Novel autonomic neurotransmitters and upper gastrointestinal function

Pharmac. Ther,Vol. 38, pp. 429-452, 1988

0163-7258/88 $0.00 + 0.50 Copyright © 1988 Pergamon Press pie

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Specialist Subject Editor: C. BELL

NOVEL

AUTONOMIC

UPPER

NEUROTRANSMITTERS

GASTROINTESTINAL

AND

FUNCTION

H. C. MCKIRDY Departments of Surgery and Pharmacology, University Hospital of Wales, Cardiff, CF4 4XN, U.K.

1. INTRODUCTION The embryological 'foregut' or upper alimentary tract extends to the choledochoduodenal junction (Hamilton et al., 1952) and encompasses the region to be covered in this review. For convenience, the upper alimentary tract will be considered in three main sections: (1) the smooth muscle portion of the oesophagus and the oesophagogastric junction, (2) the stomach and gastroduodenal junction and (3) the biliary tract and choledochoduodenal junction. It is proposed to deal only with the effects of novel autonomic transmitters on the control of motility. Historical perspectives of the development of ideas concerning peripheral non-adrenergic, non-cholinergic (NANC) inhibitory and excitatory nerves and their transmitters is covered in several fairly recent reviews (Burnstock, 1986a,b; Furness and Costa, 1980; Gillespie, 1982) and transmission in the enteric plexuses has been reviewed by Gershon (1981), Grundy (1985), North (1982) and Wood (1987). Nineteen autonomic neurotransmitters have been proposed (Burnstock, 1986a,b) and a further substance, histamine, has not been definitely excluded as a possible autonomic neurotransmitter (Costa et al., 1987). Several active substances may be released by one neurone and more than one substance may be involved in the transmission process at a single neuroeffector junction, the phenomenon of co-transmission (Burnstock, 1986a). The principal transmitter (which may be, e.g. an amine) may be released along with a purine or related substances and/or peptides and it would seem that the roles played by these other neurally-released substances are still not entirely clear. They may act as true transmitters, i.e. have a postsynaptic action via discrete channels as demonstrated for adenosine triphosphate (ATP) (Benham and Tsien, 1987) or as neuromodulators, or both, or have some trophic function (Burnstock, 1985; Lundberg and Hokfelt, 1986; Hokfelt et al., 1987). The criteria for identifying a neurotransmitter have been reviewed (Bell, 1983). Despite the approaches of different laboratories over the past two decades to tackling various facets of the problem of NANC transmitter identification (e.g. in vitro studies on nerve stimulation and the effect of added agents; extraction and biochemical analyses; electrophysiological studies; anatomical, histochemical and immunofluorescence studies) the identity of enteric NANC transmitters still has not been definitely established. However, it has recently been suggested that the active principle in the tissue extract which was thought to contain a non-adrenergic, non-cholinergic inhibitory (NANCI) transmitter (Ambache et al., 1975) is nitric oxide (Martin et al., 1988). Identification may be complicated by the possibility that one nerve may release more than one NANC transmitter and that different regions of the gastrointestinal tract in the same species may employ different NANC transmitters (Costa et aL, 1986). One factor which in the past may have caused scepticism about NANCI responses is the unphysiological nature of NANC nerve activation by electrical field stimulation. However, in the guinea-pig small intestine the physiological stimulus of distension has been shown to reflexly activate intrinsic non-adrenergic, non-cholinergic inhibitory (NANCI) 429

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neurones to the circular muscle (Hirst and McKirdy, 1974), thereby excluding the possibility of an artefactual basis for the NANCI response. Substances which may have to be considered as novel autonomic neurotransmitters in mammals will be considered in three main groups: (a) biologically active amines, (b) purines and related substances and (c) peptides. A fourth group, the amino acids, has not been considered separately since only GABA (gamma-amino butyric acid) has so far been shown of importance in the enteric nervous system (Costa et al., 1987). Emphasis will be given to studies on human tissue. As these are difficult to obtain and to work with~ results are relatively sparse. Hence evidence from any species which may throw light on novel autonomic transmitters in the gastrointestinal tract will be considered. The review was completed in August, 1987. 2. THE LOWER OESOPHAGUS AND OESOPHAGOGASTRIC JUNCTION 2. l. FUNCTIONAL CONSIDERATIONS

2.1.1. N o r m a l Motility

The main motor function of the oesophagus is transport by peristaltic waves of luminal contents to the stomach. Three main theories of oesophageal peristalsis have been offered (Janssens et al., 1978; Christensen, 1983, 1987; Roman and Gonella, 1987). In the oldesl theory the emphasis is on extrinsic nerve command from central programming; in the second theory oesophageal peristalsis is considered basically myogenic. Recent electrophysiological evidence does not support this myogenic theory of peristalsis (Crist et al.. 1987). The concept of 'descending inhibition' (Bayliss and Starling, 1899; Hirst and McKirdy, 1974) is integral to only the third of these theories where the emphasis is or intramural enteric plexus activity (Diament and E1-Sharkawy, 1977). In the past, evidence for the existence of descending inhibition in the body of the oesophagus has been lacking because no evidence of relaxation preceding the contraction of primary or secondar5 peristalsis could be detected with radiology or manometry. However, even in the guinea. pig small intestine, where descending inhibition has been demonstrated electrophysio. logically in response to distension (Hirst and McKirdy, 1974), no mechanical correlate o: the electrical response has been detected by mechanical recording or direct observatiot (Trendelenburg, 1917), presumably because inherent tone (sustained resting tension) is low Tone has to be raised, artificially if necessary, to demonstrate mechanical relaxation. Thu: the inability to detect mechanical relaxation during peristalsis does not necessarily impl! absence of descending inhibition. 2.1.2. Innervation

The 'polarity' (i.e. normal overall transport of contents in the aboral direction) of th, oesophagus, like that of the remainder of the digestive tract, can be explained by th, intrinsic descending inhibitory and excitatory pathways of the enteric plexuses involved fi the peristaltic reflexes (Hirst et al,, 1975). This same enteric neural circuitry has bee1 proposed in the third theory of oesophageal peristalsis (Diament and E1-Sharkawy, 1977 (see above). Clearly the influence of extrinsic nerves and the basic properties of the muscl, layers must play roles of varying importance in different regions of the digestive tract. I1 the oesophagus, the role of extrinsic nerves in propulsion is extremely complex but the va~ can be sectioned without removing the ability of the smooth muscle portion of th oesophagus to exhibit peristaltic contractions, presumably mediated by intrinsic nerve (Ingelfinger, 1958; Ryan et al., 1977; Reynolds et al., 1985). The final neurones of the intrinsic pathways to the circular muscle of the guinea-pi: small intestine appear to be: (a) non-adrenergic, non-cholinergic inhibitory (NANCI' (b) cholinergic excitatory (Hirst and McKirdy, 1974; Bauer and Kuriyama, 1982) anq (c) non-adrenergic, non-cholinergic excitatory (NANCE) (Bywater et al., 1981). Experi ments on transmural stimulation of the human oesophageal body and gastric fundu

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suggest that similar types of neurones innervate the circular muscle since relaxations are resistant to guanethidine and contractions are reduced but not abolished by atropine (Bennett and Stockley, 1975; McKirdy and Marshall, 1985); but the non-adrenergic, non-cholinergic (NANC) transmitters may of course be different from those in guinea-pig small intestine. However, in the human oesophagogastric junction, only NANCI and NANCE nerves appear to innervate the circular muscle since the contraction is unaffected by atropine (McKirdy and Marshall, 1985). Results on the human oesophagus have been obtained from in vitro studies on muscle strips. Such studies are open to a variety of criticisms and are unfashionable at present. They can, however, reveal the sensitivity and reaction of muscle not only to neurally released substances (either on extrinsic nerve or field stimulation) but also to exogenously applied putative transmitters. Three features of such studies should be stressed. First, if the tissue responds to the added substance within a reasonable range of concentration (up to about 10 p M), receptors for the substance are assumed to be present. However, the presence of a receptor does not necessarily mean there is a transmitter available (Goyal and Rattan, 1978). The second point concerns the technique of electrical field stimulation. If a pulse width of 0.14).3 msec is used for electrical field stimulation, all responses are rapidly abolished by the nerve blocking agent tetrodotoxin and this has been taken to indicate selective stimulation of nerves. With longer pulse widths, responses are not completely abolished by tetrodotoxin (King et al., 1977) and the significance of these tetrodotoxin resistant responses is unclear. Field stimulation may facilitate transmitter release from nerve varicosities as well as initiating propagated nerve impulses (Stj~irne, 1977). The third point concerns multiple possible sites of action on nerve and muscle of added agents (Burks, 1987). It may be difficult to determine whether a candidate substance is acting directly on smooth muscle or acting indirectly by releasing N A N C transmitters. Analysis of the site of drug action should theoretically be simpler in the in vitro than the in vivo situation, but this is seldom the case owing to the presence of enteric plexuses and difficulty in denervating a tissue, even with the help of tetrodotoxin. In the human oesophagus it is quite easy to dissect out partial-thickness circular muscle bundles avoiding the ganglia of the myenteric plexus or longitudinal muscle, and it has been hoped that such strips contain only the circular muscle and its final nerve pathways with synapses en passage. However, analysis of the site of drug action may be difficult, even in ganglion-free preparations. For example, in human taenia coli catecholamines may release NANCI transmitter (Stocktey and Bennett, 1977); in the cat oesophagus it has been suggested that catecholamines may release acetylcholine from nerve endings as their effect is blocked by atropine but not by tetrodotoxin (Gonella et al., 1980). Recent evidence suggests that dopamine in particular may release a VIP (vasoactive intestinal polypeptide)-like substance from nerve endings in the opossum oesophagus (Daniel et al., 1987). Perhaps the best approach to complete denervation will be the use of isolated smooth muscle cells (Makhlouf, 1987). By far the most extensively investigated oesophageal tissues, body and sphincter, are those of the opossum (Christensen, 1983, 1987; Goyal and Rattan, 1978) but human tissues behave similarly in vitro in response to electrical field stimulation and to many added agents (Burleigh, 1979; McKirdy and Marshall, 1985). The longitudinal and circular muscle (Bennett and Stockley, 1975) and the muscularis mucosae (Hughes, 1955; Percy and Christensen, 1984) each show distinctive features in vitro. The circular muscle has a rich NANCI and NANCE innervation, whereas the longitudinal muscle has only sparse NANCI innervation (Bennett and Stockley, 1975). The opossum muscularis mucosae has no NANC innervation (Percy and Christensen, 1984), but the muscularis mucosae of the human oesophagus does not seem to have been studied. 2.1.3. Sphincteric Region

Although everyone understands what is loosely meant by the term 'sphincter', it is difficult to find an adequate definition as each different sphincter has different functional

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properties. However, it would seem that muscle at some junctional regions of the digestive tract may differ fundamentally from the muscle above and below. The search for common factors in the various junctional regions reveals some possibly unique features of innervation, particularly the apparent density of N A N C innervation. Circular muscle strips in vitro from the oesophagogastric junction show a high level of isometric myogenic tone and a prominent component of relaxation to electrical field stimulation. On the other hand, circular muscle strips from above and below this region show only low tone and a prominent component of contraction on cessation of electrical field stimulation (the 'off-response' or 'after-contraction') and tone has to be raised artificially to reveal the preceding N A N C I response (McKirdy and Marshall, 1985). The high resting tone level, together with differences in the length/tension relationship, in innervation and in responses to various added substances may reflect in vitro differences between spincteric and non-sphincteric muscle. Possible criteria for identification of sphincteric muscle in vitro have been outlined (Fisher and Cohen, 1973). The identification of sphincter muscle may pose problems in vivo as well as in vitro. On manometry a high-pressure zone can be identified in the region of the human oesophagogastric junction, but disagreement exists over its length. Using the rapid pull-through technique [which may itself cause muscle spasm (Vantrappen and Hellemans, 1980)] a high-pressure zone of several cm length has been reported (Clark and Cuschieri, 1980) whereas using the station pull-through technique (for details see Davies et al., 1983) a high-pressure zone of approximately 1 cm length can be identified in the normal subject at approximately 1-2 cm below the diaphragm. The high-pressure zone may correspond to the region of high resting tone which can be identified in vitro.

2.2. PUTATIVETRANSMITTERS As in other regions of the alimentary tract, the longitudinal muscle of the human lower oesophagus appears to be more sensitive in vitro to some substances (e.g. acetylcholine and histamine) than is the circular muscle and this may reflect differences in innervation of the two layers. The circular layer has been studied more intensively, and it is more interesting in having a denser N A N C innervation. 2.2.1. Amines It would seem highly unlikely that an amine could be either a NANCI or a N A N C E transmitter since the response to electrical field stimulation is resistant to guanethidine, which would be expected to block release of dopamine and probably also of 5-hydroxytryptamine (5-HT) (Burks, 1987). However, amines are abundant in the enteric plexuses and may act as neurotransmitters or neuromodulators or both. In this review dopamine, 5-HT and histamine will be considered, despite their unlikely candidacy for a transmitter role in the final pathway to the muscle. A small reduction in amplitude of the after-contraction in strips from the body of the oesophagus is usually observed in the presence of guanethidine, and quite marked reductions in amplitude are produced by high concentrations of phentolamine, ergometrine and haloperidol, and also the 5-HT blockers cyproheptadine and cinanserin, none of which affect the preceding relaxation. These results are difficult to interpret because of the non-specific range of concentrations employed, but do seem to suggest that the after-contraction is unlikely to be a 'rebound' phenomenon or a consequence of the relaxation. Furthermore metoclopramide, a prokinetic agent (McCallum, 1985), produces a marked potentiation of the after-contraction without affecting the preceding relaxation. Quite commonly, in preparations from the oesophagogastric junction, an after-contraction gradually becomes smaller during equilibration and then ceases to be recorded without much alteration in the preceding relaxation. All of these features appear to suggest that the after-contraction is not directly dependent on the preceding relaxation (McKirdy and Marshall, 1985). In the study of McKirdy and Marshall (1985), dopamine had variable

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433

effects on muscle strips when added to the bath. Of ten strips with spontaneous or carbachol-induced tone, five relaxed and five contracted. The response to histamine was also variable, but contraction was converted to relaxation by the histamine H2 blocker cimetidine. The response to 5-HT was consistently contraction but exhibited quite marked tachyphylaxis. It was not possible to determine the site of action, whether nerve or muscle, of any of these amines but the phenomena of variable effect and tachyphylaxis would seem to support the view that they can be excluded as possible candidates for a transmitter role at the neuroeffector junction. It has been assumed that a neurotransmitter should have reproducible effects on the target tissue at concentrations ranging from 1 nM to about 10/IM.

2.2.2. Purines and Related Substances Circular muscles from human lower oesophagus and the oesophagogastric junction are quite insensitive to adenosine triphosphate (ATP) (McKirdy and Marshall, 1985). Of 16 preparations, seven relaxed and five contracted in response to ATP 2-200/~ M. The other four showed no response. Relaxation was found in strips (with high tone) from the region of oesophagogastric junction. Adenosine appeared to be no more effective. However, dipyridamole, which is thought to potentiate the effect of purines by blocking uptake of adenosine (Cohen, 1974), markedly prolonged the relaxation to electrical field stimulation and abolished the after-contraction. If the response to electrical field stimulation represents the algebraic sum of the effects of released neurotransmitters, this observation could be interpreted as evidence supporting an adenosine compound as the NANCI transmitter. But dipyridamole also apparently blocks prostanoid synthesis and an equally valid interpretation would implicate a prostanoid as the NANCE transmitter (Marshall et al., 1982). The evidence for a transmitter role for ATP in the human oesophagus and oesophagastric junction is weak. Evidence is against the purinergic nature of the NANCI transmitter in opossum lower oesophageal sphincter (Rattan and Goyal, 1980a).

2.2.3. Peptides The important families of gut peptides are: (a) the VIP, secretin, glucagon group; (b) gastrin, CCK-PZ (cholecystokinin-pancreozymin); (c) the opioid peptides; (d) the tachykinins, e.g. substance P; (e) the bombesins; and (f) neuropeptide Y and pancreatic polypeptide (PP). The possibility that neurotensin, somatostatin and galanin also belong in families cannot be excluded (Dockray, 1987). In the human oesophageal circular muscle strips in vitro VIP produces relaxation when tone is present and reduces the after-contraction if tone is low or absent. However, the relaxation to electrical field stimulation is still detectable in some strips in the presence of VIP (0.3 pM). This has been considered as an argument against a NANCI transmitter role for VIP as desensitisation of receptors might have been expected, preventing any further activation of receptors. In the absence of a specific blocking agent, VIP cannot be excluded and remains the most likely candidate for the NANCI transmitter role (McKirdy and Marshall, 1985). The related substance glucagon (up to 0.2 #M) has no effect and Boots secretin produces contraction. However, Boots secretin is contaminated with CCK (Johnson et al., 1982) as well as other possible substances and these may be responsible for producing the contraction. Synthetic secretin up to 0.2 #M is ineffective. Boots pancreozymin and CCK-octapeptide (0.1-1/~ M) each produce contraction which is detectable in the presence of tetrodotoxin. Pentagastrin (2/~M) also produces a modest increase in tone. Leucine and methionine-enkephalin up to concentrations of 22/~M have no effects on the circular strips from human oesophagus or oesophagogastric junction. Morphine itself has no effect until very high concentrations (3-120 #M) are attained and its effects are

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H.C. McKIRDY

variable, with contraction in some preparations and relaxation in others (McKirdy and Marshall, 1985). None of the other peptides has been tested on the human oesophagus in vitro. The amino acid transmitter GABA up to 0.1/~M was without effect on oesophageal strips. In the opossum lower oesophageal sphincter, enkephalins are excitatory and endorphin,~ inhibitory (Rattan and Goyal, 1980b): the enkephalins may act as neuromodulators in the lower oesophageal sphincter (Uddman et al., 1980). Bombesin is excitatory (Mukhopadhyay and Kunnemann, 1979), substance P is excitatory (Reynolds et al., 1984) and somatostatin has no effect on resting sphincter pressure (Bybee et al., 1979). Neurotensi~ produces relaxation of the sphincter (Goedert, 1984). Galanin produces contraction of the lower oesophageal sphincter, probably by a direct effect on the muscle and an indirecl effect by selectively inhibiting NANC transmitters (Kaczmarek et al., 1987). Some of these peptides would have to be excluded as possible NANC transmitter candidates and specific blocking agents would help with their exclusion. It would seem thal NANCI neurones are involved in peristalsis (Crist et al., 1984) and are responsible fox lower oesophageal sphincter opening, while NANCE neurones may be involved ir sphincter closure. VIP remains the most likely NANCI candidate, and substance P would seem a possible NANCE candidate since sphincteric closure and contraction is reduced by a substance P antagonist (Reynolds et al., 1984). 3. THE STOMACH 3.1. FUNCTIONALCONSIDERATIONS 3.1.1. N o r m a l M o t i l i t y

The stomach can be divided structurally and functionally into two main zones, the bod) and the antrum, separated in life by the incisura angularis. This physiological dividing line which has seen a recent revival of interest (Moore et al., 1986) separates the 'hopper' frorc the 'mill'. Cannon apparently thought the incisura angularis was a muscle pacemakex region (R. C. Garry, personal communication) though modern teaching locates the pacemaker more proximally in the body of the stomach (e.g. Kelly, 1980). The properties of gastric muscle seem to be tailored towards the main functions of filling (mainly fundu,, and body) and emptying (mainly antrum and pylorus) as revealed both by electrical and mechanical activity (Morgan et al., 1981; Szurszewski, 1987) and length/tension relation. ship (Szurszewski, 1987; Schulze-Delrieu and Shirazi, 1987). Superimposed on these basic muscle properties is a complex array of nervous mechanisms, e.g. intrinsic reflexes 'receptive relaxation', long and short extrinsic reflexes, enterogastric and corpo-antra reflexes (e.g. Martinson, 1965; Jansson, 1969; Daniel and Wiebe, 1966; Andrews anc Lawes, 1982, 1984; Kreulen et al., 1983; Galligan et al., 1986; Roman and Gonella, 1987 Szurszewski, 1987; Deponti et al., 1987) as well as probable hormonal mechanisms (e.g Lim, 1933; Hunt and Ramsbottom, 1967; Dozois and Kelly, 1971; Debas et al., 1975 Sweeting, 1986; Keinke et al., 1987). Numerous reviews of gastric filling and emptying arc available (Hunt and Knox, 1968; Blair, 1974; Cooke, 1975; Kelly, 1980; Heading, 1980 McHugh, 1983; Dubois, 1983; Dubois and Castell, 1984; Ehrlein et al., 1984; Grundy 1985; Wulschke et al., 1986; Meyer, 1987). 3.1.2. Innervation

It has long been recognised that the nervous mechanisms involved in receptive relaxation (Cannon and Lieb, 191 l) of the gastric fundus and body through the vagus arc a continuation of the relaxing mechanism of the oesophagogastric junction (Langley~ 1898), though the mechanism can also be set off by distension of the stomach itselt (Abrahamsson and Jansson, 1973). The vagal mediation of an inhibitory non-cholinergic response in the stomach was extensively investigated in the past (e.g. Brown and Garry 1932).

Novel autonomic neurotransmitters

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Before the arrival of the H2 blockers (Black et al., 1972) the vagus was of special interest to the gastric surgeon (e.g. Lawson and Hutchinson, 1973; Wilbur and Kelly, 1973) and although surgery for peptic ulcer is less common now, vagotomy is still performed despite its complications (Holle et al., 1986; Woodward, 1987). A KROC review of vagal mechanisms and possible transmitter agents is available (see Ahlman and Dahlstrom, 1983; Rehfeld, 1983; and also Lundberg et al., 1979) and many other publications deal with the specific reflex mechanisms (e.g. Martinson, 1965; Edin, 1980; Andrews and Lawes, 1982, 1984; Grundy and Scratcherd, 1984). A most interesting recent suggestion is that the vagus may have a permissive rather than a commanding role (Grundy et al., 1986) and that intrinsic reflexes may be more important than previously thought (Ohta et al., 1985; Deloof and Rousseau, 1985; Schulze-Delrieu et al., 1986; Schulze-Delrieu, 1986). According to this view, the basic controlling mechanisms lie in the enteric plexuses and the role of the vagus is in facilitating their activation.

3.1.3. Sphincteric Region

Despite fairly intensive investigation for more than a century, the role of the pylorus in regulating gastric outflow is still unclear (see reviews on gastric emptying quoted above and Edwards and Rowlands, 1968; Schulze-Delrieu and Wall, 1983; Ruckebusch and Malbert, 1986; Meyer, 1987). The pyloric 'sphincter' does not rest in a tonically contracted state like the sphincteric regions of the oesophagogastric junction and anorectum (Atkinson et al., 1951) but shows intermittent phasic contractions as demonstrated by manometry in the fasted stomach (Dooley et al., 1985). These contractions can be observed routinely during endoscopy and barium studies and can apparently occur independently of antral (Johnson, 1981; Eyre-Brook et al., 1983) or duodenal contractions (Mir et al., 1979). A small zone of the human distal pyloric sphincter shows /n vitro features of sphincteric muscle, viz. high resting tone and prominent relaxation to electrical field stimulation (Anuras et al., 1974; Schulze-Delrieu and Shirazi, 1983), and similar findings have been reported for the cat pylorus (Bertiger et al., 1987).

3.1.4. In vitro Studies on Innervation Once again it has been hoped that in vitro studies with either longitudinal or circular muscle bundles, as free as possible from ganglia, and subjected to electrical field stimulation and added agents, might reveal some information on the final neuronal pathways to the muscle layers. In circular muscle bundles of the gastric fundus, proximal body and distal antrum isometric tone was low or absent and strips contracted on electrical field stimulation (0.3 msec pulses at 10 Hz for 5 sec). These contractions were reduced, but not abolished, by atropine (Bennett and Stockley, 1975; McKirdy, unpublished). Although many explanations for atropine resistance have been offered in the past (Ambache, 1955; Goldenberg and Burns, 1968; Gillespie, 1968), it now seems likely that this small atropine-resistant component might suggest a NANCE innervation, and substance P has been suggested as the NANCE transmitter released by field stimulation (Neii et al., 1983) and during peristalsis (Yokoyama and North, 1983). In strips from the proximal gastric fundal region, or 'cardia', an off-response or after-contraction is sometimes recorded (Fig. 1) (McKirdy and Marshall, 1985). In the presence of high concentrations of acetylcholine or carbachol, the contraction to electrical field stimulation is replaced by a biphasic response comprising contraction followed by relaxation, or vice versa, or a triphasic response with contraction, relaxation then contraction. Furthermore, the relaxation and the after-contraction, when present, are resistant to prolonged exposure to guanethidine. These high concentrations of cholinergic agonists increase isometric tone, and this is important in bringing about an alteration in the quality and pattern of the response to field stimulation. Other agents which increase tone may also alter the quality of the response to electrical field stimulation. However, high

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H . C . MCKIRDY [- 2.5 (g)

t~

-0

B

Carbachol 5.5 ~M

I

I

lOs Fla. 1. Response to electrical field stimulation of a gastric fundal strip obtained from the region immediately below the oesophagogastric junction. A shows the response at the control level of tone, which is low. The latency to the take-off of the after-contraction is the longest which has been recorded in our strips. B is approximately 15 rain later, during which time carbachol has been added to the bath to give a concentration of 5.5 pM. Tone is increased but still falling in the presence of carbachol. The initial contraction during the period of stimulation is now hardly detectable, having been replaced by a relaxation (from McKirdy and Marshall, 1985, with permission).

concentrations of acetylcholine or carbachol would also be expected to produce desensitisation of cholinergic receptors and block any cholinergic responses. Assuming the response to electrical field stimulation represents the algebraic sum of the effects of released neurotransmitters, a speculative and simplistic explanation of the response to electrical field stimulation in the gastric fundus (Fig. 1) is as follows: the three transmitter agents, acetylcholine (excitatory), the NANCI transmitter and the NANCE transmitter would be released virtually simultaneously, but the acetylcholine acts more swiftly than the NANCI substance, which is the most potent of the three but is destroyed rapidly, thus allowin~ the NANCE effects to become apparent. Clearly, if tone is low, no relaxation by the NANCI agent will be detectable and only an initial contraction and an after-contractior will be recorded (Fig. 1A). It would seem that in the proximal gastric fundus (cardial electrical field stimulation may release acetylcholine (producing the initial contraction), NANCI agent and probably a NANCE agent. In circular muscle strips from the antrum and body the NANCI and NANCE response, are smaller and less reliably detectable than those in strips of similar dimensions from the oesophagus, oesophagogastric junction and proximal gastric fundus. Thus the NAN( innervation of the gastric body and antrum appears to be fairly sparse compared with th~ oesophagus and its junction with the stomach (Bennett and Stockley, 1975). Thc predominantly inhibitory response to field stimulation in the human distal pylori~ sphincter does not seem to have been analysed pharmacologically, though it is abolishec by tetrodotoxin (Schulze-Delrieu and Shirazi, 1983). However, in the dog the pylork relaxation is mediated by NANCI nerves (Mir et al., 1979). The longitudinal layer of the stomach, as elsewhere in the upper alimentary tract, has even more sparse NAN( innervation than the circular muscle, with acetylcholine being the predominant (excitatory transmitter (Bennett and Stockley, 1975). The third oblique layer of the muscularis exterm of the stomach does not appear to have been studied regarding possible NAN( innervation. The muscularis mucosae of the dog stomach has a rich NANCI innervatior (Angel et al., 1983). 3.2. PUTATIVETRANSMITTERS 3.2.1. A m i n e s As explained above in the section on the oesophagus, it is highly unlikely that an amin~ could be one of the NANC transmitters. Despite its unlikely candidacy, dopamine wa: once considered as a possible nerve transmitter in gastric relaxation (Valenzuela, 1976) While dopamine reduces the amplitude of contraction to field stimulation in human gastri~

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strips, neither domperidone nor metoclopramide, which are thought to be dopamine antagonists, appear to have any effect on the relaxation to field stimulation (McKirdy, unpublished). The circular muscle of the body of the human stomach and both longitudinal and circular layers of the antrum are insensitive to 5-HT. The longitudinal muscle of the body of the stomach contracts in response to 5-HT and this appears to be a direct effect on the muscle (Fishlock et al., 1965). Human pyloric muscle strips appear to be insensitive to 5-HT up to 1/~M. However, some 5-HT blockers enhance gastric emptying in both the human and the guinea-pig, and it would seem likely that 5-HT is involved at some stage in the control of gastric emptying (Costall et al., 1987). Circular muscle strips from the body of the human stomach contract on exposure to histamine whereas circular strips from the antrum show predominantly relaxation. Longitudinal and circular strips of pylorus were less sensitive (Bennett and Whitney, 1966) but relaxation has been reported (D'Mello and Bennett, 1981) and histamine reduced the amplitude of contraction in response to field stimulation of human pyloric strips. In the guinea-pig stomach histamine has a direct excitatory effect on the muscle as well as an indirect effect by inhibiting N A N C I transmission (Ono and Suzuki, 1987). In the cat, histamine produces contraction of the pylorus through H~ receptors on nerve and muscle, with little or no effect on either antral or duodenal muscle (Biancani et al., 1981). Disagreement exists over the involvement of H2-receptors in gastric emptying (see Scarpignato and Heading, 1987). These observations on the insensitivity to and variable effects of amines seem consistent with the view that none is likely to be a N A N C transmitter for the final nerve pathway to the muscle. 3.2.2. Purines and R e l a t e d Substances The stomach was one of the first organs to be implicated in the purinergic hypothesis of N A N C I transmission, as ATP was shown to be released from guinea-pig and toad stomachs on stimulation of vagal N A N C I fibres (Burnstock et al., 1970). However, strips from body of human stomach, antrum and pylorus are remarkably insensitive to ATP up to 300/~M. At this order of concentration, ATP produces a small contraction at low levels of tone and a small relaxation when tone was raised artificially. Perhaps some other substance may be released with the ATP and act synergistically to potentiate the effects. For example, neuropeptide Y (Lundberg et al., 1982; Allen et al., 1987) may be released along with ATP and noradrenaline in the vas deferens (see Gabella, 1987) and in the rat anococcygeus (Edwardson et al., 1985). 3.2.3. Peptides VIP has inhibitory effects on isolated strips of human gastric muscle (Ludtke et al., 1984) and inhibits both the mechanical and spontaneous electrical activity of canine antral muscle (Morgan et al., 1978). VIP has been proposed as the NANCI transmitter in rat fundus (DeBeurme and Lefebvre, 1987) and in canine gastric muscularis mucosae (Angel et al., 1983). However, no effect was detected on gastric circular muscle in response to other peptides of the same family group, viz. secretin (Ludtke et al., 1984) and glucagon. The lack of effect of glucagon, even when tone was raised artificially, seems surprising as it is routinely used to produced relaxation of the stomach during barium studies (Kreel, 1975). Nevertheless, VIP is still considered the most likely candidate as a neural mediator of gastric relaxation (Grider et al., 1985) and pyloric relaxation (Edin, 1980). The effects of the gastrin-CCK-PZ family of peptides on the stomach is extremely complex, partly because of indirect effects via nerves which may be cholinergic or N A N C , as well as the direct excitatory effects on muscle of both gastrin (Szurszewski, 1975; Strunz et al., 1979) and CCK (Yanagashi and Debas, 1978; Scheurer et al., 1983; Kuwahara et al., 1986; Kantoh et al., 1987). Despite causing an increased force of antral contraction

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(Cooke et al., 1972) pentagastrin and gastrin slow gastric emptying (Hunt and Ramsbottom, 1967; Dozois and Kelly, 1971; MacGregor et al., 1978). CCK relaxes the antrum and contracts the pylorus (Johnson, 1981); the delay in gastric emptying by CCK, with oi without secretin (Chey et al., 1970), may explain (Debaset al., 1975; Walsh, 1987) th~ 'enterogastrone'-mediated inhibitory effects produced by fat in the duodenum (Lira, 1933) The combination of CCK and secretin has also been shown to enhance pyloric contractior (Fisher et al., 1973). Morphine in the intact subject has long been thought to inhibit gastric emptying ant to cause pylorospasm. The opiate receptor blocker naloxone antagonises the effect o: enkephalins (which contract antral muscle), yet naloxone hastens gastric emptying (Fran~ et al., 1982). Contraction of the pylorus, mediated by NANC nerves, is abolished b~ naloxone, suggesting that vagal excitation of the pylorus is mediated by enkephalinergk nerves (Edin et al., 1980). Indeed the enkephalins which produce atropine-resistan contraction seem possible candidates for NANCE transmitters in the cat stomac[ (Lefebvre et al., 1986). In human gastric and pyloric strips in vitro, however, morphine naloxone and methionine- and leucine-enkephalin, up to l #M, were quite ineffectiw against the response to field stimulation (McKirdy, unpublished). Substance P (Pernow, 1983) has an excitatory effect on human gastric smooth muscl, (Ludtke et al., 1984) and it accelerates gastric emptying (Mangel and Koegel, 1984) Excitation produced by substance P on guinea-pig isolated gastric smooth muscle cells i mediated by a receptor separate from that used by acetylcholine (Louie and Owyang 1984), but in the cat pylorus the contraction to substance P is blocked by atropine (Lidberl et al., 1982). In the rat stomach the contractile effects of substance P are mediated indirectl' via nerves, whereas neurokinin A acts directly on the muscle (Holzer-Petsche et al., 1986 and a tachykinin antagonist inhibits gastric emptying (Holzer et al., 1986). However antagonists do not seem to have been tested on the NANC responses in vitro. In human strips bombesin and neurotensin are excitatory and motilin and the gastri inhibitory peptide hormone GIP are without effect (Ludtke el al., 1984). Bombesil augments antral contractility in the dog (Mayer et al., 1982). Peptide YY inhibits gastri emptying in the physiological range in the human (Savage et al., 1987). The peptide galanin (Ekblad et al., 1985; Bishop et al., 1985; Fox et al., 1986) does ne appear so far to have been tested on the stomach. The amino acid GABA had no effect on gastric or pyloric strips in vitro up to 1 g M GABA-containing nerves, however, are present in rat stomach (Hills et al., 1987). Some of these peptides may be involved in NANC transmission mechanisms, but muc more work needs to be done. In particular, specific blocking agents need to be develope and tested; some progress is being made with substance P analogue antagonists (Taylc and Bywater, 1986). VIP seems the most likely candidate for the NANCI transmitter t the muscle. NANCI mechanisms are involved in receptive relaxation and gastric fillin~ and possibly in pyloric relaxation. Substance P seems perhaps a good candidate for th NANCE transmitter to the muscle and may be involved in gastric contraction an emptying. 4. THE BILIARY TRACT 4.1. FUNCTIONALCONSIDERATIONS 4.1.1. N o r m a l Motility The main factors involved in the control of bile flow are: (a) the rate and secretor pressure of hepatic bile production, (b) the activity and resistance of the sphincter of Od~ and the cystic duct and (c) the state of contraction of the gall bladder, which store concentrates and alters the composition of the bile. The biliary tract is a low-pressuv low-flow system; pressure in the common duct is only some 5-10 cm H20 above duodem pressure (Hogan et al., 1983). Flow in the common duct is thought to be passive, and o maximal contraction of the gall-bladder pressures of only 15-26 cm H20 are achiever

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4.1.2. Innervation It is traditionally taught that the biliary tract is under predominantly hormonal rather than neural control; increased cholecystokinin-pancreozymin (CCK-PZ) secretion contracts the gall-bladder and relaxes the sphincter of Oddi (Ivy, 1934). Yet vagotomy also affects gall-bladder filling (Johnson and Boyden, 1952) and emptying (e.g. Rudick and Hutchinson, 1965). A concept of 'reciprocal innervation' (Meltzer, 1917) (by which contraction of the gall-bladder was associated with relaxation of the sphincter of Oddi) was once proposed, but was subsequently replaced by the theory of hormonal control (see Magee, 1965). Evidence is accumulating, however, to suggest that the orthodox view of gall-bladder evacuation being primarily controlled by hormonal mechanisms may require re-evaluation (Magee et al., 1984) and that control of the gall-bladder involves an interaction of both neural and humoral or hormonal factors (Ryan, 1987). The gallbladder, like the rest of the biliary tract, has a fairly rich innervation (Burnett et al., 1964; Baumgarten and Lange, 1969; Kyosolo and Rechardt, 1973; Kyosolo, 1977; Cai and Gabella, 1983a,b, 1984), but histological appearance does not reveal the relative proportions of afferent and efferent nerves. The gall-bladder, like the rest of the biliary tract, must have a fairly rich sensory innervation, as anyone who has suffered from cholecystitis or biliary colic will testify. Interestingly, Sutherland (1967) found only Dogiel type II neurones in the gall-bladder and suggested that these are therefore effector neurones. Yet the AH cell which Hirst and McKirdy (1974) considered a sensory neurone has the features of a Dogiel type II cell (Katayama et al., 1986) and a Dogiel type I cell has been shown to innervate the circular muscle (Wattchow et al., 1987), suggesting that is must be an effector neurone. This problem remains unresolved and the function of the gall-bladder ganglion cells (which are fairly sparsely distributed compared with the rest of the digestive tract) remains unclear.

4.1.3. Gall-Bladder

Like other hollow organs the gall-bladder accommodates varying volumes with little change in pressure (Ryan and Cohen, 1976a). How much of this adaptation is due to passive physical properties of the hollow viscus and how much to mechanisms, neural or humoral, which produce reduction in tone of the muscle is, as elsewhere in the gut, unknown (McKirdy, 1978). However, a component of muscle relaxation due to activity of NANCI nerves, probably important for rapid adaptation, has been reported (A1Hassani and Davison, 1979). This rapid adaptation would therefore be similar to the receptive relaxation of the stomach. The gall-bladder which has been studied most extensively in vitro is that of the guinea-pig (Ryan and Cohen, 1976b; Davison et al., 1978; Naughton et al., 1983; Shook and Burks, 1986) but disagreement persists over the nature of inhibitory neural mechanisms (Davison et al., 1978; Doggrell and Scott, 1980; Naughton et al., 1983), since what appears to be a typical NANCI response to field stimulation is resistant to tetrodotoxin (Crossley and Gillespie, 1983). Despite being one of the most commonly removed organs in surgical practice, the human gall-bladder has not been studied as thoroughly as that of the guinea-pig. Although gall-stones may be present, the wall of the excised gall-bladder often appears macroscopically normal and microscopically shows only mild chronic inflammation. However, the muscle layer is very thin and interwoven with connective tissue. It is thus difficult to dissect out individual muscle bundles. Hence it has been found more convenient and quicker to use strips of whole-wall thickness for in vitro studies, although whether the presence of the mucosa influences the behaviour of strips in vitro is not known. Two strips which had been adjacent in situ may behave differently in response to field stimulation in vitro; it has not been possible to correlate these differences with presence or absence of the mucosa. Using such whole thickness strips (l cm long by 1-2 mm broad) and isometric recording (McKirdy, unpublished observations), the findings of Mack and Todd (1968) were

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confirmed in virtually all but one respect. Mack and Todd (1968) used isotonic recording and reported that gall-bladder strips developed a high level of tone, yet in the isometric recording set-up tone (sustained resting tension) was low. Only during phasic contractions, which were recorded in a minority of strips, was any significant level of tension attained. This observation may reflect the difference between a lightly loaded isotonic and the isometric recording system (Csapo, 1960). Contractions obtained in response to electrical field stimulation (0.3 msec pulses at 10 H2 for 5 sec) were reduced but not abolished by atropine (3 ktM), suggesting a NANCE component. In the presence of atropine plus guanethidine (10 ~tM) and CCK octapeptidc (1 ~ M) or caerulein (0.1 #M) to raise the tone, the response to electrical field stimulatio~ was either abolished or significantly reduced, depending on the tone level attained. In twc out of thirteen strips a small component of relaxation became detectable in response tc field stimulation. This was abolished by tetrodotoxin, suggesting that the human gall. bladder may receive a very sparse NANCI innervation (McKirdy, unpublished). Alterna. tively the human gall-bladder may receive no NANCI innervation (A. S. Clanachan personal communication). 4.1.4. Cystic Duct

The cystic duct would be interesting to study in vitro as Scott and Otto (1979) have shown that an extremely small amount of muscle is capable of sphincter-like activity Unfortunately, however, this region usually becomes damaged during removal of th~ human gall-bladder and is generally unsuitable for in vitro study. The canine cystic duc is less sensitive than the gall-bladder to CCK (Courtney et al., 1983), and although it~ muscle responds to a variety of pharmacological agents its innervation does not appea: to have been analysed (Ryan, 1987). 4.1.5. Bile Duct As noted above, the human common bile duct has very little longitudinal muscle in it wall, but this still gives some contractile responses in vitro to very high concentrations o Boots secretin and CCK-PZ (Toouli and Watts, 1972). It is unknown whether or not an' NANC innervation is present. 4.1.6. Duodenum The duodenal intrinsic nervous system is probably fairly similar to that of the remainde of the small intestine (Taylor and Bywater, 1988). However, the duodenum is involved il many extrinsic reflex pathways and in hormonal control mechanisms. The contraction a human duodenal circular muscle to electrical field stimulation in vitro is converted to relaxation or a biphasic response in the presence of high concentrations of carbachol an, this response is guanethidine-resistant, suggesting the presence of NANCI and possibl NANCE innervation in addition to cholinergic innervation. The NANC innervation wa sparser in longitudinal strips (McKirdy, unpublished). The NANC innervation of the ra duodenum has been extensively investigated in vitro (Manzini et al., 1985; Maggi et al 1986) and projections of peptide-containing neurones have recently been demonstrated i rat enteric nervous system (Ekblad et al., 1987). 4.1.7. Sphincter o f Oddi The human sphincter of Oddi has now been extensively investigated by manometry an the findings reviewed by Hogan et al. (1983), Toouli (1984), Lebovics et al. (1986) an Ryan (1987). In vitro and in situ studies have been carried out in guinea-pig (e.g. Brook and Halliday, 1975; Harada et al., 1986), opossum (e.g. Toouli et al., 1981; Becker et al 1982; Coelho et al., 1986; Bauer et al., 1986), cat (Persson, 1972; Behar and Biancani, 198 and 1984) and dog (Tansy et al., 1974). As far as can be ascertained only one in vitr

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investigation of human sphincter of Oddi has been reported (McKirdy et al., 1987). Some strips from the choledochal sphincter region do show features considered typical of sphincteric muscle, and the innervation appears to be cholinergic, NANCI and possibly NANCE, since contractions to electrical field stimulation are reduced but not abolished by atropine (1/~M) and relaxations are resistant to guanethidine (3 pM). 4.2. PUTATIVE TRANSMITTERS

4.2.1. A m i n e s Human gall-bladder strips were found to be unresponsive to dopamine (up to 1/IM) and to 5-HT; histamine is excitatory and its effect is reduced by the H~ blocker mepyramine (Mack and Todd, 1968). The sensitivity to histamine is increased in diseased gall-bladders (Lennon et al., 1984). In guinea-pig gall-bladder H~ receptors mediate excitation and H2 receptors inhibition (Impicciatore, 1978). The canine cystic duct also has excitatory H~ and inhibitory H2 receptors (Clanachan et al., 1982a). The human cystic duct and common bile duct do not appear to have been examined for the effects of these amines. Strips from human choledochal sphincter which show tone relax in response to dopamine (2 pM), the effect being similar to that of noradrenaline (McKirdy et al., 1987). Both 5-HT and histamine (1-10 #M) produce contractions in strips showing low tone, but tachyphylaxis has prevented the localisation of their site of action to nerve or muscle. In the cat sphincter of Oddi, 5-HT produces a biphasic response comprising contraction followed by relaxation, which is explained by a direct excitatory effect on the muscle, an indirect excitatory effect via cholinergic neurones and an indirect inhibitory effect via NANCI neurones (Behar and Biancani, 1983). Histamine has a direct excitatory effect on the muscle of the opossum sphincter of Oddi and an indirect inhibitory effect via NANCI neurones (Toouli et al., 1981). 4.2.2. Purines and R e l a t e d Substances The human gall-bladder is insensitive to ATP up to 100/~M. At higher concentrations ATP produces a contraction at low levels of tone. When tone was raised with CCK or caerulein, ATP at 300/~M to 1 mM produces a detectable relaxation. Reports on the sensitivity of guinea-pig gall-bladder to ATP differ considerably. Davison et al. (1978), using a whole organ preparation, detected relaxation at 1-2/~ M ATP, whereas Naughton et al. (1983), using strips, found it necessary to use 300/~M-1 mM ATP to produce relaxation. The reason for this difference is not clear, though it may be related to in vitro technique. In some tissues ATP may exert its effect via enteric ganglion cells (Fox et al., 1986) and these were not always uniformly distributed throughout the organ and may be relatively deficient in some gall-bladder strips: these strips may therefore be less responsive to ATP. The importance of ATP in the guinea-pig gall-bladder is therefore still uncertain. It is released on electrical stimulation (Takahashi et al., 1987a) but its source has not been established. The lack of sensitivity to ATP would seem to preclude an important NANC transmitter role in the human gall-bladder. The human sphincter of Oddi is insensitive to ATP up to 300/tM (McKirdy et al., 1987), again suggesting that ATP, unless accompanied by some agent which potentiates its effect, is unlikely to be an important NANC transmitter. 4.2.3. Peptides The human gall-bladder is remarkably insensitive to VIP; no effects are detectable up to 1/~ M, irrespective to tone level, in the presence of CCK or caerulein (Feeley et al., 1984). At higher concentrations (10/zM) of VIP a small contraction, or an increase in the amplitude of spontaneous contractions, was detectable (McKirdy, unpublished). VIP-containing nerves have been visualised in human gall-bladder (Sundler et al., 1977) and VIP is released from cat gall-bladder on vagal stimulation (Bj6rck et al., 1986). VIP is inhibitory

442

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in cat (Jansson, 1978) and guinea-pig gall-bladder at high concentrations (0.1-1 #M) (Feeley et al., 1984, and compare Ryan and Ryave, 1978). The lack of sensitivity to, and lack of inhibitory effect of, VIP on the human gall-bladder seems surprising, and is different not only from oesophageal and gastric strips but also from sphincter of Oddi in the same species. VIP 0.2pM produces relaxation of human sphincter of Oddi strips which developed tone and in this tissue would be a possible candidate for a NANCI transmitter role, in the absence of a suitable blocking agent to exclude it (McKirdy et al., 1987). VIP or the related peptide histidine isoleucine (PHI) (Tatemoto and Mutt, 1981; Blank et al., 1986) are also candidates for NANCI transmitters in the canine and opossum sphincter of Oddi (Bauer et al., 1987). The VIP-related substances secretin (synthetic) and glucagon up to 1/~ M are ineffective in human gall-bladder and sphincter of Oddi (McKirdy et al., 1987). Boots secretin has excitatory effects on human gall-bladder (Mack and Todd, 1968), bile duct (Toouli and Watts, 1972) and sphincter of Oddi (McKirdy et al., 1987) but this excitatory effect is probably due to contamination with motilin and CCK or other impurities. The actions of the CCK/gastrin group of peptides on the biliary tract are complex, again due to an interaction of direct effects on muscle with those indirect effects via nervous tissue (Behar and Biancani, 1980, 1987; Takahashi et al., 1984; Strah et al., 1986; Harada et al., 1986; Takahashi et al., 1987b). A further problem may exist in vivo because of an interaction of peptides with each other (Ryan and Cohen, 1976b) and with neural activity (Davison and F6sel, 1975; Foesel and Sewing, 1978), though the importance of effects via neural structures has been questioned (Yau et al., 1973). Certainly the human biliary tract would appear to be much less sensitive to CCK in vitro than would be expected from in vivo observations. This difference in sensitivity appear,, to have been noted first by Mack and Todd (1968) who found the effective concentration of Boots pancreozymin on gall-bladder strips to be at least fifty times higher than those effective in vivo. Careful study by Keane et al. (1986) showed the EDs0 for CCK-octapeptide on human gall-bladder strips to be 676 nM with a range of 209-2187 nM, whereas th~ plasma levels of CCK lie in the range 1-16pM (Weiner et al., 1981; Liddle et al., 1984: Kellow et al., 1987; Sweeting, 1986) and basal gastrin levels lie in the range 50-70 pM (Sweeting, 1986). Again, the guinea-pig gall-bladder is more sensitive in vitro, with 50% of maximum contraction at 1 nM CCK. The canine cystic duct is considerably less sensitiw than the gall-bladder to CCK (Courtney et al., 1983), indicating regional differences ir sensitivity within the biliary tract. Human gall-bladder strips, which show modest contraction to CCK-octapeptid~ 0.1-1 p M, are markedly stimulated by similar concentrations of the related substanc~ caerulein from amphibian skin (Bertaccini et al., 1968), which is reckoned to be at leas sixteen times more potent than CCK (Banfield, 1975). This contraction was still presen in tetrodotoxin-treated strips, suggesting that CCK receptors are present in the muscle Human gall-bladder strips were unaffected by pentagastrin up to 1 #M (McKirdy unpublished). Human sphincter of Oddi is insensitive to CCK octapeptide up to 1 pM (McKirdy e al., 1987), though higher concentrations produce a modest contraction. Boots pancreo zymin in high concentration produces contraction of human sphincter of Oddi (McKird2 et al., 1987) and human bile duct (Toouli and Watts, 1972). In the opossum sphincter of Oddi CCK-octapeptide has no direct effect on the musch in vitro, and its effect in vivo may be mediated via neural structures (Bauer et al., 1986) Good evidence has been obtained for a transmitter role for CCK in the central nervou system (Rehfeld, 1980) but its role in the enteric nervous system is not clear. Sensitivity differences between the in vivo and the in vitro experiments are apparent ix the case of CCK (and also with opioids), and these differences are difficult to explain Alteration in receptors from the in vivo to the in vitro situation (Fox et al., 1983) am predominance of effect on neural structures which may be absent in the in vitro strip (McKirdy et al., 1987) are possibilities which have been suggested. Neural receptors appea

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to differ from muscle receptors for CCK and disagreement exists over their relative importance in the sphincter ofOddi (Lin, 1975; Takahashi et al., 1984; Harada et al., 1986, Sarles, 1986; Behar and Biancani, 1987). Human gall-bladder strips are insensitive to morphine (Mack and Todd, 1968) and to leucine- and methionine-enkephalin. Human sphincter of Oddi is insensitive to morphine up to 30/~ M, leucine- and methionine-enkephalin and naloxone up to 10/~M (McKirdy et al., 1987). This lack of sensitivity is in sharp contrast to the in vivo situation where the human sphincter of Oddi is exquisitely sensitive to morphine, a phenomenon which is utilised in a number of tests derived from the Nardi-type morphine provocation test (e.g. Choy et al., 1984). In the hog bile duct and sphincter of Oddi, morphine may produce its excitatory effects by releasing histamine (Ehrenpreis et al., 1987). In cat sphincter of Oddi, the enkephalins are thought to have a physiological role in regulation of basal motor activity. They exert an excitatory effect via cholinergic and tryptaminergic neurones and this effect is blocked by naloxone (Behar and Biancani, 1984). Tachykinins cause contraction of the guinea-pig gall-bladder by a direct effect on muscle (Yau, 1985) and the order of potency is as follows: Substance K > Kassinin > Eledoisin ,> Physalaemin > Substance P, which is the same order of potencies reported for the NK-2 receptor in radioligand binding assays (Shook and Burks, 1986). Substance P is excitatory in the opossum sphincter of Oddi in vitro (Bauer et al., 1986). The human gall-bladder is insensitive to pancreatic polypeptide in vitro whereas relaxation is reported in vivo (Pomeranz et al., 1983). Peptide YY has been reported to cause contraction of the gall-bladder of the guinea-pig and cat (Tatemoto and Mutt, 1981; Tatemoto, 1982) but to have no effect on either basal or CCK-stimulated gall-bladder contractions in dogs or humans (Adrian et al., 1985; Lluis et al., 1985). In the dog gall-bladder neurotensin is excitatory (Sakamoto et al., 1983) and the peptide hormone motilin is also excitatory (Takahashi et al., 1982). The amino acid transmitter GABA had no effect on human gall-bladder up to 1 #M (McKirdy, unpublished). Clearly many of these peptides would have to be excluded as possible candidates for the role of NANC transmitters. Much work remains to be done and, in particular, blocking agents need to be developed to help in their exclusion. 5. POSSIBLE CLINICAL RELEVANCE Motility disorders of the gastrointestinal tract cause considerable morbidity. Most of these disorders are minor and self-limiting but 'irritable bowel syndrome', which may be due to gut peptide imbalance (Sj61und and Ekman, 1987), is expensive in terms of clinical time and investigative resources (Read, 1987). Of the more serious motility problems, smooth muscle spasm, neuromuscular incoordination and/or failure of muscle to relax properly may be underlying causes. The common factor here may be failure of the NANCI mechanism. At the other end of the spectrum, failure of coordination of contraction leads to paralysis of the organ and stasis. Clearly a deeper understanding of the NANC mechanisms and the transmitters involved may allow development of better drugs to treat these conditions. Some conditions characterised by failure of muscle relaxation, which require surgical intervention at present (e.g. congenital hypertrophic pyloric stenosis and some instances of achalasia of the cardia and biliary dyskinesia), could perhaps become amenable to treatment by appropriate drug therapy in the future if NANC mechanisms become better understood. Some progress is already being made. The abnormalities of peptide-containing nerve fibres in infantile pyloric stenosis have recently been studied and loss of a variety of neuropeptides in the abnormal region found (Wattchow et al., 1987). Hopefully, further investigation may throw some light on the active inhibitory agent(s). Perhaps similar studies might reveal the neural lesion in achalasia of the cardia. Fuller understanding of the control mechanisms of the lower oesophageal sphincter might even allow better treatment of the common problem of reflux.

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Oesophageai spasm presents a major problem in the diagnosis of non-cardiac chest pain and, once diagnosed, in treatment. Probably because of a c o m m o n innervation of heart and oesophagus, oesophageal angina may be indistinguishable from cardiac angina. Several provocative tests have been used to establish an oesophageal cause in patients with normal cardiac investigations (including coronary angiography). Ergometrine (Davies eJ al., 1982), edrophonium (Richter et al., 1985) and bethanechol (Nostrant et al., 1986) ar~ all in use. The treatment of oesophageal spasm is quite unsatisfactory. Nitrates and calciurr blocking agents are in current use and are effective, as can be demonstrated durin~ manometry, but many patients cannot tolerate the side-effects. Once again an understanding of N A N C mechanisms may lead to a more rational approach to treatment. 'Biliary dyskinesia' or sphincter of Oddi 'dysmotility' or spasm is often considered th~ cause of 'postcholecystectomy syndrome', which is a recurrence of the patient's typica' biliary tract pain following removal of the gall-bladder. In the past this sometimes led tc a further operation with exploration of the c o m m o n bile duct and division of the sphinctel of Oddi, though this is now, if necessary, performed endoscopically. The diagnosis car sometimes be made with the Nardi-type morphine provocation test and/or m a n o m e t r y o: the sphincter of Oddi (Lebovics et al., 1986) and hopefully this is another area where more satisfactory drug treatment might become available with a clearer understanding o N A N C I mechanisms. Further, it would be helpful to be able to produce maximal sphincte~ of Oddi relaxation when trying to flush out retained stones via a T-tube in the c o m m o n duct 6. S U M M A R Y The evidence for, and possible roles of, inhibitory and excitatory non-adrenergic non-cholinergic ( N A N C ) nerves supplying smooth muscle, and the effects of putatiw transmitter candidates are considered for each of three main regions of the uppe: gastrointestinal tract: (A) the smooth muscle portion of the oesophagus and the oeso phagogastric junction, (B) the stomach (fundus, body and antrum) and gastroduodena junction and (C) the biliary tract and choledochoduodenal junction. The major points fron human tissues are as follows: 1. Inhibitory ( N A N C I ) nerves appear to be present in the muscularis externa o oesophagus, stomach and duodenum, with greater density in the circular than in th~ longitudinal muscle. 2. N A N C I nerves are present in high density at the oesophagogastric and choledo choduodenal junctions. They may also be present at the gastroduodenal junction. Th, gall-bladder may have a very sparse N A N C I innervation. 3. Excitatory ( N A N C E ) nerves appear to be present throughout the upper gastrointes tinal tract. 4. Many candidates need at present to be considered for the role of N A N C I transmitter(s) in the human upper gastrointestinal tract but substance P still seems a likel'. contender for this role. 5. Fewer candidates are at present generally available for the role of N A N C transmitter(s), with VIP and ATP being leading contenders. However, in the human uppe gastrointestinal tract the evidence for A T P is not good, and VIP still remains the favourit, candidate except in the gall-bladder, where its role remains to be elucidated. Acknowledgements--I am very grateful to Dr. T. C. Muir for his helpful criticisms and suggestions and to Mr~

Sue Forster for her skillful typing. REFERENCES ABRAHAMSSON,M. and JANSSON,G. (1973) Vago-vagal gastro-gastric relaxation in the cat. Acta Physiol. Scant

88: 289-295. ADRIAN,T. E., SAVAGE,A. P., SAGOR,G. R., ALLEN,J. M., BAKARESE-HAMILTON,A. J., TATEMOTO,K., POLAr J. M. and BLOOM,S. R. (1985) The effect of peptide YYM on gastric, pancreatic and biliary function i humans. Gastroenterol. 89: 494-499.

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