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 oxidesynthase (NOS). However, the precise role of oxidants in the regulation of NOS structure and functionremains unknown. Recently, it has been shown that under ischemic conditions oxygen radical generatingenzymes, such as xanthine oxidase (XO), also form NO through reduction of nitrite or nitrate. However, thenature and role of NOS-independent pathways of NO formation in the modulation of postischemic injury isunclear. Therefore, this project will characterize these processes of NO formation and their oxidantinteractions. Studies will be performed first at the enzyme level; then in endothelial cells, followed bystudies in isolated heart models and finally in vivo models of coronary occlusion and reflow. This projecthas the following 5 specific aims. 1) To characterize mechanisms by which 'O2~ and 'O2~ -derived oxidantsaffect the structure and function of human endothelial NOS (eNOS). 2) To determine the mechanism bywhich -O2' and 'O2 -derived oxidants alter eNOS structure and function in the isolated postischemic heartand 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 mediatedpathways of NO generation in the isolated postischemic heart and the mechanisms that regulate thisprocess. 5) In vivo testing and EPR/NMR coimaging of novel therapeutics to inhibit oxidant injury, restoreNOS function and confer myocardial protection. For these aims; EPR, electrochemical, andchemiluminescence measurements of oxygen radicals, NO, and NO derived species will be performedalong with characterization of the function, structure and modification of the critical NO generatingenzymes. Overall, this project will determine the interactions between oxygen radicals and the pathways ofNO generation that occur in the process of postischemic injury, and lead to the development of optimalstrategies to salvage heart muscle at risk.
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