Pharmacology of Neuromuscular Function

Pharmacology of neuromuscular function

Second Edition

William C. Bowman, BPharm, PhD, DSc, FIBiOl, FPS, FRSE, HonFFARCS

Professor of Pharmacology and Vice Principal, University of Strathclyde, Glasgow, UK

Wright

Table of Contents

Cover image

Title page

Copyright

Preface

Preface to the first edition

Chapter 1: Striated muscle

Publisher Summary

Innervation of striated muscle

Different types of muscle fibres

Chapter 2: Excitable membranes

Publisher Summary

Properties of excitable membranes

Some membrane-active drugs and toxins

Chapter 3: Neuromuscular transmission: prejunctional events

Publisher Summary

Introduction

Acetylcholine synthesis

Acetylcholine storage

Acetylcholine release

Chapter 4: Pharmacological manipulation of prejunctional events

Publisher Summary

Introduction

Inhibition of acetylcholine synthesis

Drugs that inhibit vesicular loading: vesamicol and tetraphenylboron

Agents and factors that modify acetylcholine release

Nicotinic and muscarinic receptors on the nerve endings

Chapter 5: Neuromuscular transmission: postjunctional events

Publisher Summary

Development of the receptor concept

Postjunctional acetylcholine receptors

Receptor function

Acetylcholine noise

Patch clamp

Channel characteristics

Kinetic analysis of agonist-receptor interaction

The endplate response

Summary of postjunctional events

Receptor desensitization

Acetylcholine receptors of denervated muscle

Receptors in myasthenia gravis

Termination of acetylcholine action: acetylcholinesterase

Anticholinesterase drugs

Chapter 6: Neuromuscular-blocking agents

Publisher Summary

Introduction

Non-depolarizing acetylcholine antagonists

Curarizing agents from natural sources

Mechanism of action of tubocurarine

Characteristics of block produced by tubocurarine

The basis of clinical monitoring

Structure–action relations

Clinically used non-depolarizing neuromuscular-blocking drugs

Pharmacokinetics

Interactions between two non-depolarizing neuromuscular-blocking drugs

‘The Priming Principle’

Unwanted effects

Unwanted drug interactions involving neuromuscular-blocking agents

Reversal agents

Some potential new non-depolarizing neuromuscular-blocking agents

Depolarizing blocking drugs

Variations in depolarization block

Multiply innervated muscle fibres

Succinyldicholine (suxamethonium)

Chapter 7: Muscle contraction

Publisher Summary

Fibre structure

Contraction

Drugs affecting muscle contractility

References

Index

Copyright

Wright

is an imprint of Butterworth Scientific

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First published 1980 by John Wright & Sons Ltd

Second edition 1990

© Butterworth & Co (Publishers) Ltd, 1990

British Library Cataloguing in Publication Data

Bowman, W. C. (William Cameron)

Pharmacology of neuromuscular function. – 2nd. ed.

1. Man. Muscles. Nerves. Drug therapy

I. Title

616.7′4061

ISBN 0-7236-0913-6

Library of Congress Cataloging in Publication Data

Bowman, W. C.

Pharmacology of neuromuscular function/William C. Bowman. – 2nd ed.

p. cm.

Includes bibliographical references.

ISBN 0-7236-0913-6

1. Neuromuscular blocking agents. 2. Neuromuscular transmission – Effect of drugs on. 3. Anesthetics-Physiological effect.

I. Title.

[DNLM: 1. Muscles-drug effects. 2. Neural Transmission. 3. Neuromuscular Blocking Agents-pharmacology. 4. Neuromuscular Junction-drug effects. 5. Neuromuscular Junction-physiology. QV 140 B787p]

RD83.5.B68 1990

615′.781–dc20

DNLM/DC 89-70552

for Library of Congress CIP

Composition by Genesis Typesetting, Borough Green, Sevenoaks, Kent

Printed and bound in Great Britain by Courier International Ltd., Tiptree, Essex

Preface

Much has happened in the 10 years since the first edition. The accelerating development of new skills and techniques, especially in the fields of protein chemistry, recombinant DNA technology, receptor antibody studies, new imaging techniques in electron microscopy, rapid biochemical mixing methods, and advances in electrophysiological recording techniques, have led to huge forward leaps in our detailed knowledge of the mysteries of the nicotinic acetylcholine receptor, and indeed of the whole transmission process. At the time of the first edition, the neuromuscular blocking drugs vecuronium (ORG NC 45) and atracurium (BW 33A), were no more than promising new compounds, barely leaving the laboratory to enter early clinical trials. Now they are established components of the anaesthetist’s armamentarium, their wide and safe use being backed by a vast literature in the clinical anaesthesiology press. Developments from them are now undergoing trials, and some of these show promise as potential muscle relaxants of the future. At a more academic level, we can now interfere, using the drug vesamicol, with the loading of acetylcholine into vesicles in the nerve endings, and advances in the study of marine and other venoms and toxins have provided additional tools for probing the functions of ion channels and other structures in highly specific ways. My attempt to deal with all of these advances has, not surprisingly, led to the considerable increase in the size of this volume and the huge increase in the number of references that it has been necessary to quote.

At first sight, a few of the drugs and toxins described might appear irrelevant to the practising anaesthetist, since they have no known clinical use. However, this is a narrow and restricted view for several reasons. Who knows to what use such substances might be put tomorrow? For example, ten years ago, botulinum toxin had no known clinical use, yet recently it has had some success in the control of blepharospasm and strabismus. In any case, anaesthetists might well be called upon to assist with life-saving treatments in cases of poisoning with such toxins. Furthermore, no drug, clinically useful or otherwise, is absolutely specific in its actions. The actions of drugs that are presently regarded as no more than pharmacological curiosities, not only illustrate additional sites of pharmacological attack but may also explain what would otherwise be mysterious side-effects of clinically useful drugs.

A great many colleagues and friends in anaesthesiology (and even some in pharmacology) have asked for, and encouraged me to produce, this updated version. I never fail to be impressed by anaesthesiologists’ voracious search for detailed pharmacological knowledge. I hope this attempt will do something towards temporarily assuaging that appetite.

While I was preparing this edition, a friend and colleague, Dr. David Savage, tragically and prematurely died. David Savage was the chemist closely associated with the design and development of both pancuronium and vecuronium. Many anaesthetists knew David well, and I would like to dedicate this volume to his memory.

I am grateful to my secretary Margaret Perry not only for uncomplainingly typing every word, and modifying it and remodifying it in her word processor in accordance with my whims, but also for helping me to read and check the proofs and the references.

W.C. Bowman

October 1989

Preface to the first edition

The origin of this monograph lies in an invitation from John Norman and Joe Whitwam to contribute an article for Topical Reviews in Anaesthesia, of which they are the Editors. My subject turned out to be vast, and despite condensing the description as much as I could and quoting reviews instead of individual papers where possible, my contribution took on a Topsy-like quality. Eventually I tried to quell my conscience after the manner of Horace: Brevis esse laboro obscurus fio. However, the final article turned out to be obviously too long for the review journal, and I am grateful to Roy Baker, of John Wright & Sons Ltd., the Managing Editor, for accepting it as a monograph when he could justifiably have discarded it out of hand.

It might well be considered presumptuous of me, in the security of my ivory tower, to write for practising anaesthetists about the drugs that they use, when of course I have never even administered a drug to a human patient. I was, nevertheless, encouraged to go ahead by my many anaesthetist friends, and by my numerous contacts with anaesthetists over the years. It seems to me that above all other members of the medical profession anaesthetists are intensely interested not only in what their drugs do but also in the science underlying the ways in which they do it. Perhaps more than any others, anaesthetists are faced with the immediate consequences of the effects of the drugs that they use, and so they are necessarily more aware of the relationship between the dose of a drug and the size of the response it produces. Herein, I think, lies the basis of the special relationship between pharmacologists and anaesthetists. Both are very conscious of the fact that it is only by a thorough understanding of their mechanisms of action, and of their pharmacokinetics and metabolism, that drugs can be used effectively and safely.

If this monograph helps to whet the appetite of any anaesthetists for more knowledge of this kind, it will have served its purpose. Although the monograph has been written primarily with anaesthetists in mind, I dare to hope that some others with an interest in drugs, including students, might find something of value in it.

My own enthusiasm for the pharmacology of muscle was awakened by Professor Eleanor Zaimis, from whom I learned a great deal. She will not agree with everything that I have written, but it gives me great pleasure to dedicate this small volume to her.

W.C. Bowman

June, 1980

Chapter 1

Striated muscle

Publisher Summary

This chapter focuses on striated muscles, which are innervated by somatic efferent nerve fibers that are fast-conducting myelinated group A axons with cell bodies in the motor nuclei of the cranial nerves in the brain stem or in the anterior horns of grey matter in the spinal cord. These peripheral neurons are known as the lower motoneurons to distinguish them from the upper motoneurons involved in the central control of striated muscle movement. Muscle fibers that receive their innervation at a focal point on their membranes are described as focally innervated. Most mammalian muscle fibers are of this type. The focal motor endplates of muscle fibers innervated by spinal nerves are usually midway between the origin and the insertion of the muscle fibers. Some very long muscle fibers may possess two or three foci of innervation; such muscle fibers are still regarded as focally innervated. Multiply innervated fibers receive a dense innervation all over their membranes from many nerve endings. These fibers are common in certain muscles of birds, some snakes, amphibians, and fish. Fibers with a Fibrillenstruktur have a distinct fibrillar appearance owing to the regular arrangement and uniform width of their myofibrils, which are distinctly separated one from another by sarcoplasm.

Innervation of striated muscle

Striated muscles are innervated by somatic efferent nerve fibres which are fast-conducting myelinated group A axons with cell bodies in the motor nuclei of the cranial nerves in the brain stem, or in the anterior horns of grey matter in the spinal cord. These peripheral neurones are known as the lower motoneurones to distinguish them from the upper motoneurones involved in the central control of striated muscle movement. The axons of the lower motoneurones pass without interruption from the central nervous system to the muscles, where each axon branches extensively, the branching occurring at nodes of Ranvier. Through its extensive branching, a single axon innervates many muscle fibres (Figure 1.1). The lower motoneurone, together with the muscle fibres it innervates, form a functional unit called a motor unit (Liddell and Sherrington, 1925; Sherrington, 1930; Buchtal, 1960). The muscle fibres belonging to any one motor unit are probably all of the same type (i.e. fast pale, fast red or slow red intermediate, as defined below) (Edström and Kugelberg, 1968; Kugelberg and Edström, 1968) but they usually do not form a compact group; rather they lie scattered throughout the muscle. The characteristics of the units are determined by the type of muscle fibres they contain. Some units participate mainly in relatively rare, quick movements and contract rapidly; they are easily fatigued and are designated type FF. Others contribute to the maintenance of posture, contract slowly and are fatigue resistant (type S). Others are both fast and fatigue resistant and are designated type FR (Burke, 1981; Hennig and Lømo, 1985). The number of muscle fibres within a motor unit differs according to the delicacy of the movement that the muscle is capable of producing. For example, in the small muscles that move the fingers and the external rectus muscles of the eye, each nerve axon supplies only 5−15 muscle fibres, whereas in the large limb and back muscles, which are less capable of fine delicate movements, each axon supplies over a thousand muscle fibres (Buchtal, 1960).

Figure 1.1 Diagram of a motor unit containing focally innervated muscle fibres. A motor endplate is enlarged as the inset on the left, and a unit of this is further enlarged in Figure 1.2. (From Bowman, W. C. and Rand, M. J. Textbook of Pharmacology, 2nd edn, Blackwell Scientific Publications, Oxford, 1980, with permission)

As it aproaches a muscle fibre, the terminal branch of an axon loses its myelin sheath, and, where it makes contact with the muscle fibre, it breaks up into a number of short twigs (telodendria) that lie in gutters, the junctional clefts, in the muscle fibre membrane. The surface of the junctional cleft in apposition to the nerve ending is thrown into folds, the junctional folds, forming so-called secondary clefts. At its narrowest, the junctional gap between the plasma membranes of nerve ending (axolemma) and muscle fibre (sarcolemma) is about 60 nm wide. The gap includes a layer of ill-defined material, the basement membrane, or basal lamina. The basement membrane is a material rich in mucopolysaccharides and with some of the characteristics of collagen. It fills the whole of the junctional gap and extends into the secondary clefts formed by the junctional folds. It probably functions both as a structural support and as a selective filter which must allow for the rapid diffusion of acetylcholine. Hirokawa and Heuser (1982) prepared deep-etch images of frog neuromuscular junctions quick frozen directly from life, and obtained an unusually distinct view of the basement membrane which resembled barbed wire lying in the junctional cleft. Its appearance arose from the wisps of material that extend from it to both pre- and postjunctional membranes. The basement membrane contains much of the acetylcholinesterase of the junction embedded within it.

The terminal Schwann cells (teloglia) of the axon twigs form ‘lids’ to the junctional clefts so that the neuromuscular junctions are enclosed. In the junctional region of the muscle fibre there is an accumulation of sarcoplasm containing many mitochondria, ribosomes and nuclei. The whole junctional structure is traditionally called the motor endplate (Krause, 1863), while the specialized postjunctional membrane, together with its immediately underlying structures, is called the sole plate. There is some confusion of terminology, however, as many authors, especially English-speaking authors, use the term ‘motor endplate’ to designate only the postjunctional membrane, in which case motor endplate and sole plate become synonymous. Figures 1.1 and 1.2 illustrate the general arrangement of the neuromuscular junction. For detailed descriptions, reviews and original papers may be consulted (Bowden and Duchen, 1976; Desaki and Uehara, 1981; Waser, 1983).

Figure 1.2 Diagram of neuromuscular junction enlarged from the motor endplate of Figure 1.1. The axon terminal contains mitochondria, microtubules and acetylcholine-containing vesicles

Focally and multiply innervated muscle fibres

Muscle fibres that receive their innervation at a focal point on their membranes are described as focally innervated (Figure 1.1). Most mammalian muscle fibres are of this type; the focal motor endplates of muscle fibres innervated by spinal nerves are usually about midway between the origin and the insertion of the muscle fibres. Some very long muscle fibres may possess two or three foci of innervation, probably supplied by the same axon; such muscle fibres are still regarded as focally innervated. Focally innervated muscle fibres are innervated by fast-conducting axons of the Aα group. While remaining within this group, axons supplying focally innervated slow-contracting intermediate muscle fibres conduct slightly more slowly than do those innervating fast-contracting muscle fibres. The motor endplates on fast pale muscle fibres form a conspicuous elevation and are sometimes called en plaque endplates; there are several junctional clefts and an associated arborization of nerve ending twigs making up the composite endplate and the clefts are thrown into deep junctional folds. Motor endplates on fast red fibres are more simple, with few junctional folds, and those on slow red intermediate fibres fall between these extremes. The different types of muscle fibre referred to here (fast pale, fast red and slow red intermediate) are defined below.

Multiply innervated fibres receive a dense innervation all over their membranes from many nerve endings. For example, it has been estimated that each multiply innervated fibre of the chicken anterior latissimus dorsi muscle has about 80 motor endplates (Ginsborg, 1960b). Multiply innervated fibres are common in certain muscles of birds (Ginsborg, 1960b; Shehata and Bowden, 1960; Koenig, 1970), some snakes (Hess, 1965), amphibia (Katz and Kuffler, 1941; Kuffler and Vaughan Williams, 1953) and fish (Nakajima, 1969; Korneliussen, 1973). In mammals, including man, the extraocular muscles (Gerebtzoff, 1959; Kupfer, 1960; Hess, 1961b; Zenker and Anzenbacher, 1964; Bach-y-Rita and Ito, 1966; Teräväinen, 1968, Teräväinen, 1972), the intrinsic laryngeal muscles (Keene, 1961; Manolov, Penev and Itchev, 1963), the striated muscle in the upper oesophagus and in the middle ear (Csillik, 1965), and the facial muscles (Bowden and Mahran, 1956; Kadanoff, 1956) contain a proportion of multiply innervated fibres. In many instances, the multiple endplates on any one multiply innervated muscle fibre appear to be innervated by the same neurone, but in others there is probably polyneuronal innervation. The axons innervating multiply innervated fibres are usually of the Aγ group. Each terminal axon branch ends in a number of small swellings resembling a bunch of grapes and known as en grappe terminations. The endplates are of the relatively simple type with smooth junctional clefts.

Muscle fibres in the fetus are multiply innervated but during postnatal development mammalian muscle fibres undergo an orderly process of elimination of neuromuscular junctions, whereby each loses all but one of the multiple inputs with which it is endowed at birth (Brown, Jansen and Van Essen, 1976). Experimental procedures that increase or decrease neuromuscular activity cause a corresponding increase or decrease in the overall rate of elimination of junctions (Thompson, 1986), but the underlying mechanism is not yet understood. The elimination process ensures that each motoneurone forms an independent motor unit containing a certain number of muscle fibres.

Different types of muscle fibres

Fibrillenstruktur and Felderstruktur

These two terms, now somewhat obsolete, nevertheless usefully describe the two main differences in appearance of different types of muscle fibres under the light microscope. For a brief historical review see Bowman and Marshall (1971). Fibres with a Fibrillenstruktur have a distinct fibrillar appearance owing to the regular arrangement and uniform width of their myofibrils which are distinctly separated one from another by sarcoplasm. Fibres with a Felderstruktur, on the other hand, have a more granular and indefinite appearance owing to the irregular width of their myofibrils and to the fact that the myofibrils are less distinctly separated from each other. Electron microscopy has shown that the most important structural difference between the two types of fibres relates to their transverse tubular systems (T tubules) and sarcoplasmic reticula, as varying the lengths of the muscle fibres at the time of fixation can cause an apparent conversion of some of the characteristics of the myofibrils formerly thought typical of a Felderstruktur or a Fibrillenstruktur (Yeh, Huang and Feng, 1963). In a fibre with a Felderstruktur, the T tubules and sarcoplasmic reticulum are less well developed, less regular in arrangement, and make less frequent contacts with each other than is the case in a Fibrillenstruktur fibre (Hess, 1961a, Hess, 1967; Page, 1965, Page, 1969; Page and Slater, 1965; Peachey, 1965; Mayr, 1966; Ezerman and Ishikawa, 1967).

Muscle fibres with a Felderstruktur respond to excitation with a slow and maintained shortening or tension increase, in contrast to the rapid and brief contractile response of muscle fibres with a Fibrillenstruktur. For example, the contraction velocity of the anterior latissimus dorsi muscle of the domestic fowl (which contains only Felderstruktur fibres) is five to seven times slower than that of the posterior latissimus dorsi muscle of the same species (which contains only Fibrillenstruktur fibres) (Ginsborg, 1960a; Page and Slater, 1965; Hník et al., 1967; Page, 1969). The different arrangements and numbers of contacts between T tubules and sarcoplasmic reticulum in the two types of fibre are compatible with their different contraction velocities. Because of their differing speeds of contraction, fibres with a Fibrillenstruktur are variously described as fast, twitch, tetanic or phasic fibres, whereas fibres with a Felderstruktur are described as slow or tonic fibres. Perhaps the best terms to distinguish the two types are ‘twitch’ and ‘tonic’; the terms ‘fast and ‘slow’ may be confusing as they are merely relative terms and, as described below, there are slow (i.e. slow red intermediate) fibres in mammalian muscles which are, nevertheless, twitch fibres with a Fibrillenstruktur. The majority of mammalian muscle fibres possess a Fibrillenstruktur, but a few fibres with a Felderstruktur have been identified in some mammalian muscles, for example in the diaphragms of rat and rabbit (Krüger, 1950; Günther, 1952), and in more specialized muscles such as the extraocular muscles (Hess, 1967). In general, fibres with a Fibrillenstruktur are focally innervated, but at least one exception to this generalization is known. In amphibians, reptiles and birds, the muscle of the iris is striated muscle (Geberg, 1884; Hoffmann, 1890). In the pigeon and the domestic fowl, the iris muscle fibres possess a Fibrillenstruktur, yet they are multiply innervated (Hess, 1966, Hess, 1969; Pilar and Vaughan, 1969a, b). In accordance with their Fibrillenstruktur the fibres have a fast contraction speed. No exception to the rule that fibres with a definite Felderstruktur receive a dense multiple innervation is known.

Fast twitch pale, fast twitch red and slow twitch red intermediate fibres

As has been emphasized above, with few exceptions, mammalian muscles are composed almost entirely of focally innervated twitch fibres. Such twitch fibres, on both morphological and histochemical grounds, can be classified into three main subtypes which have been variously termed by different authors. Close (1972) summarizes the different properties of the three subtypes and advances logical reasons for using the terms at the head of this paragraph. This classification was originally proposed by Barnard et al. (1971) and is the one used throughout this monograph. [Alternative names to those given at the head of this paragraph, and in the same order, include white, red and medium fibres; A, B and C fibres or A, C and B fibres; Classes I, II and III, or II, II and I or IIB, IIA and I fibres; see Close (1972) for a summary of the confusing terminology.]

Both the fast red and the slow red intermediate fibres have a high myoglobin content, which accounts for their colour. However, those designated fast red fibres resemble the pale fibres in their rapid rates of contraction and relaxation, whereas the red intermediate fibres are relatively slow in contracting and relaxing. As described later, the rate of contraction is determined by the rate at which thick myosin and thin actin myofilaments slide past one another, and this in turn is determined by the rate of hydrolysis of ATP by myosin ATPase. The activity of myosin ATPase is the main determinant of contraction speed and, accordingly, there is greater activity of this enzyme in fast-contracting than in slow-contracting muscle fibres (Bárány, 1967; Buller, Mommaerts and Seraydarian, 1969; Goldspink, Larson and Davies, 1970). Myosin ATPase is activated by calcium ions and the calcium-binding protein troponin from fast-contracting muscle fibres has a higher affinity for this ion than has that from slow-contracting fibres. The rate of resequestration of calcium ions by the sarcoplasmic reticulum and by mitochondria is an important factor determining the rate of relaxation (which invariably matches the rate of contraction). Fast-contracting (and therefore fast-relaxing) muscle fibres contain more sarcoplasmic reticulum than do slow-contracting fibres, and the sarcoplasmic reticulum from fast-contracting fibres sequesters calcium ions more avidly (Harigaya, Ogawa and Sugita, 1968; Sréter, 1969, Sréter, 1970; Yamamoto, Takanji and Nagai, 1970; Fiehn and Peter, 1971; Luff and Atwood, 1971). Mitochondria are more numerous in fast red and slow red intermediate fibres than in fast pale fibres, and mitochondria probably have a more important calcium-sequestering role in those fibres in which they are more numerous. Certainly this appears to be so in the slow-contracting cat soleus muscle (E. Zaimis, personal communication). Whether calcium sequestration is by sarcoplasmic reticulum or by mitochondria, the process is presumably more rapid in muscle fibres that relax more rapidly.

The speed of contraction of a muscle as a whole depends on the proportions of the fibre types of which it is composed. Fast-contracting muscles contain a higher proportion of fast pale or fast red fibres, or both, whereas slow-contracting muscles contain a high proportion of slow red intermediate fibres. Fast twitch pale fibres are adapted for short-term powerful phasic activity and predominate in flexor muscles, such as the tibialis anterior and flexor digitorum longus, and in superficial extensor muscles, such as the extensor digitorum longus and the gastrocnemius. Fast twitch red fibres are adapted for sustained phasic activity and predominate in muscles such as the diaphragm. Slow twitch red intermediate fibres are adapted for economical low-speed sustained tonic activity. They predominate in deep extensor muscles, such as the soleus and the crureus, that are mainly involved in the maintenance of posture. The time from the start of the contraction to peak tension in an isometric twitch (the maximal response to a single stimulus) of a fast-contracting muscle of a medium-sized mammal, such as the cat, is around 20 ms. Slow-contracting limb muscles in the cat have contraction times around 70 ms. Muscles composed of about equal proportions of fast-contracting and slow-contracting fibres have intermediate contraction speeds. The smaller the animal species, the faster the speeds of contraction of all its mucles, but the ratio of contraction times for the slowest and the fastest limb muscles is approximately constant at about 3.5:1 for all mammalian laboratory animals (Buller, 1970). In the cat and the guinea-pig, the soleus muscles are entirely composed of slow red intermediate fibres (Henneman and Olson, 1965; Macphedran, Wjuerker and Henneman, 1965; Karpati and Engel, 1967; Guth, 1968; Brooke and Kaiser, 1969; Barnard, Rimazewska and Wieckowski, 1971; Barnard, Wieckowski and Chiu, 1971), and these muscles therefore provide suitable models for studying the actions of drugs on this type of fibre. There are few experimentally convenient muscles known to be composed entirely of either fast pale or fast red fibres, although several, such as the cat and guinea-pig flexor digitorum longus muscles (Olson and Swett, 1966; Barnard, Rimazewska and Wieckowski, 1971; Barnard, Wieckowski and Chiu, 1971) are largely composed of a mixture of both. In man, most muscles appear to contain all three types of fibre but, judging by their contraction speeds, the triceps surae group, for example, contains a greater proportion of slow intermediate fibres than does the tibialis anterior group (Marsden and Meadows, 1968). A clear demonstration of the existence of all three types in the human gastrocnemius muscle has been described (Garnett et al., 1978). In the early stages of development, all limb muscles are slow contracting and differentiation into fast or slow types occurs over a few weeks, the time from the onset to the completion of the change being shorter in small species than in large ones. In different species, the differentiation takes place at different times during ontogeny; for example, it is mainly prenatal in the guinea-pig, mainly postnatal in the rat, and partly prenatal and partly postnatal in the cat (Banu, 1922; Denny-Brown, 1929; Buller, Eccles and Eccles, 1960; Close, 1964, 1965a, b, 1972; Buller and Lewis, 1965; Close and Hoh, 1967).

Buller, Eccles and Eccles (1960) cut and transposed the nerves that supplied two adjacent muscles in the kitten leg. One, the soleus, was destined to become slow (i.e. to be composed only of slow intermediate fibres) and the other (flexor digitorum longus) to become fast (i.e. to be composed of a predominance of fast fibres). In fact, when functional cross-innervation had occurred, each muscle exhibited contraction characteristics opposite to those of the normal contralateral muscles. Cross-innervation of adult fast and slow muscles also led to a reversal of their properties, although the transformation was incomplete (Close, 1972). A number of biochemical differences between fast and slow muscle fibres were also found to be reversed by cross-innervation (Close, 1972). The implication of these experiments is that in large measure the type of innervation determines the muscle fibre phenotype. In addition, however, there are some hereditary characteristics that are not neurally determined (Butler, Cosmos and Brierley, 1982; Sanes, 1987). The nature of the neural influence, whether it be simply the pattern of activation by nerve impulses or some specific trophic chemical elaborated and released by the nerve, has not yet been unequivocally solved. Certainly, the pattern of neural activity appears to be an important influence (Jolesz and Sréter, 1981), but it is probably not the whole story (Salviati, Biasia and Aloisi, 1986).

Chapter 2

Excitable membranes

Publisher Summary

This chapter discusses the properties of excitable membranes. The permeability of an excitable membrane—such as that of a nerve or muscle fiber—to ions is controlled by the potential difference across it. A fall in membrane potential of 15 mV or so causes the sudden opening of so-called sodium gates, which guard selective sodium channels. The concentration gradient and the electrical gradient for Na+ are from outside to inside. As soon as the sodium channels open, an inward sodium current is set up. The influx of Na+ opposes the resting membrane potential, causing a further depolarization and the opening of more sodium channels in a positive feedback or regenerative manner. In this way, all of the sodium channels rapidly open in response to an effective stimulus. It is a characteristic of sodium channels in excitable membranes that the activating stimulus has the secondary effect of causing a conformational change in the channel proteins in a way that the channel becomes occluded. The effect is called inactivation. Because of inactivation, any one channel opens only once during a depolarizing stimulus. No matter how prolonged that stimulus is, the channel converts to the inactivated state and does not reopen. Channel inactivation and dissipation of the sodium ions causes reinstatement of the resting membrane potential, and this restores the channel and the gating molecule to their resting states.

Properties of excitable membranes

As this volume is concerned to some extent with drugs that affect membrane potentials or the conduction of action potentials in nerve endings and muscle fibres, it is as well to review briefly, and in a general way, some of the properties of excitable membranes. Much of our basic knowledge is derived from experiments by A. L. Hodgkin, A. F. Huxley and R. D. Keynes on frog muscle fibres and giant axons of cephalopods, and it is reviewed lucidly and in detail by Katz (1966).

Membrane potential

Figure 2.1 represents a cylinder of a hypothetical cell with an excitable membrane, and shows the distribution of the main intracellular and extracellular ions. Potassium ions and large organic anions labelled A- (e.g. hexosephosphates, ATP, proteins) are more concentrated inside the cell, whereas sodium ions and chloride ions are more concentrated without. The uneven distribution of sodium and potassium ions is maintained by an active transport mechanism, the sodium-potasium pump, which utilizes the cell’s metabolic energy in the form of ATP, and involves the enzyme system, transport ATPase (Na+/K+-dependent ATPase). Each enzyme molecule extends across the membrane. At its outer surface it has binding sites for K+, Mg²+ and ouabain and at its inner surface it binds Na+ and vanadate ions. Na+ is transported outwards and K+ inwards. Mg²+ is necessary to activate the enzyme, and ouabain and vanadate inhibit enzyme activity. If it is assumed for the moment that Na+-K+ exchange occurs on a 1:1 basis, then one cation is merely exchanged for another so that the pump does not directly affect the membrane potential; that is, it is non-electrogenic. In fact, in many tissues, including nerve and muscle, part of the pump expels 3Na+ in exchange for 2K+ so that the pump directly contributes to the membrane potential to a small extent and is therefore electrogenic. Nevertheless, its main function is to maintain the concentration gradients on which the membrane potential depends, and for the purposes of the present discussion the pump itself is considered not to be directly electrogenic. There is no pump for chloride ions in peripheral nerve. The uneven distribution of Cl- (high outside, low inside) arises because the membrane is permeable to it and the anion is distributed largely in accordance with the potential difference across the membrane.

Figure 2.1 The ionic basis of the resting membrane potential. The diagram represents a portion of an axon, with hypothetical ionic concentrations on either side of its membrane given in mmol/litre. The arrows show the directions of the concentration gradients. Where the arrows do not cross the membrane, the membrane is assumed, for simplicity, to be impermeable to the ion concerned. A- represents large organic ions such as hexosephosphates and ATP. A microelectrode is shown inserted through the membrane, and the potential difference between it and a second electrode in the extracellular fluid is recorded on the cathode ray oscilloscope. Under such circumstances, a membrane potential of about 85 mV (inside negative) would be recorded. EK calculated from the Nernst equation (61 × log 140/5) is 88 mV and is therefore slightly larger than the true membrane potential measured with a microelectrode

If, for simplicity, it is assumed that the membrane is permeable to K+, but not to Na+ and A-, then K+ will tend to move down its concentration gradient and there will be a small flux of K+ through the membrane from inside to outside; its anions will be left behind. Hence, the membrane will separate a few charges of opposite sign and will be polarized as illustrated in Figure 2.1, being positive on the outside with respect to the inside. The pores or channels through which K+ diffuses are known as leakage channels to distinguish them from the various other K+ channels present in the membrane.

The movement or flux of K+ through the membrane along its concentration gradient is opposed by the build-up of positive charge on the outside (like charges repel each other). At equilibrium the chemical force derived from the concentration gradient that tends to move K+ from inside to outside is balanced by the electrical force that repels further movement. Situations of this sort were studied and analysed by the German physical chemist Walter Nernst in 1888, who derived an equation based on basic thermodynamic principles for calculating the potential difference across the membranes (Em), in this case the K+ equilibrium potential (EK). Those who are not at ease with thermodynamics can nevertheless see that the Nernst equation includes factors relating to concentration gradient and to electrical forces. Those concerned to know how the equation is derived may consult any appropriate textbook.

According to the Nernst equation:

where R is the gas constant, F is Faraday’s constant, T is the temperature in degrees Kelvin and z is the valency of the ion concerned. For univalent ions and a body temperature of 37ºC (310K), and converting to logarithms to the base 10, the equation takes the simplified form shown below. Because it is conventional to refer to the potential of the inside of the cell relative to the outside, the concentration gradient in the equation is turned upside down to give a negative value; thus:

Substitution of the concentrations of potassium ions in Figure 2.1 gives a value of −88 mV and this would approximate reasonably closely to the true value detected by an intracellular microelectrode and measured on an oscilloscope as illustrated in Figure 2.1. In fact, the true membrane potential is usually a little less than that calculated from the Nernst equation, because in reality the resting membrane is not totally impermeable to sodium ions, nor completely permeable to potassium ions. The membrane of a frog muscle fibre, for example, is about one seventy-fifth as permeable to Na+ as to K+ (Katz, 1966). A modified equation known as the Goldman-Hodgkin-Katz equation takes the permeabilities to Na+ and K+ into account and gives values for frog muscle and cephalopod axons that agree more closely with the measured potentials.

According to the Goldman-Hodgkin-Katz equation:

where P, the permeability of a membrane to an ion, is defined as the net flux of that ion divided by the product of the concentration difference of the ion across the membrane times the area of membrane.

The lower the permeability to Na+ and the greater to K+, the closer Em approaches EK. This is the situation in glial cells, but in nerve and muscle cells the small permeability to Na+ opposes EK and subtracts a few mV from the resting membrane potential as suggested in Figure 2.1. (i.e. EK = 88 mV; measured Em = 85 mV).

Most of the experimental data on nerve are derived from giant axons from cephalopods, but it is believed that an essentially similar ionic basis underlies the membrane potentials in mammalian axons, including their fine terminal branches. Several mammalian muscles, including human muscles taken at biopsy, have been studied with intracellular microelectrodes. As stated above, there is evidence that a 1:1 ratio of Na+ to K+ ions shifted by a non-electrogenic pump in muscle fibres is an oversimplification. A proportion of the membrane potential is, in fact, produced by an electrogenic component of pump activity. This electrogenic component of pump activity appears to be under the trophic influence of the nerve; it disappears, and the membrane potential falls, after denervation (Bray et al., 1976).

The Na+/K+-dependent ATPase that catalyses the electrogenic component of pump activity appears to be more sensitive to inhibition by cardiac glycosides than the enzyme involved in non-electrogenic pumping. Small doses of cardiac glycosides, such as ouabain, therefore cause a depolarization which is similar in extent to that produced by denervation, and which is absent after denervation (Clausen and Flatman, 1977).

Action potentials

An action potential is a self-propagating wave of reversed membrane potential that passes along a nerve or muscle fibre. It is initially triggered either from a sensory receptor or by a voluntary decision made in higher centres of the brain, which are continuously active. Eventually the action potential reaches the lower motoneurones and is transmitted to the muscle fibres.

The permeability of an excitable membrane, such as that of a nerve or muscle fibre, to ions is controlled by the potential difference across it. A fall in membrane potential of 15 mV or so causes the sudden opening of so-called ‘sodium gates’ that guard selective sodium channels. The concentration gradient and the electrical gradient for Na+ are from outside to inside (Figure 2.1), and as soon as the sodium channels open an inward sodium current (INa) is set up. The influx of Na+ opposes the resting membrane potential causing a further depolarization and hence the opening of more sodium channels in a positive feedback or regenerative manner. In this way, all of the sodium channels rapidly open in response to an effective stimulus. Obviously no more can open in response to a stronger stimulus, and since each can open only once, the action potential is ‘all-or-nothing’; that is to say that, under constant environmental conditions (e.g. temperature), all effective stimuli produce action potentials of equal amplitude. The increase in permeability to Na+ is such that it is then relevant to substitute the intra- and extracellular concentrations of Na+ into the Nernst equation to calculate ENa. If this is done for the concentrations given in Figure 2.1 (ENa = 61 log 100/10 = 61 × log 10 = +61 mV) it will be seen that the membrane potential would pass through zero to become positive on the inside with respect to the outside. In fact, the change in membrane potential that constitutes the action potential does approach the sodium equilibrium potential calculated from the Nernst equation. An active region of membrane with reversed transmembrane potential, lying adjacent to a resting region of membrane, would cause local circuit electric currents to flow through the surrounding extra- and intracellular fluids. These local circuit currents flowing through the region of membrane immediately ahead of the active region cause a voltage drop across the membrane resistance which opposes the resting potential; that is, the membrane is slightly depolarized at this point. The sodium gates at this point are thereby opened, and the consequent increase in sodium conductance sets up the action potential in the adjacent, previously resting, region. In this way, the action potential propagates from point to point along the membrane as illustrated in the diagram of Figure 2.2.

Figure 2.2 Action potential propagation. (a) Resting axon. Membrane polarized, positive on the outside with respect to the inside. (b), (c) and (d) Region of reversed potential (i.e. an action potential) propagates from right to left. Reversed potential is a consequence of the opening of voltage-operated Na+ channels. Local circuit currents (b and c) flow ahead of the active region and excite the region ahead by opening Na+ channels. The action potential is terminated because the Na+ channels become inactivated and the Na+ ions are dissipated. The restored membrane potential then closes the Na+ channels in their resting conformation. (Note that delayed rectifier K+ channels are not included in the mechanism illustrated, cf. Figure 2.4)

It is a characteristic of sodium channels in excitable membranes that the activating stimulus has the secondary effect of causing a conformational change in the channel proteins such that the channel becomes occluded. The effect is called inactivation. The gate has not yet closed, but the channel no longer permits inward Na+ flux. Because of inactivation, any one channel opens only once during a depolarizing stimulus. No matter how prolonged that stimulus, the channel converts to the inactivated state and does not reopen. Channel inactivation and dissipation of the sodium ions causes reinstatement of the resting membrane potential and this then restores the channel and the gating molecule to their resting states. Channel inactivation accounts for the refractory period of nerve. During the relatively refractory period, the channel gradually becomes re-excitable. Figure 2.3 gives a simplified view of the operation of a sodium channel. The channel is thought to be guarded by a gating molecule that can be open or shut according to the electric field across the membrane. As it is sensitive to the transmembrane voltage, the gating molecule itself is presumably charged. When the electric field changes, the gating molecule moves and this opens the channel. The movement of the gate itself causes a small current flow which, with sensitive apparatus, can be detected as the so-called gating current at the start of membrane excitation.

Figure 2.3 The diagram illustrates: (a) the resting state with channel closed; (b) the partially depolarized membrane with the voltage-sensitive gating molecule starting to respond by realigning its dipole moment with respect to the electric field; (c) the rotation of the gate results in a small displacement of positive charge from near the inner surface to near the outer surface, so that there is a reduction in the total charge separation across the membrane. In a voltage-clamp experiment, a small extra outward current (the gating current) would flow to maintain the voltage across the membrane. The opened gate allows inward Na+ current to flow and this reverses the polarity of the membrane, which causes the opening of more sodium channels in a regenerative manner. (d) The large and abrupt change in electric field has caused a conformational change in the channel protein that prevents it