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Abstract
Objective and Methods Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO· production from endothelium has been extensively characterized, little is known about endothelium-derived O-·2. In the present study, we determined the O-·2 production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy.
Results An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O-·2, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O-·2 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 O-·2 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 O-·2 production. Finally, the amount of O-·2 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.
