期刊
FREE RADICAL BIOLOGY AND MEDICINE
卷 29, 期 5, 页码 388-396出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/S0891-5849(00)00336-1
关键词
superoxide anion; lucigenin; chemiluminescence; cytochrome c; electron spin resonance; endothelial cell; free radicals
Endothelium produces oxygen-derived free radicals (nitric oxide, NO.; superoxide anion, O-2(.-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O-2(.-) production, particularly due to the difficulty in assessing O-2(.-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O-2(.-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O-2(.-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O-2(.-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O-2(.-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 mu m rather than at 100 mu m appears more suitable to detect O-2(.-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O-2(.-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O-2(.-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed. (C) 2000 Elsevier Science Inc.
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