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Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and alpha-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 +/- 9% (P > 0.05) and 176 +/- 13% (P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.

We examined the role of CCK in bombesin-induced pancreatic growth in rats using the CCK receptor antagonist L-364,718. Rats (155 +/- 1 g, 8-10 per group) received subcutaneous injections every 8 h for 5 days with bombesin (0.6, 1.7 and 5 nmol/kg) or bombesin in combination with L-364,718 (1 mg/kg). After 5 days the pancreas was removed and pancreatic weight, protein content, DNA, amylase and chymotrypsin contents were determined. Bombesin produced a significant increase (48-475%) of pancreatic weight, tissue contents of protein, DNA, amylase and chymotrypsinogen (F = 82, P less than 0.001). When a large dose of bombesin (5 nmol/kg) was combined with L-364,718 a significant inhibition (up to 70%) of all tissue parameters was observed (P less than 0.001). L-364,718 did not affect the growth response to a small dose of bombesin (0.6 nmol/kg). Plasma CCK levels 15 min after a single injection of bombesin (0.6, 1.7 and 5 nmol/kg) were significantly increased in response to the 5 nmol/kg dose (2.0 +/- 0.7 to 3.4 +/- 0.8 pM, F = 6.9, P less than 0.01). No increases of CCK plasma levels were found in response to the 0.6 and 1.7 nmol/kg doses of bombesin, corresponding to the lack of effects of L-364,718 on growth parameters at these doses. Measuring the time-course of CCK plasma levels after a single injection of 5 nmol/kg bombesin revealed an increase from basal values of 1.4 +/- 0.3 pM to maximal levels of 3.5 +/- 0.5 pM after 15 min (F = 7.1, P less than 0.001). Values returned to basal after 60 min. These results suggest that low doses of bombesin act directly at the acinar cell or through release of non-CCK growth factors whereas high doses of bombesin act in part through CCK release.

Recent synthesis of specific, potent bombesin receptor antagonists allows examination of the role of bombesin-like peptides in physiological processes in vivo. We characterized effects of [D-Phe6]bombesin(6-13)-methyl-ester (BME) on pancreatic enzyme secretion stimulated by the C-terminal decapeptide of gastrin releasing peptide (GRP-10), food intake, and diversion of bile-pancreatic juice in rats. In isolated pancreatic acini, BME had no agonistic effects on amylase secretion but competitively inhibited responses to GRP-10, yielding a pA2 value of 8.89 +/- 0.19. In conscious rats with gastric, jugular vein, bile-pancreatic, and duodenal cannulas, basal enzyme secretion (bile-pancreatic juice recirculated) was not affected by the antagonist. Maximal amylase response to GRP-10 (0.5 nmol/kg/h) was inhibited dose dependently by BME, reaching 97% inhibition at a dose of 400 nmol/kg/h. The dose response curve of amylase secretion stimulated by GRP-10 was shifted to the right by 40 nmol/kg/h BME, but maximal amylase response was unaltered, suggesting competitive inhibition in vivo. Liquid food intake and bile-pancreatic juice diversion caused substantial increases in amylase secretion; neither response was altered during administration of 400 pmol/kg/h BME. These results demonstrate that BME is a potent, competitive antagonist of pancreatic responses to bombesin-like peptides in vitro and in vivo. Lack of effect of BME on basal pancreatic secretion or responses to liquid food intake or diversion of bile-pancreatic juice in rats suggests that endogenous bombesin-like peptides do not act either directly or indirectly to mediate these responses.

The role of gastrin in regulating pancreatic growth and its interaction with secretin is unclear. We determined the dose-response relationships of pentagastrin for gastric and pancreatic secretion. Doses that stimulated gastric and pancreatic secretion were given every 8 h for 3 or 5 days; trophic responses of pancreas, oxyntic gland area, duodenum, and colon were compared. Interaction of secretin and pentagastrin on pancreatic growth was measured after 5 days of treatment. Pentagastrin was 250 times less potent for stimulation of pancreatic versus gastric secretion. A submaximal dose of pentagastrin for stimulating pancreatic enzyme secretion doubled pancreatic deoxyribonucleic acid synthesis after 3 days. Pentagastrin caused dose-related increases in pancreatic weight after 3 and 5 days. Pentagastrin had no effect on weight, deoxyribonucleic acid synthesis, or deoxyribonucleic acid content of oxyntic gland area, duodenum, or colon. Secretin had additive effects on pentagastrin-induced pancreatic growth. The pancreas appears to be more sensitive than other organs to trophic effects of pentagastrin. Secretin has additive rather than inhibitory effects on pentagastrin-induced pancreatic growth.

Plasma levels of neurotensin are increased by ingestion of fat, making this peptide a candidate for mediation of pancreatic adaptation to dietary fat. We examined the effects of doses of neurotensin on pancreatic secretion and growth to determine whether doses stimulating secretion also increased pancreatic growth and lipase content in rats. Because neurotensin inhibits gastric secretion in other species, we also measured its effects on gastric secretion and growth. In conscious rats, neurotensin (33, 100, and 300 micrograms kg-1 subcutaneously in gelatin) produced dose-related increases in pancreatic amylase output and decreases in basal gastric secretion. Duration of acid inhibition by neurotensin was longer than stimulation of amylase secretion. Chronic administration of the same doses of neurotensin to groups of rats every 8 h for 5 days produced small but statistically significant trophic effects on the pancreas. The highest dose of neurotensin significantly increased pancreatic weight (16%) and content of DNA (12%), protein (17%), and chymotrypsinogen (60%) but did not affect amylase or lipase content. There were no effects of neurotensin on any measurement of oxyntic or pyloric gland area growth. We conclude that although neurotensin stimulates both pancreatic secretion and growth, it is not the mediator of fat-induced pancreatic adaptation.