Oxidants and Nitric Oxide in Post-Ischemic Heart Injury

缺血后心脏氧自由基生成增加，导致一氧化氮(NO)改变 生产和缺血后损伤。使用电子顺磁共振(EPR)和其他技术， 我们直接测量了缺血后心脏中氧自由基和NO的产生机制。 从这些和其他最近的研究中，已经表明氧化剂改变了一氧化氮的生成 合酶(NOS)。然而，氧化剂在一氧化氮合酶结构和功能调节中的确切作用 仍然不为人知。最近的研究表明，在缺血条件下氧自由基的产生 酶，如黄嘌呤氧化酶(XO)，也通过还原亚硝酸盐或硝酸盐形成NO。然而， NO形成的一氧化氮合酶非依赖性通路在脑缺血后损伤调控中的性质和作用 不清楚。因此，本项目将描述这些NO生成过程及其氧化剂 互动。研究将首先在酶的水平上进行；然后在内皮细胞上，然后是 研究离体心模型，最后研究冠状动脉闭塞和复流的活体模型。这个项目 有以下5个具体目标。1)表征‘O2~和’O2~-衍生氧化剂的机理 影响人内皮型一氧化氮合酶(ENOS)的结构和功能。2)通过以下方式确定机制 O_2“和O_2衍生的氧化剂改变缺血后心脏eNOS的结构和功能 并评估恢复eNOS功能的方法。3)研究亚硝酸盐的基本作用机制， 硝酸盐或有机硝酸盐介导NO的产生。4)表征亚硝酸盐或硝酸盐介导的作用 离体心缺血后NO生成途径及其调控机制 进程。5)体内试验和EPR/核磁共振共成像的新疗法，以抑制氧化损伤，恢复 一氧化氮合酶在心肌保护中的作用。为了实现这些目标：EPR、电化学和 将进行氧自由基、NO和NO衍生物种的化学发光测量 以及临界NO生成的功能、结构和修饰的表征 酵素。总体而言，这个项目将确定氧自由基之间的相互作用和 无一代发生在缺血后损伤的过程中，并导致最优发展 挽救处于危险中的心肌的策略。

Oxygen radical generation is increased in the postischemic heart and leads to altered nitric oxide (NO) production and postischemic injury. Using Electron Paramagnetic Resonance (EPR) and other techniques, we directly measured the mechanisms of oxygen radical and NO generation in the postischemic heart. From these and other recent studies, it has been shown that oxidants alter NO generation from nitric oxide synthase (NOS). However, the precise role of oxidants in the regulation of NOS structure and function remains unknown. Recently, it has been shown that under ischemic conditions oxygen radical generating enzymes, such as xanthine oxidase (XO), also form NO through reduction of nitrite or nitrate. However, the nature and role of NOS-independent pathways of NO formation in the modulation of postischemic injury is unclear. Therefore, this project will characterize these processes of NO formation and their oxidant interactions. Studies will be performed first at the enzyme level; then in endothelial cells, followed by studies in isolated heart models and finally in vivo models of coronary occlusion and reflow. This project has the following 5 specific aims. 1) To characterize mechanisms by which 'O2~ and 'O2~ -derived oxidants affect the structure and function of human endothelial NOS (eNOS). 2) To determine the mechanism by which -O2" and 'O2 -derived oxidants alter eNOS structure and function in the isolated postischemic heart and evaluate approaches to restore eNOS function. 3) To characterize fundamental mechanisms of nitrite, nitrate or organic nitrate mediated NO generation. 4) To characterize the role of nitrite or nitrate mediated pathways of NO generation in the isolated postischemic heart and the mechanisms that regulate this process. 5) In vivo testing and EPR/NMR coimaging of novel therapeutics to inhibit oxidant injury, restore NOS function and confer myocardial protection. For these aims; EPR, electrochemical, and chemiluminescence measurements of oxygen radicals, NO, and NO derived species will be performed along with characterization of the function, structure and modification of the critical NO generating enzymes. Overall, this project will determine the interactions between oxygen radicals and the pathways of NO generation that occur in the process of postischemic injury, and lead to the development of optimal strategies to salvage heart muscle at risk.