Human coronary and peripheral arteries show endothelial dysfunction in a variety of conditions, including atherosclerosis, hypercholesterolemia, smoking, and hypertension. This dysfunction manifests as a loss of endothelium-dependent vasodilatiation to acetylcholine infusion or sheer-stress, and is typically associated with decreased generation of nitric oxide by the endothelium. Vitamin C, or ascorbic acid, when acutely infused or chronically ingested, improves endothelium-dependent vasodilatation present in these cllinical conditions. The mechanism by which ascorbic acid improves endothelial function is unknown, although it has been attributed to an antioxidant effect of the vitamin to enhance the synthesis or prevent the breakdown of nitric oxide. Accordingly, the overall goal of this proposal is to define the mechanism(s) by which ascorbic acid can affect nitric oxide synthesis and action at the cellular level. These studies will be carried out in primary culture of endothelial and arterial smooth muscle cells, so that intracellular substrates, co-factors, and oxidant stress can be manipulated at different intracellular ascorbic acid concentrations. Studies in the first aim establish how ascorbic acid is taken up, recycled, and exported by endothelial cells. Normal and optimal intracellular ascorbate concentrations will be determined, and these will be related to the ability of endothelial cells to recycle and move the vitamin out of the blood stream. In the second aim, the role of ascorbic acid in preventing endogenous and exogenous oxidant stress in endothelial cells will be assessed, and this will be related to effects on endothelial cell nitric oxide synthase activity. The ability of ascorbic acid to scavenge superoxide, recycle membrane alpha-tocopherol, and to protect against endothelial cell damage by oxidized LDL will be considered in light of how the vitamin preserves nitric oxide synthesis or action. Studies in the third aim will define mechanism(s) by which ascorbate enhances endothelial cell nitric oxide synthase and guanylate cyclase activities. Major areas for study will be the ability of ascorbic acid to 1) diminish feedback inhibition and inhibitory S-nitrosation of nitric oxide synthase, 2) enhance the function of tetrahydrobiopterin to activate nitric oxide synthase, and to 3) sensitize guanylate cyclase to nitric oxide.
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