The mechanisms of ethanol-induced hypertension are unknown. Abnormalities in the L-arginine:nitric oxide (NO) system have been implicated in various forms of hypertension. In rats, ethanol acutely inhibits endothelium- dependent vasorelaxation which may potentiate the vascular smooth muscle pressor effect of ethanol and contribute to cause hypertension. Chronically, ethanol enhances endothelium-dependent vasorelaxation thus inducing endothelium-dependent tolerance to its pressor effects which may protect against the development of hypertension. After very prolonged exposure to ethanol however, this tolerance appears to be lost and hypertension develops. Our preliminary studies in cultured bovine endothelial cells indicate that short (20 min) exposure to ethanol at low concentrations (25-50 mM) enhances and at high concentrations (100-300 mM) inhibits the NO synthase (NOS) response to agonists, whereas prolonged ethanol treatment (24-96 hr) markedly potentiates it. Therefore, our central hypothesis is that ethanol disrupts the endothelial L-arginine:NO pathway leading to abnormalities in endothelial function that may eventually cause hypertension. We propose two mechanisms for this: 1) ethanol disrupts the phosphoinositide (PI) signaling system which normally regulates NOS activity, and 2) ethanol alters NOS levels or its affinity to cofactors. This proposal focuses on the study of the effects of ethanol on the endothelial L-arginine:NO system and on the signal transduction processes that determine its activity. A first goal of this proposal is to characterize in detail the acute and chronic effects of ethanol on the activity of the endothelial NOS. NOS activity will be determined by measuring the conversion of L-arginine to L-citrulline in intact endothelial cells and crude homogenates, and the formation of cyclic GMP in a bioassay of reporter cells cocultured with endothelium. This goal will include comparative studies between bovine and human endothelium. A second goal is to study the effects of ethanol on the various mediators of the endothelial PI signaling system. This goal involves the investigation of phospholipase C, protein kinase C activity, PI hydrolysis, calcium mobilization and membrane phospholipids. Finally, we propose to examine the effects of ethanol on the levels of the constitutive endothelial NOS and its affinity to cofactors. For this goal, we will measure NOS protein and mRNA levels, and perform kinetic studies of NOS activity. We will use bovine and human endothelial cells exposed to ethanol in culture as our experimental model. Disruption of the endothelial L-arginine:NO pathway by ethanol is likely to cause substantial alterations in endothelium- dependent vasorelaxation and blood pressure control. Therefore, the results of these studies may have important implications for our understanding of the pathogenesis of ethanol-induced hypertension in humans.
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