The purpose of this application is to investigate biochemical mechanisms of anoxic cellular injury as a model of acute tubular necrosis or myocardial infarction in the broader clinical realm. Specific interests are directed to a preparation of anoxic isolated renal tubular epithelial and vascular endothelial cells as relates to: 1) generation of oxygen free-radicals, which are exceedingly injurious to cell membranes and intracellular organelles; 2) effect of disposal of free radical with enzymes (superoxide dismutase and catalase) or scavengers (mannitol); and 3) effect of blockade of anoxic-induced degradation of ATP to hypoxanthine, a substrate for a free radical generating system. Resultant alterations in membrane phospholipid composition will be detected by high pressure liquid chromatography and by generation of polyunsaturated fatty acid conjugated dienes. Concomitant alterations in membrane permeability with cell swelling and changes in parameters of cell viability (Trypan blue exclusion, ATP levels, protein synthesis) will also be evaluated.
