■ 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

Abstract

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