Protein kinase C (PKC) has been hypothesized to be involved in the regulation of tonic contraction in vascular smooth muscle. The data supporting this are indirect and are derived mainly from studies characterizing the contractile effects of certain phorbol esters which are presumably specific activators of PKC. In addition, studies of hormone-response coupling and PKC activation in response to Ca2+-dependent activators in other cell types support the concept that Ca2+-dependent contractile agonists could activate PKC in vascular smooth muscle. The broad goals of the proposed research are to determine if PKC activation in vascular smooth muscle occurs as a consequence of stimulation by physiologically relevant stimuli, and if so, whether PKC activation is involved in the regulation of contractile function. Specifically, the proposed studies include experiments to directly estimate PKC activation in response agonists or membrane depolarization in intact functional preparations of arterial smooth muscle. PKC activation will be assessed by: 1) measuring the translocation of activated PKC from cytosol to membranes; 2) measuring the phosphorylation of specific PKC substrates; and, 3) by measuring accumulation of the endogenous activator, diacylglycerol. The involvement of PKC in regulating contractile function will be tested by correlating PKC activity with mechanical indices of activation, by assessing the effects of putative PKC inhibitors on PKC activity and function, and by determining the mechanisms by which specific activators of PKC (phorbol esters) contract vascular smooth muscle. The role of extracellular and intracellular Ca2+ in regulating PKC activation and/or metabolism to PKM, the irreversibly activated proteolytic fragment of PKC, will be determined. Details regarding this aspect of signal transduction in normal vascular smooth muscle may ultimately be important in understanding pathology associated with vascular diseases such as hypertension or atherosclerosis.
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