The proposal is to study the interrelationships between the sites where oxygen free radicals are produced within specific lung cells and the sites where antioxidant defensive enzymes are located. Alterations in the rates of production of oxygen free radicals in response to both in vivo and in vitro exposures to hyperoxia will be investigated. Induction of antioxidant enzymes and changes in antioxidant enzyme distributions within cells in response to injury caused by exposure to hyperoxia will be correlated with changes in the site specific rate of production of oxygen radicals. These studies will be focused on cells from lung airways (tracheal epithelial cells and Clara cells) and from the gas exchange parenchyma (alveolar type II epithelial cells and endothelial cells). These cells were chosen because of significant differences in function, structure and sensitivity to hyperoxic-mediated injury. The proposed studies will help define the basis for why specific cells are uniquely resistant or sensitive to hyperoxic- mediated injury. The results will provide important new information needed to design pharmacologic therapy directed at reducing the rates of toxic oxygen radical species being produced at key target sites or pharmacologic therapy designed to augment antioxidant defenses in those cells where these defenses are inadequate against the enhanced stress produced by hyperoxic exposure. The primary techniques to be used include electron microscopic immunocytochemical localization of antioxidant enzymes using cryoultrathin sections and protein-A gold immunolabeling. Cell isolation techniques and culture will be used to prepare isolated cell fractions for biochemical analysis of sites and relative rates of production of oxygen radicals. The analysis techniques will include both electron spin resonance spectroscopy and chemical trapping techniques which can be followed spectrophotometrically.
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