Superoxide Generation from eNOS: The Role of Pterins

描述（由申请人提供）：内皮功能障碍（艾德）是动脉粥样硬化和其他心血管疾病（CVD）病理生理学中的关键事件。艾德的根本原因尚未完全确定，尽管一氧化氮（NO）和活性氧（ROS）的不平衡产生是因果关系。NO的酶促产生依赖于内皮中的最佳基础四氢生物蝶呤（BH 4）水平。通过遗传操作或药物补充增加辅助因子的可用性已被证明是有益的，尽管对内皮中控制BH 4的机制知之甚少。这一更新的广泛目标旨在弥合知识上的差距，并基于这样的假设，即通过脂质过氧化产物和ROS改变BH 4代谢对正常NO/ROS通量和内皮生理学具有重要影响，有利于与动脉粥样硬化形成相关的表型变化。目前的建议是建立在三个发现：（i）无BH 4的内皮型一氧化氮合酶（eNOS）产生约100 nmol超氧化物/min/mg蛋白质，其被BH 4抑制（微摩尔范围）;（ii）BH 4消耗内皮细胞增加超氧化物的生产钙依赖性机制;（iii）4-羟基-2-壬烯醛（HNE）是BH 4合成的有效抑制剂，消耗BH 4和NO，并增加内皮细胞中超氧化物的产生。评估eNOS产生超氧化物增加的后果对疾病中NO和ROS氧化剂信号传导的影响至关重要。具体而言，我们将：1）建立HNE和过氧化物增加非偶联eNOS依赖性超氧化物的机制; 2）研究HNE和过氧化物消耗BH 4对增加的线粒体-超氧化物释放和功能障碍的影响; 3）阐明BH 4在限制氧化损伤、线粒体功能障碍和细胞表型变化中的作用。在执行本提案时，我们将使用已建立的内皮细胞培养物以及从GTPCH转基因小鼠中分离的细胞来检查BH 4的内源性变化在内皮反应中的影响，所述内皮反应与eNOS的保护、“再偶联”和/或额外的抗氧化活性有关。总的来说，这项建议的目的是了解这些基本机制，为开发新的和改进的预防和治疗动脉粥样硬化和心血管疾病的策略提供基础。

DESCRIPTION (provided by applicant): Endothelial dysfunction (ED) is a critical event in the pathophysiology of atherosclerosis and other cardiovascular diseases (CVD). The underlying causes of ED have not been fully established, although unbalanced production of nitric oxide (NO) and reactive oxygen production (ROS) are causally involved. Enzymatic production of NO is dependent on optimal basal tetrahydrobiopterin (BH4) levels in the endothelium. Increased availability of the cofactor by genetic manipulation or pharmacological supplementation has been shown to be beneficial, although the mechanisms controlling BH4 in the endothelium are poorly understood. The broad objectives of this renewal is designed to bridge the gap in knowledge and is based upon the hypothesis that altering BH4 metabolism by lipid peroxidation products and ROS has important consequences in normal NO/ROS fluxes and endothelial physiology favoring phenotypical changes associated with atherogenesis. The present proposal is built on three findings: (i) BH4-free endothelial nitric oxide synthase (eNOS) generates ~100 nmoles superoxide/min/mg protein which is inhibited by BH4 (micromolar range); (ii) BH4 depletion in endothelial cells increases superoxide production by calcium-dependent mechanisms; (iii) 4-hydroxy-2-nonenal (HNE) is a potent inhibitor of BH4 synthesis, depletes BH4 and NO, and increases superoxide production in endothelial cells. The evaluation of the consequences of increased superoxide production from eNOS is critical to the implications of NO and ROS oxidant signaling in disease. Specifically we will: 1) establish the mechanisms increasing uncoupled eNOS-dependent superoxide by HNE and peroxides; 2) investigate the influence of BH4 depletion by HNE and peroxides on increased mitochondrial-superoxide release and dysfunction; 3) elucidate the role of BH4 in limiting oxidative damage, mitochondrial dysfunction and changes in cell phenotype. In the execution of this proposal we will use established endothelial cell cultures and also cells isolated from GTPCH-transgenic mice to examine the influence of endogenous variation in BH4 in the endothelial responses which have been linked to protection, "re-coupling" of eNOS and/or additional antioxidant activity. Generally this proposal is aimed at understanding these fundamental mechanisms that should provide the basis for developing new and improved strategies in the prevention and treatment of atherosclerosis and CVD.