Modulatory Role of Nitric Oxide on Superoxide-Dependent Luminol Chemiluminescence

Abstract

Reactive oxygen species are involved in luminol chemiexcitation induced in biological systems, but the contribution of nitrogen-derived oxidants in the process still remains unclear. Herein, we report that luminol chemiluminescence (LCL) induced by a superoxide[formula]- and hydrogen peroxide (H2O2)-generating system (2–25 mU/ml xanthine oxidase plus acetaldehyde and oxygen) was markedly inhibited by nitric oxide ([formula]NO) added either as bolus (0–10 μm) or a continuous flow (0–10 μm/min). However, the inhibition of LCL was followed by an overshoot in light emission after most[formula]NO was consumed or the infusion stopped and was due to reactions of remaining peroxynitrite, the product of the reaction between[formula]and[formula]NO, with luminol. Nitric oxide also inhibited peroxynitrite- and glucose oxidase-induced LCL, but no overshoot was observed. On the other hand, a continuous flux of pure peroxynitrite, at 2 to 10 μm/min, induced LCL with quantum yields close to those obtained by identical micromolar fluxes of[formula], while peroxynitrite formed from the decomposition of the sydnonimine SIN-1 yielded 76% of the chemiluminescence obtained with authentic peroxynitrite. Peroxynitrite- induced LCL was 80 and 55% inhibitable by SOD and catalase, respectively, showing that there were[formula]and H2O2-dependent routes of chemiexcitation. The hydroxyl radical scavengers dimethyl sulfoxide, mannitol, and ethanol and the metal chelator diethylenetriaminepentaacetic acid did not inhibit peroxynitrite-induced LCL while desferrioxamine was an efficient inhibitor of light emission by reaction with an activated state of peroxynitrous acid which is responsible of performing the initial one-electron oxidation of luminol. Our results are consistent with a dual role of[formula]NO in[formula]-induced LCL: (I) formation of peroxynitrite which in turn promotes the light-emitting route and (II) reaction with luminol radical intermediates directing the system toward a dark pathway. These considerations are of critical importance when analyzing cell- and tissue-derived LCL in[formula]NO-,[formula]-, and peroxynitrite-producing systems.