Redox-activation of vascular stores of NO by vitamin C

描述(申请人提供)：动脉粥样硬化早期 内皮依赖性松弛受损。潜在的内皮细胞 功能障碍被认为与一氧化氮的生物利用度不足有关 (否)。这种效应背后的机制仍不确定，但可能包括 NO与自由基的反应和底物的耗尽。因为没有发挥作用 在动脉粥样硬化形成中的关键作用，重要的是确定是否没有 生物利用度可以提高。最近的临床研究表明，维生素C 抗坏血酸可通过增强内源性血管内皮细胞功能逆转内皮功能障碍 NO介导的血管松弛。然而，这背后的分子机制 效果还不是很清楚。目前的概念是抗坏血酸的行为 作为一种抗氧化剂，通过节约细胞内的硫醇或清除 氧化应激增强时产生的超氧自由基。初步 本申请中提供的数据提出了另一种假设 包含上述两个概念。我们提出了一个独特的角色 抗坏血酸在血管系统中的基础上发现，除了 血管松弛，内皮型一氧化氮合酶产生的部分一氧化氮 (ENOS)以稳定的NO加合物的形式储存在组织中。而当 这种组织储存的NO的存在早已经被认识到，其 潜在的生理和临床意义尚未得到检验。 我们观察到抗坏血酸通过以下方式在体外松弛血管主动脉环 氧化还原激活组织储存以释放NO。我们的初步数据表明 NO以以下形式结合于内皮细胞和平滑肌细胞 S-亚硝硫醇，其中被裂解的细胞内增加减少 谷胱甘肽。建立在这些观察基础上的耐人寻味的新奇假设是 维生素C通过允许NO的释放逆转内皮功能障碍 从一个预制的血管池中。使用生化/功能组合 为了解决上述问题，我们提出了以下三个具体目标 假设：1.研究抗坏血酸诱导血管松弛的机制 及其与细胞氧化还原状态的关系；2.鉴定化学物质 NO在血管系统中的性质和定位，并决定了 控制其稳定性和生物活性以产生NO的因素；以及3.至 探讨抗坏血酸在维持血管内环境稳定中的作用 使用不同的内皮功能障碍动物模型。来自这些的结果 预计调查将大大加强我们对 一氧化氮合酶依赖性和非依赖性一氧化氮在内皮细胞中的作用 功能障碍。此外，它们应该提供对 抗坏血酸不仅具有抗氧化性质，而且是维生素C的基础 与氧化应激增强相关的疾病状态的补充剂 如高血压、高胆固醇血症和糖尿病。

DESCRIPTION (provided by applicant): 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.