Transmission by post-ganglionic axons of the autonomic nervous system: The importance of the specialized neuroeffector junction

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Pergamon

Neuroscience Vol. 73, No. 1, pp. 7-23, 1996 Copyright © 1996IBRO.Publishedby ElsevierScienceLtd Printed in Great Britain.All rights reserved S0306-4522(96)00031-0 0306-4522/96$15.00+ 0.00

COMMENTARY TRANSMISSION BY POST-GANGLIONIC AXONS OF THE AUTONOMIC NERVOUS SYSTEM: THE IMPORTANCE OF THE SPECIALIZED NEUROEFFECTOR JUNCTION G. D. S. HIRST, J. K. CHOATE, H. M. COUSINS, F. R. EDWARDS and M. F. KLEMM Department of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia 1. INTRODUCTION 2. STRUCTURE OF AUTONOMIC AND ENTERIC NEUROEFFECTOR JUNCTIONS 2.1. Historical aspects 2.2. Sympathetic neuroeffector junctions 2.3. Parasympathetic neuroeffector junctions 2.4. Enteric neuroeffector junctions 2.5. Conclusions 3. ACTIONS AND RELEASE OF INDIVIDUAL QUANTA OF TRANSMITTER 3.1. Post-junctional actions 3.2. Probability of release of transmitter at individual autonomic neuroeffector junctions 4. RESPONSES PRODUCED BY NEURALLY RELEASED AND ADDED TRANSMITTERS 4.1. General comments 4.2. Vagal stimulation and added acetylcholine on cardiac pacemaker cells 4.3. Sympathetic stimulation and added catecholamines on cardiac pacemaker cells 4.4. Catecholamines and sympathetic nerves innervating arteries, veins and the dilator layer of the iris 4.5. ATP and sympathetic nerves innervating systemic arteries and vasa deferentia 4.6. Acetylcholine and excitatory neuroeffector transmission in the intestine 5. CONCLUDING REMARKS 6. REFERENCES

1.

INTRODUCTION

Many organs are innervated by projections from the autonomic nervous system. Invariably the organs consist of many individual cells which are electrically coupled to neighbouring cells to form electrical syncytia. As examples, blood vessels, such as arteries, arterioles and veins, are made up of many vascular smooth muscle cells orientated roughly at right angles to the major axes of the vessels: in each vessel, a small proportion of the current injected into an individual cell escapes across the membrane of that cell but the majority flows into neighbouring cells. 7°'1°1'137Individual cardiac myocytes are coupled to nearby cells, so enabling action potentials to conduct readily from cell to cell. TM Similarly, individual intestinal muscle cells are coupled to their neighbours to form electrical syncytia which behave electrically in much the same way as do single large cells. ~ Abbreviations: ACh, acetylcholine; ATP, adenosinc-5'-

triphosphate; cAMP, cyclic AMP; EJC, excitatory junctional current; EJP, excitatory junction potential; IP3, inositol (1,4,5)trisphosphate; IBMX, iso-butylmethylxanthine; NA, noradrenaline; VIP, vasoactive intestinal peptide.

7 8 8 9 10 11 i1 11 11 12 13 13 13 15 16 17 18 19 19

The autonomic nervous system is divided into the sympathetic, parasympathetic and enteric divisions. The sympathetic division is derived from the thoracolumbar outflow whilst the parasympathetic division arises from the craniosacral outflows. The enteric nervous system is an independent network of nerves which resides entirely within the intestine. In each division ganglion cells give rise to ganglionic axons which run in axon bundles into the tissues they innervate. Individual axons branch near the cells that they innervate and the fine branches become varicose. Each varicosity contains numerous vesicles which store transmitter substances. Most, but not all, sympathetic varicosities release a catecholamine, either noradrenaline (NA) or adrenaline as their transmitter. Most parasympathetic varicosities release acetylcholine (ACh). Some varicosities of either division have been suggested to release adenosine-5'-triphosphate (ATP) in addition to their classical transriaitter substance. In the enteric nervous system, most excitatory projections release ACh whilst the inhibitory projections have been proposed to release a number of substances which include nitric oxide or related precursor, 119 ATP, or

8

G.D.S. Hirst et al.

vasoactive intestinal peptide (VIP) and related peptides? 2 In the somatic nervous system transmitters are released as performed packets or quanta, from the stores of vesicles present in individual nerve terminals. After release transmitters interact with pools of receptors located in the synaptic clefts close to their points of release. In tissues innervated by the autonomic nervous system it was thought that transmitters acted more like local hormones. A moderate proportion of varicosities were thought to lie close by the membranes of target cells with the remainder lying varying distances away. Transmitters would then be released at various distances from the cells they innervate. This implies that the responses produced by individual quanta of transmitter would vary in amplitude depending upon the separation between varicosity and effector cell membrane. Rather than acting in high concentration at the point of release, transmitters would diffuse through the extracellular space and interact with receptors distributed widely over the membranes of target cells. Receptors with the highest affinity for transmitter would be of greatest physiological importance with added and neurally released transmitters activating identical receptors to produce similar responses. However, recent structural observations show that a large proportion of varicosities form organized neuroeffector contacts. Moreover the notion that transmitters act as local hormones does not fit with many recent analyses of the process of neuroeffector transmission in a range of organs. These studies have shown that added and neurally released transmitters often act quite differently, which suggests that neurally released transmitters interact with specialized junctional receptors: these have different properties from extrajunctional receptors that are most readily activated by applied transmitter. 2. STRUCTUREOF AUTONOMICAND ENTERIC NEUROEFFECTORJUNCTIONS 2.1. Historical aspects

The relationship between axon bundles, individual axons and varicosities with their effector cells has

been studied extensively using electron microscopy. The composition of axon bundles varies from single axons with or without associated Schwann cell, to axon bundles containing up to 100 profiles. Larger axon bundles and most small bundles are partially surrounded by Schwann cells which occasionally run between the axons. Bundles usually consist of varicosities and fine intervaricose axons (diameter 0.1-0.4pm); the latter contain neurotubules. Varicosities have larger profiles than do intervaricose axons; unlike intervaricose axons they contain synaptic vesicles and mitochondria. In most studies, the relationship between individual varicosities and target cells has been examined by viewing random sections of tissue. With this approach some varicosity membranes, exposed by a break in the Schwann cell, are seen to lie within 20 nm of the effector cell membranes, others are seen to lie distant from an effector cell. In many blood vessels, about half of the varicosities were seen to form close contacts with nearby smooth muscle cells. In arteries and veins these neuroeffector junctions had cleft widths in the range 60-200 n m . 2'4'18'31'32'35'40'92'105"123"126 However, in both tissues, the remaining varicosities were seen to lie some distance from the nearest cell membrane. The results of these studies are summarized in Table 1. Similar morphological studies have been conducted on many other organs innervated by the autonomic nervous system. Each reported that although some junctions had small neuroeffector cleft widths, others had large separations between the membranes of the nerve terminals and the effector cells. Thus Merillees ~°2 stated that all terminal axons in the guinea-pig vas deferens were within 50 nm of a smooth muscle cell, with many as close as 25 nm, and that many en passage (non-terminal) varicosities lay within 50-100 nm of a smooth muscle cell but that several varicosities were up to 250 nm distant from the nearest muscle cell. Intimate contacts (20 nm) have also been reported in the cat myometrium, the human ureter, TM the guinea-pig seminal vesicle j°8 and the rat urinary bladder. 57'1°8Again in each of these organs, exposed varicosities were also

Table 1. Distribution of neuroeffector cleft widths determined from analysis of randomly viewed sections of vascular smooth muscle preparations Vessel Rabbit ear artery Rat mesenteric arterioles Rat mesenteric arteries Dog mesenteric arteries Rabbit pulmonary artery Rabbit facial vein Untreated Relaxed Contracted Myocardial arterioles Myocardial venules Mesenteric veins

Mean cleft width (nm) mean _ S.E.M.

Range (nm)

Reference

500 + 81 10if400 300-700 705 1900

80-1000 80 1000 60-2000 1000~000

116 40 40 3 139

250 __+41 260 + 37 390 + 28 252 _+25 171 + 14 197 + 18

50-750 50-1000 50-1000 75-750 46-490 50-850

122 51 85

Neuroeffector junctions Table 2. Properties of sympathetic neuroeffector junctions in blood vessels determined by analysis of successive sections of tissue using the electron microscope

Tissue Rabbit juxtaglomerula arterioles Afferent Efferent Guinea-pig submucosal arterioles Guinea-pig mesenteric veins

Percentage of varicosities forming junctions 70 69 1.26 + 0.29 90 98

Area occupied by Varicosity volume junctions (#m 2) (pm 3) mean _+S.E.M. mean _+S.E.M.) 0.48 + 0.32 (n = 4) 0.38 __+0.05 (n = 6) 1.2 _+0.25 (n = 11) 0,64 _+0.08 (n = 47)

0.52 + 0.25 (n = 8) 0.36 __+0.13 (n = 8)

Reference

95 95 98

(n = 11) 0.81 + 0.15 (n = 17)

85

found to lie well away from the nearest muscle cell. However, the relationship between varicosities and With cardiac tissues, intimate contacts (20 nm), close target cells cannot be determined reliably by viewing contacts ( < 100 nm) and junctions with separations random sections of tissue. This procedure assumes > 1 0 0 n m , have been noted in a number of that the relationship between a varicosity and the species. 31'34'47'82'84'11°'131'141Intimate contacts, some of target tissue observed in a single section is retained which contained localized clusters of vesicles near the throughout the entire outline of that varicosity. This region of close apposition, TM and less organized neu- has been shown not to be the case when successive roeffector junctions have been noted in the iris of a sections through an individual varicosity were examnumber of species. 4s'~°9'121'135Gabella54 reported that ined. With this approach most varicosities which lose 50% of the varicosities in the guinea-pig sphincter their Schwann cell coat are seen to form a close pupillae were separated from muscle cells by 20 nm. apposition with a nearby cell. A similar pattern emerges in intestinal muscle. 2.2. Sympathetic neuroeffector junctions Gabella53found that vesiculated nerve processes were loosely associated with smooth muscle cells and The organization of sympathetic neuroeffector cytoplasmic extensions of interstitial cells in the junctions in a number of arterioles and small muscuguinea-pig ileum. A few varicosities approached in- lar arteries from different beds in different animals terstitial cells to within 20 nm, while others were has been determined using serial sections of tiswithin 100 nm of smooth muscle cells. 55 Gabella56 sue. 94'9~98 More recently these studies have been describes intestinal neuromuscular junctions with extended to include sympathetic neuroeffector juncclefts of 80 nm or more filled with intervening basal tions in guinea-pig mesenteric veins,85 in toad cardiac lamina; occasionally neuroeffector junctions with pacemaker cells, 84 in guinea-pig sinoatrial nodes, 34 cleft widths of 15 nm, which lacked intervening basal and in rat iris dilator muscles.63 lamina were observed. Cleft widths in the circular Sympathetic varicosities innervating blood vessels muscle of rat duodenum varied from 20 to 500 nm; in are found in axon bundles which contain from 1 to the muscularis mucosa they were over 500nm. 14° 100 axons. Axon bundles are situated in the adventiBennett and Rogers 12 found some varicosities lay as tia, close to the medioadventitial border. Many of the close as 100 nm to muscle cells of the rat taenia coll. axons are intervaricose, i.e. they are < 0 . 2 # m in Llewellyn-Smith et al. 93 have also described organized diameter and contain many neurotubules. Varicosineuroeffector junctions which contain vesiculated ties are usually between 0.3 and 1 #m in diameter and nerve profiles lying from 50 to 200 nm away from contain synaptic vesicles and mitochondria. Symlongitudinal muscle cells of the guinea-pig ileum, pathetic varicosities contain three types of synaptic some muscle cells have rare foot like processes which vesicles; small clear and small granular vesicles and extend to within 15-20 nm of nerve fibre bundles. large granular vesicles. 58 The small vesicles are Finally Zhou and Komuro 144 illustrated close con- thought to contain NA in mammals and adrenaline tacts between varicosities and interstitial cells. in amphibians, the large vesicles are thought to Together these observations indicate that the sep- contain neuropeptides. The presence of adrenaline or aration between autonomic varicosities and effector NA in small granular vesicles of varicosities innervatcells is variable. Since it had been suggested that all ing blood vessels has been confirmed by 5-hydroxyvaricosities which are exposed through the Schwann dopamine incubation.85'98 When consecutive sections of a number of blood cell, regardless of the distance from the effector cell, are capable of releasing transmitter ~°°'~°3this gave rise vessels were examined the majority of varicosities to the view that autonomic nerves bathed the tissue which become exposed through a break in the in transmitter in much the same way as one would do Schwann cell were found to form specialized neuroby adding transmitter substance to the extracellular muscular junctions with vascular smooth muscle cells (see Table 2). At each junction, the varicosity medium.

G . D . S . Hirst et al.

10

membrane was separated from the smooth muscle by a gap of < 100 nm which was filled with a single layer of basal lamina. Catecholamine containing vesicles were found to concentrate near the area of contact. 85'9~98Statistical analysis of vesicle distribution in junctions formed on mesenteric veins demonstrated a significantly greater density of small vesicles in the volume immediately above the contact, compared with areas away from the contact. 85 Although it was clear that vascular sympathetic junctions lacked detectable post-junctional specializations, approximately 25% of junctions had demonstrable presynaptic membrane specializations. These appeared as small electron-dense patches on the presynaptic membrane which were closely associated with a few synaptic vesiclesY'96'97 The dimensions of sympathetic neuroeffector junctions found in blood vessels are presented in Table 2. Sympathetic neuromuscular junctions which are similar to those found in blood vessels have been identified in the pacemaker region of the toad heart 84 and in the sinoatrial node of the guinea-pig heart. 34 In these tissues both sympathetic and parasympathetic varicosities are present. These can be distinguished by using a modified chromaffin procedure 58 which causes a dense precipitate to form in catecholamine containing vesicles. In amphibian and mammalian cardiac pacemaker tissue the majority (88% and 90%, respectively) of sympathetic varicosities followed in serial sections, which became exposed through a gap in the Schwann cell, formed specialized neuromuscular junctions with the cardiac muscle cells. The features of these junctions are the same as those described for blood vessels. Again the density of small synaptic vesicles immediately above the specialized junctions was found to be significantly greater than the density of vesicles in other parts of the varicosities. In contrast the larger peptide containing vesicles are usually situated away from the region of close apposition. 34'84 However, unlike neuroeffector junctions in blood vessels presynaptic membrane specializations were not detected in cardiac tissue, even after prolonged osmium fixation or treatment with phosphotungstic acid, which exposes such specializations at other synapses) 5 The dimensions of sympathetic neu-

roeffector junctions found in cardiac muscle are given in Table 3. When the structure of sympathetic neuroeffector junctions in the rat iris dilator was determined, it was found that approximately one-third of varicosities made specialized contacts with myoepithelial cells of the dilator and another third with melanophores in the stroma; 46 the remaining varicosities failed to form organized neuroeffector junctions. 63 The organized junctions were as described previously; their dimensions are presented in Table 3. In the sympathetic varicosities which became exposed through a gap in the Schwann cell but failed to make neuroeffector contact with either melanophores or myoepithelial cells, the distribution of synaptic vesicles was found to be homogeneous. Thus unlike the organized junctions, vesicles were not found to cluster towards the exposed varicosity membrane. 63 The physiological function of non-contacting varicosities is unclear. Olsen H5 proposed that as well as innervating blood vessels, melanophores and smooth muscle, efferent fibres in the iris might release substances into the aqueous humour. Alternatively Richardson TM suggested that varicosities situated at some distance from an effector tissue might be the site for uptake, synthesis and storage of transmitters. In summary, each study which has examined successive sections of tissue has indicated that most sympathetic varicosities which lose part of their Schwann cell sheath, form organized neuroeffector junctions. 2.3. Parasympathetic neuroeffector junctions Only a few studies have determined the entire structure of parasympathetic neuroeffector junctions. In cardiac pacemaker cells of toads 84 and guineapigs 34 and rat iris dilator muscle, 63 parasympathetic varicosities, other than the lack of dense cored synaptic vesicles seen after chromaffin fixation, were found to be similar to sympathetic varicosities. Most varicosities that became exposed through a gap in their Schwann cell wrap formed organized neuroeffector junctions. At the junctions, the varicosity membrane was separated from a nearby muscle cell by a single layer of basal lamina, < 100 nm thick) 4'63'84 Small synaptic vesicles again concentrated near the area of

Table 3. Properties of sympathetic neuroeffector junctions on cardiac muscle and in structures associated with the rat iris dilator muscle, determined by analysis of successive sections of tissue using the electron microscope

Tissue

Percentage of Area occupied by varicosities forming junctions (~m 2) junctions mean _ S.E.M.

Toad sinus venosus

88

Guinea-pig sinoatrial node

90

Rat iris dilator myoepithelium

26

Rat iris dilator melanophores

35

0.436 _+0.07 (n = 25) 0.15 _ 0.03 (n = 9) 0.23 __+0.07 (n = 6) 0.21 _ 0.05 (n = 8)

Varicosity volume

(#m 3) mean _ S.E.M.

Reference

0.68 _ 0.39 (n = 4) 0.31 4- 0.06 (n = 9) 0.21 + 0.07 (n = 4)

84 34 63 63

11

Neuroeffector junctions Table 4. Properties of parasympathetic neuroeffector junctions on cardiac muscle and in structures associated with the rat iris dilator muscle, determined by analysis of successive sections of tissue using the electronmicroscope Percentage of varicosities forming junctions

Area occupied by junctions (~m 2) mean + S.E.M.

Variscosity volume ~ m 3) mean + S.E.M.

Toad sinus venosus

96

Guinea-pig sinoatrial node

85

Rat iris dilator myoepithelium

36

0.60 + 0.16 (n = 9) 0.48 + 0.07 (n = 13) 0.36 _ 0.09 (n = 14)

Rat iris dilator melanophores

71

0.453 + 0.06 (n = 28) 0.210 __+0.04 (n = 13) 0.50 _+0.12 (n = 14) 0.44 _+0.08 (n = 27)

Tissue

contact. The larger vesicles, which in the toad are thought to contain the neuropeptide somatostatin, 3° were again located some distance from the contact region. The dimensions of parasympathetic neuroeffector junctions found in cardiac and dilator muscles are shown in Table 4. As with many sympathetic terminals, presynaptic membrane specializations were again not detected in parasympathetic varicosities. Parasympathetic neuroeffector junctions, very similar to those formed by sympathetic varicosities, were identified in the myoepithelium of the rat iris dilator. The dimensions of varicosities and contacts are shown in Table 4. A proportion of parasympathetic varicosities (18%) made neuroeffector junctions only with myoepithelial cells whereas others made junctions with myoepithelial cells and with melanophores. Yet others formed junctions only with melanophores. Again a high percentage (18%) of exposed varicosities failed to make neuroeffector junctions with any effector tissue. 63 Likewise at the organized neuroeffector junctions vesicles were clustered towards the area of contact whereas in the varicosities that failed to make a contact, vesicles were homogeneously distributed throughout the varicosity.63 Very recently the relationship between parasympathetic varicosities and bladder smooth muscle cells has been determined from an examination of consecutive sections of tissue. 57 Again in this tissue many varicosities which lose a part of their Schwann cell coating were found to form close appositions with nearby muscle cells. 2.4. Enteric neuroeffector junctions The innervation of the longitudinal muscle of the guinea-pig ileum has been examined by viewing consecutive sections of tissue, s6 This muscle layer is some five to 10 cells thick and the innervation, which is predominantly cholinergic, is confined to the tertiary plexus lying over the inner surface of the longitudinal muscle layer: 2 This study has again found that many varicosities lying in the tertiary plexus form organized neuroeffector junctions with longitudinal muscle cells. Two types of neuromuscular junctions are present. One type is formed by small varicosities,

Reference 84 34 63 63

giving rise to junctions with small areas of junctional contact. The other junctions more closely resemble the junctions found in tissues innervated by the other divisions of the autonomic nervous system: they are formed by larger varicosities and have larger areas of junctional contact. Approximately 25% of these latter junctions are seen to have presynaptic membrane specializations which resemble those found at sympathetic neuromuscular junctions. 86 2.5. Conclusion These studies have indicated that most autonomic varicosities which are not totally enclosed by a Schwann cell sheath, form organized neuroeffector junctions with nearby target cells. These junctions have many features traditionally associated with synaptic structures found in the central nervous system and at skeletal neuromuscular junctions. A part of the varicosity approaches the target cell membrane and forms a junction which has a neuroeffector gap of 20-80 nm. Often the cleft is filled with a single layer of basal lamina. Vesicles invariably are concentrated towards the region of close apposition. In many tissues varicosities contain presynaptic specializations which resemble the structures associated with transmitter release at many synapses. Unlike many, but not all, synapses postsynaptic specializations are not detected in tissues innervated by the autonomic system. 3. ACTIONSAND RELEASEOF INDIVIDUALQUANTA OF TRANSMITTER

3.1. Post-junctional actions At many synapses quanta of transmitter are released at irregular intervals in the absence of nerve stimulation. Where the transmitter interacts with receptors that are directly coupled to sets of ligand gated channels, each quantum of transmitter evokes a small change in the membrane potential of the postsynaptic cell. Hence miniature excitatory or inhibitory synaptic potentials are recorded from a range of neurons and miniature end-plate potentials are recorded from skeletal neuromuscular junctions. Miniature junction potentials that correspond to the spontaneous release of quanta of transmitter are recorded from only a limited number of tissues which

12

G . D . S . Hirst et al.

are innervated by the autonomic or enteric nervous systems. In systemic arteries, arterioles and vasa deferentia, which are innervated by the sympathetic nervous system, spontaneous miniature excitatory junction potentials (sEJPs) are readily detected. In these tissues quanta of sympathetic transmitter activate sets of ligand gated channels. Stimulating the sympathetic nerves to these tissues initiates, after a brief latency, an excitatory junction potential (EJP). 24,66 Although these EJPs have durations of about a second, the underlying excitatory junctional currents (EJCs) are brief. 23,28"7° Typically an EJC is calculated to have a rising phase lasting about 10 ms and a decay phase lasting 100-150ms. 28'7°,72 Direct measurements of the time courses of EJCs, using a voltage clamp technique 5° or an extracellular recording technique 22 provide very similar values. Although the decay phases of such EJCs last for much longer than they do at most synapses, their rapid rising phases indicate that sets of ligand gated channels are being activated. As EJCs start 1-2 ms after a nerve action potential invades a varicosity, neither a Gprotein nor a second messenger are involved in their initiation. If the separation between the membrane of an individual varicosity and the nearest post-junctional membrane varied, the size of the responses produced by identical quanta released from each varicosity would vary from varicosity to varicosity. As the distance over which the transmitter diffuses increases, the amount of transmitter reaching the post-junctional membrane decreases very rapidly, with the amount of transmitter reaching a point falling as a power of the separation. Junctions with small separations between varicosity and target membranes would produce large quantal responses: those with large separations would produce small quantal responses. Thus the amplitudes of quantal responses, i.e. sEJPs, should vary widely. Some should be large and some should be barely distinguishable from the recording noise. When the first recordings were made from vas deferens this was found to be the case. Many sEJPs were recorded from a single cell, although a number of large sEJPs were detected the majority of potentials had amplitudes that were similar to the recording noiseY However, as has been pointed out, tissues, like the vas deferens, which are innervated by the autonomic nervous system, are made up of many cells coupled together as electrical syncytia. 1~ This means that when a recording is made at a point, events which occur some distance from that point will be attenuated in amplitude. Thus if identically sized quantal currents were produced at a number of separate varicosities, a quantal event occurring at a neuroeffector junction near the electrode would produce a large potential change at the recording site. The same quantal event occurring at a neuroeffector junction some distance from the electrode would produce a smaller potential change at the recording site and those occurring further from the recording

electrode would produce smaller signals with their amplitudes becoming similar to the recording noise. As many more junctions lie some distance from the electrode than lie close by the electrode, the amplitude distributions of sEJPs will be skewed with most signals having amplitudes similar to the noise level. Evidently in complex multicellular preparations, the interpretation of sEJP amplitude histograms is difficult. These ambiguities of interpretation would be removed if recordings could be made from a single isolated innervated cell; recordings from preparations in which this is possible have not been described. A compromise is to make recordings from a collection of coupled cells which is sufficiently small to be isopotential. When this was done, using short segments of arteriole which contained some 100 to 200 individual smooth muscle cells, successive sEJPS had similar time courses and a unimodal amplitude distribution. 72 Such distributions would only be found if the separation between each varicosity and the muscle cell was very similar. 3.2. Probability of release of transmitter at individual autonomic neuroeffector junctions It is important to stress that comments about the release of transmitter at individual autonomic neuroeffector junctions can only be made if the response to the release of a single quantum of transmitter can be detected. When a quantum of transmitter activates sets of ligand gated channels a signal is produced which can be detected using either extracellular or intracellular recording techniques. This is the case at some sympathetic and parasympathetic neuroeffector junctions where a secondary transmitter, possibly ATP, activates sets of ligand gated channels. At virtually all other junctions where the primary transmitter is either a catecholamine or ACh, the released transmitter interacts with receptors which are more remotely coupled to sets of ion channels. These pathways may involve the perturbation of a Gprotein, the formation of a second messenger or both. At such junctions, spontaneous excitatory or inhibitory junction potentials which correspond to the release of individual quanta of transmitter are not detected. Thus a statement about the release of NA/adrenaline or ACh from individual autonomic varicosities is not possible at this stage. Where the release of transmitter from a small pool of varicosities can be monitored using electrophysiological techniques, it has become apparent that the likelihood that a varicosity will release a quantum of transmitter with each nerve impulse is very low. When recordings were made from electrically short segments of arteriole it was found that the sizes of individual sEJPs were quite similar to those of evoked EJPs. Thus each nerve impulse released one, two and sometimes three quanta of transmitter. However, the histological studies carried out on the preparations indicated that the segments of arteriole were innervated by some 100 to 200 individual varicosities.

Neuroeffector junctions Together the observations suggested either that only a few varicosities were capable of releasing transmitter or that the probability that an individual varicosity releases transmitter per impulse was very l o w . 72 The application of extracellular recording techniques to arteries and preparations of vas deferens confirmed that when an impulse invades a large pool of varicosities only a few quanta of transmitter are released. The low probability of release was not associated with failures of successive impulses to propagate into the nerve terminal: each stimulus initiates an action potential which invades each v a r i c o s i t y . 22'23'38 Similarly "shaped" quantal responses were selected and were taken to represent the release of a quantum of transmitter from a single release point. These experiments showed that release from an individual varicosity was intermittent, Subsequently the release of transmitter from an individual varicosity was characterized using more refined techniques which allow recordings to be made from single varicosities. These studies found that the probability of release varies from varicosity to varicosity. Some varicosities have appreciable probabilities of release whereas others have very low probabilities of release. 91'99 Together the observations on sympathetic neuroeffector transmission in systemic arteries, arterioles and vasa deferentia indicate that individual quanta of transmitter activate sets of ligand gated channels to produce a brief flow of inward current. The responses produced by successive quanta of transmitter released from the same varicosity are similar in amplitude. 91 All of the responses produced by single quanta released from a large pool of varicosities have similar amplitudes and time c o u r s e s . 72 Clearly some form of organization must occur between individual varicosities and target cell membranes with this pattern repeating from varicosity to varicosity. This idea is well supported by the morphological studies discussed previously. 4. RESPONSES PRODUCED BY NEURALLY RELEASED AND ADDED TRANSMITTERS

4.1. General comments

Sympathetic, parasympathetic and enteric varicosities contain a bewildering array of potential transmitter substances. These include neuropeptides such as neuropeptide Y, substance P, VIP, enkephalins and many conventional transmitters. The role of neuropeptides is unclear. They may function as transmitter substances. Although their release is readily detectable, in most cases either trains of repetitive stimuli or higher than normal levels of sensory stimulation are required to produce clear-cut peptidergic responses. Since the vesicles in which they are stored tend to accumulate away from the organized neuroeffector junctions formed by peripheral varicosities, 34'84it seems likely that these substances will be released in the extracellular space and act widely. These observations might suggest that their release

13

mechanisms and actions differ from those of the more classical transmitters: perhaps they are released by the sustained moderate increase in the internal concentration of calcium ions, [Ca2+]i , within nerve terminals that follows repetitive nerve activity. Peptidergic responses are preceded by long latent periods and the responses themselves frequently persist for many tens of minutes. On the other hand, it is clear that virtually all peripheral varicosities are capable of releasing transmitters which produce much shorter term changes in the activity of the organs. Presumably these transmitters are released by the high local [Ca2+]~ that occurs near presynaptic release points on varicosities after an action potential has invaded the nerve terminal. This review will concentrate on the actions of the transmitters involved in these more transient effects. The three major groups of transmitter substances released by sympathetic and parasympathetic varicosities are the catecholamines, NA or adrenaline, ACh and perhaps ATP. In the enteric nervous system whilst it is clear that many excitatory projections to both the circular and longitudinal muscle layers utilize ACh as their major excitatory transmitter the identity of inhibitory transmitter substances remains much less clear. Transmitters suggested to be released by enteric inhibitory fibres include ATP, the peptide VIP 52 and nitric oxide) 19 If each of the conventional transmitters was released at variable distances from the target cell membrane and diffused freely in the extracellular space, one would expect the responses produced by nerve stimulation and applied transmitter to be similar. Perhaps the time courses of the responses might differ because of the differing rates of application of transmitter, but the responses should have similar ionic bases and similar pharmacological behaviours. At a superficial level added and neurally released transmitters almost invariably act to produce similar changes in organ activity. In the heart, either vagus nerve stimulation or added ACh cause a bradycardia, sympathetic nerve stimulation or added catecholamine cause a tachycardia. In blood vessels, sympathetic nerve stimulation and the addition of either NA or ATP each cause vasoconstriction. In the intestine, excitatory nerve stimulation or added ACh increase intestinal motility. However, when the changes in membrane potential associated with each of these responses are examined it is frequently apparent that different mechanisms are activated. The following sections will discuss the differences and similarities of the responses produced by neurally released and added transmitters at a number of neuroeffector junctions. 4.2. Vagal stimulation and added aeetylcholine on cardiac pacemaker cells It is well documented that added ACh causes an increase in the K + conductance of the membranes of

14

G.D.S. Hirst

cardiac muscle cells, m ACh activates muscarinic receptors which are coupled by G-proteins to sets of K + selective channels. 61 These channels show inward rectification and are blocked by B a 2 + . 106'112 Thus when applied to pacemaker cells, ACh increases the peak diastolic potential and shortens the duration of individual action potentials. When sufficient ACh is applied to prevent the generation of pacemaker action potentials, the membrane potential settles to a potential negative of the peak diastolic potential. When applied to quiescent pacemaker cells, often those in which beating has been prevented by blocking cardiac muscle calcium channels, applied ACh causes a hyperpolarization. 26,59'6°More recently it has been shown that added ACh inhibits the flow of pacemaker current provided by a set of cation selective hyperpolarization activated channels. 42 Such channels open slowly when the membranes of pacemaker cells are brought negative of - 4 0 mV.142 They allow the inward movement of Na + and the effiux of K + so moving the membrane potential towards 0 mV. 41 The resulting current is termed either i h to symbolize its activation at hyperpolarized potentials 142 or if to symbolize the "funny" nature of its activation characteristicsfl The current ih undoubtedly contributes to pacemaking activity since reducing or increasing the amplitude of this current decreases or increases, respectively, the rate of generation of pacemaker action potentials. However, it is agreed that the current is not essential for pacemaking activity since complete inhibition of ih allows pacemaking activity to continue albeit at a reduced rate. 39 It has been generally assumed that the vagally released ACh activates the same pathways as those activated by added ACh, causing bradycardia either by increasing the K ÷ conductance of pacemaker cells or by both increasing the K ÷ conductance and by reducing the flow of the pacemaker current ih. HOWever, this assumption had never been tested. Hutter and Trautwein, 75 in their pioneering paper, suggested that vagal Stimulation produced bradycardia either by reducing a pacemaker current or by activating an inhibitory current. Not surprisingly, when it was found that added ACh did indeed activate a K ÷ conductance, the general view was taken that neurally released ACh would act in the same way. Support for the idea was found in studies where responses to applied and vagally released ACh were compared in arrested preparations: each caused hyperpolarizations that were abolished by muscarinic antagonists. 6° When ACh was applied by ionophoresis it was found that responses could be obtained whatever the position of the ionophoretic pipette. This led to the view that there was no special accumulation of post-junctional receptors near parasympathetic nerve terminals. 6° The finding that neurally released and added AChproduced different sequences of membrane potential changes, associated with different changes in mem-

et al.

brane conductance, was made fortuitously. In cane toads, a component of the response to high frequency vagal stimulation results from the release of somatostatin, this response being particularly apparent after atropinization. 3° As a prelude to analysing this peptidergic response, several responses to vagal stimulation were recorded in the absence of muscarinic blockade. It became apparent that neurally released and added ACh caused different sequences of membrane potential changes. 27 The responses to vagal stimulation and added ACh were each readily blocked by muscarinic receptor antagonists: this indicates that ACh is released by vagal stimulation and that it subsequently activates a muscarinic receptor. 26 However, vagal stimulation reduced the rate of generation of pacemaker action potentials by increasing the peak diastolic potential only slightly, slowing the rate of diastolic depolarization and leaving the duration of individual action potentials unchanged. In fact the same sequence of potential changes were described by Hutter and Trautwein. 7s The responses to vagal stimulation were quite different from those produced by added ACh in the same tissues. The bradycardia produced by added ACh was associated with a large increase in peak diastolic potential and a shortening of the duration of individual pacemaker action potentials. When vagal stimulation was sufficiently intense to stop the generation of pacemaker action potentials, the membrane potential settled at a level positive to that of the peak diastolic potential: when pacemaking was stopped by applied ACh the membrane potential settled at a value negative to that of the peak diastolic potential. 26 In amphibian pacemaker preparations, where beating was stopped using an organic calcium antagonist, both vagal stimulation and bath applied ACh caused hyperpolarizations which were blocked by muscarinic antagonists. 26 However, the addition of barium ions, Ba 2+, had little effect on the hyperpolarizations produced by vagal stimulation but blocked those produced by applied ACh, presumably by blocking the K + channels which are activated by muscarinic antagonists. 26 The two responses were further distinguished: during maintained vagal stimulation the membrane resistance increased, whereas in the presence of applied ACh the membrane resistance fell. 26 In an attempt to better mimic the effects of nerve stimulation, ACh was applied by ionophoresis. Although ionophoretically applied ACh produced similar hyperpolarizations to those produced by vagal stimulation, the ionophoretic potentials were again sensitive to Ba 2+ , whereas those resulting from vagus nerve stimulation were not. 2° Moreover, it was invariably found that ionophoretic potentials had slower time courses than did the vagal responses: an analysis of these data suggested that neurally released and added ACh activated separate pathways with distinct kinetics. 2° Finally, an analysis of the membrane potential changes recorded from beating and arrested preparations during vagal inhibition, using a

Neuroeffector junctions computer simulation, suggested that all of the response to vagal stimulation could be explained if neuronally released ACh activated a set of receptors which caused a selective suppression of the Na ÷ conductances involved in pacemaking activity. 45 Although the effects of applied ACh could be explained by the activation of a K ÷ conductance, those of neurally released ACh could not. 45 Similar experiments were carried out on sinoatrial node cells of the guinea-pig. 29 Vagal stimulation likewise reduced the rate of generation of pacemaker action potentials by slowing the rate of diastolic depolarization and leaving the duration of individual action potentials unchanged. A similar sequence of membrane potential changes had previously been described in rabbit heart by Toda and West. TM In contrast, the responses produced by added ACh were associated with large increases in peak diastolic potential and a shortening of the duration of individual pacemaker action potentials. During vagal arrest the membrane potential settled at a level positive to that of the peak diastolic potential: when applied ACh stopped the generation of pacemaker action potentials and the membrane potential settled at a value negative to that of the peak diastolic potentialfl 9 In arrested mammalian pacemaker cells of the guineapig sinoatrial node, we have recently found that vagal stimulation evokes membrane hyperpolarizations 8° and that quite similar hyperpolarizations can be produced by the local application of ACh. Again the responses to vagal stimulation were little affected by adding Ba 2÷ to the physiological saline: the peak amplitude of hyperpolarizations evoked by vagal stimulation (10 Hz for 5 s) in control solutions was 10.3 + 2.0mV and in 1 mM Ba 2÷ containing solutions was 9.0+2.6mV; n =4. In these preparations, the matched responses to applied ACh were partly resistant to Ba 2+ addition, the mean amplitude of control responses was 9.7 + 1.1 mV; the mean amplitude of responses in Ba 2÷ containing solutions was 3.4 + 0.5mV; n = 8. (J. Choate and G. D. S. Hirst, unpublished observations). Together the studies suggest that in both toad and guinea-pig pacemaker cells, neurally released ACh activates different sets of muscarinic receptors from those readily activated by applied ACh. Presumably those activated by neurally released ACh are located at the parasympathetic neuroeffector junctions found in these tissues. When activated the "junctional" receptors may well cause the formation of an unidentified second messenger substance which diffuses through the pacemaker cell syncytium and reduces the flow of pacemaker Na + current (for discussion see Ref. 20). Since pacemaker cells undoubtedly possess muscarinic receptors coupled to sets of K ÷ selective channels and these do not appear to be activated during vagal stimulation, little ACh must escape from the neuroeffector junctions: These observations suggest that the "extrajunctional" muscarinic receptors which are coupled to sets of K+-selective chan-

15

nels in pacemaker cells have little role in the control of heart rate. However, it is important to note that this does not imply that muscarinic receptors coupled to a K + conductance have an extrajunctional location throughout the entire heart. Indeed in conducting tissues of the heart, neurally released ACh appears to change cardiac excitability by increasing g~¢.27.75 4.3. Sympathetic stimulation and added catecholamines on cardiac pacemaker cells Sympathetic nerves which innervate mammalian hearts release NA, in amphibian hearts they release adrenaline. In mammalian and amphibian hearts added catecholamines activate beta-adrenoceptors and cause tachycardias. In mammals the predominant sub-type of receptor activated is the betas-type, 43 while in amphibians the predominant sub-type resembles a mammalian betae-type. 2'3 In mammalian hearts tachycardias produced by sympathetic nerve stimulation are readily blocked by beta-adrenoceptor antagonists. '~5 In contrast in amphibian hearts the tachycardia caused by sympathetic nerve stimulation persists in the presence of high concentrations of beta- (and alpha-) adrenoceptor antagonists. ~°7 Although different types of receptors are activated, in both amphibian and mammalian pacemaker cells, the sequences of membrane potential changes recorded during sympathetic nerve stimulation are very similar. 21'33'75'125 Sympathetic nerve stimulation produces a two component tachycardia. During the first component, the rate of diastolic depolarization is increased and the amplitudes of successive pacemaker action potentials are either unchanged or slightly reduced, otherwise the time courses of action potentials are unchanged. 21'33 In both classes when the generation of pacemaker action potentials is blocked by adding an organic calcium antagonist to the physiological saline, sympathetic nerve stimulation initiates an EJP with the same temporal characteristics as the first phase of tachycardia recorded from the beating preparation.2~ '33 The tachycardias produced by added catecholamines in beating amphibian and mammalian pacemaker cells are also very similar. 2''33However, in both classes, the membrane potential changes produced by added catecholamines differ from those recorded after sympathetic nerve stimulation. 2~'33'133Added catecholamines combine with sets of beta-adrenoceptors which in turn activate adenylate-cyclase. The increased levels of cyclic AMP (cAMP) cause phosphorylation of many of the voltage-dependent channels active during pacemaking activity. As a result, voltage-dependent Ca 2+ channels allow increased Ca 2+ entry. The kinetic behaviour of delayed rectifier K ÷ channels is changed leading to an increase in the rate of repolarization. The activation potential of hyperpolarization activated cation selective channels is moved to more positive potentials. 6~ Together these changes lead to an increased rate of

16

G . D . S . Hirst et al.

discharge of pacemaker action potentials with their peak diastolic potentials being more negative, the amplitude of the action potentials being greatly increased and the duration of individual action potentials being reduced. 21'33 As one would expect, the responses to added catecholamines are mimicked by forskolin, which directly activates adenylate cyclase and by iso-butylmethylxanthine (IBMX) which inhibits phosphodiesterases and causes cAMP to accumulate. 61 In amphibians both the tachycardias and the underlying EJPs recorded from arrested preparations, persist in the presence of beta- (and alpha-) adrenoceptor antagonists but are abolished by dihydroergotamine. 2l EJPs are readily mimicked either by bath application or ionophoretic application of adrenaline: again these responses persist in the presence of beta-adrenoceptor antagonists and such responses are abolished by dihydroergotamineJ 9 These observations suggest that neurally released adrenaline activates a novel adrenoceptor. Although EJPs are mimicked by applied ATP these responses are not abolished by dihydroergotamine, indicating that the activation of a purine receptor is not responsible for the sympathetic tachycardia. 21 However, EJPs and responses to added adrenaline are blocked by purinoceptor desensitization. 21 One explanation for this observation is that during junctional transmission ATP has a co-operative role during the activation of the novel adrenoceptors, perhaps analogous to that of glycine during glutaminergic transmission at synapses in the CNS (see Ref. 83). In mammals, sympathetic tachycardias and the underlying EJPs, are abolished by betal-selective adrenoceptor antagonists) 3 EJPs are mimicked by NA and these receptors are also abolished by beta~-adrenoceptor antagonists. 33 These observations suggest that in both mammalian and amphibian cardiac pacemaker cells there are sets of adrenoceptors which, when activated, initiate EJPs in preparations whose voltage-dependent channels are inactive. Presumably the receptors are responsible for the initiation of a sympathetic tachycardia. The responses to sympathetic nerve stimulation are not mimicked by forskolin or IBMX. Similarly, EJPs are neither mimicked nor enhanced by phosphodiesterase inhibitionJ 9'33 Thus the second messenger pathway activated by neuronally released catecholamines does not involve cAMP. Together these observations indicate that neurally released catecholamines selectively activate a population of junctional receptors. In amphibians, where it is easy to pharmacologically distinguish junctional and extrajunctional receptors to catecholamines, the idea that adrenaline is largely restricted to the neuroeffector junctions can be easily tested. This can be done by blocking the neuronal uptake of adrenaline using desmethylimipramine (DMI). When this was done, the responses to sympathetic nerve stimulation were greatly potentiated but the increased component

of the response was blocked by a beta-adrenoceptor antagonist. 19 Thus DMI allowed the escape of adrenaline from junctions so that it now had access to extrajunctional beta-adrenoceptors. The finding that beta-adrenoceptor blockade returned the responses to their control values suggests that during normal transmission, at each junction where transmitter is released, the post-junctional receptors are saturated. Thus the size of a response is restricted by the number of junctional receptors available at each junction. 19 Similar suggestions have been made for some central and peripheral synapses. 69,79 In summary, two distinct pathways can be activated by added and neurally released catecholamines. One pathway involves cAMP and is presumably activated by circulating catecholamines. The other is linked to an unidentified second messenger pathway and is activated by neurally released catecholamines. It seems likely that sympathetic transmission occurs at the organized neuroeffector junctions detected on cardiac pacemaker cells. It is not clear to what extent these observations apply to other chambers of the heart. However it should be noted that in amphibian atria much of the force response produced by sympathetic nerve stimulation is resistant to betaadrenoceptor blockade. 1°7 Similarly in mammalian atria although sympathetic nerve stimulation produces an inotropic response this is not associated with a change in the time course of atrial action potentials. 33 4.4. Catecholamines and sympathetic nerves innervating arteries, veins and the dilator layer o f the iris In many systemic arteries and arterioles sympathetic nerve stimulation produces constrictions which result partly from the activation of alpha-adrenoceptors and partly from the involvement of non-alphaadrenoceptors. 66 The consensus view is that the non-alpha-component results from the activation of purinoceptors. 89'128 In pulmonary arteries and veins, sympathetic nerve stimulation produces constrictions which result only from the activation of alphaadrenoceptors. 129,13° Similar constrictions are also recorded from the dilator layer of the iris following sympathetic nerve activity.63 This section of the commentary will restrict itself to the responses of target tissues which involve the activation of alpha-adrenoceptors. Those involving the activation of non-alphaadrenoceptors will be dealt with in the subsequent section. Applied N A often closely mimics the action of neurally released NA at the sympathetic neuroeffector junctions where an alpha-adrenoceptor is involved. In the portal vein, sympathetic nerve stimulation increases the rate of discharge of action potentials and causes a contraction. These responses which result from the release of N A are readily abolished by alpha-adrenoceptor antagonists and

Neuroeffector junctions are well mimicked by applied N A . 74'136 Similarly in mesenteric veins, sympathetic nerve stimulation produces a long-lasting EJP which is well mimicked by ionophoretically applied N A . 129 In pulmonary arteries, sympathetic nerve stimulation produces a longlasting depolarization and contraction; added NA, however, causes a maximal constriction without a depolarization. ~3° In the dilator layer of the iris sympathetic nerve stimulation evokes a long-lasting EJP that results from the activation of alphalBadrenoceptors. 63 These responses are potentiated by blocking the neuronal uptake of NA without any obvious change in the nature of the responses. 63 At many of these junctions it appears likely that the alpha-adrenoceptor is linked to a pathway which involves inositol 1,4,5-trisphosphate (IP3). 13 In resting preparations, a pulsatile release of Ca 2+ occurs spontaneously. Since the membranes of these cells contain Ca 2+ activated Cl- channels each pulse of Ca 2+ causes the opening of several C a 2+ activated CI- channels and an ongoing discharge of transient depolarizations is detected. 63'137'm Sympathetic nerve activity triggers IP 3 production, an acceleration in the pulsatile rate of Ca 2÷ release from intracellular stores, a contraction and associated EJP result. Not surprisingly, if the EJP is abolished by removal of [C1-]o the contraction persists.63 None of the electrophysiological data collected to date suggest that there is any special accumulation of adrenoceptors near the neuroeffector junctions formed by sympathetic nerve terminals. Very similar responses to those produced by nerve stimulation are produced by added NA. However, it is noticeable that maximal contractions of pulmonary arteries when produced by added NA are not associated with membrane depolarizations whereas responses to nerve stimulation are accompanied by slow EJPs. 13° Whether this simply represents high concentrations of NA near points of release and a uniformly low concentration of transmitter over the muscle surface during bath application is not known. Although adrenoceptors may be evenly distributed, it seems likely purinoceptors have an uneven distribution in veins and in pulmonary arteries. In both tissues applied ATP readily causes vasoconstriction, 67 in veins this is accompanied by substantial membrane depolarization. 67 Since the sympathetic nerves are reported to release ATP but depolarizations resistant to alpha-adrenoceptor blockade are not detected, one must assume that purinoceptors are absent from the neuroeffector junctions found in these tissues. In summary, in tissues where neurally released catecholamines activate alpha-adrenoceptors, even though organized sympathetic neuroeffector junctions exist there is no clear evidence for the existence of junctional and extrajunctional adrenoceptors with differing properties. However, some specialization must occur as purinoceptors appear to be excluded from the junctional regions of many pulmonary and venous blood vessels.

17

4.5. A TP and sympathetic nerves innervating systemic arteries and vasa deferentia As noted above, stimulation of the sympathetic innervation to systemic arteries evokes constrictions which are partly resistant to alpha-adrenoceptor blockade (see also Ref. 68). Similar responses that are resistant to alpha-adrenoceptor blockade are also generated by preparations of vasa deferentia. In arterioles, it was originally suggested that the resistance to alpha-adrenoceptor blockade occurred because a population of non-alpha-, non-betaadrenoceptors were located near sympathetic nerve terminals and these were activated by neurally released catecholamines. 7~'73 Subsequently, it was shown that non-alpha-, non-beta-adrenoceptors were found in mesenteric arteries but not in mesenteric veins, 67 a distribution which correlates with the involvement and lack of involvement of non-alphaadrenoceptor mechanisms in the contractile responses to sympathetic nerve stimulation. 67 However, the finding that agents which desensitize purinoceptors and so abolish EJPs failed to block the responses to activation of non-alpha-, non-beta-adrenoceptors ruled out this hypothesis. 67 The second suggestion is that sympathetic nerves released two transmitters NA a n d A T P . 49'89'128 According to this view N A activates post-junctional, and pre-junctional alpha-adrenoceptors. Thus the alpha-adrenoceptor mediated component of contraction is abolished by the appropriate adrenoceptor antagonists. ATP activates post-junctional purinoceptors and initiates an EJP. When the EJPs are large enough to activate voltage-dependent Ca 2+ channels, Ca 2+ entry occurs and a contraction results. The purinoceptor component of contraction is abolished either by purinoceptor desensitization, ~27 application of suramin, a compound said to block purinoceptors 44 or by blocking the voltage dependent C a 2+ channels activated by membrane depolarization. ~4 However, if ATP is the transmitter at these junctions some form of purinoceptor specialization must exist. It has been pointed out that EJPs result from the activation of ligand gated channels, with junctional currents starting some 1-2 ms after invasion of the terminals by nerve impulses. 22 When ATP is applied close to a cell by ionophoresis, the responses have minimum latencies of 40-70 ms 8 which cannot be attributed to diffusional delays.H8 Moreover, much of the' current flowing following the application of ATP is carried by Ca 2+ with sufficient Ca 2+ entering after the blockade of voltage-dependent Ca 2÷ channels to trigger a contraction. ~2° This is in direct contrast to the contraction triggered by sympathetic nerve stimulation.14'~2° Voltage clamp studies indicate that sEJCs have peak amplitudes of about 0.1 nA? ° In comparison, the channels activated by applied ATP have a small conductance and allow only small currents to flow.l° Clearly many such channels would have to be activated to produce an sEJC. As it is

18

G.D.S. Hirst et al.

agreed that the sympathetic vesicles of peripheral sympathetic varicosities contain only about 50 molecules of ATP 9° it seems likely that a maximum of 50 channels could be activated when a sympathetic vesicle discharges its contents. Presumably the low conductance channels are not activated by neurally released ATP, rather a separate subset of high conductance ATP channels must exist. Finally, although suramin is very effective in some tissues, including vasa deferentia, in blocking the response to nerve stimulation, it is very ineffective in blocking the responses to applied ATP in vasa deferentia. 12° Together these observations suggest that applied ATP most readily activates a set receptors that are linked to low conductance indirectly coupled channels which have a relatively high selectivity to Ca 2+. If ATP is the transmitter at these junctions, sets of ligand gated high conductance channels must be located near the points of transmitter release. 4.6. Aeetyleholine and excitatory neuroeffeetor transmission in the intestine

Excitatory nerve stimulation evokes EJPs in both the longitudinal and circular muscle layers of the mammalian intestine. These in turn trigger the opening of voltage-dependent Ca 2+ channels in intestinal muscle, causing action potential generation and triggering contraction of the muscle layers. TM Provided that only single stimuli are applied, the responses are readily abolished by muscarinic receptor antagonists, indicating that ACh is being released and is activating muscarinic receptors. 5'78'143 In the longitudinal muscle layer of the guinea-pig ileum, applied ACh activates muscarinic receptors which in turn cause an increase in [Ca2+]i by two main routes. Firstly, applied ACh leads to the opening of sets of non-selective cation channels which allow the influx of Na + and Ca 2+ and the efflux of K + . TM However, in normal physiological solutions the amount of Ca 2÷ entering the muscle cells via these channels is negligible. 77,H7The depolarization following the activation of non-selective cation channels causes the opening of voltage-dependent Ca 2+ channels, thus triggering action potential generation and muscle contraction. 16 Secondly applied ACh causes [CaZ+]~ to increase by releasing Ca 2+ from an intracellular storey This pathway involves the formation of IP 3 and the pulsatile release of Ca 2+ from intracellular stores. 17'87'88 In addition to activating the contractile elements within the intestinal muscle cells, the pulse of Ca 2+ also causes the synchronous activation of about 100 Ca 2+ activated K + channels located in the membranes of these cells. 9'17'114As a result sporadic increases in K + conductance are detected causing transient membrane hyperpolarizations. Such membrane hyperpolarizations can be blocked by the application of Ba 2+ to the bathing solution or by the specific Ca 2+ activated K + channel blocker, charybdotoxin. 62'1°4 Thus applied ACh causes a complex sequence of membrane potential

changes. A membrane depolarization results from the activation of non-selective cation channels. Transient membrane hyperpolarizations, resulting from the irregular increases in K ÷ conductance that follow the pulsatile release of Ca 2÷ from an intracellular store, are superimposed upon the membrane depolarization. The membrane depolarization activates voltage-dependent Ca 2+ channels, so triggering muscle action potentials and longitudinal muscle contraction. Stimulation of enteric cholinergic nerves with single transmural stimuli initiates EJPs, which like the membrane depolarizations produced by the ionophoretic application of ACh, start after a long latency (500-600 ms). Therefore both result from the activation of complex pathways rather than from the activation of ligand gated channels. EJPs trigger Ca 2÷ muscle action potentials 6 and muscle contraction. The application of one of a range of organic Ca 2+ antagonists abolishes muscle action potentials to reveal EJPs. These EJPs continue to evoke contractions. Moreover, when the changes in muscle [Ca2+]i were measured in fura-2 loaded preparations, it was found that EJPs were associated with increases in [Ca2+]i with the increases being similar to those caused by individual muscle action potentials? 7 This observation, along with the finding that the EJP and the transient increase in [Ca2+]i activated by neurally released ACh occur simultaneously, raises the possibility that the channels activated during an EJP have a high Ca 2÷ selectivity. 37 Neurally released ACh and ACh released from an ionophoretic pipette, appear to activate different membrane channels to produce a membrane depolarization. Firstly, the depolarizations produced by ionophoretically applied ACh are interrupted by transient membrane hyperpolarizations whereas in marked contrast the EJPs invariably had smooth rising and falling phases. This suggests that the increase in [Ca2~]i caused by neurally released ACh involves a different pathway to that activated by applied ACh; one that does not involve IP 3 and the associated pulsatile release of Ca 2+ from an intracellular store. Secondly, the responses to nerve stimulation and applied ACh display different sensitivities to a range of organic Ca 2+ antagonists. High concentrations of verapamil, nicardipine or diltiazem abolish the EJP, the increase in [CaZ+]i and the associated contraction of the muscle layer by a postsynaptic action (see Ref. 81). In contrast, the membrane depolarization produced by the ionophoretic application of ACh is reduced in amplitude but is not abolished. Given that these Ca 2+ antagonists do not block muscarinic non-selective cation channels, 76 the simplest explanation is that ionophoretically applied ACh causes a depolarization by activating two sets of muscarinic receptors. One set is linked to nonselective cation channels while the second is linked to channels which are readily blocked by high concentrations of selected organic C a 2+ antagonists.

Neuroeffector junctions Presumably neurally released ACh has access only to the muscarinic receptors which are linked to the ion channels blocked by organic Ca 2÷ antagonists. The failure of neurally released ACh to mimic applied ACh suggests that cholinergic neuroeffector transmission occurs at the organized neuroeffector junctions found in intestinal muscle, s6 The idea that neurally released ACh is restricted to neuroeffector junctions can be easily tested by preventing the hydrolysis of ACh using cholinesterase inhibitors, such as eserine. When this was done, EJPs which had previously been blocked with either nicardipine, verapamil or diltiazem, could readily be restored. The restored responses typically had both a longer latency and a longer time course than did EJPs recorded in control solutions from the same tissues. Furthermore, the restored membrane depolarizations produced by neurally released ACh were associated with smaller increases in muscle [Ca2+]i and smaller contractions than those associated with control EJPs. 36 Together, the observations suggest that ACh released from enteric cholinergic nerves does not activate all of the sub-types of muscarinic receptors present in the intestine. Thus, in control solutions, neurally released ACh activates a set of junctional receptors which cause an increase in [Ca2+]i by a pathway which does not involve the pulsatile release of Ca 2÷ from intracellular stores. Secondly, neurally released ACh does not normally activate the muscarinic receptors linked to non-selective cation channels. However, these receptors, which must have an extrajunctional location, could be activated by neurally released ACh after cholinesterase inhibition. Whether or not the two populations of muscarinic receptors have pharmacologically distinct properties remains to be determined.

19 5. CONCLUDING REMARKS

In many tissues innervated by either the autonomic or enteric nervous systems communication seems to occur at discrete junctional contacts. When sections of tissue, selected at random, are viewed with an electron microscope, in most tissues a proportion of autonomic or enteric varicosities are seen to form close appositions with nearby cells. When the structure of the neuroeffector junctions has been determined by viewing sequential ultrathin sections of tissue, it has become apparent that the number of junctions formed was severely underestimated. In many preparations neurally released transmitters and the same transmitters applied by superfusion do not produce identical responses. We suggest that neuraUy released transmitters activate pools of specialized receptors which are located at the organized neuroeffector junctions found in these preparations. When transmitter is released it is restricted to the neuro-effector cleft and is transiently present in a very high concentration. Little transmitter escapes from the cleft but any that does so may activate the higher affinity extrajunctional receptors. Unfortunately the extrajunctional receptors are those most readily examined in pharmacological experiments and studies using single isolated cells. This pattern of behaviour has also been observed at the skeletal neuromuscular junction. Neurally released transmitter activates sets of receptors which are coupled to high conductance cation selective channels whereas receptors located away from the points of transmitter release are coupled to low conductance cation selective channels. 64

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