SUPEROXIDE-MEDIATED REDUCTION OF THE NITROXIDE GROUP CAN PREVENT DETECTION OF NITRIC-OXIDE BY NITRONYL NITROXIDES

Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated (NO)-N-. chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without det ectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN conce ntration. Model experiments using hypoxanthine/xanthine oxidase in aqu eous solution and KO2 in dimethylsulfoxide as sources of O-2(.-) revea led that there is a rapid reduction of nitronyl nitroxides by superoxi de. The second order rate constant for the reaction of the water solub le NN with O2(.-) was determined to be 8.8 . 10(5) M(-1)s(-1), which i s more than two orders of magnitude higher than the value reported pre viously for reaction with (NO)-N-. (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the p resence of glutathione, ascorbic acid or rabbit liver microsomes. Inco rporation of both nitronyl and imino nitroxides into liposomes strongl y decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposoma l encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.