ACTIVATION OF PROTEIN KINASE C IN VASCULAR SMOOTH MUSCLE

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Protein kinase C, a phospholipid- and calcium-dependent enzyme, is thought to be important in the signal transduction process of hormones which activate phospholipase C and release diacylglycerol. Recent studies have suggested that activation of protein kinase C may play a central role in maintaining the tonic (sustained) state of contraction seen in vascular smooth muscle. Preliminary studies have demonstrated that the vasodilator, bradykinin and the vasoconstrictor, angiotensin II, both increase intracellular calcium and diacylglycerol formation consistent with activation of phospholipase C, yet have different effects on membrane-bound protein kinase C activity in cultured vascular smooth muscle cells (cvsmc). This suggests that regulation of protein kinase C activity may be a fundamental mechanism whereby hormones modulate vascular reactivity. The goal of the present proposal is to further explore the mechanisms which account for these differences between angiotensin II and bradykinin. The most likely explanation is that these hormones act on different phospholipid pools to release diacylglycerols (or other factors) which possess different capacities to activate protein kinase C. The studies outlined in this proposal will use quantitative enzyme assays as well as radiolabelling of endogenous phospholipids to characterize the effects of angiotensin II and bradykinin on different phospholipid pools. In addition, gas chromatography, argentation thin layer chromatography, and high pressure liquid chromatography will be used to determine the molecular species of diacyglycerol released after exposure to these hormones. Finally, a hormone-sensitive plasma membrane preparation will be prepared to allow further characterization of the factors which regulate the activity of cytosolic and membrane-bound protein kinase C. The results of these studies will provide significant new information on the transmembrane signalling system for bradykinin, and the mechanism(s) by which hormones regulate protein kinase C activity in vascular smooth muscle cells. Ultimately, these studies will provide a better understanding of the cellular basis of hormone- induced vasoconstriction and vasodilitation and will provide new insights into the physiology of vascular reactivity and the pathophysiology of hypertension.