This proposal is designed to elucidate in detail signal transduction pathways activated by pancreatic secretagogues and thus has direct relevance to both the physiology and pathophysiology of the exocrine pancreas. Pancreatic secretagogues such as cholecystokinin, neuromedin C and acetylcholine initiate a signaling cascade resulting in an increase in [Ca 2+]i. This increase in [Ca2+]i is primarily the result of Ca2+ release from an intracellular storage site. The elevation in [Ca2+]i carries important temporal and spatial information, unique to each agonist, which serves as a primary signal in the control of digestive enzyme secretion from pancreatic acinar cells. This proposal will focus on understanding how ligands utilizing the same general transduction pathway achieve signal specificity. It is hypothesized that signal specificity is a result of the significant molecular diversity which exists in all elements of the transduction system, such that stimulation with a particular secretagogue results in activation of particular subset of these signaling proteins. The individual proteins responsible for transducing this signal from receptor occupation on the plasma membrane to the release of Ca2+ from the intracellular store will be defined. The individual G-protein alpha/betagamma subunits and phospholipase C beta isozymes expressed and functionally significant in pancreatic acinar cells and the pancreatic cell-line AR4-2J will be elucidated by immunoblotting and PCR. Specific interactions between alphaq family subunits and betagamma subunits will be investigated by co-immunoprecipitation and the constituent subunits compared to the as/betagamma heterotrimer. Experiments will be performed to elucidate the mechanism of phospholipase-C beta activation in acinar cells; it is hypothesized that both G protein alpha and betagamma subunits are involved. In single rat pancreatic acini, individual G-protein alpha subunits, betagamma subunits and PLC-beta isozymes will be selectively antagonized by utilizing specific antibodies and antisense oligonucleotides. The measurement of phosphoinositide hydrolysis and fluctuations in [Ca2+]i measured by fluorescence digital imaging will be utilized as a readout of the coupling of secretagogues to their respective transduction mechanisms. The association of G-subunits with the PLC-beta effector enzyme on stimulation will be investigated by a co-immunoprecipitation protocol. These techniques will determine if stimulation by a specific secretagogue results in activation of a unique subset of the signaling proteins in acinar cells leading to the specific pattern of Ca2+ signaling observed on stimulation by individual secretagogues.
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