALS; see Chapter 19).

Therefore, it may be that O2– accumulates in motor neurons and kills them in at least on e form of this progressive, fatal disease. Two

other forms of SOD encoded by at least one different gene are also found in humans.

Neutrophils also discharge the enzyme myeloperoxidase, which catalyzes the conversion of Cl

–

, Br

–

, I

–

, and SCN

–

to the corresponding

acids (HOCl, HOBr, etc). These acids are also potent oxidants. Because Cl

–

is present in greatest abundance in body fluids, the principal

product is HOCl.

In addition to myeloperoxidase and defensins, neutrophil granules contain an elastase, t wo metalloproteinases that attack collagen, and a

variety of other proteases that help destroy invading organisms. These enzymes act in a cooperative fashion with the O2–, H

, and

HOCl formed by the action of the NADPH oxidase and myeloperoxidase to produce a killing zone around the activated neutrophil. This

zone is effective in killing invading organisms, but in certain diseases (eg, rheumatoid ar thritis) the neutrophils may also cause local

destruction of host tissue.

The movements of the cell in phagocytosis, as well as migration to the site of infection, in volve microtubules and microfilaments (see

Chapter 1). Proper function of the microfilaments involves the interaction of the actin th ey contain with myosin-1 on the inside of the

cell membrane (see Chapter 1).

Like neutrophils, eosinophils have a short half-life in the circulation, are attracted to the surface of endothelial cells by selectins, bind to

integrins that attach them to the vessel wall, and enter the tissues by diapedesis. Like neutr ophils, they release proteins, cytokines, and

chemokines that produce inflammation but are capable of killing invading organisms. H owever, eosinophils have some selectivity in the

way in which they respond and in the killing molecules they secrete. Their maturation an d activation in tissues is particularly stimulated

by IL-3, IL-5, and GM-CSF (see below). They are especially abundant in the mucosa of the gastrointestinal tract, where they defend

against parasites, and in the mucosa of the respiratory and urinary tracts. Circulating eo sinophils are increased in allergic diseases such as

asthma and in various other respiratory and gastrointestinal diseases.

Basophils also enter tissues and release proteins and cytokines. They resemble but a re not identical to mast cells, and like mast cells they

contain histamine (see below). They release histamine and other inflammatory mediators when activated by binding of specific antigens

to cell-fixed IgE molecules, and are essential for immediate-type hypersensitivity reaction s. These range from mild urticaria and rhinitis

to severe anaphylactic shock. The antigens that trigger IgE formation and basophil (and mast cell) activation are innocuous to most

individuals, and are referred to as allergens.

MAST CELLS

Mast cells are heavily granulated cells of the connective tissue that are abundant in tiss ues that come into contact with the external

environment, such as beneath epithelial surfaces. Their granules contain proteoglycans, histamine, and many proteases. Like basophils,

they degranulate when allergens bind to IgE molecules directed against them that previo usly coat the mast cell surface. They are

involved in inflammatory responses initiated by immunoglobulins IgE and IgG (see belo w). The inflammation combats invading

parasites. In addition to this involvement in acquired immunity, they release TNFα in resp onse to bacterial products by an antibody-

independent mechanism, thus participating in the nonspecific innate immunity that combats infections prior to the development of an

adaptive immune response (see following text). Marked mast cell degranulation produc es clinical manifestations of allergy up to and

including anaphylaxis.

MONOCYTES

Monocytes enter the blood from the bone marrow and circulate for about 72 hours. T hey then enter the tissues and become tissue

macrophages (Figure 3–1). Their life span in the tissues is unknown, but bone marrow transplantation data in humans suggest that they

persist for about 3 months. It appears that they do not reenter the circulation. Some of th em end up as the multinucleated giant cells seen

in chronic inflammatory diseases such as tuberculosis. The tissue macrophages include t he Kupffer cells of the liver, pulmonary alveolar

macrophages (see Chapter 35), and microglia in the brain, all of which come from the circulation. In the past, they have been called the

reticuloendothelial system, but the general term tissue macrophage system seems more appropriate.

Macrophages are activated by cytokines released from T lymphocytes, among others. A ctivated macrophages migrate in response to

chemotactic stimuli and engulf and kill bacte

Macrophages

Pseudopods

Bacteria

FIGURE 3–1 Macrophages contacting bacteria and preparing to engulf them. Fig ure is a colorized version of a scanning electron

micrograph.

ria by processes generally similar to those occurring in neutrophils. They play a key ro le in immunity (see below). They also secrete up to

100 different substances, including factors that affect lymphocytes and other cells, pros taglandins of the E series, and clot-promoting

factors.

GRANULOCYTE & MACROPHAGE COLONY-STIMULATING FACTORS

The production of white blood cells is regulated with great precision in healthy individua ls, and the production of granulocytes is rapidly

and dramatically increased in infections. The proliferation and self-renewal of hematop oietic stem cells (HSCs) depends on stem cell

factor (SCF). Other factors specify particular lineages. The proliferation and ma turation of the cells that enter the blood from the

marrow are regulated by glycoprotein growth factors or hormones that cause cells in o ne or more of the committed cell lines to

proliferate and mature (Table 3–1). The regulation of erythrocyte production by erythropo ietin is discussed in Chapter 39. Three

additional factors are called colony-stimulating factors (CSFs), because they cause appropr iate single stem cells to proliferate in soft

agar, forming colonies in this culture medium. The factors stimulating the production of committed stem cells include granulocyte–

macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), and macrophage CSF (M -CSF). Interleukins IL-1 and IL-6 followed by

IL-3 (Table 3–1) act in sequence to convert pluripotential uncommitted stem cells to committe d progenitor cells. IL-3 is also known as

multi-CSF. Each of the CSFs has a predominant action, but all the CSFs and interleukin s also have other overlapping actions. In

addition, they activate and sustain mature blood cells. It is interesting in this regard that th e genes for many of these factors are located

together on the long arm of chromosome 5 and may have originated by duplication of an ancestral gene. It is also interesting that basal

hematopoiesis is normal in mice in which the GMCSF gene is knocked out, indicating tha t loss of one factor can be compensated for by

others. On the other hand, the absence of GM-CSF causes accumulation of surfactant i n the lungs (see Chapter 35).

As noted in Chapter 39, erythropoietin is produced in part by kidney cells and is a circu lating hormone. The other factors are produced

by macrophages, activated T cells, fibroblasts, and endothelial cells. For the most part, th e factors act locally in the bone marrow

(Clinical Box 3–1).

LYMPHOCYTES

Lymphocytes are key elements in the production of immunity (see below). After birth, some lymphocytes are formed in the bone

marrow. However, most are formed in the lymph nodes (Figure 3–2), thymus, and sp leen from precursor cells that originally came from

the bone marrow and were processed in the thymus or bursal equivalent (see below). Lymphocytes enter the bloodstream for the most

part via the lymphatics. At

TABLE 3–1 Hematopoietic growth factors.

Cytokine IL-1

IL-3

IL-4

IL-5

IL-6

IL-11

Erythropoietin

Cell Lines

Stimulated

Erythrocyte

Granulocyte Megakaryocyte Monocyte

Erythrocyte

Granulocyte Megakaryocyte Monocyte

Basophil

Eosinophil

Erythrocyte

Granulocyte Megakaryocyte Monocyte

Erythrocyte

Granulocyte Megakaryocyte Erythrocyte

SCF

G-CSF Erythrocyte

Granulocyte Megakaryocyte Monocyte

Granulocyte

GM-CSF Erythrocyte

M-CSF Granulocyte Megakaryocyte Monocyte

Thrombopoietin Megakaryocyte

Cytokine Source Multiple cell types

T lymphocytes

T lymphocytes T lymphocytes Endothelial cells

Fibroblasts Macrophages Fibroblasts Osteoblasts

Kidney

Kupffer cells of liver Multiple cell types

Endothelial cells Fibroblasts

Monocytes

Endothelial cells Fibroblasts

Monocytes

T lymphocytes Endothelial cells Fibroblasts

Monocytes

Liver, kidney

any given time, only about 2% of the body lymphocytes are in the peripheral blood. M ost of the rest are in the lymphoid organs. It has

been calculated that in humans, 3.5 × 10

lymphocytes per day enter the circulation via the thoracic duct alone; however, this cou nt

includes cells that reenter the lymphatics and thus traverse the thoracic duct more than on ce. The effects of adrenocortical hormones on

the lymphoid organs, the circulating lymphocytes, and the granulocytes are discussed in Chapter 22.

Cortical follicles, B cells

Key: IL = interleukin; CSF = colony stimulating factor; G = granulocyte; M = macrophage; SCF = stem cel l factor.

Reproduced with permission from McPhee SJ, Lingappa VR, Ganong WF (editors): Pathophysio logy of Disease, 4th ed. McGraw-Hill, 2003.

CLINICAL BOX 3–1 Disorders of Phagocytic Function

More than 15 primary defects in neutrophil function have been described, along with a t least 30 other conditions in which there is a

secondary depression of the function of neutrophils. Patients with these diseases are pro ne to infections that are relatively mild when only

the neutrophil system is involved, but which can be severe when the monocyte-tissue m acrophage system is also involved. In one

syndrome (neutrophil hypomotility), actin in the neutrophils does not polymerize norma lly, and the neutrophils move slowly. In another,

there is a congenital deficiency of leukocyte integrins. In a more serious disease (chron ic granulomatous disease), there is a failure to

generate O2– in both neutrophils and monocytes and consequent inability to kill m any phagocytosed bacteria. In severe congenital

glucose 6-phosphate dehydrogenase deficiency, there are multiple infections because o f failure to generate the NADPH necessary for

O2– production. In congenital myeloperoxidase deficiency, microbial killing p ower is reduced because hypochlorous acid is not formed.

Paracortex, T cells

Medullary cords, plasma cells

FIGURE 3–2 Anatomy of a normal lymph node. (After Chandrasoma. Rep roduced with permission from McPhee SJ, Lingappa VR, Ganong WF [editors]:

Pathophysiology of Disease, 4th ed. McGraw-Hill, 2003.)

IMMUNITY

OVERVIEW

Insects and other invertebrates have only innate immunity. T his system is triggered by receptors that bind sequences of sugars, fats, or

amino acids in common bacteria and activate various defense mechanisms. The receptor s are coded in the germ line, and their

fundamental structure is not modified by exposure to antigen. The activated defenses in clude, in various species, release of interferons,

phagocytosis, production of antibacterial peptides, activation of the complement system, and several proteolytic cascades. Even plants

release antibacterial peptides in response to infection. In vertebrates, innate immunity is a lso present, but is complemented by adaptive

or acquired immunity, a system in which T and B lymphocytes are activated by very specific antigens. In both innate and acquired

immunity, the receptors involved recognize the shape of antigens, not their specific chem ical composition. In acquired immunity,

activated B lymphocytes form clones that produce more antibodies which attack foreign proteins. After the invasion is repelled, small

numbers persist as memory cells so that a second exposure to the same antigen provoke s a prompt and magnified immune attack. The

genetic event that led to acquired immunity occurred 450 million years ago in the ancest ors of jawed vertebrates and was probably

insertion of a transposon into the genome in a way that made possible the generation of the immense repertoire of T cell receptors that

are present in the body.

In vertebrates, including humans, innate immunity provides the first line of defense aga inst infections, but it also triggers the slower but

more specific acquired immune response (Figure 3–3). In vertebrates, natural and acq uired immune mechanisms also attack tumors and

tissue transplanted from other animals.

Once activated, immune cells communicate by means of cytokines and chemokines. The y kill viruses, bacteria, and other foreign cells by

secreting other cytokines and activating the complement system.

CYTOKINES

Cytokines are hormonelike molecules that act—generally in a paracrine fashion—to reg ulate immune responses. They are secreted not

only by lymphocytes and macrophages but by endothelial cells, neurons, glial cells, and other types of cells. Most of the cytokines were

initially named for their actions, for example, B cell-differentiating factor, B cell-stimulat ing factor 2. However, the nomenclature has

since been rationalized by international agreement to that of the interleukins. For exa mple, the name of B cell-differentiating factor was

changed to interleukin-4. A number of cytokines selected for their biological and clinica l relevance are listed in Table 3–2, but it would

be beyond the scope of this text to list all cytokines, which now number more than 100.

Many of the receptors for cytokines and hematopoietic growth factors (see above), as well as the receptors for prolactin

Plasma cell

γδ

T cell Chemokines T

N M IL-4

Bacteria Viruses Tumors Naive T cell

APC

1 Cytotoxic

lymphocyte

FIGURE 3–3 How bacteria, viruses, and tumors trigger innate immunity and initiate the acquired immune response. Arrows

indicate mediators/cytokines that act on the target cell shown and/or pathways o f differentiation. APC, antigen-presenting cell; M,

monocyte; N, neutrophil; T

1 and T

2, helper T cells type 1 and type 2, respectively.

TABLE 3–2 Examples of cytokines and their clinical relevance.

Cytokine Cellular Sources Major Activities Clinical Relevance

Interleukin-1 Macrophages

Activation of T cells and macrophages; promotion of inflammation

Implicated in the pathogenesis of septic shock, rheumatoid arthritis, and atherosclero sis

Interleukin-2 Type 1 (TH1) helper T cells

Activation of lymphocytes, natural killer cells, and macrophages

Used to induce lymphokine-activated killer cells; used in the treatment of metastatic r enal-cell carcinoma, melanoma, and various other

tumors

Interleukin-4 Type 2 (TH2) helper T cells,

mast cells, basophils, and eosinophils

Activation of lymphocytes, monocytes, and IgE class switching

As a result of its ability to stimulate IgE production, plays a part in mast-cell sensitization an d thus in allergy and in defense against

nematode infections

Interleukin-5 Type 2 (TH2) helper T cells, mast cells, and eosinophils Differentiation of eosinophils

Monoclonal antibody against interleukin-5 used to inhibit the antigen-induced late-ph ase eosinophilia in animal models of allergy

Interleukin-6 Type 2 (TH2) helper T cells

and macrophages

Activation of lymphocytes; differentiation of B cells; stimulation of the production of acu te-phase proteins

Overproduced in Castleman’s disease; acts as an autocrine growth factor in myelom a and in mesangial proliferative glomerulonephritis

Interleukin-8 T cells and macrophages

Chemotaxis of neutrophils, basophils, and T cells

Levels are increased in diseases accompanied by neutrophilia, making it a potent ially useful marker of disease activity

Interleukin-11 Bone marrow stromal cells

Stimulation of the production of acutephase proteins

Used to reduce chemotherapy-induced thrombocytopenia in patients with cancer

Interleukin-12 Macrophages and B cells

Stimulation of the production of interferon γ by type 1 (TH1) helper T cells and by natural killer cells ; induction of type 1 (TH1) helper T

cells

May be useful as an adjuvant for vaccines

Tumor necrosis factor α

Macrophages, natural killer cells, T cells, B cells, and mast cells

Promotion of inflammation Treatment with antibodies against tumor necrosis facto r α beneficial in rheumatoid arthritis

Lymphotoxin (tumor necrosis factor β) Type 1 (TH1) helper T cells and B cells

Promotion of inflammation Implicated in the pathogenesis of multiple sclerosis and insulin -dependent diabetes mellitus

Transforming growth factor β

T cells, macrophages, B cells, and mast cells

Immunosuppression May be useful therapeutic agent in multiple sclerosis and myasthen ia gravis

Granulocyte

macrophage colonystimulating factor T cells, macrophages, natural killer cells, and B ce lls Promotion of the growth of granulocytes and

monocytes

Used to reduce neutropenia after chemotherapy for tumors and in ganciclovir-treated patien ts with AIDS; used to stimulate cell

production after bone marrow transplantation

Interferonα Virally infected cells

Induction of resistance of cells to viral infection

Used to treat AIDS-related Kaposi sarcoma, melanoma, chronic hepatitis B infection, an d chronic hepatitis C infection

Interferonβ Virally infected cells

Induction of resistance of cells to viral infection

Used to reduce the frequency and severity of relapses in multiple sclerosis

Interferonγ Type 1 (TH1) helper T cells

and natural killer cells Activation of macrophages; inhibition of type 2 (TH2) helper T cells

Used to enhance the killing of phagocytosed bacteria in chronic granulomatous disease

Reproduced with permission from Delves PJ, Roitt IM: The immune system. First of two parts. N Engl J Med 2000;343:37.

(see Chapter 25), and growth hormone (see Chapter 24) are members of a cytokine-r eceptor superfamily that has three subfamilies

(Figure 3–4). The members of subfamily 1, which includes the receptors for IL-4 and IL-7, are homodimers. The members of subfamily

2, which includes the receptors for IL-3, IL-5, and IL-6, are heterodimers. The recep tor for IL-2 and several other cytokines is unique in

that it consists of a heterodimer plus an unrelated protein, the so-called Tac antigen. The other members of subfamily 3 have the same γ

chain as IL2R. The extracellular domain of the homodimer and heterodimer subunits al l contain four conserved cysteine residues plus a

conserved Trp-Ser-X-Trp-Ser domain, and although the

Erythropoietin G-CSF

IL-4

IL-7

Growth hormone PRL

IL-3

Sharedβ

IL-2 GM-CSF

subunit

IL-4 IL-5 IL-7 IL-6

IL-9

IL-11

Shared gp130

IL-15 LIF

subunit

OSM

CNTF

ECF

Cytoplasm

Subfamily 1 β β Subfamily 2 Subfamily 3

FIGURE 3–4 Members of one of the cytokine receptor superfamilies, showing shared structural elements. Note that all the subunits

except the

subunit in subfamily 3 have four conserved cysteine residues (open boxes at top) and a Trp-Ser-X-Trp-Ser motif (pink).

Many subunits also contain a critical regulatory domain in their cytoplasmic port ions (green). CNTF, ciliary neurotrophic factor; LIF,

leukemia inhibitory factor; OSM, oncostatin M; PRL, prolactin. (Modified from D’Andrea AD: Cytokine rec eptors in congenital hematopoietic disease.

N Engl J Med 1994;330:839.)

intracellular portions do not contain tyrosine kinase catalytic domains, they activate cytop lasmic tyrosine kinases when ligand binds to

the receptors.

The effects of the principal cytokines are listed in Table 3–2. Some of them have system ic as well as local paracrine effects. For example,

IL-1, IL-6, and tumor necrosis factor α cause fever, and IL-1 increases slow-wave sle ep and reduces appetite.

Another superfamily of cytokines is the chemokine family. Chemokines are substances that attract neutrophils (see previous text) and

other white blood cells to areas of inflammation or immune response. Over 40 have no w been identified, and it is clear that they also play

a role in the regulation of cell growth and angiogenesis. The chemokine receptors are G protein-coupled receptors that cause, among

other things, extension of pseudopodia with migration of the cell toward the source of t he chemokine.

THE COMPLEMENT SYSTEM

The cell-killing effects of innate and acquired immunity are mediated in part by a system of more than 30 plasma proteins originally

named the complement system because they “complemented” the effects of antibodies. Three different pathways or enzyme cascades

activate the system: the classic pathway, triggered by immune complexes; the mannose-binding lectin pathway, triggered when this

lectin binds mannose groups in bacteria; and the alternative or properdin pathw ay, triggered by contact with various viruses, bacteria,

fungi, and tumor cells. The proteins that are produced have three functions: They help kill invading organisms by opsonization,

chemotaxis, and eventual lysis of the cells; they serve in part as a bridge from innate to a cquired immunity by activating B cells and

aiding immune memory; and they help dispose of waste products after apoptosis. Cell ly sis, one of the principal ways the complement

system kills cells, is brought about by inserting proteins called perforins into their cell membranes. These create holes, which permit

free flow of ions and thus disruption of membrane polarity.

INNATE IMMUNITY

The cells that mediate innate immunity include neutrophils, macrophages, and natural killer (NK) cells, large lymphocytes that are not

T cells but are cytotoxic. All these cells respond to lipid and carbohydrate sequences un ique to bacterial cell walls and to other substances

characteristic of tumor and transplant cells. Many cells that are not professional immuno cytes may nevertheless also contribute to innate

immune responses, such as endothelial and epithelial cells. The activated cells produce th eir effects via the release of cytokines, as well

as, in some cases, complement and other systems.

An important link in innate immunity in Drosophila is a receptor protein name d toll, which binds fungal antigens and triggers activation

of genes coding for antifungal proteins. An expanding list of toll-like receptors (TLRs) have now been identified in humans. One of

these, TLR4, binds bacterial lipopolysaccharide and a protein called CD14, and this initia tes a cascade of intracellular events that activate

transcription of genes for a variety of proteins involved in innate immune responses. Th is is important because bacterial

lipopolysaccharide produced by gram-negative organisms is the cause of septic shock. TLR2 mediates the response to microbial

lipoproteins, TLR6 cooperates with TLR2 in recognizing certain peptidoglycans, and TL R9 recognizes the DNA of certain bacteria.

ACQUIRED IMMUNITY

As noted previously, the key to acquired immunity is the ability of lymphocytes to produ ce antibodies (in the case of B cells) or cell-

surface receptors (in the case of T cells) that are specific for one of the many millions o f foreign agents that may invade the body. The

antigens stimulating production of T cell receptors or antibodies are usually proteins and polypeptides, but antibodies can also be formed

against nucleic acids and lipids if these are presented as nucleoproteins and lipoproteins, and antibodies to smaller molecules can be

produced experimentally if the molecules are bound to protein. Acquired immunity has two components: humoral immunity and cellular

immunity. Humoral immunity is mediated by circulating immunoglobulin antibodies in th e γ-globulin fraction of the plasma proteins.

Immunoglobulins are produced by differentiated forms of B lymphocytes known as plasma cells, and they activate the complement

system and attack and neutralize antigens. Humoral immunity is a major defense against bacterial infections. Cellular immunity is

mediated by T lymphocytes. It is responsible for delayed allergic reactions and rejection of transplants of foreign tissue. Cytotoxic T

cells attack and destroy cells that have the antigen which activated them. They kill by inse rting perforins (see above) and by initiating

apoptosis. Cellular immunity constitutes a major defense against infections due to viruses , fungi, and a few bacteria such as the tubercle

bacillus. It also helps defend against tumors.

DEVELOPMENT OF THE IMMUNE SYSTEM

During fetal development, and to a much lesser extent during adult life, lymphocyte pre cursors come from the bone marrow. Those that

populate the thymus (Figure 3–5) become transformed by the environment in this orga n into T lymphocytes. In birds, the precursors that

populate the bursa of Fabricius, a lymphoid structure near the cloaca, become transform ed into B lymphocytes. There is no bursa in

mammals, and the transformation to B lymphocytes occurs in bursal equivalents, t hat is, the fetal liver and, after birth, the bone

marrow. After residence in the thymus or liver, many of the T and B lymphocytes migr ate to the lymph nodes.

T and B lymphocytes are morphologically indistinguishable but can be identified by mar kers on their cell membranes. B cells

differentiate into plasma cells and memory B cells. There are three major types of T ce lls: cytotoxic T cells, helper T cells, and

memory T cells. There are two subtypes of helper T cells: T helper 1 (TH1) cells secrete IL-2 and γ- interferon and are concerned

primarily with cellular immunity; T helper 2 (TH2) cells secrete IL-4 and IL-5 and inter act primarily with B cells in relation to humoral

immunity. Cytotoxic T cells destroy transplanted and other foreign cells, with their devel opment aided and directed by helper T cells.

Markers on the surface of lymphocytes are assigned CD (clusters of differentiation) n umbers on the basis of their reactions to a

Memory T cells

Thymus T lymphocytes

Cytotoxic T cells

(mostly CD8 T cells) Cellular immunity

Bone marrow lymphocyte precursors Helper T cells (CD4 T cells)

B lymphocytes Bursal equivalent (liver, bone marrow) Plasma cells

IgG

IgA HumoralIgM

immunity

IgD

IgE

Memory B cells

FIGURE 3–5 Development of the system mediating acquired immunity.

panel of monoclonal antibodies. Most cytotoxic T cells display the glycoprotein CD8, an d helper T cells display the glycoprotein CD4.

These proteins are closely associated with the T cell receptors and may function as core ceptors. On the basis of differences in their

receptors and functions, cytotoxic T cells are divided into αβ and γδ types (see below). Natural killer cells (see above) are also cytotoxic

lymphocytes, though they are not T cells. Thus, there are three main types of cytotoxic lymphocytes in the body: αβ T cells, γδ T cells,

and NK cells.

MEMORY B CELLS & T CELLS

After exposure to a given antigen, a small number of activated B and T cells persist as m emory B and T cells. These cells are readily

converted to effector cells by a later encounter with the same antigen. This ability to pro duce an accelerated response to a second

exposure to an antigen is a key characteristic of acquired immunity. The ability persists f or long periods of time, and in some instances

(eg, immunity to measles) it can be lifelong.

After activation in lymph nodes, lymphocytes disperse widely throughout the body and are especially plentiful in areas where invading

organisms enter the body, for example, the mucosa of the respiratory and gastrointestin al tracts. This puts memory cells close to sites of

reinfection and may account in part for the rapidity and strength of their response. Che mokines are involved in guiding activated

lymphocytes to these locations.

Langerhans dendritic cells in the skin. Macrophages and B cells themselves, and likely m any other cell types, can also function as APCs.

In APCs, polypeptide products of antigen digestion are coupled to protein products of the major histocompatibility complex (MHC)

genes and presented on the surface of the cell. The products of the MHC genes are ca lled human leukocyte antigens (HLA).

The genes of the MHC, which are located on the short arm of human chromosome 6, encode glycoproteins and are divided into two

classes on the basis of structure and function. Class I antigens are composed of a 45-kD a heavy chain associated noncovalently with β

microglobulin encoded by a gene outside the MHC (Figure 3–6). They are found on a ll nucleated cells. Class II antigens are

heterodimers made up of a 29- to 34-kDa αα to 28-kDa β chain. They are present in a ntigen-presenting cells, including B cells, and in

activated T cells.

The class I MHC proteins (MHC-I proteins) are coupled primarily to peptide fragment s generated from proteins synthesized within cells.

The peptides to which the host is not tolerant (eg, those from mutant or viral proteins) ar e recognized by T cells. The digestion of these

proteins occurs in

ANTIGEN RECOGNITION

The number of different antigens recognized by lymphocytes in the body is extremely large. The repertoire develops initially without

exposure to the antigen. Stem cells differentiate into many million different T and B lymp hocytes, each with the ability to respond to a

particular antigen. When the antigen first enters the body, it can bind directly to the appr opriate receptors on B cells. However, a full

antibody response requires that the B cells contact helper T cells. In the case of T cells, t he antigen is taken up by an antigen-presenting

cell and partially digested. A peptide fragment of it is presented to the appropriate recep tors on T cells. In either case, the cells are

stimulated to divide, forming clones of cells that respond to this antigen (clonal selection). Ef fector cells are also subject to negative

selection, during which lymphocyte precursors that are reactive with self antige ns are normally deleted. This results in immune

tolerance. It is this latter process that presumably goes awry in autoimmune diseases, wh ere the body reacts to and destroys cells

expressing normal proteins, with accompanying inflammation that may lead to tissue des truction.

ANTIGEN PRESENTATION

Antigen-presenting cells (APCs) include specialized cells called dendritic cells in the lymph node s and spleen and the

C C

proteasomes, complexes of proteolytic enzymes that may be produced by genes in the MH C group, and the peptide fragments appear

to bind to MHC proteins in the endoplasmic reticulum. The class II MHC proteins (MH C-II proteins) are concerned primarily with

peptide products of extracellular antigens, such as bacteria, that enter the cell by endocy tosis and are digested in the late endosomes.

FIGURE 3–6 Structure of human histocompatibility antigen HLA-A2. T he antigen-binding pocket is at the top and is formed by the

α1

and

α2

parts of the molecule. The

α3

portion and the associated

β2

- microglobulin (

β2

m) are close to the membrane. The extension of

the C terminal from

α3

that provides the transmembrane domain and the small cytoplasmic portion of the molecule hav e been omitted.

(Reproduced with permission from Bjorkman PJ et al: Structure of the human histocompatibility antigen HLA-A2. Nature 1987;329:506.)

CD4

Class II Class I MHC

CD8

MHC

T CELL RECEPTORS

The MHC protein–peptide complexes on the surface of the antigen-presenting cells bin d to appropriate T cells. Therefore, receptors on

the T cells must recognize a very wide variety of complexes. Most of the receptors on c irculating T cells are made up of two polypeptide

units designated α and β. They form heterodimers that recognize the MHC proteins and the antigen fragments with which they are

combined (Figure 3–7). These cells are called αβ T cells. About 10% of the circulating T cells have two different polypeptides designated

γ and δ in their receptors, and they are called γδ T cells. These T cells are prominent in the mucosa of the gastrointestinal tract, and there

is evidence that they form a link between the innate and acquired immune systems by w ay of the cytokines they secrete (Figure 3–3).

CD8 occurs on the surface of cytotoxic T cells that bind MHC-I proteins, and CD4 oc curs on the surface of helper T cells that bind

MHC-II proteins (Figure 3–8). The CD8 and CD4 proteins facilitate the binding of the MHC proteins to the T cell receptors, and they

also foster lymphocyte development, but how they produce these effects is unsettled. Th e

TCR TCR

FIGURE 3–8 Diagrammatic summary of the structure of CD4 and CD8, and their re lation to MHC-I and MHC-II proteins. Note

that CD4 is a single protein, whereas CD8 is a heterodimer.

activated CD8 cytotoxic T cells kill their targets directly, whereas the activated CD4 helpe r T cells secrete cytokines that activate other

lymphocytes.

The T cell receptors are surrounded by adhesion molecules and proteins that bind to co mplementary proteins in the antigen-presenting

cell when the two cells transiently join to form the “immunologic synapse” that permits T cell activation to occur. It is now generally

accepted that two signals are necessary to produce activation. One is produced by the b inding of the digested antigen to the T cell

receptor. The other is produced by the joining of the surrounding proteins in the “syna pse.” If the first signal occurs but the second does

not, the T cell is inactivated and becomes unresponsive.

Antigen-presenting

Cytoplasm

cell membrane

ECF β

m α

MHC molecular complex α

Antigen fragment

Constant regions

Variable regions

S–S ECF

+ +

Cytoplasm β α

T cell receptor heterodimer (α:β) T cell

membrane

FIGURE 3–7 Interaction between antigen-presenting cell (top) and

αβ

T lymphocyte (bottom). The MHC proteins (in this case,

MHC-I) and their peptide antigen fragment bind to the

and

units that combine to form the T cell receptor.

B CELLS

As noted above, B cells can bind antigens directly, but they must contact helper T cells to produce full activation and antibody formation.

It is the TH2 subtype that is mainly involved. Helper T cells develop along the TH2 linea ge in response to IL-4 (see below). On the other

hand, IL-12 promotes the TH1 phenotype. IL-2 acts in an autocrine fashion to cause a ctivated T cells to proliferate. The role of various

cytokines in B cell and T cell activation is summarized in Figure 3–9.

The activated B cells proliferate and transform into memory B cells (see above) and plasma cells. The plasma cells secrete large

quantities of antibodies into the general circulation. The antibodies circulate in the globuli n fraction of the plasma and, like antibodies

elsewhere, are called immunoglobulins. The immunoglobulins are actually the se creted form of antigen-binding receptors on the B cell

membrane.

MHC class II

Macrophage (antigen

presenting

cell)

Antigenbinding site

2 IL-1 Fab

CH1

CD4 TCR C

SS SS

Cytokine

induced IL-2R activation

IL-2 Complement

CH2binding

Fc Macrophage

binding

3 Hinge

Activated B cell

4 CD4

Activated

T cell

Inflammation

IL-2R

Cytotoxic and delayed T cell hypersensitivity

CD8

Antibodyproducing cell MHC class I

Cell death

FIGURE 3–10 Typical immunoglobulin G molecule. Fab, portion of the molecule tha t is concerned with antigen binding; Fc, effector

portion of the molecule. The constant regions are pink and purple, and the variable regions are orange. The constant segment of the

heavy chain is subdivided into C

1, C

2, and C

3. SS lines indicate intersegmental disulfide bonds. On the right side, the C labels are

omitted to show regions J

, D, and J

FIGURE 3–9 Summary of acquired immunity. (1) An antigenpresent ing cell ingests and partially digests an antigen, then presents part

of the antigen along with MHC peptides (in this case, MHC II peptides on the cell surfa ce). (2) An “immune synapse” forms with a naive

CD4 T cell, which is activated to produce IL-2. (3) IL-2 acts in an autocrine fashion to cause the cell to multiply, forming a clone. (4)

The activated CD4 cell may promote B cell activation and production of plasma cells or it m ay activate a cytotoxic CD8 cell. The CD8

cell can also be activated by forming a synapse with an MCH I antigen-presenting cell. (Reprodu ced with permission from McPhee SJ, Lingappa

VR, Ganong WF [editors]: Pathophysiology of Disease, 4th ed. McGraw-Hill, 2003.)

IMMUNOGLOBULINS

Circulating antibodies protect their host by binding to and neutralizing some protein toxin s, by blocking the attachment of some viruses

and bacteria to cells, by opsonizing bacteria (see above), and by activating complement. Five general types of immunoglobulin

antibodies are produced by the lymphocyte–plasma cell system. The basic component o f each is a symmetric unit containing four

polypeptide chains (Figure 3–10). The two long chains are called heavy chain s, whereas the two short chains are called light chains.

There are two types of light chains, k and λ, and eight types of heavy chains. The chain s are joined by disulfide bridges that permit

mobility, and there are intrachain disulfide bridges as well. In addition, the heavy chains are flexible in a region called the hinge. Each

heavy chain has a variable (V) segment in which the amino acid sequence is highly var iable, a diversity (D) segment in which the amino

acid segment is also highly variable, a joining (J) segment in which the sequence is mode rately variable, and a constant (C) segment in

which the sequence is constant. Each light chain has a V, a J, and a C segment. The V s egments form part of the antigen-binding sites

(Fab portion of the molecule [Figure 3–10]). The Fc portion of the molecule is the effe ctor portion, which mediates the reactions

initiated by antibodies.

Two of the classes of immunoglobulins contain additional polypeptide components (Tab le 3–3). In IgMs, five of the basic

immunoglobulin units join around a polypeptide called the J chain to form a pentamer. I n IgAs, the secretory immunoglobulins, the

immunoglobulin units form dimers and trimers around a J chain and a polypeptide that c omes from epithelial cells, the secretory

component (SC).

In the intestine, bacterial and viral antigens are taken up by M cells (see Chapter 27) an d passed on to underlying aggregates of lymphoid

tissue (Peyer’s patches), where they activate naive T cells. These lymphocytes then form B cells that infiltrate mucosa of the

gastrointestinal, respiratory, genitourinary, and female reproductive tracts and the breas t. There they secrete large amounts of IgAs when

exposed again to the antigen that initially stimulated them. The epithelial cells produce the SC, which acts as a receptor for and binds the

IgA. The resulting secretory immunoglobulin passes through the epithelial cell and is sec reted by exocytosis. This system of secretory

immunity is an important and effective defense mechanism.

GENETIC BASIS OF DIVERSITY

IN THE IMMUNE SYSTEM

The genetic mechanism for the production of the immensely large number of different configurations of immunoglobulins produced by

human B cells is a fascinating biologic problem.

TABLE 3–3 Human immunoglobulins.

Immunoglobulin Function Heavy Chain Additional Chain

Plasma Concentration

Structure (mg/dL)

IgG Complement activation γ1

γ2

γ3

γ4

Monomer 1000

IgA Localized protection in external secretions (tears, intestinal secretions, etc)

J, SC Monomer; dimer with J or SC 200 chain; trimer with J chain

IgM Complement activation μ J Pentamer with J chain 120

IgD Antigen recognition by B cells δ Monomer 3

IgE Reagin activity; releases histamine from basophils and mast cells

In all instances, the light chains are k or γ

ε Monomer 0.05

Diversity is brought about in part by the fact that in immune globulin molecules there are two kinds of light chains and eight kinds of

heavy chains. As noted previously, there are areas of great variability (hypervariable reg ions) in each chain. The variable portion of the

heavy chains consists of the V, D, and J segments. In the gene family responsible for th is region, there are several hundred different

coding regions for the V segment, about 20 for the D segment, and 4 for the J segmen t. During B cell development, one V coding

region, one D coding region, and one J coding region are selected at random and reco mbined to form the gene that produces that

particular variable portion. A similar variable recombination takes place in the coding re gions responsible for the two variable segments

(V and J) in the light chain. In addition, the J segments are variable because the gene se gments join in an imprecise and variable fashion

(junctional site diversity) and nucleotides are sometimes added (junctional insertion diver sity). It has been calculated that these

mechanisms permit the production of about 10

different immunoglobulin molecules. Additional variability is added by somatic

mutation.

Similar gene rearrangement and joining mechanisms operate to produce the diversity in T cell receptors. In humans, the α subunit has a

V region encoded by 1 of about 50 different genes and a J region encoded by 1 of a nother 50 different genes. The β subunits have a V

region encoded by 1 of about 50 genes, a D region encoded by 1 of 2 genes, and a J region encoded by 1 of 13 genes. These variable

regions permit the generation of up to an estimated 10

different T cell receptors (Clinical Box 3–2 and Clinical Box 3–3).

A variety of immunodeficiency states can arise from defects in these various stages of B and T lymphocyte maturation. These are

summarized in Figure 3–12.

PLATELETS

Platelets are circulating cells that are important mediators of hemostasis. While not immun e cells, per se, they often participate in the

response to tissue injury in cooperation with inflammatory cell types (see below). They have a ring of microtubules around their

periphery and an extensively invaginated membrane with an intricate canalicular system in contact with the ECF. Their membranes

contain receptors for collagen, ADP, vessel wall von Willebrand factor (see below), an d fibrinogen. Their cytoplasm contains actin,

myosin, glycogen, lysosomes, and two types of granules: (1) dense granules, which co ntain the nonprotein substances that are secreted in

response to platelet activation, including serotonin, ADP, and other adenine nucleotides; and (2) α-granules, which contain secreted

proteins other than the hydrolases in lysosomes. These proteins include clotting factors a nd plateletderived growth factor (PDGF).

PDGF is also produced by macrophages and endothelial cells. It is a dimer made up of A and B subunit polypeptides. Homodimers (AA

and BB), as well as the heterodimer (AB), are produced. PDGF stimulates wound heali ng and is a potent mitogen for vascular smooth

muscle. Blood vessel walls as well as platelets contain von Willebrand factor, which, in a ddition to its role in adhesion, regulates

circulating levels of factor VIII (see below).

When a blood vessel wall is injured, platelets adhere to the exposed collagen and von Willebrand facto r in the wall via receptors on the

platelet membrane. Von Willebrand factor is a very large circulating molecule that is pro duced by endothelial cells. Binding produces

platelet activations which release the contents of their granules. The released ADP acts o n the ADP receptors in the platelet membranes

to produce further accumulation of more platelets (platelet aggregation). Humans have a t least three different types of platelet ADP

receptors: P2Y

, P2Y

, and P2X

. These are obviously attractive targets for drug development, and several new inhibitor s have shown

promise in the prevention of heart attacks and strokes. Aggregation is also fostered by platelet-activating factor (PAF), a cytokine

secreted by neutrophils and monocytes as well as platelets. This compound also has infla mmacytes as well as platelets. This compound

also has inflamma acetylglyceryl-3-phosphorylcholine, which is produced from membr ane lipids. It acts via a G protein-coupled receptor

CLINICAL BOX 3–2 CLINICAL BOX 3–3 Autoimmunity

Sometimes the processes that eliminate antibodies against self antigens fail and a variety o f different autoimmune diseases are

produced. These can be B cell- or T cell-mediated and can be organ-specific or system ic. They include type 1 diabetes mellitus

(antibodies against pancreatic islet B cells), myasthenia gravis (antibodies against nicotinic cholinergic receptors), and multiple sclerosis

(antibodies against myelin basic protein and several other components of myelin). In so me instances, the antibodies are against receptors

and are capable of activating those receptors; for example, antibodies against TSH recep tors increase thyroid activity and cause Graves’

disease (see Chapter 20). Other conditions are due to the production of antibodies agai nst invading organisms that cross-react with

normal body constituents (molecular mimicry). An example is rheumatic f ever following a streptococcal infection; a portion of cardiac

myosin resembles a portion of the streptococcal M protein, and antibodies induced by th e latter attack the former and damage the heart.

Some conditions may be due to bystander effects, in which inflammation sensitizes T cells in the neighborhood , causing them to

become activated when otherwise they would not respond. However, much is still unce rtain about the pathogenesis of autoimmune

disease.

increase the production of arachidonic acid derivatives, including thromboxane A

. The role of this compound in the balance between

clotting and anticlotting activity at the site of vascular injury is discussed in Chapter 32.

Platelet production is regulated by the colony-stimulating factors that control the product ion of megakaryocytes, plus thrombopoietin, a

circulating protein factor. This factor, which facilitates megakaryocyte maturation, is pro duced constitutively by the liver and kidneys,

and there are thrombopoietin receptors on platelets. Consequently, when the number of platelets is low, less is bound and more is

available to stimulate production of platelets. Conversely, when the number of platelets is high, more is bound and less is available,

producing a form of feedback control of platelet production. The amino terminal portio n of the thrombopoietin molecule has the platelet-

stimulating activity, whereas the carboxyl terminal portion contains many carbohydrate r esidues and is concerned with the bioavailability

of the molecule.

When the platelet count is low, clot retraction is deficient and there is poor constriction o f ruptured vessels. The resulting clinical

syndrome (thrombocytopenic purpura) is characterized by easy bruisability and m ultiple subcutaneous hemorrhages. Purpura may also

occur when the platelet count is normal, and in some of these cases, the circulating plate lets are abnormal (thrombasthenic purpura).

Individuals with thrombocytosis are predisposed to thrombotic events.

Tissue Transplantation

The T lymphocyte system is responsible for the rejection of transplanted tissue. When tis sues such as skin and kidneys are transplanted

from a donor to a recipient of the same species, the transplants “take” and function for a while but then become necrotic and are

“rejected” because the recipient develops an immune response to the transplanted tissue. This is generally true even if the donor and

recipient are close relatives, and the only transplants that are never rejected are those fro m an identical twin. A number of treatments

have been developed to overcome the rejection of transplanted organs in humans. The goal of treatment is to stop rejection without

leaving the patient vulnerable to massive infections. One approach is to kill T lymphocyt es by killing all rapidly dividing cells with drugs

such as azathioprine, a purine antimetabolite, but this makes patients susceptible to infectio ns and cancer. Another is to administer

glucocorticoids, which inhibit cytotoxic T cell proliferation by inhibiting production of IL -2, but these cause osteoporosis, mental

changes, and the other facets of Cushing syndrome (see Chapter 22). More recently, i mmunosuppressive drugs such as cyclosporine or

tacrolimus (FK-506) have found favor. Activation of the T cell receptor normally increases intracellular Ca

, which acts via

calmodulin to activate calcineurin (Figure 3-11). Calcineurin dephosphorylates the trans cription factor NF-AT, which moves to the

nucleus and increases the activity of genes coding for IL-2 and related stimulatory cyto kines. Cyclosporine and tacrolimus prevent the

dephosphorylation of NF-AT. However, these drugs inhibit all T cell-mediated immune responses, and cyclosporine causes kidney

damage and cancer. A new and promising approach to transplant rejection is the produ ction of T cell unresponsiveness by using drugs

that block the costimulation that is required for normal activation (see text). Clinically eff ective drugs that act in this fashion could be of

great value to transplant surgeons.

INFLAMMATION &

WOUND HEALING

LOCAL INJURY

Inflammation is a complex localized response to foreign substances such as bacteria or in some instances to internally produced

substances. It includes a sequence of reactions initially involving cytokines, neutrophils, adhesion molecules, complement, and IgG.

PAF, an agent with potent inflammatory effects, also plays a role. Later, monocytes and lymphocytes are involved. Arterioles in the

inflamed area dilate, and capillary permeability is increased (see Chapters 33 and 34). W hen the inflammation occurs in or just under the

skin (Figure 3–13), it

T cell receptor

B by increasing the production of I

, and this is probably the main basis of their anti-inflammatory action (see Chapter 22).

CAM

Calcineurin

TCLBP

CsABP

NF-AT

IL-2 gene activation Nucleus

FIGURE 3–11 Action of cyclosporine (CsA) and tacrolimus (TCL) in lymphocytes. BP, bi nding protein; CAM, calmodulin.

SYSTEMIC RESPONSE TO INJURY

Cytokines produced in response to inflammation and other injuries also produce system ic responses. These include alterations in plasma

acute phase proteins, defined as proteins whose concentration is increased or decreased by at le ast 25% following injury. Many of the

proteins are of hepatic origin. A number of them are shown in Figure 3–14. The cause s of the changes in concentration are incompletely

understood, but it can be said that many of the changes make homeostatic sense. Thus, for example, an increase in C-reactive protein

activates monocytes and causes further production of cytokines. Other changes that oc cur in response to injury include somnolence,

negative nitrogen balance, and fever.

is characterized by redness, swelling, tenderness, and pain. Elsewhere, it is a key compo nent of asthma, ulcerative colitis, and many other

diseases.

Evidence is accumulating that a transcription factor, nuclear factorκB, plays a key role in the inflammatory response. NF

B is a

heterodimer that normally exists in the cytoplasm of cells bound to I

, which renders it inactive. Stimuli such as cytokines, viruses,

and oxidants separate NF

B from I

, which is then degraded. NF

B moves to the nucleus, where it binds to the DNA of the genes for

numerous inflammatory mediators, resulting in their increased production and secretion . Glucocorticoids inhibit the activation of

WOUND HEALING

When tissue is damaged, platelets adhere to exposed matrix via integrins that bind to coll agen and laminin (Figure 3–13). Blood

coagulation produces thrombin, which promotes platelet aggregation and granule releas e. The platelet granules generate an inflammatory

response. White blood cells are attracted by selectins and bind to integrins on endothelia l cells, leading to their extravasation through the

blood vessel walls. Cytokines released by the white blood cells and platelets up-regulate integrins on macrophages, which migrate to the

area of injury, and on fibroblasts and epithelial cells, which mediate wound healing

Pluripotent stem cell Autosomal

recessive SCID

BONE MARROW Lymphoid progenitor

THYMUS X-linked SCID pre-B cell

X-linked agammaglobulinemia Immature T cell

B cell MHC class I deficiency MHC class II deficiency

Hyper-IgM syndrome CD8

cell CD4 cell

IgM IgG IgA IgE FIGURE 3–12 Sites of congenital blockade of B and T lymphocyte maturation in various immunodeficiency

states. SCID, severe combined immune deficiency. (Modified from Rosen FS, Cooper MD, Wedgwood RJP : The primary immunodeficiencies. N Engl J

Med 1995;333:431.)

Fibrin clot

Neutrophil

Macrophage

TGFβ1 Platelet TGF

αplug FGF

VEGF PDGF BB

IGFTGFβ1

PDGF AB

Blood vessel

VEGF

Neutrophil

FGF-2

Fibroblast

FIGURE 3–13 Cutaneous wound 3 days after injury, showing the mult iple cytokines and growth factors affecting the repair

process. VEGF, vascular endothelial growth factor. For other abbreviations, se e Appendix. Note the epidermis growing down under the

fibrin clot, restoring skin continuity. (Modified from Singer AJ, Clark RAF: Cutaneous wo und healing. N Engl J Med 1999;341:738.)

and scar formation. Plasmin aids healing by removing excess fibrin. This aids the migra tion of keratinocytes into the wound to restore

the epithelium under the scab. Collagen proliferates, producing the scar. Wounds gain 20% of their ultimate strength in 3 weeks and later

gain more strength, but they never reach more than about 70% of the strength of norm al skin.

30,100

30,000

700

600

C-reactive protein

500

Serum amyloid A 400

300

Haptoglobin

200

Fibrinogen

100

Albumin

Transferrin 0 714 21

Time after inflammatory stimulus (d) FIGURE 3–14 Time course of changes in some major acute phase proteins. C3, C3 component

of complement. (Modified and reproduced with permission from Gitlin JD, Colten HR: Molecular biology of ac ute phase plasma proteins. In Pick F, et al [editors]:

Lymphokines, vol 14, pages 123–153. Academic Press, 1987.)

CHAPTER SUMMARY

■ Immune and inflammatory responses are mediated by several different cell types— granulocytes, lymphocytes, monocytes, mast cells,

tissue macrophages, and antigen presenting cells— that arise predominantly from the bo ne marrow and may circulate or reside in

connective tissues.

■ Granulocytes mount phagocytic responses that engulf and destroy bacteria. These are accompanied by the release of reactive oxygen

species and other mediators into adjacent tissues that may cause tissue injury.

■ Mast cells and basophils underpin allergic reactions to substances that would be trea ted as innocuous by nonallergic individuals.

■ A variety of soluble mediators orchestrate the development of immunologic effector cells and their subsequent immune and

inflammatory reactions.

■ Innate immunity represents an evolutionarily conserved, primitive response to stere otypical microbial components.

■ Acquired immunity is slower to develop than innate immunity, but long-lasting and more effective.

■ Genetic rearrangements endow B and T lymphocytes with a vast array of receptors capab le of recognizing billions of foreign antigens.

■ Self-reactive lymphocytes are normally deleted; a failure of this process leads to autoimmune disease. Disease can also result from

abnormal function or development of granulocytes and lymphocytes. In these latt er cases, deficient immune responses to microbial

threats usually result.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. In normal hu man blood

A) the eosinophil is the most common type of white blood cell. B) there are more lymph ocytes than neutrophils.

C) the iron is mostly in hemoglobin.

D) there are more white cells than red cells.

E) there are more platelets than red cells.

2. Lymphocytes

A) all originate from the bone marrow after birth. B) are unaffected by hormones.

C) convert to monocytes in response to antigens.

D) interact with eosinophils to produce platelets.

E) are part of the body’s defense against cancer.

3. The ability of the blood to phagocytose pathogens and mount a respiratory burst is increas ed by

A) interleukin-2 (IL-2).

B) granulocyte colony-stimulating factor (G-CSF). C) erythropoietin.

D) interleukin-4 (IL-4).

E) interleukin-5 (IL-5).

4. Cells responsible for innate immunity are activated most commonly by

A) glucocorticoids.

B) pollen.

C) carbohydrate sequences in bacterial cell walls.

D) eosinophils.

E) cytoplasmic proteins of bacteria.

CHAPTER RESOURCES

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Samstein B, Emond JC: Liver transplant from living related donors. Annu Rev Med 2 001;52:147.

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Tedder TF, et al: The selectins: Vascular adhesion molecules. FASEB J 1995;9:866.

Tilney NL: Transplant: From Myth to Reality. Yale University Press, 2003.

Walport MJ: Complement. (Two parts) N Engl J Med 2001;344:1058, 1140.

SECTION II PHYSIOLOGY OF NERVE & MUSCLE CELLS CH APTER

Excitable Tissue: Nerve 4

OBJEC TIV ES

After studying this chapter, you should be able to:

Name the parts of a neuron and their functions.

■

Name the various types of glia and their functions.

■

Describe the chemical nature of myelin, and summarize the differences in the ways in which u nmyelinated and myelinated neurons

conduct impulses.

■

Define orthograde and retrograde axonal transport and the molecular motors involved in each.

■

Describe the changes in ionic channels that underlie electrotonic potentials, the ac tion potential, and repolarization.

■

List the various nerve fiber types found in the mammalian nervous system.

■

Describe the function of neurotrophins.

INTRODUCTION

The human central nervous system (CNS) contains about 10

(100 billion) neurons. It also contains 10–50 times this number of glial

cells. The CNS is a complex organ; it has been calculated that 40% of the human genes partic ipate, at least to a degree, in its formation.

The neurons, the basic building blocks of the nervous system, have evolved from prim itive neuroeffector cells that respond to various

stimuli by contracting. In more complex animals, contraction has become the specialized function of muscle cells, whereas integration

and transmission of nerve impulses have become the specialized functions of neurons. This chapter describes the cellular components of

the CNS and the excitability of neurons, which involves the genesis of electrical signals that enable neu rons to integrate and transmit

impulses (action potentials, receptor potentials, and synaptic potentials).

CELLULAR ELEMENTS IN THE CNS

GLIAL CELLS

For many years following their discovery, glial cells (or glia) were viewed as CNS con nective tissue. In fact, the word glia is Greek for

glue. However, today theses cells are recognized for their role in communication within the CNS in partnership with neurons. Unlike

neurons, glial cells continue to undergo cell division in adulthood and their ability to pro liferate is particularly noticeable after brain

injury (eg, stroke).

There are two major types of glial cells in the vertebrate nervous system: microglia and m acroglia. Microglia are scavenger cells that

resemble tissue macrophages and remove debris resulting from injury, infection, and d isease (eg, multiple sclerosis, AIDS-related

dementia, Parkinson disease, and Alzheimer disease). Microglia arise from macrophage s outside of the nervous system and are

physiologically and embryologically unrelated to other neural cell types.

There are three types of macroglia: oligodendrocytes, Schwann cells, and astrocytes (F igure 4–1). Oligodendrocytes and Schwann cells

are involved in myelin formation around axons in the CNS and peripheral nervous sys tem, respectively. Astrocytes, which are found

throughout the brain, are of two subtypes. Fibrous astrocytes, which contain many intermediate filaments, ar e found primarily in white

matter. Protoplasmic astrocytes are found in gray matter and have a granular cy toplasm. Both types send processes to blood vessels,

where they induce capillaries to form the tight junctions making up the blood–brain barrier. They als o send processes that

NEURONS

Neurons in the mammalian central nervous system come in many different shapes and s izes. Most have the same parts as the typical

spinal motor neuron illustrated in Figure 4–2. The cell body (soma) contains the nucleus and is the metabolic center of the neuron.

Neurons have several processes called dendrites that extend outward from the cell bod y and arborize extensively. Particularly in the

cerebral and cerebellar cortex, the dendrites have small knobby projections called dendritic spines. A typical neuron also has a long

fibrous axon that originates from a somewhat thickened area of the cell body, the axon hillock. The first portion of the axon is called the

initial segment. The axon divides into presynaptic terminals, each ending in a n umber of synaptic knobs which are also called

terminal buttons or boutons. They contain granules or vesicles in which the syn aptic transmitters secreted by the nerves are stored.

Based on the number of processes that emanate from their cell body, neurons can be c lassified as unipolar, bipolar, and multipolar

(Figure 4–3).

A Oligodendrocyte B Schwann cell C Astrocyte

Oligodendrocyte Perineural in white matter

oligodendrocytes Nodes of Ranvier Capillary

End-foot

Neuron Layers

of myelin

Axons

envelop synapses and the surface of nerve cells. Protoplasmic astrocytes have a membr ane potential that varies with the external K

concentration but do not generate propagated potentials. They produce substances that are tropic to neurons, and they help maintain the

appropriate concentration of ions and neurotransmitters by taking up K

and the neurotransmitters glutamate and γ-aminobutyrate

(GABA).

Schwann

End-foot

cell

Fibrous astrocyte

Nucleus

Inner

tongue

Axon

Neuron

FIGURE 4–1 The principal types of glial cells in the nervous system. A) Oligodendrocytes are small with relatively few processes.

Those in the white matter provide myelin, and those in the gray matter support neurons. B) Sch wann cells provide myelin to the

peripheral nervous system. Each cell forms a segment of myelin sheath about 1 mm lon g; the sheath assumes its form as the inner tongue

of the Schwann cell turns around the axon several times, wrapping in concen tric layers. Intervals between segments of myelin are the

nodes of Ranvier. C) Astrocytes are the most common glia in the CNS and are characte rized by their starlike shape. They contact both

capillaries and neurons and are thought to have a nutritive function. They are also involved in forming the blood–brain barrier. (From

Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill, 20 00.)

Cell body (soma)

Initial segment

of axon Node of Ranvier Schwann cell

Axon hillock

Nucleus

Terminal buttons

Dendrites

FIGURE 4–2 Motor neuron with a myelinated axon. A m otor neuron is comprised of a cell body (soma) with a nucleus, several

processes called dendrites, and a long fibrous axon that originates from the axon hillock. The first portion of the axon is called the initial

segment. A myelin sheath forms from Schwann cells and surrounds the axon except at its en ding and at the nodes of Ranvier. Terminal

buttons (boutons) are located at the terminal endings.

A Unipolar cell B Bipolar cell C Pseudo-unipolar cell

Dendrite Dendrites Peripheral axon to skin and

muscle

Cell body

Axon

Cell body Cell body Single bifurcated process

Axon Central axon

Invertebrate neuron Bipolar cell of retina Axon terminals Ganglion cell of dorsal root

D Three types of multipolar cells

Dendrites Apical

dendrite Cell body Cell body

Basal dendrite Axon

Dendrites

Cell body

Axon Axon

Motor neuron of spinal cord

Pyramidal cell of hippocampus Purkinje cell of cerebellum

FIGURE 4–3 Some of the types of neurons in the mammalian nervous system . A) Unipolar neurons have one process, with different

segments serving as receptive surfaces and releasing terminals. B) Bipolar neurons have t wo specialized processes: a dendrite that carries

information to the cell and an axon that transmits information from the cell. C) Some sensory neu rons are in a subclass of bipolar cells

called pseudo-unipolar cells. As the cell develops, a single process splits into two, both of which function as axons—one going to skin or

muscle and another to the spinal cord. D) Multipolar cells have one axon and many dendri tes. Examples include motor neurons,

hippocampal pyramidal cells with dendrites in the apex and base, and cerebellar Purkinje cells with an extensive dendritic tree in a single

plane. (From Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed . McGraw-Hill, 2000.)

The conventional terminology used for the parts of a neuron works well enough for s pinal motor neurons and interneurons, but there are

problems in terms of “dendrites” and “axons” when it is applied to other types of neuro ns found in the nervous system. From a

functional point of view, neurons generally have four important zones: (1) a receptor, or dendritic zone, where multiple local potential

changes generated by synaptic connections are integrated; (2) a site where propagated action potentials are generated (the initial segment

in spinal motor neurons, the initial node of Ranvier in cutaneous sensory neurons); (3) an axonal process that transmits propagated

impulses to the nerve endings; and (4) the nerve endings, where action potentials cause the release of synaptic transmitters. The cell body

is often located at the dendritic zone end of the axon, but it can be within the axon (eg, auditory neurons) or attached to the side of the

axon (eg, cutaneous neurons). Its location makes no difference as far as the receptor f unction of the dendritic zone and the transmission

function of the axon are concerned.

The axons of many neurons are myelinated, that is, they acquire a sheath of myelin, a protein–lipid complex that is wrapped around the

axon (Figure 4–2). In the peripheral nervous system, myelin forms when a Schwann c ell wraps its membrane around an axon up to 100

times (Figure 4–1). The myelin is then compacted when the extracellular portions of a m embrane protein called protein zero (P

) lock to

the extracellular portions of P

in the apposing membrane. Various mutations in the gene for P

cause peripheral neuropathies; 29

different mutations have been described that cause symptoms ranging from mild to seve re. The myelin sheath envelops the axon except

at its ending and at the nodes of Ranvier, periodic periodic μm constrictions that are about 1 mm apart (Figure 4 –2). The insulating

function of myelin is discussed later in this chapter. Not all neurons are myelinated; some are unmyelinated, that is, simply surrounded

by Schwann cells without the wrapping of the Schwann cell membrane that produces m yelin around the axon.

In the CNS of mammals, most neurons are myelinated, but the cells that form the myelin are oligodendrocytes rather than Schwann cells

(Figure 4–1). Unlike the Schwann cell, which forms the myelin between two nodes of Ranvier on a single neuron, oligodendrocytes emit

multiple processes that form myelin on many neighboring axons. In multiple sclerosis, a crippling autoimmune disease, patchy

destruction of myelin occurs in the CNS (see Clinical Box 4–1). The loss of myelin is as sociated with delayed or blocked conduction in

the demyelinated axons.

CLINICAL BOX 4–1 Demyelinating Diseases

Normal conduction of action potentials relies on the insulating properties of myelin. Thus, defects in myelin can have major adverse

neurological consequences. One example is multiple sclerosis (MS), an autoim mune disease that affects over 3 million people

worldwide, usually striking between the ages of 20 and 50 and affecting women about twice as often as men. The cause of MS appears to

include both genetic and environmental factors. It is most common among Caucasians li ving in countries with temperate climates,

including Europe, southern Canada, northern United States, and southeastern Australia. Environmental triggers include early exposure to

viruses such as Epstein-Barr virus and those that cause measles, herpes, chicken pox, o r influenza. In MS, antibodies and white blood

cells in the immune system attack myelin, causing inflammation and injury to the sheath a nd eventually the nerves that it surrounds. Loss

of myelin leads to leakage of K

through voltage-gated channels, hyperpolarization, and failure to conduct action poten tials. Typical

physiological deficits range from muscle weakness, fatigue, diminished coordination, slu rred speech, blurred or hazy vision, bladder

dysfunction, and sensory disturbances. Symptoms are often exasperated by increased b ody temperature or ambient temperature.

Progression of the disease is quite variable. In the most common form, transient episode s appear suddenly, last a few weeks or months,

and then gradually disappear. Subsequent episodes can appear years later, and eventua lly full recovery does not occur. Others have a

progressive form of the disease in which there are no periods of remission. Diagnosing MS is very difficult and generally is delayed until

multiple episodes occur with deficits separated in time and space. Nerve conduction tests can detect slowed conduction in motor and

sensory pathways. Cerebral spinal fluid analysis can detect the presence of oligoclonal b ands indicative of an abnormal immune reaction

against myelin. The most definitive assessment is magnetic resonance imaging (MRI) to visualize multiple scarred (sclerotic) areas in

the brain. Although there is no cure for MS, some drugs (eg, β-interferon) that suppre ss the immune response reduce the severity and

slow the progression of the disease.

AXONAL TRANSPORT

Neurons are secretory cells, but they differ from other secretory cells in that the secreto ry zone is generally at the end of the axon, far

removed from the cell body. The apparatus for protein synthesis is located for the most part in the cell body, with transport of proteins

and polypeptides to the axonal ending by axoplasmic flow. Thus, the cell body maintains the function al and anatomic integrity of the

axon; if the axon is cut, the part distal to the cut degenerates (wallerian degeneration). Or thograde transport occurs along

microtubules that run along the length of the axon and requires two molecular motors, d ynein and kinesin (Figure 4–4). Orthograde

transport moves from the cell body toward the axon terminals. It has both fast and slow components; fast axonal transport occurs at

about 400 mm/day, and slow axonal transport occurs at 0.5 to 10 mm/day. Retrograde transpo rt, which is in the opposite direction

(from the

FIGURE 4–4 Axonal transport along microtubules by dynein and kinesin. Fast and slow axonal orthograde transport occurs along

microtubules that run along the length of the axon from the cell body to the terminal. Re trograde transport occurs from the terminal to

the cell body. (From Widmaier EP, Raff H, Strang KT: Vander’s Human Physiology. McGraw -Hill, 2008.)

nerve ending to the cell body), occurs along microtubules at about 200 mm/day. Synap tic vesicles recycle in the membrane, but some

used vesicles are carried back to the cell body and deposited in lysosomes. Some materi als taken up at the ending by endocytosis,

including nerve growth factor (NGF) and various viruses, are also transported back to the cell body . A potentially important exception

to these principles seems to occur in some dendrites. In them, single strands of mRNA tr ansported from the cell body make contact with

appropriate ribosomes, and protein synthesis appears to create local protein domains.

EXCITATION & CONDUCTION

Nerve cells have a low threshold for excitation. The stimulus may be electrical, chemical , or mechanical. Two types of physicochemical

disturbances are produced: local, nonpropagated potentials called, depending on their lo cation, synaptic, generator, or electrotonic

potentials; and propagated potentials, the action potentials (or nerve impulses). These are the only electrical responses of neurons and

other excitable tissues, and they are the main language of the nervous system. They are due to changes in the conduction of ions across

the cell membrane that are produced by alterations in ion channels. The electrical events in neurons are rapid, being measured in

milliseconds (ms); and the potential changes are small, being measured in millivolts (mV).

The impulse is normally transmitted (conducted) along the axon to its termination . Nerves are not “telephone wires” that transmit

impulses passively; conduction of nerve impulses, although rapid, is much slower than th at of electricity. Nerve tissue is in fact a

relatively poor passive conductor, and it would take a potential of many volts to produc e a signal of a fraction of a volt at the other end of

a meter-long axon in the absence of active processes in the nerve. Conduction is an act ive, self-propagating process, and the impulse

moves along the nerve at a constant amplitude and velocity. The process is often compa red to what happens when a match is applied to

one end of a trail of gunpowder; by igniting the powder particles immediately in front o f it, the flame moves steadily down the trail to its

end as it is extinguished in its progression.

Mammalian neurons are relatively small, but giant unmyelinated nerve cells exist in a num ber of invertebrate species. Such cells are

found, for example, in crabs (Carcinus), cuttlefish (Sepia), and squid (Loligo). The fu ndamental properties of neurons were first

determined in these species and then found to be similar in mammals. The neck region o f the muscular mantle of the squid contains

single axons up to 1 mm in diameter. The fundamental properties of these long axons a re similar to those of mammalian axons.

RESTING MEMBRANE POTENTIAL

When two electrodes are connected through a suitable amplifier and placed on the surf ace of a single axon, no potential difference is

observed. However, if one electrode is inserted into the interior of the cell, a constant potential difference is observed, with the inside

negative relative to the outside of the cell at rest. A membrane potential results from sepa ration of positive and negative charges across

the cell membrane (Figure 4–5). In neurons, the resting membrane potential is usually ab out –70 mV, which is close to the equilibrium

potential for K

(Figure 4–6).

In order for a potential difference to be present across a membrane lipid bilayer, two c onditions must be met. First, there must be an

unequal distribution of ions of one or more species across the membrane (ie, a concent ration gradient). Two, the membrane must be

permeable to one or more of these ion species. The permeability is provided by the exis tence of channels or pores in the bilayer; these

channels are usually permeable to a single species of ions. The resting membrane poten tial represents an equilibrium situation at which

the driving force for the membrane-permeant ions down their concentration gradients across the membrane is equal and opposite to the

driving force for these ions down their electrical gradients.

–

– + –

Equal

–

+,–

–

+ +

– –

+ – +– +

– –

–

Extracellular side

–

Cytoplasmic side +

– –

––

Equal

+ –

+,– – – – + +

–

– –

– +–

FIGURE 4–5 This membrane potential results from separation of positive and negati ve charges across the cell membrane. The

excess of positive charges (red circles) outside the cell and negative charges (blue circles ) inside the cell at rest represents a small

fraction of the total number of ions present. (From Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill, 2000.).

(a)

+30

3 5

2 7 –70 1 6

(b)

Gated Na

channel

Gated K

channel K

600

300 P

1 0 1 234 Time (ms)

FIGURE 4–6 The changes in (a) membrane potential (mV) and (b) relative membrane permeability (P) to Na+ and K+ during an action

potential. (From Widmaier EP, Raff H, Strang KT: Vander’s Human Physiology. McGraw-Hill, 2008.)

In neurons, the concentration of K

is much higher inside than outside the cell, while the reverse is the case for Na

. This concentration

difference is established by the Na

-K

ATPase. The outward K

concentration gradient results in passive movement of K

out of the

cell when K

-selective channels are open. Similarly, the inward Na

concentration gradient results in passive movement of Na

into the

cell when Na

-selective channels are open. Because there are more open K

channels than Na

channels at rest, the membrane

permeability to K

is greater. Consequently, the intracellular and extracellular K

concentrations are the prime determinants of the

resting membrane potential, which is therefore close to the equilibrium potential for K

. Steady ion leaks cannot continue forever

without eventually dissipating the ion gradients. This is prevented by the Na

-K

ATPase, which actively moves Na

and K

against

their electrochemical gradient.

IONIC FLUXES DURING

THE ACTION POTENTIAL

The cell membranes of nerves, like those of other cells, contain many different types of ion channels. Some of these are voltage-gated

and others are ligand-gated. It is the behavior of these channels, and particularly Na

and K

channels, which explains the electrical

events in nerves.

The changes in membrane conductance of Na

and K

that occur during the action potentials are shown in Figure 4–6. The conductance

of an ion is the reciprocal of its electrical resistance in the membrane and is a measure of the membrane permeability to that ion. In

response to a depolarizing stimulus, some of the voltage-gated Na

channels become active, and when the threshold potential is

reached, the voltage-gated Na

channels overwhelm the K

and other channels and an action potential results (a positive feedback loop).

The membrane potential moves toward the equilibrium potential for Na

(+60 mV) but does not reach it during the action potential,

primarily because the increase in Na

conductance is short-lived. The Na

channels rapidly enter a closed state called the inactivated

state and remain in this state for a few milliseconds before returning to the resting state , when they again can be activated. In addition,

the direction of the electrical gradient for Na

is reversed during the overshoot because the membrane po tential is reversed, and this

limits Na

influx. A third factor producing repolarization is the opening of voltage-gated K

channels. This opening is slower and more

prolonged than the opening of the Na

channels, and consequently, much of the increase in K

conductance comes after the increase in

conductance. The net movement of positive charge out of the cell due to K

efflux at this time helps complete the process of

repolarization. The slow return of the K

channels to the closed state also explains the after-hyperpolarization, fol lowed by a return to

the resting membrane potential. Thus, voltage-gated K

channels bring the action potential to an end and cause closure of their gates

through a negative feedback process. Figure 4–7 shows the sequential feedba ck control in voltagegated K

and Na

channels during the

action potential.

Decreasing the external Na

concentration reduces the size of the action potential but has little effect on the resting m embrane potential.

The lack of much effect on the resting membrane potential would be predicted, since th e permeability of the membrane to Na

at rest is

relatively low. Conversely, increasing the external K

concentration decreases the resting membrane potential.

Although Na

enters the nerve cell and K

leaves it during the action potential, the number of ions involved is minute relative to the total

numbers present. The fact that the nerve gains Na

and loses K

during activity has been demonstrated experimentally, but significant

differences in ion concentrations can be measured only after prolonged, repeated stimu lation.

Other ions, notably Ca

, can affect the membrane potential through both channel movement and membrane int eractions. A decrease in

extracellular Ca

concentration increases the excitability of nerve and muscle cells by decreasing the amo unt of depolarization

necessary to initiate the changes in the Na

and K

conductance that produce the action potential. Conversely, an increase in

extracellular Ca

concentration can stabilize the membrane by decreasing excitability.

DISTRIBUTION OF ION CHANNELS IN MYELINATED NEURONS

The spatial distribution of ion channels along the axon plays a key role in the initiation an d regulation of the action potential. Voltage-

gated Na

channels are highly concentrated in the nodes of Ranvier and the initial segment in mye linated neurons. The initial segment

and, in sensory neurons, the first node of Ranvier are the sites where impulses are nor mally generated, and the other nodes of Ranvier

are the sites to which the impulses jump during saltatory conduction. The number of Na

channels per square micrometer of membrane

in myelinated mammalian neurons has been estimated to be 50–75 in the cell body, 350– 500 in the initial segment, less than 25 on the

surface of the myelin, 2000–12,000 at the nodes of Ranvier, and 20–75 at the axon te rminals. Along the axons of unmyelinated neurons,

the number is about 110. In many myelinated neurons, the Na

channels are flanked by K

channels that are involved in repolarization.

“ALL-OR-NONE” LAW

It is possible to determine the minimal intensity of stimulating current (threshold intensity) that, acting fo r a given duration, will just

produce an action potential. The threshold intensity varies with the duration; with weak s timuli it is long, and with strong stimuli it is

short. The relation between the strength and the duration of a threshold stimulus is called the strength–duration curve. Slowly rising

currents fail to fire the nerve because the nerve adapts to the applied stimulus, a process called adaptation.

(a) Start

Depolarizing stimulus

Opening of voltage-gated Na

channels Stop

Inactivation

of Na

channels

Depolarization of membrane potential

Positive feedback

Increased P

Increased flow of Na

into

the cell

(b) Start

Depolarization of membrane by Na

influx Opening of voltage-gated K

channels

Repolarization of membrane potential

Negative feedback

Increased P

Increased flow of K

out of the cell

FIGURE 4–7 Feedback control in voltage-gated ion channels in the membrane . (a) Na

channels exert positive feedback. (b) K

channels exert negative feedback. (From Widmaier EP, Raff H, Strang KT: Vander’s Huma n Physiology. McGraw-Hill, 2008.)

Once threshold intensity is reached, a full-fledged action potential is produced. Further increases in the intensity of a stimulus produce

no increment or other change in the action potential as long as the other experimental co nditions remain constant. The action potential

fails to occur if the stimulus is subthreshold in magnitude, and it occurs with constant am plitude and form regardless of the strength of

the stimulus if the stimulus is at or above threshold intensity. The action potential is therefo re “all or none” in character and is said to

obey the all-or-none law.

change that rises sharply and decays exponentially with time. The magnitude of this resp onse drops off rapidly as the distance between

the stimulating and recording electrodes is increased. Conversely, an anodal current pro duces a hyperpolarizing potential change of

similar duration. These potential changes are called electrotonic potentials. A s the strength of the current is increased, the response is

greater due to the increasing addition of a local response of the membrane (Figure 4–8). Finally, at 7–15 mV of depolarization (potential

of –55 mV), the firing level is reached and an action potential occurs.

ELECTROTONIC POTENTIALS, LOCAL RESPONSE, & FIRING LEVEL

Although subthreshold stimuli do not produce an action potential, they do have an effec t on the membrane potential. This can be

demonstrated by placing recording electrodes within a few millimeters of a stimulating el ectrode and applying subthreshold stimuli of

fixed duration. Application of such currents leads to a localized depolarizing potential

CHANGES IN EXCITABILITY DURING ELECTROTONIC POTENTIAL S & THE ACTION POTENTIAL

During the action potential, as well as during electrotonic potentials and the local respons e, the threshold of the neuron to stimulation

changes. Hyperpolarizing responses elevate the threshold, and depolarizing potentials lo wer it as they move

Propagated

action potential Spike

potential Firing level −55

Local response Resting membrane potential

70 −

0.5 1.0 1.5 ms

After-depolarization

After-hyperpolarization

Local

response Period of latent addition

Supernormal period −85

FIGURE 4–8 Electrotonic potentials and local response. The ch anges in the membrane potential of a neuron following application of

stimuli of 0.2, 0.4, 0.6, 0.8, and 1.0 times threshold intensity are shown super imposed on the same time scale. The responses below the

horizontal line are those recorded near the anode, and the responses above the line are those recorded near the cathode. The stimulus of

threshold intensity was repeated twice. Once it caused a propagated action potential (top line), an d once it did not.

Refractory period

Subnormal period

Time

FIGURE 4–9 Relative changes in excitability of a nerve cell membra ne during the passage of an impulse. Note that excitability is

the reciprocal of threshold. (Modified from Morgan CT: Physiological Psychology. McGraw-Hill, 1943.)

the membrane potential closer to the firing level. During the local response, the threshold is lowered, but during the rising and much of

the falling phases of the spike potential, the neuron is refractory to stimulation. This re fractory period is divided into an absolute

refractory period, corresponding to the period from the time the firing level is reached until repolarization is about one-third complete,

and a relative refractory period, lasting from this point to the start of afterdepolarization. During the absolute re fractory period, no

stimulus, no matter how strong, will excite the nerve, but during the relative refractory p eriod, stronger than normal stimuli can cause

excitation. During after-depolarization, the threshold is again decreased, and during aft er-hyperpolarization, it is increased. These

changes in threshold are correlated with the phases of the action potential in Figure 4–9 .

SALTATORY CONDUCTION

Conduction in myelinated axons depends on a similar pattern of circular current flow. H owever, myelin is an effective insulator, and

current flow through it is negligible. Instead, depo

ECF ++ ++ –– ++ + – – – – + + – – – Axon

– – – – + + – – – ++ ++ –– ++ +

ELECTROGENESIS OF

THE ACTION POTENTIAL

The nerve cell membrane is polarized at rest, with positive charges lined up along the ou tside of the membrane and negative charges

along the inside. During the action potential, this polarity is abolished and for a brief per iod is actually reversed (Figure 4–10). Positive

charges from the membrane ahead of and behind the action potential flow into the area of negativity represented by the action potential

(“current sink”). By drawing off positive charges, this flow decreases the polarity of th e membrane ahead of the action potential. Such

electrotonic depolarization initiates a local response, and when the firing level is reached , a propagated response occurs that in turn

electrotonically depolarizes the membrane in front of it.

Active Inactive node node

ECF _ +

Myelin

Axon + _ _ +

Direction of propagation

FIGURE 4–10 Local current flow (movement of positive charges) around an im pulse in an axon. Top: Unmyelinated axon.

Bottom: Myelinated axon. Positive charges from the membrane ahead of and behind the actio n potential flow into the area of negativity

represented by the action potential (“current sink”). In myelinated axons, depolarization jumps from one node of Ranvier to the next

(salutatory conduction).

larization in myelinated axons jumps from one node of Ranvier to the next, with the cur rent sink at the active node serving to

electrotonically depolarize the node ahead of the action potential to the firing level (Figu re 4–10). This jumping of depolarization from

node to node is called saltatory conduction. It is a rapid process that allows myelinated axons t o conduct up to 50 times faster than the

fastest unmyelinated fibers.

+ + − + + + + + + + +

_ _ + _ _ _ _ _ _ _ _

+ + + + − + + + + + +

_ _ _ _ + _ _ _ _ _ _

ORTHODROMIC & ANTIDROMIC

CONDUCTION

An axon can conduct in either direction. When an action potential is initiated in the midd le of it, two impulses traveling in opposite

directions are set up by electrotonic depolarization on either side of the initial current sin k. In the natural situation, impulses pass in one

direction only, ie, from synaptic junctions or receptors along axons to their termination. Such conduction is called orthodromic.

Conduction in the opposite direction is called antidromic. Because synapses, unlike ax ons, permit conduction in one direction only, an

antidromic impulse will fail to pass the first synapse they encounter and die out at that po int.

BIPHASIC ACTION POTENTIALS

The descriptions of the resting membrane potential and action potential outlined above a re based on recording with two electrodes, one in

the extracellular space and the other inside it. If both recording electrodes are placed on the surface of the axon, there is no potential

difference between them at rest. When the nerve is stimulated and an impulse is conduc ted past the two electrodes, a characteristic

sequence of potential changes results. As the wave of depolarization reaches the electro de nearest the stimulator, this electrode becomes

negative relative to the other electrode (Figure 4–11). When the impulse passes to the p ortion of the nerve between the two electrodes,

the potential returns to zero, and then, as it passes the second electrode, the first electrod e becomes positive relative to the second. It is

conventional to connect the leads in such a way that when the first electrode becomes n egative relative to the second, an upward

deflection is recorded. Therefore, the record shows an upward deflection followed by an isoelectric interval and then a downward

deflection. This sequence is called a biphasic action potential (Figure 4–11).

PROPERTIES OF MIXED NERVES

Peripheral nerves in mammals are made up of many axons bound together in a fibrous envelope called the epineurium. Potential

changes recorded extracellularly from such nerves therefore represent an algebraic su mmation of the all-or-none action potentials of

many axons. The thresholds of the individual axons in the nerve and their distance from the stimulat

+ + + + + + + + − + +

_ _ _ _ − _ _ _ + _ _

+ + + + + + + + + + −

_ _ _ _ _ _ _ _ _ _ +

Nerve

mV Time

FIGURE 4–11 Biphasic action potential. Both recording electrodes are on th e outside of the nerve membrane. It is conventional to

connect the leads in such a way that when the first electrode becomes negative relative t o the second, an upward deflection is recorded.

Therefore, the record shows an upward deflection followed by an isoelectric interval a nd then a downward deflection.

ing electrodes vary. With subthreshold stimuli, none of the axons are stimulated and no response occurs. When the stimuli are of

threshold intensity, axons with low thresholds fire and a small potential change is observ ed. As the intensity of the stimulating current is

increased, the axons with higher thresholds are also discharged. The electrical response increases proportionately until the stimulus is

strong enough to excite all of the axons in the nerve. The stimulus that produces excitati on of all the axons is the maximal stimulus, and

application of greater, supramaximal stimuli produces no further increase in the size of the observed potential.

NERVE FIBER TYPES & FUNCTION

After a stimulus is applied to a nerve, there is a latent period before the start of the action potential. This inte rval corresponds to the time

it takes the impulse to travel along the axon from the site of stimulation to the recording e lectrodes. Its duration is proportionate to the

distance between the stimulating and recording electrodes and inversely proportionate to the speed of conduction. If the duration of the

latent period and the distance between the stimulating and recording electrodes are know n, axonal conduction velocity can be

calculated.

Erlanger and Gasser divided mammalian nerve fibers into A, B, and C groups, further subdividing the A group into α, β, γ, and δ fibers.

In Table 4–1, the various fiber types are listed

TABLE 4–1 Nerve fiber types in mammalian nerve.

Fiber Type Function Fiber

Diameter (

m) Conduction Spike Absolute Refractory Velocity (m/s) Duration (ms) Period (ms)

Proprioception; somatic motor 12–20 70–120

Touch, pressure 5–12 30–70 0.4–0.5 0.4–1

Motor to muscle spindles 3–6 15–30

Pain, cold, touch 2–5 12–30

B Preganglionic autonomic <3 3–15 1.2 1.2

Dorsal root Pain, temperature, some mechano-reception 0.4–1.2 0.5–2 2 2

Sympathetic Postganglionic sympathetic 0.3–1.3 0.7–2.3 2 2

A and B fibers are myelinated; C fibers are unmyelinated.

with their diameters, electrical characteristics, and functions. By comparing the neurolog ic deficits produced by careful dorsal root

section and other nerve-cutting experiments with the histologic changes in the nerves, th e functions and histologic characteristics of each

of the families of axons responsible for the various peaks of the compound action pote ntial have been established. In general, the greater

the diameter of a given nerve fiber, the greater its speed of conduction. The large axon s are concerned primarily with proprioceptive

sensation, somatic motor function, conscious touch, and pressure, while the smaller axo ns subserve pain and temperature sensations and

autonomic function. The dorsal root C fibers conduct some impulses generated by touc h and other cutaneous receptors in addition to

impulses generated by pain and temperature receptors.

Further research has shown that not all the classically described lettered components are homogeneous, and a numerical system (Ia, Ib,

II, III, IV) has been used by some physiologists to classify sensory fibers. Unfortunate ly, this has led to confusion. A comparison of the

number system and the letter system is shown in Table 4–2.

In addition to variations in speed of conduction and fiber diameter, the various classes o f fibers in peripheral nerves differ in their

sensitivity to hypoxia and anesthetics (Table 4–3). This fact has clinical as well as physio logic significance. Local anesthetics depress

transmission in the group C fibers before they affect group A touch fibers. Conversely , pressure on a nerve can cause loss of conduction

in large-diameter motor, touch, and pressure fibers while pain sensation remains relative ly intact. Patterns of this type are sometimes

seen in individuals who sleep with their arms under their heads for long periods, causin g compression of the nerves in the arms. Because

of the association of deep sleep with alcoholic intoxication, the syndrome is most commo n on weekends and has acquired the interesting

name Saturday night or Sunday morning paralysis.

NEUROTROPHINS

TROPHIC SUPPORT OF NEURONS

A number of proteins necessary for survival and growth of neurons have been isolate d and studied. Some of these neurotrophins are

products of the muscles or other structures that the neurons innervate, but others are pr oduced by astrocytes. These proteins bind to

receptors at the endings of a neuron. They are internalized and then transported by ret rograde transport to the neuronal cell body, where

they foster the production of proteins associated with neuronal development, growth, a nd survival. Other neurotrophins are produced in

neurons and transported in an anterograde fashion to the nerve ending, where they ma intain the integrity of the postsynaptic neuron.

TABLE 4–2 Numerical classification sometimes used for sensory neurons.

Number Fiber Origin Type

Ia Muscle spindle, annulo-spiral ending A α

Ib Golgi tendon organ A α

II Muscle spindle, flower-spray ending; touch, A β pressure

III Pain and cold receptors; some touch receptors A

IV Pain, temperature, and other receptors Dorsal root C

TABLE 4–3 Relative susceptibility of mammalian A, B, and C nerve fibers to condu ction block produced by various agents.

Most Least Susceptibility to: Susceptible Intermediate Susceptible

Hypoxia B A C

Pressure A B C

Local anesthetics C B A

RECEPTORS

Four established neurotrophins and their three high-affinity receptors are listed in Table 4–4. Each of these trk receptors dimerizes, and

this initiates autophosphorylation in the cytoplasmic tyrosine kinase domains of the recep tors. An additional low-affinity NGF receptor

that is a 75-kDa protein is called p75

NTR

. This receptor binds all four of the listed neurotrophins with equal affinity. There is som e

evidence that it can form a heterodimer with trk A monomer and that the dimer has incr eased affinity and specificity for NGF. However,

it now appears that p75

NTR

receptors can form homodimers that in the absence of trk receptors cause apoptosis, a n effect opposite to the

usual growth-promoting and nurturing effects of neurotrophins.

ACTIONS

The first neurotrophin to be characterized was NGF, a protein growth factor that is necessary for the growth and maintenance of

sympathetic neurons and some sensory neurons. It is present in a broad spectrum of an imal species, including humans, and is found in

many different tissues. In male mice, there is a particularly high concentration in the sub mandibular salivary glands, and the level is

reduced by castration to that seen in females. The factor is made up of two α, two β, an d two γ subunits. The β subunits, each of which

has a molecular mass of 13,200 Da, have all the nerve growth-promoting activity, the α subunits have trypsinlike activity, and the γ

subunits are serine proteases. The function of the proteases is unknown. The structure of the β unit of NGF resembles that of insulin.

TABLE 4–4 Neurotrophins.

Neurotrophin Receptor

Nerve growth factor (NGF) trk A

Brain-derived neurotrophic factor (BDNF) trk B

Neurotrophin 3 (NT-3) trk C, less on trk A and trk B

Neurotrophin 4/5 (NT-4/5) trk B

CLINICAL BOX 4–2 Axonal Regeneration

Peripheral nerve damage is often reversible. Although the axon will degenerate distal to the damage, connective elements of the so-

called distal stump often survive. Axonal sprouting occurs from the proximal st ump, growing toward the nerve ending. This results

from growth-promoting factors secreted by Schwann cells that attract axons toward the distal stump. Adhesio n molecules of the

immunoglobulin superfamily (eg, NgCAM/L1) promote axon growth along cell membr anes and extracellular matrices. Inhibitory

molecules in the perineurium assure that the regenerating axons grow in a correct trajec tory. Denervated distal stumps are able to

upregulate production of neurotrophins that promote growth. Once the regenerate d axon reaches its target, a new functional connection

(eg, neuromuscular junction) is formed. Regeneration allows for considerable, althoug h not full, recovery. For example, fine motor

control may be permanently impaired because some motor neurons are guided to an in appropriate motor fiber. Nonetheless, recovery of

peripheral nerves from damage far surpasses that of central nerve pathways. The prox imal stump of a damaged axon in the CNS will

form short sprouts, but distant stump recovery is rare, and the damaged axons are unlik ely to form new synapses. This is because CNS

neurons do not have the growth-promoting chemicals needed for regeneration. In fac t, CNS myelin is a potent inhibitor of axonal

growth. In addition, following CNS injury several events—astrocytic prolifera tion, activation of microglia, scar formation,

inflammation, and invasion of immune cells—provide an inappropriate environment for regeneration. T hus, treatment of brain and

spinal cord injuries frequently focuses on rehabilitation rather than reversing the nerve damage. New research is aiming to identify ways

to initiate and maintain axonal growth, to direct regenerating axons to reconnect with the ir target neurons, and to reconstitute original

neuronal circuitry.

NGF is picked up by neurons and is transported in retrograde fashion from the ending s of the neurons to their cell bodies. It is also

present in the brain and appears to be responsible for the growth and maintenance of c holinergic neurons in the basal forebrain and

striatum. Injection of antiserum against NGF in newborn animals leads to near total destr uction of the sympathetic ganglia; it thus

produces an immunosympathectomy. There is evidence that the maintenance of neurons by NGF is due to a reduction in apoptosis.

Brain-derived neurotrophic factor (BDNF), neurotrophin 3

(NT-3), NT-4/5, and NGF each maintain a different pattern of neurons, although there is some overlap. Disruption of NT-3

by gene knockout causes a marked loss of cutaneous mechanoreceptors, even in heter ozygotes. BDNF acts rapidly and can actually

depolarize neurons. BDNF-deficient mice lose peripheral sensory neurons and have se vere degenerative changes in their vestibular

ganglia and blunted long-term potentiation.

OTHER FACTORS AFFECTING

NEURONAL GROWTH

The regulation of neuronal growth is a complex process. Schwann cells and astrocytes produce ciliary neurotrophic factor (CNTF).

This factor promotes the survival of damaged and embryonic spinal cord neurons and may prove to be of value in treating human

diseases in which motor neurons degenerate. Glial cell line-derived neurotrophic facto r (GDNF) maintains midbrain dopaminergic

neurons in vitro. However, GDNF knockouts have dopaminergic neurons that appear normal, but they have no kidneys and fail to

develop an enteric nervous system. Another factor that enhances the growth of neuron s is leukemia inhibitory factor (LIF). In addition,

neurons as well as other cells respond to insulinlike growth factor I (IGF-I) an d the various forms of transforming growth factor

(TGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF).

Clinical Box 4–2 compares the ability to regenerate neurons after central and periphera l nerve injury.

CHAPTER SUMMARY

■ There are two main types of microglia and macroglia. Microglia are scavenger cells. M acroglia include oligodendrocytes, Schwann

cells, and astrocytes. The first two are involved in myelin formation; astrocytes produce substances that are tropic to neurons, and they

help maintain the appropriate concentration of ions and neurotransmitters.

■ Neurons are composed of a cell body (soma) which is the metabolic center of the neuron, dendrites that extend outward from the cell

body and arborize extensively, and a long fibrous axon that originates from a somewhat thick ened area of the cell body, the axon hillock.

■ The axons of many neurons acquire a sheath of myelin, a protein–lipid complex th at is wrapped around the axon. Myelin is an effective

insulator, and depolarization in myelinated axons jumps from one node of Ranvier to the nex t, with the current sink at the active node

serving to electrotonically depolarize to the firing level the node ahead of the action po tential.

■ Orthograde transport occurs along microtubules that run the length of the axon and req uires molecular motors, dynein, and kinesin.

■ Two types of physicochemical disturbances occur in neurons: local, nonprop agated potentials (synaptic, generator, or electrotonic

potentials) and propagated potentials (action potentials).

■ In response to a depolarizing stimulus, voltage-gated Na

channels become active, and when the threshold potential is reached, an

action potential results. The membrane potential moves toward the equilibrium potential f or Na

. The Na

channels rapidly enter a

closed state (inactivated state) before returning to the resting state. The direction of the elec trical gradient for Na

is reversed during the

overshoot because the membrane potential is reversed, and this limits Na

influx. Voltage-gated K

channels open and the net movement

of positive charge out of the cell helps complete the process of repolarization. The slow return of the K

channels to the closed state

explains afterhyperpolarization, followed by a return to the resting membrane potential.

■ Nerve fibers are divided into different categories based on axonal diameter, cond uction velocity, and function.

■ Neurotrophins are produced by astrocytes and transported by retrograde transport to the neuronal cell body, where they foster the

production of proteins associated with neuronal development, growth, and survival.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. The distance from between one stimulating electrode to

recording electrode is 4.5 cm. When the axon is stimulated, the latent period is 1.5 ms. W hat is the conduction velocity of the axon? A)

15 m/s

B) 30 m/s

C) 40 m/s

D) 67.5 m/s

E) This cannot be determined from the information given.

2. Which of the following has the slowest conduction velocity? A) Aα fibers

B) Aβ fibers

C) Aγ fibers

D) B fibers

E) C fibers

3. A man falls into a deep sleep with one arm under his head. This arm is paraly zed when he awakens, but it tingles, and pain sensation

in it is still intact. The reason for the loss of motor function without loss of pain sensatio n is that in the nerves to his arm, A) A fibers are

more susceptible to hypoxia than B fibers. B) A fibers are more sensitive to pressure th an C fibers. C) C fibers are more sensitive to

pressure than A fibers. D) motor nerves are more affected by sleep than sensory nerv es. E) sensory nerves are nearer the bone than

motor nerves and

hence are less affected by pressure.

4. Which part of a neuron has the highest concentration of Na

channels per square millimeter of cell membrane?

A) dendrites

B) cell body near dendrites

C) initial segment

D) axonal membrane under myelin

E) none of the above

5. Which of the following statements about nerve growth factor is not true?

A) It is made up of three polypeptide subunits.

B) It facilitates the process of apoptosis.

C) It is necessary for the growth and development of the sympathetic nervous system.

D) It is picked up by nerves from the organs they innervate. E) It is present in the brain .

CHAPTER RESOURCES

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Boron WF, Boulpaep EL: Medical Physiology, Elsevier, 2005.

Bradbury EJ, McMahon SB: Spinal cord repair strategies: Why do they work? Nat Rev Neurosc i 2006;7:644.

Catterall WA: Structure and function of voltage-sensitive ion channels. Science 1988; 242:6 49.

Hille B: Ionic Channels of Excitable Membranes, 3rd ed. Sinauer Associates, 2001.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural Science,4th ed. McG raw-Hill, 2000.

Nicholls JG, Martin AR, Wallace BG: From Neuron to Brain: A Cellular and Molecular App roach to the Function of the Nervous

System, 4th ed. Sinauer Associates, 2001.

Thuret S, Moon LDF, Gage FH: Therapeutic interventions after spinal cord injur y. Nat Rev Neurosci 2006;7:628.

Volterra A, Meldolesi J: Astrocytes, from brain glue to communication elements: T he revolution continues. Nat Rev Neurosci

2005;6:626.

Widmaier EP, Raff H, Strang KT: Vander’s Human Physiology. McGraw-Hill, 2008.

Excitable Tissue: Muscle

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Differentiate the major classes of muscle in the body.

■

Describe the molecular and electrical makeup of muscle cell excitation– contraction cou pling.

■

Define thick and thick filaments and how they slide to create contraction.

■

Differentiate the role(s) for Ca

in skeletal, cardiac, and smooth muscle contraction.

■

Appreciate muscle cell diversity.

INTRODUCTION

Muscle cells, like neurons, can be excited chemically, electrically, and mechanically to pr oduce an action potential that is transmitted

along their cell membranes. Unlike neurons, they respond to stimuli by activating a cont ractile mechanism. The contractile protein

myosin and the cytoskeletal protein actin are abundant in muscle, where they are the pri mary structural components that bring about

contraction.

Muscle is generally divided into three types: skeletal, cardiac, and smooth , although smooth muscle is not a homogeneous single

category. Skeletal muscle makes up the great mass of the somatic musculature. It has we ll-developed cross-striations, does not normally

contract in the absence of nervous stimulation, lacks anatomic and functional connection s between individual muscle fibers, and is

generally under voluntary control. Cardiac muscle also has cross-striations, but it is func tionally syncytial and, although it can be

modulated via the autonomic nervous system, it can contract rhythmically in the absence of external innervation owing to the presence in

the myocardium of pacemaker cells that discharge spontaneously (see Chapter 30). Sm ooth muscle lacks cross-striations and can be

further subdivided into two broad types: unitary (or visceral) smooth muscle and multiu nit smooth muscle. The type found in most

hollow viscera is functionally syncytial and contains pacemakers that discharge irregular ly. The multiunit type found in the eye and in

some other locations is not spontaneously active and resembles skeletal muscle in graded contractile ability.

SKELETAL MUSCLE MORPHOLOGY

ORGANIZATION

Skeletal muscle is made up of individual muscle fibers that are the “building blocks” of th e muscular system in the same sense that the

neurons are the building blocks of the nervous system. Most skeletal muscles begin and end in tendons, and the muscle fibers are

arranged in parallel between the tendinous ends, so that the force of contraction of the units is additive. Each muscle fiber is a single cell

that is multinucleated, long, cylindrical, and surrounded by a cell membrane, the sarcolemma (Figure 5–1). There are no syncytial

bridges between cells. The muscle fibers are made up of myofibrils, which are divisible into individual filaments. These myofilaments

contain several proteins that together make up the contractile machinery of the skeletal m uscle.

Terminal cistern Transverse tubules

Sarcoplasmic reticulum

Sarcolemma

(muscle fiber membrane)

Filaments

Mitochondrion

Myofibril

B Z disk Sarcomere Z disk

Thin filament (F-actin)

Tropomyosin Troponin

Actin

Thick filament (myosin)

FIGURE 5–1 Mammalian skeletal muscle. A single muscle fi ber surrounded by its sarcolemma has been cut away to show individual

myofibrils. The cut surface of the myofibrils shows the arrays of thick and thin filamen ts. The sarcoplasmic reticulum with its transverse

(T) tubules and terminal cisterns surrounds each myofibril. The T tubules invaginate from the sa rcolemma and contact the myofibrils

twice in every sarcomere. Mitochondria are found between the myofibrils and a basal la mina surrounds the sarcolemma. (Reproduced with

permission from Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill , 2000.)

The contractile mechanism in skeletal muscle largely depends on the proteins myosin-II, actin, tro pomyosin, and troponin. Troponin is

made up of three subunits: troponin I, troponin T, and trop onin C. Other important proteins in muscle are involved in main taining the

proteins that participate in contraction in appropriate structural relation to one another an d to the extracellular matrix.

A band I band H band Z line M line

FIGURE 5–2 Electron micrograph of human gastrocnemius muscle. The various bands and lines are identified at the top (

13,500).

(Courtesy of Walker SM, Schrodt GR.)

STRIATIONS

Differences in the refractive indexes of the various parts of the

muscle fiber are responsible for the characteristic cross-stria

tions seen in skeletal muscle when viewed under the micro

scope. The parts of the cross-striations are frequently

identified by letters (Figure 5–2). The light I band is divided by

the dark Z line, and the dark A band has the lighter H band in

its center. A transverse M line is seen in the middle of the H

band, and this line plus the narrow light areas on either side of

it are sometimes called the pseudo-H zone. The area between

two adjacent Z lines is called a sarcomere. The orderly arrange

ment of actin, myosin, and related proteins that produces this

pattern is shown in Figure 5–3. The thick filaments, which are

about twice the diameter of the thin filaments, are made up of myosin; the thin filaments are made up of actin, tropomyosin, and

troponin. The thick filaments are lined up to form the A bands, whereas the array of th in filaments extends out of the A band and into the

less dense staining I bands. The lighter H bands in the center of the A bands are the reg ions where, when the muscle is relaxed, the thin

filaments do not overlap the thick filaments. The Z lines allow for anchoring of the thin filaments. If a transverse section through the A

band is examined under the electron microscope, each thick filament is seen to be surro unded by six thin filaments in a regular hexagonal

pattern.

The form of myosin found in muscle is myosin-II, with two globular heads and a long tail. The heads of the myosin

Sarcomere A band

Actin

Myosin

Actin

Z line Relaxed Z line Contracted

Z line Thick Thin AB

M line

Tropomyosin IC

Troponin IC T

Actin

Actin Myosin

CD FIGURE 5–3 A) Arrangement of thin (actin) and thick (myosin) filaments in skeletal muscle (compare to Figure 5–2). B) Sliding of

actin on myosin during contraction so that Z lines move closer together. C) Detail of re lation of myosin to actin in an individual

sarcomere, the functional unit of the muscle. D) Diagrammatic representation of the ar rangement of actin, tropomyosin, and troponin of

the thin filaments in relation to a myosin thick filament. The globular heads of myo sin interact with the thin filaments to create the

contraction. Note that myosin thick filaments reverse polarity at the M line in th e middle of the sarcomere, allowing for contraction. (C

and D are modified with permision from Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science , 4th ed. McGraw-Hill, 2000.)

molecules form cross-bridges with actin. Myosin contains heavy chains and light chains , and its heads are made up of the light chains

and the amino terminal portions of the heavy chains. These heads contain an actin-bind ing site and a catalytic site that hydrolyzes ATP.

The myosin molecules are arranged symmetrically on either side of the center of the sar comere, and it is this arrangement that creates the

light areas in the pseudo-H zone. The M line is the site of the reversal of polarity of the myosin molecules in each of the thick filaments.

At these points, there are slender cross-connections that hold the thick filaments in prope r array. Each thick filament contains several

hundred myosin molecules.

The thin filaments are polymers made up of two chains of actin that form a long double helix. Tropomyosin molecules are long filaments

located in the groove between the two chains in the actin (Figure 5–3). Each thin filame nt contains 300 to 400 actin molecules and 40 to

60 tropomyosin molecules. Troponin molecules are small globular units located at interv als along the tropomyosin molecules. Each of

the three troponin subunits has a unique function: Troponin T binds the troponin compo nents to tropomyosin; troponin I inhibits the

interaction of myosin with actin; and troponin C contains the binding sites for the Ca

that helps to initiate contraction.

Some additional structural proteins that are important in skeletal muscle function include actinin, titin, and desmin. Actinin binds actin

to the Z lines. Titin, the largest known protein (with a molecular mass near 3,000,000 D a), connects the Z lines to the M lines and

provides scaffolding for the sarcomere. It contains two kinds of folded domains that pr ovide muscle with its elasticity. At first when the

muscle is stretched there is relatively little resistance as the domains unfold, but with furth er stretch there is a rapid increase in resistance

that protects the structure of the sarcomere. Desmin adds structure to the Z lines in part b y binding the Z lines to the plasma membrane.

Although these proteins are important in muscle structure/ function, by no means do the y represent an exhaustive list.

SARCOTUBULAR SYSTEM

The muscle fibrils are surrounded by structures made up of membranes that appear in electron photomicrographs as vesicles and tubules.

These structures form the sarcotubular system, which is made up of a T system and a sarcoplasmic reticulum. The T system of

transverse tubules, which is continuous with the sarcolemma of the muscle fiber, forms a grid perforated by the individual muscle fibrils

(Figure 5–1). The space between the two layers of the T system is an extension of the e xtracellular space. The sarcoplasmic reticulum,

which forms an irregular curtain around each of the fibrils, has enlarged terminal ciste rns in close contact with the T system at the

junctions between the A and I bands. At these points of contact, the arrangement of the central T system with a cistern of the

sarcoplasmic reticulum on either side has led to the use of the term triads to describe the sy stem. The T system, which is continuous with

the sarcolemma, provides a path for the rapid transmission of the action potential from t he cell membrane to all the fibrils in the muscle.

The sarcoplasmic reticulum is an important store of Ca

and also participates in muscle metabolism.

DYSTROPHIN–GLYCOPROTEIN COMPLEX

The large dystrophin protein (molecular mass 427,000 Da) forms a rod tha t connects the thin actin filaments to the transmembrane

protein β-dystroglycan in the sarcolemma by smaller proteins in the cytoplasm, syntrophins . β-dystroglycan is connected to merosin

(merosin refers to laminins that contain the α2 subunit in their trimeric makeup) in the ex tracellular matrix by α-dystroglycan (Figure 5–

4). The dystroglycans are in turn associated with a complex of four transmembrane gly coproteins: α-, β-, γ-, and δ-sarcoglycan. This

dystrophin–glycoprotein complex adds strength to the muscle by providing a sca ffolding for the fibrils and connecting them to the

extracellular environment. Disruption of the tightly choreographed structure can lead to several different pathologies, or muscular

dystrophies (see Clinical Box 5–1).

ELECTRICAL PHENOMENA

& IONIC FLUXES

ELECTRICAL CHARACTERISTICS

OF SKELETAL MUSCLE

The electrical events in skeletal muscle and the ionic fluxes that underlie them share distin ct similarities to those in nerve, with

quantitative differences in timing and magnitude. The resting membrane potential of ske letal muscle is about –90 mV. The action

potential lasts 2 to 4 ms and is conducted along the muscle fiber at about 5 m/s. The abso lute refractory period is 1 to 3 ms long, and the

after-polarizations, with their related changes in threshold to electrical stimulation, are rel atively prolonged. The initiation of impulses at

the myoneural junction is discussed in the next chapter.

ION DISTRIBUTION & FLUXES

The distribution of ions across the muscle fiber membrane is similar to that across the ne rve cell membrane. Approximate values for the

various ions and their equilibrium potentials are shown in Table 5–1. As in nerves, depo larization is largely a manifestation of Na

influx, and repolarization is largely a manifestation of K

efflux.

CONTRACTILE RESPONSES

It is important to distinguish between the electrical and mechanical events in skeletal musc le. Although one response

β1 γ1

Laminin 2

Functionally important

carbohydrate side chains Sarcoglycan

complex

Dystroglycans

Sarcospan Dystrophin

F-Actin

Syntrophins

FIGURE 5–4 The dystrophin–glycoprotein complex. Dystrophin co nnects F-actin to the two members of the dystroglycan (DG)

complex, α and β-dystroglycan, and these in turn connect to the merosin subunit of lamin in 211 in the extracellular matrix. The

sarcoglycan complex of four glycoproteins, α-, β-, γ-, and δ-sarcoglycan, sarcospan, and syn tropins are all associated with the

dystroglycan complex. There are muscle disorders associated with loss, abnormalities, or both o f the sarcoglycans and merosin.

(Reproduced with permission from Kandel ER, Scwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill, 2000.)

does not normally occur without the other, their physiologic bases and characteristics ar e different. Muscle fiber membrane

depolarization normally starts at the motor end plate, the specialized structure under the m otor nerve ending. The action potential is

transmitted along the muscle fiber and initiates the contractile response.

THE MUSCLE TWITCH

A single action potential causes a brief contraction followed by relaxation. This respons e is called a muscle twitch. In Figure 5–5, the

action potential and the twitch are plotted on the same time scale. The twitch starts about 2 ms after the start of depolarization of the

membrane, before repolarization is complete. The duration of the twitch varies with the type of muscle being tested. “Fast” muscle

fibers, primarily those concerned with fine, rapid, precise movement, have twitch durat ions as short as 7.5 ms. “Slow” muscle fibers,

principally those involved in strong, gross, sustained movements, have twitch durations up to 100 ms.

MOLECULAR BASIS OF CONTRACTION

The process by which the contraction of muscle is brought about is a sliding of the thin filaments over the thick filaments. Note that this

shortening is not due to changes in the actual lengths of the thick and thin filaments, rath er, by their increased overlap within the muscle

cell. The width of the A bands is constant, whereas the Z lines move closer together wh en the muscle contracts and farther apart when it

relaxes (Figure 5–3).

The sliding during muscle contraction occurs when the myosin heads bind firmly to acti n, bend at the junction of the head with the neck,

and then detach. This “power stroke” depends on the simultaneous hydrolysis of ATP. Myosin-II molecules are dimers that have two

heads, but only one attaches to actin at any given time. The probable sequence of event s of the power stroke is outlined in Figure 5–6. In

resting muscle, troponin I is bound to actin and tropomyosin and covers the sites where myosin heads interact with actin. Also at rest, the

myosin head contains tightly bound ADP. Following an action potential cytosolic Ca

is increased and free Ca

binds to troponin C.

This binding results in a weakening of the troponin I interaction with actin and exposes the actin binding site for myosin to

text continues on p. 100

CLINICAL BOX 5–1

Disease of Muscle

Muscular Dystrophies

The term muscular dystrophy is applied to diseases that cause progressive weakness of skeletal muscle. About 50 such diseases have

been described, some of which include cardiac as well as skeletal muscle. They range f rom mild to severe and some are eventually fatal.

They have multiple causes, but mutations in the genes for the various components of the dystrophin–glycoprotein complex are a

prominent cause. The dystrophin gene is one of the largest in the body, and mutations c an occur at many different sites in it. Duchenne

muscular dystrophy is a serious form of dystrophy in which the dystrophin protein is absen t from muscle. It is X-linked and usually

fatal by the age of 30. In a milder form of the disease, Becker muscular dystrop hy, dystrophin is present but altered or reduced in

amount. Limb-girdle muscular dystrophies of various types are associated with mutation s of the genes coding for the sarcoglycans or

other components of the dystrophin–glycoprotein complex.

Metabolic Myopathies

Mutations in genes that code for enzymes involved in the metabolism of carbohydrates, fats, and proteins to CO

and H

O in muscle and

the production of ATP can cause metabolic myopathies (eg, McArdle syndrome) . Metabolic myopathies all

TABLE 5–1 Steady-state distribution of ions in the intracellular and extracellular compartm ents of mammalian skeletal muscle,

and the equilibrium potentials for these ions.

100

Concentration (mmol/L) 0

Ion

Intracellular Extracellular Fluid Fluid Equilibrium Potential (mV)

12 145 +65 30

155 4 –95 0 H

–5

3.8

–5

–32

–

3.8 120 –90 0 5 10 15 20 25 ms

HCO3– 8 27 –32

–

155 0 …

Membrane potential = –90 mV

–

represents organic anions. The value for intracellular Cl

–

is calculated from the membrane potential, using the Nernst equation.

have in common exercise intolerance and the possibility of muscle breakdown due to ac cumulation of toxic metabolites.

Ion Channel Myopathies

In the various forms of clinical myotonia, muscle relaxation is prolonged after voluntary contraction. The molecular bases of myotonias

are due to dysfunction of channels that shape the action potential. Myotonia dystrophy is caused by an autosomal dominant mutation that

leads to overexpression of a K

channel (although the mutation is not at the K

channel). A variety of myotonias are associated with

mutations in Na

channels (eg, hyperkalemic periodic paralysis, paramyotonia congenita, or Na

channel congenita) or Cl

–

channels (eg,

dominant or recessive myotonia congenita).

Malignant hyperthermia is another disease related to dysfunctional muscle ion channels. Patients with malignant hyperthermia can

respond to general anesthetics such as halothane by eliciting rigidity in the muscles and a quick increase in body temperature. This

disease has been traced to a mutation in RyR, the Ca

release channel in the sarcoplasmic reticulum. The mutation results in an

inefficient feedback mechanism to shut down Ca

release after stimulation of the RyR, and thus, increased contractility and heat

generation.

FIGURE 5–5 The electrical and mechanical responses of a mammalian skel etal muscle fiber to a single maximal stimulus. The

electrical response (mV potential change) and the mechanical response (T, tension in ar bitrary units) are plotted on the same abscissa

(time). The mechanical response is relatively long-lived compared to the electrical resp onse that initiates contraction.

Troponin

Thin

filament

ADP Myosin

Actin

Tropomyosin Thick

filament

ADP

Exposed binding site

Longitudinal force

ADP

ATP

P i ADP

E FIGURE 5–6 Power stroke of myosin in skeletal muscle. A) At rest, myosin heads are bound to adenosine diphosphate and are said

to be in a “cocked” position in relation to the thin filament, which does not hav e Ca

bound to the troponin–tropomyosin complex. B)

bound to the troponin–tropomyosin complex induced a conformational change in the thin filame nt that allows for myosin heads to

cross-bridge with thin filament actin. C) Myosin heads rotate, move the attached actin and shorten t he muscle fiber, forming the power

stroke. D) At the end of the power stroke, ATP binds to a now exposed site, and causes a detac hment from the actin filament. E) ATP is

hydrolyzed into ADP and inorganic phosphate (P

) and this chemical energy is used to “re-cock” the myosin head. (Modified with permission

from Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McG raw-Hill, 2000.)

allow for formation of myosin/actin cross-bridges. Upon formation of the cross-bridge , ADP is released, causing a conformational

change in the myosin head that moves the thin filament relative to the thick filament, com prising the crossbridge “power stroke.” ATP

quickly binds to the free site on the myosin, which leads to a detachment of the myosin h ead from the thin filament. ATP is hydrolyzed

and inorganic phosphate (P

) released, causing a “re-cocking” of the myosin head and completing the cycle. As lon g as Ca

remains

elevated and sufficient ATP is available, this cycle repeats. Many myosin heads cycle at or near the same time, and they cycle

repeatedly, producing gross muscle contraction. Each power stroke shortens the sarcom ere about 10 nm. Each thick filament has about

500 myosin heads, and each head cycles about five times per second during a rapid co ntraction.

The process by which depolarization of the muscle fiber initiates contraction is called ex citation–contraction coupling. The action

potential is transmitted to all the fibrils in the fiber via the T system (Figure 5–7). It trigge rs the release of Ca

from the terminal

cisterns, the lateral sacs of the sarcoplasmic reticulum next to the T system. Depolarization of the T tubule membrane activates the

sarcoplasmic reticulum via dihydropyridine receptors (DHPR), named for the drug dih ydropyridine, which blocks them (Figure 5–8).

DHPR are voltage-gated Ca

channels in the T tubule membrane. In cardiac muscle, influx of Ca

via these channels triggers the

release of Ca

stored in the sarcoplasmic reticulum (calciuminduced calcium release) by activating the ryanodine receptor (RyR). The

RyR is named after the plant alkaloid ryanodine that was used in its discovery. It is a liga nd-gated Ca

channel with Ca

as its natural

ligand. In skeletal muscle, Ca

entry from the extracellular fluid (ECF) by this route is not required for Ca

release. Instead, the DHPR

that serves as the voltage sensor unlocks release of Ca

from the nearby sarcoplasmic reticulum via physical interaction with the RyR.

The released Ca

is quickly amplified through calciuminduced calcium release. Ca

is reduced in the muscle cell by the sarcoplasmic

or endoplasmic reticulum Ca

ATPase (SERCA) pump. The SERCA pump uses energy from ATP hydrolysis to rem ove Ca

from the

cytosol back into the terminal cisterns, where it is stored until released by the next action potential. Once the Ca

concentration outside

the reticulum has been lowered sufficiently, chemical interaction between myosin and ac tin ceases and the muscle relaxes. Note that ATP

provides the energy for both contraction (at the myosin head) and relaxation (via SERC A). If transport of Ca

into the reticulum is

inhibited, relaxation does not occur even though there are no more action potentials; the resulting sustained contraction is called a

contracture.

TYPES OF CONTRACTION

Muscular contraction involves shortening of the contractile elements, but because muscl es have elastic and viscous elements in series

with the contractile mechanism, it is possible

Steps in contraction

Discharge of motor neuron

Release of transmitter (acetylcholine) at motor end-plate

Binding of acetylcholine to nicotinic acetylcholine receptors

Increased Na

and K

conductance in end-plate membrane

Generation of end-plate potential

Generation of action potential in muscle fibers

Inward spread of depolarization along T tubules

Release of Ca

from terminal cisterns of sarcoplasmic reticulum and diffusion to thick and thin filaments

Binding of Ca

to troponin C, uncovering myosin-binding sites on actin

Formation of cross-linkages between actin and myosin and sliding of thin on thick filam ents, producing movement

Steps in relaxation

pumped back into sarcoplasmic reticulum

Release of Ca

from troponin

Cessation of interaction between

actin and myosin

Steps 1–6 in contraction are discussed in Chapter 4. FIGURE 5–7 Flow of info rmation that leads to muscle contraction.

Dihydropyridine receptor A Extracellular space

+ + + +

NH2

COOH Cytoplasm COOH

Pivot Recorder

Lumen of SR

To stimulator

Ryanodine receptor

FIGURE 5–8 Relation of the T tubule (TT) to the sarcoplasmic reticulum in C a

transport. In skeletal muscle, the voltage-gated

dihydropyridine receptor in the T tubule triggers Ca

release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR).

Upon sensing a voltage change, there is a physical interaction between the sarcolemmal-bound DHPR and the SR-bound RyR. This

interaction gates the RyR and allows for Ca

release from the SR.

Force

transducer

Recorder

for contraction to occur without an appreciable decrease in the length of the whole mus cle (Figure 5–9). Such a contraction is called

isometric (“same measure” or length). Contraction against a constant load with a decrea se in muscle length is isotonic (“same tension”).

Note that because work is the product of force times distance, isotonic contractions do w ork, whereas isometric contractions do not. In

other situations, muscle can do negative work while lengthening against a constant weig ht.

SUMMATION OF CONTRACTIONS

The electrical response of a muscle fiber to repeated stimulation is like that of nerve. The fiber is electrically refractory only during the

rising phase and part of the falling phase of the spike potential. At this time, the contracti on initiated by the first stimulus is just

beginning. However, because the contractile mechanism does not have a refractory pe riod, repeated stimulation before relaxation has

occurred produces additional activation of the contractile elements and a response that is added to the contraction already present. This

phenomenon is

FIGURE 5–9 A) Muscle preparation arranged for recording isotonic contracti ons. B) Preparation arranged for recording isometric

contractions. In A, the muscle is fastened to a writing lever that swings on a pivot. In B, it is attached to an electronic transducer that

measures the force generated without permitting the muscle to shorten.

known as summation of contractions. The tension developed during summation is considerably greater th an that during the single

muscle twitch. With rapidly repeated stimulation, activation of the contractile mechanism o ccurs repeatedly before any relaxation has

occurred, and the individual responses fuse into one continuous contraction. Such a res ponse is called a tetanus (tetanic contraction). It

is a complete tetanus when no relaxation occurs between stimuli and an incomplete tetanus when periods of incomplete relaxation take

place between the summated stimuli. During a complete tetanus, the tension developed is about four times that developed by the

individual twitch contractions. The development of an incomplete and a complete tetanus in response to stimuli of increasing frequency

is shown in Figure 5–10.

FIGURE 5–10 Tetanus. Isometric tension of a single muscle fiber during continuou sly increasing and decreasing stimulation frequency.

Dots at the top are at intervals of 0.2 s. Note the development of incomplete and then co mplete tetanus as stimulation is increased, and

the return of incomplete tetanus, then full response, as stimulation frequency is de creased.

The stimulation frequency at which summation of contractions occurs is determined by the twitch duration of the particular muscle being

studied. For example, if the twitch duration is 10 ms, frequencies less than 1/10 ms (100 /s) cause discrete responses interrupted by

complete relaxation, and frequencies greater than 100/s cause summation.

RELATION BETWEEN MUSCLE LENGTH & TENSION & VELOCITY O F CONTRACTION

Both the tension that a muscle develops when stimulated to contract isometrically (the total tension ) and the passive tension exerted by

the unstimulated muscle vary with the length of the muscle fiber. This relationship can be studied in a whole skeletal muscle preparation

such as that shown in Figure 5–9. The length of the muscle can be varied by changing the distance between its two attachments. At each

length, the passive tension is measured, the muscle is then stimulated electrically, and the t otal tension is measured. The difference

between the two values at any length is the amount of tension actually generated by the contractile process, the active tension. The

records obtained by plotting passive tension and total tension against muscle length are s hown in Figure 5–11. Similar curves are

obtained when single muscle fibers are studied. The length of the muscle at which the ac tive tension is maximal is usually called its

resting length. The term comes originally from experiments demonstrating that th e length of many of the muscles in the body at rest is

the length at which they develop maximal tension.

The observed length–tension relation in skeletal muscle is explained by the sliding filame nt mechanism of muscle contraction. When the

muscle fiber contracts isometrically, the tension developed is proportional to the number of crossbridges between the actin and the

myosin molecules. When muscle is stretched, the overlap between actin and myosin is re duced and the number of cross-linkages is

therefore reduced. Conversely, when the muscle is appreciably shorter than resting len gth, the distance the thin filaments can move is

reduced.

The velocity of muscle contraction varies inversely with the load on the muscle. At a giv en load, the velocity is maximal at the resting

length and declines if the muscle is shorter or longer than this length.

FIBER TYPES

Although skeletal muscle fibers resemble one another in a general way, skeletal muscle is a heterogeneous tissue made up of fibers that

vary in myosin ATPase activity, contractile speed, and other properties. Muscles are fre quently classified into two types, “slow” and

“fast.” These muscles can contain a mixture of three fiber types: type I (or SO for slow -oxidative); type IIA (FOG for fast-oxidative-

glycolytic); or type IIB (FG for fast glycolytic). Some of the properties associated with type I, type IIA, and type IIB fibers are

summarized in Table 5–2. Although this classification scheme is valid for muscles across many mammalian species, there are significant

variations of fibers within and between muscles. For example, type I fibers in a given m uscle can be larger than type IIA fibers from a

different muscle in the same animal. Many of the differences in the fibers that make up muscles stem from differences in the proteins

within them. Most of these are encoded by multigene families. Ten different isoforms of the myosin heavy chains (MHCs) have been

characterized. Each of the two types of light chains also have isoforms. It appears that t here is only one form of actin, but multiple

isoforms of tropomyosin and all three components of troponin.

Resting length

Total tension

Active tension 10

ENERGY SOURCES & METABOLISM

Muscle contraction requires energy, and muscle has been called “a machine for conver ting chemical energy into mechanical work.” The

immediate source of this energy is ATP, and this is formed by the metabolism of carboh ydrates and lipids.

Passive tension

0 01 23 4 5

Increase in muscle length (cm)

FIGURE 5–11 Length–tension relationship for the human triceps muscle. The passive t ension curve measures the tension exerted by

this skeletal muscle at each length when it is not stimulated. The total tension curve represe nts the tension developed when the muscle

contracts isometrically in response to a maximal stimulus. The active tension is the difference b etween the two.

PHOSPHORYLCREATINE

ATP is resynthesized from ADP by the addition of a phosphate group. Some of the ene rgy for this endothermic reaction is supplied by

the breakdown of glucose to CO

and H

O, but there also exists in muscle another energy-rich phosphate compound that can su pply this

energy for short periods. This compound is phosphorylcreatine, which is hydrolyzed to cr eatine and phosphate groups with the release

of considerable energy (Figure 5–12). At rest, some ATP in the mitochondria transfers its phosphate to creatine, so that a

phosphorylcreatine

TABLE 5–2 Classification of fiber types in skeletal muscles.

Type 1 Type IIA Type IIB

Other names Fast, Oxidative, Glycolytic (FOG) Slow, Oxidative (SO) Fast, Glycolytic (FG )

Color Red Red White

Myosin ATPase Activity Fast Slow Fast

-pumping capacity of sarcoplasmic reticulum High Moderate High

Diameter Large Small Large

Glycolytic capacity High Moderate High

Oxidative capacity Moderate High Low

Associated Motor Unit Type Fast Resistant to Fatigue (FR) Slow (S) Fast Fatigable (FF)

Membrane potential = –90 mV

Oxidative capacity Moderate High Low

store is built up. During exercise, the phosphorylcreatine is hydrolyzed at the junction b etween the myosin heads and actin, forming ATP

from ADP and thus permitting contraction to continue.

CARBOHYDRATE & LIPID BREAKDOWN

At rest and during light exercise, muscles utilize lipids in the form of free fatty acids as th eir energy source. As the intensity of exercise

increases, lipids alone cannot supply energy fast enough and so use of carbohydrate b ecomes the predominant component in the muscle

fuel mixture. Thus, during exercise, much of the energy for phosphorylcreatine and A TP resynthesis comes from the breakdown of

glucose to CO

and H

O. Glucose in the bloodstream enters cells, where it is degraded through a series of che mical reactions to

pyruvate. Another source of intracellular glucose, and consequently of pyruvate, is gly cogen, the carbohydrate polymer that is especially

abundant in liver and skeletal muscle. When adequate O

is present, pyruvate enters the citric acid cycle and is metabolized—through

this cycle and the so-called respiratory enzyme pathway—to CO

and H

O. This process is called aerobic glycolysis. The metabolism of

glucose or glycogen to CO

and H

O forms large quantities of ATP from ADP. If O

supplies are insufficient, the pyruvate formed from

glucose does not enter the tricarboxylic acid cycle but is reduced to lactate. This process of anaerobic glycolysis is associated with the

net production of much smaller quantities of energy-rich phosphate bonds, but it does n ot require the presence of O

. A brief overview of

the various reactions involved in supplying energy to skeletal muscle is shown in Figure 5–13.

ATP + H

O ADP + H

+ 7.3 kcal H

N Rest HN PO

Phosphorylcreatine + ADP Creatine + ATP H

+ —

C + ATP H

+—

C +

ADP Glucose + 2 ATP (or glycogen + 1 ATP) CH

NCH

COO

−

Exercise CH

NCH

COO

−

Creatine Phosphorylcreatine

Anaerobic

2 Lactic acid + 4 ATP Glucose + 2 ATP (or glycogen + 1 ATP)

HN C

—

O Oxygen 6 CO

+ 6 H

O + 40 ATP

—

N CH

Creatinine

FIGURE 5–12 Creatine, phosphorylcreatine, and creatinine cycling in muscle. During periods of high activity, cycling of

phosphorylcreatine allows for quick release of ATP to sustain muscle activity.

FIGURE 5–13

by hydrolysis of 1 mol of ATP and reactions responsible for resynthesis of ATP . The amount of ATP formed per mole of free fatty acid

(FFA) oxidized is large but varies with the size of the FFA. For example, complete oxid ation of 1 mol of palmitic acid generates 140 mol

of ATP. FFA Oxygen CO

+ H

O + ATP

ATP turnover in muscle cells. Energy released

THE OXYGEN DEBT MECHANISM

During exercise, the muscle blood vessels dilate and blood flow is increased so that the a vailable O

supply is increased. Up to a point,

the increase in O

consumption is proportional to the energy expended, and all the energy needs are met by aerobic processes. However,

when muscular exertion is very great, aerobic resynthesis of energy stores cannot keep pace with their utilization. Under these

conditions, phosphorylcreatine is still used to resynthesize ATP. In addition, some ATP s ynthesis is accomplished by using the energy

released by the anaerobic breakdown of glucose to lactate. Use of the anaerobic pathw ay is self-limiting because in spite of rapid

diffusion of lactate into the bloodstream, enough accumulates in the muscles to eventuall y exceed the capacity of the tissue buffers and

produce an enzyme-inhibiting decline in pH. However, for short periods, the presence of an anaerobic pathway for glucose breakdown

permits muscular exertion of a far greater magnitude than would be possible without it. For example, in a 100-m dash that takes 10 s,

85% of the energy consumed is derived anaerobically; in a 2-mi race that takes 10 min , 20% of the energy is derived anaerobically; and

in a longdistance race that takes 60 min, only 5% of the energy comes from anaerobic metabolism.

After a period of exertion is over, extra O

is consumed to remove the excess lactate, replenish the ATP and phosphorylcreatine sto res,

and replace the small amounts of O

that were released by myoglobin. The amount of extra O

consumed is proportional to the extent to

which the energy demands during exertion exceeded the capacity for the aerobic synth esis of energy stores, ie, the extent to which an

oxygen debt was incurred. The O

debt is measured experimentally by determining O

consumption after exercise until a constant, basal

consumption is reached and subtracting the basal consumption from the total. The amou nt of this debt may be six times the basal O

consumption, which indicates that the subject is capable of six times the exertion that wou ld have been possible without it.

RIGOR

When muscle fibers are completely depleted of ATP and phosphorylcreatine, they dev elop a state of rigidity called rigor. When this

occurs after death, the condition is called rigor mortis. In rigor, almost all of the myosin heads attach to actin but in an abnormal, fixed,

and resistant way.

storage in phosphate bonds is a small factor. Consequently, heat production is considera ble. The heat produced in muscle can be

measured accurately with suitable thermocouples.

Resting heat, the heat given off at rest, is the external manifestation of basal metabo lic processes. The heat produced in excess of resting

heat during contraction is called the initial heat. This is made up of activation heat, the heat that muscle produces whenever it is

contracting, and shortening heat, which is proportionate in amount to the distance the musc le shortens. Shortening heat is apparently

due to some change in the structure of the muscle during shortening.

Following contraction, heat production in excess of resting heat continues for as long as 30 min. This recovery heat is the heat liberated

by the metabolic processes that restore the muscle to its precontraction state. The recover y heat of muscle is approximately equal to the

initial heat; that is, the heat produced during recovery is equal to the heat produced duri ng contraction.

If a muscle that has contracted isotonically is restored to its previous length, extra heat in addition to recovery heat is produced

(relaxation heat). External work must be done on the muscle to return it to its previous length, and relaxation heat is mainly a

manifestation of this work.

PROPERTIES OF SKELETAL

MUSCLES IN THE

INTACT ORGANISM

EFFECTS OF DENERVATION

In the intact animal or human, healthy skeletal muscle does not contract except in respon se to stimulation of its motor nerve supply.

Destruction of this nerve supply causes muscle atrophy. It also leads to abnormal excita bility of the muscle and increases its sensitivity to

circulating acetylcholine (denervation hypersensitivity; see Chapter 6). Fine, irregular co ntractions of individual fibers (fibrillations)

appear. This is the classic picture of a lower motor neuron lesion. If the mo tor nerve regenerates, the fibrillations disappear. Usually,

the contractions are not visible grossly, and they should not be confused with fasciculations, which are jerky, visible contractions of

groups of muscle fibers that occur as a result of pathologic discharge of spinal motor n eurons.

HEAT PRODUCTION IN MUSCLE

Thermodynamically, the energy supplied to a muscle must equal its energy output. The energy output appears in work done by the

muscle, in energy-rich phosphate bonds formed for later use, and in heat. The overall mechanical efficiency of skeletal muscle (work

done/total energy expenditure) ranges up to 50% while lifting a weight during isotonic contraction and is essentially 0% during isometric

contraction. Energy

THE MOTOR UNIT

Because the axons of the spinal motor neurons supplying skeletal muscle each branch t o innervate several muscle fibers, the smallest

possible amount of muscle that can contract in response to the excitation of a single moto r neuron is not one muscle fiber but all the fibers

supplied by the neuron. Each single motor neuron and the muscle fibers it innervates co nstitute a motor unit. The number of muscle

fibers in a motor unit varies. In muscles such as those of the hand and those concerned with motion of the eye (ie, muscles concerned

with fine, graded, precise movement), each motor unit innervates very few (on the ord er of three to six) muscle fibers. On the other hand,

values of 600 muscle fibers per motor unit can occur in human leg muscles. The group of muscle fibers that contribute to a motor unit

can be intermixed within a muscle. That is, although they contract as a unit, they are not necessarily “neighboring” fibers within the

muscle.

Each spinal motor neuron innervates only one kind of muscle fiber, so that all the musc le fibers in a motor unit are of the same type. On

the basis of the type of muscle fiber they innervate, and thus on the basis of the duration of their twitch contraction, motor units are

divided into S (slow), FR (fast, resistant to fatigue), and FF (fast, fatigable) units. Interes tingly, there is also a gradation of innervation of

these fibers, with S fibers tending to have a low innervation ratio (ie, small units) and FF fibers tending to have a high innervation ratio

(ie, large units). The recruitment of motor units during muscle contraction is not random , rather it follows a general scheme, the size

principle. In general, a specific muscle action is developed first by the recruitment of S muscle units that contract relatively slowly to

produce controlled contraction. Next, FR muscle units are recruited, resulting in more p owerful response over a shorter period of time.

Lastly, FF muscle units are recruited for the most demanding tasks. For example, in mus cles of the leg, the small, slow units are first

recruited for standing. As walking motion is initiated, their recruitment of FR units increa ses. As this motion turns to running or jumping,

the FF units are recruited. Of course, there is overlap in recruitment, but, in general, this principle holds true.

The differences between types of muscle units are not inherent but are determined by, among other things, their activity. When the nerve

to a slow muscle is cut and the nerve to a fast muscle is spliced to the cut end, the fast ne rve grows and innervates the previously slow

muscle. However, the muscle becomes fast and corresponding changes take place in its muscle protein isoforms and myosin ATPase

activity. This change is due to changes in the pattern of activity of the muscle; in stimulatio n experiments, changes in the expression of

MHC genes and consequently of MHC isoforms can be produced by changes in the p attern of electrical activity used to stimulate the

muscle. More commonly, muscle fibers can be altered by a change in activity initiated th rough exercise (or lack thereof). Increased

activity can lead to muscle cell hypertrophy, which allows for increase in contractile stre ngth. Type IIA and IIB fibers are most

susceptible to these changes. Alternatively, inactivity can lead to muscle cell atrophy and a loss of contractile strength. Type I fibers—

that is, the ones used most often—are most susceptible to these changes.

ELECTROMYOGRAPHY

Activation of motor units can be studied by electromyography, the process of recordin g the electrical activity of muscle on an

Biceps

500 μV

Triceps

0.5 s

FIGURE 5–14 Electromyographic tracings from human biceps and triceps muscles during alternate flexion and extension of the

elbow. Note the alternate activation and rest patterns as one muscle is used for flexio n and the other for extension. Electrical activity of

stimulated muscle can be recorded extracellularly, yielding typical excitation responses after stim ulation.

(Courtesy of Garoutte BC.)

oscilloscope. This may be done in unanaesthetized humans by using small metal disks on the skin overlying the muscle as the pick-up

electrodes or by using hypodermic needle electrodes. The record obtained with such el ectrodes is the electromyogram (EMG). With

needle electrodes, it is usually possible to pick up the activity of single muscle fibers. The measured EMG depicts the potential

difference between the two electrodes, which is altered by the activation of muscles in b etween the electrodes. A typical EMG is shown

in Figure 5–14.

It has been shown by electromyography that little if any spontaneous activity occurs in t he skeletal muscles of normal individuals at rest.

With minimal voluntary activity a few motor units discharge, and with increasing volunta ry effort, more and more are brought into play

to monitor the recruitment of motor units. Gradation of muscle res ponse is therefore in part a function of the number of motor units

activated. In addition, the frequency of discharge in the individual nerve fibers plays a role, the tension developed during a tetanic

contraction being greater than that during individual twitches. The length of the muscle i s also a factor. Finally, the motor units fire

asynchronously, that is, out of phase with one another. This asynchronous firing cause s the individual muscle fiber responses to merge

into a smooth contraction of the whole muscle. In summary, EMGs can be used to quic kly (and roughly) monitor abnormal electrical

activity associated with muscle responses.

THE STRENGTH OF SKELETAL MUSCLES

Human skeletal muscle can exert 3 to 4 kg of tension per square centimeter of cross-se ctional area. This figure is about the same as that

obtained in a variety of experimental animals and seems to be constant for mammalian sp ecies. Because many of the muscles in humans

have a relatively large cross-sectional area, the tension they can develop is quite large. T he gastrocnemius, for example, not only

supports the weight of the whole body during climbing but resists a force several times this great when the foot hits the ground during

running or jumping. An even more striking example is the gluteus maximus, which can exert a tension of 1200 kg. The total tension that

could be developed if all muscles in the body of an adult man pulled together is approx imately 22,000 kg (nearly 25 tons).

BODY MECHANICS

Body movements are generally organized in such a way that they take maximal advanta ge of the physiologic principles outlined above.

For example, the attachments of the muscles in the body are such that many of them are normally at or near their resting length when

they start to contract. In muscles that extend over more than one joint, movement at one joint may compensate for movement at another

in such a way that relatively little shortening of the muscle occurs during contraction. Ne arly isometric contractions of this type permit

development of maximal tension per contraction. The hamstring muscles extend from th e pelvis over the hip joint and the knee joint to

the tibia and fibula. Hamstring contraction produces flexion of the leg on the thigh. If th e thigh is flexed on the pelvis at the same time,

the lengthening of the hamstrings across the hip joint tends to compensate for the shorte ning across the knee joint. In the course of

various activities, the body moves in a way that takes advantage of this. Such factors as momentum and balance are integrated into body

movement in ways that make possible maximal motion with minimal muscular exertion. O ne net effect is that the stress put on tendons

and bones is rarely over 50% of their failure strength, protecting them from damage.

In walking, each limb passes rhythmically through a support or stance phase when the foot is on the ground and a swing phase when the

foot is off the ground. The support phases of the two legs overlap, so that two periods of double support occur during each cycle. There

is a brief burst of activity in the leg flexors at the start of each step, and then the leg is sw ung forward with little more active muscular

contraction. Therefore, the muscles are active for only a fraction of each step, and wal king for long periods causes relatively little

fatigue.

A young adult walking at a comfortable pace moves at a velocity of about 80 m/min an d generates a power output of 150 to 175 W per

step. A group of young adults asked to walk at their most comfortable rate selected a ve locity close to 80 m/min, and it was found that

they had selected the velocity at which their energy output was minimal. Walking more r apidly or more slowly took more energy.

CARDIAC MUSCLE MORPHOLOGY

The striations in cardiac muscle are similar to those in skeletal muscle, and Z lines are pre sent. Large numbers of elongated mitochondria

are in close contact with the muscle fibrils. The muscle fibers branch and interdigitate, bu t each is a complete unit surrounded by a cell

membrane. Where the end of one muscle fiber abuts on another, the membranes of bo th fibers parallel each other through an extensive

series of folds. These areas, which always occur at Z lines, are called intercalated d isks (Figure 5–15). They provide a strong union

between fibers, maintaining cell-to-cell cohesion, so that the pull of one contractile cell ca n be transmitted along its axis to the next.

Along the sides of the muscle fibers next to the disks, the cell membranes of adjacent fib ers fuse for considerable distances, forming gap

junctions. These junctions provide low-resistance bridges for the spread of excitation fr om one fiber to another. They permit cardiac

muscle to function as if it were a syncytium, even though no protoplasmic bridges are p resent between cells. The T system in cardiac

muscle is located at the Z lines rather than at the A–I junction, where it is located in mamm alian skeletal muscle.

ELECTRICAL PROPERTIES

RESTING MEMBRANE

& ACTION POTENTIALS

The resting membrane potential of individual mammalian cardiac muscle cells is about –8 0 mV. Stimulation produces a propagated

action potential that is responsible for initiating contraction. Although action potentials var y among the cardiomyocytes in different

regions of the heart (discussed in later chapters), the action potential of a typical ventricu lar cardiomyocyte can be used as an example

(Figure 5–16). Depolarization proceeds rapidly and an overshoot of the zero potential is present, as in skeletal muscle and nerve, but this

is followed by a plateau before the membrane potential returns to the baseline. In mamm alian hearts, depolarization lasts about 2 ms, but

the plateau phase and repolarization last 200 ms or more. Repolarization is therefore no t complete until the contraction is half over.

As in other excitable tissues, changes in the external K

concentration affect the resting membrane potential of cardiac muscle, whereas

changes in the external Na

concentration affect the magnitude of the action potential. The initial rapid depolarization and the overshoot

(phase 0) are due to opening of voltage-gated Na

channels similar to that occurring in nerve and skeletal muscle (Figure 5–17). The

initial rapid repolarization (phase 1) is due to closure of Na

channels and opening of one type of K

channel. The subsequent prolonged

plateau (phase 2) is due to a slower but prolonged opening of voltage-gated Ca

channels. Final repolarization (phase 3) to the resting

membrane potential (phase 4) is due to closure of the Ca

channels and a slow, delayed increase of K

efflux through various types of

channels. Cardiac myocytes contain at least two types of Ca

channels (T- and Ltypes), but the Ca

current is due mostly to

opening of the slower L-type Ca

channels.

Intercalated disk

Nucleus 10 μm FIBER

2μm

Capillary N

Fibrils Sarcolemma

Sarcoplasmic reticulum

T system

Terminal cistern N

FIBRIL

Intercalated disk

SARCOMERE Mitochondria

Electron photomicrograph of cardiac muscle. Note the similarity of the A-I regions seen in the skeletal FIGURE 5–15 Cardiac muscle.

muscle EM of Figure 3-2. The fuzzy thick lines are intercalated disks and function simil arly to the Z-lines but occur at cell membranes (

12,000). (Reproduced with permission from Bloom W, Fawcett DW: A Textbook of Histology, 10th ed. Saunde rs, 1975.) B) Artist interpretation of cardiac

muscle as seen under the light microscope (top) and the electron microscope (bo ttom). Again, note the similarity to skeletal muscle

structure. N, nucleus.

(Reproduced

with permission from Braunwald E, Ross J, Sonnenblick EH: Mechanisms of contraction of the normal and failing heart. N Engl

J Med 1967;277:794. Courtesy of Little, Brown.)

MECHANICAL PROPERTIES

CONTRACTILE RESPONSE

The contractile response of cardiac muscle begins just after the start of depolarization an d lasts about 1.5 times as long as the action

potential (Figure 5–16). The role of Ca

in excitation– contraction coupling is similar to its role in skeletal muscle (see above).

However, it is the influx of extracellular Ca

through the voltage-sensitive DHPR in the T system that triggers calcium-induced calciu m

release through the RyR at the sarcoplasmic reticulum. Because there is a net influx of C a

during activation, there is also a more

prominent role for plasma membrane Ca

ATPases and the Na

/Ca

exchanger in recovery of intracellular Ca

concentrations.

Specific effects

+20

Action potential

S recorded with

surface electrode 2 0

Action potential recorded intracellularly

0.5 g 4 −90

INa

ARP

RRP

Mechanical response

0 100 200 300 ms

FIGURE 5–16 Comparison of action potentials and contractile response of a mammalian cardiac muscle fiber in a typical

ventricular cell. In the top-most trace, the most commonly viewed surface action potential recor ding can be seen and it is broken down

into four regions: Q, R, S, and T. In the middle trace, the intracellular recording of the action potential shows the quick depolarization

and extended recovery. In the bottom trace, the mechanical response is matched to th e extracellular and intracellular electrical activities.

Note that in the absolute refractory period (ARP), the cardiac myocyte cannot be excited , whereas in the relative refractory period (RRP)

minimal excitation can occur.

ICa

0 200 Time (ms)

FIGURE 5–17 Dissection of the cardiac action potential. Top: The ac tion potential of a cardiac muscle fiber can be broken down into

several phases: 0, depolarization; 1, initial rapid repolarization; 2, plateau phase; 3, late ra pid repolarization; 4, baseline. Bottom:

Diagrammatic summary of Na

, Ca

, and cumulative K

currents during the action potential. As is convention, inward currents are

downward, and outward currents are upward.

of drugs that indirectly alter Ca

concentrations are discussed in Clinical Box 5–2.

During phases 0 to 2 and about half of phase 3 (until the membrane potential reaches a pproximately –50 mV during repolarization),

cardiac muscle cannot be excited again; that is, it is in its absolute refractory period. It remains re latively refractory until phase 4.

Therefore, tetanus of the type seen in skeletal muscle cannot occur. Of course, tetanizat ion of cardiac muscle for any length of time

would have lethal consequences, and in this sense, the fact that cardiac muscle cannot b e tetanized is a safety feature.

ISOFORMS

Cardiac muscle is generally slow and has relatively low ATPase activity. Its fibers are de pendent on oxidative metabolism and hence on

a continuous supply of O

. The human heart contains both the α and the β isoforms of the myosin heavy chain (α MHC and β MHC). β

MHC has lower myosin ATPase activity than α MHC. Both are present in the atria, with the α isoform predominating, whereas the β

isoform predominates in the ventricle. The spatial differences in expression contribute to the well-coordinated contraction of the heart.

CLINICAL BOX 5–2 Glycolysidic Drugs & Cardiac Contractions

Oubain and other digitalis glycosides are commonly used to treat failing hearts. These dr ugs have the effect of increasing the strength of

cardiac contractions. Although there is discussion as to full mechanisms, a working hyp othesis is based on the ability of these drugs to

inhibit the Na, K ATPase in cell membranes of the cardiomyocytes. The block of the Na , K ATPase in cardiomyocytes would result in

an increased intracellular Na

concentration. Such an increase would result in a decreased Na

influx and hence Ca

efflux via the Na

exchange antiport during the Ca

recovery period. The resulting intracellular Ca

concentration increase in turn increases the

strength of contraction of the cardiac muscle. With this mechanism in mind, these drugs can also be quite toxic. Overinhibition of the Na,

K ATPase would result in a depolarized cell that could slow conduction, or even sponta neously activate. Alternatively, overly increased

concentration could also have ill effects on cardiomyocyte physiology.

CORRELATION BETWEEN MUSCLE FIBER LENGTH & TENSION

The relation between initial fiber length and total tension in cardiac muscle is similar to tha t in skeletal muscle; there is a resting length at

which the tension developed on stimulation is maximal. In the body, the initial length of th e fibers is determined by the degree of

diastolic filling of the heart, and the pressure developed in the ventricle is proportionate to the volume of the ventricle at the end of the

filling phase (Starling’s law of the heart). The developed tension (Figure 5–18) increases as the diasto lic volume increases until it

reaches a maximum, then tends to decrease. However, unlike skeletal muscle, the decre ase in developed tension at high degrees of

stretch is not due to a decrease in the number of cross-bridges between actin and myos in, because even severely dilated hearts are not

stretched to this degree. The decrease is due instead to beginning disruption of the myoc ardial fibers.

The force of contraction of cardiac muscle can be also increased by catecholamines, an d this increase occurs without a change in muscle

length. This positive ionotropic effect of catecholamines is mediated via innervated β

-adrenergic receptors, cyclic AMP, and their

effects on Ca

homeostasis. The heart also contains noninnervated β

-adrenergic receptors, which also act via cyclic AMP, but their

ionotropic effect is smaller and is maximal in the atria. Cyclic AMP activates protein kina se A, and this leads to phosphorylation of the

voltage-dependent Ca

channels, causing them to spend more time in the open state. Cyclic AMP also increases the active transport of

to the sarcoplasmic reticulum, thus accelerating relaxation and consequently shortening systole. This is important when the cardiac

rate is increased because it permits adequate diastolic filling (see Chapter 31).

METABOLISM

Mammalian hearts have an abundant blood supply, numerous mitochondria, and a high content of myoglobin, a muscle pigment that can

function as an O

storage mechanism. Normally, less than 1% of the total energy liberated is provided by anaerobic metabolism. During

hypoxia, this figure may increase to nearly 10%; but under totally anaerobic conditions , the energy liberated is inadequate to sustain

ventricular contractions. Under basal conditions, 35% of the caloric needs of the huma n heart are provided by carbohydrate, 5% by

ketones and amino acids, and 60% by fat. However, the proportions of substrates utiliz ed vary greatly with the nutritional state. After

ingestion of large amounts of glucose, more lactate and pyruvate are used; during prolo nged starvation, more fat is used. Circulating free

fatty acids normally account for almost 50% of the lipid utilized. In untreated diabetics, t he carbohydrate utilization of cardiac muscle is

reduced and that of fat is increased.

SMOOTH MUSCLE MORPHOLOGY

Smooth muscle is distinguished anatomically from skeletal and cardiac muscle because it lacks visible cross-striations. Actin and

myosin-II are present, and they slide on each other to produce contraction. However, they are not arranged in regular arrays, as in

skeletal and cardiac muscle, and so the striations are absent. Instead of Z lines, there are dense bodies in the cytoplasm and attached to

the cell membrane, and these are bound by α-actinin to actin filaments. Smooth muscle a lso contains tropomyosin, but troponin appears

to be absent. The isoforms of actin and myosin differ from those in skeletal muscle. A s arcoplasmic reticulum is present, but it is less

extensive than those observed in skeletal or cardiac muscle. In general, smooth muscles contain few mitochondria and depend, to a large

extent, on glycolysis for their metabolic needs.

270

240

Systolic

intraventricular pressure

210

180

150

Developed tension 120

Diastolic

intraventricular pressure

0 10 20 30 40 50 60 70 Diastolic volume (mL)

FIGURE 5–18 Length–tension relationship for cardiac muscle. Comparison of the systolic intraventricular pressure (top trace) and

diastolic intraventricular pressure (bottom trace) display the developed tension in the car diomyocyte. Values shown are for canine heart.

TYPES

There is considerable variation in the structure and function of smooth muscle in differe nt parts of the body. In general, smooth muscle

can be divided into unitary (or visceral) smooth muscle and multiunit smooth muscle . Unitary smooth muscle occurs in large sheets,

has many low-resistance gap junctional connections between individual muscle cells, and functions in a syncytial fashion. Unitary

smooth muscle is found primarily in the walls of hollow viscera. The musculature of the intestine, the uterus, and the ureters are

examples. Multiunit smooth muscle is made up of individual units with few (or no) gap j unctional bridges. It is found in structures such

as the iris of the eye, in which fine, graded contractions occur. It is not under voluntary control, but it has many functional similarities to

skeletal muscle. Each multiunit smooth muscle cell has en passant endings of nerve fiber s, but in unitary smooth muscle there are en

passant junctions on fewer cells, with excitation spreading to other cells by gap junctions . In addition, these cells respond to hormones

and other circulating substances. Blood vessels have both unitary and multiunit smooth m uscle in their walls.

ELECTRICAL & MECHANICAL ACTIVITY

Unitary smooth muscle is characterized by the instability of its membrane potential and b y the fact that it shows continuous, irregular

contractions that are independent of its nerve supply. This maintained state of partial con traction is called tonus, or tone. The membrane

potential has no true “resting” value, being relatively low when the tissue is active and hi gher when it is inhibited, but in periods of

relative quiescence values for resting potential are on the order of –20 to –65 mV. Smo oth muscle cells can display divergent electrical

activity (eg, Figure 5–19). There are slow sine wave-like fluctuations a few millivolts in magnitude and spikes that sometimes overshoot

the zero potential line and sometimes do not. In many tissues, the spikes have a duration of about 50 ms, whereas in some tissues the

action potentials have a prolonged plateau during repolarization, like the action potentials in cardiac muscle. As in the other muscle

types, there are significant contributions of K

, Na

, and Ca

channels and Na, K ATPase to this electrical activity. However,

discussion of contributions to individual smooth muscle types is beyond the scope of this text.

Because of the continuous activity, it is difficult to study the relation between the electrica l and mechanical events in unitary smooth

muscle, but in some relatively inactive preparations, a single spike can be generated. In such preparations the excitation–contraction

coupling in unitary smooth muscle can occur with as much as a 500-ms delay. Thus, it i s a very slow process compared with that in

skeletal and cardiac muscle, in which the time from initial depolarization to initiation of co ntraction is less than 10 ms. Unlike unitary

smooth muscle, multiunit smooth muscle is nonsyncytial and contractions do not spread w idely through it. Because of this, the

contractions of multiunit smooth muscle are more discrete, fine, and localized than those of unitary smooth muscle.

50 mV

4 s

FIGURE 5–19 Electrical activity of individual smooth muscle cells in the gu inea pig taenia coli. Left: Pacemaker-like activity with

spikes firing at each peak. Right: Sinusoidal fluctuation of membra ne potential with firing on the rising phase of each wave. In other

fibers, spikes can occur on the falling phase of sinusoidal fluctuations and there can be mixtures o f sinusoidal and pacemaker potentials

in the same fiber.

MOLECULAR BASIS OF CONTRACTION

As in skeletal and cardiac muscle, Ca

plays a prominent role in the initiation of contraction of smooth muscle. However, the s ource of

increase can be much different in unitary smooth muscle. Depending on the activating stimulus, Ca

increase can be due to influx

through voltage- or ligand-gated plasma membrane channels, efflux from intracellular stores through the RyR, efflux from intracellular

stores through the inositol trisphosphate receptor (IP

R) Ca

channel, or via a combination of these channels. In addition, the lack of

troponin in smooth muscle prevents Ca

activation via troponin binding. Rather, myosin in smooth muscle must be phosphorylat ed for

activation of the myosin ATPase. Phosphorylation and dephosphorylation of myosin als o occur in skeletal muscle, but phosphorylation is

not necessary for activation of the ATPase. In smooth muscle, Ca

binds to calmodulin, and the resulting complex activates

calmodulin-dependent myosin light chain kinase. This enzyme catalyzes the ph osphorylation of the myosin light chain on serine at

position 19. The phosphorylation increases the ATPase activity.

Myosin is dephosphorylated by myosin light chain phosphatase in the cell. However, dephosphorylation of myosin light chain kinase

does not necessarily lead to relaxation of the smooth muscle. Various mechanisms are in volved. One appears to be a latch bridge

mechanism by which myosin cross-bridges remain attached to actin for some time after the cytoplasmic Ca

concentration falls. This

produces sustained contraction with little expenditure of energy, which is especially imp ortant in vascular smooth muscle. Relaxation of

the muscle presumably occurs when the Ca

-calmodulin complex finally dissociates or when some other mechanism comes into play .

The events leading to contraction and relaxation of unitary smooth muscle are summariz ed in Figure 5–20. The events in multiunit

smooth muscle are generally similar.

Unitary smooth muscle is unique in that, unlike other types of muscle, it contracts when stretched in the absence of any extrinsic

innervation. Stretch is followed by a decline in membrane potential, an increase in the fr equency of spikes, and a general increase in tone.

If epinephrine or norepinephrine is added to a preparation of intestinal smooth muscle arranged for recording of intracellular potentials in

vitro, the membrane potential usually becomes larger, the spikes decrease in frequency, and the muscle relaxes (Figure 5–21).

Norepinephrine is the chemical mediator released at noradrenergic nerve endings, and stimulation of the noradrenergic nerves to the

preparation produces inhibitory potentials. Acetylcholine has an effect opposite to that o f norepinephrine on the membrane potential and

contractile activity of intestinal smooth muscle. If acetylcholine is added to the fluid bathin g a smooth muscle preparation in vitro, the

membrane potential decreases and the spikes become more frequent. The muscle becom es more active, with an increase in tonic tension

and the number of rhythmic contractions. The effect is mediated by phospholipase

CLINICAL BOX 5–3

Binding of acetylcholine to muscarinic receptors

Increased influx of Ca

into the cell

Activation of calmodulin-dependent myosin light chain kinase

Phosphorylation of myosin

Increased myosin ATPase activity and binding of myosin to actin

Contraction

Dephosphorylation of myosin by myosin light chain phosphatase

Relaxation, or sustained contraction due to the latch bridge and other mechanisms

FIGURE 5–20 Sequence of events in contraction and relaxation of smooth muscl e. Flow chart illustrates many of the molecular

changes that occur from the initiation of contraction to its relaxation. Note the distinct dif ferences from skeletal and cardiac muscle

excitation.

Common Drugs That Act on Smooth Muscle

Overexcitation of smooth muscle in the airways, such as that observed during an asthm a attack, can lead to bronchoconstriction. Inhalers

that deliver drugs to the conducting airway are commonly used to offset this smooth mu scle bronchoconstriction, as well as other

symptoms in the asthmatic airways. The rapid effects of drugs in inhalers are related to smooth muscle relaxation. Rapid response inhaler

drugs (eg, ventolin, albuterol, sambuterol) frequently target β-adrenergic receptors in t he airway smooth muscle to elicit a relaxation.

Although these β-adrenergic receptor agonists targeting the smooth muscle do not treat all symptoms associated with bronchial

constriction (eg, inflammation and increased mucus), they are quick and frequently allo w for sufficient opening of the conducting airway

to restore airflow, and thus allow for other treatments to reduce airway obstruction.

Smooth muscle is also a target for drugs developed to increase blood flow. As discussed in the text, NO is a natural signaling molecule

that relaxes smooth muscle by raising cGMP. This signaling pathway is naturally down-r egulated by the action of phosphodiesterase

(PDE), which transforms cGMP into a nonsignaling form, GMP. The drugs sild enafil, tadalafil, and vardenafil are all specific inhibitors

of PDE V, an isoform found mainly in the smooth muscle in the corpus cavernosum of the penis (see Chapter 25). Thus, oral

administration of these drugs can block the action of PDE V, increasing blood flow in a very limited region in the body and offsetting

erectile dysfunction.

C, which produces IP

and allows for Ca

release through IP

receptors. In the intact animal, stimulation of cholinergic nerves causes

release of acetylcholine, excitatory potentials, and increased intestinal contractions.

0 Acetylcholine, parasympathetic stimulation, cold, stretch

Like unitary smooth muscle, multiunit smooth muscle is very sensitive to circulating chem ical substances and is normally activated by

chemical mediators (acetylcholine and norepinephrine) released at the endings of its mo tor nerves. Norepinephrine in particular tends to

persist in the muscle and to cause repeated firing of the muscle after a single stimulus rat her than a single action potential. Therefore, the

contractile response produced is usually an irregular tetanus rather than a single twitch. When a single twitch response is obtained, it

resembles the twitch contraction of skeletal muscle except that its duration is 10 times as lo ng.

−50

Membrane potential

Epinephrine, sympathetic stimulation

FIGURE 5–21 Effects of various agents on the membrane potential of intestinal smoot h muscle. Drugs and hormones can alter

firing of smooth muscle action potentials by raising (top trace) or lowering (bottom trace) resting membrane potential.

RELAXATION

In addition to cellular mechanisms that increase contraction of smooth muscle, there are cellular mechanisms that lead to its relaxation

(Clinical Box 5–3). This is especially important in smooth muscle that surrounds the bloo d vessels to increase blood flow. It was long

known that endothelial cells that line the inside of blood cells could release a substance th at relaxed smooth muscle (endothelial derived

relaxation factor,

EDRF). EDRF was later identified as the gaseous second messenger molecule, nitric oxide (NO). NO produced in endothelial cells is

free to diffuse into the smooth muscle for its effects. Once in muscle, NO directly activa tes a soluble guanylate cyclase to produce

another second messenger molecule, cyclic guanosine monophosphate (cGMP). This molecule can activate cGMP-sp ecific protein

kinases that can affect ion channels, Ca

homeostasis, or phosphatases, or all of those mentioned, that lead to smooth muscle rela xation

(see Chapters 7 and 33).

The consequences of plasticity can be demonstrated in humans. For example, the tensio n exerted by the smooth muscle walls of the

bladder can be measured at different degrees of distention as fluid is infused into the bl adder via a catheter. Initially, tension increases

relatively little as volume is increased because of the plasticity of the bladder wall. Howev er, a point is eventually reached at which the

bladder contracts forcefully (see Chapter 38).

FUNCTION OF THE NERVE

SUPPLY TO SMOOTH MUSCLE

The effects of acetylcholine and norepinephrine on unitary smooth muscle serve to emp hasize two of its important properties: (1) its

spontaneous activity in the absence of nervous stimulation, and (2) its sensitivity to chem ical agents released from nerves locally or

brought to it in the circulation. In mammals, unitary muscle usually has a dual nerve sup ply from the two divisions of the autonomic

nervous system. The function of the nerve supply is not to initiate activity in the muscle b ut rather to modify it. Stimulation of one

division of the autonomic nervous system usually increases smooth muscle activity, wher eas stimulation of the other decreases it.

However, in some organs, noradrenergic stimulation increases and cholinergic stimulati on decreases smooth muscle activity; in others,

the reverse is true.

FORCE GENERATION &

PLASTICITY OF SMOOTH MUSCLE

Smooth muscle displays a unique economy when compared to skeletal muscle. Despite a pproximately 20% of the myosin content and a

100-fold difference in ATP use when compared with skeletal muscle, they can generat e similar force per crosssectional area. One of the

tradeoffs of obtaining force under these conditions is the noticeably slower contractions when compared to skeletal muscle. There are

several known reasons for these noticeable changes, including unique isoforms of myo sin and contractile-related proteins expressed in

smooth muscle and their distinct regulation (discussed above). The unique architecture o f the smooth cell and its coordinated units also

likely contribute to these changes.

Another special characteristic of smooth muscle is the variability of the tension it exerts a t any given length. If a unitary smooth muscle

is stretched, it first exerts increased tension. However, if the muscle is held at the greater length after stretching, the tension gradually

decreases. Sometimes the tension falls to or below the level exerted before the muscle w as stretched. It is consequently impossible to

correlate length and developed tension accurately, and no resting length can be assigne d. In some ways, therefore, smooth muscle

behaves more like a viscous mass than a rigidly structured tissue, and it is this property th at is referred to as the plasticity of smooth

muscle.

CHAPTER SUMMARY

■ There are three main types of muscle cells: skeletal, cardiac, and smooth.

■ Skeletal muscle is a true syncytium under voluntary control. Skeletal muscles receiv e electrical stimuli from neurons to elicit

contraction: “excitation–contraction coupling.” Action potentials in muscle cells are devel oped largely through coordination of Na

, K

and Ca

channels. Contraction in skeletal muscle cells is coordinated through Ca

regulation of the actomyosin system that gives the

muscle its classic striated pattern under the microscope.

■ There are several different types of skeletal muscle fibers (I, IIA, IIB) t hat have distinct properties in terms of protein makeup and force

generation. Skeletal muscle fibers are arranged into motor units of like fibers within a m uscle. Skeletal motor units are recruited in a

specific pattern as the need for more force is increased.

■ Cardiac muscle is a collection of individual cells (cardiomyocytes) that are linked as a syncytium by gap junctional communication.

Cardiac muscle cells also undergo excitation– contraction coupling. Pacemaker cells in th e heart can initiate propagated action potentials.

Cardiac muscle cells also have a striated, actomyosin system that underlies contraction.

■ Smooth muscle cells are largely under control of the autonomic nervous system.

■ There are two broad categories of smooth muscle cells: unitary and multiunit. Unitary smooth muscle contraction is synchronized by

gap junctional communication to coordinate contraction among many cells. Multiunit smo oth muscle contraction is coordinated by motor

units, functionally similar to skeletal muscle.

■ Smooth muscle cells contract through an actomyosin system, but do not have w ell-organized striations. Unlike skeletal and cardiac

muscle, Ca

regulation of contraction is primarily through phosphorylation–dephosphorylation reactions .

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed . 1. The action potential of skeletal muscle

A) has a prolonged plateau phase.

B) spreads inward to all parts of the muscle via the T tubules.

C) causes the immediate uptake of Ca

into the lateral sacs of the sarcoplasmic reticulum.

D) is longer than the action potential of cardiac muscle.

E) is not essential for contraction.

2. The functions of tropomyosin in skeletal muscle include A) sliding on actin to produc e shortening.

B) releasing Ca

after initiation of contraction.

C) binding to myosin during contraction.

D) acting as a “relaxing protein” at rest by covering up the sites where myosin bin ds to actin.

E) generating ATP, which it passes to the contractile mechanism.

3. The cross-bridges of the sarcomere in skeletal muscle are made

up of

A) actin.

B) myosin.

C) troponin.

D) tropomyosin.

E) myelin.

4. The contractile response in skeletal muscle

A) starts after the action potential is over.

B) does not last as long as the action potential.

C) produces more tension when the muscle contracts isometrically than when the muscle contracts isotonically.

D) produces more work when the muscle contracts isometrically than when the muscle contracts isotonically.

E) decreases in magnitude with repeated stimulation. 5. Gap junctions

A) are absent in cardiac muscle.

B) are present but of little functional importance in cardiac muscle.

C) are present and provide the pathway for rapid spread of excitation from one cardia c muscle fiber to another.

D) are absent in smooth muscle.

E) connect the sarcotubular system to individual skeletal muscle cells.

CHAPTER RESOURCES

Alberts B, et al: Molecular Biology of the Cell, 5th ed. Garland Science, 2007.

Fung YC: Biomechanics, 2nd ed. Springer, 1993.

Hille B: Ionic Channels of Excitable Membranes, 3rd ed. Sinaver Associates, 2001.

Horowitz A: Mechanisms of smooth muscle contraction. Physiol Rev 1996;76: 967.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural Science, 4th ed. Mc Graw-Hill, 2000.

Sperelakis N (editor): Cell Physiology Sourcebook, 3rd ed. Academic Press, 2001 .

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Synaptic & Junctional Transmission

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Describe the main morphologic features of synapses.

■

Distinguish between chemical and electrical transmission at synapses.

■

Define convergence and divergence in neural networks, and

discuss their implications.

■

Describe fast and slow excitatory and inhibitory postsynaptic potentials, outline the ionic fluxes that underlie them, and explain how

the potentials interact to generate action potentials.

■

Define and give examples of direct inhibition, indirect inhibition, presynaptic inhibition, and postsynaptic inhibition.

■

Describe the neuromuscular junction, and explain how action potentials in the m otor neuron at the junction lead to contraction of the

skeletal muscle.

■

Define and explain denervation hypersensitivity.

INTRODUCTION

The all-or-none type of conduction seen in axons and skeletal muscle has been discusse d in Chapters 4 and 5. Impulses are transmitted

from one nerve cell to another cell at synapses (Figure 6–1). These are the junctions where the axon or some other portion of one cell

(the presynaptic cell) terminates on the dendrites, soma, or axon of another neuron (Figure 6–2) or, in some cases, a muscle or gland cell

(the postsynaptic cell). Cell-to-cell communication occurs across either a chemical or electrical synapse. At chemical synapses, a

synaptic cleft separates the terminal of the presynaptic cell from the postsynaptic cell. An im pulse in the presynaptic axon causes

secretion of a chemical that diffuses across the synaptic cleft and binds to receptors on t he surface of the postsynaptic cell. This triggers

events that open or close channels in the membrane of the postsynaptic cell. In electrical synapses, the membranes of the presynaptic and

postsynaptic neurons come close together, and gap junctions form between the cells (se e Chapter 2). Like the intercellular junctions in

other tissues, these junctions form low-resistance bridges through which ions can pass w ith relative ease. There are also a few conjoint

synapses in which transmission is both electrical and chemical. Regardless of the type of synapse, transmission is not a simple jumping

of an action potential from the presynaptic to the postsynaptic cell. The effects of discha rge at individual synaptic endings can be

excitatory or inhibitory, and when the postsynaptic cell is a neuron, the summation of al l the excitatory and inhibitory effects determines

whether an action potential is generated. Thus, synaptic transmission is a complex proce ss that permits the grading and adjustment of

neural activity necessary for normal function. Because most synaptic transmission is che mical, consideration in this chapter is limited to

chemical transmission unless otherwise specified.

Transmission from nerve to muscle resembles chemical synaptic transmission from one neuron to another. The neuromuscular

junction, the specialized area where a motor nerve terminates on a skeletal muscle fib er, is the site of a stereotyped transmission

process. The contacts between autonomic neurons and smooth and cardiac muscle are less specialized, and transmission in these

locations is a more diffuse process. These forms of transmission are also considered in this chapter.

115

3 4

FIGURE 6–1 Synapses on a typical motor neuron. The neuron has dendrites (1), an axon (2), and a prominent nucleus (3). Note that

rough endoplasmic reticulum extends into the dendrites but not into the axon. Many diff erent axons converge on the neuron, and their

terminal boutons form axodendritic (4) and axosomatic (5) synapses. (6) Myelin sheath. (Reproduced with permission from Krstic RV:

Ultrastructure of the Mammalian Cell. Springer, 1979.)

SYNAPTIC TRANSMISSION:

FUNCTIONAL ANATOMY

TYPES OF SYNAPSES

The anatomic structure of synapses varies considerably in the different parts of the mam malian nervous system. The ends of the

presynaptic fibers are generally enlarged to form terminal boutons (synaptic kn obs) (Figure 6–2). In the cerebral and cerebellar cortex,

endings are commonly located on dendrites and frequently on dendritic spines, which are small knobs projecting from dendrites (Figure

6–3). In some instances, the terminal branches of the axon of the presynaptic neuron f orm a basket or net around the soma of the

postsynaptic cell (basket cells of the cerebellum and autonomic ganglia). In other locatio ns, they intertwine with the dendrites of the

postsynaptic cell (climbing fibers of the cerebellum) or end on the dendrites directly (ap ical dendrites of cortical pyramidal cells). Some

end on axons of postsynaptic neurons (axoaxonal endings). On average, each neuron divides to form over 2000 synaptic endings, and

because the human central nervous system (CNS) has 10

neurons, it follows that there are about 2 × 10

synapses. Ob

FIGURE 6–2 Electron photomicrograph of synaptic knob (S) ending on the s haft of a dendrite (D) in the central nervous system.

P, postsynaptic density; M, mitochondrion. (

56,000). (Courtesy of DM McDonald.)

viously, therefore, communication between neurons is extremely complex. It should be noted as well that synapses are dynamic

structures, increasing and decreasing in complexity and number with use and experienc e.

It has been calculated that in the cerebral cortex, 98% of the synapses are on dendrites and only 2% are on cell bodies. In the spinal cord,

the proportion of endings on dendrites is less; there are about 8000 endings on the den drites of a typical spinal neuron and about 2000 on

the cell body, making the soma appear encrusted with endings.

PRESYNAPTIC & POSTSYNAPTIC STRUCTURE & FUNCTION

Each presynaptic terminal of a chemical synapse is separated from the postsynaptic struc ture by a synaptic cleft that is 20 to 40 nm wide.

Across the synaptic cleft are many neurotransmitter receptors in the postsynaptic membr ane, and usually a postsynaptic thickening called

the postsynaptic density (Figures 6–2 and 6–3). The postsynaptic density is an o rdered complex of specific receptors, binding proteins,

and enzymes induced by postsynaptic effects.

Inside the presynaptic terminal are many mitochondria, as well as many membrane-encl osed vesicles, which contain neurotransmitters.

There are three kinds of synaptic vesicles: small, clear synaptic vesicles that c ontain acetylcholine, glycine, GABA, or glutamate; small

vesicles with a dense core that contain catecholamines; and large vesicles with a dense co re that contain

Presynaptic cell

Microtubules

Mitochondria

Clear vesicles

Postsynaptic density

Active zone

Dendritic spine

Axodendritic Postsynaptic cell

Dendrite

Axodendritic

Soma

Axosomatic

Axon

Axo-axonal

Axodendritic, axoaxonal, and axosomatic

FIGURE 6–3

synapses. Many presynaptic neurons terminate on dendritic spines, as sho wn at the top, but some also end directly on the shafts of

dendrites. Note the presence of clear and granulated synaptic vesicles in endings and clustering of clear vesicles at active zones.

neuropeptides. The vesicles and the proteins contained in their walls are synthesized in th e neuronal cell body and transported along the

axon to the endings by fast axoplasmic transport. The neuropeptides in the large dense- core vesicles must also be produced by the

protein-synthesizing machinery in the cell body. However, the small clear vesicles and t he small dense-core vesicles recycle in the nerve

ending. These vesicles fuse with the cell membrane and release transmitters through exo cytosis and are then recovered by endocytosis to

be refilled locally. In some instances, they enter endosomes and are budded off the end osome and refilled, starting the cycle over again.

The steps involved are shown in Figure 6–4. More commonly, however, the synaptic v esicle discharges its contents through a small hole

in the cell membrane, then the opening reseals rapidly and the main vesicle stays inside th e cell (kiss-and-run discharge). In this way, the

full endocytotic process is short-circuited.

The large dense-core vesicles are located throughout the presynaptic terminals that cont ain them and release their neuropeptide contents

by exocytosis from all parts of the terminal. On the other hand, the small vesicles are loc ated near the synaptic cleft and fuse to the

membrane, discharging their contents very rapidly into the cleft at areas of membrane th ickening called active zones (Figure 6–3). The

active zones contain many proteins and rows of calcium channels.

The Ca

that triggers exocytosis of transmitters enters the presynaptic neurons, and transmitter re lease starts within 200 μs. Therefore, it

is not surprising that the voltage-gated Ca

channels are very close to the release sites at the active zones. In addition, for the transm itter

to be effective on the postsynaptic neuron requires proximity of release to the postsyna ptic receptors. This orderly organization of the

synapse depends in part on neurexins, proteins bound to the membrane of the presyn aptic neuron that bind neurexin receptors in the

membrane of the postsynaptic neuron. In many vertebrates, neurexins are produced b y a single gene that codes for the α isoform.

However, in mice and humans they are encoded by three genes, and both α and β isof orms are produced. Each of the genes has two

regulatory regions and extensive alternative splicing of their mRNAs. In this way, over 1000 different neurexins are produced. This raises

the possibility that the neurexins not only hold synapses together, but also provide a mec hanism for the production of synaptic

specificity.

As noted in Chapter 2, vesicle budding, fusion, and discharge of contents with subsequ ent retrieval of vesicle membrane are fundamental

processes occurring in most, if not all, cells. Thus, neurotransmitter secretion at synapse s and the accompanying membrane retrieval are

specialized forms of the general processes of exocytosis and endocytosis. The details of the processes by which synaptic vesicles fuse

with the cell membrane are still being worked out. They involve the v-snare protein synaptobrevin in the v esicle membrane locking

with the t-snare protein syntaxin in the cell membrane; a multiprotein complex regulate d by small GTPases such as rab3 is also

involved in the process (Figure 6–5). The synapse begins in the presynaptic and not in the postsynaptic cell. The one-way gate at the

synapses is necessary for orderly neural function.

Clinical Box 6–1 describes the how neurotoxins can disrupt transmitter release in either the CNS or at the neuromuscular junction.

ELECTRICAL EVENTS IN

POSTSYNAPTIC NEURONS

EXCITATORY & INHIBITORY

POSTSYNAPTIC POTENTIALS

Penetration of an α-motor neuron is a good example of the techniques used to study po stsynaptic electrical activity. It is

NT Early endosome

NT uptake Budding Endosome fusion H+

Translocation Translocation

Docking

Plasma

membrane

ATP

Priming Fusion/

Endocytosis

exocytosis

4 Ca2+ Ca2+ ?

Synaptic cleft

Ca2+

FIGURE 6–4 Small synaptic vesicle cycle in presynaptic nerve terminals. Vesicles bud off the early endosome and then fill with

neurotransmitter (NT; top left). They then move to the plasma membrane, dock, and be come primed. Upon arrival of an action potential

at the ending, Ca

influx triggers fusion and exocytosis of the granule contents to the synaptic cleft. The v esicle wall is then coated with

clathrin and taken up by endocytosis. In the cytoplasm, it fuses with the early e ndosome, and the cycle is ready to repeat. (Reproduced with

permission from Sdhof TC: The synaptic vesicle cycle: A cascade of proteinprotein interactions. Nature 19 95;375:645. Copyright by Macmillan Magazines.)

achieved by advancing a microelectrode through the ventral portion of the spinal cord. Puncture of a cell membrane is signaled by the

appearance of a steady 70-mV potential difference between the microelectrode and an electrode outside the cell. The cell can be

identified as a spinal motor neuron by stimulating the appropriate ventral root and obser ving the electrical activity of the cell. Such

stimulation initiates an antidromic impulse (see Chapter 4) that is conducted to the soma

Neuron:

Synaptic vesicle Plasma membrane

NSF

α/γ SNAPs Synaptobrevin

Syntaxin

rab3

munc18/

SNAP

rbSec1

GTP

FIGURE 6–5 Main proteins that interact to produce synaptic vesicle docking and fusi on in nerve endings. (Reproduced with permission

from Ferro-Novick S, John R: Vesicle fusion from yeast to man. Nature 1994;370:191. Copyright by Macmillan Magazines.)

and stops at this point. Therefore, the presence of an action potential in the cell after anti dromic stimulation indicates that the cell that has

been penetrated is an α-motor neuron. Stimulation of a dorsal root afferent (sensory ne uron) can be used to study both excitatory and

inhibitory events in α-motor neurons (Figure 6–6).

When an impulse reaches the presynaptic terminals, an interval of at least 0.5 ms, the synaptic delay, occurs before a response is

obtained in the postsynaptic neuron. It is due to the time it takes for the synaptic mediator to be released and to act on the membrane of

the postsynaptic cell. Because of it, conduction along a chain of neurons is slower if ma ny synapses are in the chain than if there are

only a few. Because the minimum time for transmission across one synapse is 0.5 ms, it is also possible to determine whether a given

reflex pathway is monosynaptic or polysynaptic (contains more than one synapse) by m easuring the delay in transmission from the

dorsal to the ventral root across the spinal cord.

A single stimulus applied to the sensory nerves characteristically does not lead to the for mation of a propagated action potential in the

postsynaptic neuron. Instead, the stimulation produces either a transient partial depolariz ation or a transient hyperpolarization. The initial

depolarizing response produced by a single stimulus to the proper input begins about 0 .5 ms after the afferent impulse enters the spinal

cord. It reaches its peak 11.5 ms later and then declines exponentially. During this poten tial, the excitability of the neuron to other stimuli

is increased, and consequently the potential is called an excitatory postsynaptic pote ntial (EPSP) (Figure 6–6).

CLINICAL BOX 6–1 Botulinum and Tetanus Toxins

Several deadly toxins which block neurotransmitter release are zinc endopeptidases that cleave and hence inactivate proteins in the

fusion–exocytosis complex. Tetanus toxin and botulinum toxins B, D, F, and G act on synaptobrevin, and botulinum toxin C acts on

syntaxin. Botulinum toxins A and B act on SNAP-25. Clinically, tetanus toxin causes spa stic paralysis by blocking presynaptic

transmitter release in the CNS, and botulism causes flaccid paralysis by blocking the rele ase of acetylcholine at the neuromuscular

junction. On the positive side, however, local injection of small doses of botulinum toxin (botox) has proved effective in the treatment of

a wide variety of conditions characterized by muscle hyperactivity. Examples include in jection into the lower esophageal sphincter to

relieve achalasia and injection into facial muscles to remove wrinkles.

A Stretch reflex circuit for knee jerk

Quadriceps (extensor) Muscle spindle Sensory neuron

Spinal cord

Hamstring (flexor)

Extensor motor

neuron

Flexor motor neuron Inhibitory interneuron

B Experimental setup for recording from cells in the circuit

Current passing

Recording

Extracellular stimulating electrodes Sensory neuron

Extensor

motor neuron Recording

EPSP

Ia afferent fibers from muscle

spindles of

quadriceps

Motor neuron

Sensory neuron

EPSP

Recording Current passing Inhibitory interneurons Recording

Flexor

motor

neuron

Ia afferent fibers from muscle

spindles of

quadriceps

EPSP

IPSP

IPSP Motor neuron Interneuron

FIGURE 6–6 Excitatory and inhibitory synaptic connections mediating the str etch reflex provide an example of typical circuits

within the CNS. A) The stretch receptor sensory neuron of the quadriceps musc le makes an excitatory connection with the extensor

motor neuron of the same muscle and an inhibitory interneuron projecting to fl exor motor neurons supplying the antagonistic hamstring

muscle. B) Experimental setup to study excitation and inhibition of the extensor motor neuron. T op panel shows two approaches to elicit

an excitatory (depolarizing) postsynaptic potential or EPSP in the extensor motor neuron–electri cal stimulation of the whole Ia afferent

nerve using extracellular electrodes and intracellular current passing through an electro de inserted into the cell body of a sensory neuron.

Bottom panel shows that current passing through an inhibitory interneuron elicits an inh ibitory (hyperpolarizing) postsynaptic potential

or IPSP in the flexor motor neuron. (From Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill, 2000.)

The EPSP is produced by depolarization of the postsynaptic cell membrane immediately under the presynaptic ending. The excitatory

transmitter opens Na

or Ca

ion channels in the postsynaptic membrane, producing an inward current. The area of current flow thus

created is so small that it does not drain off enough positive charge to depolarize the wh ole membrane. Instead, an EPSP is inscribed.

The EPSP due to activity in one synaptic knob is small, but the depolarizations produced by each of the active knobs summate.

EPSPs are produced by stimulation of some inputs, but stimulation of other inputs produ ces hyperpolarizing responses. Like the EPSPs,

they peak 11.5 ms after the stimulus and decrease exponentially. During this potential, th e excitability of the neuron to other stimuli is

decreased; consequently, it is called an inhibitory postsynaptic potential

(IPSP) (Figure 6–6). –

An IPSP can be produced by a localized increase in Cl transport. When an inhibitory sy naptic knob becomes active,

the released transmitter triggers the opening of Cl

–

channels in the area of the postsynaptic cell membrane under the knob. Cl

–

moves

down its concentration gradient. The net effect is the transfer of negative charge into th e cell, so that the membrane potential increases.

The decreased excitability of the nerve cell during the IPSP is due to movement of the m embrane potential away from the firing level.

Consequently, more excitatory (depolarizing) activity is necessary to reach the firing lev el. The fact that an IPSP is mediated by Cl

–

can

be demonstrated by repeating the stimulus while varying the resting membrane potential of the postsynaptic cell. When the membrane

potential is at E

, the potential disappears (Figure 6–7), and at more negative membrane potentials, it bec omes positive (reversal

potential).

Because IPSPs are net hyperpolarizations, they can be produced by alterations in other ion channels in the neuron. For example, they can

be produced by opening of K

channels, with movement of K

out of the postsynaptic cell, or by closure of Na

or Ca

channels.

–40 mV

5 mV

–60 mV RMP

–70 mV E

–90 mV E

–100 mV 5 ms

FIGURE 6–7 IPSP is due to increased Cl influx during stimulation. This can be demonstrated by repeating the stimulus while varying

the resting membrane potential (RMP) of the postsynaptic cell. When the membrane pot ential is at E

, the potential disappears, and at

more negative membrane potentials, it becomes positive (reversal potential).

A Temporal summation Recording

B Spatial summation

Recording

Synaptic current A A Axon Axon

A AAB

2 × 10–10 A

Synaptic potential

TEMPORAL & SPATIAL SUMMATION

Summation may be temporal or spatial (Figure 6–8). Temporal summation occurs if repeated affe rent stimuli cause new EPSPs before

previous EPSPs have decayed. A longer time constant for the EPSP allows for a greater opportunity for summation. When activity is

present in more than one synaptic knob at the same time, spatial summation occurs and activity in one synaptic knob summates with

activity in another to approach the firing level. The EPSP is therefore not an allor-none response but is proportionate in size to the

strength of the afferent stimulus.

Spatial summation of IPSPs also occurs, as shown by the increasing size of the response , as the strength of an inhibitory afferent volley

is increased. Temporal summation of IPSPs also occurs.

Long time Long length constant constant (100 ms) V

(1 mm) V

2 mV

Short time Short length

constant constant

(20 ms) V

(0.33 mm) V

2 mV

25 ms FIGURE 6–8 Central neurons integrate a variety of synaptic inputs through te mporal and spatial summation. A) The time

constant of the postsynaptic neuron affects the amplitude of the depolarization caused b y consecutive EPSPs produced by a single

presynaptic neuron. B) The length constant of a postsynaptic cell affects the amplitude of two EPSPs produced by two presynaptic

neurons, A and B. (From Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science , 4th ed. McGraw-Hill, 2000.)

SLOW POSTSYNAPTIC POTENTIALS

In addition to the EPSPs and IPSPs described previously, slow EPSPs and IPSPs have be en described in autonomic ganglia, cardiac and

smooth muscle, and cortical neurons. These postsynaptic potentials have a latency of 10 0 to 500 ms and last several seconds. The slow

EPSPs are generally due to decreases in K

conductance, and the slow IPSPs are due to increases in K

conductance.

GENERATION OF THE ACTION

POTENTIAL IN THE

POSTSYNAPTIC NEURON

The constant interplay of excitatory and inhibitory activity on the postsynaptic neuron p roduces a fluctuating membrane potential that is

the algebraic sum of the hyperpolarizing and depolarizing activity. The soma of the neu ron thus acts as a sort of integrator. When the 10

to 15 mV of depolarization sufficient to reach the firing level is attained, a propagated s pike results. However, the discharge of the

neuron is slightly more complicated than this. In motor neurons, the portion of the cell w ith the lowest threshold for the production of a

fullfledged action potential is the initial segment, the portion of the axon at and just beyond th e axon hillock. This unmyelinated

segment is depolarized or hyperpolarized electrotonically by the current sinks and sour ces under the excitatory and inhibitory synaptic

knobs. It is the first part of the neuron to fire, and its discharge is propagated in two dir ections: down the axon and back into the soma.

Retrograde firing of the soma in this fashion probably has value in wiping the slate clea n for subsequent renewal of the interplay of

excitatory and inhibitory activity on the cell.

drites. There, each can become associated with a single ribosome in a dendritic spine an d produce proteins, which alters the effects of

input from individual synapses on the spine. Changes in dendritic spines have been imp licated in motivation, learning, and long-term

memory.

ELECTRICAL TRANSMISSION

At synaptic junctions where transmission is electrical, the impulse reaching the presynapt ic terminal generates an EPSP in the

postsynaptic cell that, because of the low-resistance bridge between the two, has a much shorter latency than the EPSP at a synapse

where transmission is chemical. In conjoint synapses, both a short-latency response and a longer-latency, chemically mediated

postsynaptic response take place.

INHIBITION & FACILITATION AT SYNAPSES

DIRECT & INDIRECT INHIBITION

Inhibition in the CNS can be postsynaptic or presynaptic. Postsynaptic inhibition during the course of an IPSP is called direct

inhibition because it is not a consequence of previous discharges of the postsyna ptic neuron. There are various forms of indirect

inhibition, which is inhibition due to the effects of previous postsynaptic neuron discharge. For example, the postsynaptic cell can be

refractory to excitation because it has just fired and is in its refractory period. During af terhyperpolarization it is also less excitable. In

spinal neurons, especially after repeated firing, this after-hyperpolarization may be larg e and prolonged.

FUNCTION OF THE DENDRITES

For many years, the standard view has been that dendrites are simply the sites of curren t sources or sinks that electrotonically change the

membrane potential at the initial segment; that is, they are merely extensions of the soma t hat expand the area available for integration.

When the dendritic tree of a neuron is extensive and has multiple presynaptic knobs en ding on it, there is room for a great interplay of

inhibitory and excitatory activity.

It is now well established that dendrites contribute to neural function in more complex w ays. Action potentials can be recorded in

dendrites. In many instances, these are initiated in the initial segment and conducted in a retrograde fashion, but propagated action

potentials are initiated in some dendrites. Further research has demonstrated the malleabil ity of dendritic spines. Dendritic spines appear,

change, and even disappear over a time scale of minutes and hours, not days and mon ths. Also, although protein synthesis occurs mainly

in the soma with its nucleus, strands of mRNA migrate into the den

POSTSYNAPTIC INHIBITION IN THE SPINAL CORD

Various pathways in the nervous system are known to mediate postsynaptic inhibition, a nd one illustrative example is presented here.

Afferent fibers from the muscle spindles (stretch receptors) in skeletal muscle project di rectly to the spinal motor neurons of the motor

units supplying the same muscle (Figure 6–6). Impulses in this afferent fiber cause EPSP s and, with summation, propagated responses in

the postsynaptic motor neurons. At the same time, IPSPs are produced in motor neuron s supplying the antagonistic muscles which have

an inhibitory interneuron interposed between the afferent fiber and the motor neuron. Therefore, activity in the afferent fibers from the

muscle spindles excites the motor neurons supplying the muscle from which the impulse s come, and inhibits those supplying its

antagonists (reciprocal innervation).

PRESYNAPTIC INHIBITION &

FACILITATION

Another type of inhibition occurring in the CNS is presynaptic inhibition, a process med iated by neurons whose terminals are on

excitatory endings, forming axoaxonal synapses (Figure 6–3). The neurons responsible for postsynaptic and presynaptic inhibition are

compared in Figure 6–9. Three mechanisms of presynaptic inhibition have been describ ed. First, activation of the presynaptic receptors

increases Cl

–

conductance, and this has been shown to decrease the size of the action potentials reach ing the excitatory ending (Figure 6–

10). This in turn reduces Ca

entry and consequently the amount of excitatory transmitter released. Voltage-gated K

channels are also

opened, and the resulting K

efflux also decreases the Ca

influx. Finally, there is evidence for direct inhibition of transmitter release

independent of Ca

influx into the excitatory ending.

The first transmitter shown to produce presynaptic inhibition was GABA. Acting via GA BA

receptors, GABA increases Cl

–

conductance. GABA

receptors are also present in the spinal cord and appear to mediate presynaptic inhibitio n via a G protein that

produces an increase in K

conductance. Baclofen, a GABA

agonist, is effective in the treatment of the spasticity of spinal cord injury

and multiple sclerosis, particularly when administered intrathecally via an implanted pump . Other transmitters also mediate presynaptic

inhibition by G protein-mediated effects on Ca

channels and K

channels.

Conversely, presynaptic facilitation is produced when the action potential is prolonged (Figure 6–10) and the Ca

channels are open

for a longer period. The molecular events responsible for the production of presynapt ic facilitation mediated by serotonin in the sea snail

Aplysia have been worked out in detail. Serotonin released at an axoaxonal ending incr eases intraneuronal cAMP levels, and the

resulting phosphorylation

Presynaptic inhibition Postsynaptic inhibition

Motor neuron

FIGURE 6–9 Arrangement of neurons producing presynaptic and postsynap tic inhibition. The neuron producing presynaptic

inhibition is shown ending on an excitatory synaptic knob. Many of these neurons ac tually end higher up along the axon of the excitatory

cell.

Presynaptic action

potential EPSP in postsynaptic neuron

current in presynaptic neuron

Presynaptic inhibition

Presynaptic facilitation

FIGURE 6–10 Effects of presynaptic inhibition and facilitation on the action potential a nd the Ca

current in the presynaptic

neuron and the EPSP in the postsynaptic neuron. In each case, the solid lin es are the controls and the dashed lines the records

obtained during inhibition or facilitation. (Modified from Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill,

2000.)

of one group of K

channels closes the channels, slowing repolarization and prolonging the action potential .

ORGANIZATION OF

INHIBITORY SYSTEMS

Presynaptic and postsynaptic inhibition are usually produced by stimulation of certain sy stems converging on a given postsynaptic

neuron (afferent inhibition). Neurons may also inhibit themselves in a negative feedbac k fashion (negative feedback inhibition). For

instance, each spinal motor neuron regularly gives off a recurrent collateral that synaps es with an inhibitory interneuron, which

terminates on the cell body of the spinal neuron and other spinal motor neurons (Figure 6–11). This particular inhibitory neuron is

sometimes called a Renshaw cell after its discoverer. Impulses generated in the motor ne uron activate the inhibitory interneuron to

secrete inhibitory mediators, and this slows or stops the discharge of the motor neuron. Similar inhibition via recurrent collaterals is seen

in the cerebral cortex and limbic system. Presynaptic inhibition due to descending pathw ays that terminate on afferent pathways in the

dorsal horn may be involved in the gating of pain transmission.

Another type of inhibition is seen in the cerebellum. In this part of the brain, stimulation of basket cells produces IPSPs in the Purkinje

cells. However, the basket cells and the Purkinje

A Motor neuron X

Motor neuron

Inhibitory interneuron C

Axon FIGURE 6–12 Simple nerve net. Neurons A, B, and C have excitatory endin gs on neurons X, Y, and Z.

FIGURE 6–11 Negative feedback inhibition of a spinal motor neuron v ia an inhibitory interneuron (Renshaw cell).

cells are excited by the same parallel-fiber excitatory input. This arrangement, which ha s been called feed-forward inhibition,

presumably limits the duration of the excitation produced by any given afferent volley.

SUMMATION & OCCLUSION

As noted above, the axons of most neurons discharge onto many other neurons. Conv ersely, any given neuron receives input from many

other neurons (convergence).

In the hypothetical nerve net shown in Figure 6–12, neurons A and B converge on X , and neuron B diverges on X and Y. A stimulus

applied to A or to B will set up an EPSP in X. If A and B are stimulated at the same time and action potentials are produced, two areas of

depolarization will be produced in X and their actions will sum. The resultant EPSP in X will be twice as large as that produced by

stimulation of A or B alone, and the membrane potential may well reach the firing level of X. The effect of the depolarization caused by

the impulse in A adds to that due to activity in B, and vice versa; spatial summation has ta ken place. In this case, Y has not fired, but its

excitability has been increased, and it is easier for activity in neuron C to fire Y during t he EPSP. Y is therefore said to be in the

subliminal fringe of X. More generally stated, neurons are in the subliminal fringe if they are not discharged by an afferent volley (not

in the discharge zone) but do have their excitability increased. The neurons that ha ve few active knobs ending on them are in the

subliminal fringe, and those with many are in the discharge zone. Inhibitory impulses sh ow similar temporal and spatial facilitation and

subliminal fringe effects.

If action potentials are produced repeatedly in neuron B, X and Y will discharge as a r esult of temporal summation of the EPSPs that are

produced. If C is stimulated repeatedly, Y and Z will discharge. If B and C are fired re peatedly at the same time, X, Y, and Z will

discharge. Thus, the response to stimulation of B and C together is not as great as the su m of responses to stimulation of B and C

separately, because B and C both end on neuron Y. This decrease in expected respons e, due to presynaptic fibers sharing postsynaptic

neurons, is called occlusion.

NEUROMUSCULAR

TRANSMISSION:

NEUROMUSCULAR JUNCTION

ANATOMY

As the axon supplying a skeletal muscle fiber approaches its termination, it loses its myeli n sheath and divides into a number of terminal

boutons, or endfeet (Figure 6–13). The endfeet contain many small, clear vesicles that c ontain acetylcholine, the transmitter at these

junctions. The endings fit into junctional folds, which are depressions in the mo tor end plate, the thickened portion of the muscle

membrane at the junction. The space between the nerve and the thickened muscle memb rane is comparable to the synaptic cleft at

synapses. The whole structure is known as the neuromuscular, or myoneural, junctio n. Only one nerve fiber ends on each end plate,

with no convergence of multiple inputs.

SEQUENCE OF EVENTS

DURING TRANSMISSION

The events occurring during transmission of impulses from the motor nerve to the musc le are somewhat similar to those occurring at

neuron-to-neuron synapses (Figure 6–14). The impulse arriving in the end of the moto r neuron increases the permeability of its endings

to Ca

. Ca

enters the endings and triggers a marked increase in exocytosis of the acetylcholine-co ntaining vesicles. The acetylcholine

diffuses to the

(a)

(b) Motor nerve fiber

Myelin

Axon terminal

Schwann cell

Synaptic vesicles (containing ACh) Active zone

Sarcolemma

Synaptic cleft

Junctional Nucleus of muscle fiber

Region of

folds

sarcolemma

with ACh receptors

FIGURE 6–13 The neuromuscular junction. (a) Scanning ele ctronmicrograph showing branching of motor axons with terminals

embedded in grooves in the muscle fiber’s surface. (b) Structure of a neuro muscular junction.

(From Widmaier EP, Raff H, Strang KT: Vanders

Human Physiology.

McGraw-Hill, 2008.)

muscle-type nicotinic acetylcholine receptors, which are concentrated at the tops of the j unctional folds of the membrane of the motor

end plate. Binding of acetylcholine to these receptors increases the Na

and K

conductance of the membrane, and the resultant influx of

produces a depolarizing potential, the end plate potential. The current sink created by this local potential depolarizes the adjacent

muscle membrane to its firing level. Acetylcholine is then removed from the synaptic cle ft by acetylcholinesterase, which is present in

high concentration at the neuromuscular junction. Action potentials are generated on eith er side of the end plate and are conducted away

from the end plate in both directions along the muscle fiber. The muscle action potential, in turn, initiates muscle contraction, as

described in Chapter 5.

END PLATE POTENTIAL

An average human end plate contains about 15 to 40 million acetylcholine receptors. Ea ch nerve impulse releases about 60

acetylcholine vesicles, and each vesicle contains about 10,000

molecules of the neurotransmitter. This amount is enough to activate about 10 times the n umber of acetylcholine receptors needed to

produce a full end plate potential. Therefore, a propagated response in the muscle is re gularly produced, and this large response obscures

the end plate potential. However, the end plate potential can be seen if the tenfold safety factor is overcome and the potential is reduced

to a size that is insufficient to fire the adjacent muscle membrane. This can be accomplish ed by administration of small doses of curare, a

drug that competes with acetylcholine for binding to muscle-type nicotinic acetylcholine receptors. The response is then recorded only at

the end plate region and decreases exponentially away from it. Under these conditions, end plate potentials can be shown to undergo

temporal summation.

QUANTAL RELEASE OF TRANSMITTER

Small quanta (packets) of acetylcholine are released randomly from the nerve cell mem brane at rest. Each produces a minute

depolarizing spike called a miniature end plate potential, which is about 0.5 mV in amplitude. The size of the quanta of acetylcholine

released in this way varies directly with the Ca

concentration and inversely with the Mg

concentration at the end plate. When a nerve

impulse reaches the ending, the number of quanta released increases by several orders of magnitude, and the result is the large end plate

potential that exceeds the firing level of the muscle fiber.

Quantal release of acetylcholine similar to that seen at the myoneural junction has been o bserved at other cholinergic synapses, and

quantal release of other transmitters probably occurs at noradrenergic, glutaminergic, a nd other synaptic junctions.

Two diseases of the neuromuscular junction, myasthenia gravis and Lambert-Eaton syn drome, are described in Clinical Box 6–2 and

Clinical Box 6–3, respectively.

Motor neuron action potential

enters voltage-gated channels

++ +

–– –

– –

–

Acetylcholine

vesicle

7 Propagated action potential in muscle plasma membrane

Voltage-gated Na

channels

Acetylcholine

+ +

+ + +

release

entry

–

–––

+ +

–

– –

– – –

–

8 Acetylcholine degradation Acetylcholine receptor

Muscle fiber action potential Acetylcholinesterase initiation

Motor end plate

Local current between

depolarized end plate and adjacent muscle plasma membrane

FIGURE 6–14 Events at the neuromuscular junction that lead to an action po tential in the muscle fiber plasma membrane.

Although potassium exits the muscle cell when Ach receptors are open, sodium entry an d depolarization dominate. (From Widmaier EP, Raff

H, Strang KT: Vanders Human Physiology. McGraw-Hill, 2008.)

NERVE ENDINGS IN SMOOTH & CARDIAC MUSCLE

ANATOMY

The postganglionic neurons in the various smooth muscles that have been studied in deta il branch extensively and come in close contact

with the muscle cells (Figure 6–15). Some of these nerve fibers contain clear vesicles an d are cholinergic, whereas others contain the

characteristic dense-core vesicles that contain norepinephrine. There are no recognizab le end plates or other postsynaptic specializations.

The nerve fibers run along the membranes of the muscle cells and sometimes groove th eir surfaces. The multiple branches of the

noradrenergic and, presumably, the cholinergic neurons are beaded with enlargements (varicosities) and contain synaptic vesicles

(Figure 6–15). In noradrenergic neurons, the varicosities are about 5 μm apart, with u p to 20,000 varicosities per neuron. Transmitter is

apparently liberated at each varicosity, that is, at many locations along each axon. This a rrangement permits one neuron to innervate

many effector cells. The type of contact in which a neuron forms a synapse on the sur face of another neuron or a smooth muscle cell and

then passes on to make similar contacts with other cells is called a synapse en pass ant.

In the heart, cholinergic and noradrenergic nerve fibers end on the sinoatrial node, the atrioventricular node, and the bundle of His.

Noradrenergic fibers also innervate the ventricular muscle. The exact nature of the end ings on nodal tissue is not known. In the ventricle,

the contacts between the noradrenergic fibers and the cardiac muscle fibers resemble th ose found in smooth muscle.

JUNCTIONAL POTENTIALS

In smooth muscles in which noradrenergic discharge is excitatory, stimulation of the no radrenergic nerves produces discrete partial

depolarizations that look like small end plate potentials and are called excitatory ju nction potentials (EJPs). These potentials summate

with repeated stimuli. Similar EJPs are seen in tissues excited by cholinergic discharges. I n tissues inhibited by noradrenergic stimuli,

hyperpolarizing inhibitory junction potentials (IJPs) are produced by stimulation of th e noradrenergic nerves. Junctional potentials

spread electrotonically.

CLINICAL BOX 6–2 CLINICAL BOX 6–3 Myasthenia Gravis

Myasthenia gravis is a serious and sometimes fatal disease in which skeletal musc les are weak and tire easily. It occurs in 25 to 125 of

every 1 million people worldwide and can occur at any age but seems to have a bimod al distribution, with peak occurrences in

individuals in their 20s (mainly women) and 60s (mainly men). It is caused by the form ation of circulating antibodies to the muscle type

of nicotinic acetylcholine receptors. These antibodies destroy some of the recep tors and bind others to neighboring receptors, triggering

their removal by endocytosis. Normally, the number of quanta released from the motor nerve terminal declines with successive repetitive

stimuli. In myasthenia gravis, neuromuscular transmission fails at these low levels of qua ntal release. This leads to the major clinical

feature of the disease– muscle fatigue with sustained or repeated activity. There are two major forms of the disease. In one form, the

extraocular muscles are primarily affected. In the second form, there is a generalized w eakness of skeletal muscles. Weakness improves

after a period of rest or after administration of acetylcholinesterase inhibitors. Cholinesterase inhibitors prevent metabolism of

acetylcholine and can thus compensate for the normal decline in released neurotransmitt ers during repeated stimulation. In severe cases,

all muscles, including the diaphragm, can become weak and respiratory failure and dea th can ensue. The major structural abnormality in

myasthenia gravis is the appearance of sparse, shallow, and abnormally wide or absent synaptic clefts in the motor end plate. Studies

show that the postsynaptic membrane has a reduced response to acetylcholine and a 70 –90% decrease in the number of receptors per end

plate in affected muscles. Patients with mysathenia gravis have a greater than normal ten dency to also have rheumatoid arthritis, systemic

lupus erythematosus, and polymyositis. About 30% of mysathenia gravis patients have a maternal relative with an autoimmune disorder.

These associations suggest that individuals with myasthenia gravis share a genetic predisp osition to autoimmune disease. The thymus

may play a role in the pathogenesis of the disease by supplying helper T cells sensitized against thymic proteins that cross-react with

acetylcholine receptors. In most patients, the thymus is hyperplastic, and 10–15% have thymomas. Thymectomy is indicated if a

thymoma is suspected. Even in those without thymoma, thymectomy induces remission in 35% and improves symptoms in another 45%

of patients.

DENERVATION HYPERSENSITIVITY

When the motor nerve to skeletal muscle is cut and allowed to degenerate, the muscle gr adually becomes extremely sensitive to

acetylcholine. This denervation hypersensitivity or super

Lambert–Eaton Syndrome

Another condition that resembles myasthenia gravis is the relatively rare condition called Lambert–Eaton Syndrome (LEMS). In this

condition, muscle weakness is caused by an autoimmune attack against one of the Ca

channels in the nerve endings at the

neuromuscular junction. This decreases the normal Ca

influx that causes acetylcholine release. Proximal muscles of the lower

extremities are primarily affected, producing a waddling gait and difficulty raising the a rms. Repetitive stimulation of the motor nerve

facilitates accumulation of Ca

in the nerve terminal and increases acetylcholine release, leading to an increase in musc le strength. This

is in contrast to myasthenia gravis in which symptoms are exasperated by repetitive stimu lation. About 40% of patients with LEMS also

have cancer, especially small cell cancer of the lung. One theory is that antibodies that h ave been produced to attack the cancer cells may

also attack Ca

channels, leading to LEMS. LEMS has also been associated with lymphosarcoma, malig nant thymoma, and cancer of

the breast, stomach, colon, prostate, bladder, kidney, or gall bladder. Clinical signs usua lly precede the diagnosis of cancer. A syndrome

similar to LEMS can occur after the use of aminoglycoside antibiotics, which also impair Ca

channel function.

sensitivity is also seen in smooth muscle. Smooth muscle, unlike skeletal muscle, does not atrophy when denervated, but it becomes

hyperresponsive to the chemical mediator that normally activates it. A good example of denervation hypersensitivity is the response of

the denervated iris. If the postganglionic sympathetic nerves to one iris are cut in an exp erimental animal and, after several weeks,

norepinephrine (the transmitter released by sympathetic postganglionic neurons) is injec ted intravenously, the denervated pupil dilates

widely. A much smaller, less prolonged response is observed on the intact side.

The reactions triggered by section of an axon are summarized in Figure 6–16. Hyperse nsitivity of the postsynaptic structure to the

transmitter previously secreted by the axon endings is a general phenomenon, largely d ue to the synthesis or activation of more

receptors. There is in addition orthograde degeneration (wallerian degeneration) and retrogr ade degeneration of the axon stump to the

nearest collateral (sustaining collateral). A series of changes occur in the cell b ody that include a decrease in Nissl substance

(chromatolysis). The nerve then starts to regrow, with multiple small branches projecting along the path the axon previously followed

(regenerative sprouting). Axons sometimes grow back to their original targets, especiall y in locations like the neuromuscular junction.

However, nerve regeneration is generally limited because axons often become entangle d in the area of tissue damage at the site where

they were disrupted. This

Autonomic

nerve fiber

Varicosity Sheet of

cells

Mitochondrion

Synaptic vesicles

Varicosities

FIGURE 6–15 Endings of postganglionic autonomic neurons on smooth muscl e. Neurotransmitter, released from varicosities along

the branched axon, diffuses to receptors on smooth muscle cell plasma memb ranes. (From Widmaier EP, Raff H, Strang KT: Vanders Human

Physiology. McGraw-Hill, 2008.)

difficulty has been reduced by administration of neurotro

phins. For example, sensory neurons torn when dorsal nerve

roots are avulsed from the spinal cord regrow and form func

tional connections in the spinal cord if experimental animals

are treated with NGF, neurotrophin 3, or GDNF.

Hypersensitivity is limited to the structures immediately

innervated by the destroyed neurons and fails to develop in neu

rons and muscle farther downstream. Suprasegmental spinal cord lesions do not lead to hypersensitivity of the paralyzed skeletal muscles

to acetylcholine, and destruction of the preganglionic autonomic nerves to visceral struc tures does not cause hypersensitivity of the

denervated viscera. This fact has practical implications in the treatment of diseases due to spasm of the blood vessels in the extremities.

For example, if the upper extremity is sympathectomized by removing the upper part of the ganglionic chain and the stellate ganglion,

Axon branch Receptor

(sustaining collateral)

the hypersensitive smooth muscle in the vessel walls is stimulated by circulating norepinep hrine, and episodic vasospasm continues to

occur. However, if preganglionic sympathectomy of the arm is performed by cutting th e ganglion chain below the third ganglion (to

interrupt ascending preganglionic

Retrograde

degeneration

Receptor

hypersensitive

fibers) and the white rami of the first three thoracic nerves, no hypersensitivity results.

Site of in jury Denervation hypersensitivity has multiple causes. As noted in Chapter 2, a deficiency of a given chemical messenger genX

erally produces an upregulation of its receptors. Another factor is lack of reuptake of secreted neurotransmitters. Retrograde

reaction:

Regenerative

sprouting

chromatolysis

Orthograde

(wallerian)

degeneration

CHAPTER SUMMARY

FIGURE 6–16 Summary of changes occurring in a neuron

and the structure it innervates when its axon is crushed or cut at

the point marked X. Hypersensitivity of the postsynaptic structure to

the transmitter previously secreted by the axon occurs largely due to

the synthesis or activation of more receptors. There is both orthograde

(wallerian) degeneration from the point of damage to the terminal and

retrograde degeneration of the axon stump to the nearest collateral

(sustaining collateral). Changes also occur in the cell body, including

chromatolysis. The nerve starts to regrow, with multiple small branch

es projecting along the path the axon previously followed (regenera

tive sprouting).

■ Presynaptic terminals are separated from the postsynaptic structure by a synaptic cleft. The postsynaptic membrane contains many

neurotransmitter receptors and usually a postsynaptic thickening called the postsynaptic density.

■ At chemical synapses, an impulse in the presynaptic axon causes secretion of a chemical that diffuses across the synaptic cleft and

binds to postsynaptic receptors, triggering events that open or close channels in the me mbrane of the postsynaptic cell. At electrical

synapses, the membranes of the presynaptic and postsynaptic neurons come close toget her, and gap junctions form low-resistance

bridges through which ions pass with relative ease from one neuron to the next.

■ A neuron receives input from many other neurons (convergence), and a neuron branches to innervate many other neurons (divergence).

■ An EPSP is produced by depolarization of the postsynaptic cell after a latency of 0 .5 ms; the excitatory transmitter opens Na

or Ca

ion channels in the postsynaptic membrane, producing an inward current. An IPS P is produced by a hyperpolarization of the postsynaptic

cell; it can be produced by a localized increase in Cl

–

transport. Slow EPSPs and IPSPs occur after a latency of 100 to 500 ms in

autonomic ganglia, cardiac and smooth muscle, and cortical neurons. The slow EPSPs are d ue to decreases in K

conductance, and the

slow IPSPs are due to increases in K

conductance.

■ Postsynaptic inhibition during the course of an IPSP is called direct inhibition. Indir ect inhibition is due to the effects of previous

postsynaptic neuron discharge; for example, the postsynaptic cell cannot be activated during its r efractory period. Presynaptic inhibition

is a process mediated by neurons whose terminals are on excitatory endings, formi ng axoaxonal synapses; in response to activation of

the presynaptic terminal. Activation of the presynaptic receptors can increase Cl

–

conductance, decreasing the size of the action

potentials reaching the excitatory ending, and reducing Ca

entry and the amount of excitatory transmitter released.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. Fast inhibitor y postsynaptic potentials (IPSPs)

A) are a consequence of decreased Cl

–

conductance. B) occur in skeletal muscle.

C) can be produced by an increase in Na

conductance. D) can be produced by an increase in Ca

conductance. E) interact with other

fast and slow potentials to move the

membrane potential of the postsynaptic neuron toward or away from the firing level.

2. Fast excitatory postsynaptic potentials (EPSPs)

A) are a consequence of decreased Cl

–

conductance. B) occur in skeletal muscle.

C) can be produced by an increase in Na

conductance. D) can be produced by a decrease in Ca

conductance. E) all of the above

3. Initiation of an action potential in skeletal muscle by stimulating its motor ner ve

A) requires spatial facilitation.

B) requires temporal facilitation.

C) is inhibited by a high concentration of Ca

at the neuromuscular junction.

D) requires the release of norepinephrine.

E) requires the release of acetylcholine.

4. A 35-year-old woman sees her physician to report muscle weakness in the extraocu lar eye muscles and muscles of the extremities. She

states that she feels fine when she gets up in the morning, but the weakness begin s soon after she becomes active. The weakness is

improved by rest. Sensation appears normal. The physician treats her with an anticholinesterase inhibitor, and she notes immediate return

of muscle strength. Her physician diagnoses her with

A) Lambert–Eaton syndrome.

B) myasthenia gravis.

C) multiple sclerosis.

D) Parkinson disease.

E) muscular dystrophy.

CHAPTER RESOURCES

Boron WF, Boulpaep EL: Medical Physiology, Elsevier, 2005. Hille B: Ionic Channels of Excitable Membranes, 3rd ed. Sinauer

Associates, 2001.

Jessell TM, Kandel ER: Synaptic transmission: A bidirectional and a

self-modifiable form of cellcell communication. Cell

1993;72(Suppl):1.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural

Science, 4th ed. McGraw-Hill, 2000.

McPhee SJ, Ganong WF: Pathophysiology of Disease. An Introduction

to Clinical Medicine, 5th ed. McGraw-Hill, 2006.

Squire LR, et al (editors): Fundamental Neuroscience, 3rd ed.,

Academic Press, 2008.

Unwin N: Neurotransmitter action: Opening of ligand-gated ion

channels. Cell 1993; 72(Suppl):31.

Van der Kloot W, Molg J: Quantal acetylcholine release at the

vertebrate neuromuscular junction. Physiol Rev 1994;74:899.

Neurotransmitters & Neuromodulators

OBJEC TIV ES

After studying this chapter, you should be able to:

List neurotransmitters and the principal sites in the nervous system at which they

■

are released.

■

Describe the receptors for catecholamines, acetylcholine, 5-HT, amino acids, and opio ids.

■

Summarize the steps involved in the biosynthesis, release, action, and removal from the synap tic cleft of the various synaptic

transmitters.

■

Define opioid peptide, list the principal opioid peptides in the body, and name the

CH APTER

precursor molecules from which they originate.

INTRODUCTION

The fact that transmission at most synapses is chemical is of great physiologic and pharm acologic importance. Nerve endings have been

called biological transducers that convert electrical energy into chemical energy. In broa d terms, this conversion process involves the

synthesis of the neurotransmitters, their storage in synaptic vesicles, and their release by the nerve impu lses into the synaptic cleft. The

secreted transmitters then act on appropriate receptors on the membrane of the postsyna ptic cell and are rapidly removed from the

synaptic cleft by diffusion, metabolism, and, in many instances, reuptake into the presyn aptic neuron. Some chemicals released by

neurons have little or no direct effects on their own but can modify the effects of neuro transmitters. These chemicals are called

neuromodulators. All these processes, plus the postreceptor events in the postsynaptic neu ron, are regulated by many physiologic

factors and at least in theory can be altered by drugs. Therefore, pharmacologists (in th eory) should be able to develop drugs that regulate

not only somatic and visceral motor activity but also emotions, behavior, and all the othe r complex functions of the brain.

CHEMICAL TRANSMISSION

OF SYNAPTIC ACTIVITY

CHEMISTRY OF TRANSMITTERS

One suspects that a substance is a neurotransmitter if it is unevenly distributed in the nerv ous system and its distribution parallels that of

its receptors and synthesizing and catabolizing enzymes. Additional evidence includes d emonstration that it is released from appropriate

brain regions in vitro and that it produces effects on single target neurons when applied to their membranes by means of a micropipette

(microiontophoresis). Many transmitters and enzymes involved in their synthesis and ca tabolism have been localized in nerve endings by

immunohistochemistry, a technique in which antibodies to a given substance are labeled an d applied to brain and other tissues. The

antibodies bind to the substance, and the location of the substance is then determined by locating the label with the light microscope or

electron microscope. In situ hybridization histochemistry, which permits localization of the mRNAs for pa rticular synthesizing

enzymes or receptors, has also been a valuable tool.

Identified neurotransmitters and neuromodulators can be divided into two major catego ries: small-molecule transmitters and large-

molecule transmitters. Small-molecule transmitters

129 include monoamines (eg, acetylcholine, serotonin, histamine), catecholamines (dopamine, norepin ephrine, and epinephrine), and

amino acids (eg, glutamate, GABA, glycine). Large-molecule transmitter s include a large number of peptides called neuropeptides

including substance P, enkephalin, vasopressin, and a host of others. In general, neuro peptides are colocalized with one of the small-

molecule neurotransmitters (Table 7–1).

There are also other substances thought to be released into the synaptic cleft to act as eith er a transmitter or modulator of synaptic

transmission. These include purine derivatives like adenosine and adenosine triphosphat e (ATP) and a gaseous molecule, nitric oxide

(NO).

Figure 7–1 shows the biosynthesis of some common smallmolecule transmitters released by neurons in the central or peripheral nervous

system. Figure 7–2 shows the location of major groups of neurons that contain norepin ephrine, epinephrine, dopamine, and

acetylcholine. These are some of the major neuromodulatory systems.

RECEPTORS

Cloning and other molecular biology techniques have permitted spectacular advances in knowledge about the structure and function of

receptors for neurotransmitters and other chemical messengers. The individual receptor s, along with their

TABLE 7–1 Examples of colocalization of small-molecule transmitters with n europeptides.

Small-Molecule Transmitter

Neuropeptide

Monoamines

Acetylcholine Enkephalin, calcitonin-gene-related peptide,

galanin, gonadotropin-releasing hormone, neurotensin, somatostatin, substance P, vasoactive intestinal polypeptide

Serotonin

Catecholamines

Cholecystokinin, enkephalin, neuropeptide Y, substance P, vasoactive intestinal polypep tide

Dopamine Cholecystokinin, enkephalin, neurotensin

Norepinephrine Enkephalin, neuropeptide Y, neurotensin, somatostatin, vasopressin

Epinephrine

Amino Acids

Enkephalin, neuropeptide Y, neurotensin, substance P

Glutamate Substance P

Glycine Neurotensin

GABA Cholecystokinin, enkephalin, somatostatin, substance P, thyrotropin-releasing horm one

ligands, are discussed in the following parts of this chapter. However, five themes have emerged that should be mentioned in this

introductory discussion.

First, in every instance studied in detail to date, it has become clear that each ligand has m any subtypes of receptors. Thus, for example,

norepinephrine acts on α

and α

receptors, and three of each subtype have been cloned. In addition, there are β

, β

, and β

receptors.

Obviously, this multiplies the possible effects of a given ligand and makes its effects in a given cell more selective.

Second, there are receptors on the presynaptic as well as the postsynaptic elements for m any secreted transmitters. These presyna ptic

receptors, or autoreceptors, often inhibit further secretion of the ligand, providing feedback control. For example, norepinephrine acts

on α

presynaptic receptors to inhibit norepinephrine secretion. However, autoreceptors can also facilitate the release of

neurotransmitters.

Third, although there are many ligands and many subtypes of receptors for each ligand , the receptors tend to group in large families as

far as structure and function are concerned. Many receptors act via trimeric G proteins and protein kinases to produce their effects.

Others are ion channels. The receptors for a group of selected, established neurotransm itters and neuromodulators are listed in Table 7–2,

along with their principal second messengers and, where established, their net effect on ion channels. It should be noted that this table is

an oversimplification. For example, activation of α

-adrenergic receptors decreases intracellular cAMP concentrations, but there is

evidence that the G protein activated by α

-adrenergic presynaptic receptors also acts directly on Ca

channels to inhibit norepinephrine

release by decreasing Ca

increases.

Fourth, receptors are concentrated in clusters in postsynaptic structures close to the endi ngs of neurons that secrete the neurotransmitters

specific for them. This is generally due to the presence of specific binding proteins for them. In the case of nicotinic acetylcholine

receptors at the neuromuscular junction, the protein is rapsyn, and in the case of excitatory glutamatergic receptors, a family of PB2-

binding proteins is involved. GABA

receptors are associated with the protein gephyrin, which also binds glyci ne receptors, and

GABA

receptors are bound to the cytoskeleton in the retina by the protein MAP-1B. At least in the case of GABA

receptors, the

binding protein gephyrin is located in clumps in the postsynaptic membrane. With activit y, the free receptors move rapidly to the

gephyrin and bind to it, creating membrane clusters. Gephyrin binding slows and restric ts their further movement. Presumably, during

neural inactivity, the receptors are unbound and move again.

Fifth, prolonged exposure to their ligands causes most receptors to become unresponsiv e, that is, to undergo desensitization. This can be

of two types: homologous desensitization, with loss of responsiveness only to th e particular ligand and maintained responsiveness of the

cell to other ligands; and heterologous desensitization, in which the cell becomes unresponsive to other ligands as well. Desensitization

in β- adrenergic receptors has been studied in considerable detail. One form involves p hosphorylation of the carboxyl terminal

FIGURE 7–1 Biosynthesis of some common small molecule transmitters. (Reprodu ced with permission from Boron WF, Boulpaep EL: Medical

Physiology. Elsevier, 2005.)

FIGURE 7–2 Four diffusely connected systems of central neurons using modu latory transmitters. A) Norepinephrine-containing

neurons. B) Serotonin-containing neurons. C) D opamine-containing neurons. D) Acetylcholine-containing n eurons. (Reproduced with

permission from Boron WF, Boulpaep EL: Medical Physiology. Elsevier, 2005.)

region of the receptor by a specific β-adrenergic receptor

kinase (β-ARK) or binding β-arrestins. Four β-arrestins have

been described in mammals. Two are expressed in rods and

cones of the retina and inhibit visual responses. The other two,

β-arrestin 1 and β-arrestin 2, are more ubiquitous. They

desensitize β-adrenegic receptors, but they also inhibit other

heterotrimeric G protein-coupled receptors. In addition, they

foster endocytosis of ligands, adding to desensitization.

REUPTAKE

Neurotransmitters are transported from the synaptic cleft back into the cytoplasm of the neurons that secreted them, a process referred to

as reuptake (Figure 7–3). The high-affinity reuptake systems employ two families of transporter pr oteins. One family has 12

transmembrane domains and cotransports the transmitter with Na

and Cl

–

. Members of this family include transporters for

norepinephrine, dopamine, serotonin, GABA, and glycine, as well as transporters for p roline, taurine, and the acetylcholine precursor

choline. In addition, there

TABLE 7–2 Mechanism of action of selected small-molecule transmitters. Transmitte r

Monoamines Acetylcholine

Serotonin

Receptor Second Messenger Net Channel Effects

Nicotinic M

5HT

↑Na

, K

↑ IP

, DAG ↑Ca

↓Cyclic AMP ↑K

↓Cyclic AMP

↓Cyclic AMP ↓K

, DAG ↓K

↑

↑IP

, DAG

↑Na

↑Cyclic AMP Catecholamines Dopamine

, D

Norepinephrine α

↑Cyclic AMP

↓Cyclic AMP ↑K

, ↓Ca

Cyclic AMP↓

↑IP

, DAG ↓K

+ +

, ↓Ca

↓Cyclic AMP ↑K ↑Cyclic AMP

↑Cyclic AMP

Amino Acids

Glutamate Metabotropic

GABA

Glycine Ionotropic

AMPA, Kainate NMDA

GABA

Glycine

↑Na

, K

↑Na

, K

,Ca

↑Cl

–

↑IP

, DAG ↑K

,↓Ca

↑Cl

–

Eleven subtypes identified; all decrease cAMP or increase IP

and DAG, except one, which increases cAMP.

may be an epinephrine transporter. The other family is made up of at least three transpo rters that mediate glutamate uptake by neurons

and two that transport glutamate into astrocytes. These glutamate transporters are couple d to the cotransport of Na

and the

countertransport of K

, and they are not dependent on Cl

–

transport. There is a debate about their structure, and they may have 6, 8, or

10 transmembrane domains. One of them transports glutamate into glia rather than neur ons (see Chapter 4).

There are in addition two vesicular monoamine transporters, VMAT1 and VMAT2, tha t transport neurotransmitters from the cytoplasm

to synaptic vesicles. They are coded by different genes but have extensive homology. B oth have a broad specificity, moving dopamine,

norepinephrine, epinephrine, serotonin, and histamine from the cytoplasm into secretor y granules. Both are inhibited by reserpine, which

accounts for the marked monoamine depletion produced by this drug. Like the neurotr ansmitter membrane transporter family, they have

12 transmembrane domains, but they have little homology to the other transporters. The re is also a vesicular GABA transporter (VGAT)

that moves GABA and glycine into vesicles and a vesicular acetylcholine transporter.

Reuptake is a major factor in terminating the action of transmitters, and when it is inhibite d, the effects of transmitter release are

increased and prolonged. This has clinical consequences. For example, several effectiv e antidepressant drugs are inhibitors of the

reuptake of amine transmitters, and cocaine is believed to inhibit dopamine reuptake. Glu tamate uptake into neurons and glia is

important because glutamate is an excitotoxin that can kill cells by overstimulating them (s ee Clinical Box 7–1). There is evidence that

during ischemia

Neuron Presynaptic

Glial cellterminal 4

1 Amino acid precursor Transmitter

synthesis NTT

K+

2 5

VMAT 3 H

−

Second

+ Other

messengers

receptors

Postsynaptic terminal

Neuron

FIGURE 7–3 Fate of monoamines secreted at synaptic junctions. In each monoamine-secreting neuron, the monoamine is synthesized

in the cytoplasm and the secretory granules (1) and its concentration in secretory granules is maintained (2) by the two vesicular

monoamine transporters (VMAT). The monoamine is secreted by exocytosis of the granules (3), and it acts (4) on receptors (Y-shaped

structures labeled R). Many of these receptors are postsynaptic, but some are presynaptic and some are located on glia. In addition, there

is extensive reuptake into the cytoplasm of the presynaptic terminal (5) via the mo noamine neurotransmitter transporter (NTT) for the

monoamine that is synthesized in the neuron. (Reproduced with permission from Hoffman BJ, et al: Distribution of monoamine neurotransmitter transporters

in the rat brain. Front Neuroendocrinol 1998;19:187.)

and anoxia, loss of neurons is increased because glutamate reuptake is inhibited.

SMALL-MOLECULE TRANSMITTERS

Synaptic physiology is a rapidly expanding, complex field that cannot be covered in de tail in this book. However, it is appropriate to

summarize information about the principal neurotransmitters and their receptors.

MONOAMINES

Acetylcholine

Acetylcholine, which is the acetyl ester of choline, is largely enclosed in small, clear syna ptic vesicles in high concentration in the

terminal boutons of neurons that release acetylcholine (cholinergic neurons). Synthesis of ac etylcholine involves the reaction of choline

with acetate (Figure 7–1). Acetylcholine is the transmitter at the neuromuscular junction, in autonomic ganglia, and in postganglionic

parasympathetic nerve-target organ junctions and some postganglionic sympathetic nerv e-target junctions. It is also found within the

brain, including the basal forebrain complex and pontomesencephalic cholinergic comp lex (Figure 7–2). These systems may be involved

in regulation of sleep-wake states, learning, and memory.

Cholinergic neurons actively take up choline via a transporter (Figure 7–4). Choline is also synthesized in neurons. The acetate is

activated by the combination of acetate groups with reduced coenzyme A. The reaction between active acetate (acetyl-coenzyme A,

acetyl-CoA) and choline is catalyzed by the enzyme choline acetyltransferase. This enzyme is foun d in high concentration in the

cytoplasm of cholinergic

CLINICAL BOX 7–1

Cholinergic neuron

Excitotoxins

Glutamate is usually cleared from the brain’s extracellular fluid by Na

-dependent uptake systems in neurons and glia, keeping only

micromolar levels of the chemical in the extracellular fluid despite millimolar levels inside neurons. However, excessive levels of

glutamate occur in response to ischemia, anoxia, hypoglycemia, or trauma. Glutamate an d some of its synthetic congeners are unique in

that when they act on neuronal cell bodies, they can produce so much Ca

influx that neurons die. This is the reason why

microinjection of these excitotoxins is used in research to produce discrete lesions that destro y neuronal cell bodies without affecting

neighboring axons. Evidence is accumulating that excitotoxins play a significant role in t he damage done to the brain by a stroke. When

a cerebral artery is occluded, the cells in the severely ischemic area die. Surrounding pa rtially ischemic cells may survive but lose their

ability to maintain the transmembrane Na

gradient. The elevated levels of intracellular Na

prevent the ability of astrocytes to remove

glutamate from the brain’s extracellular fluid. Therefore, glutamate accumulates to the p oint that excitotoxic damage and cell death

occurs in the penumbra, the region around the completely infarcted area.

nerve endings. Acetylcholine is then taken up into synaptic vesicles by a vesicular transp orter, VAChT.

Cholinesterases

Acetylcholine must be rapidly removed from the synapse if repolarization is to occur. T he removal occurs by way of hydrolysis of

acetylcholine to choline and acetate, a reaction catalyzed by the enzyme acetylcholinesterase. This enzyme is also called true or specific

cholinesterase. Its greatest affinity is for acetylcholine, but it also hydrolyzes other ch oline esters. There are a variety of esterases in the

body. One found in plasma is capable of hydrolyzing acetylcholine but has different pr operties from acetylcholinesterase. It is therefore

called pseudocholinesterase or nonspecific cholinesterase. The plasma moiety is partly under endocr ine control and is affected by

variations in liver function. On the other hand, the specific cholinesterase molecules are clustered in the postsynaptic membrane of

cholinergic synapses. Hydrolysis of acetylcholine by this enzyme is rapid enough to exp lain the observed changes in Na

conductance

and electrical activity during synaptic transmission.

Acetylcholine Receptors

Historically, acetylcholine receptors have been divided into two main types on the basis of their pharmacologic properties. Muscarine,

the alkaloid responsible for the toxicity of

Acetyl-CoA +

Choline

ACh

Choline ACh

ASE

Postsynaptic tissue

FIGURE 7–4 Biochemical events at cholinergic endings. ACh, acetylcholine; ASE, acetylcholinesterase; X, recept or.

toadstools, has little effect on the receptors in autonomic ganglia but mimics the stimulator y action of acetylcholine on smooth muscle

and glands. These actions of acetylcholine are therefore called muscarinic actions, and the receptors involved are muscarinic

cholinergic receptors. They are blocked by the drug atropine. In sympathetic ganglia, small am ounts of acetylcholine stimulate

postganglionic neurons and large amounts block transmission of impulses from pregang lionic to postganglionic neurons. These actions

are unaffected by atropine but mimicked by nicotine. Consequently, these actions of ac etylcholine are nicotinic actions and the receptors

are nicotinic cholinergic receptors. Nicotinic receptors are subdivided into th ose found in muscle at neuromuscular junctions and those

found in autonomic ganglia and the central nervous system. Both muscarinic and nicotin ic acetylcholine receptors are found in large

numbers in the brain.

The nicotinic acetylcholine receptors are members of a superfamily of ligand-gated ion channels that also includes the GABA

and

glycine receptors and some of the glutamate receptors. They are made up of multiple su bunits coded by different genes. Each nicotinic

cholinergic receptor is made up of five subunits that form a central channel which, whe n the receptor is activated, permits the passage of

and other cations. The 5 subunits come from a menu of 16 known subunits, α

–α

, β

–β

, γ, δ, and ε, coded by 16 different genes.

Some of the receptors are homomeric—for example, those that contain five α

subunits—but most are heteromeric. The muscle type

nicotinic receptor found in the fetus is made up of two α

subunits, a β

subunit, a γ subunit, and a δ subunit (Figure 7–5). In adult

mammals, the γ subunit is replaced by a δ subunit, which decreases the channel open tim e but increases its

No ACh bound: Two ACh molecules bound: Channel closed Channel open

ACh

Na+

FIGURE 7–5 Three-dimensional model of the nicotinic acetylcholine-gated ion channel. The receptor–channel complex consists

of five subunits, all of which contribute to forming the pore. When two molecules of acetylcho line bind to portions of the

-subunits

exposed to the membrane surface, the receptor–channel changes conformation. This opens the pore in the portion of the channel

emnbedded in the lipid bilayer, and both K

and Na

flow through the open channel down their electrochemical gradient. (From Kandel ER ,

Schwartz JH, Jessell TM [editors]: Principles of Neural Science, 4th ed. McGraw-Hill, 2000.)

conductance. The nicotinic cholinergic receptors in autonomic ganglia are heteromers th at usually contain α

subunits in combination

with others, and the nicotinic receptors in the brain are made up of many other subunits . Many of the nicotinic cholinergic receptors in

the brain are located presynaptically on glutamate-secreting axon terminals, and they fac ilitate the release of this transmitter. However,

others are postsynaptic. Some are located on structures other than neurons, and some s eem to be free in the interstitial fluid, that is, they

are perisynaptic in location.

Each α subunit has a binding site for acetylcholine, and when an acetylcholine molecul e binds to each of them, they induce a

confirmational change in the protein so that the channel opens. This increases the condu ctance of Na

and other cations, and the resulting

influx of Na

produces a depolarizing potential. A prominent feature of neuronal nicotinic cholinerg ic receptors is their high

permeability to Ca

Muscarinic cholinergic receptors are very different from nicotinic cholinergic receptor s. Five types, encoded by five separate genes, have

been cloned. The exact status of M

is uncertain, but the remaining four receptors are coupled via G proteins to adenylyl cy clase, K

channels, and/or phospholipase C (Table 7–2). The nomenclature of these receptors ha s not been standardized, but the receptor

designated M

in Table 7–2 is abundant in the brain. The M

receptor is found in the heart. The M

receptor is found in pancreatic acinar

and islet tissue, where it mediates increased secretion of pancreatic enzymes and insulin. The M

and M

receptors are associated with

smooth muscle.

Serotonin

Serotonin (5-hydroxytryptamine; 5-HT) is present in highest concentration in blood pla telets and in the gastrointestinal tract, where it is

found in the enterochromaffin cells and the myenteric plexus. It is also found within the brain stem in the midline raphé nuclei which

project to portions of the hypothalamus, the limbic system, the neocortex, the cerebellum , and the spinal cord (Figure 7–2).

Serotonin is formed in the body by hydroxylation and decarboxylation of the essential amino acid tryptophan (Figures 7–1 and 7–6).

After release from serotonergic neurons, much of the released serotonin is recaptured by an active reuptake mechanism and inactivated

by monoamine oxidase (MAO) to form 5-hydroxyindoleacetic acid (5-HIAA). This s ubstance is the principal urinary metabolite of

serotonin, and urinary output of 5-HIAA is used as an index of the rate of serotonin m etabolism in the body.

Tryptophan hydroxylase in the human CNS is slightly different from the tryptophan hy droxylase in peripheral tissues, and is coded by a

different gene. This is presumably why knockout of the TPH1 gene, which codes for tryptophan hydroxylase in peripheral tissues, has

much less effect on brain serotonin production than on peripheral serotonin production .

As described in Clinical Box 7–2, there is evidence for a relationship between behavior and brain serotonin content.

Serotonergic Receptors

The number of cloned and characterized serotonin receptors has increased rapidly. Th ere are at least seven types of 5-HT

Serotonergic neuron

CLINICAL BOX 7–2

-Tryptophan

5-HTP

MAO 5-HIAA

5-HT

Reuptake 5-HT

Postsynaptic tissue

FIGURE 7–6 Biochemical events at serotonergic synapses. Biochemical events at ser otonergic synapses. Biochemical events at

serotonergic synapses. HIAA, 5-hydroxyindoleacetic acid; X, serotonin receptor. F or clarity, the presynaptic receptors have been

omitted.

receptors (from 5-HT

through 5-HT

receptors). Within the

5-HT

group are the 5-HT

, 5-HT

, and

5-HT

subtypes. Within the 5-HT

group there are 5-HT

5-HT

, and 5-HT

subtypes. There are two 5-HT

subtypes: 5-HT

and 5-HT

. Most of these are G protein-coupled receptors and

affect adenylyl cyclase or phospholipase C (Table 7–2). However, the 5-HT

receptors, like nicotinic cholinergic receptors, are ligand-

gated ion channels. Some of the serotonin receptors are presynaptic, and others are po stsynaptic.

5-HT

receptors mediate platelet aggregation and smooth muscle contraction. Mice in which th e gene for 5-HT

receptors has been

knocked out are obese as a result of increased food intake despite normal responses to leptin, and they are prone to fatal seizures. 5-HT

receptors are present in the gastrointestinal tract and the area postrema and are related to vomiting. 5-HT

receptors are also present in

the gastrointestinal tract, where they facilitate secretion and peristalsis, and in the brain. 5 -HT

and 5-HT

receptors in the brain are

distributed throughout the limbic system, and the 5-HT

receptors have a high affinity for antidepressant drugs.

Histamine

Histaminergic neurons have their cell bodies in the tuberomammillary nucleus of the pos terior hypothalamus, and their axons project to

all parts of the brain, including the cerebral cortex and the spinal cord. Histamine is also found in cells in the gastric mucosa and in

heparin-containing cells called mast cells that are plentiful in the anterior and posterior lobes of the pituitary gland as well as at body

surfaces.

Role of Serotonin in Mood & Behavior

The hallucinogenic agent lysergic acid diethylamide (LSD) is a serotonin agonist that p roduces its effects by activating 5-HT

receptors

in the brain. The transient hallucinations and other mental aberrations produced by this d rug were discovered when the chemist who

synthesized it inhaled some by accident. Its discovery called attention to the correlation b etween behavior and variations in brain

serotonin content. Psilocin (and its phosphorylated form, psilocybin), a substance fo und in certain mushrooms, and N,N-

dimethyltryptamine (DMT) are also hallucinogenic and, like serotonin, are deriv atives of tryptamine. 2,5-Dimethoxy-4-

methylamphetamine (DOM) and mescaline and its congeners, the other true hal lucinogens, are phenylethylamines rather than

indolamines. However, all these hallucinogens appear to exert their effects by binding to 5-HT

receptors. 3,4-

Methylenedioxymethamphetamine, a drug known as MDMA or ecstasy, is a popular dru g of abuse. It produces euphoria, but this is

followed by difficulty in concentrating, depression, and, in monkeys, insomnia. The dr ug causes release of serotonin followed by

serotonin depletion; the euphoria may be due to the release and the later symptoms to the depletion.

Drugs that increase extracellular norepinephrine levels in the brain elevate mood, and d rugs that decrease extracellular norepinephrine

levels cause depression. However, individuals with congenital dopamine β-hydroxylase (DBH) deficiency are normal as far as mood is

concerned. Drugs that inhibit norepinephrine reuptake were of considerable value in th e treatment of depression, but these drugs also

inhibit serotonin reuptake. It is also known that the primary serotonin metabolite 5-HIAA is l ow in CSF of depressed individuals. Drugs

such as fluoxetine (Prozac), which inhibit serotonin reuptake without affecting norepinephrine reuptake, are effective as

antidepressants. Thus, the focus in treating clinical depression has shifted from norepine phrine to serotonin.

Histamine is formed by decarboxylation of the amino acid histidine (Figure 7–1). Histam ine is converted to methylhistamine or,

alternatively, to imidazoleacetic acid. The latter reaction is quantitatively less important in h umans. It requires the enzyme diamine

oxidase (histaminase) rather than MAO, even though MAO catalyzes the oxidation of m ethylhistamine to methylimidazoleacetic acid.

The three known types of histamine receptors—H

, H

, and H

—are all found in both peripheral tissues and the brain. Most, if not all,

of the H

receptors are presynaptic, and they mediate inhibition of the release of histamine and o ther transmitters via a G protein. H

receptors activate phospholipase C, and H

receptors increase the intracellular cAMP concentration. The function of this diffuse

histaminergic system is unknown, but evidence links brain histamine to arousal, sexual b ehavior, blood pressure, drinking, pain

thresholds, and regulation of the secretion of several anterior pituitary hormones.

CATECHOLAMINES

Norepinephrine & Epinephrine

The chemical transmitter present at most sympathetic postganglionic endings is norepinep hrine. It is stored in the synaptic knobs of the

neurons that secrete it in characteristic small vesicles that have a dense core (granulated v esicles; see above). Norepinephrine and its

methyl derivative, epinephrine, are secreted by the adrenal medulla, but epinephrine is n ot a mediator at postganglionic sympathetic

endings. As discussed in Chapter 6, each sympathetic postganglionic neuron has multipl e varicosities along its course, and each of these

varicosities appears to be a site at which norepinephrine is secreted.

There are also norepinephrine-secreting and epinephrinesecreting neurons in the brain . Norepinephrine-secreting neurons are properly

called noradrenergic neurons, although the term adrenergic neurons is also applied. However, it seems appropriate to reserve the

latter term for epinephrine-secreting neurons. The cell bodies of the norepinephrine-co ntaining neurons are located in the locus ceruleus

and other medullary and pontine nuclei (Figure 7–2). From the locus ceruleus, the axon s of the noradrenergic neurons form the locus

ceruleus system. They descend into the spinal cord, enter the cerebellum, and asc end to innervate the paraventricular, supraoptic, and

periventricular nuclei of the hypothalamus, the thalamus, the basal telencephalon, and th e entire neocortex.

Biosynthesis & Release of Catecholamines

The principal catecholamines found in the body—norepinephrine, epinephrin e, and dopamine—are formed by hydroxylation and

decarboxylation of the amino acid tyrosine (Figure 7–1). Some of the tyrosine is forme d from phenylalanine, but most is of dietary

origin. Phenylalanine hydroxylase is found primarily in the liver (see Clinical Bo x 7–3). Tyrosine is transported into catecholamine-

secreting neurons and adrenal medullary cells by a concentrating mechanism. It is conv erted to dopa and then to dopamine in the

cytoplasm of the cells by tyrosine hydroxylase and dopa decarboxylase. The decarboxylase, which is also called aromatic L-amino

acid decarboxylase, is very similar but probably not identical to 5-hydroxytryptophan d ecarboxylase. The dopamine then enters the

granulated vesicles, within which it is converted to norepinephrine by dopamine β-hydroxylase (DBH). L-Dopa is the isomer involved,

but the norepinephrine that is formed is in the D configuration. The rate-limiting step in synthesis is the conversion of tyrosine to dopa.

Tyrosine hydroxylase, which catalyzes this step, is subject to feedback inhibition by dop amine and norepinephrine, thus providing

internal control of the synthetic process. The cofactor for tyrosine hydroxylase is

CLINICAL BOX 7–3 Phenylketonuria

Phenylketonuria is a disorder characterized by severe mental deficiency and the accumulation in the blood, tissues, and urine of large

amounts of phenylalanine and its keto acid derivatives. It is usually due to de creased function resulting from mutation of the gene for

phenylalanine hydroxylase. This gene is located on the long arm of chromoso me 12. Catecholamines are still formed from tyrosine,

and the cognitive impairment is largely due to accumulation of phenylalanine and its der ivatives in the blood. Therefore, it can be treated

with considerable success by markedly reducing the amount of phenylalanine in the die t. The condition can also be caused by

tetrahydrobiopterin (BH4) deficiency. Because BH4 is a cofactor for tyrosine hydr oxylase and tryptophan hydroxylase, as well as

phenylalanine hydroxylase, cases due to tetrahydrobiopterin deficiency have catechola mine and serotonin deficiencies in addition to

hyperphenylalaninemia. These cause hypotonia, inactivity, and developmental problems . They are treated with tetrahydrobiopterin,

levodopa, and 5-hydroxytryptophan in addition to a low-phenylalanine diet. BH4 is als o essential for the synthesis of nitric oxide (NO)

by nitric oxide synthase. Severe BH4 deficiency can lead to impairment of NO formatio n, and the CNS may be subjected to increased

oxidative stress.

tetrahydrobiopterin, which is converted to dihydrobiopterin when tyrosine is con verted to dopa.

Some neurons and adrenal medullary cells also contain the cytoplasmic enzyme phenylethanolamine-Nmethyltransferase (PNMT),

which catalyzes the conversion of norepinephrine to epinephrine. In these cells, norepi nephrine apparently leaves the vesicles, is

converted to epinephrine, and then enters other storage vesicles.

In granulated vesicles, norepinephrine and epinephrine are bound to ATP and associat ed with a protein called chromogranin A. In some

but not all noradrenergic neurons, the large granulated vesicles also contain neuropepti de Y. Chromogranin A is a 49-kDa acid protein

that is also found in many other neuroendocrine cells and neurons. Six related chro mogranins have been identified. They have been

claimed to have multiple intracellular and extracellular functions. Their level in the plasma is elevated in patients with a variety of tumors

and in essential hypertension, in which they probably reflect increased sympathetic activ ity. However, their specific functions remain

unsettled.

The catecholamines are transported into the granulated vesicles by two vesicular transpo rters, and these transporters are inhibited by the

drug reserpine.

Catecholamines are released from autonomic neurons and adrenal medullary cells by ex ocytosis. Because they are present in the

granulated vesicles, ATP, chromogranin A, and the dopamine β hydroxylase that is not mem brane-bound are released with

norepinephrine and epinephrine. The half-life of circulating dopamine β-hydroxylase is much longer than that of the catecholamines, and

circulating levels of this substance are affected by genetic and other factors in addition t o the rate of sympathetic activity.

Noradrenergic

neuron

Dopa Tyrosine

Dopamine

Catabolism of Catecholamines

Norepinephrine, like other amine and amino acid transmitters, is removed from the syna ptic cleft by binding to postsynaptic receptors,

binding to presynaptic receptors (Figure 7–3), reuptake into the presynaptic neurons, o r catabolism. Reuptake is a major mechanism in

the case of norepinephrine, and the hypersensitivity of sympathetically denervated struc tures is explained in part on this basis. After the

noradrenergic neurons are cut, their endings degenerate with loss of reuptake in them. Consequently, more norepinephrine from other

sources is available to stimulate the receptors on the autonomic effectors.

Epinephrine and norepinephrine are metabolized to biologically inactive products by ox idation and methylation. The former reaction is

catalyzed by MAO and the latter by catechol-Omethyltransferase (COMT). MAO is located on the o uter surface of the mitochondria.

It has two isoforms, MAO-A and MAO-B, which differ in substrate specificity and sen sitivity to drugs. Both are found in neurons. MAO

is widely distributed, being particularly plentiful in the nerve endings at which catecholam ines are secreted. COMT is also widely

distributed, particularly in the liver, kidneys, and smooth muscles. In the brain, it is prese nt in glial cells, and small amounts are found in

postsynaptic neurons, but none is found in presynaptic noradrenergic neurons. Conse quently, catecholamine metabolism has two

different patterns.

Extracellular epinephrine and norepinephrine are for the most part O-methylated, and m easurement of the concentrations of the O-

methylated derivatives normetanephrine and metanephrine in the urine is a good index of the rate of secretion of norepinephrine and

epinephrine. The O-methylated derivatives that are not excreted are largely oxidized, an d 3-methoxy-4-hydroxymandelic acid

(vanillylmandelic acid, VMA) is the most plentiful catecholamine metabolite in the urine. Small amounts of the O-methylated

derivatives are also conjugated to sulfate and glucuronide.

In the noradrenergic nerve terminals, on the other hand, some of the norepinephrine is constantly being converted by intracellular MAO

(Figure 7–7) to the physiologically inactive deaminated derivatives, 3,4-dihydroxymand elic acid (DOMA) and its corresponding glycol

(DHPG). These are subsequently converted to their corresponding O-methyl derivativ es, VMA and 3-methoxy-4-hydroxyphenylglycol

(MHPG).

α & β Receptors

Epinephrine and norepinephrine both act on α and β receptors, with norepinephrine ha ving a greater affinity for α-adrenergic

MAO

Deaminated

derivatives

Reuptake NE

COMT

Normetanephrine

Postsynaptic tissue

FIGURE 7–7 Biochemical events at noradrenergic endings. NE, norepinephrine; COMT, c atecholOmethyltransferase; MAO,

monoamine oxidase; X, receptor. For clarity, the presynaptic receptors have b een omitted. Note that MAO is intracellular, so that

norepinephrine is being constantly deaminated in noradrenergic endings. COMT acts primarily on secreted norepinephrine.

receptors and epinephrine for β-adrenergic receptors. As noted previously, the α a nd β receptors are typical G protein-coupled receptors,

and each has multiple forms. They are closely related to the cloned receptors for dopam ine and serotonin and to muscarinic acetylcholine

receptors.

Clonidine lowers blood pressure when administered centrally. It is an α

agonist and was initially thought to act on presynaptic α

receptors, reducing central norepinephrine discharge. However, its structure resembles that of imidazoline, and it binds to imidazoline

receptors with higher affinity than to α

adrenergic receptors. A subsequent search led to the discovery that imidazoline recepto rs occur

in the nucleus tractus solitarius and the ventrolateral medulla. Administration of imidazolin es lowers blood pressure and has a depressive

effect. However, the full significance of these observations remains to be explored.

Dopamine

In certain parts of the brain, catecholamine synthesis stops at dopamine (Figure 7–1) wh ich can then be secreted into the synaptic cleft.

Active reuptake of dopamine occurs via a Na

- and Cl

–

-dependent dopamine transporter. Dopamine is metabolized to inactive

compounds by MAO and COMT in a tabolized to inactive compounds by MAO and C OMT in a Dihydroxyphenylacetic acid (DOPAC)

and homovanillic acid (HVA) are conjugated, primarily to sulfate.

Dopaminergic neurons are located in several brain regions including the nigrostriatal system, which p rojects from the substantia nigra

to the striatum and is involved in motor control, and the mesocortical system, which a rises primarily in the ventral tegmental area

(Figure 7–2). The mesocortical system projects to the nucleus accumbens and limbic sub cortical areas, and it is involved in reward

behavior and addiction. Studies by PET scanning in normal humans show that a steady loss of dopamine receptors occurs in the basal

ganglia with age. The loss is greater in men than in women.

Dopamine Receptors

Five different dopamine receptors have been cloned, and several of these exist in multip le forms. This provides for variety in the type of

responses produced by dopamine. Most, but perhaps not all, of the responses to these r eceptors are mediated by heterotrimeric G

proteins. One of the two forms of D

receptors can form a heterodimer with the somatostatin SST5 receptor, further increasi ng the

dopamine response menu. Overstimulation of D

receptors is thought to be related to schizophrenia (see Clinical Box 7–4). D

receptors

are highly localized, especially to the nucleus accumbens (Figure 7–2). D

receptors have a greater affinity than the other dopamine

receptors for the “atypical” antipsychotic drug clozapine, which is effective in schizophre nia but produces fewer extrapyramidal side

effects than the other major tranquilizers do.

EXCITATORY & INHIBITORY

AMINO ACIDS

Glutamate

The amino acid glutamate is the main excitatory transmitter in the brain and spinal cord, and it has been c alculated that it is the

transmitter responsible for 75% of the excitatory transmission in the brain. Glutamate is formed by reductive amination of the Krebs

cycle intermediate α-ketoglutarate in the cytoplasm. The reaction is reversible, but in glut aminergic neurons, glutamate is concentrated in

synaptic vesicles by the vesicle-bound transporter BPN1. The cytoplasmic store of glutamine is enriched by three transporters that

import glutamate from the interstitial fluid, and two additional transporters carry glutamat e into astrocytes, where it is converted to

glutamine and passed on to glutaminergic neurons. The interaction of astrocytes and glu taminergic neurons is shown in Figure 7–8.

Released glutamate is taken up by astrocytes and converted to glutamine, which passes b ack to the neurons and is converted back to

glutamate, which is released as the synaptic transmitter. Uptake into neurons and astrocy tes is the main mechanism for removal of

glutamate from synapses.

Glutamate Receptors

Glutamate receptors are of two types: metabotropic receptors and ionotropic receptors. The metabo tropic receptors are G protein-

coupled receptors that increase intracellular IP

and

CLINICAL BOX 7-4 Schizophrenia

Schizophrenia is an illness that involves deficits of multiple brain systems that alter a n individual’s inner thoughts as well as their

interactions with others. Individuals with schizophrenia suffer from hallucinations, delus ions, and racing thoughts (positive symptoms);

and they experience apathy, difficulty dealing with novel situations, and little spontaneity or motivation (negative symptoms).

Worldwide, about 1–2% of the population lives with schizophrenia. A combination of genetic, biological, cultural, and psychological

factors contributes to the illness. A large amount of evidence indicates that a defect in the mesocortical system is responsible for the

development of at least some of the symptoms of schizophrenia. Attention was initially fo cused on overstimulation of limbic D

dopamine receptors. Amphetamine, which causes release of dopamine as well as norepine phrine in the brain, causes a

schizophrenialike psychosis; brain levels of D

receptors are said to be elevated in schizophrenics; and there is a clear positive

correlation between the antischizophrenic activity of many drugs and their ability to bloc k D

receptors. However, several recently

developed drugs are effective antipsychotic agents but bind D

receptors to a limited degree. Instead, they bind to D

receptors, and there

is active ongoing research into the possibility that these receptors are abnormal in individ uals with schizophrenia.

Lactate Capillary

Glucose

34 ATP

Gln

Gln Lactate

Glu ATP

Glu Glu ATP

3Na

+ 3Na

2K+

Glutaminergic synapse

Astrocyte

FIGURE 7–8 The glutamate–glutamine cycle through glutaminergic neurons and as trocytes. Glutamate released into the synaptic

cleft is taken up by a Na

-dependent glutamate transporter, and in the astrocyte it is converted to glutamine. The glutamin e enters the

neuron and is converted to glutamate. Glucose is transported out of capillaries and enters astrocytes and neurons. In astrocytes, it is

metabolized to lactate, producing two ATPs. One of these powers the conversion of glut amate to glutamine, and the other is used by

– K

ATPase to transport three Na

out of the cell in exchange for two K

. In neurons, the glucose is metabolized further through

the citric acid cycle, producing 34 ATPs.

DAG levels or decrease intracellular cAMP levels. Eleven subtypes have been identified (Table 7–2). They are both presynaptic and

postsynaptic, and they are widely distributed in the brain. They appear to be involved in the production of synaptic plasticity, particularly

in the hippocampus and the cerebellum. Knockout of the gene for one of these recepto rs, one of the forms of mGluR1, causes severe

motor incoordination and deficits in spatial learning.

The ionotropic receptors are ligand-gated ion channels that resemble nicotinic cholinerg ic receptors and GABA and glycine receptors.

There are three general types, each named for the congeners of glutamate to which the y respond in maximum fashion. These are the

kainate receptors (kainate is an acid isolated from seaweed), AMPA receptors (for αα hydroxy-5-methylisoxazole-4-propionate), and

NMDA receptors (for Nmethyl-D-aspartate). Four AMPA, five kainate, and s ix NMDA subunits have been identified, and each is coded

by a different gene. The receptors were initially thought to be pentamers, but some may be tetramers, and their exact stoichiometry is

unsettled.

The kainate receptors are simple ion channels that, when open, permit Na

influx and K

efflux. There are two populations of AMPA

receptors: one is a simple Na

channel and one also passes Ca

. The balance between the two in a given synapse can be shifted by

activity.

The NMDA receptor is also a cation channel, but it permits passage of relatively large a mounts of Ca

, and it is unique in several ways

(Figure 7–9). First, glycine facilitates its function by binding to it, and glycine appears to be essential for its normal response to

glutamate. Second, when glutamate binds to it, it opens, but at normal membrane potentia ls, its channel is blocked by a Mg

ion. This

block is removed only when the neuron containing the receptor is partially depolarized by activation of AMPA or other channels that

produce rapid depolarization via other synaptic circuits. Third, phencyclidine and ketam ine, which produce amnesia and a feeling of

dissociation from the environment, bind to another site inside the channel. Most target ne urons for glutamate have both AMPA and

NMDA receptors. Kainate receptors are located presynaptically on GABA-secreting n erve endings and postsynaptically at various

localized sites in the brain. Kainate and AMPA receptors are found in glia as well as neu rons, but it appears that NMDA receptors occur

only in neurons.

The concentration of NMDA receptors in the hippocampus is high, and blockade of th ese receptors prevents long-term potentiation, a

long-lasting facilitation of transmission in neural pathways following a brief period of h igh-frequency stimulation. Thus, these receptors

may well be involved in memory and learning.

GABA

GABA is the major inhibitory mediator in the brain, including being responsible for pre synaptic inhibition. GABA, which exists as β-

aminobutyrate in the body fluids, is formed

L-Glutamate Ca

K + Glycine

Extracellular MK-801

Intracellular

Channel blocker

Open ion channel

Closed ion channel

FIGURE 7–9 Diagrammatic representation of the NMDA receptor. When glyc ine and glutamate bind to the receptor, the closed ion

channel (left) opens, but at the resting membrane potential, the channel is bloc ked by Mg

(right). This block is removed if partial

depolarization is produced by other inputs to the neuron containing the receptor, and C a

and Na

enter the neuron. Blockade can also

be produced by the drug dizocilpine maleate (MK-801).

by decarboxylation of glutamate (Figure 7–1). The enzyme that catalyzes this reaction is glutamate decarboxylase (GAD), which is

present in nerve endings in many parts of the brain. GABA is metabolized primarily by transamination to succinic semialdehyde and

thence to succinate in the citric acid cycle. GABA transaminase (GABA-T) is the enzyme that catalyzes the transamination. Pyridoxal

phosphate, a derivative of the B complex vitamin pyridoxine, is a cofactor for GAD an d GABA-T. In addition, there is an active

reuptake of GABA via the GABA transporter. A vesicular GABA transporter (VGAT ) transports GABA and glycine into secretory

vesicles.

GABA Receptors

Three subtypes of GABA receptors have been identified: GABA

, GABA

, and GABA

. The GABA

and GABA

receptors are

widely distributed in the CNS, whereas in adult vertebrates the GABA

receptors are found almost exclusively in the retina. The

GABA

and GABA

receptors are ion channels made up of five subunits surrounding a pore, like the nicot inic acetylcholine receptors

and many of the glutamate receptors. In this case, the ion is Cl

–

(Figure 7–10). The GABA

receptors are metabotropic and are coupled

to heterotrimeric G proteins that increase conductance in K

channels, inhibit adenylyl cyclase, and inhibit Ca

influx. Increases in Cl

–

influx and K

efflux and decreases in Ca

influx all hyperpolarize neurons, producing an IPSP. The G protein mediation of GAB A

receptor effects is unique in that a G protein heterodimer, rather than a single protein, is involved.

The GABA

receptors are relatively simple in that they are pentamers of three ρ subunits in various combinations. On

GABA

Extracellular

K+ Ca

α α

γ γ

−

Cl−

COOH

Intracellular

FIGURE 7–10 Diagram of GABA

and GABA

receptors, showing their principal actions. The G protein that mediates the effects

of GABA

receptors is a heterodimer. (Reproduced with permission from Bowery NG, Brown DA: T he cloning of GABA

receptors. Nature 1997;386:223.

the other hand, the GABA

receptors are pentamers made up of various combinations of six α subunits, four β, fo ur γ, one δ, and one ε.

This endows them with considerably different properties from one location to another.

An observation of considerable interest is that there is a chronic low-level stimulation of GABA

receptors in the CNS that is aided by

GABA in the interstitial fluid. This background stimulation cuts down on the “noise” cau sed by incidental discharge of the billions of

neural units and greatly improves the signal-to-noise ratio in the brain. It may be that this GABA discharge declines with advancing age,

resulting in a loss of specificity of responses of visual neurons. Support for this hypoth esis comes from studies in which microinjection of

GABA in older monkeys resulted in restoration of the specificity of visual neurons.

The increase in Cl

–

conductance produced by GABA

receptors is potentiated by benzodiazepines, drugs that have marked anti-anxiety

activity and are also effective muscle relaxants, anticonvulsants, and sedatives. Benzodia zepines bind to the α subunits. Diazepam and

other benzodiazepines are used throughout the world. At least in part, barbiturates and alcohol also act by facilitating Cl

–

conductance

through the Cl

–

channel. Metabolites of the steroid hormones progesterone and deoxycorticosterone b ind to GABA

receptors and

increase Cl

–

conductance. It has been known for many years that progesterone and deoxycorticos terone are sleep-inducing and

anesthetic in large doses, and these effects are due to their action on GABA

receptors.

A second class of benzodiazepine receptors is found in steroid-secreting endocrine gla nds and other peripheral tissues, and hence these

receptors are called peripheral benzodiazepine receptors. They may be involved in s teroid biosynthesis, possibly performing a

function like that of the StAR protein in moving steroids into the mitochondria. Another possibility is a role in the regulation of cell

proliferation. Peripheral-type benzodiazepine receptors are also present in astrocytes in the brain, and they are found in brain tumors.

Glycine

Glycine has both excitatory and inhibitory effects in the CNS. When it binds to NMDA receptors, it makes them more sensitive. It

appears to spill over from synaptic junctions into the interstitial fluid, and in the spinal cor d, for example, this glycine may facilitate pain

transmission by NMDA receptors in the dorsal horn. However, glycine is also respons ible in part for direct inhibition, primarily in the

brain stem and spinal cord. Like GABA, it acts by increasing Cl

–

conductance. Its action is antagonized by strychnine. The clinical

picture of convulsions and muscular hyperactivity produced by strychnine emphasizes the importance of postsynaptic inhibition in

normal neural function. The glycine receptor responsible for inhibition is a Cl

–

channel. It is a pentamer made up of two subunits: the

ligand-binding α subunit and the structural β subunit. Recently, solid evidence has been presented that three kinds of neurons are

responsible for direct inhibition in the spinal cord: neurons that secrete glycine, neurons that secrete GABA, and neurons that secrete

both. Presumably, neurons that secrete only glycine have the glycine transporter GLYT 2, those that secrete only GABA have GAD, and

those that secrete glycine and GABA have both. This third type of neuron is of special interest because the neurons seem to have glycine

and GABA in the same vesicles.

Anesthesia

Although general anesthetics have been used for millennia, little has been understood ab out their mechanisms of action. However, it now

appears that alcohols, barbiturates, and many volatile inhaled anesthetics as well act on io n channel receptors and specifically on

GABA

and glycine receptors to increase Cl

–

conductance. Regional variation in anesthetic actions in the CNS seems to parallel the

variation in subtypes of GABA

receptors. Other inhaled anesthetics do not act by increasing GABA receptor activity, b ut appear to act

by inhibiting NMDA and AMPA receptors instead.

In contrast to general anesthetics, local anesthetics produce anesthesia by blocking cond uction in peripheral nerves via reversibly binding

to and inactivating Na

channels. Na

influx through these channels normally causes depolarization of nerve cell membranes and

propagation of impulses toward the nerve terminal. When depolarization and propagati on are interrupted, the individual loses sensation

in the area supplied by the nerve.

pled receptors. Activation of the substance P receptor causes activation of phospholipas e C and increased formation of IP

and DAG.

Substance P is found in high concentration in the endings of primary afferent neurons in the spinal cord, and it is probably the mediator

at the first synapse in the pathways for pain transmission in the dorsal horn. It is also fou nd in high concentrations in the nigrostriatal

system, where its concentration is proportional to that of dopamine, and in the hypothala mus, where it may play a role in neuroendocrine

regulation. Upon injection into the skin, it causes redness and swelling, and it is probably the mediator released by nerve fibers that is

responsible for the axon reflex. In the intestine, it is involved in peristalsis. It has recently been reported that a centrally active NK-1

receptor antagonist has antidepressant activity in humans. This antidepressant effect take s time to develop, like the effect of the

antidepressants that affect brain monoamine metabolism, but the NK-1 inhibitor does no t alter brain monoamines in experimental

animals. The functions of the other tachykinins are unsettled.

LARGE-MOLECULE TRANSMITTERS: NEUROPEPTIDES

Substance P & Other Tachykinins

Substance P is a polypeptide containing 11 amino acid residues that is found in the intesti ne, various peripheral nerves, and many parts

of the CNS. It is one of a family of six mammalian polypeptides called tachykinins that d iffer at the amino terminal end but have in

common the carboxyl terminal sequence of Phe-X-Gly-LeuMet-NH

, where X is Val, His, Lys, or Phe. The members of the family are

listed in Table 7–3. There are many related tachykinins in other vertebrates and in inver tebrates.

The mammalian tachykinins are encoded by two genes. The neurokinin B gene encodes only o ne known polypeptide, neurokinin B.

The substance P/neurokinin A gene encodes the remaining five polypeptides. Three are formed by alternative processing of the

primary RNA and two by posttranslational processing.

There are three neurokinin receptors. Two of these, the substance P and the neuropep tide K receptors, are G protein-cou

Opioid Peptides

The brain and the gastrointestinal tract contain receptors that bind morphine. The search for endogenous ligands for these receptors led to

the discovery of two closely related pentapeptides (enkephalins; Table 7–4) that bind to th ese opioid receptors.

TABLE 7–4 Opioid peptides and their precursors.

Precursor Opioid

Peptides Structures

Proenkephalin Tyr-Gly-Gly-Phe-Met Met

enkephalin

Leu

enkephalin Tyr-Gly-Gly-Phe-Leu

Octapeptide Tyr-Gly-Gly-Phe-Met-Arg-GlyLeu

Heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe

TABLE 7–3 Mammalian tachykinins.

Proopiomelanocortin β-Endorphin Tyr-Gly-Glu-Phe-Met-Thr-Ser

Lys-Ser-Gln-Thr-Pro-Leu-ValThr-Leu-Phe-Lys-Asn-Ala-Ile-ValLys-Asn-Ala-His-L ys-Lys-Gly-Gln

Gene Polypeptide Products

SP/NKA Substance P Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lle

Receptors

Substance P (NK-1) Prodynorphin Dynorphin 1–8

Neurokinin A

Neuropeptide K Neuropeptide K (NK-2)

Dynorphin 1–17

Tyr-Gly-Gly-Phe-Leu-Arg-Arglle-Arg-Pro-Lys-Leu-Lys-TrpAsp-Asn-Gln

Neuropeptide α α-Neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-LysTyr-Pro-Lys

Neurokinin A (3–10)

NKB Neurokinin B Neurokinin B (NK-3)

-Neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-LysTyr-Pro

One contains methionine (met-enkephalin), and one contains leucine (leu-enkephalin). These and oth er peptides that bind to opioid

receptors are called opioid peptides. The enkephalins are found in nerve endings i n the gastrointestinal tract and many different parts of

the brain, and they appear to function as synaptic transmitters. They are found in the su bstantia gelatinosa and have analgesic activity

when injected into the brain stem. They also decrease intestinal motility.

Like other small peptides, the opioid peptides are synthesized as part of larger precurso r molecules. More than 20 active opioid peptides

have been identified. Unlike other peptides, however, the opioid peptides have a numb er of different precursors. Each has a prepro form

and a pro form from which the signal peptide has been cleaved. The three precursors that have been characterized, and the opioid

peptides they produce, are shown in Table 7–4. Proenkephalin was first identified in the adrenal medulla , but it is also the precursor for

met-enkephalin and leu-enkephalin in the brain. Each proenkephalin molecule contains four met-enkephalins, one leuenkephalin, one

octapeptide, and one heptapeptide. Proopiomelanocortin, a large precur sor molecule found in the anterior and intermediate lobes of the

pituitary gland and the brain, contains β-endorphin, a polypeptide of 31 amino acid res idues that has metenkephalin at its amino terminal.

There are separate enkephalin-secreting and β endorphin-secreting systems of neurons in the brain. β-Endorphin is also secreted into the

bloodstream by the pituitary gland. A third precursor molecule is prodynorphin, a protein that contains three leuenkephalin residues

associated with dynorphin and neoendorphin. Dynorphin 1-17 is found in the duoden um and dynorphin 1-8 in the posterior pituitary and

hypothalamus. Alpha- and β-neoendorphins are also found in the hypothalamus. The r easons for the existence of multiple opioid peptide

precursors and for the presence of the peptides in the circulation as well as in the brain and the gastrointestinal tract are presently

unknown.

Enkephalins are metabolized primarily by two peptidases: enkephalinase A, which splits the Gly-Phe bond, and enkephalinase B, which

splits the Gly-Gly bond. Aminopeptidase, which splits the Tyr-Gly bond, also contribute s to their metabolism.

Opioid receptors have been studied in detail, and three are now established: μ, κ, and δ . They differ in physiologic effects (Table 7–5),

distribution in the brain and elsewhere, and affinity for various opioid peptides. All thre e are G proteincoupled receptors, and all inhibit

adenylyl cyclase. In mice in which the μ receptors have been knocked out, morphine f ails to produce analgesia, withdrawal symptoms,

and self-administration of nicotine. Selective knockout of the other system fails to produ ce this blockade. Activation of μ receptors

increases K

conductance, hyperpolarizing central neurons and primary afferents. Activation of κ r eceptors and δ receptors closes Ca

channels.

The affinities of individual ligands for the three types of receptors are summarized in Fi gure 7–11. Endorphins bind only to μ receptors,

the main receptors that mediate analgesia. Other opioid peptides bind to multiple opioid r eceptors.

TABLE 7–5 Physiologic effects produced by stimulation of opiate receptors.

Receptor Effect

μ Analgesia

Site of action of morphine

Respiratory depression

Constipation

Euphoria

Sedation

Increased secretion of growth hormone and prolactin

Meiosis

κ Analgesia

Diuresis

Sedation

Meiosis

Dysphoria

δ Analgesia

Other Polypeptides

Numerous other polypeptides are found in the brain. For example, somatostatin is foun d in various parts of the brain, where it apparently

functions as a neurotransmitter with effects on sensory input, locomotor activity, and co gnitive function. In the hypothalamus, this

growth hormone-inhibiting hormone is secreted into the portal hypophysial vessels; in th e endocrine pancreas, it inhibits insulin

secretion and the secretion of other pancreatic hormones; and in the gastrointestinal trac t, it is an important inhibitory gastrointestinal

regulator. A family of five different somatostatin receptors have been

Endomorphins

Dynorphins β-Endorphin Enkephalins

κ δ

FIGURE 7–11 Opioid receptors. The ligands for the κ, μ, and δ receptors are sh own with the width of the arrows proportionate to the

Macmillan Magazines.)

identified (SSTR1 through SSTR5). All are G protein-coupled receptors. They inhibit a denylyl cyclase and exert various other effects on

intracellular messenger systems. It appears that SSTR2 mediates cognitive effects and inh ibition of growth hormone secretion, whereas

SSTR5 mediates the inhibition of insulin secretion.

Vasopressin and oxytocin are not only secreted as hormones but also are present in ne urons that project to the brain stem and spinal cord.

The brain contains bradykinin, angiotensin II, and endothelin. The gastrointestinal horm ones VIP, CCK-4, and CCK-8 are also found in

the brain. There are two kinds of CCK receptors in the brain, CCK-A and CCK-B. CC K-8 acts at both binding sites, whereas CCK-4 acts

at the CCK-B sites. Gastrin, neurotensin, galanin, and gastrin-releasing peptide are also found in the gastrointestinal tract and brain.

Neurotensin and VIP receptors have been cloned and shown to be G protein-coupled receptors. The hypothalamus contains both gastrin

17 and gastrin 34. VIP produces vasodilation and is found in vasomotor nerve fibers. The functions of these peptides in the nervous

system are unknown.

Calcitonin gene-related peptide (CGRP) is a polypeptide that exists in two forms in ra ts and humans: CGRPα and CGRPβ. In humans,

these two forms differ by only three amino acid residues, yet they are encoded by diff erent genes. In rats, and presumably in humans,

CGRPβ is present in the gastrointestinal tract, whereas CGRPβ is found in primary affer ent neurons, neurons that project which taste

impulses to the thalamus, and neurons in the medial forebrain bundle. It is also present a long with substance P in the branches of

primary afferent neurons that end near blood vessels. CGRP-like immunoreactivity is p resent in the circulation, and injection of CGRP

causes vasodilation. CGRPα and the calcium-lowering hormone calcitonin are both prod ucts of the calcitonin gene. In the thyroid gland,

splicing produces the mRNA that codes for calcitonin, whereas in the brain, alternative splicing produces the mRNA that codes for

CGRPα. CGRP has little effect on Ca

metabolism, and calcitonin is only a weak vasodilator.

Neuropeptide Y is a polypeptide containing 36 amino acid residues that acts on at le ast two of the four known G proteincoupled

receptors: Y

, Y

, and Y

. Neuropeptide Y is found throughout the brain and the autonomic nervous system. W hen injected into the

hypothalamus, this polypeptide increases food intake, and inhibitors of neuropeptide Y synthesis decrease food intake. Neuropeptide Y-

containing neurons have their cell bodies in the arcuate nuclei and project to the parave ntricular nuclei.

OTHER CHEMICAL TRANSMITTERS Purine & Pyrimidine Transmitters

After extended debate, it now seems clear that ATP, uridine, adenosine, and adenosine metabolites are neurotransmitters or

neuromodulators. Adenosine is a neuromodulator that acts as a general CNS depressan t and has additional widespread effects throughout

the body. It acts on four receptors: A

, A

, and A

. All are G protein-coupled receptors and increase (A

and A

) or decrease

and A

) cAMP concentrations. The stimulatory effects of coffee and tea are due to blockade o f adenosine receptors by caffeine and

theophylline. Currently, there is considerable interest in the potential use of A

antagonists to decrease excessive glutamate release and

thus to minimize the effects of strokes.

ATP is also becoming established as a transmitter, and it has widespread receptor-mediat ed effects in the body. It appears that soluble

nucleotidases are released with ATP, and these accelerate its removal after it has produc ed its effects. ATP has now been shown to

mediate rapid synaptic responses in the autonomic nervous system and a fast response i n the habenula. ATP binds to P2X receptors

which are ligand-gated ion channel receptors; seven subtypes (P2X

–P2X

) have been identified. P2X receptors have widespread

distributions throughout the body; for example, P2X

and P2X

receptors are present in the dorsal horn, indicating a role for ATP in

sensory transmission. ATP also binds to P2Y receptors which are G proteincoupled rec eptors. There are eight subtypes of P2Y receptors:

P2Y

, P2Y

, and P2Y

Cannabinoids

Two receptors with a high affinity for Δ

-tetrahydrocannabinol (THC), the psychoactive ingredient in marijuana, have been clon ed. The

receptor triggers a G protein-mediated decrease in intracellular cAMP levels and is com mon in central pain pathways as well as in

parts of the cerebellum, hippocampus, and cerebral cortex. The endogenous ligand for the receptor is anandamide, a derivative of

arachidonic acid. This compound mimics the euphoria, calmness, dream states, drowsine ss, and analgesia produced by marijuana. There

are also CB

receptors in peripheral tissues, and blockade of these receptors reduces the vasodilator effect of anandamide. However, it

appears that the vasodilator effect is indirect. A CB

receptor has also been cloned, and its endogenous ligand may be

palmitoylethanolamide (PEA). However, the physiologic role of this compound is unsettled.

Gases

Nitric oxide (NO), a compound released by the endothelium of blood vessels as endoth elium-derived relaxing factor (EDRF), is also

produced in the brain. It is synthesized from arginine, a reaction catalyzed in the brain b y one of the three forms of NO synthase. It

activates guanylyl cyclase and, unlike other transmitters, it is a gas, which crosses cell me mbranes with ease and binds directly to

guanylyl cyclase. It may be the signal by which postsynaptic neurons communicate with presynaptic endings in long-term potentiation

and long-term depression. NO synthase requires NADPH, and it is now known that NA DPH-diaphorase (NDP), for which a

histochemical stain has been available for many years, is NO synthase.

Other Substances

Prostaglandins are derivatives of arachidonic acid found in the nervous system. They are present in nerve-ending fractions of brain

homogenates and are released from neural tissue in vitro. A putative prostaglandin tran sporter with 12 membrane-spanning domains has

been described. However, prostaglandins appear to exert their effects by modulating r eactions mediated by cAMP rather than by

functioning as synaptic transmitters.

Many steroids are neuroactive steroids; that is, they affect brain fun ction, although they are not neurotransmitters in the usual sense.

Circulating steroids enter the brain with ease, and neurons have numerous sex steroid a nd glucocorticoid receptors. In addition to acting

in the established fashion by binding to DNA (genomic effects), some steroids seem to a ct rapidly by a direct effect on cell membranes

(nongenomic effects). The effects of steroids on GABA receptors have been discusse d previously. Evidence has now accumulated that

the brain can produce some hormonally active steroids from simpler steroid precursors , and the term neurosteroids has been coined to

refer to these products. Progesterone facilitates the formation of myelin, but the exact ro le of most steroids in the regulation of brain

function remains to be determined.

CHAPTER SUMMARY

■ Neurotransmitters and neuromodulators are divided into two major categories: small-mo lecule transmitters (monoamines,

catecholamines, and amino acids) and large-molecule transmitters (neuropeptides). Usu ally neuropeptides are colocalized with one of the

small-molecule neurotransmitters.

■ Monoamines include acetylcholine, serotonin, and histamine. Catecholami nes include norepinephrine, epinephrine, and dopamine.

Amino acids include glutamate, GABA, and glycine.

■ Acetylcholine is found at the neuromuscular junction, in autonomic ganglia, and in postganglionic parasympathetic nervetarget organ

junctions and some postganglionic sympathetic nerve-target junctions. It is also found in the basal forebrain complex and

pontomesencephalic cholinergic complex. There are two major types of cholinergic receptors: muscarinic (G protein-coupled receptors)

and nicotinic (ligand-gated ion channel receptors).

■ Serotonin (5-HT) is found within the brain stem in the midline raphé nuclei w hich project to portions of the hypothalamus, the limbic

system, the neocortex, the cerebellum, and the spinal cord. There are at least seven types of 5-HT receptors, and many of these contain

subtypes. Most are G protein-coupled receptors.

■ Norepinephrine-containing neurons are in the locus ceruleus and other medullary and p ontine nuclei. Some neurons also contain

PNMT, which catalyzes the conversion of norepinephrine to epinephrine. Epinephrine and norepinephrine act on α and β receptors, with

norepinephrine having a greater affinity for α-adrenergic receptors and epinephrin e for β-adrenergic receptors. They are G protein-

coupled receptors, and each has multiple forms.

■ The amino acid glutamate is the main excitatory transmitter in the CNS. There are two ma jor types of gluatamate receptors:

metabotropic (G protein-coupled receptors) and ionotropic (ligand-gated ion channels recep tors, including kainite, AMPA, and NMDA).

■ GABA is the major inhibitory mediator in the brain. Three subtypes of GABA rec eptors have been identified: GABA

and GABA

(ligand-gated ion channel) and GABA

(G proteincoupled). The GABA

and GABA

receptors are widely distributed in the CNS.

■ There are three types of G protein-coupled opioid receptors (μ, κ, and δ) that dif fer in physiological effects, distribution in the brain

and elsewhere, and affinity for various opioid peptides.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. Which of the following is a ligand-gated ion channel? A) VIP

receptor

B) norepinephrine receptor

C) GABA

receptor

D) GABA

receptor

E) metabotropic glutamate receptor

2. Which of the following synaptic transmitters is not a peptide, polypeptide, or p rotein?

A) substance P

B) met-enkephalin

C) β-endorphin

D) serotonin

E) dynorphin

3. Activation of which of the following receptors would be expected to decrease anxie ty?

A) nicotinic cholinergic receptors

B) glutamate receptors

C) GABA

receptors

D) glucocorticoid receptors

E) α

-adrenergic receptors

4. Which of the following receptors is coupled to a heterotrimeric G protein?

A) glycine receptor

B) GABA

receptor

C) nicotinic acetylcholine receptor at myoneural junction D) 5-HT

receptor

E) ANP receptor

5. Which of the following would not be expected to enhance noradrenergic tra nsmission?

A) A drug that increases the entry of arginine into neurons. B) A drug that enhances ty rosine hydroxylase activity. C) A drug that

enhances dopamine β-hydroxylase activity. D) A drug that inhibits monoamine oxidase .

E) A drug that inhibits norepinephrine reuptake.

CHAPTER RESOURCES

Boron WF, Boulpaep EL: Medical Physiology. Elsevier, 2005. Cooper JR, Bloom FE, Roth RH: The Biochemical Basis of

Neuropharmacology, 8th ed. Oxford University Press, 2002. Fink KB, Göthert M: 5-HT recep tor regulation of neurotransmitter

release. Pharmacol Rev 2007;59:360.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural

Science, 4th ed. McGraw-Hill, 2000.

Monaghan DT, Bridges RJ, Cotman CW: The excitatory amino acid

receptors: Their classes, pharmacology, and distinct properties in

the function of the central nervous system. Ann Rev Pharmacol

Toxicol 1989;29:365.

Nadeau SE, et al: Medical Neuroscience, Sauders, 2004.

Olsen RW: The molecular mechanism of action of general

anesthetics: Structural aspects of interactions with GABA

receptors. Toxicol Lett 1998;100:193.

Owens DF, Kriegstein AR: Is there more to GABA than synaptic

inhibition? Nat Rev Neurosci 2002;3:715.

Squire LR, et al (editors): Fundamental Neuroscience, 3rd ed.

Academic Press, 2008.

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Properties of Sensory Receptors

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Describe the classification of sensory receptors.

■

Name the types of sensory receptors found in the skin, and discuss their relation to touc h, cold, warmth, and pain.

■

Define generator potential.

■

Explain the essential elements of sensory coding.

INTRODUCTION

Information about the internal and external environment activates the CNS via a variety of sensory receptors. These receptors are

transducers that convert various forms of energy in the environment into action potentia ls in neurons. The characteristics of these

receptors, the way they generate impulses in afferent neurons, and the general principl es or “laws” that apply to sensation are considered

in this chapter. Emphasis is placed on receptors mediating the sensation of touch, and lat er chapters focus on other sensory processes.

We learn in elementary school that there are “five senses,” but this dictum takes into acc ount only some of the senses that reach our

consciousness. In addition, some sensory receptors relay information that does not reac h consciousness. For example, the muscle

spindles provide information about muscle length, and other receptors provide informa tion about arterial blood pressure, the temperature

of the blood in the head, and the pH of the cerebrospinal fluid. The list of senses in Tab le 8–1 is somewhat simplified. The rods and

cones, for example, respond maximally to light of different wavelengths, and three diff erent types of cones are present, one for each of

the three primary colors. There are five different modalities of taste: sweet, salt, sour, bi tter, and umami. Sounds of different pitches are

heard primarily because different groups of hair cells in the cochlea are activated maxim ally by sound waves of different frequencies.

Whether these various responses to light, taste, and sound should be considered separa te senses is a semantic question that in the present

context is largely academic.

SENSE RECEPTORS

& SENSE ORGANS

It is worth noting that the term receptor is used in physiology to refer not only to sensory recept ors but also, in a very different sense, to

proteins that bind neurotransmitters, hormones, and other substances with great affinity and specificity as a first step in initiating specific

physiologic responses.

CLASSIFICATION OF

SENSORY RECEPTORS

Numerous attempts have been made to classify sensory receptors, but none has been en tirely successful. One classification divides them

into (1) teleceptors (“distance receivers”), which are concerned with events at a distanc e; (2) exteroceptors, which are concerned with the

external environment near at hand; (3) interoceptors, which are concerned with the inte rnal

149

TABLE 8–1 Principle sensory modalities.

Sensory System Modality Stimulus Energy Receptor Class Receptor Cell Types

Somatosensory Touch Tap, flutter 5–40 Hz Cutaneous mechanoreceptor Meissner cor puscles

Somatosensory Touch Motion Cutaneous mechanoreceptor Hair follicle receptors

Somatosensory Touch Deep pressure, vibration 60–300 Hz

Cutaneous mechanoreceptor Pacinian corpuscles

Somatosensory Touch Touch, pressure Cutaneous mechanoreceptor Merkel cells

Somatosensory Touch Sustained pressure Cutaneous mechanoreceptor Ruffini corpusc les

Somatosensory Proprioception Stretch Mechanoreceptor Muscle spindles

Somatosensory Proprioception Tension Mechanoreceptor Golgi tendon organ

Somatosensory Temperature Thermal Thermoreceptor Cold and warm receptors

Somatosensory Pain

Chemical, thermal, and mechanical

Chemoreceptor, thermoreceptor, and mechanoreceptor Polymodal receptors or chemic al, thermal, and mechanical nociceptors

Somatosensory Itch Chemical Chemoreceptor Chemical nociceptor

Visual Vision Light Photoreceptor Rods, cones

Auditory Hearing Sound Mechanoreceptor Hair cells (cochlea)

Vestibular Balance Angular acceleration Mechanoreceptor Hair cells (semicircular cana ls)

Vestibular Balance Linear acceleration, gravity Mechanoreceptor Hair cells (otolith org ans)

Olfactory Smell Chemical Chemoreceptor Olfactory sensory neuron

Gustatory Taste Chemical Chemoreceptor Taste buds

environment; and (4) proprioceptors, which provide information about the position of the body in space at any given instant. However,

the conscious component of proprioception (“body image”) is actually synthesized from information coming not only from receptors in

and around joints but also from cutaneous touch and pressure receptors.

Other special terms are frequently used to identify sensory receptors. The cutaneous re ceptors for touch and pressure are

mechanoreceptors. Potentially harmful stimuli such as pain, extreme heat, and extreme cold are said to be mediated by nociceptors.

The term chemoreceptor is used to refer to receptors stimulated by a change in the chemical composition of the environment in which

they are located. These include receptors for taste and smell as well as visceral receptors such as those sensitive to changes in the plasma

level of O

, pH, and osmolality. Photoreceptors are those in the rods and con es in the retina that respond to light.

SENSE ORGANS

Sensory receptors can be specialized dendritic endings of afferent nerve fibers, and th ey are often associated with nonneural cells that

surround it, forming a sense organ. Touch and pressure are sensed by four types o f mechanoreceptors (Figure 8–1). Meissner

corpuscles are dendrites encapsulated in connective tissue and respond to changes in tex ture and slow vibrations. Merkel cells are

expanded dendritic endings, and they respond to sustained pressure and touch. Ruffin i corpuscles are enlarged dendritic endings with

elongated capsules, and they respond to sustained pressure. Pacinian corpuscles consist of unmyelinated dendritic endings of a sensory

nerve fiber, 2 μm in diameter, encapsulated by concentric lamellae of connective tissue that give the organ the appearance of a cocktail

onion. Theses receptors respond to deep pressure and fast vibration.

The Na

channel BNC1 is closely associated with touch receptors. This channel is one of the degenerins, so called because when they

are hyperexpressed, they cause the neurons they are in to degenerate. However, it is n ot known if BNC1 is part of the receptor complex

or the neural fiber at the point of initiation of the spike potential. The receptor may be o pened mechanically by pressure on the skin.

Some sensory receptors are not specialized organs but rather are free nerve endings. P ain and temperature sensations arise from

unmyelinated dendrites of sensory neurons located around hair follicles throughout the glaborous and hairy skin as well as deep tissue.

A Modality Touch

Receptors

Meissner’s Merkel Pacinian corpuscle cells corpuscle Ruffini endings

B Location

Receptive field

C Intensity and time course

Neural

spike train

Stimulus

FIGURE 8–1 Sensory systems encode four elementary attributes of stimuli: m odality, location (receptive field), intensity, and

duration (timing). A) The human hand has four types of mechanoreceptors; their combined act ivation produces the sensation of contact

with an object. Selective activation of Merkel cells and Ruffini endings causes sensation of steady pressure; selective activation of

Meissner’s and Pacinian corpuscles causes tingling and vibratory sensation. B) Location of a stim ulus is encoded by spatial distribution

of the population of receptors activated. A receptor fires only when the skin close to its sensory terminals is touched. These receptive

fields of mechanoreceptors (shown as red areas on fingertips) differ in size and respo nse to touch. Merkel cells and Meissner’s corpuscles

provide the most precise localization as they have the smallest receptive fields and are most sens itive to pressure applied by a small

probe. C) Stimulus intensity is signaled by firing rates of individual receptors; duration of stimu lus is signaled by time course of firing.

The spike trains indicate action potentials elicited by pressure from a small probe at the cen ter of each receptive field. Meissner’s and

Pacinian corpuscles adapt rapidly, the others adapt slowly. (From Kandel ER, Schwartz JH, Jessell TM [ed itors]: Principles of Neural Science, 4th ed.

McGraw-Hill, 2000.)

GENERATION OF IMPULSES IN CUTANEOUS RECEPTORS

PACINIAN CORPUSCLES

The way receptors generate action potentials in the sensory nerves that innervate them v aries with the complexity of the sense organ. In

the skin, the Pacinian corpuscle has been studied in some detail. As noted above, the Pac inian corpuscles are touch receptors. Because of

their relatively large size and accessibility, they can be isolated, studied with microelectrod es, and subjected to microdissection. The

myelin sheath of the sensory nerve begins inside the corpuscle (Figure 8–2). The first n ode of Ranvier is also located inside, whereas the

second is usually near the point at which the nerve fiber leaves the corpuscle.

GENERATOR POTENTIALS

Recording electrodes can be placed on the sensory nerve as it leaves a Pacinian corpus cle and graded pressure applied to the corpuscle.

When a small amount of pressure is applied, a nonpropagated depolarizing potential res embling an EPSP is recorded. This is called the

generator potential or receptor potential (Figure 8–2). As the pressure is increased, the magn itude of the receptor potential increases.

When the magnitude of the generator potential is about 10 mV, an action potential is gen erated in the sensory nerve. As the pressure is

further increased, the generator potential becomes even larger and the sensory nerve f ires repetitively.

SOURCE OF THE GENERATOR POTENTIAL

By microdissection techniques, it has been shown that removal of the connective tissue l amellas from the unmyelinated nerve

e e

dcb

a a

3 4

dcb abcd a

FIGURE 8–2 Demonstration that the generator potential in a Pacinian corpus cle originates in the unmyelinated nerve terminal.

(1) The electrical responses to a pressure of 1

(record a), 2

(b), 3

(c), and 4

(d) were recorded. The strongest stimulus produced an

action potential in the sensory nerve (e). (2) Similar responses persisted after remo val of the connective tissue capsule, except that the

responses were more prolonged because of partial loss of adaptation. (3) The generator r esponses persisted but the action potential was

absent when the first node of Ranvier was blocked by pressure or with narcotics (arrow). (4) All responses disappeared when the sensory

nerve was cut and allowed to degenerate before the experiment.

ending in a Pacinian corpuscle does not abolish the generator potential. When the first n ode of Ranvier is blocked by pressure or

narcotics, the generator potential is unaffected but conducted impulses are abolished (Fi gure 8–2). When the sensory nerve is sectioned

and the nonmyelinated terminal is allowed to degenerate, no generator potential is forme d. These and other experiments have established

that the generator potential is produced in the unmyelinated nerve terminal. The recepto r therefore converts mechanical energy into an

electrical response, the magnitude of which is proportionate to the intensity of the stimulu s. The generator potential in turn depolarizes

the sensory nerve at the first node of Ranvier. Once the firing level is reached, an actio n potential is produced and the membrane

repolarizes. If the generator potential is great enough, the neuron fires again as soon as it repolarizes, and it continues to fire as long as

the generator potential is large enough to bring the membrane potential of the node to th e firing level. Thus, the node converts the graded

response of the receptor into action potentials, the frequency of which is proportionate to the magnitude of the applied stimuli.

SENSORY CODING

Converting a receptor stimulus to a recognizable sensation is termed sensory coding. All sen sory systems code for four elementary

attributes of a stimulus: modality, location, intensity, and duration. Modality is the type of energy tr ansmitted by the stimulus. Location

is the site on the body or space where the stimulus originated. Intensity is signaled by the response amp litude or frequency of action

potential generation. Duration refers to the time from start to end of a response in the receptor. The se attributes of sensory coding are

shown for the modality of touch in Figure 8–1.

MODALITY

Humans have four basic classes of receptors based on their sensitivity to one predomin ant form of energy: mechanical, thermal,

electromagnetic, or chemical. The particular form of energy to which a receptor is mos t sensitive is called its adequate stimulus. The

adequate stimulus for the rods and cones in the eye, for example, is light (an example o f electromagnetic energy). Receptors do respond

to forms of energy other than their adequate stimuli, but the threshold for these nonspec ific responses is much higher. Pressure on the

eyeball will stimulate the rods and cones, for example, but the threshold of these recepto rs to pressure is much higher than the threshold

of the pressure receptors in the skin.

LOCATION

The term sensory unit is applied to a single sensory axon and all its peri pheral branches. These branches vary in number but may be

numerous, especially in the cutaneous senses. The receptive field of a sensory unit is the spatial distribution from which a stimulus

produces a response in that unit (Figure 8–1). Representation of the senses in the skin is punctate. If the skin is carefully mapped,

millimeter by millimeter, with a fine hair, a sensation of touch is evoked from spots over lying these touch receptors. None is evoked

from the intervening areas. Similarly, temperature sensations and pain are produced by stimulation of

CLINICAL BOX 8–1 Two-Point Discrimination

The size of the receptive fields for light touch can be measured by the two-point threshold test. In this procedure, the two points on a

pair of calipers are simultaneously positioned on the skin and one determines the minimu m distance between the two caliper points that

can be perceived as separate points of stimulation. This is called the two-point discriminatio n threshold. If the distance is very small,

each caliper point is touching the receptive field of only one sensory neuron. If the dist ance between stimulation points is less than this

threshold, only one point of stimulation can be felt. Thus, the two-point discrimination th reshold is a measure of tactile acuity. The

magnitude of two-point discrimination thresholds varies from place to place on the body and is smallest where touch receptors are most

abundant. Stimulus points on the back, for instance, must be separated by at least 65 mm before they can be distinguished as separate,

whereas on the fingertips two stimuli are recognized if they are separated by as little as 2 mm. Blind individuals benefit from the tactile

acuity of fingertips to facilitate the ability to read Braille; the dots forming Braille symbols are separated by 2.5 mm. Two-point

discrimination is used to test the integrity of the dorsal column (medial lemnisc us) system, the central pathway for touch and

proprioception.

the skin only over the spots where the receptors for these modalities are located. In the cornea and adjacent sclera of the eye, the surface

area supplied by a single sensory unit is 50–200 mm

. Generally, the areas supplied by one unit overlap and interdigitate with the areas

supplied by others.

One of the most important mechanisms that enable localization of a stimulus site is lateral inhibition. Information from sensory neurons

whose receptors are at the peripheral edge of the stimulus is inhibited compared to infor mation from the sensory neurons at the center of

the stimulus. Thus, lateral inhibition enhances the contrast between the center and periph ery of a stimulated area and increases the ability

of the brain to localize a sensory input. Lateral inhibition underlies two-point discr imination (see Clinical Box 8–1).

INTENSITY

The intensity of sensation is determined by the amplitude of the stimulus applied to the re ceptor. This is illustrated in Figure 8–3. As a

greater pressure is applied to the skin, the receptor potential in the mechanoreceptor inc reases (not shown), and the frequency of the

action potentials in a single axon transmitting information to the CNS is also increased. In addition to increasing the firing rate in a single

axon, the greater intensity of stimulation also will recruit more receptors into the receptiv e field.

It has long been taught that the magnitude of the sensation felt is proportional to the log o f the intensity of the stimulus (Weber–Fechner

law). It now appears, however, that a power function more accurately describes this relation. In other words,

R = KS

where R is the sensation felt, S is the intensity of the stimulus, and, for any specific senso ry modality, K and A are constants. The

frequency of the action potentials generated in a sensory nerve fiber is also related to th e intensity of the initiating stimulus by a power

function. An example of this relation is shown is shown in Figure 8–4, in which the calc ulated exponent is 0.52. However, the relation

between direct stimulation of a sensory nerve and the sensation felt is linear. Consequen tly, it appears that for any given sensory

modality, the relation between sensation and stimulus intensity is determined primarily by the properties of the peripheral receptors.

DURATION

When a maintained stimulus of constant strength is applied to a receptor, the frequency of the action potentials in its sensory nerve

declines over time. This phenomenon is known as adaptation or desensitization. The degre e to which adaptation occurs varies from one

sense to another. Receptors can be classified into rapidly adapting (phasic) receptors and slowly adapti ng (tonic) receptors. This is

illustrated for different types of touch receptors in Figure 8–1. Meissner and Pacinian c orpuscles are examples of rapidly adapting

receptors, and Merkel cells and Ruffini endings are examples of slowly adapting recep tors. Other examples of slowly adapting receptors

are muscle spindles and nociceptors. Different types of sensory adaptation appear to ha ve some value to the individual. Light touch

would be distracting if it were persistent; and, conversely, slow adaptation of spindle inp ut is needed to maintain posture. Similarly, input

from nociceptors provides a warning that would lose its value if it adapted and disappea red.

SENSORY INFORMATION

The speed of conduction and other characteristics of sensory nerve fibers vary, but ac tion potentials are similar in all nerves. The action

potentials in the nerve from a touch receptor, for example, are essentially identical to tho se in the nerve from a warmth receptor. This

raises the question of why stimulation of a touch receptor causes a sensation of touch an d not of warmth. It also raises the question of

how it is possible to tell whether the touch is light or heavy.

LAW OF SPECIFIC NERVE ENERGIES

The sensation evoked by impulses generated in a receptor depends in part on the specif ic part of the brain they ultimately activate. The

specific sensory pathways are discrete from sense

Afferent neuron

Skin

Glass probe

180

120

Time

FIGURE 8–3 Relationship between stimulus and impulse frequency in an affere nt fiber. Action potentials in an afferent fiber from a

mechanoreceptor of a single sensory unit increase in frequency as branches of the aff erent neuron are stimulated by pressure of

increasing magnitude. (From Widmaier EP, Raff H, Strang KT: Vander’s Human Physiology. McGraw-Hil l, 2008.)

100

R = 9.4 S

0.52

2.0 1.9

80 1.8

1.7

1.6

1.5

1.4

50 1.3

40 1.2

1.1

1.0

0.9

.2 .3 .4 .5 .6 .7 .8 .9 1.0 1.11.2 1.31.4 1.51.6 1.71.81.9 2.0 2.1 Log S

10 20 30 40 50 60 70 80 90 100 S (% maximum stimulus)

FIGURE 8–4 Relation between magnitude of touch stimulus (S) and frequency of action potentials in sensory nerve fibers (R).

Dots are individual values from cats and are plotted on linear coordinates (left) and log–log c oordinates (right). The equation shows the

calculated power function relationship between R and S. (Reproduced, with per mission, from Werner G, Mountcastle VB: Neural activity in

mechanoreceptive cutaneous afferents. Stimulus–response relations, Weber functions, and information tr ansmission. J Neurophysiol 1965;28:359.)

CLINICAL BOX 8–2 CLINICAL BOX 8–3 Vibratory Sensibility

Vibratory sensibility

Vibratory sensibility Hz) tuning fork to the skin on the fingertip, tip of the toe, or bony prominences o f the toes. The normal response is

a “buzzing” sensation. The sensation is most marked over bones. The term pallesthesia is also us ed to describe this ability to feel

mechanical vibrations. The receptors involved are the receptors for touch, especially Pacinian corpuscles, but a time factor is also

necessary. A pattern of rhythmic pressure stimuli is interpreted as vibration. The impulse s responsible for the vibrating sensation are

carried in the dorsal columns. Degeneration of this part of the spinal cord occurs in poorly controlled diabetes, pernicious anemia,

vitamin B

deficiencies, or early tabes dorsalis. Elevation of the threshold for vibratory stimuli is an early symptom of this

degeneration. Vibratory sensation and proprioception are closely related; when one is d iminished, so is the other.

Stereognosis

Stereognosis is the perception of the form and nature of an object without looking at it. Normal per sons can readily identify objects such

as keys and coins of various denominations. This ability depends on relatively intact touc h and pressure sensation and is compromised

when the dorsal columns are damaged. The inability to identify an object by touch is cal led tactile agnosia. It also has a large cortical

component; impaired stereognosis is an early sign of damage to the cerebral cortex and sometimes occurs in the absence of any

detectable defect in touch and pressure sensation when there is a lesion in the parietal lob e posterior to the postcentral gyrus.

Stereoagnosia can also be expressed by the failure to identify an object by sight (visual agnosia) , the inability to identify sounds or

words (auditory agnosia) or color (color agnosia), or the inability to identify the loca tion or position of an extremity (position

agnosia).

organ to cortex. Therefore, when the nerve pathways from a particular sense organ a re stimulated, the sensation evoked is that for which

the receptor is specialized no matter how or where along the pathway the activity is initia ted. This principle, first enunciated by Müller in

1835, has been given the rather cumbersome name of the law of specific nerve energie s. For example, if the sensory nerve from a

Pacinian corpuscle in the hand is stimulated by pressure at the elbow or by irritation from a tumor in the brachial plexus, the sensation

evoked is touch. Similarly, if a fine enough electrode could be inserted into the appropr iate fibers of the dorsal columns of the spinal

cord, the thalamus, or the postcentral gyrus of the cerebral cortex, the sensation produc ed by stimulation would be touch. The general

principle of specific nerve energies remains one of the cornerstones of sensory physio logy.

LAW OF PROJECTION

No matter where a particular sensory pathway is stimulated along its course to the cortex , the conscious sensation produced is referred to

the location of the receptor. This principle is called the law of projection. Cortical stimulation e xperiments during neurosurgical

procedures on conscious patients illustrate this phenomenon. For example, when the co rtical receiving area for impulses from the left

hand is stimulated, the patient reports sensation in the left hand, not in the head.

RECRUITMENT OF SENSORY UNITS

As the strength of a stimulus is increased, it tends to spread over a large area and gener ally not only activates the sense organs

immediately in contact with it but also “recruits” those in the surrounding area. Furtherm ore, weak stimuli activate the receptors with the

lowest thresholds, and stronger stimuli also activate those with higher thresholds. Some o f the receptors activated are part of the same

sensory unit, and impulse frequency in the unit therefore increases. Because of overlap and interdigitation of one unit with another,

however, receptors of other units are also stimulated, and consequently more units fire. In this way, more afferent pathways are

activated, which is interpreted in the brain as an increase in intensity of the sensation.

NEUROLOGICAL EXAM

The sensory component of a neurological exam includes an assessment of various sens ory modalities including touch, proprioception,

vibratory sense, and pain. Clinical Box 8–2 describes the test for vibratory sensibility. C ortical sensory function can be tested by placing

familiar objects in a patient’s hands and asking him or her to identify it with the eyes clos ed (see Clinical Box 8–3).

CHAPTER SUMMARY

■ Sensory receptors are commonly classified as mechanoreceptors, nociceptors, che moreceptors, or photoreceptors.

■ Touch and pressure are sensed by four types of mechanoreceptors: Meissner’ s corpuscles (respond to changes in texture and slow

vibrations), Merkel’s cells (respond to sustained pressure and touch), Ruffini corpuscles (respond to sustained pressure), and Pacinian

corpuscles (respond to deep pressure and fast vibrations).

■ Nociceptors and thermoreceptors are free nerve endings on unmyelinated or ligh tly myelinated fibers in hairy and glaborous skin and

deep tissues.

■ The generator or receptor potential is the nonpropagated depolarizing potential rec orded in a sensory organ after an adequate stimulus

is applied. As the stimulus is increased, the magnitude of the receptor potential increases. Whe n it reaches a critical threshold, an action

potential is generated in the sensory nerve.

■ Converting a receptor stimulus to a recognizable sensation is termed sensory codin g. All sensory systems code for four elementary

attributes of a stimulus: modality, location, intensity, and duration.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. Pacinian corp uscles are

A) a type of thermoreceptor.

B) usually innervated by Aδ nerve fibers.

C) rapidly adapting touch receptors.

D) slowly adapting touch receptors.

E) nociceptors.

2. Adaptation to a sensory stimulus produces

A) a diminished sensation when other types of sensory stimuli are withdrawn.

B) a more intense sensation when a given stimulus is applied repeatedly.

C) a sensation localized to the hand when the nerves of the brachial plexus are stimulate d.

D) a diminished sensation when a given stimulus is applied repeatedly over time.

E) a decreased firing rate in the sensory nerve from the receptor when one’s attention is directed to another matter. 3. Sensory systems

code for the following attributes of a stimulus:

A) modality, location, intensity, and duration

B) threshold, receptive field, adaptation, and discrimination

C) touch, taste, hearing, and smell

D) threshold, laterality, sensation, and duration

E) sensitization, discrimination, energy, and projection 4. In which of the following is th e frequency of stimulation not linearly related to

the strength of the sensation felt?

A) sensory area of the cerebral cortex

B) specific projection nuclei of the thalamus

C) lateral spinothalamic tract

D) dorsal horn

E) cutaneous receptors

5. Which of the following receptors and sense organs are incorrectly

paired?

A) rods and cones : eye

B) receptors sensitive to sodium : taste buds

C) hair cells : olfactory epithelium

D) receptors sensitive to stretch : carotid sinus

E) glomus cells : carotid body

6. Which best describes the law of specific nerve energies?

A) No matter where a particular sensory pathway is stimulated along its course to the c ortex, the conscious sensation produced is

referred to the location of the receptor.

B) A nerve can only be stimulated by electrical energy.

C) Receptors can respond to forms of energy other than their adequate stimuli, but the th reshold for these nonspecific responses is much

higher.

D) For any given sensory modality, the specific relationship between sensation and stimulu s intensity is determined by the properties of

the peripheral receptors.

E) The sensation evoked by impulses generated in a receptor depends in part on the specific part of the brain they ultimately activate.

7. Which of the following does not contain cation channels that are

activated by mechanical distortion, producing depolarization?

A) olfactory receptors

B) Pacinian corpuscles

C) hair cells in cochlea

D) hair cells in semicircular canals

E) hair cells in utricle

CHAPTER RESOURCES

Barlow HB, Mollon JD (editors): The Senses. Cambridge University Press, 1982 .

Bell J, Bolanowski S, Holmes MH: The structure and function of Pacinian corp uscles: A review. Prog Neurobiol 1994;42:79.

Haines DE (editor): Fundamental Neuroscience for Basic and Clinical Applications, 3rd ed. Elsevier, 2006.

Iggo A (editor): Handbook of Sensory Physiology. Vol 2, Somatosensory System . Springer-Verlag, 1973.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural Science, 4th ed. Mc Graw-Hill, 2000.

Mountcastle VB: Perceptual Neuroscience. Harvard University Press, 1999.

Squire LR, et al (editors): Fundamental Neuroscience, 3rd ed. Academic Press, 20 08.

Reflexes

CH APTER

OBJEC TIV ES

After studying this chapter, you should be able to:

Describe the components of a reflex arc.

■

Describe the muscle spindles and their role in the stretch reflex.

■

Describe the Golgi tendon organs and analyze their function as part of a feedback system that maintains muscle force.

■

Define reciprocal innervation, inverse stretch reflex, clonus, and lengthening reaction.

INTRODUCTION

The basic unit of integrated reflex activity is the reflex arc. This arc consists of a sense organ, an afferent neuron, one or more synapses

within a central integrating station, an efferent neuron, and an effector. In mammals, th e connection between afferent and efferent

somatic neurons is generally in the brain or spinal cord. The afferent neurons enter via the dorsal roots or cranial nerves and have their

cell bodies in the dorsal root ganglia or in the homologous ganglia on the cranial nerves . The efferent fibers leave via the ventral roots or

corresponding motor cranial nerves. The principle that in the spinal cord the dorsal roo ts are sensory and the ventral roots are motor is

known as the Bell–Magendie law.

Activity in the reflex arc starts in a sensory receptor with a receptor potential whose mag nitude is proportional to the strength of the

stimulus (Figure 9–1). This generates all-ornone action potentials in the afferent nerve, the number of action potentials being proportional

to the size of the generator potential. In the central nervous system (CNS), the response s are again graded in terms of excitatory

postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) at the synap tic junctions. All-or-none responses are

generated in the efferent nerve. When these reach the effector, they again set up a gra ded response. When the effector is smooth muscle,

responses summate to produce action potentials in the smooth muscle, but when the effe ctor is skeletal muscle, the graded response is

always adequate to produce action potentials that bring about muscle contraction. The co nnection between the afferent and efferent

neurons is usually in the CNS, and activity in the reflex arc is modified by the multiple in puts converging on the efferent neurons or at

any synaptic station within the reflex loop.

The simplest reflex arc is one with a single synapse between the afferent and efferent n eurons. Such arcs are monosynaptic, and ref lexes

occurring in them are called monosynaptic reflexes. Reflex arcs in which one or more interneuron is in terposed between the afferent

and efferent neurons are called polysynaptic reflexes. There can be anywhere from two to hundre ds of synapses in a polysynaptic reflex

arc.

157

Sense organ Afferent Synapse neuron

Efferent Neuromuscular Muscle neuron junction

Generator potential Action EPSPs potentials (and IPSPs) Action Endplate Action potentia ls potentials potentials

FIGURE 9–1 The reflex arc. Note that at the receptor and in the CNS a nonpr opagated graded response occurs that is proportionate to

the magnitude of the stimulus. The response at the neuromuscular junction is a lso graded, though under normal conditions it is always

large enough to produce a response in skeletal muscle. On the other hand, in the portion s of the arc specialized for transmission (afferent

and efferent axons, muscle membrane), the responses are all-or-none action potentials.

MONOSYNAPTIC REFLEXES: THE STRETCH REFLEX

When a skeletal muscle with an intact nerve supply is stretched, it contracts. This respons e is called the stretch reflex. The stimulus that

initiates the reflex is stretch of the muscle, and the response is contraction of the muscle b eing stretched. The sense organ is a small

encapsulated spindlelike or fusiform shaped structure called the muscle spindle, located within the fleshy part of the muscle. The

impulses originating from the spindle are transmitted to the CNS by fast sensory fibers th at pass directly to the motor neurons which

supply the same muscle. The neurotransmitter at the central synapse is glutamate. The str etch reflex is the best known and studied

monosynaptic reflex and is typified by the knee jerk reflex (see Clinical Box 9–1).

STRUCTURE OF MUSCLE SPINDLES

Each muscle spindle has three essential elements: (1) a group of specialized intrafusal mu scle fibers with contractile polar ends and a

noncontractile center, (2) large diameter myelinated afferent nerves (types Ia and II) o riginating in the central portion of the intrafusal

fibers, and (3) small diameter myelinated efferent nerves supplying the polar contractile regions of the intrafusal fibers (Figure 9–2A). It

is important to understand the relationship of these elements to each other and to the mus cle itself to appreciate the role of this sense

organ in signaling changes in the length of the muscle in which it is located. Changes in muscle length are associated with changes in

joint angle; thus muscle spindles provide information on position (ie, proprioception).

The intrafusal fibers are positioned in parallel to the extrafusal fib ers (the regular contractile units of the muscle) with the ends of the

spindle capsule attached to the tendons at either end of the muscle. Intrafusal fibers do n ot contribute to the overall contractile force of

the muscle, but rather serve a pure sensory function. There are two types of intrafusal fibers in mammalian muscle spindles. The first type

contains many nuclei in a dilated central area and is called a nuclear bag fiber (Figure 9–2B). There are two subtypes of nuclear bag

fibers, dynamic and static. Typically, there are two or three nuclear bag fibers per spindle. The s econd intrafusal fiber type, the nuclear

chain fiber, is thinner and shorter and lacks a definite bag. Each spindle has about five of these fibers.

There are two kinds of sensory endings in each spindle, a single primary (group Ia) ending a nd up to eight secondary (group II)

endings. The Ia afferent fiber wraps around the center of the dynamic and static nuclear bag fib ers and nuclear chain fibers. Group II

sensory fibers are located adjacent to the centers of the static nuclear bag and nuclear c hain fibers; these fibers do not innervate the

dynamic nuclear bag fibers. Ia afferents are very sensitive to the velocity of the change in muscle length during a stretch (dynamic

response); thus they provide information about the speed of movements and allow for qu ick corrective movements. The steady-state

(tonic) activity of group Ia and II afferents provide information on steady-state length of the muscle (static response). The top trace in

Figure 9–2C shows the dynamic and static components of activity in a Ia afferent durin g muscle stretch. Note that they discharge most

rapidly while the muscle is being stretched (shaded area of graphs) and less rapidly dur ing sustained stretch.

The spindles have a motor nerve supply of their own. These nerves are 3–6 μm in diam eter, constitute about 30% of the fibers in the

ventral roots, and are called γ-motor neurons. There are two types of γ-motor neurons: dynamic, which supply the dynamic nuclear bag

fibers and static, which supply the static nuclear bag fibers and the nuclear chain fib ers. Activation of dynamic γ-motor neurons

increases the dynamic sensitivity of the group Ia endings. Activation of the static γ- moto r neurons increases the tonic level of activity in

both group Ia and II endings, decreases the dynamic sensitivity of group Ia afferents, and can prevent silencing of Ia afferents during

muscle stretch (Figure 9–2C).

CLINICAL BOX 9–1 Knee Jerk Reflex

Tapping the patellar tendon elicits the knee jerk, a stretch reflex of the quadriceps femoris mus cle, because the tap on the tendon

stretches the muscle. A similar contraction is observed if the quadriceps is stretched man ually. Stretch reflexes can also be elicited from

most of the large muscles of the body. Tapping on the tendon of the triceps brachii, for example, causes an extensor response at the

elbow as a result of reflex contraction of the triceps; tapping on the Achilles tendon cau ses an ankle jerk due to reflex contraction of the

gastrocnemius; and tapping on the side of the face causes a stretch reflex in the masseter . The knee jerk reflex is an example of a deep

tendon reflex (DTR) in a neurological exam and is graded on the following s cale: 0 (absent), 1+ (hypoactive), 2+ (brisk, normal), 3+

(hyperactive without clonus), 4+ (hyperactive with mild clonus), and 5+ (hyperactive w ith sustained clonus). Absence of the knee jerk

can signify an abnormality anywhere within the reflex arc, including the muscle spindle , the Ia afferent nerve fibers, or the motor

neurons to the quadriceps muscle. The most common cause is a peripheral neuropathy from such things as diabetes, alcoholism, and

toxins. A hyperactive reflex can signify an interruption of corticospinal and other desce nding pathways that influence the reflex arc.

A Muscle spindle B Intrafusal fibers of the muscle spindle

Static nuclear bag fiber

C Response of Ia sensory fiber to selective activation of motor neurons

200

Intrafusal muscle

fibers

Dynamic nuclear bag fiber

Nuclear

chain fiber Dynamic response Steady-state response 0 Stretch alone

200 Capsule

Sensory endings II

Afferent axons

Stimulate static gamma fiber Static 200

Efferent axons

Dynamic

Gamma motor

endings 0

Stimulate dynamic gamma fiber 6

0 0.2 s

FIGURE 9–2 Mammalian muscle spindle. A) Diagrammatic repre sentation of the main components of mammalian muscle spindle

including intrafusal muscle fibers, afferent sensory fiber endings, and efferent motor f ibers (γ-motor neurons). B) Three types of

intrafusal muscle fibers: dynamic nuclear bag, static nuclear bag, and nuclear chain fibers. A single Ia afferent fiber innervates all three

types of fibers to form a primary sensory ending. A group II sensory fiber inne rvates nuclear chain and static bag fibers to form a

secondary sensory ending. Dynamic γ-motor neurons innervate dynamic bag fibers; s tatic γ-motor neurons innervate combinations of

chain and static bag fibers. C) Comparison of discharge pattern of Ia afferent activ ity during stretch alone and during stimulation of static

or dynamic γ-motor neurons. Without γ-stimulation, Ia fibers show a small dynamic re sponse to muscle stretch and a modest increase in

steady-state firing. When static γ-motor neurons are activated, the steadystate response i ncreases and the dynamic response decreases.

When dynamic γ-motor neurons are activated, the dynamic response is markedly increased b ut the steady-state response gradually

returns to its original level. (From Kandel ER, Schwartz JH, Jessell TM [editors]: Principles of N eural Science, 4th ed. McGraw-Hill, 2000.)

CENTRAL CONNECTIONS

OF AFFERENT FIBERS

Ia fibers end directly on motor neurons supplying the extrafusal fibers of the same mus cle (Figure 9–3). The time between the application

of the stimulus and the response is called the reaction time. In humans, the reaction time f or a stretch reflex such as the knee jerk is 19–

24 ms. Weak stimulation of the sensory nerve from the muscle, known to stimulate only Ia fibers, causes a contractile response with a

similar latency. Because the conduction velocities of the afferent and efferent fiber type s are known and the distance from the muscle to

the spinal cord can be measured, it is possible to calculate how much of the reaction time was taken up by conduction to and from the

spinal cord. When this value is subtracted from the reaction time, the remainder, called t he central delay, is the time taken for the reflex

activity to traverse the spinal cord. In humans, the central delay for the knee jerk is 0.6– 0.9 ms, and figures of similar magnitude have

been found in experimental animals. Because the minimal synaptic delay is 0.5 ms, only one synapse could have been traversed.

Muscle spindles also make connections that cause muscle contraction via polysynaptic pa thways, and the afferents involved are probably

those from the secondary endings. However, group II fibers also make monosynaptic connections to the motor neurons and make a small

contribution to the stretch reflex.

Dorsal root

Interneuron releasing inhibitory mediator

Motor neuron Ib fiber

from Ia fiber

Golgi fromtendon muscle

Ventral rootorgan

spindle

Motor endplate on extrafusal fiber

FIGURE 9–3 Diagram illustrating the pathways responsible for the stretch reflex and the inverse stretch reflex. Stretch stimulates

the muscle spindle, which activates Ia fibers that excite the motor neuron. Stretch also stimulates the Golgi tendon organ, which activates

Ib fibers that excite an interneuron that releases the inhibitory mediator glycine. With strong stretch, the resulting hyperpolarization of

the motor neuron is so great that it stops discharging.

FUNCTION OF MUSCLE SPINDLES

When the muscle spindle is stretched, its sensory endings are distorted and receptor pote ntials are generated. These in turn set up action

potentials in the sensory fibers at a frequency proportional to the degree of stretching. B ecause the spindle is in parallel with the

extrafusal fibers, when the muscle is passively stretched, the spindles are also stretched, referred to as “loading the spindle.” This

initiates reflex contraction of the extrafusal fibers in the muscle. On the other hand, the s pindle afferents characteristically stop firing

when the muscle is made to contract by electrical stimulation of the α-motor neurons to th e extrafusal fibers because the muscle shortens

while the spindle is unloaded (Figure 9–4).

Thus, the spindle and its reflex connections constitute a feedback device that operates to maintain muscle length; if the muscle is

stretched, spindle discharge increases and reflex shortening is produced, whereas if the muscle is shortened without a change in γ-motor

neuron discharge, spindle afferent activity decreases and the muscle relaxes. Dynamic a nd static responses of muscle spindle afferents

influence physiological tremor (see Clinical Box 9–2).

EFFECTS OF γ-MOTOR

NEURON DISCHARGE

Stimulation of γ-motor neurons produces a very different picture from that produced by stimulation of the extrafusal fibers. Such

stimulation does not lead directly to detectable contraction of the muscles because the intr afusal fibers are not strong enough or plentiful

enough to cause shortening. However, stimulation does cause the contractile ends of the intrafusal fibers to shorten and therefore

stretches the nuclear bag portion of the spindles, deforming the endings and initiating im pulses in the Ia fibers (Figure 9–4). This in turn

can lead to reflex contraction of the muscle. Thus, muscle can be made to contract via s timulation of the α-motor neurons that innervate

the extrafusal fibers or the γ-motor neurons that initiate contraction indirectly via the stre tch reflex.

If the whole muscle is stretched during stimulation of the γ- motor neurons, the rate of discharge in the Ia fibers is further increased

(Figure 9–4). Increased γ-motor neuron activity thus increases spindle sensitivity during stretch.

In response to descending excitatory input to spinal motor circuits, both α- and γ-motor neurons are activated. Because of this “α–γ

coactivation,” intrafusal and extrafusal fibers shorten together, and spindle afferent acti vity can occur throughout the period of muscle

contraction. In this way, the spindle remains capable of responding to stretch and reflex ly adjusting α-motor neuron discharge.

Spindle

Tendon

Extrafusal fiber Sensory nerve

Impulses in sensory nerve Muscle at rest

Muscle stretched

CLINICAL BOX 9–2 Physiological Tremor

The response of the Ia sensory fiber endings to the dynamic (phasic) as well as the stati c events in the muscle is important because the

prompt, marked phasic response helps to dampen oscillations caused by conduction del ays in the feedback loop regulating muscle

length. Normally a small oscillation occurs in this feedback loop. This physiologic tremor has a low am plitude (barely visible to the

naked eye) and a frequency of approximately 10 Hz. Physiological tremor is a normal phenomenon which affects everyone while

maintaining posture or during movements. However, the tremor would be worse if it w ere not for the sensitivity of the spindle to

velocity of stretch. It can become exaggerated in some situations such as when we are a nxious or tired or because of drug toxicity.

Numerous factors contribute to the genesis of physiological tremor. It is likely dependen t on not only central (inferior olive) sources but

also from peripheral factors including motor unit firing rates, reflexes, and mechanical resonance.

Muscle contracted

Increased γ efferent discharge

Increased γ efferent

discharge—muscle stretched

FIGURE 9–4 Effect of various conditions on muscle spindle discharge. When the whole muscle is stretched, the muscle spindle is

also stretched and its sensory endings are activated at a frequency proportional to the d egree of stretching (“loading the spindle”).

Spindle afferents stop firing when the muscle contracts (“unloading the spindle”). Stimulation of

-motor neurons cause the contractile

ends of the intrafusal fibers to shorten. This stretches the nuclear bag region, initiating imp ulses in sensory fibers. If the whole muscle is

stretched during stimulation of the

-motor neurons, the rate of discharge in sensory fibers is further increased.

CONTROL OF γ-MOTOR

NEURON DISCHARGE

The γ-motor neurons are regulated to a large degree by descending tracts from a nu mber of areas in the brain. Via these pathways, the

sensitivity of the muscle spindles and hence the threshold of the stretch reflexes in vario us parts of the body can be adjusted and shifted

to meet the needs of postural control.

Other factors also influence γ-motor neuron discharge. Anxiety causes an increased disch arge, a fact that probably explains the

hyperactive tendon reflexes sometimes seen in anxious patients. In addition, unexpected movement is associated with a greater efferent

discharge. Stimulation of the skin, especially by noxious agents, increases γ-motor neur on discharge to ipsilateral flexor muscle spindles

while decreasing that to extensors and produces the opposite pattern in the opposite limb . It is well known that trying to pull the hands

apart when the flexed fingers are hooked together facilitates the knee jerk reflex (Jend rassik’s maneuver), and this may also be due to

increased γ-motor neuron discharge initiated by afferent impulses from the hands.

RECIPROCAL INNERVATION

When a stretch reflex occurs, the muscles that antagonize the action of the muscle involv ed (antagonists) relax. This phenomenon is said

to be due to reciprocal innervation. Impulses in the Ia fibers from the muscle spindles of the p rotagonist muscle cause postsynaptic

inhibition of the motor neurons to the antagonists. The pathway mediating this effect is b isynaptic. A collateral from each Ia fiber passes

in the spinal cord to an inhibitory interneuron that synapses on a motor neuron supplyin g the

Nerve fiber Tendon bundles

Organ of Golgi, showing ramification of nerve fibrils Muscular fibers

FIGURE 9–5 Golgi tendon organ. (Reproduced, with permission, from Goss CM [editor]: Gray’s An atomy of the Human Body, 29th ed. Lea & Febiger,

1973.)

antagonist muscles. This example of postsynaptic inhibition is discussed in Chapter 6, and the pathway is illustrated in Figure 6–6.

INVERSE STRETCH REFLEX

Up to a point, the harder a muscle is stretched, the stronger is the reflex contraction. Ho wever, when the tension becomes great enough,

contraction suddenly ceases and the muscle relaxes. This relaxation in response to stron g stretch is called the inverse stretch reflex or

autogenic inhibition.

The receptor for the inverse stretch reflex is in the Golgi tendon organ (Figure 9–5). This organ consists of a netlike collection of

knobby nerve endings among the fascicles of a tendon. There are 3–25 muscle fibers per tendon organ. The fibers from the Golgi tendon

organs make up the Ib group of myelinated, rapidly conducting sensory nerve fibers. Stimulation of these Ib fibers leads to the

production of IPSPs on the motor neurons that supply the muscle from which the fibers arise. The Ib fibers end in the spinal cord on

inhibitory interneurons that in turn terminate directly on the motor neurons (Figure 9–3) . They also make excitatory connections with

motor neurons supplying antagonists to the muscle.

Because the Golgi tendon organs, unlike the spindles, are in series with the muscle fiber s, they are stimulated by both passive stretch and

active contraction of the muscle. The threshold of the Golgi tendon organs is low. The d egree of stimulation by passive stretch is not

great because the more elastic muscle fibers take up much of the stretch, and this is why it takes a strong stretch to produce relaxation.

However, discharge is regularly produced by contraction of the muscle, and the Golgi tendon organ thus functions as a transducer in a

feedback circuit that regulates muscle force in a fashion analogous to the spindle feedba ck circuit that regulates muscle length.

The importance of the primary endings in the spindles and the Golgi tendon organs in r egulating the velocity of the muscle contraction,

muscle length, and muscle force is illustrated by the fact that that section of the afferent n erves to an arm causes the limb to hang loosely

in a semiparalyzed state. The organization of the system is shown in Figure 9–6. The int eraction of

Interneuronal

control

signal

Inter

neurons Force feedback Tendon organs

α Control − signal +

α Internal disturbances

Efferent signal +

Muscle External forces

Muscular + Muscle force

Load

length −

Length and velocity

-Dynamic

feedback

control

signal

γd

γ -Static

control

signal

γs

Spindles

FIGURE 9–6 Block diagram of peripheral motor control system. The dashe d line indicates the nonneural feedback from muscle that

limits length and velocity via the inherent mechanical properties of muscle. γ

, dynamic γ-motor neurons; γ

, static γ-motor neurons.

(Reproduced, with permission, from Houk J in: Medical Physiology, 13th ed. Mount-Castle VB [e ditor]. Mosby, 1974.)

CLINICAL BOX 9–3 Clonus

A characteristic of states in which increased γ-motor neuron discharge is present is clonus. This neurologic sign is the occurrence of

regular, repetitive, rhythmic contractions of a muscle subjected to sudden, maintained str etch. Only sustained clonus with five or more

beats is considered abnormal. Ankle clonus is a typical example. This is initiated by brisk , maintained dorsiflexion of the foot, and the

response is rhythmic plantar flexion at the ankle. The stretch reflex–inverse st retch reflex sequence may contribute to this response.

However, it can occur on the basis of synchronized motor neuron discharge without G olgi tendon organ discharge. The spindles of the

tested muscle are hyperactive, and the burst of impulses from them discharges all the mo tor neurons supplying the muscle at once. The

consequent muscle contraction stops spindle discharge. However, the stretch has been m aintained, and as soon as the muscle relaxes it is

again stretched and the spindles stimulated. Clonus may also occur after disruption of de scending cortical input to a spinal glycinergic

inhibitory interneuron called the Renshaw cell. This cell receives excitatory input from α-motor neuron s via an axon collateral (and in

turn it inhibits the same). In addition, cortical fibers activating ankle flexors contact Rens haw cells (as well as type Ia inhibitory

interneurons) that inhibit the antagonistic ankle extensors. This circuitry prevents reflex stimulation of the extensors when flexors are

active. Therefore, when the descending cortical fibers are damaged (upper motor neuron lesion), the inhibition of antagonists is absent.

The result is repetitive, sequential contraction of ankle flexors and extensors (clonus). C lonus may be seen in patients with amyotrophic

lateral sclerosis, stroke, multiple sclerosis, spinal cord damage, and hepatic encephalopat hy.

ation is clearly seen. Passive flexion of the elbow, for example, meets immediate resistan ce as a result of the stretch reflex in the triceps

muscle. Further stretch activates the inverse stretch reflex. The resistance to flexion sudd enly collapses, and the arm flexes. Continued

passive flexion stretches the muscle again, and the sequence may be repeated. This sequ ence of resistance followed by give when a limb

is moved passively is known as the clasp-knife effect because of its resemblanc e to the closing of a pocket knife. It is also known as the

lengthening reaction because it is the response of a spastic muscle to lengthening.

POLYSYNAPTIC REFLEXES:

THE WITHDRAWAL REFLEX

Polysynaptic reflex paths branch in a complex fashion (Figure 9–7). The number of sy napses in each of their branches varies. Because of

the synaptic delay at each synapse, activity in the branches with fewer synapses reaches the motor neurons first, followed by activity in

the longer pathways. This causes prolonged bombardment of the motor neurons from a single stimulus and consequently prolonged

responses. Furthermore, some of the branch pathways turn back on themselves, permi tting activity to reverberate until it becomes unable

to cause a propagated transsynaptic response and dies out. Such reverberating circuits are common in the brain and spinal cord.

WITHDRAWAL REFLEX

The withdrawal reflex is a typical polysynaptic reflex that occurs in response to a usuall y painful stimulation of the skin or subcutaneous

tissues and muscle. The response is flexor muscle contraction and inhibition of extensor muscles, so that the body part stimulated is

flexed and withdrawn from the stimulus. When a strong stimulus is applied to a limb, the response includes not only flexion and

withdrawal of that limb but also

spindle discharge, tendon organ discharge, and reciprocal innervation determines the r ate of discharge of α-motor neurons (see Clinical

Box 9–3).

Sensory

B Cneuron

MUSCLE TONE

The resistance of a muscle to stretch is often referred to as its tone or tonus. If the motor nerve to a muscle is cut, the m uscle offers very

little resistance and is said to be flaccid. A hypertonic (spastic) muscle is one in wh ich the resistance to stretch is high because of

hyperactive stretch reflexes. Somewhere between the states of flaccidity and spasticity is the ill-defined area of normal tone. The

muscles are generally hypotonic when the rate of γ-motor neuron discharge is low and hypertonic when it is high.

When the muscles are hypertonic, the sequence of moderate stretch → muscle contract ion, strong stretch → muscle relax

Motor neuron

FIGURE 9–7 Diagram of polysynaptic connections between afferent and efferent neurons in the spinal cord. The dorsal root fiber

activates pathway A with three interneurons, pathway B with four interneurons, and pathway C with four interneurons. Note that one of

the interneurons in pathway C connects to a neuron that doubles back to othe r interneurons, forming reverberating circuits.

extension of the opposite limb. This crossed extensor response i s properly part of the withdrawal reflex. Strong stimuli in experimental

animals generate activity in the interneuron pool that spreads to all four extremities. This is difficult to demonstrate in normal animals but

is easily demonstrated in an animal in which the modulating effects of impulses from the brain have been abolished by prior section of

the spinal cord (spinal animal). For example, when the hind limb of a spinal cat is pinched, the stimulate d limb is withdrawn, the

opposite hind limb extended, the ipsilateral forelimb extended, and the contralateral fore limb flexed. This spread of excitatory impulses

up and down the spinal cord to more and more motor neurons is called irradiati on of the stimulus, and the increase in the number of

active motor units is called recruitment of motor units.

IMPORTANCE OF THE

WITHDRAWAL REFLEX

Flexor responses can be produced by innocuous stimulation of the skin or by stretch of the muscle, but strong flexor responses with

withdrawal are initiated only by stimuli that are noxious or at least potentially harmful to t he animal. These stimuli are therefore called

nociceptive stimuli. Sherrington pointed out the survival value of the withdrawal response. Flexi on of the stimulated limb gets it away

from the source of irritation, and extension of the other limb supports the body. The pa ttern assumed by all four extremities puts the

animal in position to run away from the offending stimulus. Withdrawal reflexes are prepotent; that is, they preempt the spinal

pathways from any other reflex activity taking place at the moment.

Many of the characteristics of polysynaptic reflexes can be demonstrated by studying th e withdrawal reflex. A weak noxious stimulus to

one foot evokes a minimal flexion response; stronger stimuli produce greater and great er flexion as the stimulus irradiates to more and

more of the motor neuron pool supplying the muscles of the limb. Stronger stimuli also cause a more prolonged response. A weak

stimulus causes one quick flexion movement; a strong stimulus causes prolonged flexion and sometimes a series of flexion movements.

This prolonged response is due to prolonged, repeated firing of the motor neurons. Th e repeated firing is called after-discharge and is

due to continued bombardment of motor neurons by impulses arriving by complicated and circuitous polysynaptic paths.

As the strength of a noxious stimulus is increased, the reaction time is shortened. Spatial and temporal facilitation occurs at synapses in

the polysynaptic pathway. Stronger stimuli produce more action potentials per second in the active branches and cause more branches to

become active; summation of the EPSPs to the firing level therefore occurs more rapidly .

FRACTIONATION & OCCLUSION

Another characteristic of the withdrawal response is the fact that supramaximal stimulatio n of any of the sensory nerves from a limb

never produces as strong a contraction of the flexor muscles as that elicited by direct ele ctrical stimulation of the muscles themselves.

This indicates that the afferent inputs fractionate the motor neuron p ool; that is, each input goes to only part of the motor neuron pool

for the flexors of that particular extremity. On the other hand, if all the sensory inputs a re dissected out and stimulated one after the

other, the sum of the tension developed by stimulation of each is greater than that produ ced by direct electrical stimulation of the muscle

or stimulation of all inputs at once. This indicates that the various afferent inputs share so me of the motor neurons and that occlusion

occurs when all inputs are stimulated at once.

GENERAL PROPERTIES

OF REFLEXES

It is apparent from the preceding description of the properties of monosynaptic and po lysynaptic reflexes that reflex activity is

stereotyped and specific in terms of both the stimulus and the response; a particular stimu lus elicits a particular response. The fact that

reflex responses are stereotyped does not exclude the possibility of their being modified by experience. Reflexes are adaptable and can

be modified to perform motor tasks and maintain balance. Descending inputs from high er brain regions play an important role in

modulating and adapting spinal reflexes.

ADEQUATE STIMULUS

The stimulus that triggers a reflex is generally very precise. This stimulus is called the adequate stimulus for the particular reflex. A

dramatic example is the scratch reflex in the dog. This spinal reflex is adequately stimula ted by multiple linear touch stimuli such as

those produced by an insect crawling across the skin. The response is vigorous scratch ing of the area stimulated. If the multiple touch

stimuli are widely separated or not in a line, the adequate stimulus is not produced and n o scratching occurs. Fleas crawl, but they also

jump from place to place. This jumping separates the touch stimuli so that an adequate sti mulus for the scratch reflex is not produced. It

is doubtful if the flea population would survive long without the ability to jump.

FINAL COMMON PATH

The motor neurons that supply the extrafusal fibers in skeletal muscles are the efferent s ide of many reflex arcs. All neural influences

affecting muscular contraction ultimately funnel through them to the muscles, and they a re therefore called the final common paths.

Numerous inputs converge on them. Indeed, the surface of the average motor neuron and its dendrites accommodates about 10,000

synaptic knobs. At least five inputs go from the same spinal segment to a typical spinal m otor neuron. In addition to these, there are

excitatory and inhibitory inputs, generally relayed via interneurons, from other levels o f the spinal cord and multiple long-descending

tracts from the brain. All of these pathways converge on and determine the activity in th e final common paths.

CENTRAL EXCITATORY

& INHIBITORY STATES

The spread up and down the spinal cord of subliminal fringe effects from excitatory sti mulation has already been mentioned. Direct and

presynaptic inhibitory effects can also be widespread. These effects are generally trans ient. However, the spinal cord also shows

prolonged changes in excitability, possibly because of activity in reverberating circuits o r prolonged effects of synaptic mediators. The

terms central excitatory state and central inhibitory state have been used to describe pr olonged states in which excitatory influences

overbalance inhibitory influences and vice versa. When the central excitatory state is ma rked, excitatory impulses irradiate not only to

many somatic areas of the spinal cord but also to autonomic areas. In chronically parap legic humans, for example, a mild noxious

stimulus may cause, in addition to prolonged withdrawal-extension patterns in all four lim bs, urination, defecation, sweating, and blood

pressure fluctuations (mass reflex).

CHAPTER SUMMARY

■ A reflex arc consists of a sense organ, an afferent neuron, one or more synapses w ithin a central integrating station, an efferent neuron,

and an effector response.

■ A muscle spindle is a group of specialized intrafusal muscle fibers with contractile p olar ends and a noncontractile center that is located

in parallel to the extrafusal muscle fibers and is innervated by types Ia and II afferent f ibers and γ-motor neurons. Muscle stretch

activates the muscle spindle to initiate reflex contraction of the extrafusal muscle fibers in the same muscle (stretch reflex).

■ A Golgi tendon organ is a netlike collection of knobby nerve endings among the fa scicles of a tendon that is located in series with

extrafusal muscle fibers and innervated by type Ib afferents. They are stimulated by both passive stretch and active contraction of the

muscle to relax the muscle (inverse stretch reflex) and function as a transducer to regulate mu scle force.

■ A collateral from an Ia afferent branches to terminate on an inhibitory interneuron that synapses on an antagonistic muscle (reciprocal

innervation) to relax that muscle when the agonist contracts. Clonus is the occurrence of regular, rhythmic contractions of a muscle

subjected to sudden, maintained stretch. A sequence of increased resistance followed by reduced resistance when a limb is moved

passively is known as the lengthening reaction.

MULTIPLE-CHOICE QUESTIONS

For all questions, select the single best answer unless otherwise directed. 1. The inverse stretch reflex

A) has a lower threshold than the stretch reflex.

B) is a monosynaptic reflex.

C) is a disynaptic reflex with a single interneuron inserted

between the afferent and efferent limbs.

D) is a polysynaptic reflex with many interneurons inserted between the afferent and effe rent limbs.

E) requires the discharge of central neurons that release acetylcholine.

2. When γ-motor neuron discharge increases at the same time as

α-motor neuron discharge to muscle,

A) prompt inhibition of discharge in spindle Ia afferents takes place.

B) the contraction of the muscle is prolonged.

C) the muscle will not contract.

D) the number of impulses in spindle Ia afferents is smaller than when α discharge alone i s increased.

E) the number of impulses in spindle Ia afferents is greater than when α discharge alone is increased.

3. Which of the following is not characteristic of a reflex?

A) Modification by impulses from various parts of the CNS

B) May involve simultaneous contraction of some muscles and relaxation of others

C) Chronically suppressed after spinal cord transection

D) Always involves transmission across at least one synapse

E) Frequently occurs without conscious perception 4. Withdrawal reflexes are not

A) initiated by nociceptive stimuli.

B) prepotent.

C) prolonged if the stimulus is strong.

D) an example of a flexor reflex.

E) accompanied by the same response on both sides of the body.

CHAPTER RESOURCES

Haines DE (editor): Fundamental Neuroscience for Basic and Clinical Applications, 3rd e d. Elsevier, 2006.

Hulliger M: The mammalian muscle spindle and its central control. Rev Physiol Bio chem Pharmacol 1984;101:1.

Hunt CC: Mammalian muscle spindle: Peripheral mechanisms. Physiol Rev 1990; 70: 643.

Jankowska E: Interneuronal relay in spinal pathways from proprioceptors. Prog Neuro biol 1992;38:335.

Kandel ER, Schwartz JH, Jessell TM (editors): Principles of Neural Science, 4th ed. Mc Graw-Hill, 2000.

Lundberg A: Multisensory control of spinal reflex pathways. Prog Brain Res 1979;50:11.

Matthews PBC: Mammalian Muscle Receptors and Their Central Actions, Williams & Wilkins, 1972.

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SECTION III CENTRAL &

PERIPHERAL NEUROPHYSIOLOGY

CH APTER

Pain & Temperature 10

OBJEC TIV ES

After studying this chapter, you should be able to:

Name the types of peripheral nerve fibers and receptor types that mediate

■

warmth, cold, and nociception.

■

Explain the difference between pain and nociception.

■

Explain the differences between fast and slow pain and acute and chronic pain.

■

Explain hyperalgesia and allodynia.

■

Describe and explain referred pain.

INTRODUCTION

One of the most common reasons an individual seeks the advice of a physician is becau se he or she is in pain. Pain was called by

Sherrington, “the physical adjunct of an imperative protective reflex.” Painful stimuli gen erally initiate potent withdrawal and avoidance

responses. Pain differs from other sensations in that it sounds a warning that something is wrong, preempts other signals, and is

associated with an unpleasant affect. It turns out to be immensely complex because whe n pain is prolonged and tissue is damaged, central

nociceptor pathways are sensitized and reorganized.

NOCICEPTORS &

THERMORECEPTORS

Pain and temperature sensations arise from unmyelinated dendrites of sensory neurons located around hair follicles throughout the

glabrous and hairy skin as well as deep tissue. Impulses from nociceptors (pain) are transmitted via two fiber types. One system

comprises thinly myelinated Aδ fibers (2–5 μm in diameter) which conduct at rates of 1 2–30 m/s. The other is unmyelinated C fibers

(0.4–1.2 μm in diameter) which conduct at low rates of 0.5–2 m/s. Thermorecepto rs also span these two fiber types. Cold receptors are

on dendritic endings of Aδ fibers and C fibers, whereas warmth (heat) receptors are o n C fibers.

Mechanical nociceptors respond to strong pressure (eg, from a sharp object). Thermal n ociceptors are activated by skin temperatures

above 45 °C or by severe cold. Chemically sensitive nociceptors respond to various ag ents like bradykinin, histamine, high acidity, and

environmental irritants. Polymodal nociceptors respond to combinations of these stimuli.

167

Mapping experiments show that the skin has discrete coldsensitive and heat-sensitive spo ts. There are 4 to 10 times as many cold-

sensitive as heat-sensitive spots. The threshold for activation of warmth receptors is 30 °C, and th ey increase their firing rate up to 46

°C. Cold receptors are inactive at temperatures of 40 °C, but then steadily increase t heir firing rate as skin temperature falls to about 24

°C. As skin temperature further decreases, the firing rate of cold receptors decreases u ntil the temperature reaches 10 °C. Below that

temperature, they are inactive and the cold becomes an effective local anesthetic.

Because the sense organs are located subepithelially, it is the temperature of the subcutan eous tissues that determines the responses. Cool

metal objects feel colder than wooden objects of the same temperature because the meta l conducts heat away from the skin more rapidly,

cooling the subcutaneous tissues to a greater degree.

A major advance in this field has been the cloning of three thermoreceptors and nocice ptors. The receptor for moderate cold is the cold

and menthol-sensitive receptor 1 (CMR 1). Two types of vanilloid receptors respond to noxious heat (VR1 and VRL-1). Vanillins are

a group of compounds, including capsaicin, that cause pain. The VR1 receptors respon d not only to capsaicin but also to protons and to

potentially harmful temperatures above 43 °C. VRL-1, which responds to temperatur es above 50 °C but not to capsaicin, has been

isolated from C fibers. There may be many types of receptors on single peripheral C fi ber endings, so single fibers can respond to many

different noxious stimuli. However, the different properties of the VR1 and the VRL-1 receptors make it likely that there are many

different nociceptor C fibers systems as well.

CMR1, VR1, and VRL1 are members of the transient receptor potential (TRP) f amily of excitatory ion channels. VR1 has a PIP

binding site, and when the amount of PIP

bound is decreased, the sensitivity of the receptors is increased. Aside from the fact tha t

activation of the cool receptor causes an influx of Ca

, little is known about the ionic basis of the initial depolarization they produce. In

the cutaneous receptors in general, depolarization could be due to inhibition of K

channels, activation of Na

channels, or inhibition of

the Na

–K

pump, but the distinction between these possibilities has not been made.

CLASSIFICATION OF PAIN

For scientific and clinical purposes, pain is defined by the International Association for t he Study of Pain (IASP) as, “an unpleasant

sensory and emotional experience associated with actual or potential tissue damage, or d escribed in terms of such damage.” This is to be

distinguished from the term nociception which the IASP defines as the unconscious activity indu ced by a harmful stimulus applied to

sense receptors.

Pain is sometimes classified as fast and slow pain. A painful stimulus causes a “bright,” sh arp, localized sensation (fast pain) followed

by a dull, intense, diffuse, and unpleasant feeling (slow pain). Evidence suggests that fast pain is due to activity in the

CLINICAL BOX 10–1 Itch & Tickle

Itching (pruritus) is not much of a problem for normal individuals, but severe itchi ng that is difficult to treat occurs in diseases such as

chronic renal failure, some forms of liver disease, atopic dermatitis, and HIV infection. Especially in areas where many naked endings of

unmyelinated nerve fibers occur, itch spots can be identified on the skin by careful map ping. In addition, itch-specific fibers have been

demonstrated in the ventrolateral spinothalamic tract. This and other evidence implicate th e existence of an itch-specific path. Relatively

mild stimulation, especially if produced by something that moves across the skin, produc es itch and tickle. Scratching relieves itching

because it activates large, fast-conducting afferents that gate transmission in the dorsal ho rn in a manner analogous to the inhibition of

pain by stimulation of similar afferents. It is interesting that a tickling sensation is usually r egarded as pleasurable, whereas itching is

annoying and pain is unpleasant. Itching can be produced not only by repeated local m echanical stimulation of the skin but also by a

variety of chemical agents. Histamine produces intense itching, and injuries cause its liberation in the skin. However, in most instances

of itching, endogenous histamine does not appear to be the responsible agent; doses of histamine that are too small to produce itching still

produce redness and swelling on injection into the skin, and severe itching frequently o ccurs without any visible change in the skin. The

kinins cause severe itching.

A δ pain fibers, whereas slow pain is due to activity in the C pain fibers. Itch and tickle are related to pain sensation (see Clinical Box

10–1).

Pain is frequently classified as physiologic or acute pain and pathologic or chronic pain, which includes inflammatory pain and

neuropathic pain. Acute pain typically has a sudden onset and recedes during the h ealing process. Acute pain can be considered as

“good pain” as it serves an important protective mechanism. The withdrawal reflex is an example of this protective role of pain.

Chronic pain can be considered “bad pain” because it persists long after recovery from an injury and is often refractory to common

analgesic agents, including nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates . Chronic pain can result from nerve injury

(neuropathic pain) including diabetic neuropathy, toxin-induced nerve damage, and ischemia. Causalgia is a type of neuropathic pain

(see Clinical Box 10–2).

Pain is often accompanied by hyperalgesia and allodynia. Hyperalgesia is an exaggerated response to a noxious stimulus, whereas

allodynia is a sensation of pain in response to an innocuous stimulus. An example of the latter is the painful sensation from a warm

shower when the skin is damaged by sunburn.

CLINICAL BOX 10–2 Neuropathic Pain

Neuropathic pain may occur when nerve fibers are injured. Commonly, it is excruciating and a d ifficult condition to treat. It occurs in

various forms in humans. For example, in causalgia, spontaneous burning pain occurs long after seemingly trivial injuries. The pain is

often accompanied by hyperalgesia and allodynia. Reflex sympathetic dystrophy is often present as well. In this condition, the skin in

the affected area is thin and shiny, and there is increased hair growth. Research in anim als indicates that nerve injury leads to sprouting

and eventual overgrowth of noradrenergic sympathetic nerve fibers into the dorsal roo t ganglia of the sensory nerves from the injured

area. Sympathetic discharge then brings on pain. Thus, it appears that the periphery has been short-circuited and that the relevant altered

fibers are being stimulated by norepinephrine at the dorsal root ganglion level. Alpha-a drenergic blockade produces relief of causalgia-

type pain in humans, though for unknown reasons α

-adrenergic blockers are more effective than α

-adrenergic blocking agents.

Treatment of painful sensory neuropathy is a major challenge and current therapies are often inadequate.

Hyperalgesia and allodynia signify increased sensitivity of nociceptive afferent fibers. F igure 10–1 shows how chemicals released at the

site of injury can further activate nociceptors leading to inflammatory pain. Injured cells release chemicals such as K

that depolarize

nerve terminals, making nociceptors more responsive. Injured cells also release bradyk inin and Substance P, which can further sensitize

nociceptive terminals. Histamine is released from mast cells, serotonin (5-HT) from plate lets, and prostaglandins from cell membranes,

all contributing to the inflammatory process and they activate or sensitize the nociceptors . Some released substances act by releasing

another one (eg, bradykinin activates both Aδ and C fibers and increases synthesis and release of prostaglandins). Prostaglandin E

cyclooxygenase metabolite of arachidonic acid) is released from damaged cells and pro duces hyperalgesia. This is why aspirin and other

NSAIDs (inhibitors of cyclooxygenase) alleviate pain.

DEEP PAIN

The main difference between superficial and deep sensibility is the different nature of th e pain evoked by noxious stimuli. This is

probably due to a relative deficiency of Aδ nerve fibers in deep structures, so there is l ittle rapid, bright pain. In addition, deep pain and

visceral pain are poorly localized, nauseating, and frequently associated with sweating a nd changes in blood pressure. Pain can be

elicited experimentally from the periosteum and ligaments by injecting hypertonic saline i nto them. The pain produced in this fashion

initiates reflex contraction of nearby skeletal muscles. This reflex contraction is similar to the muscle spasm associated with injuries to

bones, tendons, and joints. The steadily contracting muscles become ischemic, and ische mia stimulates the pain receptors in the muscles

(see Clinical Box 10–3). The pain in turn initiates more spasm, setting up a vicious cycle .

VISCERAL PAIN

In addition to being poorly localized, unpleasant, and associated with nausea and autono mic symptoms, visceral pain often radiates or is

referred to other areas.

Mast cell

CGRP Substance P

Histamine Bradykinin

Lesion

5-HT

Prostaglandin

Dorsal root

ganglion neuron

CGRP

Substance P Blood vessel Spinal cord

FIGURE 10–1 In response to tissue injury, chemical mediators can sensitize an d activate nociceptors. These factors contribute to

hyperalgesia and allodynia. Tissue injury releases bradykinin and prostaglandins that se nsitize or activate nociceptors, which in turn

releases substance P and calcitonin gene-related peptide (CGRP). Substance P acts on m ast cells to cause degranulation and release

histamine, which activates nociceptors. Substance P causes plasma extravasation and CGRP dilates blood vessels; the resulting edema

causes additional release of bradykinin. Serotonin (5-HT) is released from plate lets and activates nociceptors. (From Kandel ER, Schwartz JH,

Jessell TM [editors]: Principles of Neural Science. McGraw-Hill, 2000.)

CLINICAL BOX 10–3 Muscle Pain

If a muscle contracts rhythmically in the presence of an adequate blood supply, pain do es not usually result. However, if the blood

supply to a muscle is occluded, contraction soon causes pain. The pain persists after the contraction until blood flow is reestablished.

These observations are difficult to interpret except in terms of the release during contrac tion of a chemical agent (Lewis’s “P factor”)

that causes pain when its local concentration is high enough. When the blood supply is r estored, the material is washed out or

metabolized. The identity of the P factor is not settled, but it could be K

. Clinically, the substernal pain that develops when the

myocardium becomes ischemic during exertion (angina pectoris) is a classic exam ple of the accumulation of P factor in a muscle.

Angina is relieved by rest because this decreases the myocardial O

requirement and permits the blood supply to remove the factor.

Intermittent claudication, the pain produced in the leg muscles of persons with occ lusive vascular disease, is another example. It

characteristically comes on while the patient is walking and disappears on stopping. Visc eral pain, like deep somatic pain, initiates reflex

contraction of nearby skeletal muscle. This reflex spasm is usually in the abdominal wall and makes the abdominal wall rigid. It is most

marked when visceral inflammatory processes involve the peritoneum. However, it can occur without such involvement. The spasm

protects the underlying inflamed structures from inadvertent trauma. Indeed, this reflex spasm is sometimes called “guarding.”

The autonomic nervous system, like the somatic, has afferent components, central integr ating stations, and effector pathways. The

receptors for pain and the other sensory modalities present in the viscera are similar to th ose in skin, but there are marked differences in

their distribution. There are no proprioceptors in the viscera, and few temperature and touch receptors. Nociceptors are present, although

they are more sparsely distributed than in somatic structures.

Afferent fibers from visceral structures reach the CNS via sympathetic and parasympat hetic nerves. Their cell bodies are located in the

dorsal roots and the homologous cranial nerve ganglia. Specifically, there are visceral a fferents in the facial, glossopharyngeal, and vagus

nerves; in the thoracic and upper lumbar dorsal roots; and in the sacral roots (Figure 10 –2). There may also be visceral afferent fibers

from the eye in the trigeminal nerve.

As almost everyone knows from personal experience, visceral pain can be very severe . The receptors in the walls of the hollow viscera

are especially sensitive to distention of these organs. Such distention can be produced ex perimentally in the gastrointestinal tract by

inflation of a swallowed balloon attached to a tube. This produces pain that waxes and w anes (intestinal colic) as the intestine contracts

and relaxes on the balloon. Similar colic is produced in intestinal obstruction by the contr actions of the dilated intestine above the

obstruction. When a visceral organ is inflamed or hyperemic, relatively minor stimuli ca use severe pain. This is probably a form of

hyperalgesia.

REFERRED PAIN

Irritation of a visceral organ frequently produces pain that is felt not at that site but in so me somatic structure that may be a considerable

distance away. Such pain is said to be referred to the somatic structure. Obviously, kno wledge of referred pain and the common sites of

pain referral from each of the viscera is of great importance to the physician. Perhaps t he bestknown example is referral of cardiac pain

to the inner aspect of the left arm. Other examples include pain in the tip of the shoulder caused by irritation of the central portion of the

diaphragm and pain in the testicle due to distention of the ureter. Additional instances ab ound in the practices of medicine, surgery, and

dentistry. However, sites of reference are not stereotyped, and unusual reference sites occur with considerable frequency. Cardiac pain,

for instance, may be referred to the right arm, the abdominal region, or even the back and neck.

When pain is referred, it is usually to a structure that developed from the same embryon ic segment or dermatome as the structure in

which the pain originates. This principle is called the dermatomal rule. For example, the heart and the arm have the same segmental

origin, and the testicle has migrated with its nerve supply from the primitive urogenital ri dge from which the kidney and ureter have

developed.

The basis for referred pain may be convergence of somatic and visceral pain fibers on the same second-order neurons in the dorsal horn

that project to the thalamus and then to the somatosensory cortex (Figure 10–3). This is called the convergence–projection theory.

Somatic and visceral neurons converge in lamina I–VI of the ipsilateral dorsal horn, bu t neurons in lamina VII receive afferents from

both sides of the body—a requirement if convergence is to explain referral to the side opposite that of the source of pain. The somatic

nociceptive fibers normally do not activate the second-order neurons, but when the vis ceral stimulus is prolonged, facilitation of the

somatic fiber endings occurs. They now stimulate the secondorder neurons, and of cou rse the brain cannot determine whether the

stimulus came from the viscera or from the area of referral.

Glossopharyngeal nerve

Superior laryngeal nerve Vagus

THORACIC PAIN LINE Splanchnic nerves

(T7–9)

R splanchnic

nerves (T7–9)

Duodenum and jejunum (splanchnic nerves) Upper thoracic vagal rami

Brachial plexus

i (

(Intercostal nerves)

Lower

splanch

nic nerves (T10–L1)

Ureter

(T11–L1)

Peripheral

Central

diaphragm

(intercostal nerves)

(phrenic nerve)

Small intestine splanchnic nerves

e t a l peritoneum(T9–11) r i

Somatic nerves (T11–L1)

Fundus (T11–L1)

PELVIC lleum

PAIN LINE

Trigone

Prostate Cervix andUrethra upper vagina

(pelvic nerves, S2–4)

(S2–4)

Parasympathetic

Testicle

rami (S2–4)

(sacral nerves, S2–4) (genitofemoral nerves, L1–2) (spermatic plexus, T10)