Faculty Bibliography

To determine the relative contributions of neural reflexes and intestinal hormones to the inhibition of gastric acid secretion by intestinal fat, rats with an extrinsically denervated, transplanted segment of jejunum and those with an innervated segment of jejunum were studied. Postoperatively, meal-stimulated gastric acid secretion was measured by extragastric titration. When secretion reached a plateau, graded doses of oleic acid or saline were instilled into the jejunal segments. In both groups, acid secretion was inhibited by intrajejunal fat but not saline. At doses of 0.4 and 0.08 mmol oleic acid, there was a 25% and 17% greater maximal inhibition of plateau acid response in the innervated rats than in the transplanted rats, presumably because of the neural contribution. To examine the hormonal mediators, the effects of a somatostatin monoclonal antibody and a cholecystokinin A receptor antagonist (L-364,718) on fat-induced inhibition of gastric acid secretion were studied in transplanted rats. Treatment of the patients with transplants with a somatostatin monoclonal antibody (2.18 mg IV) or L-364,718 (1 mg/kg IV) reduced the fat-induced inhibition of acid secretion by 95% and 28%, respectively. In conclusion, both neural and hormonal mechanisms mediate fat-induced inhibition of gastric acid secretion, with the hormonal mechanism predominating. Somatostatin, and to a lesser extent cholecystokinin, contribute to the hormonal mechanism.

Cell surface peptidases degrade enkephalins and thereby restrict the number of molecules available to activate receptors. The effects of peptidase inhibitors on degradation of enkephalins and on enkephalin-stimulated contraction of gastric smooth muscle cells were examined. Muscle cells dispersed from the guinea pig stomach degraded [Tyr1-3H] [Leu5]enkephalin (41.6 +/- 9.0% degradation at 60 min incubation, mean +/- SD, n = 4 animals). Amastatin (10 microM, an aminopeptidase inhibitor) inhibited degradation by 72.1 +/- 1.5% The residual peptidase activity was inhibited by phosphoramidon (1 microM, an endopeptidase EC 3.4.24.11 inhibitor) by 58.0 +/- 11.0%. [Tyr1-125I] [Met5]enkephalin was similarly degraded. Phosphoramidon (1 microM) inhibited the degradation of the aminopeptidase-resistant peptide [Tyr1-3H] [D-Ala2]-[Leu5]enkephalin by greater than 95%. [Met5]enkephalin, incubated with cells for 30 s, stimulated contraction [50% maximal contraction (EC50) 120 +/- 50 nM, n = 6]. Pretreatment of cells with phosphoramidon alone, amastatin alone, or phosphoramidon plus amastatin, caused 20-fold (EC50 6.5 +/- 1.1 nM), 2-fold (EC50 63 +/- 23 nM), and 100-fold (EC50 1.1 +/- 0.3 nM) increase in potency of [Met5]enkephalin, respectively. The results show that endopeptidase EC 3.4.24.11 and aminopeptidases contribute to degradation of enkephalins by gastric muscle cells. The rapidity and magnitude of the potentiating effects of the inhibitors on enkephalin-stimulated contraction suggest a close physical relationship between the peptidases and the enkephalin receptors.

Purified mast cell carboxypeptidase cleaved the C-terminal leucines from Leu5-enkephalin (Leu-ENK), neurotensin (NT), and kinetensin (KT), with Km values of 36, 16, and 15 microM, and kcat values of 44, 51, and 53 s-1, respectively. To better predict potential in vivo hydrolysis products generated by mast cell proteases, these peptides were incubated with released skin mast cell supernatants. Leu5-enkephalin was hydrolyzed only by carboxypeptidase. Kinetensin was cleaved by tryptase, chymase, and carboxypeptidase to yield KT(1-3), KT(1-7), KT(1-8), KT(4-7), and KT(4-8), the last two peptides by the concerted action of two of the proteases. NT(1-11) and NT(1-12) were generated from neurotensin by chymase and carboxypeptidase, respectively.

Calcitonin gene-related peptide (CGRP) and substance P (SP) are released from sensory nerves upon exposure to irritating stimuli. Neutral endopeptidase (NEP), a membrane-bound peptidase, cleaves many peptides including SP, thereby limiting their biological actions. Recombinant NEP cleaved CGRP1 approximately 88-fold less rapidly than it cleaved SP. The slow cleavage by NEP of CGRP compared to SP suggests that this enzyme is likely to have weaker physiologic effects on CGRP than have been demonstrated for SP.

The mechanism by which calcitonin gene-related peptide (CGRP) inhibits exocrine secretion from the rat pancreas was examined in the isolated, vascularly perfused pancreas and in vitro using freshly isolated pancreatic acini. CGRP (10(-10)-10(-7) M) inhibited the volume and protein output from the perfused pancreas, stimulated by a mixture of the cholecystokinin octapeptide CCK8 (10(-10) M) and secretin (10(-8) M). The inhibition by CGRP was dose related and maximal at 10(-8) M (P less than 0.05). CGRP (10(-8) M) failed to inhibit amylase secretion from isolated pancreatic acini, stimulated by graded concentrations of CCK8 (10(-13)-10(-8) M). This implies an indirect mechanism of inhibition. The mechanism of inhibition was investigated in the isolated, vascularly perfused pancreas using tetrodotoxin, atropine and hexamethonium (all 10(-7) M). Tetrodotoxin and atropine but not hexamethonium prevented the inhibition of volume and protein secretion by CGRP (10(-8) M) (P less than 0.05). Tetrodotoxin, atropine and hexamethonium were without effect on exocrine secretion stimulated by CCK8 and secretin (controls). These results indicate that CGRP inhibits pancreatic exocrine secretion by an indirect, neurally mediated mechanism involving cholinergic-muscarinic transmission.

The mechanism of inhibition of pancreatic exocrine secretion by somatostatin is unknown. We hypothesized that somatostatin acts indirectly, via intrinsic pancreatic neurons, to inhibit pancreatic exocrine secretion. To test this hypothesis, amylase and volume outputs in response to secretin (10(-8) mol/L) and cholecystokinin octapeptide (CCK) (10(-8) mol/L) were studied in the rat isolated, perfused, pancreas model. Somatostatin (10(-7) mol/L) significantly inhibited amylase output by 48% compared with control (352 +/- 57 v 676 +/- 85 U/30 min, P less than .05 by ANOVA). Blockade of axonal neuronal transmission by tetrodotoxin (10(-7) mol/L) completely abolished the inhibitory effect of somatostatin (992 +/- 53 U/30 min). Similar effects were seen on volume output. The inhibitory effect of somatostatin on amylase output was not affected by cholinergic receptor blockade with atropine (328 +/- 65 U/30 min) or by sympathetic ganglionic blockade with hexamethonium (360 +/- 68 U/30 min). This suggests that the intrinsic pancreatic neurons responsible for the inhibitory effect of somatostatin are peptidergic. The possibility that somatostatin acts directly on the acinar cell to inhibit exocrine secretion was tested by incubating varying doses of somatostatin (10(-12) to 10(-7) mol/L) with isolated pancreatic acini in the presence of graded concentrations of CCK (10(-12) to 10(-10) mol/L). In this model, CCK alone is a potent stimulant of amylase release, with a Km of 6 X 10(-12) mol/L and a Vmax of 22 +/- 3% total amylase. In this model, somatostatin had no inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene-related peptide (CGRP) stimulates release of peptides derived from pro-somatostatin (Pro-S) into the general circulation. The purpose of this investigation was to elucidate the origin and molecular heterogeneity of Pro-S-derived peptides secreted in response to CGRP. Catheters were placed into the vena cava and veins draining the gastric fundus and corpus, antrum, and small intestine of anesthetized pigs. Human CGRP I was infused into the descending aorta at 0.2, 0.4, 0.8, and 1.6 micrograms.kg-1.h-1 for consecutive 30-min intervals. Blood was collected from the venous catheters after each period. S-28 was separated from Pro-S, S-14, and S-13 by immunoaffinity chromatography and peptides were quantified by radioimmunoassay. CGRP primarily evoked release of peptides measured collectively as Pro-S, S-14, and S-13 into venous blood draining the fundus and corpus, and concentrations were significantly elevated above basal at 0.8 and 1.6 micrograms.kg-1.h-1 CGRP (P less than 0.05). Basal concentrations of S-28, Pro-S, S-14, and S-13 in blood from the antrum and small intestine were not significantly elevated by CGRP. In conclusion, CGRP stimulated release of Pro-S-derived peptides from the gastric fundus and corpus but not from the antrum or small intestine.

Peptidases degrade neuropeptides and thereby limit the duration and extent of their influence. This investigation examined the importance of peptidases in the degradation of the neuropeptide enkephalin in the stomach wall of the rat. Metabolism of [Leu5]- and [D-Ala2][Leu5]enkephalin by gastric membranes was examined in vitro. Degradation of [Tyr1-3H][Leu5]enkephalin was studied in the gastric submucosa of anesthetized and conscious rats in vivo by using a catheter to deliver peptide to tissues and implanted dialysis fibers to collect the metabolites. Specific inhibitors were used to assess the contribution of particular enzymes. [Leu5]- and [Tyr1-3H][Leu5]enkephalin were metabolized by membranes and in the stomach wall by hydrolysis of the Tyr1-Gly2 bond. Degradation was inhibited by the aminopeptidase inhibitor amastatin (10(-5) M in vitro, 10 nmol in vivo). Inhibitors of endopeptidase-24.11 (phosphoramidon) and angiotensin-converting enzyme (captopril) did not inhibit degradation. Metabolism of the aminopeptidase-resistant analogue [D-Ala2][Leu5]enkephalin by membranes was unaffected by amastatin and weakly inhibited by phosphoramidon affected by amastatin and weakly inhibited by phosphoramidon and captopril. A carboxypeptidase removed the COOH-terminal leucine residue and made a substantial contribution to degradation of both peptides by gastric membranes.