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

Objective and Methods Endothelium produces oxygen-derived free radicals whi ch play a major role in Vessel wall physiology and pathology. Whereas NO. p roduction from endothelium has been extensively characterized, little is kn own 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 resonan ce (ESR) spectroscopy. Results An ESR adduct DMPO-OH detected in the supernatant of BAEC after sti mulation 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- a nd dose-dependent manner. The coenzymes NADH and NADPH both increased the E SR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmalei mide I inhibited the A23187-stimulated O-2(-.) production, suggesting the i nvolvement of protein kinase C. These signals were not altered L-NAME, a NO -synthase inhibitor, suggesting that the endogenous production of NO. did n ot alter O-2(-.) production. Finally, the amount of O-2(-.) generated by A2 3187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same con ditions.