ENDOGENOUS XANTHINE-OXIDASE DOES NOT SIGNIFICANTLY CONTRIBUTE TO VASCULAR ENDOTHELIAL PRODUCTION OF REACTIVE OXYGEN SPECIES

The contribution of xanthine oxidoreductase (XDH + XO) to the extracel lular release of hydrogen peroxide (H2O2) and intracellular H2O2 conce ntration in cultured bovine aortic endothelial cells (BAEC) was determ ined. Intracellular H2O2 concentration was measured by the aminotriazo le-mediated inactivation of catalase, while extracellular H2O2 release was measured by the horseradish peroxidase-mediated oxidation of p-hy droxyphenyl acetic acid to a fluorescent dimer. Supplementation of rea ction systems with xanthine did not increase H2O2 production by cells. Inhibition of XO activity with allopurinol did not decrease either in tracellular concentrations or the extracellular release of H2O2. Simil arly, inactivation of XO by culture of cells with tungsten did not hav e any effect on intracellular levels of H2O2, while it increased extra cellular release of H2O2 by 86 and 103% from cells cultured in Medium 199 (M199) and Dulbecco's modified Eagle's medium (DMEM), respectively . Cells cultured in DMEM had an average of 8 times greater XDH + XO sp ecific activity, compared to M199 cultured cells, and had a threefold greater rate of release of H2O2 than M199-grown cells. However, DMEM-c ultured cells did not have a greater rate of myxothiazole-resistant re spiration, suggesting that this increase in H2O2 release comes from so urces other than XO. These results show that cellular XO does not cont ribute significantly to basal H2O2 production in bovine endothelial ce lls. Analysis of XDH + XO activity of endothelial cells derived from v essels of various species showed a relatively low specific activity of this potential oxidant source in human-derived cells compared with ce lls cultured from other species such as rodents. (C) 1994 Academic Pre ss, Inc.