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Evidence against cholecystokinin mediation of basal and bombesin-stimulated pancreatic secretion in the rat
GASTROENTEROLOGY
1988;95:151-5
Evidence Against Cholecystokinin Mediation of Basal and BombesinStimulated Pancreatic Secretion in the Rat JAMES
R. WISNER,
Jr., SUSUMU
OZAWA,
Deuartment of Medicine, Section of Gastroenterology. of Medicine, Los Angeles, California
Studies in dogs suggest that homhesin-stimulated pancreatic exocrine function is mediated via endogenous cholecystokinin. We studied (a) the shortterm effects of homhesin on pancreatic juice volume and protein output in unconscious rats and (h) whether a potent cholecystokinin-receptor antagoaffects the pancreatic exocrine renist, L-364,718, sponse to homhesin. A 4-h i.v. infusion of low-dose (0.2 nmol/kg h) or high-dose (1.0 nmol/kg h) hombesin elicited significant increases in pancreatic juice volume and protein output, which were unaltered by treatment with L-364,718 at a dose capable of fully suppressing cholecystokininoctapeptide-stimulated pancreatic juice volume and protein output. We conclude that the effects of exogenously administered bombesin on the exocrine pancreas in the rat are not mediated via release of endogenous cholecystokinin.
ombesin. a tetradecapeptide originally isolated from the skin of the European disglossid frogs Bombina bombina and Bombina variegata variegata (l-3), is a potent pancreatic secretagogue in rodents (e.g.. mice, rats, guinea pigs], dogs, and humans (3-14). Exogenously administered bombesin evokes cholecystokinin (CCK) release in the dog in vivo (3,10,12,15) and proglumide, a CCK-receptor antagonist, inhibits the stimulatory effects of bombesin on the canine pancreas in vivo (16). These latter findings have led to the suggestion that bombesin exerts its principal effects on pancreatic function in the dog via an indirect mechanism involving the release of CCK (16). Whether similar in vivo mechanisms for bombesin actions on pancreatic function are operative in other species is unclear at present. In this study in unconscious rats we examined (a] the effects of exogenously administered bombesin and cholecystokinin-octapeptide (CCK-8) on pancre-
B
and IAN G. RENNER
llniversity
of Southern California School
atic volume and protein output and (b) whether a highly potent and specific peripheral CCK-receptor antagonist, L-364,718, attenuates the stimulatory effects of bombesin on pancreatic juice flow and protein output. The in vitro and in vivo pharmacologic actions of L-364,718 have been described recently by Chang and Lotti (I 7). Materials
and Methods
Chemicals Synthetic bombesin (bombesin tetradecapeptide, lot # 607373) was purchased from CalBiochem (San Diego, Calif.) and CCK-8 (Sincalide, lot # 7L77160) was obtained from E.K. Squibb & Sons, Inc. (Princeton, N.J.). Compound L-364,718 (3S-[-]-N-[2,3-dihydro-l-methyl-2-oxo-5-phenyllH-2,4-benzodiazepine-3-yl]-lH-indole-2-carboxamide, Lot # OOODOO~) was supplied by Dr. Ben Evans of the Merck Sharp & Dohme Research Laboratories [West Point, Pa.). Bombesin and CCK-8 were solubilized immediately before use in a solution of 0.20/;, bovine serum albumin in normal saline. A working stock solution of L-364,728 (1.0 mgiml in polyethylene glycol-4OOiglycerol. 5:1 vol/vol) was prepared and stored at 4°C until used. ‘The concentration of L-364,718 for i.v. injections was prepared immediately before use by mixing the stock solution with distilled water for a final injection volume of 1.0 ml/kg body wt.
Animals
and
Surgical
Procedures
IMale Wistar rats (100-120 g body wt) purchased from Simonsen Laboratories (Gilroy, Calif.) were deprived of food but not water for 18 h before the experiments. Animals were anesthetized by an i.p. injection of urethane (750 mgikg) followed by an identical dose adminis-
Abbreviations used in this paper: CCK, cholecystokinin; CCK-8, cholecystokinin-octapeptide; PPJ, pure pancreatic juice. B 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
152 WISNERETAL.
Table
GASTROENTEROLOGYVol.
I. Total Outputs of Pure Pancreatic Juice and 4-Hour [Stimulated) Periods in Unconscious L-364,718’
Treatment
group
Controls L-364,718b 1.0mgikg i.v. bolus Bombesin 0.2 nmolikg h 0.2nmolikg. h + L-364,718 1.0nmol/kg . h 1.0nmol/kg . h + L-364,718 CCK-8 1.0nmolikg . h 1.0nmol/kg .h + L-364,718
95,No. 1
Protein During Initial, 2-Hour Basal (Unstimulated) and Rats Treated With Bombesin, Cholecystokinin-Octapeptide, PPJ volume (~1)
Final, and
PPJ protein (mg)
No. of rats
2-h basal
8
36.85 6.7
73.32 13.3
0.689t 0.076
1.332 0.34
5
29.8-r4.8
69.9+ 10.0
0.6882 0.089
1.82t 0.36
5 5 5 5
28.6k 27.8* 33.92 35.42
226 " 206 + 472 2 446 "
0.587" 0.693+ 0.725+ 0.7482
8.39? 7.99c 18.3k 20.5t
8 5
33.5t 3.6 27.2t 4.3
3.4 5.5 4.4 5.2
4-h final
2-h basal
63.3" 55.6" 40.0'." 33.3'.."
183 + 26.6" 63.0+ 14.0
0.086 0.064 0.109 0.149
0.636t 0.057 0.598" 0.067
4-h final
2.82 2.90' 0.67"," 1.83c,d
10.72 2.33" 1.36k 0.25
PPJ, pure pancreatic juice. a All animals received an initial, 2-h i.v. infusion of normal saline CCK-8, cholecystokinin-octapeptide; followed by a 4-h infusion of either 0.2% bovine serum albumin in normal saline (controls), bombesin, or CCK-8. b Where applicable, L364,718was administered as a single, l.O-mg/kg body wt i.v. bolus injection at the end of the 2-h infusion of normal saline. ’ p < 0.001 compared with the corresponding final 4-h value in control rats. d p < 0.001compared with 4-h values in rats infused with 0.2nmol of bombesin/kg . h.
tered by a subcutaneous route 10 min later (18,19). For collection of pure pancreatic juice (PPJ), the common bile-pancreatid duct was cannulated according to the procedure of Petersen and Grossman (18) as previously described by our laboratory (20). A right subcostal laparotomy was performed, the bile duct was ligated distal to the liver, and the common bile-pancreatic duct was cannulated transduodenally to a depth of 0.5 cm with PE-10 polyethylene tubing. The left external jugular vein was catheterized with PE-50 tubing and infusions of test substances were conducted for 4 h after a 2-h basal infusion of normal saline using a Sage syringe infusion pump (Model 355) at a flow rate of 0.5 ml/h. Animals were unconscious throughout the 6-h period and body temperatures were maintained at 37 t 1°C (mean +- SEM) using a heating pad and monitoring temperatures with a rectal thermometer (20).
animal) at hourly intervals for 6 h as previously described (20). The hourly PPJ volume was determined gravimetritally and samples were stored frozen at -20°C until analyzed for protein content (20). The protein content in PPJ samples was determined according to the method of Lowry et al. (21).
Statistical
Analyses
Statistical analysis of data was accomplished by Student’s two-tailed t-test (22). For more complex intercomparisons of the data, analysis of variance and Duncan’s new multiple range test were used (23). A probability value of ~0.05 between comparisons was considered to be statistically significant.
Results Experimental
Design
All rats
received an initial 2-h continuous i.v. infusion of normal saline (basal PPJ collection) and were then segregated into one control group and seven experimental groups for the remaining 4 h of the experiment. Control rats subsequently received a 4-h i.v. infusion of 0.2% bovine serum albumin in normal saline while three experimental groups received an i.v. infusion of either bombesin (0.2 or 1.0 nmolikg hr) or CCK-8 (1.0 nmolikg h). The remaining four groups were given an iv. bolus injecti6n of L-364,718 at a dose of 1.0 mgikg immediately after the 2-h basal experimental period and were then infused for 4 h with either 0.2% bovine serum albumin in normal saline, bombesin (0.2 or 1.0 nmolikg h), or CCK-8 (1.0 nmolikg h).
Sample
Collection
Procedures
and Analyses
Pure pancreatic juice was collected under paraffin oil (in preweighed plastic cups placed 1-2 cm below the
Basal, Bombesin-Stimulated, and Cholecystokinin-Octapeptide-Stimulated Pancreatic Secretion The total outputs of PPJ and PPJ protein during the 6-h experiment are given in Table 1.The initial 2-h basal PPJ volumes and protein outputs in all groups did not differ significantly from one another. A subsequent 4-h i.v. infusion of rats with bombesin at either 0.2 or 1.0 nmol/kg h evoked significant elevations in PPJ outputs of approximately threefold and sixfold, respectively, compared with the 4-h output in control rats infused with 0.2% bovine serum albumin in normal saline (Table 1). The 4-h total PPJ protein outputs were also significantly elevated by sixfold and la-fold over control values in animals infused with the low and high doses of bombesin, respectively. The 4-h output of
lulv
1988
both PPJ and PPJ protein in rats intravenously infused with 1.0 nmol of CCK-8ikg . h was also significantly elevated over control values by -2.5-fold and eightfold, respectively.
Effectsof L-364,718 on Bombesin-Stimulated and Cholecystokinin-OctapeptideStimulated Pancreatic Secretion The 4-h outputs of PPJ and PPJ protein in rats that were given a single i.v. bolus injection of 1.0 mgikg of L-364,718 after the initial 2-h basal collection period did not differ from the corresponding control values (Table 1). The 1.0 mgikg i.v. bolus CCK-8-stimudose of L-364,718 totally suppressed lated outputs of PPJ volume and PPJ protein to control animal levels. No effects of L-364,718 were evident on these parameters in rats infused for 4 h with either low or high doses of bombesin (Table 1).
Discussion In this study a continuous 4-h i.v. infusion of unconscious rats with either a low or high dose of bombesin resulted in a marked stimulation of both PPJ volume and protein output. The pancreatic response to CCK-8 stimulation was totally abolished by treatment with the CCK-receptor antagonist L-364,718. By contrast, stimulation of pancreatic exocrine function by either low- or high-dose bombesin was unaltered by L-364,718. The 4-h output of PPJ and protein in rats not treated with either bombesin or CCK-8 was not altered by L-364,718 treatment. These findings indicate that the effects of bombesin on the exocrine pancreas in rats are not mediated by endogenous CCK. Furthermore, the ability of L-364,718 to fully inhibit the actions of exogenous CCK-8 on the pancreas in rats and of CCK in dogs (24), coupled with the findings of a lack of an effect of L-364,718 on basal PPJ flow and protein output in rats (this study) and dogs (24), suggests that endogenous CCK does not significantly contribute to basal pancreatic exocrine function. The doses of bombesin selected in this study were similar to those used by Nealon et al. in dogs (16) and by Chowdhury et al. (25) and Linari et al. (26) in rats to study the effects of bombesin on pancreatic function. Our rationale for using a single l.O-mgikg dose of L-364,718 in this study was based on prior findings that [a) a 1 .O-mgikg dose of the compound is lo-fold higher than that required to effectively block CCK-8-induced gastric emptying in mice and gallbladder contractions in guinea pigs (17) and (b) a l.O-mg/kg i.v. bolus injection of L-364,718 given 1 h before a continuous i.v. infusion of a supramaximal dose of cerulein (a potent CCK analogue) totally
BLOCKAD
OF PANCREATIC;
SECRE:TA(;OGIJES
IN RATS
153
abolished cerulein-induced acute panc,reatitis over a 6-h experimental period (unpublished observations). This latter result strongly suggests that L-364,718 has a relatively long circulating half-life in the rat and that a 1.0-mgikg dose of the compound can fully block the actions of markedly elevated CCK analogue levels on the rat pancreas in vivo. Work by Nealon et al. in dogs treated in vivo with bombesin has shown that CCK-receptor blockade with proglumide significantly attenuates bombesin (16). stimulation of pancreatic exocrine function These authors concluded that bombesin effects on the canine pancreas in vivo are primarily mediated via release of CCK, but proglumide failed to fully abolish bombesin-stimulated pancreatic protein secretion (16). These observations, coupled with the fact that proglumide is a relatively weak and nonspecific CCK-receptor antagonist (27,28), weaken the conclusion by Nealon et al. (16) that bombesin effects on the canine pancreas are principally mediated by CCK. Our finding that L-364,718 does not alter bombesin-stimulated pancreatic exocrine secretion in rats strongly suggests that the actions of bombesin on the pancreas in the rat, in contrast to dogs, is not mediated via CCK. These conclusions are strengthened by observations of Chowdhury et al. (25) that bombesin in the rat, in contrast to dogs and humans (8,9,12,29), does not evoke elevations in circulating CCK. Furthermore, direct stimulatory actions of both bombesin nonapeptide and bombesin on in vitro pancreatic protein secretion have been demonstrated in rodents as well as humans (4-7.14). Additionally, specific plasma membrane receptors for bombesin have been identified in rodents (30,31)and humans (32),and the in vitro stimulatory actions of bombesin on dispersed acini from guinea rats, guinea pigs, and mice are not suppressed by various analogues of the CCK-receptor antagonist proglumide (33). Although the mechanism for the bombesinenhanced PPJ volume observed in this study is not clear, in vivo studies in the dog by Miyata et al. (9) have shown that bombesin stimulates ,gastrin release and gastric acid production and that bombesinmediated gastrin release and gastric (acid secretion elevate circulating secretin levels. As duodenal acidification was shown to result in elevations in circulating secretin levels and enhanced pancreatic volume and bicarbonate release in the dog, these workers postulated that bombesin effects on the pancreas are at least partially mediated by endogenous secretin (9). Furthermore, Nealon et al. showed that bombesin markedly stimulates pancreatic bicarbonate release in the dog, and this effect was almost
154
WISNER
ET AL.
totally abolished by proglumide (16).As proglumide has both CCK- and gastrin-receptor antagonist properties (27,28),the observation that proglumide blocked bombesin-stimulated bicarbonate output in dogs could be consistent with an effect of a bombesin-stimulated endogenous secretin release secondary to gastrin-stimulated gastric acid production. In studies by us (manuscript in preparation), pyloric ligation in the unconscious, bombesin-stimulated rat failed to alter the PPJ volume, suggesting that the effect of bombesin on PPJ output in the rat is mediated by factors other than gastric acid-induced increases in endogenous secretin.
GASTROENTEROLOGY
15.
16.
17.
18.
19.
References 1. Anastasi A, Erspamer V, Bucci M. Isolation and structure of bombesin and alytesin, two analogous peptides from the skin of the European amphibians Bombina and Alytes. Experientia 1971;27:166-7. P. Bombesin and bombesin-like peptides of the 2. Melchiorri amphibian skin. In: Bloom SR, ed. Gut hormones. Edinburgh: Churchill-Livingstone, 1978:869-22. 3. Erspamer V. Peptides of the amphibian skin active on the gut. II. Bombesin-like peptides: isolation, structure and basic functions. In: Glass J, ed. Gastrointestinal hormones. New York: Raven, 1980;343-61. 4. Deschodt-Lankman M, Robberecht P, DeNeef P, Lammens M, Christophe J. In vitro action of bombesin and bombesin-like peptides on amylase secretion, calcium efflux, and adenylate cyclase activity in the rat pancreas. J Clin Invest 1976;58:8918. 5. Iwatsuki M, Peter OH. In vitro action of bombesin on amylase secretion, membrane potential, and membrane resistance in rat and mouse pancreatic acinar cells: a comparison with other secretagogues. J Clin Invest 1976;58:891-8. 6. Uhlemann ER, Rottman AJ, Gardner JD. Actions of peptides from amphibian skin on amylase release from dispersed pancreatic acini. Am J Physiol 1979:236:E571-6. 7. Howard JM, Jensen RT. Bombesin-induced residual stimulation of amylase release from mouse pancreatic acini. Am J Physiol 1985;248:G196-9. 8. Konturek SJ, Droll R, Tessler J. Effect of bombesin and related peptides on the release and actions of intestinal hormones on pancreatic secretion. J Physiol 1976;257:663-72. 9. Miyata M, Rayford PL, Thompson JC. Hormonal (gastrin, secretin, cholecystokinin) and secretory effects of bombesin and duodenal acidification in dogs. Surgery 1980;87:209-15. TJ, Ghatei MA, Bloom SR, et al. A qualitative 10. McDonald comparison of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrinreleasing peptide (GRP). Regul Pept 1981;2:293-304. G, Erspamer V, Melchiorri P, Sopranza N. Gastrin 11. Bertacinni release by bombesin in the dog. Br J Pharmacol 1974;52:21925. HR, Curtis PJ, Rayford PL. Thompson JC. Effect of 12. Fender bombesin on serum gastrin and cholecystokinin in dogs. Surg Forum 1976;27:414-6. MV, Niebel W, Lamer C, et al. Effects of truncal 13. Singer vagotomy and antrectomy on bombesin-stimulated pancreatic secretion, release of gastrin, and pancreatic polypeptide in the anesthetized dog. Dig Dis Sci 1981;26:871-87. JL, et al. Studies on human 14. Susini C, Estaval A, Scemama pancreatic acini: action of secretagogues on amylase release
20.
21.
22. 23. 24.
25.
26.
27.
and celhdar cyclic AMP accumulation. Pancreas 1986;1:1249. Erspamer V, Melchiorri P. Actions of bombesin on secretions and motility of the gastrointestinal tract. In: Thompson JC, ed. Gastrointestinal hormones. Austin, Texas: Ilniversity of Texas Press, 1974;575-89. Nealon WH, Beauchamp D, Townsend CM Jr, Thompson JC. Role of cholecystokinin in canine pancreatic exocrine response to bombesin stimulation. Am J Surg 1987;153:96-101. Chang RSL, Lotti VJ. Biochemical and pharmacological characteristics of an extremely potent and selective nonpeptide cholecystokinin receptor antagonist. Proc Nat1 Acad Sci USA 1986;83:4923-6. Petersen H, Grossman MI. Pancreatic exocrine secretion in anesthetized and conscious rats. Am J Physiol 1977;233: E530-3. Renner IG, Wisner JR Jr, Rinderknecht H. The protective effects of exogenous secretin on ceruletide-induced acute pancreatitis in the rat. J Clin Invest 1988;72:1981-92. Renner IG, Wisner JR Jr, Lavigne BC. Partial restoration of pancreatic function by exogenous secretin in rats with ceruletide-induced acute pancreatitis. Dig Dis Sci 1986;31: 305-13. Lowry OH. Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75, Adler HL, Roessler EB. Introduction to probability and statistics. San Francisco: WH Freeman, 1968. Steele RGD, Torie GH. Principles and procedures of statistics. New York: McGraw-Hill, 1967. Pendleton RG. Bendesky RJ, Schaffer L, Nolan TE. Gould RJ, Clineschmidt BV. Roles of endogenous cholecystokinin in biliary. pancreatic and gastric function: studies with L364,718. a specific cholecystokinin receptor antagonist. ] Pharmacol Exper Ther 1987;241:110-6. Chowdhury P, Ami M, Rayford PL. Effect of cadmium and bombesin on exocrine pancreatic secretions and plasma levels of gastrin and cholecystokinin (CCK) in rats. Ann Clin Lab Sci 1986;16:479-87. Linari G, Linari MB, Lutoslawska G. The action of bombesin and litorin on pancreatic secretion in the rat. Rendic Gastroenter01 1977;9:179-84. Hahne WF, Jensen RT, Lemp GF, Gardner JD, Proglumide and benzotript: receptor
28
members
of a different
antagonists.
Proc Nat1 Acad
408. Loewe CJ, Grider JR, Vlahcevic gastrin/CCK receptor antagonist atic enzyme
29
Vol. 95, No. 1
secretion
class
of cholecystokinin
Sci USA 1981;78:6304-
ZR. Effect of proglumide, a on gastric acid and pancre-
in vivo (abstr).
Gastroenterology
1983;
84:1232. Ghatei MA, Jung RT, Stevenson JC, et al. Bombesin: action on gut hormones and calcium in man. J Clin Endocrinol Metab
1982;54:980-5. 30. Jensen RT, Moody T, Pert C, Rivier JE, Gardner JD. Interaction of bombesin and litorin with specific membrane receptors on pancreatic
acinar
cells.
Proc
Natl
Acad
Sci USA
1978;75:
6139-43. 31. Jensen RT, Gardner JD. Identification and characterization receptors for secretagogues on pancreatic acinar cells. Proc 1981;40:2487-96. 32. Scemama JL, Zahidi A, Fourmy D. et al. interaction [l251]-Tyr”-bombesin with specific receptors on normal man pancreatic membranes. Regul Pept 1986;13:125-32.
of Fed of hu-
33. Jensen RT, Zhou Z-C. Murphy RB, et al. Structural features of various proglumide-related cholecystokinin receptor antagonists. Am J Physiol 1986:251:G839-46.
luly 1988
Received Address Ilnivtxsity 2025
Zonal
BLOCKADE
August 31, 1987. Arxepted February 8, 1988. requests for reprints to: Dr. James R. Wisner, Jr., of Southern California School of Medicine, HMR 902. Avenue, Los Angeles, California YUO33.
OF PANCREATIC
S~~CRI:TA~;OGIIE:S
IN RATS
This work was supported in part by National Institutes Health grant DHHS 1 ROl AlM34322-03. The authors thank Kuth McLaughlin and Daniel Green expert technical assistance.
155
of for
1988;95:151-5
Evidence Against Cholecystokinin Mediation of Basal and BombesinStimulated Pancreatic Secretion in the Rat JAMES
R. WISNER,
Jr., SUSUMU
OZAWA,
Deuartment of Medicine, Section of Gastroenterology. of Medicine, Los Angeles, California
Studies in dogs suggest that homhesin-stimulated pancreatic exocrine function is mediated via endogenous cholecystokinin. We studied (a) the shortterm effects of homhesin on pancreatic juice volume and protein output in unconscious rats and (h) whether a potent cholecystokinin-receptor antagoaffects the pancreatic exocrine renist, L-364,718, sponse to homhesin. A 4-h i.v. infusion of low-dose (0.2 nmol/kg h) or high-dose (1.0 nmol/kg h) hombesin elicited significant increases in pancreatic juice volume and protein output, which were unaltered by treatment with L-364,718 at a dose capable of fully suppressing cholecystokininoctapeptide-stimulated pancreatic juice volume and protein output. We conclude that the effects of exogenously administered bombesin on the exocrine pancreas in the rat are not mediated via release of endogenous cholecystokinin.
ombesin. a tetradecapeptide originally isolated from the skin of the European disglossid frogs Bombina bombina and Bombina variegata variegata (l-3), is a potent pancreatic secretagogue in rodents (e.g.. mice, rats, guinea pigs], dogs, and humans (3-14). Exogenously administered bombesin evokes cholecystokinin (CCK) release in the dog in vivo (3,10,12,15) and proglumide, a CCK-receptor antagonist, inhibits the stimulatory effects of bombesin on the canine pancreas in vivo (16). These latter findings have led to the suggestion that bombesin exerts its principal effects on pancreatic function in the dog via an indirect mechanism involving the release of CCK (16). Whether similar in vivo mechanisms for bombesin actions on pancreatic function are operative in other species is unclear at present. In this study in unconscious rats we examined (a] the effects of exogenously administered bombesin and cholecystokinin-octapeptide (CCK-8) on pancre-
B
and IAN G. RENNER
llniversity
of Southern California School
atic volume and protein output and (b) whether a highly potent and specific peripheral CCK-receptor antagonist, L-364,718, attenuates the stimulatory effects of bombesin on pancreatic juice flow and protein output. The in vitro and in vivo pharmacologic actions of L-364,718 have been described recently by Chang and Lotti (I 7). Materials
and Methods
Chemicals Synthetic bombesin (bombesin tetradecapeptide, lot # 607373) was purchased from CalBiochem (San Diego, Calif.) and CCK-8 (Sincalide, lot # 7L77160) was obtained from E.K. Squibb & Sons, Inc. (Princeton, N.J.). Compound L-364,718 (3S-[-]-N-[2,3-dihydro-l-methyl-2-oxo-5-phenyllH-2,4-benzodiazepine-3-yl]-lH-indole-2-carboxamide, Lot # OOODOO~) was supplied by Dr. Ben Evans of the Merck Sharp & Dohme Research Laboratories [West Point, Pa.). Bombesin and CCK-8 were solubilized immediately before use in a solution of 0.20/;, bovine serum albumin in normal saline. A working stock solution of L-364,728 (1.0 mgiml in polyethylene glycol-4OOiglycerol. 5:1 vol/vol) was prepared and stored at 4°C until used. ‘The concentration of L-364,718 for i.v. injections was prepared immediately before use by mixing the stock solution with distilled water for a final injection volume of 1.0 ml/kg body wt.
Animals
and
Surgical
Procedures
IMale Wistar rats (100-120 g body wt) purchased from Simonsen Laboratories (Gilroy, Calif.) were deprived of food but not water for 18 h before the experiments. Animals were anesthetized by an i.p. injection of urethane (750 mgikg) followed by an identical dose adminis-
Abbreviations used in this paper: CCK, cholecystokinin; CCK-8, cholecystokinin-octapeptide; PPJ, pure pancreatic juice. B 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
152 WISNERETAL.
Table
GASTROENTEROLOGYVol.
I. Total Outputs of Pure Pancreatic Juice and 4-Hour [Stimulated) Periods in Unconscious L-364,718’
Treatment
group
Controls L-364,718b 1.0mgikg i.v. bolus Bombesin 0.2 nmolikg h 0.2nmolikg. h + L-364,718 1.0nmol/kg . h 1.0nmol/kg . h + L-364,718 CCK-8 1.0nmolikg . h 1.0nmol/kg .h + L-364,718
95,No. 1
Protein During Initial, 2-Hour Basal (Unstimulated) and Rats Treated With Bombesin, Cholecystokinin-Octapeptide, PPJ volume (~1)
Final, and
PPJ protein (mg)
No. of rats
2-h basal
8
36.85 6.7
73.32 13.3
0.689t 0.076
1.332 0.34
5
29.8-r4.8
69.9+ 10.0
0.6882 0.089
1.82t 0.36
5 5 5 5
28.6k 27.8* 33.92 35.42
226 " 206 + 472 2 446 "
0.587" 0.693+ 0.725+ 0.7482
8.39? 7.99c 18.3k 20.5t
8 5
33.5t 3.6 27.2t 4.3
3.4 5.5 4.4 5.2
4-h final
2-h basal
63.3" 55.6" 40.0'." 33.3'.."
183 + 26.6" 63.0+ 14.0
0.086 0.064 0.109 0.149
0.636t 0.057 0.598" 0.067
4-h final
2.82 2.90' 0.67"," 1.83c,d
10.72 2.33" 1.36k 0.25
PPJ, pure pancreatic juice. a All animals received an initial, 2-h i.v. infusion of normal saline CCK-8, cholecystokinin-octapeptide; followed by a 4-h infusion of either 0.2% bovine serum albumin in normal saline (controls), bombesin, or CCK-8. b Where applicable, L364,718was administered as a single, l.O-mg/kg body wt i.v. bolus injection at the end of the 2-h infusion of normal saline. ’ p < 0.001 compared with the corresponding final 4-h value in control rats. d p < 0.001compared with 4-h values in rats infused with 0.2nmol of bombesin/kg . h.
tered by a subcutaneous route 10 min later (18,19). For collection of pure pancreatic juice (PPJ), the common bile-pancreatid duct was cannulated according to the procedure of Petersen and Grossman (18) as previously described by our laboratory (20). A right subcostal laparotomy was performed, the bile duct was ligated distal to the liver, and the common bile-pancreatic duct was cannulated transduodenally to a depth of 0.5 cm with PE-10 polyethylene tubing. The left external jugular vein was catheterized with PE-50 tubing and infusions of test substances were conducted for 4 h after a 2-h basal infusion of normal saline using a Sage syringe infusion pump (Model 355) at a flow rate of 0.5 ml/h. Animals were unconscious throughout the 6-h period and body temperatures were maintained at 37 t 1°C (mean +- SEM) using a heating pad and monitoring temperatures with a rectal thermometer (20).
animal) at hourly intervals for 6 h as previously described (20). The hourly PPJ volume was determined gravimetritally and samples were stored frozen at -20°C until analyzed for protein content (20). The protein content in PPJ samples was determined according to the method of Lowry et al. (21).
Statistical
Analyses
Statistical analysis of data was accomplished by Student’s two-tailed t-test (22). For more complex intercomparisons of the data, analysis of variance and Duncan’s new multiple range test were used (23). A probability value of ~0.05 between comparisons was considered to be statistically significant.
Results Experimental
Design
All rats
received an initial 2-h continuous i.v. infusion of normal saline (basal PPJ collection) and were then segregated into one control group and seven experimental groups for the remaining 4 h of the experiment. Control rats subsequently received a 4-h i.v. infusion of 0.2% bovine serum albumin in normal saline while three experimental groups received an i.v. infusion of either bombesin (0.2 or 1.0 nmolikg hr) or CCK-8 (1.0 nmolikg h). The remaining four groups were given an iv. bolus injecti6n of L-364,718 at a dose of 1.0 mgikg immediately after the 2-h basal experimental period and were then infused for 4 h with either 0.2% bovine serum albumin in normal saline, bombesin (0.2 or 1.0 nmolikg h), or CCK-8 (1.0 nmolikg h).
Sample
Collection
Procedures
and Analyses
Pure pancreatic juice was collected under paraffin oil (in preweighed plastic cups placed 1-2 cm below the
Basal, Bombesin-Stimulated, and Cholecystokinin-Octapeptide-Stimulated Pancreatic Secretion The total outputs of PPJ and PPJ protein during the 6-h experiment are given in Table 1.The initial 2-h basal PPJ volumes and protein outputs in all groups did not differ significantly from one another. A subsequent 4-h i.v. infusion of rats with bombesin at either 0.2 or 1.0 nmol/kg h evoked significant elevations in PPJ outputs of approximately threefold and sixfold, respectively, compared with the 4-h output in control rats infused with 0.2% bovine serum albumin in normal saline (Table 1). The 4-h total PPJ protein outputs were also significantly elevated by sixfold and la-fold over control values in animals infused with the low and high doses of bombesin, respectively. The 4-h output of
lulv
1988
both PPJ and PPJ protein in rats intravenously infused with 1.0 nmol of CCK-8ikg . h was also significantly elevated over control values by -2.5-fold and eightfold, respectively.
Effectsof L-364,718 on Bombesin-Stimulated and Cholecystokinin-OctapeptideStimulated Pancreatic Secretion The 4-h outputs of PPJ and PPJ protein in rats that were given a single i.v. bolus injection of 1.0 mgikg of L-364,718 after the initial 2-h basal collection period did not differ from the corresponding control values (Table 1). The 1.0 mgikg i.v. bolus CCK-8-stimudose of L-364,718 totally suppressed lated outputs of PPJ volume and PPJ protein to control animal levels. No effects of L-364,718 were evident on these parameters in rats infused for 4 h with either low or high doses of bombesin (Table 1).
Discussion In this study a continuous 4-h i.v. infusion of unconscious rats with either a low or high dose of bombesin resulted in a marked stimulation of both PPJ volume and protein output. The pancreatic response to CCK-8 stimulation was totally abolished by treatment with the CCK-receptor antagonist L-364,718. By contrast, stimulation of pancreatic exocrine function by either low- or high-dose bombesin was unaltered by L-364,718. The 4-h output of PPJ and protein in rats not treated with either bombesin or CCK-8 was not altered by L-364,718 treatment. These findings indicate that the effects of bombesin on the exocrine pancreas in rats are not mediated by endogenous CCK. Furthermore, the ability of L-364,718 to fully inhibit the actions of exogenous CCK-8 on the pancreas in rats and of CCK in dogs (24), coupled with the findings of a lack of an effect of L-364,718 on basal PPJ flow and protein output in rats (this study) and dogs (24), suggests that endogenous CCK does not significantly contribute to basal pancreatic exocrine function. The doses of bombesin selected in this study were similar to those used by Nealon et al. in dogs (16) and by Chowdhury et al. (25) and Linari et al. (26) in rats to study the effects of bombesin on pancreatic function. Our rationale for using a single l.O-mgikg dose of L-364,718 in this study was based on prior findings that [a) a 1 .O-mgikg dose of the compound is lo-fold higher than that required to effectively block CCK-8-induced gastric emptying in mice and gallbladder contractions in guinea pigs (17) and (b) a l.O-mg/kg i.v. bolus injection of L-364,718 given 1 h before a continuous i.v. infusion of a supramaximal dose of cerulein (a potent CCK analogue) totally
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abolished cerulein-induced acute panc,reatitis over a 6-h experimental period (unpublished observations). This latter result strongly suggests that L-364,718 has a relatively long circulating half-life in the rat and that a 1.0-mgikg dose of the compound can fully block the actions of markedly elevated CCK analogue levels on the rat pancreas in vivo. Work by Nealon et al. in dogs treated in vivo with bombesin has shown that CCK-receptor blockade with proglumide significantly attenuates bombesin (16). stimulation of pancreatic exocrine function These authors concluded that bombesin effects on the canine pancreas in vivo are primarily mediated via release of CCK, but proglumide failed to fully abolish bombesin-stimulated pancreatic protein secretion (16). These observations, coupled with the fact that proglumide is a relatively weak and nonspecific CCK-receptor antagonist (27,28), weaken the conclusion by Nealon et al. (16) that bombesin effects on the canine pancreas are principally mediated by CCK. Our finding that L-364,718 does not alter bombesin-stimulated pancreatic exocrine secretion in rats strongly suggests that the actions of bombesin on the pancreas in the rat, in contrast to dogs, is not mediated via CCK. These conclusions are strengthened by observations of Chowdhury et al. (25) that bombesin in the rat, in contrast to dogs and humans (8,9,12,29), does not evoke elevations in circulating CCK. Furthermore, direct stimulatory actions of both bombesin nonapeptide and bombesin on in vitro pancreatic protein secretion have been demonstrated in rodents as well as humans (4-7.14). Additionally, specific plasma membrane receptors for bombesin have been identified in rodents (30,31)and humans (32),and the in vitro stimulatory actions of bombesin on dispersed acini from guinea rats, guinea pigs, and mice are not suppressed by various analogues of the CCK-receptor antagonist proglumide (33). Although the mechanism for the bombesinenhanced PPJ volume observed in this study is not clear, in vivo studies in the dog by Miyata et al. (9) have shown that bombesin stimulates ,gastrin release and gastric acid production and that bombesinmediated gastrin release and gastric (acid secretion elevate circulating secretin levels. As duodenal acidification was shown to result in elevations in circulating secretin levels and enhanced pancreatic volume and bicarbonate release in the dog, these workers postulated that bombesin effects on the pancreas are at least partially mediated by endogenous secretin (9). Furthermore, Nealon et al. showed that bombesin markedly stimulates pancreatic bicarbonate release in the dog, and this effect was almost
154
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totally abolished by proglumide (16).As proglumide has both CCK- and gastrin-receptor antagonist properties (27,28),the observation that proglumide blocked bombesin-stimulated bicarbonate output in dogs could be consistent with an effect of a bombesin-stimulated endogenous secretin release secondary to gastrin-stimulated gastric acid production. In studies by us (manuscript in preparation), pyloric ligation in the unconscious, bombesin-stimulated rat failed to alter the PPJ volume, suggesting that the effect of bombesin on PPJ output in the rat is mediated by factors other than gastric acid-induced increases in endogenous secretin.
GASTROENTEROLOGY
15.
16.
17.
18.
19.
References 1. Anastasi A, Erspamer V, Bucci M. Isolation and structure of bombesin and alytesin, two analogous peptides from the skin of the European amphibians Bombina and Alytes. Experientia 1971;27:166-7. P. Bombesin and bombesin-like peptides of the 2. Melchiorri amphibian skin. In: Bloom SR, ed. Gut hormones. Edinburgh: Churchill-Livingstone, 1978:869-22. 3. Erspamer V. Peptides of the amphibian skin active on the gut. II. Bombesin-like peptides: isolation, structure and basic functions. In: Glass J, ed. Gastrointestinal hormones. New York: Raven, 1980;343-61. 4. Deschodt-Lankman M, Robberecht P, DeNeef P, Lammens M, Christophe J. In vitro action of bombesin and bombesin-like peptides on amylase secretion, calcium efflux, and adenylate cyclase activity in the rat pancreas. J Clin Invest 1976;58:8918. 5. Iwatsuki M, Peter OH. In vitro action of bombesin on amylase secretion, membrane potential, and membrane resistance in rat and mouse pancreatic acinar cells: a comparison with other secretagogues. J Clin Invest 1976;58:891-8. 6. Uhlemann ER, Rottman AJ, Gardner JD. Actions of peptides from amphibian skin on amylase release from dispersed pancreatic acini. Am J Physiol 1979:236:E571-6. 7. Howard JM, Jensen RT. Bombesin-induced residual stimulation of amylase release from mouse pancreatic acini. Am J Physiol 1985;248:G196-9. 8. Konturek SJ, Droll R, Tessler J. Effect of bombesin and related peptides on the release and actions of intestinal hormones on pancreatic secretion. J Physiol 1976;257:663-72. 9. Miyata M, Rayford PL, Thompson JC. Hormonal (gastrin, secretin, cholecystokinin) and secretory effects of bombesin and duodenal acidification in dogs. Surgery 1980;87:209-15. TJ, Ghatei MA, Bloom SR, et al. A qualitative 10. McDonald comparison of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrinreleasing peptide (GRP). Regul Pept 1981;2:293-304. G, Erspamer V, Melchiorri P, Sopranza N. Gastrin 11. Bertacinni release by bombesin in the dog. Br J Pharmacol 1974;52:21925. HR, Curtis PJ, Rayford PL. Thompson JC. Effect of 12. Fender bombesin on serum gastrin and cholecystokinin in dogs. Surg Forum 1976;27:414-6. MV, Niebel W, Lamer C, et al. Effects of truncal 13. Singer vagotomy and antrectomy on bombesin-stimulated pancreatic secretion, release of gastrin, and pancreatic polypeptide in the anesthetized dog. Dig Dis Sci 1981;26:871-87. JL, et al. Studies on human 14. Susini C, Estaval A, Scemama pancreatic acini: action of secretagogues on amylase release
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and celhdar cyclic AMP accumulation. Pancreas 1986;1:1249. Erspamer V, Melchiorri P. Actions of bombesin on secretions and motility of the gastrointestinal tract. In: Thompson JC, ed. Gastrointestinal hormones. Austin, Texas: Ilniversity of Texas Press, 1974;575-89. Nealon WH, Beauchamp D, Townsend CM Jr, Thompson JC. Role of cholecystokinin in canine pancreatic exocrine response to bombesin stimulation. Am J Surg 1987;153:96-101. Chang RSL, Lotti VJ. Biochemical and pharmacological characteristics of an extremely potent and selective nonpeptide cholecystokinin receptor antagonist. Proc Nat1 Acad Sci USA 1986;83:4923-6. Petersen H, Grossman MI. Pancreatic exocrine secretion in anesthetized and conscious rats. Am J Physiol 1977;233: E530-3. Renner IG, Wisner JR Jr, Rinderknecht H. The protective effects of exogenous secretin on ceruletide-induced acute pancreatitis in the rat. J Clin Invest 1988;72:1981-92. Renner IG, Wisner JR Jr, Lavigne BC. Partial restoration of pancreatic function by exogenous secretin in rats with ceruletide-induced acute pancreatitis. Dig Dis Sci 1986;31: 305-13. Lowry OH. Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75, Adler HL, Roessler EB. Introduction to probability and statistics. San Francisco: WH Freeman, 1968. Steele RGD, Torie GH. Principles and procedures of statistics. New York: McGraw-Hill, 1967. Pendleton RG. Bendesky RJ, Schaffer L, Nolan TE. Gould RJ, Clineschmidt BV. Roles of endogenous cholecystokinin in biliary. pancreatic and gastric function: studies with L364,718. a specific cholecystokinin receptor antagonist. ] Pharmacol Exper Ther 1987;241:110-6. Chowdhury P, Ami M, Rayford PL. Effect of cadmium and bombesin on exocrine pancreatic secretions and plasma levels of gastrin and cholecystokinin (CCK) in rats. Ann Clin Lab Sci 1986;16:479-87. Linari G, Linari MB, Lutoslawska G. The action of bombesin and litorin on pancreatic secretion in the rat. Rendic Gastroenter01 1977;9:179-84. Hahne WF, Jensen RT, Lemp GF, Gardner JD, Proglumide and benzotript: receptor
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members
of a different
antagonists.
Proc Nat1 Acad
408. Loewe CJ, Grider JR, Vlahcevic gastrin/CCK receptor antagonist atic enzyme
29
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secretion
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ZR. Effect of proglumide, a on gastric acid and pancre-
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84:1232. Ghatei MA, Jung RT, Stevenson JC, et al. Bombesin: action on gut hormones and calcium in man. J Clin Endocrinol Metab
1982;54:980-5. 30. Jensen RT, Moody T, Pert C, Rivier JE, Gardner JD. Interaction of bombesin and litorin with specific membrane receptors on pancreatic
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6139-43. 31. Jensen RT, Gardner JD. Identification and characterization receptors for secretagogues on pancreatic acinar cells. Proc 1981;40:2487-96. 32. Scemama JL, Zahidi A, Fourmy D. et al. interaction [l251]-Tyr”-bombesin with specific receptors on normal man pancreatic membranes. Regul Pept 1986;13:125-32.
of Fed of hu-
33. Jensen RT, Zhou Z-C. Murphy RB, et al. Structural features of various proglumide-related cholecystokinin receptor antagonists. Am J Physiol 1986:251:G839-46.
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Received Address Ilnivtxsity 2025
Zonal
BLOCKADE
August 31, 1987. Arxepted February 8, 1988. requests for reprints to: Dr. James R. Wisner, Jr., of Southern California School of Medicine, HMR 902. Avenue, Los Angeles, California YUO33.
OF PANCREATIC
S~~CRI:TA~;OGIIE:S
IN RATS
This work was supported in part by National Institutes Health grant DHHS 1 ROl AlM34322-03. The authors thank Kuth McLaughlin and Daniel Green expert technical assistance.
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