Faculty Bibliography

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.

The purpose of this investigation was to examine the metabolism and inactivation of gastrin releasing peptide 10 (GRP10) by endopeptidase-24.11 prepared from the stomach wall. GRP10 was metabolized in vitro by gastric endopeptidase-24.11. The metabolites were purified by high-pressure liquid chromatography and identified as (1-8) GRP10 and (9-10) GRP10 by amino acid analysis, indicating hydrolysis of the His8-Leu9 bond. The intravenous administration of GRP10 to conscious dogs stimulated gastrin release, gastric acid secretion, pancreatic protein secretion and pancreatic bicarbonate secretion. Incubation of GRP10 with endopeptidase-24.11 significantly diminished the biological activity of the digests compared to control digests containing heat-inactivated enzyme. This effect was abolished by the enzyme inhibitor phosphoramidon. It is concluded that endopeptidase-24.11 from the stomach metabolizes and inactivates GRP10.

The coexistence of calcitonin gene-related peptide (CGRP) and somatostatin (SS) within the stomach and pancreas and the potent inhibitory effects of both peptides on exocrine secretions from these organs suggest that they are functionally related. To assess the potential role of SS in the mediation of CGRP action, the effects of intravenous human CGRP (64, 132, and 264 pmol.kg-1.h-1) and somatostatin-14 (SS-14; 100, 400, and 800 pmol.kg-1.h-1) on plasma levels of SS immunoreactivity (SS-IR) and on pentagastrin-stimulated gastric and pancreatic secretion were compared in conscious dogs. CGRP caused significant inhibition of gastric acid (85-102%), pancreatic protein (63-86%), and pancreatic bicarbonate (74-89%) outputs and a simultaneous dose-related rise (40-102 fmol/ml) in plasma SS-IR. Cessation of CGRP infusion resulted in prompt return of plasma SS-IR to basal levels and an increase in gastric and pancreatic secretion. Although CGRP is a potent releasor of SS into the circulation, its inhibitory action on gastric acid secretion cannot be explained solely by a rise in plasma SS-IR. In the pancreas, in contrast to the stomach, inhibition appears to be more closely related to a rise in circulating level of SS-IR.

The purpose of this investigation is to examine the metabolism and inactivation of human and porcine gastrin 17 (nonsulfated) (G-17) and cholecystokinin octapeptide (sulfated) (CCK-8) by gastric endopeptidase 24.11. Endopeptidase 24.11 was isolated by immunoaffinity chromatography using a monoclonal antibody to the kidney enzyme. Peptides were incubated with endopeptidase 24.11. The digests were either fractionated by reverse-phase high-pressure liquid chromatography and the products identified by amino acid analysis or they were used for bioassays. Digests of human gastrin were assayed for stimulation of acid secretion in the anesthetized rat, and cholecystokinin digests were assayed for the stimulation of amylase secretion from isolated rat pancreatic acini. Human G-17 was degraded by cleavage of the Trp4-Leu5,Ala11-Tyr12,Gly13-Trp14,Trp14 -Met15, and Asp16-Phe17-NH2 bonds, and the fragments (1-16), (1-13), (1-11), (1-4), (5-11), (5-13), (12-13), (12-14), (14-16), and (17-NH2) were identified. Porcine G-17 was degraded by hydrolysis of the Ala11-Tyr12,Gly13-Trp14, and Asp16-Phe17-NH2 bonds producing (1-16), (1-13), (1-11), (12-13), (14-16), and (17-NH2) fragments. CCK-8 was degraded by hydrolysis of the Gly4-Trp5 and Asp7-Phe8-NH2 bonds, and the fragments (1-7), (1-4), (5-7), (5-8), and (8-NH2) were identified. There was a progressive decline in the biological activity with incubation time.