RELEASE OF HYDROGEN-PEROXIDE IN RESPONSE TO HYPOXIA-REOXYGENATION - ROLE OF AN NAD(P)H OXIDASE-LIKE ENZYME IN ENDOTHELIAL-CELL PLASMA-MEMBRANE

The dynamics and mechanisms of extracellular release of hydrogen perox ide (H2O2) from bovine pulmonary artery endothelial cells (EC) subject ed to anoxia, hypoxia, and hypoxia followed by reoxygenation were exam ined using various inhibitors of enzymatic systems in intact cells and by direct measurement of H2O2 production from isolated EC plasma memb ranes. Extracellular H2O2 was measured with a fluorometric assay. EC e xposed to hypoxia (3% O-2) and anoxia (0% O-2) released less H2O2 (29. 6 +/- 1.3% and 4.2 +/- 0.7%, respectively) compared with EC exposed to normoxia (20% O-2). The extracellular release of H2O2 from EC previou sly exposed to hypoxia for 24 h increased immediately after reoxygenat ion (20% O-2) to 272 +/- 48%, as compared with EC exposed continuously to normoxia (100% release), Inhibition of xanthine oxidase (XO) by al lopurinol did not reduce the release of H2O2 from cells exposed to nor moxia or hypoxia followed by reoxygenation. Furthermore, inhibitors of cyclooxygenase (indomethacin), phospholipase A(2) (quinacrine and chl orpromazine), nitric oxide synthase (L-arginine analogs), the mitochon drial electron transport chain (rotenone and cyanide), and cytochrome P-450 (methoxypsoralen) had no or minimal effect on this release. On t he other hand, inhibitors of protein kinase C (calphostin and staurosp orine) and NADPH oxidase (diphenyliodonium) reduced the release of H2O 2 from EC in a dose-dependent manner in both exposure groups. In separ ate experiments, plasma membranes isolated from EC were found to produ ce H2O2 in the presence of NADH or NADPH as electron donors, This was inhibited by diphenyliodonium but not by allopurinol. These results su ggest that the baseline production of H2O2 by EC and the increase in H 2O2 released from EC in response to hypoxia followed by reoxygenation is not due to XO activity, but rather to a membrane-bound enzyme simil ar to NADPH oxidase. This effect may be mediated through protein kinas e C activity.