Detection of superoxide anion release by endothelial cells using reduced cytochrome c UV-visible absorption, ESR spin trapping, hydroethidine fluorescence and lucigenin-enhanced chemiluminescence techniques

Reduced cytochrome c UV-visible absorption, ESR spin trapping, hydroethidin e fluorescence and lucigenin-enhanced chemiluminescence techniques, were us ed to study O-2(-) production from A23187-stimulated bovine aortic endothel ial cells (BAEC), All detected O-2(-) release but some rack of sensitivity and specificity in probes and enzymes occurred, Cytochrome c prepared with trichloroacetic acid (TCA), TCA-free or acetylated (C2506, C7752, C4186) pr ovided similar superoxide-dismutase (SOD)-inhibitable absorbances at 20 muM . At a higher concentration (100 muM) TCA-prepared cytochrome c could no lo nger detect O-2(-) production whereas TCA-free cytochrome c detected less O -2(-) at 100 muM than at 20 muM. Catalase C-40 was not contaminated with SO D-like impurities, Lucigenin provided high artefactual amounts of O-2(-) pr oduction from stimulated BAEC in the presence of NADH or NADPH. Moreover, e ven at a low concentration (5 muM) lucigenin and NADH (or NADPH) decreased endothelium-dependent relaxation induced by acetylcholine to 50 %, The use of lucigenin is not recommended.