J. Phy8iol. (1967), 189, pp. 317-327 317 With 4 text-figure8 Printed in Great Britain INHIBITION OF GASTROINTESTINAL MOVEMENT BY SYMPATHETIC NERVE STIMULATION: THE SITE OF ACTION BY M. D. GERSHON From the Department of Pharmacology, University of Oxford (Received 8 August 1966) SUMMARY 1. The mechanism of sympathetic nervous inhibition of gastrointestinal movement was investigated in order to determine whether the primary action of sympathetic nerve stimulation was on ganglia or on the smooth muscle itself. 2. The effect of sympathetic nerve stimulation did not resemble the effect of ganglionic blockade or that of removing all nervous tone with tetrodotoxin. 3. The same frequency of sympathetic nerve stimulation to the rabbit jejunum which inhibited the myogenic spontaneous activity blocked excitatory responses to transmural post-ganglionic cholinergic nerve stimulation and antagonized contractions in response to drugs acting directly on the smooth muscle. 4. Acetylcholine output was measured as an indicator of post-ganglionic nervous activity. In contrast to hexamethonium, stimulation of sympathetic nerves at frequencies of stimulation sufficient to relax the muscle did not reduce the output of acetylcholine. 5. Vagus nerves and sympathetic nerves to the guinea-pig stomach were stimulated individually and simultaneously in the presence of hyoscine. The relaxant responses to stimulation of the two nerves summated when they were stimulated simultaneously, indicating that the transganglionic vagal pathway was not blocked. 6. It is concluded that the effect of sympathetic nerve stimulation is due to the direct action of the released noradrenaline on the smooth muscle. INTRODUCTION It is now well established that sympathetic nerves to the gastrointestinal tract inhibit movement of the gut through the release of the chemical transmitter, noradrenaline (Finkleman, 1930; Brown, Davies & Gillespie, 1958). The site of action of the released transmitter has not yet been
318 M. D. GERSHON established. Recent histochemical studies of the innervation of the gut have indicated that adrenergic fibres are largely confined to the region of the myenteric plexus (Norberg, 1964; Jacobowitz, 1965). Since catecholamines affect ganglionic transmission (Marrazzi, 1939; Biilbring, 1944) this morphological observation has been taken to indicate that sympathetic inhibition of gastrointestinal movement is exerted primarily at the myenteric ganglia and not by an action of the released catecholamines on the smooth muscle itself (Norberg, 1964; Norberg & Sj6qvist, 1966). The experiments to be described were undertaken in an attempt to localize the site of action of the sympathetic transmitter. It was found that sympathetic nerves acted on the smooth muscle directly, and that their effects differed from those produced by removal of all nervous tone. No influence of sympathetic nerves on ganglionic transmission was found. Relaxation occurred without reduction of the output of acetylcholine. The results indicate that the site of action of the transmitter released by sympathetic nerve stimulation is the smooth muscle of the gut. METHODS Guinea-pigs and rabbits of either sex were stunned and bled. The stomach and oesophagus of the guinea-pig was dissected together with the vagi and the coeliac axis and its branches. The vagi were then separated from the oesophagus which was ligated near the stomach and cut away. The pancreas and other tissues surrounding the vessels were removed. The stomach was cannulated through the pylorus, the contents were washed out with saline, and the stomach was filled with 25 ml. saline. The preparation was set up in a 100 ml. organ-bath filled with Krebs solution, bubbled with 95% 02 and 5% C02 at 370 C. Intralulminal pressure was recorded as described by Paton & Vane (1963). The vagi and the coeliac axis were passed through insulated platinum ring electrodes. The nerves were stimulated with trains of square wave pulses for a fixed time of 10 sec. The pulse duration was 10 mse and the strength of stimulation was supramaximal. The frequency was adjusted so that stimulation of either the vagi or the perivascular nerves gave equal responses (see Results). Two stimulators were used so that the nerves could be stimulated either separately or simultaneously The solution always contained hyoscine (10-7 g/ml.) to prevent contraction due to stimulation of cholinergic nerves (Paton & Vane, 1963). Guinea-pig ileum and rabbit jejunum were removed from the animals with mesenteric vessels attached. Both preparations were set up in 10 ml. Krebs solution, bubbled with 95% 02 and 5% C02 at 370 C. A mesenteric artery was drawn through insulated platinum ring electrodes for stimulation of perivascular nerves (Finkleman, 1930). Another platinum electrode was introduced into the lumen of the intestine in order to stimulate the preparations between this electrode and another free in the bath (transmural stimulation; Paton, 1955). For transmural stimulation the pulse duration was 0 1 msec and the stimulus strength was again supramaximal. Once more, two stimulators were used so that the preparations could be stimulated either transmurally or through the perivascular nerves, separately or simultaneously. Contractions of the longitudinal muscle of both preparations were recorded either auxotonically with a frontal writing lever on a smoked drum or isometrically with a semi-conductor strain gauge on a paper recorder (Hellige). The output of acetylcholine from the rabbit jejunum and the guinea-pig ileuim in the presence of eserine (10-6 g/ml.) was assayed on the guinea-pig ileum as described by Paton
SYMPATHETIC INHIBITION OF GUT (1957). The Krebs solution used for the assay always contained morphine (10-5 g/ml.), eserine (2 x 10-9 g/ml.) and mepyramine (3 x 10-9 g/ml.). Donor preparations were set up in a 10 ml. organ-bath. A polythene cannula was inserted into the anal end to drain off intralumninal contents. The oral end was tied off so that the lumen did not communicate with the bath. Drugs used were acetylcholine bromide, eserine (physostigmine) sulphate, hexamethonium bromide, histamine acid phosphate, hyoscine hydrobromide, (-)-noradrenaline bitartrate and crystalline tetrodotoxin. RESULTS 319 In all preparations (-)-noradrenaline produced responses which were similar to those produced by perivascular nerve stimulation. These responses could be best seen in the guinea-pig stomach preparation which has a high tone and lacks spontaneous activity. The effects of perivascular nerve stimulation and the application of (-)-noradrenaline, were compared with that of removing all nervous activity by tetrodotoxin. Tetrodotoxin blocks the conduction of nerve action potentials and so abolishes nervemediated effects in innervated smooth muscle preparations (Gershon, 1966), but it has no direct effect on the smooth muscle itself (Toida & Osa, 1965; Biilbring & Tomita, 1966). In contrast to (-)-noradrenaline and sympathetic nerve stimulation, tetrodotoxin (10-7 g/ml.) never relaxed the stomach. It also failed to have any effect on the responses of the stomach to applied (-)-noradrenaline. It did not change the log dose-response relationship for (-)-noradrenaline even when applied in a concentration 10 times larger than that required to block the response of the stomach to stimulation of the vagus and the sympathetic nerves (Gershon, 1966). These observations, illustrated in Fig. 1, indicate that the removal of all nervous activity differs in its effects from sympathetic nerve stimulation and that no part of the response to (-)-noradrenaline depended upon the pre-existence of a nervous tone. Stimulation of perivascular nerves to the rabbit jejunum caused relaxation of the longitudinal muscle and inhibition of spontaneous activity (Finkleman, 1930). Similar responses to stimulation of the perivascular nerves could be obtained, from preparations of guinea-pig ileum, but only if the tone of the preparations was first raised with histamine (10-8 g/ml.). Preparations of either kind occasionally contracted during stimulation of the perivascular nerves and, more frequently, contracted after cessation of stimulation. Contraction during stimulation could be abolished by adding hyoscine (0I g/ml.) to the bath, indicating that some cholinergic fibres were also stimulated. Such preparations were discarded. In the other preparations, inhibitory responses to perivascular nerve stimulation were unaffected either by hyoscine (10-7g/ml.) or by hexamethonium (5 X 10-5 g/ml.) (Wilson, 1962), although hexamethonium occasionally increased the magnitude of the spontaneous contractions of the rabbit
320 M. D. GERSHON jejunum (Fig. 2a and b) (Birmingham & Wilson, 1965). Since the inhibitory responses of the preparations which were studied were unaffected by hyoscine, it is unlikely that acetylcholine was released from perivascular nerves in amounts large enough to affect the results. In both preparations perivascular nerve stimulation inhibited contractions of the longitudinal muscle to histamine (10-9 g-10-7 g/ml.) in the presence of hyoscine (10-7 g/ml.), which was added to rule out possible cholinergic influence, and to acetylcholine (10-9-10-7 g/ml.) (Fig. 2e andf). 5 min NA NA S S S NA NA 5 x10 8 5 xlo -7 5x10 8 5 x10 7 Tetrodotoxin Fig. 1. Intraluminal pressure recorded from the guinea-pig stomach. NA = (- noradrenaline; S = sympathetic (perivascular) nerve stimulation (300 pulses 1*0 msec, 30 c/s). Tetrodotoxin was added at the arrow and remained present thereafter. The preparation was washed after each addition of (-)-noradrenaline. Tetrodotoxin did not affect the tone of the stomach, nor the response to (-)-noradrenaline, but abolished responses to sympathetic nerve stimulation. Responses of both preparations to transmural stimulation were also blocked by simultaneous stimulation of the perivascular nerves. Since the responses to transmural stimulation are resistant to gangionic blockade they probably result from stimulation of post-ganglionic nerves (Paton, 1955). Stimulation of the perivascular nerves at frequencies below 5/sec was inadequate to relax spontaneous muscle tone and inhibit the pendular activity of the rabbit jejunum. Similarly, no inhibition of the response to
SYMPATHETIC INHIBITION OF GUT 321 transmural stimulation was observed if the perivascular nerves were stimulated at frequencies below 5/sec. These results are illustrated for rabbit jejunum in Fig. 2. a b c d S.~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ lo0 Ti T,tP5 I P5 P2 T10 T10 TIcTIo+ps T10TIO T1+P2 T10+P5 C6 C6 e f h Washed ' 0 Fig 2 The effect of sympathetic nerve stimulation on spontaneous activity and on the activity evoked by transmural stimulation and by drugs. The contractions of the longitudinal muscle of the rabbit jejunum are recorded isometrically. (a) The responses to transmural stimulation, T (0), and the effect of stimulating perivascular nerves, P (>), simultaneously P+ T (4Q>). The numbers refer to the frequency of stimulation per second. (b)-(d) in the presence of hexamethonium (5 x 10-5 g/ml. (C6)). (e) Effect of sympathetic nerve stimulation on tone induced by histamine 10-5 g/ml. (Hist) in the presence of hyoscine 10-7. (f) Effect of sympathetic nerve stimulation on contraction produced by acetylcholine (ACh). (g) and (h) Effect of sympathetic nerve stimulation on contraction due to the presence of eserine 10-6 g/ml. The output of acetylcholine was measured from the rabbit jejunum and guinea-pig ileum as an index of post-ganglionic nerve activity. From the reduction of the resting output of acetylcholine by hexamethonium and cocaine, Zar (1966) has estimated that about 60 % of the resting output is the result of the activity of ganglion cells. If sympathetic nerves were able to inhibit ganglia, stimulation of the perivascular nerves should, like hexamethonium, reduce the resting output of acetylcholine. In order to preserve acetylcholine, eserine (10- g/ml.) was added to the donor bath. Both the 2I Physiol. i89
322 M. D. GERSHON rabbit jejunum and guinea-pig ileum were contracted by the eserine and both could then be relaxed by stimulation of the sympathetic nerves at frequencies of stimulation greater than 5/sec (Fig. 2g and h). In both preparations the resting output of acetylcholine remained stable for at least 2 hr after the addition of eserine. The effect of stimulation of sympathetic nerves on the release of acetylcholine is shown in Table 1 and in Fig. 3. Since the lumen of the intestine did not communicate with the fluid of TABLE 1. Effect of sympathetic nerve stimulation on the resting output of acetylcholine from guinea-pig ileum (ng/g/min) 1 c/s Stimulation Stimulation of peri- of peri- Resting vascular Resting vascular Resting Expt. output nerves 5/sec output nerves 20/sec output 1 55 60 50 54 54 2 50 51 54 50 54 3 61 60 54 51 50 Consecutive collection periods were 10 min. Perivascular nerves were stimulated 5 times for alternate 1 min periods during the total 10 min collection. The intestinal segments were weighed at the conclusion of the experiments. 25-20- ;15 5 10 c/s.10 c/s 30 c/s Rest Rest Rest Rest Transmural Perivascular Perivascular Perivascular stimulation stimulation stimulation stimulation Fig. 3. Acetylcholine output from the rabbit jejunum assayed on the guinea-pig ileum. The columns represent consecutive collection periods. Sympathetic (perivascular) nerves were stimulated for alternate 1 min periods, i.e. 5 min out of each 10 min as indicated. Transmural stimulation was for a continuous period. The frequency of stimulation is indicated above the appropriate coluimns. Two different experiments from different rabbits are shown (continuous and interrupted lines).
SYMPATHETIC INHIBITION OF GUT 323 the bath, acetylcholine release from the mucosa probably did not contribute significantly to the measured acetylcholine output. The output of acetylcholine from the guinea-pig ileum was higher than that from the rabbit jejunum, but in both preparations, stimulation of the sympathetic nerves at frequencies sufficient to cause relaxation of the muscle contracted by eserine (Fig. 2g and h) failed to decrease the output of acetylcholine. When the perivascular nerves to the rabbit jejunum were stimulated at 30/sec there was some decrease in the output of acetylcholine. It is known that catecholamines reduce the output of acetylcholine from preganglionic nerve endings (Paton & Thompson, 1953) and also from post-ganglionic endings (Schaumann, 1958; Kosterlitz & Watt, 1965); so it would be expected that, if sufficient catecholamines were released by sympathetic nerve stimulation, the output of acetylcholine from the intestine would eventually be reduced. This does occur. However, the reduction of acetylcholine output was not correlated with the relaxation of the muscle. Since the muscle was relaxed with low frequencies of stimulation but the output of acetylcholine was not reduced until sympathetic nerves were stimulated in excess of 30/sec, it is unlikely that the effect played any part in the relaxation observed in the present experiments. Stimulation of either the vagus nerves or the perivascular (sympathetic) nerves relax the isolated guinea-pig stomach in the presence of hyoscine (Greeff, Kasperat & Osswald, 1962; Paton & Vane, 1963; Martinson, 1965). The inhibitory fibres which run in the vagus nerve are preganglionic (Greeff et al. 1962; Paton & Vane, 1963; Martinson, 1965; Biilbring & Gershon, 1966), distinct from the sympathetic (Martinson, 1965; Campbell, 1966) and non-adrenergic. Responses to stimulation of the vagus nerves and the perivascular nerves in the presence of hyoscine are shown in Fig. 4. The frequency of stimulation was selected so that the responses were of equal magnitude and were submaximal. When both nerves were stimulated simultaneously the relaxation produced by the combined stimulation was more than twice as large as that produced by either nerve stimulated alone. The two different inhibitory mechanisms thus appear to be synergistic. The summation of the responses was noted both when the nerves were stimulated for 10 sec simultaneously and when the sympathetic nerves were stimulated for 10 sec in the midst of a sustained 3 min period of vagal stimulation. These results could not have been obtained if stimulation of the sympathetic nerves had blocked ganglionic transmission in the inhibitory vagal pathway. 2I-2
324 M. D. GERSHON 5 min S V V V S V S V V V S V + S S Fig. 4. Intraluminal pressure recorded from the guinea-pig stomach in the presence of hyoscine (10-7 g/ml.). V = vagal stimulation, 1 lc/s. S = sympathetic (perivascular) nerve stimulation, 10 c/s. V+S = combined stimulation of both nerves. Bars indicate when the vagus nerves were stimulated continuously at 10 cls for 3 min. At the first V+S both nerves were stimulated simultaneously for 10 sec. The second time, the sympathetics were stimulated for 10 sec in the midst of a continuous 3 min period of vagal stimulation. DISCUSSION The present experiments were designed to investigate whether the inhibition of gastrointestinal movement by sympathetic nerve stimulation was due to a direct action on the smooth muscle of the gut or to an action on ganglionic transmission. Gastrointestinal movement consists, in part, of changes of the spontaneous tone and, in part, of the response to excitatory nervous activity. Both are antagonized by activation of the sympathetic nerves. The question then arises whether the spontaneous tone is also, in part, nervemediated. It was found that abolition of nervous activity by tetrodotoxin had no effect on the spontaneous tone of the guinea-pig stomach (or the rabbit jejunum, Gershon (1966)), i.e. it did not mimic the effect of sympathetic nerve stimulation. Application of noradrenaline, however, did produce relaxation similar to that caused by stimulation of sympathetic nerves. The fact that tetrodotoxin abolished all nervous responses, but did not reduce the response to noradrenaline, indicates that the noradrenaline released by sympathetic nerve stimulation acts on the muscle directly. This is supported by the observations that sympathetic nerve stimulation was equally able to relax the intestine in which the ganglia were already
S YMPATHETIC INHIBITION OF GUT 325 blocked by hexamethonium, and in which cholinergic nervous activity was blocked by hyoscine. Thus there is no need for a nervous tone to exist before the relaxation by the sympathetic transmitter or applied noradrenaline can take place. Further experiments indicated that sympathetic nerve stimulation acted on the smooth muscle directly. The effect of sympathetic nerve stimulation on intestinal movement evoked by excitatory post-ganglionic nervous stimulation was studied using transmural and perivascular stimulation of the guinea-pig and rabbit intestine. In the latter preparation sympathetic stimulation suppressed pendular activity which is resistant both to botulinum toxin (Ambache & Lessin, 1955) and tetrodotoxin (Gershon, 1966) and is generally believed to be of myogenic origin. The same frequency of sympathetic nerve stimulation which was required to suppress pendular movement also abolished contraction in response to transmural stimulation. This effect, not shared by ganglion blocking agents, can only be explained by an action distal to the ganglia. Wilson (1962) made similar observations on the guinea-pig intestine and reached a similar conclusion. Sympathetic nerve stimulation also reduced contractions produced by histamine (in the presence of hyoscine) and by acetylcholine. Since tetrodotoxin fails to antagonize the action of these drugs (Gershon, 1966), and both drugs are able to contract denervated intestinal muscle equally as well as innervated (Paton & Zar, 1965), the inhibitory action of sympathetic nerve stimulation must be exerted on the smooth muscle itself. In order to determine whether any evidence could be found for an action of sympathetic nerves on ganglia, the intestinal acetylcholine output was measured. In contrast to hexamethonium which reduces the output of acetylcholine, sympathetic nerve stimulation failed to do so in both guinea-pig ileum and rabbit jejunum at frequencies which cause relaxation. Since about 60 % of the resting output results from ganglionic activity (Zar, 1966) it is very unlikely that relaxation in response to sympathetic nerve stimulation is due to ganglionic blockade. In the isolated stomach in which preganglionic vagal fibres innervate inhibitory ganglia, it was possible to study the effect of stimulating sympathetic nerves on ganglionic transmission directly. Vagus and sympathetic nerves were stimulated separately and simultaneously in the presence of hyoscine. It was found that sympathetic nerve stimulation did not antagonize the vagal inhibitory effect, but, on the contrary, summated with it. From in vivo experiments in the cat, Kewenter (1965) has concluded that there is an inhibitory tone in the ileum which is sympathetic in nature. This conclusion was based on the observation that vagal excitation of the
326 M. D. GERSHON 326M.DGRHO ileum, but not of the jejunum, was enhanced by guanethidine and ergotamine. Because the contractile action of acetylcholine was of equal magnitude in both ileum and jejunum, Kewenter proposed that this inhibitory tone is directed against ganglionic transmission. Kewenter did not report, however, whether sympathetic nerve stimulation antagonized contractions due to acetylcholine as in the present experiments, nor whether guanethidine and ergotamine enhanced the acetylcholine contractions. Thus, while there may well be an inhibitory tone in the cat's ileum there is insufficient evidence for the conclusion that it is caused by sympathetic nervous inhibition of ganglionic transmission. In summary, no evidence of ganglionic inhibition by sympathetic nerve stimulation was found. Abolition of nervous activity produced different effects from stimulation of sympathetic nerves and stimulation of the nerves antagonized the effects produced by drugs acting directly on the smooth muscle. Thus it appears that the effect of sympathetic nerve stimulation is inhibition, not of ganglia, but of the smooth muscle itself. The histochemical observations of Norberg ( 1964) and Jacobowitz ( 1965), that the adrenergic innervation of the intestine seems to lie mostly in the myenteric plexus, at first appear to be at variance with the conclusion that the effect of sympathetic nerve stimulation is inhibition of the smooth muscle itself and not of ganglia. This should not be surprising, however, when it is considered that the cholinergic innervation of the intestine also appears to be confined to the myenteric plexus. Thus, the longitudinal muscle of the guinea-pig ileum separated from the myenteric plexus contains little or no acetylcholine, and fails to respond to either electrical stimulation with current pulses of brief duration or to drugs which act through nervous pathways (Paton & Zar, 1965; Zar, 1966). In fact, Paton (1964) in an electron microscopic examination of the longitudinal muscle could find no evidence of any innervation of the muscle itself. It thus appears that both the adrenergic and cholinergic innervation of the longitudinal muscle of the gut is confined to a region close to the myenteric plexus and that both influence the smooth muscle directly, despite the necessity for diffusion of transmitter over a relatively large distance. I would like to thank Dr Edith Biilbring for her encouragement and advice, and Miss Carole Foster for careful technical assistance. This work was supported by the Medical Research Council grant number G 964/340/B and was done during the tenure of a post-doctoral fellowship of the United States Public Health Service. REFERENCES AMBACHE, N. & LEsSIN, A. W. (1955). Classification of intestinomotor drugs by means of type D botulinum toxin. J. Physiol. 127, 449-478. BIRMINGHAM, A. T. & WILsoN, A. B. (1965). An analysis of the blocking action of dimethylphenylpiperazinium iodide on the inhibition of isolated small intestine produced by stimulation of the sympathetic nerves. Br. J. Pharmac. Chemother. 24. 375-386.
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