The aim of the proposed work is that of defining the role of the enzymes, protein kinase C (PKC) and protein kinase A (PKA), in the regulation of the sustained phase of angiotensin II- and K+-induced aldosterone secretion from bovine adrenal glomerulosa cells, and the role that PKC plays in the phenomenon of time-dependent potentiation (TDP) in response to angiotensin II (AII). A determination will be made of the different isoforms of PKC present in these cells using Western analysis with isoform-specific polyclonal antibodies. Once the isoforms have been determined, then the redistribution, translocation, and activation of the different isoforms in response to different agonists will be measured by, respectively, Western analysis of cell fractions with isoform-specific polyclonal antibodies, immunocytochemistry with confocal laser microscopy using isoform specific monoclonal antibodies, and the phosphorylation of a specific PKC substrate, MARCKS, or substrate peptides in cells prelabeled with [32P]-phosphate. The link between the pathways of calcium entry and the activation of PKC will be studies using measurements of Ca2+ influx, fura 2 fluorescence and changes in caveolae distribution by immunocytochemistry. The related roles of PKC and PKA in the regulation of aldosterone secretion will be studied by identifying the isoforms of adenyl cyclase in these cells and determining whether any of these isoforms are activated by PKC or Ca2+ in response to either K+ or AII. The activation of PKA will be determined using 8-azido-cAMP binding and by measuring the extent and the sustained phase of aldosterone secretion will be examined by comparing the activation of the isoenzymes with the expression of the mRNA for the steroid acute regulatory protein (StARP). The phosphorylation of the cAMP response element binding protein (CREB) will also be studied in response to several agonists to determine whether or not any of these events correlate in time and magnitude with the time course and magnitude of aldosterone secretion and TDP. The effect that the disruption of the actin architecture, by cytochalasin D, has on these events will also be examined. The applicant believes the new insight provided by this research into the mechanisms by which aldosterone secretion is regulated are likely to be relevant to the field of hypertension.
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