The goal of this proposal is to determine the biochemical mechanisms responsible for lung reperfusion injury. HYPOTHESES: 1. Xanthine oxidase (XO) is critically important in initiating lung reperfusion injury and changes in its gene expression, processing and translation of mRNA, and stability of the final protein product are important determinants of susceptibility to reperfusion injury. 2. Propagation of lung reperfusion injury is fostered by disturbances in intracellular energy metabolism created during ischemia and persisting through the initial stages of reperfusion. 3. Nitric oxide (NO.) plays a central role in ameliorating lung reperfusion injury. RESEARCH PLAN: The project will investigate the molecular, biochemical and physiologic basis for lung reperfusion injury.
The first aim will be to assess the molecular and cellular regulation of the xanthine: acceptor oxidoreductase system (XDH/XO) and to establish the relationship between alterations in enzyme expression and susceptibility to reperfusion injury. A hierarchy of methods ranging from in vitro to in situ to in vivo will be employed to determine the importance of cytokines and 02 tension as modulators of transcriptional regulation, protein modification and enzyme activity of XDH/XO, and susceptibility to reperfusion injury.
The second aim will be to characterize changes in intracellular energy metabolism during ischemia and reperfusion. A comprehensive assessment will characterize changes in adenine nucleotides and their catabolites during ischemia and reperfusion.
The third aim will determine the role played by NO. in lung reperfusion injury. Our preliminary experiments indicate that L-arginine which enhances NO. production, but not Nw-nitro-L-arginine, a competitive antagonist which blocks NO. production, prevents lung reperfusion injury. Studies will examine the mechanism of this protective effect focusing in particular on the ability of NO. to activate guanylate cyclase, to inhibit Fe-S enzymes and to prevent hydroxyl radical (HO.) formation catalyzed by heme bound iron by occupying the catalytic iron coordination site. Studies will also determine the importance of cytokines and 02 tension as modulators of transcriptional regulation and enzyme activity of NO synthase. SIGNIFICANCE: The proposed research will provide a better understanding of lung reperfusion injury so that methods of preventing or interrupting the process can be designed.
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