Oxygen radical s 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, NO mediated, vasoreactivity also occur, and are of critical importance in post-ischemic injury. This endothelial dysfunction is thought to be a consequence of oxygen radical derived injury. Using Electron Paramagnetic Resonance (EPR) 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. There is increasing evidence that the pathways of oxygen radical and NO generation interact, and that this interaction regulates the cellular production of these critical free radical signaling molecules. 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. Studies will be performed, first, at the enzyme level; than in endothelial cells, monocyte, and leukocytes; followed by studies in isolated heart models in the presence and absence of leukocytes. There are 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 02- and O2-derived oxidants on NO generation from NO synthase (NOS); 3) To determine the effect of NO on the pathways of oxygen radical generation and the pathogenesis of injury in the post-ischemic heart. 4) To determine the effect of O2- and O2-derived oxidants on NOS function in the post-ischemic heart. 5) To determine the effect of leukocyte derived oxidants on NOS function in the post-ischemic heart and the effect of modulating NO in leukocyte mediated injury. For each of 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 in the post-ischemic heart, and through this knowledge provide insight regarding optimal strategies to prevent post- ischemic injury.
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