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Chromogranin A and pancreastatin in serum of gastrinoma patients
258 PURIFICATION OF SECRETIN-RELEASING PEPTIDES FROM RAT SMALL INTESTINAL MUCOSA. Sztefko K, Chang TM and Chey WY. Division of Gastroenterology and Hepatology, University of Rochester Medical Center, and Isaac Gordon Center for Digestive Diseases and Nutrition. Rochester, NY 14607, U.S.A. Bioactivity of a secretin-releasing peptide tSRP) is present in duodenal acid perfusate of the rat (J. Clin. Invest. 86, 1474, 1990). We have purified t w o SRPs from the mucosa of rat upper small intestine. Segments of rat small intestine (40 cm from the pylorus) were excised and boiled in H20 for 10 min. The mucosa was homogenized and stirred in 0.5 N acetic acid at 4°C overnight and centrifuged to obtain a crude extract. The bioactivity of [he crude extract or fractions from the subsequent purification steps were determined by stimulation of exocrine pancreatic secretion of volume and bicarbonate (from 50% to > 150% over basal), and elevation of secretinqike immunoreactivity (from < 2 pM basal to > 4 pM) after intraduodenal infusion of the materials in anesthetized and pylorus ligated rats. The crude extract was filtered through an Amicon PM10 membrane and the filtrate was lyophilized. It was redissolved in H20 and mixed with methanol (80% V/V) at 4 ° C. After centrifugation, the supernate was lyophilized and chromatographed on a Sephadex G-25 SF column followed by chromatography on a column of SP-Sephadex C-25. The bioactive material eluted from the SP-Sephadex column at the end of a linear gradient of 50-800 mM NH4 + acetate was lyophilized, redissolved in .01% TFA and extracted on a C18 Sep-Pak cartridge. The extract was chromatographed by HPLC on a semipreparative C18 column to yield 2 bioactive peaks. They were purified to homogeneity by chromatography and rechromatography on an Asahipak ODP analytical column. Both purified materials also stimulated dose-dependently the release of secretin from a rat duodenal secretin cell-enriched cell preparation. Amino acid analysis of one of these peaks indicated that it was a glycine-rich peptide with a minimum M.W. of 4,100 Da. The amino acid sequence of these peptides will be determined.
ACTIONS OF CHOLECYSTOKININ ON MYENTERIC NEURONS IN T H E GASTRIC ANTRUM OF THE GUINEA-PIG. J.F. Tack, J. Janssens and G. Vantrappen. Centre for G.I. research K.U. Leuven, Belgium. It has been shown that cholecystokinin (CCK) stimulates antral contractile activity in the guineapig stomach in vitro by acting on intrinsic cholinergic neurons. However, the actions of CCK on antral neurones have not been studied directly. We used conventional intracellular recording techniques to study the actions of CCK-octapeptide on 33 myenteric neurons in the guinea-pig gastric antrum. CCK was applied either by pressure micro-ejection from micro-pipettes, or by addition to the superfusion solution. Thirteen out of 33 neurons did not respond to CCK. All remaining cells responded to CCK in one or more of the following ways: 1) In 7 neurons, application of CCK caused a brief depolarization (11.5 + 4.4 mV; 11.3 + 3.9 sec). 2) In 5 neurons, CCK caused a prolonged hyperpolarizing response (9.3 + 4.7 mV; 93.3 + 28.5 sec). 3) In 12 neurons, application of CCK caused a prolonged depolarization (5.1 + 0.6 mV; 125.9 + 9.3 sec). The brief depolarization was associated with a decreased input resistance (IR), whereas the prolonged responses were associated with an increased IR. CCK responses were seen in all subtypes of antral neurons and there was no apparent correlation between cell type and type of CCK response. The effects of CCK still occurred in the presence of 0.2 /~M tetrodotoxin, indicating a direct action on the impaled neurons. Dose-related responses occurred during application in the supeffusion solution at concentrations of 0.1 nM to 0.1 ~M. Conclusion: CCK has multiple actions on antral myenteric neurons. These findings directly confirm the neural component in the influence of CCK on antral contractility. They are also supportive of a potential role for CCK as a neurotransmitter in the gastric antrum. (Supported by NFWO Belgium)
ACTIONS OF CHOLECYSTOKININ ON MYENTERIC NEURONS IN T H E GASTRIC ANTRUM OF THE GUINEA-PIG. J.F. Tack, J. Janssens and G. Vantrappen. Centre for G.I. research K.U. Leuven, Belgium. It has been shown that cholecystokinin (CCK) stimulates antral contractile activity in the guineapig stomach in vitro by acting on intrinsic cholinergic neurons. However, the actions of CCK on antral neurones have not been studied directly. We used conventional intracellular recording techniques to study the actions of CCK-octapeptide on 33 myenteric neurons in the guinea-pig gastric antrum. CCK was applied either by pressure micro-ejection from micro-pipettes, or by addition to the superfusion solution. Thirteen out of 33 neurons did not respond to CCK. All remaining cells responded to CCK in one or more of the following ways: 1) In 7 neurons, application of CCK caused a brief depolarization (11.5 + 4.4 mV; 11.3 + 3.9 sec). 2) In 5 neurons, CCK caused a prolonged hyperpolarizing response (9.3 + 4.7 mV; 93.3 + 28.5 sec). 3) In 12 neurons, application of CCK caused a prolonged depolarization (5.1 + 0.6 mV; 125.9 + 9.3 sec). The brief depolarization was associated with a decreased input resistance (IR), whereas the prolonged responses were associated with an increased IR. CCK responses were seen in all subtypes of antral neurons and there was no apparent correlation between cell type and type of CCK response. The effects of CCK still occurred in the presence of 0.2 /~M tetrodotoxin, indicating a direct action on the impaled neurons. Dose-related responses occurred during application in the supeffusion solution at concentrations of 0.1 nM to 0.1 ~M. Conclusion: CCK has multiple actions on antral myenteric neurons. These findings directly confirm the neural component in the influence of CCK on antral contractility. They are also supportive of a potential role for CCK as a neurotransmitter in the gastric antrum. (Supported by NFWO Belgium)