The major aims of the proposed research is to more fully define the mechanisms by which CA2+ regulates cell function during such processes as the hormonal and ionic regulations of aldosterone secretion, from adrenal glomerulosa cells. Cell proliferation of cultured 3T3 cells, and leukocytes function.
Specific aims i nclude an elucidation of the temporal patterns of protein phosphorylation, inositol lipid metabolism, Ca2+ metabolism, and protein kinase C function during angiotensin II and bombesin-mediated response in, respectively, glomerulosa and 3T3 cells. The role of type II CaM-dependent protein kinase in the initial phase of cellular response; the immunocytochemical localization of the C-kinase and the possible role of M-kinase in sustained cellular response; the mechanism by which information is conveyed from cell surface to cell interior via the C-kinase branch of the Ca2+ messenger system during the sustained phase of cellular response focusing particularly on changes in Na+/H+ exchange and possible protein kinase cascades; the molecular basis of memory in the C-kinase-mediated phase of cellular response; and a reexamination of the temporal pattern of events in the cAMP messenger system during ACTH-mediate aldosterone secretion from adrenal glomerulosa cells as the system in which to explore these issues. Methods will include studies of Ca2+ fluxes and measurements of intracellular free Ca2+ using both fura 2 and aequorin as indicators; studies of the turnover of inositol lipids and inositols phosphates by both chemical and radio- isotope methods; 2-D gel electrophoresis of the phosphoproteins obtained from extracts of cells; and both light and EM immunocytochemical localization of C-kinase. The problem under investigation is of broad biological interest, and has implications for a wide range of biological phenomena. A better understanding of Ca2+ messenger system function is likely to alter our views of cell growth and proliferation, peptide and steroid hormone secretion, and smooth muscle contraction.
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