Quantification of superoxide radical formation in intact vascular tissue using a Cypridina luciferin analog as an alternative to lucigenin.
Lucigenin has been frequently used for the chemiluminescent detection of superoxide (*O-2) in intact tissue. More recent studies, however, revealed that lucigenin per se causes formation of *O-2 raising doubt about this probe to detect reliably *O-2. We therefore tested a more recently described chemiluminescence probe (2-methyl-6-phenyl-3,7-dihydroimidazol[1,2-alpha]pyrazine-3-one (CLA)) to estimate the ability of vascular tissue to generate *O-2 as an alternative to lucigenin. In a cell free system as well as in vascular tissue, CLA-enhanced chemiluminescence was dose dependently inhibited by superoxide dismutase (SOD), vitamin C and sodium nitroprusside (SNP). Electron spin resonance studies revealed that lucigenin (250 microM) but not CLA (1 microM) caused extra *O-2 production in vascular tissue. Stimulation of vessels with NADH (200 microM) increased CLA enhanced chemiluminescence, which was inhibited by low concentrations of superoxide dismutase (20U/ml). Endothelial removal as well as the nitric oxidase-synthase inhibitor increased CLA chemiluminescence in vessels. We conclude that CLA is a sensitive and specific chemiluminescence probe to detect *O-2 production in intact vascular tissue.
Division of Cardiology, University Hospital Eppendorf, Hamburg, 20246, Germany.
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Skatchkov
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Sperling
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Hink
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Anggard
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Münzel
D000166:Acridines / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D002457:Cell Extracts / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008163:Luminescent Measurements; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011719:Pyrazines / Q000378:metabolism*; D011817:Rabbits; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000166:Acridines; D002457:Cell Extracts; D011719:Pyrazines; C048193:2-methyl-6-phenyl-3,7-dihydroimidazo(1,2-a)pyrazin-3-one; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; D019323:omega-N-Methylarginine; D013482:Superoxide Dismutase; D001205:Ascorbic Acid