Rapid superoxide production by endothelial cells and their injury upon reperfusion

Cultured human umbilical vein endothelial cells (EC) were exposed to hypoxia for various time periods followed by reoxygenation, and production of both O2- and H2O2 by EC was measured using the cytochrome c and scopoletin methods, respectively. Production of significant amounts of O2- was observed as early as 1 min after reoxygenation, reached maximum at 3 min and seemed to decline thereafter. Production of H2O2 was observed slightly later than that of O2-. Endothelial cell injury during hypoxia/reoxygenation was determined by both the trypan blue dye exclusion and chromium release methods. Significant increase of EC injury was detected after reoxygenation by both methods. This injury was significantly inhibited by allopurinol but not by superoxide dismutase or catalase. These results indicate that the production of reactive oxygen metabolites by EC and EC injury occur very rapidly after reoxygenation, which has not been reported, and these should be taken in consideration for the understanding and treatment of the clinical reperfusion injury.