University of Essen, Essen, F.R.G.

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1 J. Phy8iol. (1985), 369, pp With 4 text-figure8 Printed in Great Britain GASTRIN RESPONSE TO A MEAL BEFORE AND AFTER CUTTING THE EXTRINSIC NERVES OF THE STOMACH IN THE DOG By V. E. EYSSELEIN, W. NIEBEL AND M. V. SINGER From the Division of Gastroenterology, Department of Medicine, University of Essen, Essen, F.R.G. (Received 12 March 1985) SUMMARY 1. Atropine inhibits the post-prandial gastrin release after truncal vagotomy in the dog. Whether this action of atropine is due to suppression of stimulatory cholinergic fibres in the sympathetic nerves of the stomach and the upper small intestine or due to blockade of intrinsic gastric cholinergic mechanisms is unknown. 2. Conscious dogs were fed a meat meal (35 g/kg body weight) before and after truncal vagotomy and after truncal vagotomy plus coeliac and superior mesenteric ganglionectomy. Experiments were repeated in the presence of atropine (50,sg/kg body weight, given as an i.v. bolus 60 min prior to the meal). In another set of dogs, only ganglionectomy was performed and the same experiments were done as in the first set of dogs. 3. Truncal vagotomy enhanced the post-prandial 120 min integrated plasma gastrin response by 2-6 times as compared to the response with the vagus nerves intact. Before truncal vagotomy, atropine enhanced the integrated plasma gastrin response by 2-6 times; after truncal vagotomy atropine suppressed this response by 2-3 times. 4. After truncal vagotomy, with or without atropine, additional coeliac and superior mesenteric ganglionectomy did not alter the integrated plasma gastrin response. 5. With the vagus nerves intact, ganglionectomy alone had no effect on the integrated plasma gastrin response whether or not atropine was given. 6. The finding that atropine suppresses the post-prandial plasma gastrin response to a meal after truncal vagotomy and coeliac and superior mesenteric ganglionectomy, i.e. cutting the extrinsic nerves of the stomach and the upper small intestine, suggests the existence of stimulatory cholinergic intrinsic fibres located within the stomach. INTRODUCTION The vagus nerves can both stimulate and inhibit the release of gastrin. In the dog, vagal excitation by sham feeding or by insulin hypoglycaemia causes release of gastrin which can be abolished by denervation of the antrum (Tepperman, Walsh & Preshaw, 1972), the main source for circulating gastrin. Accordingly, gastrin release by sham feeding is abolished by truncal vagotomy in the dog (Dockray & Tracy, 12-2

2 356 V. E. EYSSELEIN, W. NIEBEL AND M. V. SINGER 1980). The existence of cholinergic stimulatory fibres in the vagus nerves can be suspected by the fact that high doses of atropine suppress the stimulatory effect of insulin-induced hypoglycaemia on gastrin release in dogs with innervated antral pouches (Csendes, Walsh & Grossman, 1972). The existence of inhibitory fibres within the vagus nerves for gastrin release is suggested by the finding that cooling of the cervical vagus nerves augments the gastrin response after insulin hypoglycaemia (Cairus, Deveney & Way, 1974). Truncal vagotomy enhances the meal-induced gastrin release in the dog (Debas, Walsh & Grossman, 1976; Schafmeyer, Teichmann, Swierczek, Rayford & Thompson, 1978). Since the post-prandial gastrin response is enhanced after proximal selective vagotomy but not after antral vagotomy (in experiments with constant intragastric ph), a fundic inhibitory mechanism maintained by tonic vagal activity has been postulated (Debas, Sael, Cork, Soon-Shiong & Walsh, 1981). Atropine given prior to a meal enhances plasma gastrin release (Impicciatore, Walsh & Grossman, 1977), but since atropine suppresses gastric acid secretion, the effect of atropine on gastrin release might be also due to altered acid secretion. When the intragastric ph was kept constant at ph 6-0 by intragastric titration, a low dose of atropine had no effect on gastrin release induced by a liquid meal (Dockray & Tracy, 1980). When the vagus nerves are cut, atropine suppresses post-prandial gastrin release (Debas et al. 1976). The effects of atropine on post-prandial gastrin release after truncal vagotomy in the dog could be explained by cholinergic stimulatory fibres located within the stomach (intrinsic mechanisms) or by fibres reaching the stomach via the nervi splanchnici and the coeliac and superior mesenteric ganglia. The action of the sympathetic nervous system on gastrin release has not been studied intensively so far. Infusion of adrenaline (Vendsalu, 1960; Stadil & Rehfeld, 1973) stimulates gastrin release in man, but splanchnic nervous stimulation does not release gastrin in the pig (Olesen, Sottimano, Holst & Nielsen, 1984). The aim of the present study was to clarify the role of the vagal and splanchnic nerves in the gastrin response to a meal and to discriminate their effects from that of the intrinsic cholinergic nerves of the stomach. For these reasons, we studied the effect of atropine on the plasma gastrin response to a meal in one set of dogs before and after truncal vagotomy and truncal vagotomy plus coeliac and superior mesenteric ganglionectomy, i.e. after extrinsic denervation of the stomach, and, in another set of dogs, after coeliac and superior mesenteric ganglionectomy alone. This procedure was chosen since anatomical studies have shown that, in the dog, all sympathetic nerves of the stomach pass through these ganglia (Tiscornia, 1976). METHODS Animal preparation Mongrel dogs of both sexes, weighing kg, underwent surgery on several occasions. The following anaesthetic procedures were invariably employed. 30 min after pre-medication with acepromacin (10 mg/kg I.M.) anaesthesia was induced with sodium pentobarbitone (30 mg/kg I.v.). When necessary, supplementary doses of sodium pentobarbitone were administered during the operation procedure. In no instance did any of the dogs show any sign of pain during the operations. A tracheal tube was inserted in each animal and a Harvard respirator was used to maintain intermittent positive pressure when respiration was necessary.

3 NERVES AND GASTRIN RELEASE During the first operation, the dogs were fitted with chronic gastric fistulas using a Thomas-type cannula, modified so that the inner flange was circular rather than oval (Thomas, 1951). During experiments, a rubber tube was inserted through the gastric cannula in order to take samples of gastric content for ph measuring. This design allowed no unwanted leakage of gastric juice or contents. During the second operation, either truncal vagotomy or coeliac and superior mesenteric ganglionectomy were performed. Truncal vagotomy was performed through a lower left intercostal space. Just above the diaphragm, a segment of 2 cm was removed from each vagus nerve and any intercommunicating fibres were resected. Coeliac and superior mesenteric ganglionectomy was performed according to the method described by Marlett & Code (Marlett & Code, 1979). The ganglia were exposed through a mid-line abdominal incision. The ganglia and plexuses anterior to the aorta between and surrounding the coeliac and superior mesenteric arteries and the neural tissue surrounding the main branches of these vessels were removed. In one set of seven dogs, only coeliac and superior mesenteric ganglionectomy was performed, in another set of six dogs both truncal vagotomy and additional coeliac and superior mesenteric ganglionectomy were carried out. Plan of experiments Studies were started no sooner than 4 weeks after the first operation. Dogs were fasted 18 h before each test but had free access to water. The interval between tests was at least 48 h. After vagotomy and vagotomy plus ganglionectomy, the dogs received homogenized food for 2 days before each test to facilitate gastric emptying. Observations were made at various times, ranging from 1 week to 6 weeks after vagotomy alone and ganglionectomy alone and between 2 weeks and 2 months after vagotomy plus ganglionectomy. The general condition of the animals after vagotomy plus ganglionectomy was satisfactory. As had previous investigators (for review see Marlett & Code, 1979) we also noted diarrhoea in the dogs after ganglionectomy. Diarrhoea usually ceased after 3 weeks. The temporary weight loss of about 2-4 kg in some dogs also stopped after 2-3 weeks after the operation. At the beginning of each experiment, the gastric cannula was opened and the stomach was thoroughly rinsed with distilled water. Then a polyvinyl tube (4mm i.d.) was passed through the lumen of the cannula and advanced 5 cm into the stomach. A 19-gauge scalp needle was inserted into a leg vein and used for drawing blood starting 30 min later. The dogs were fed a mixed-meat meal (ChappyO, canned dog food, 35 g/kg body weight). The composition of the food was 7-9 % protein and 3-3% fat. In all experiments the dogs ate the entire meal within 2-3 min. In one set of dogs, the test meal was given before and after vagotomy and after vagotomy plus ganglionectomy. In another set of dogs the test meal was given before and after ganglionectomy. At each stage of innervation, the test meal was repeated in the presence of atropine. An intravenous bolus of 50 #sg/kg body weight of atropine sulphate dissolved in 5 ml 0-15 M-NaCl was given 60 min prior to the test meal. At no instance did the animals show any signs of atropine toxicity. In each experiment blood samples for radioimmunoassay of gastrin were taken 30 and 15 min and immediately before and at 15 min intervals after a test meal for another 120 min. Blood was collected in tubes containing EDTA and 2000 K.I.U. aprotinine (Trasylole). The blood samples were immediately centrifuged, the plasma saved and stored at -20 'C until radioimmunoassay of gastrin was performed. Plasma gastrin was determined by radioimmunoassay using antibody 1611 (kindly supplied by Dr J. H. Walsh, CURE, Los Angeles, CA, U.S.A.) as previously described (Eysselein, Singer, Wentz & Goebell, 1984). Verification of the effectiveness of truncal vagotomy and coeliac and superior mesenteric ganglionectomy 2-Deoxy-D-glucose (2-DG) was given as a centrally acting stimulant whose action on gastric acid secretion is mediated via the vagus nerves (Hirschowitz & Sachs, 1965). An intravenous bolus injection of 100 mg/kg body weight of 2-DG dissolved in 30 ml 0-15 M-NaCl was given before and after the different surgical procedures. Gastric juices were collected in 15 min samples during 15 min periods basally and for 1 h after giving 2-DG. Volume was measured to the nearest 0.1 ml and acid secretion was determined in 0-2 ml samples by titration with 0-2 N-NaOH to ph 7-0 on an automatic titrator (Radiometer Copenhagen). 357

4 358 V. E. E YSSELEIN, W. NIEBEL AND M. V. SINGER After completing the whole series of experiments, the dogs were killed by an intravenous injection of sodium pentobarbitone and the oesophagus examined macroscopically and histologically for possible regeneration of the vagus nerves. In no instance was incomplete dissection of the vagus nerves observed. In addition, macroscopic and histological examination of the area from which the ceoliac and mesenteric ganglia had been removed, did not reveal an incomplete dissection of the splanchnic nerves and the ganglia. Material Atropine sulphate and 2-DG were bought from Merck, Darmstadt, F.R.G. Calculaions andatt8wticl analysis Both, in the presence and absence of atropine, the test meal was given once in each dog before and after the different surgical procedures. Before statistical analysis, the mean value of the three basal plasma gastrin concentrations was calculated for each dog and these individual dog means were used to calculate the group means + s.e. of means, which are reported. These individual dog means of basal plasma gastrin concentrations were used to evaluate the effect of the different surgical procedures on basal plasma levels. As an indicator of the response to a meal, the 120 min integrated plasma gastrin response was calculated by the formula described by Taylor, Feldman, Richardson & Walsh (1978) for each dog. All statistical analyses were performed on the integrated responses for gastrin. The individual responses were used to calculate the group means+s.e. of means and two-way analysis of variance and/or the Student's t test for paired values were used to evaluate the difference between the various treatments. P values less than 0 05 % were considered significant. RESULTS Intact extrinsic nerves In the absence of atropine, plasma gastrin concentrations rose from a mean basal value of pm to a peak of pm within 15 min after eating the mixed-meat meal, declined during the following 60 min but remained elevated throughout the time of the study (Fig. 1 A). Atropine did not significantly alter basal gastrin concentrations. The post-prandial peak of plasma gastrin concentrations was delayed by about 30 min and remained thereafter at a plateau for 45 min. The 120 min integrated plasma gastrin response to the meal was significantly augmented by 2-6 times ( vs pm min). Truncal vagotomy After truncal vagotomy basal plasma gastrin levels were significantly higher than before vagotomy (26 + 9vs pm, Fig. 1 B). The 120 min integrated gastrin response to the meal was enhanced by 2-6 times ( vs pm min) being of the same magnitude of the response as before truncal vagotomy when atropine was given prior to the meal ( pm min). After truncal vagotomy, the post-prandial plasma gastrin response showed an early peak at 30 min and a late peak at 90 min which was numerically higher than the first one. When atropine was given after truncal vagotomy basal gastrin levels were comparable to those before truncal vagotomy (7+2 vs. 5+1 pm, respectively) and significantly lower than those after truncal vagotomy alone (26 ± 9 pm, Fig. 1 B). Compared to truncal vagotomy alone the early plasma gastrin response to the test meal was suppressed after additional atropine and plasma gastrin concentrations rose

5 NERVES AND GASTRIN RELEASE 359 steadily reaching the highest concentrations at 120 min after eating. After truncal vagotomy the integrated 120 min plasma gastrin response was significantly suppressed by atropine ( v pm min) and was of similar magnitude as that observed in response to the meal without atropine before truncal vagotomy. A B 100 Meal Meal 60 {jk, J11I +Atropine1 IU M N CU ~~~~~~I/Control Control Arpe 0L Time (min) Time (min) Fig. 1. Effect of atropine before (A) and after (B) truncal vagotomy on basal and post-prandial plasma gastrin concentrations (pm). Results are means+s.e. of means of seven dogs. Truncal vagotomy and coeliac and superior mesenteric ganglionectomy In dogs with previous truncal vagotomy, coeliac and superior mesenteric ganglionectomy did not significantly alter basal plasma gastrin concentrations as compared to truncal vagotomy alone (Fig. 2). Neither the time course nor the magnitude of response to the meal was significantly changed by additional ganglionectomy. The 120 min integrated plasma gastrin response was pm min after truncal vagotomy and pm mmin after truncal vagotomy plus ganglionectomy. Atropine significantly suppressed basal plasma gastrin levels (8 + 3 vs pm, Fig. 3) after truncal vagotomy plus coeliac and superior mesenteric ganglionectomy. Atropine also significantly reduced the post-prandial 120 min integrated plasma gastrin response by 2-2 times after truncal vagotomy ( pm min) and ganglionectomy ( pm min). As already observed after truncal vagotomy atropine predominantly suppressed the early post-prandial plasma gastrin response.

6 360 V. E. EYSSELEIN, W. NIEBEL AND M. V. SINGER 1100 Meal I 2 60 CL c._ wcn E a: 40, 0?t&S I 80, I After tvg. IT / I 20 - I o. _ L I m I Time (min) Fig. 2. Effect of truncal vagotomy (t.v.) alone and truncal vagotomy plus coeliac and superior mesenteric ganglionectomy (t.v.g.) on basal and post-prandial plasma gastrin concentrations (pm). Results are means+ S.E. of means of six dogs. Coeliac and superior mesenteric ganglionectomy Ganglionectomy alone did not significantly alter basal plasma gastrin concentrations and the time course and magnitude of response to the meal (Fig. 4, pm min vs pm min). With the ganglia intact, atropine significantly enhanced the post-prandial plasma gastrin response (Fig. 4, pm min vs ± 1364 pm min) as shown in another set of dogs (Fig. 1A). The time course and the magnitude of response to the meal were not altered by ganglionectomy ( pm min vs pm min). Response to 2-DG Truncal vagotomy and truncal vagotomy plus ganglionectomy significantly reduced the gastric acid response by more than 95 %. The 1 h incremental gastric acid output

7 NERVES AND GASTRIN RELEASE r- i100 k Q._ co 60 0 E I I I I I Time (min) Fig. 3. Effect of atropine on basal and post-prandial plasma gastrin concentrations (pm) after truncal vagotomy and coeliac and superior mesenteric ganglionectomy (t.v.g.). Results are means + S.E. of means of six dogs. to 2-DG was mmol before and mmol after truncal vagotomy and mmol after vagotomy plus ganglionectomy. Coeliac and superior mesenteric ganglionectomy alone did not alter significantly the gastric acid response to 2-DG ( mmol/h vs mmol/h). DISCUSSION The present study shows that, in the dog, dissection of the sympathetic nervous supply of the stomach and small intestine at the level of the coeliac and superior mesenteric ganglia has no effect on basal and food-stimulated plasma gastrin release.

8 362 V. E. EYSSELEIN, W. NIEBEL AND M. V. SINGER 180 r 150 Meal Before g- * A*, -- +atropine /,' * / \ \\,/.. \ / i._ cl C E U, a, ; After g. t \ / +atropine ' / 0,.-0 1 N. _ - * After g. 0Nl-.\ *~~~~~~~~~~~" 30 0 Before g. 0 L- L -30 I I I J 120 Time (min) Fig. 4. Basal and post-prandial plasma gastrin concentrations (pm) before and after coeliac and superior mesenteric ganglionectomy (g.) in the absence and presence of atropine. Results are means+ S.E. of means of six dogs. There was no difference in the response whether ganglionectomy was performed before or after truncal vagotomy. Both findings indicate that the extrinsic sympathetic nerves of the stomach do not play a significant role in the control of release of gastrin basally and after feeding. Since the stomach and the upper small intestine are supplied by sympathetic fibres running in the splanchnic nerves which pass through the coelical and superior mesenteric ganglia, removal of these ganglia should have disrupted all sympathetic innervation of the stomach and the small intestine (Tiscornia, 1976; Marlett & Code, 1979). Adrenergic fibres running in the vagi should also have been removed by additional truncal vagotomy. Our results are in agreement with those obtained by Graffner, Ekelund, Hakanson, Oscarson, Rosengren & Sundler (1984) who found that basal serum gastrin levels in sympathectomized rats did not differ from normal values. Atropine and truncal vagotomy enhanced the post-prandial gastrin release and this finding is consistent with the existence of cholinergic inhibitory vagal fibres for gastrin release. Removal of the inhibitory effect of low intragastric ph on gastrin release is another likely explanation as suggested by our finding that after atropine the post-prandial intragastric ph reached higher values than without atropine and was well above the threshold for inhibition of gastrin release (ph < 3 4). Thus, the

9 NERVES AND GASTRIN RELEASE intragastric ph reached a peak of min after the meal in the control animals and a peak of 6-2 (at 30 min) after additional atropine (results of two dogs). After truncal vagotomy atropine suppressed rather than enhanced gastrin release. It appears that the cholinergic inhibitory mechanisms have a different threshold for atropine since the action of the cholinergic stimulatory mechanisms is unmasked after truncal vagotomy. Our data confirm those obtained by Debas et al. (1976) who gave a higher dose of atropine (200,ug/kg as an i.v. bolus) and studied post-prandial gastrin release before and after truncal vagotomy. In man, in contrast to the dog, atropine enhances post-prandial plasma gastrin release after truncal vagotomy (Hansky & King, 1977). These different findings may be explained by the different doses of atropine used (1-2 mg atropine I.M.), the time passed by after truncal vagotomy and the species used. The reason why in the dog atropine suppresses post-prandial plasma gastrin release after truncal vagotomy is unclear. Since the cholinergic fibres reaching the stomach via the nervi vagi are cut, stimulatory cholinergic fibres located within the stomach (intrinsic system) or cholinergic fibres in the sympathetic nervous supply of the stomach may be responsible for this effect. We have excluded the possibility that cholinergic fibres running in the splanchnic nerves play a role in mediating this action of atropine, since coeliac and superior mesenteric ganglionectomy did not alter the effect of atropine after truncal vagotomy. Thus, it appears to be likely that the inhibitory action of atropine on post-prandial gastrin release after truncal vagotomy is due to intrinsic cholinergic stimulatory fibres located within the stomach. In conclusion, in the dog, the splanchnic nerves do not appear to play any major role in the release of gastrin basally and in response to a meal. The inhibitory action of atropine on post-prandial gastrin release after removal of the extrinsic (vagal and splanchnic) nerves indicates that local cholinergic nerves within the stomach mediate part of the gastrin response to a meal. The authors would like to thank G. Albertz, D. Lewin and K. Scheel for expert technical assistance, R. Munchow for drawing the Figures and B. Beutling for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Si 228/5-3 and Ey 14/2-1). 363 REFERENCES CAIRUS, D., DEVENEY, C. W. & WAY, L. W. (1974). Mechanism of the release of gastrin by insulin hypoglycaemia. Surgical Forum 25, CSENDES, A., WALSH, J. H. & GROSSMAN, M. I. (1972). Effects of atropine and of antral acidification on gastrin release and acid secretion in response to insulin and feeding in dogs. Ga8troenterology 63, DEBAS, H. T., SAEL, A. M., CORK, C. A., SOON-SHIONG, T. & WALSH, J. H. (1981). Vagal distribution for stimulation and inhibition of gastrin release. Surgical Forum XXX, DEBAS, H. T., WALSH, J. H. & GROSSMAN, M. I. (1976). After vagotomy atropine suppresses gastrin release by food. Oa8troenterology 70, DocKRAY, G. J. & TRACY, H. J. (1980). Atropine does not abolish cephalic vagal stimulation of gastrin release in dogs. Journal of Physiology 306, EYSSELEIN, V. E., SINGER, M. V., WENTZ, H. & GOEBELL, H. (1984). Action of ethanol on gastrin release in the dog. Digestive Diseases and Sciences 29, GRAFFNER, H., EKELUND, M., HAKANSON, R., OSCARSON, J., ROSENGREN, E. & SUNDLER, F. (1984). Effects of upper abdominal sympathectomy on gastric acid, serum gastrin, and catecholamines in the rat gut. Scandinavian Journal of Gastroenterology 19,

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