Quantification of superoxide radical formation in intact vascular tissue using a Cypridina luciferin analog as an alternative to lucigenin

Abstract

Lucigenin has been frequently used for the chemiluminescent detection of superoxide (.O2-) in intact tissue. More recent studies, however, revealed that lucigenin per se causes formation of .O2- raising doubt about this probe to detect reliably .O2-. We therefore tested a more recently described chemiluminescence probe (2-methyl-6-phenyl-3,7-dihydroimidazol[1,2-α] pyrazine-3-one (CLA)) to estimate the ability of vascular tissue to generate .O2- 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 μM) but not CLA (1 μM) caused extra .O2- production in vascular tissue. Stimulation of vessels with NADH (200 μM) increased CLA enhanced chemiluminescence, which was inhibited by low concentrations of superoxide dismutase (20 U/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 .O2- production in intact vascular tissue.