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.
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