Understanding pancreatic secretion is important both as it relates to the function this digestive organ and in understanding basic cellular mechanisms. Secretagogues such as acetylcholine (ACh) and cholecystokinin (CCK) act on the exocrine pancreas to increase cytoplasmic Ca2+ and release digestive enzymes. The model so far developed and being studied is that receptor activation leads to an increase in the intracellular messengers Ca2+ and diacylgycerol, which in turn activate a battery of kinases and phosphatases which mediate the secretory response. Studies of receptors, intracellular messengers, protein phosphorylation, and amylase secretion are carried out using isolated pancreatic acini. Free Ca2+ in acinar cells will be measured by microspectrofluorometery using the probe fura-2. Kinase and phosphatase activity will be studied as chemical reactions in broken cells. Emphasis will be placed, however, on evaluation of secretagogue regulated substrate protein phosphorylation in isolated acini labeled by incubation with 32P04 followed by 1- and 2 dimensional gel electrophoresis as well as cell fractionation. Measurements of cytoplasmic Ca2+ in single cells w-ill allow determination of whether Ca2+ transients are phasic or tonic and whether individual cells differ or mirror the population. The time course of changes in Ca2+ will be studied to understand the mechanism of maintained secretion and determine if a sustained elevation of Ca2+ is involved. The participation of gap junctions in coordination of Ca2+ between cells of an acinus will be evaluated. Intracellular phosphorylation of individual proteins will be studied as to time dependence, relation to changes in cytoplasmic Ca2+, and the specific kinase involved. Other questions to be studied include: Is the rise in Ca2+ necessary for all changes in protein phosphorylation? Are granule and/or plasma membrane proteins reversibly phosphorylated in a regulated manner in situ? Are particulate proteins reversibly phosphorylated and in what organelle do they reside. Finally, substrates undergoing secretagogue regulated phosphorylation will be purified, sequenced, and used to generate antibodies to better understand their functional role. These studies are also relevant to understanding of the abnormal secretion seen in pancreatic diseases such as pancreatitis and cancer.
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