The objective of this proposal is to study the mechanisms of secretion and action of secretin (S) and cholecystokinin (CCK). Four major hypotheses will be tested: l) The releases of S and CCK are mediated by lumenally active S- and CCK-releasing peptides (S-RP and CCK-RP) in the upper small intestine and by a pancreatic S-RP found in canine pancreatic juice; 2) The releases and actions of S and CCK are neurally mediated; 3) Secretin- containing endocrine cells in gastric mucosa are controlled by neuropeptides and participate in the regulatory mechanism of gastric acid secretion; 4) The release of CCK from pancreatic islets is neurally regulated and participates in regulation of pancreatic exocrine secretion. The existence of S-RP and/or CCK-RP will be proved by purification and structure determination of these peptides from rat intestinal extract or canine pancreatic juice. Synthetic peptide corresponding to these peptides will be synthesized to confirm their bioactivity and to raise polyclonal antibodies for establishing specific radioimmunoassays and histochemical localization of these peptides in the gastrointestinal tract. The physiological actions of S-RP and CCK-RP will be studied in various animal models. The involvement of neuronal factors in the releases and actions of S and CCK will be tested by studying the effects of chemical ablation of the sensory afferent neurons, immunoneutralization of various candidate neuropeptides, antagonists of neurotrasmitters or neuropeptides, and determination of specific receptors for S or CCK in the synaptosomal membranes of the submucous, myenteric and vagal nerves. Existence of mRNA of S in gastric mucosa will be shown by in situ hybridization with a specific cDNA probe and by reverse transcription-polymerase chain reaction. The function of gastric S will be tested in isolated and perfused rat stomach by studying the effects of a specific anti-S serum on secretion of gastric acid, gastrin, somatostatin and prostaglandins and by demonstrating release of S from the mucosal explants of the antrum and fundus. The existence of CCK in pancreatic islet will be confirmed by in situ hybridization with a CCK-specific cDNA probe and by studying CCK release from isolated islets. The function(s) of islet CCK will be tested by studying the effect of the specific CCK receptor antagonist, loxiglumide, on exocrine secretion from isolated and perfused rat pancreas or isolated islets. The results of these studies may lead to better understanding of the release and action mechanisms of S and CCK, of the involvement of S in defense mechanism of gastric mucosa, and the physiological and pathophysiological mechanism of pancreatic exocrine secretion, thereby leading to better treatment of pancreatic insufficiency.
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