Stimulation of pancreatic enzyme secretion by the hormone cholecystokinin (CCK) and neurotransmitter acetycholine (ACH) requires as an initial step the mobilization of intracellular calcium. However, it is unknown how calcium regulates pancreatic secretion and other biological effects of secretagogues. Like the well known """"""""second messenger"""""""" cyclic AMP, calcium may affect cellular function through and alteration in phosphorylation of specific regulatory proteins. Work performed in this laboratory has demonstrated that stimulation of pancreatic secretion by CCK and ACH is closely correlated with the rapid dephosphorylation of two soluble proteins. In addition, protein dephosphorylation has been implicated in the action of somatostatin in the pancreas and of insulin in other tissues. The purpose of this proposal is to demonstrate and/or characterize these dephosphorylation responses in isolated pancreatic acini by examining protein phosphatase regulation by calcium and extracellular agents. Protein phosphatase activity will be assayed in acinar cytosol and particulate preparations using as substrates 32P-labelled exogenous and endogenous phosphoproteins. Particular attention will be given to demonstrating calcium-activated phosphatase activity which acts on the aforementioned soluble proteins. Phosphatase regulation by extracellular agents will be evaluated with regard to three mechanisms: direct activation in cytosol, generation of a non-calcium regulatory substance at the plasma membrane, and stable modification of the phosphatase enzyme or modulator. Various purification techniques including gel filtration, ion exchange, and substrate affinity chromatography will allow further characterization of acinar phosphatases and their regulation. These chromatographic techniques, in conjunction with one and two-dimensional gel electrophoresis, will also be used to identify and isolate physiologically significant substrates of acinar protein phosphatases. Thus, by focusing on the enzymatic site of action of calcium and regulatory agents in the pancreas, this project intends to elucidate further the mechanism of pancreatic stimulus-secretion coupling.
