Gut, 1991,32,215-219 215 Effect of the cholecystokinin-receptor antagonist lorglumide on pancreatic enzyme secretion Gut: first published as 10.1136/gut.32.2.215 on 1 February 1991. Downloaded from stimulated by bombesin, food, and caerulein, giving similar plasma cholecystokinin concentrations in the dog

A J L de Jong, M V Singer, J B M J Jansen, W Niebel, LC Rovati, C B H W Lamers

Abstract including the enterohormone cholecystokinin, a This study was undertaken to determine the powerful stimulant of pancreatic enzyme role of cholecystokinin in pancreatic enzyme secretion.36 secretion stimulated by bombesin and a meal It is generally accepted that food stimulates by (a) comparing the pancreatic enzyme output pancreatic enzyme secretion through a delicate during bombesin infusion and after a meal to interplay between nerves and gastrointestinal output during caerulein infusion and (b) hormones.247 Several studies have suggested an comparing the inhibitory effect of the chole- important role for the vagal cholinergic system cystokinin-receptor antagonist lorglumide and for cholecystokinin.27 (CR-1409) on enzyme output in response to The present study was undertaken to deter- bombesin and food with the response to mine the role of cholecystokinin in the caerulein. Bombesin (90 pmollkg per h) and stimulation of pancreatic enzyme secretion by caerulein (30 pmollkg per h) were infused into bombesin and food in the dog by (a) comparing seven dogs in doses giving similar plasma the effects on pancreatic enzyme secretion of cholecystokinin peak increments as a meal similar plasma cholecystokinin concentrations (mean (SEM) 6.8 (0.8), 6-3 (1.2), and 5.7 (0.8) obtained after feeding and after the infusion of pM, respectively), together with either saline bombesin and the synthetic cholecystokinin- or 2 mg/kg per h of A lorglumide. background peptide caerulein and (b) comparing inhibition http://gut.bmj.com/ infusion of synthetic secretin 20.5 pmollkg per by the cholecystokinin-receptor antagonist CR- h was given in each experiment. In addition, 1409 (lorglumide)'9 on bombesin and food stimu- gastric acid secretion was determined in the lated pancreatic enzyme secretion to that during experiments with bombesin and caerulein infusion of caerulein. Since the dogs were infusion. Pancreatic protein responses to equipped with a gastric fistula, which was kept bombesin (1231 (247) mg/h) and food (1430 open during the studies to prevent acid entering

(220) mg/h) were similar to the responses to the duodenum, we were also able to study the on September 30, 2021 by guest. Protected copyright. caerulein (1249 (201) mg/h). Lorglumide effect of the cholecystokinin-receptor antagonist inhibited pancreatic protein output during on gastric acid during the infusions of caerulein stimulation with bombesin by 60%, after the and bombesin. meal by 45%, and during caerulein infusion by 68%. Pancreatic bicarbonate output by bombesin, caerulein, and food was inhibited Methods by lorglumide by 28%, 40%, and 38%, respec- Seven mongrel female dogs, each weighing 17- tively. In contrast, lorglumide significantly 32 kg, were fitted with a chronic duodenal fistula Department of increased acid Gastroenterology- gastric secretion from 1.12 to as described by Thomas and a gastric fistula Hepatology, University 7-98 mmol/h during bombesin infusion and using a Thomas-type cannula, modified so that of Leiden, The from 0.52 to 7.62 mmol/h during caerulein the inner flange was circular rather than oval.'0" Netherlands infusion. In conclusion, cholecystokinin plays The duodenal Thomas cannula was placed A J L de Jong an J B M JJansen important part in the stimulation of pan- opposite the main pancreatic duct, while the C B H W Lamers creatic enzyme secretion by bombesin and a accessory pancreatic duct was ligated. Studies meal in conscious dogs and it is involved in the Division of were started four weeks after surgery. Food but Gastroenterology, regulation ofgastric acid during stimulation by not water was withheld for 18 hours before each Department of Medicine, infusions of caerulein and bombesin. test. In each dog a glass cannula connected to a University ofEssen, FRG M V Singer piece of polyethylene tubing was inserted into W Niebel the main pancreatic duct and pancreatic juice Bombesin is a neuropeptide present in the was collected in ice-cold graduated conical tubes. Rotta Research Laboratories, Monza, nerves of the gastrointestinal tract including the The duodenal fistula was then closed by putting Italy duodenum and pancreas of several species.' gauze dipped in soft paraffin around the glass L C Rovati Bombesin-like peptides potently stimulate pan- cannula to avoid leakage of duodenal contents. Correspondence to: creatic enzyme secretion.2-' How bombesin acts After the gastric cannula was opened and the Professor Dr C B H W Lamers, University Hospital on the pancreas has not been fully elucidated. On stomach rinsed with distilled water a polyethy- Leiden, Building 1, C4-PO15, the one hand, receptors for bombesin have been lene tube with a diameter of 10 mm was inserted PO Box 9600, 2300 RC Leiden, The Netherlands. identified on acinar cells ofmost species' 5; on the into the stomach and the fistula was closed in a Accepted for publication other hand, bombesin is known to stimulate the similar way to the duodenal fistula. 30 April 1990 secretion ofseveral gastrointestinal polypeptides, Six experiments, separated by at least one 216 dej7ong, Singer,Jansen, Niebel, Rovati, Lamers

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. U . - s r r -60 -30 0 30 60 90 120 -60 -30 0 30 60 90 120 -60 -30 0 30 60 90 120 Time (min) Figure 1: Plasma cholecystokinin (CCK) concentrations (mean (SEM), 7 dogs) duringinfusion ofcaerulein (left), intragastric administration ofa meal (middle), and bombesin infusion (right) together with an infusion oflorglumide or saline.

week, were performed at random on each of the sulphated tyrosyl region.'2 4 Pancreatic and seven dogs: intragastric administration of a gastric juice were collected continuously and standard test meal and infusions of bombesin separated into 15 minute samples. Volume was and caerulein together with either saline or measured to the nearest 0 1 ml. Concentrations lorglumide (CR-1409; Rotta Research Labora- of protein and bicarbonate were measured as tories, Monza, Italy) were given. All studies were described previously,'5 while gastric acid con- done with an intravenous of 20.5 pmol/kg per h centrations were determined by titration with synthetic secretin (Hoechst AB, Frankfurt, 0-1 M NaOH to pH 7.0. FRG) dissolved in 0-1% dog albumin solution, to Results are expressed as mean (SEM). In the guarantee sufficiently large volumes ofpancreatic three experiments in each dog in which either juice. In preliminary studies the doses of bom- saline or lorglumide was infused before the http://gut.bmj.com/ besin (UCB, Brussels, Belgium) and caerulein -stimulants, the mean of the three results for each (Farmitalia, Milan, Italy) were determined to dog was used for further analysis. Statistical give plasma cholecystokinin concentrations analysis was done by Student's t test for paired similar to those after the meal. The meal, results. consisting of 100 g of liver extract (Murnil) containing 74-3 g protein and 17-3 g fat homo- genised in 400 ml of water, was instilled through Results on September 30, 2021 by guest. Protected copyright. the gastric fistula within 10 minutes to guarantee In the 21 experiments in the seven dogs in which reproducible responses. either saline or lorglumide was infused with the Plasma samples for measurement of chole- secretin infusion alone, no significant differences cystokinin were obtained at 15 minute intervals. were found in pancreatic protein secretion (201 Plasma cholecystokinin was measured by a (54) v 154 (45) mg/h) and bicarbonate secretion specific and sensitive radioimmunoassay as des- (2-3 (0-5) v 2-6 (0-8) mmol/h). cribed previously.2 3 Antibody T204 was used, Plasma cholecystokinin peak increments were which showed equal binding to carboxy-terminal similar in response to the three stimulants: 6-8 cholecystokinin peptides containing the (0-8) pM during bombesin infusion, 5-7 (0-8)

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-60 -30 6 30 60 90 120 -60 -30 6 30 60 90 120 -60 -3 0 30 6 90 120 Time (min) Figure 2: Pancreatic protein output (mean (SEM), 7 dogs) during infusion ofcaerulein (left), intragastric administration ofa meal (middle), and bombesin infusion (right) together with an infusion oflorglumide or saline. Effectofthe cholecystokinin-receptor antagonist lorglumide onpancreatic enzyme secretion stimulated by bombesin, food, andcaerulein 217

Caerulein Bombesin Gut: first published as 10.1136/gut.32.2.215 on 1 February 1991. Downloaded from

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0 -60 -30 0 30 60 90 120 -60 -30 0 30 60 90 12( Time (min) Figure 3: Gastric acid output (mean (SEM), 7 dogs) during infusion ofcaerulein (left) or bombesin (right) together with an infusion oflorglumide or saline.

pM after the standard test meal, and 6.3 (1 2) pM significant), afterthe testmeal to 7.3 (1 7) mmol/h during caerulein infusion (Fig 1). (p

Infusion of bombesin increased pancreatic (0.9) mmol/h. The relative inhibition of pan- http://gut.bmj.com/ protein output from 149 (39) to 1231 (247) mg/h creatic bicarbonate output by lorglumide was not (p<0005); the standard test meal increased significantly different during administration of pancreatic protein secretion from 221 (62) to the three stimuli: 28% during infusion of bom- 1430(220) mg/h (p<0O01), and caerulein infusion besin, 38% after the test meal, and 40% during increased pancreatic protein secretion from 220 caerulein infusion. (62) to 1249 (201) mg/h (p

atic protein output during bombesin infusion during caerulein infusion from 0.52 to 7.62 on September 30, 2021 by guest. Protected copyright. and after the test meal was not significantly mmol/h (p<0-05) and during bombesin admini- different from that during infusion of caerulein stration from 1-12 to 7-98 mmol/h (p<005), (Fig 2). Pancreatic bicarbonate secretion while the increase in gastric acid output before increased from 1.4 (0.4) to 5.7 (1.2) mmol/h administration of the stimulants, from 0-08 to (p<0 01) during bombesin infusion, from 2-9 0-58 mmol/h was not significant (Fig 3). (0.5) to 11.8 (1.1) mmol/h (p

secretion by 68%, inhibited the enzyme response cholecystokinin-receptor antagonist L-364, to bombesin by 60% and to the test meal by 45%. 718.23 It is noteworthy that receptors for

Although the percentage inhibition by lorglu- bombesin have not been identified on canine Gut: first published as 10.1136/gut.32.2.215 on 1 February 1991. Downloaded from mide of the pancreatic enzyme response to the acinar cells,25 precluding extrapolation of the test meal was not significantly different from that present findings to other species. to caerulein infusion, the relatively lower The finding that all three stimuli increase inhibitory effect of lorglumide on the test meal secretin-induced pancreatic bicarbonate secre- stimulated enzyme secretion (45% v 68%) tion can be partly explained by the well estab- suggests that, apart from cholecystokinin, other lished potentiation between cholecystokinin and mediators may be involved in the pancreatic secretin on bicarbonate output from the pancreas enzyme response to the meal. Among these, the in the dog.232026 The significantly greater bicar- importance ofvagal cholinergic reflexes from the bonate response to the meal than to caerulein stomach and upper small intestine to the pancreas stimulation suggests other mechanisms, possibly has been well established.'8 21 Our results con- vagal-cholinergic reflexes originating in the trast with those ofPendleton et al, which showed stomach and small intestine.23710 21 that the non-peptide cholecystokinin-receptor The finding that the bicarbonate response to antagonist L-364,718 did not affect post- bombesin is similar to that to caerulein suggests prandial pancreatic enzyme secretion in dogs, that the potentiation between cholecystokinin suggesting that endogenous cholecystokinin and secretin can fully account for this stimulation does not have a physiological role in regulating of pancreatic bicarbonate output during bom- postprandial pancreatic enzyme secretion.22 The besin. The inhibition of pancreatic bicarbonate reason for the discrepancy between that study output by lorglumide was much smaller than and ours is not apparent. The results ofour study that of enzyme secretion for all three stimuli. In are more in line with those of Konturek et al. 17 fact, only inhibition of test meal stimulated They recently showed that the pancreatic protein pancreatic bicarbonate secretion was significant, output after an intragastric meal was inhibited by whereas the inhibition of bicarbonate secretion 70% in the first hour after administration of during the infusions of bombesin and caerulein slightly lower doses of0 5 and 1[0 ,umol/kg per h just failed to be significant. This finding suggests oflorglumide than that ofabout 4 [tmol/kg per h that cholecystokinin interacts with vagal- used in the present study.'7 Interestingly, in the cholinergic mechanisms activated by intragastric study of Konturek et al the higher dose did not meal stimulation. Since the percentage inhibition provoke a more profound inhibition of meal- by lorglumide of meal stimulated bicarbonate stimulated pancreatic protein output. 17 The latter secretion (38%) was similar to that found during finding may indicate that in their studies all stimulation with caerulein (40%), the possibility cholecystokinin-receptors on the pancreas were that cholecystokinin mainly interacts with http://gut.bmj.com/ blocked by the doses of the cholecystokinin- secretin in the regulation of meal stimulated receptor antagonist administered. Dose- pancreatic bicarbonate secretion cannot be response studies with various doses of chole- excluded with certainty from the present study. cystokinin-8 and lorglumide, however, showed In the study of Konturek et al the inhibition by that the doses oflorglumide used in the test meal lorglumide of test meal stimulated bicarbonate study did not completely abolish the stimulation secretion failed to be significant in the first hour, of pancreatic protein secretion by cholecysto- but was significant in the second and third hour on September 30, 2021 by guest. Protected copyright. kinin.'7 Unfortunately, the study of Konturek et oftheir study." alt7 cannot be compared with ours in more detail An interesting finding is that gastric acid out- since no plasma cholecystokinin concentrations put during infusion of caerulein and bombesin were measured and therefore no attempt could was increased by administration of lorglumide, be made to compare the effect ofthe meal with an suggesting that cholecystokinin acts as an appropriate dose of cholecystokinin. Further- inhibitor of gastric acid secretion during stimu- more, the study design was different from ours, lation by these polypeptides. Recently, receptors since increasing doses oflorglumide were infused for cholecystokinin have been identified on after the start of the cholecystokinin infusion, somatostatin producing D-cells in the mucosa of while in our study infusion of lorglumide was the gastric body.27 It is likely that lorglumide started one hour before the test meal stimulation. inhibits the stimulation of the D-cells by chole- The finding that two recent studies using dif- cystokinin, resulting in a reduced inhibition of ferent designs have shown that the structurally acid secretion by somatostatin in the gastric unrelated cholecystokinin-receptor antagonist body. Hildebrand et al have shown that another L-364,718 also inhibited test meal stimulated structurally related cholecystokinin-receptor pancreatic enzyme secretion further supports antagonist, CR-1505, stimulated gastric acid the part that cholecystokinin plays in the regu- secretion during infusion of cholecystokinin-8, lation of postprandial pancreatic enzyme but not during infusion ofpentagastrin.28 secretion.2324 The present study suggests an important role Since the inhibitory effect of lorglumide on for cholecystokinin in the stimulation of pan- bombesin-stimulated pancreatic protein creatic enzyme secretion by bombesin and a test secretion (60%) was closely similar to that on the meal in the dog. Furthermore, endogenously enzyme secretion stimulated by caerulein, it is released cholecystokinin seems to function as an unlikely that, apart from cholecystokinin, other inhibitor of gastric acid secretion during stimu- factors contribute to the regulation ofbombesin- lation by bombesin and caerulein. stimulated pancreatic enzyme secretion. This finding is in agreement with the study of This study was supported by grant 13-37-43 from the Netherlands Foundation for Medical Research MEDIGON and grant Si Konturek et al using the non-peptide 228/7-1 from the Deutsche Forschungsgemeinschaft. Effectofthe cholecystokinin-receptor antagonist lorglumide onpancreatic enzyme secreron stimulated by bombesin, food, andcaerulein 219

We are indebted to Mrs D Levin, Mrs G Albertz, Ms K Garlipp, cholecystokinin determined by two techniques in dogs. and Mr I J Kuipers for technical assistance and to Louise Niepoth ScandJ Gastroenterol 1978; 13: 969-74. for secretarial help. 16 Hildebrand P, Beglinger C, Kohler E, Senitkar I, Gyr K. Biological effects ofa proglumide derivata as cholecystokinin antagonist in conscious dogs. Regul Pept 1987; 18: 213-20. Gut: first published as 10.1136/gut.32.2.215 on 1 February 1991. Downloaded from 1 Yanaihara N, Yanaihara C, Mochizuki T, Iwahara K, Fujita 17 Konturek SJ, Tasler J, Cieszkowski M, Szewczyk K, Hladij T, Iwanga T. Immunoreactive GRP. Peptides 1981; 2: 185- M. Effect of cholecystokinin receptor antagonist on pan- 91. creatic responses to exogenous gastrin and cholecystokinin 2 Solomon TE. Regulation of pancreatic secretion. Clin Gastro- and to meal stimuli. Gastroenterology 1988; 94: 1014-23. enterol 1984; 13: 657-78. 18 Singer MV, Solomon TE, Grossman MI. Effect ofatropine on 3 Solomon TE. Control of exocrine pancreatic secretion. In: secretion from intact and transplanted pancreas in the dog. Johnson LR, ed. Physiology ofthe gastrointestinal tract. 2nd AmJ Physiol 1980; 238: G18-22. ed. New York: Raven Press, 1987: 1172-207. 19 Singer MV, Solomon TE, Wood J, Grossman MI. Latency of 4 De Jong AJL, Singer MV, Lamers CBHW. Effect of pan- pancreatic enzyme response to intraduodenal stimulants. creatic polypeptide-antiserum on bombesin-stimulated pan- AmJ Physiol 1980; 238: G23-9. creatic exocrine secretion in dogs. Peptides 1987; 8: 973-6. 20 Singer MV, Niebel W, Gotthold S, Hoffmeister D, Goebell H. 5 Gardner JD, Jensen RT. Characterization of receptors for Extrinsic nerves as mediators of the pancreatic secretory gastrointestinal peptides. Scand J Gastroenterol 1983: 18 response to intravenous caerulein and intraduodenal (suppl 82): 7-18. tryptophan. Gastroenterology 1987; 92: 1642. 6 De Jong AJL, Klamer M, Jansen JBMJ, Lamers CBHW. 21 Niebel W, Beglinger C, Singer MV. Pancreatic bicarbonate Effect ofatropine and somatostatin on bombesin-stimulated response to HCL before and after cutting the extrinsic plasma gastrin, cholecystokinin and pancreatic polypeptide nerves of the pancreas in dogs. Am J Physiol 1988; 254: in man. Regul Pept 1987; 17: 285-93. G436-43. 7 Singer MV. Pancreatic secretory response to intestinal stimu- 22 Pendleton RG, Bendesky RJ, Schaffer L, Nolan TE, Gould lants. ScandJ7 Gastroenterol 1987; 22 (suppl 139): 1-13. RJ, Clineschmidt BV. Roles ofendogenous cholecystokinin 8 Setnikar I, Bani M, Cereda R, et al. Pharmacological charac- in biliary, pancreatic and gastric function: studies with L- terisation of a new potent and specific nonpolypeptide 364,718, a specific cholecystokinin receptor antagonist. cholecystokinin antagonist. DrugRes 1987; 37: 703-7. J PharmacolExp Ther 1987; 241: 110-6. 9 Makovec F, Bani M, Cereda R, et al. Pharmacological 23 Konturek SJ, Tasler J, Konturek JW, et al. Effects of non- properties of lorglumide as a member of a new class of peptidal CCK receptor antagonist (L-364718) on pancreatic cholecystokinin antagonists. DrugRes 1987; 37: 1265-8. responses to cholecystokinin, gastrin, bombesin, and meat 10 Thomas JE. An improved cannula for gastric and intestinal feeding in dogs. Gut 1989; 30: 110-7. fistulas. Proc Soc Exp Biol (NY) 1941; 46: 260-4. 24 Hosotani R, Chowdhury P, Rayford PL. L-364,718, a new 11 De Jong AJL, Singer MV, Lamers CBHW. Effect of rabbit CCK antagonist, inhibits postprandial pancreatic secretion anti-pancreatic polypeptide serum on postprandial pan- and PP release in dogs. DigDis Sci 1989; 34: 462-7. creatic exocrine secretion in dogs. Gastroenterology 1986; 90: 25 Bommelaer G, Rozental G, Bernier C, et al. Action of 1926-31. secretagoques on amylase release from the dog pancreatic 12 Jansen JBMJ, Lamers CBHW. Radioimmunoassay of chole- acini. Digestion 1981; 21: 248-54. cystokinin in human tissue and plasma. Clin Chim Acta 26 Beglinger C, Grossman MI, Solomon TE. Interaction between 1983; 131: 305-16. stimulants of exocrine pancreatic secretion in dogs. Am J 13 JBMJ Jansen, Lamers CBHW. Molecular forms of chole- Physiol 1984; 246: G173-9. cystokinin in plasma from normal and gastrectomized 27 Soll AH, Amirian DA, Park J, Elashoff JD, Yamada T. human subjects following a fat meal. Peptides 1987; 8: 801- Cholecystokinin potently releases somatostatin from canine 6. fundic mucosal cells in short-term culture. Am J Physiol 14 Beglinger C, Fried M, Whitehouse J, Jansen JB, Lamers CB, 1985; 248: G569-73. Gyr K. Pancreatic enzyme response to a liquid meal and to 28 Hildebrand P, Beglinger C, Kohler E, Setnikar I, Gyr K. hormonal stimulation; correlation with plasma secretin and Biological effects of a proglumide derivative as chole- cholecystokinin levels. J Clin Invest 1985; 75: 1471-6. cystokinin antagonist in conscious dogs. Regul Pept 1987; 15 Singer MV, Sarles H. Pancreatic dose-response curves to 18:213-20. http://gut.bmj.com/ on September 30, 2021 by guest. Protected copyright.