title: 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 (O2.-)- and hydrogen peroxide (H2O2)-generating system (2-25 mU/ml xanthine oxidase plus acetaldehyde and oxygen) was markedly inhibited by nitric oxide (.NO) added either as bolus (0-10 microM) or a continuous flow (0-10 microM/min). However, the inhibition of LCL was followed by an overshoot in light emission after most .NO was consumed or the infusion stopped and was due to reactions of remaining peroxynitrite, the product of the reaction between O2.- and .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 microM/min, induced LCL with quantum yields close to those obtained by identical micromolar fluxes of O2.-, 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 O2.- 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 .NO in O2.(-)-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 .NO-, O2.(-)-, and peroxynitrite-producing systems.
authors.affiliation: Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
authors.forename: L
authors.initials: L
authors.lastname: Castro
authors.forename: M N
authors.initials: MN
authors.lastname: Alvarez
authors.forename: R
authors.initials: R
authors.lastname: Radi
mesh_terms: D000166:Acridines / Q000737:chemistry; D005949:Glucose Oxidase / Q000378:metabolism; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D008165:Luminol / Q000737:chemistry*; D008956:Models, Chemical; D009566:Nitrates / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000737:chemistry; D013481:Superoxides / Q000737:chemistry*; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D000166:Acridines; D009566:Nitrates; D017382:Reactive Oxygen Species; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; C039642:peroxynitric acid; D009569:Nitric Oxide; D008165:Luminol; D005949:Glucose Oxidase; D014969:Xanthine Oxidase