Oxygen radical/nitric oxide interactions post-ischemia

氧自由基和一氧化氮（NO）在细胞信号传导和炎症中起着重要作用。近十年来的研究表明，氧自由基的生成在缺血后的心脏中增加，是缺血后损伤的重要原因。内皮依赖性血管反应性的改变也会发生，并且在缺血后损伤的发病机制中起着至关重要的作用。这种内皮功能障碍被认为是氧自由基源性损伤的结果。利用电子顺磁共振（EPR）等技术，我们直接测量和表征了缺血后心脏和组成心脏的离体细胞中氧自由基和NO生成的机制。从这些和其他最近的研究中，越来越多的证据表明，氧自由基和NO的产生途径相互作用，这种相互作用调节了这些关键自由基信号分子的细胞产生。然而，这些相互作用的确切性质以及它们如何调节体内缺血后损伤的过程尚不清楚。因此，本项目的目标是表征氧自由基和NO生成途径之间的基本相互作用，并确定这如何影响局部缺血和再流的体内模型中的缺血后损伤。研究将首先在酶水平上进行；然后是内皮细胞，肌细胞和白细胞；随后进行了冠状动脉闭塞和血流的体内模型研究。该项目有以下5个具体目标。1)测定和定量NO及其衍生物对细胞主要氧自由基生成途径的影响。2)确定O2-和O2衍生的氧化剂对心脏中存在的3种一氧化氮合酶（NOS）异构体生成NO的影响；3)确定NO对氧通路的影响；4)探讨02及其衍生氧化剂对正常及缺血后心脏NOS功能的影响。5)评价预防OX和NO介导损伤的优化治疗方法。为了这些目的；EPR、电化学和化学发光测量氧自由基、NO和NO衍生物，以及表征关键氧自由基和NO生成酶的功能、表达和修饰。总的来说，本项目将确定体内缺血后损伤过程中氧自由基和NO生成的分子和细胞途径之间的相互作用，并导致制定最佳策略来预防缺血后损伤。

Oxygen radicals and nitric oxide (NO) have important roles in cellular signaling and inflammation. Studies over the last decade have demonstrated that oxygen radical generation is increased in the post- ischemic heart and is an important cause of post-ischemic injury. Alterations in endothelial dependent, vasoreactivity also occur, and are of critical important in the pathogenesis of post-ischemic injury. This endothelial dysfunction is thought to be a consequence of oxygen radical derived injury. Using Electron Paramagnetic Resonance (EPR) and other techniques, we directly measured and characterized the mechanisms of oxygen radical and NO generation in the post-ischemic heart and in the isolated cells of which the heart is comprised. From these and other recent studies, there is increasing evidence in the pathways of oxygen radical and NO generation interact, and that this interaction regulates the cellular production of these critical free radical signaling molecules. However, the exact nature of these interaction and how they modulate the process of in vivo post-ischemic injury is unknown. Therefore, the goal of this project is to characterize the fundamental interactions between the pathways of oxygen radical and NO generation, and determine how this influences post-ischemic injury in an in vivo model of regional ischemia and reflow. Studies will be performed first at the enzyme level; then in endothelial cells, myocytes, and leukocytes; followed by studies in an in vivo model of coronary occlusion and reflow. This project has the following 5 specific aims. 1) To determine and quantitate the effect of NO and NO derived species on the major cellular pathways of oxygen radical generation. 2) To determine the effect of O2- and O2-derived oxidants on NO generation from each of the 3 nitric oxide synthase (NOS) isoforms present in the heart; 3) To determine the effect of NO on the pathways of oxygen; 4) To determine the effect of 02 and 02-derived oxidants on in vivo NOS function in the normal and post-ischemic heart. 5) To evaluated optimized therapies to prevent OX- and NO mediated injury. 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, expression and modification of the critical oxygen radical and NO generating enzymes. Overall, this project will determine the interactions between the molecular and cellular pathways of oxygen radical and NO generation that occur in the process of in vivo post-ischemic injury, and lead to the development of optimal strategies to prevent post-ischemic injury.