Early in atherosclerosis endothelium-dependent relaxation is impaired. The underlying endothelial dysfunction is thought to involve inadequate bioavailability of nitric oxide (NO). Mechanisms that underlie this effect remain uncertain but may include reaction of NO with free radicals and depletion of substrate. Since NO plays a crucial role in atherogenesis, it is important to determine if NO bioavailability can be enhanced. Recent clinical studies suggest that vitamin C (ascorbate) can reverse endothelial dysfunction by enhancing endogenous NO-mediated vasorelaxation. However, the molecular mechanisms underlying this effect are not clearly understood. The current concepts are that ascorbate acts as an antioxidant by either sparing intracellular thiols or by scavenging superoxide radicals produced during an enhanced oxidative stress. Preliminary data presented in this application suggest an alternative hypothesis that encompasses both aforementioned concepts. We propose a unique role for ascorbate in the vasculature based on the finding that, in addition to eliciting vasorelaxation, part of the NO produced from endothelial NO-synthase (eNOS) is stored in the tissue in the form of stable NO adducts. While the existence of such tissue stores of NO has been recognized earlier, its potential physiological and clinical implications have not yet been examined. We have observed that ascorbate relaxes vascular aortic rings in vitro by redox-activating tissue stores to release NO. Our preliminary data suggest that NO is bound in endothelial and smooth muscle cells in the form of S-nitrosothiols, which are cleaved by an increase in intracellular reduced glutathione. The intriguing and novel hypothesis built on these observations is that vitamin C reverses endothelial dysfunction by allowing the release of NO from a preformed vascular pooi. Using a combined biochemical/functional approach, the following three specific aims are proposed to address the above hypothesis: 1. To investigate the mechanism of ascorbate induced vasorelaxation and its relationship to the cellular redox status; 2. To identify the chemical nature and localization of NO stores in the vasculature and determine the factors that govern their stability and bioactivation to yield NO; and 3. To investigate the role of ascorbate in the maintenance of vascular homeostasis using different animal models of endothelial dysfunction. Results from these investigations are expected to significantly enhance our understanding of the role of NOS dependent and -independent NO production in endothelial dysfunction. Moreover, they should provide new insight into the actions of ascorbate beyond those of mere antioxidant nature and a rationale for vitamin C supplementation in disease states associated with an enhanced oxidative stress such as hypertension, hypercholesterolemia, and diabetes.
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