This proposal considers the basic hypothesis that specific isoforms of protein kinase C (PKC), in concert with Ca2+ and diacylglycerol (DAG), play key roles in the regulation of amylase release by isolated rat parotid cells. Having already identified PKC-alpha, -delta, and -zeta isoforms, we will characterize their subcellular sites of activation (with regard to plasma and nuclear membranes, cytoskeleton) in response to receptor agonists (carbachol, norepinephrine, substance P), Ca2+, and several phorbol esters. By taking advantage of the differences in Ca2+ sensitivity and stimulus-specific isozyme activation, we will determine whether particular PKC isozymes are linked to amylase secretion. PKC will be measured by exogenous substrate phosphorylation and isozymes determined by immunoblot (Western) analysis. Phorbol ester-induced down-regulation and selective PKC inhibitors will also be employed to analyze the functional consequences of selective inhibition of particular subtypes on evoked amylase secretion. Measurement of the RNA message using Northern analysis will identify the genes of the various PKC isoforms which are expressed in the parotid cell. To assess the functional role of DAG- induced activation of PKC, the effects of pharmacologic agents that inhibit DAG lipase and kinase on NE-, CCh-, or substance P-stimulated PKC activity (exogenous substrate phosphorylation), translocation of PKC isozymes, and amylase will be studied. Since cAMP mediates amylase release via a Ca2+-dependent process, we will also examine the effects of the beta agonist isoproterenol, forskolin, and cAMP analogs on agonist- induced translocation of PKC isoforms. These studies should fortify the concept that phosphoinositide-derived messengers (Ca2+ and diacylglycerol), together with cAMP, underlie the integrative actions of receptor agonists on amylase secretion by the parotid gland. These findings should also provide salient information concerning the role of salivary secretion in dental health.
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