HORMONAL REGULATION OF CULTURED PANCREATIC ACINAR CELLS

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We have recently developed an in vitro system of pancreatic acinar AR42J cells, a highly differentiated rat cell line. In addition, we have developed a primary monolayer culture system of mouse pancreatic acinar cells which allows studies of normal cell function over a period of several weeks. The objective of this proposal is to determine which hormones have direct regulatory effects on pancreatic acinar cell growth and differentiation, and to elucidate the mechanisms involved, by utilizing these two culture systems. We will test: hormones that regulate intracellular Ca2+, (e.g. cholecystokinin); hormones that act via cyclic AMP (e.g. secretin); hormones that act via tyrosine kinase activity (e.g. insulin); and steroid hormones. To identify hormones which regulate growth, we will measure DNA synthesis, protein and DNA content, and the nuclear labeling index. We will test the hypothesis that CCK increases the growth of pancreatic acinar cells by the same mechanisms that mediate CCK induced enzyme secretion, by evaluating the effects of other Ca2+-mediated secretagogues, and of pharmacological agents which directly increase intracellular Ca2+, or activate protein kinase C. Variant strains of AR42J cells with unique growth properties will also be developed to help elucidate the mechanism involved in CCK induced growth. To identify hormones which regulate acinar cell differentiation we will test effects on pancreatic secretory enzyme synthesis using biosynthetic labeling, immunoprecipitation, and two-dimensional gel electrophoresis. To determine the mechanisms by which these hormones affect enzyme synthesis, we will utilize in vitro translation and nucleic acid hybridization with cDNA probes to quantitate changes in the acinar cell mRNA. Nuclear run-off assays will be conducted to determine whether changes in mRNA levels represent changes in transcription, or mRNA stability. Effects on cellular morphology will be evaluated by morphometric analysis and immunocytochemistry. The longterm regulation of CCK receptors by CCK and other factors will be determined by analysis of hormone binding, internalization and synthesis.