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 d oubt 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 ab ility of vascular tissue to generate O-.(2)- as an alternative to luci genin. In a cell free system as well as in vascular tissue, CLA-enhanc ed chemiluminescence was dose dependently inhibited by superoxide dism utase (SOD), vitamin C and sodium nitroprusside (SNP). Electron spin r esonance studies revealed that lucigenin (250 mu M) but not CLA (1 mu M) caused extra O-.(2)- production in vascular tissue. Stimulation of vessels with NADH (200 mu M) 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 inh ibitor increased CLA chemiluminescence in vessels. We conclude that CL A is a sensitive and specific chemiluminescence probe to detect O-.(2) - production ire intact vascular tissue. (C) 1998 Academic Press.