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&z.rad;; 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 microm rather than at 100 microm 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.
Barbacanne
M A
MA
INSERM U397 et Laboratoire de Physiologie, et CJF-9107, Toulouse Cedex, France.
Souchard
J P
JP
Darblade
B
B
Iliou
J P
JP
Nepveu
F
F
Pipy
B
B
Bayard
F
F
Arnal
J F
JF
D000166:Acridines; D000818:Animals; D001011:Aorta; D016477:Artifacts; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence / Q000379:methods; D013113:Spin Labels; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*
D000166:Acridines; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D013113:Spin Labels; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; D000001:Calcimycin; C017245:5,5-dimethyl-1-pyrroline-1-oxide