Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells

Abstract

An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O2-*, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O2-* production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O2-* production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO* did not alter O2-* production. Finally, the amount of O2-* generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.