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A time- and dose-dependent effect of exogenous hydrogen peroxide was determined on myocardial function, structure, high energy phosphate and lipid peroxidation in the isolated perfused rat heart. Hydrogen peroxide induced a dose-dependent decrease in cardiac function whereas 200 microM hydrogen peroxide reduced +dP/dt to 50% of control value after 10 mins. The effect of 300 microM hydrogen peroxide was more severe after 15 mins; changes observed with this dose were reversible within 10 mins of perfusion, becoming irreversible after 15 mins. Lipid peroxidation and severe morphological damage were observed after 10 mins of perfusion with 300 microM hydrogen peroxide. When 16 mEq potassium ions were added in the perfusion buffer during hydrogen peroxide perfusion, the degree of tissue damage and loss of ATP were attenuated. However, lipid peroxidation was not inhibited by high potassium ions. When 0.25 microM N,N'-diphenyl-1,4-phenylenediamine, a potent antioxidant, was added to the perfusate, lipid peroxidation was totally inhibited and the degree of tissue damage was decreased. However, depletion of tissue ATP and functional deterioration were not influenced. These results suggest that hydrogen peroxide-mediated ATP loss was independent of lipid peroxidation.

Monoclonal antibodies (MAb's) to xanthine oxidase (XO) (from bovine milk) were produced by hybridoma technique. Culture supernatants were initially screened using enzyme-linked immunoabsorbant assay (ELISA). Forty four positive clones were subcloned and further characterized by ELISA and immunoblot analysis. Out of fifteen clones, which were positive in immunoblot analysis, one clone N2-26 was also positive in immunocytochemical studies. Indirect immunoperoxidase and enzyme histochemistry staining showed that XO activity is present in endothelial cells of capillaries, small blood vessels and also in interstitial cells. Electron microscopy revealed that diaminobenzidine reaction product was distributed in the cytoplasm of interstitial cells and endothelial cells of capillaries and small blood vessels. This is the first report of the presence of XO in interstitial cells and endothelial cells of small blood vessels. Allopurinol, which inhibits the xanthine oxidase activity, did not have any effect on the immunocytochemical staining. Our results in normal rat heart suggest that XO activity is confined to interstitial cells, endothelial cells of capillaries and small blood vessels.

Effects of ischemia on cell membrane of rat heart were investigated. The endothelial surface revealed the existence of ruthenium red-positive glycocalyx at the anionic site. Membrane bound enzyme as Na-K ATPase was mostly located in the inner side and pinocytotic vesicles of endothelial cell. The clumping and dispersion in glycocalyx of endothelial cells was observed in an ischemic heart and it may prove the functional disturbance of plasma membrane. A potential and functional defect with reduced activity of Na-K ATPase occurred within 1 hr of vascular ligation. The membrane dysfunction due to these molecular changes has been proved by the membrane permeability alteration as well as the intracytoplasmic localization of horseradish peroxidase as tracer.