Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques

Endothelium produces oxygen-derived free radicals (nitric oxide, NO.; super oxide anion, O-2(.-)) which play a major role in physiology and pathology o f 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 o f lucigenin-enhanced chemiluminescence. We compared four techniques of O-2( .-) assessment when its production is low. In the present study, we have co mpared ferricytochrome c reduction, electron spin resonance (ESR) spectrosc opy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enha nced chemiluminescence to assess O-2(.-) production in cultured bovine aort ic 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 si gnals evoked by postconfluent BAEC were increased 2- to 7-fold in compariso n 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, lucigeni n-enhanced chemiluminescence generated high amounts of O-2(.-). Thus, ferri cytochrome c reduction, electron spin resonance (ESR), and hydroethidine fl uorescence appear as adequate tools for the detection of extracellular endo thelium-derived O-2(.-) production, whereas lucigenin may be artifactual, e ven when a low concentration of lucigenin is employed. (C) 2000 Elsevier Sc ience Inc.