Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide

Abstract

The impact of nitric oxide (NO) synthesis on different biological cascades can rapidly change dependent on the rate of NO formation and composition of the surrounding milieu. With this perspective, we used diaminonaphthalene (DAN) and diaminofluorescein (DAF) to examine the nitrosative chemistry derived from NO and superoxide (O2-) simultaneously generated at nanomolar to low micromolar per minute rates by spermine/NO decomposition and xanthine oxidase-catalyzed oxidation of hypoxanthine, respectively. Fluorescent triazole product formation from DAN and DAF increased as the ratio of O2- to NO approached equimolar, then decreased precipitously as O2- exceeded NO. This pattern was also evident in DAF-loaded MCF-7 carcinoma cells and when stimulated macrophages were used as the NO source. Cyclic voltammetry analysis and inhibition studies by using the N2O3 scavenger azide indicated that DAN- and DAF-triazole could be derived from both oxidative nitrosylation (e.g., DAF radical + NO) and nitrosation (NO+ addition). The latter mechanism predominated with higher rates of NO formation relative to O2-. The effects of oxymyoglobin, superoxide dismutase, and carbon dioxide were examined as potential modulators of reactant availability for the O2- + NO pathway in vivo. The findings suggest that the outcome of NO biosynthesis in a scavenger milieu can be focused by O2- toward formation of NO adducts on nucleophilic residues (e.g., amines, thiols, hydroxyl) through convergent mechanisms involving the intermediacy of nitrogen dioxide. These modifications may be favored in microenvironments where the rate of O2- production is temporally and spatially contemporaneous with nitric oxide synthase activity, but not in excess of NO generation.

Fig 1.

Reacitivy of DAN with NO + O. Reactions were carried out in HX buffer as described in Methods containing DAN (30 μM) and XO to give the indicated O flux for 1 h after the addition of spermine/NO (6 μM). Shown are representative data (n = three trials) of fluorescence (triplicate mean ± SEM) monitored at λ_ex/em of 360/465 nm (gain 75) minus background.

Fig 2.

Reacitivy of DAF with NO + O. (A) XO was added to HX buffer containing DAF (1 μM) to give O flux as indicated. Spermine/NO was subsequently added forming NO as indicated. Shown are representative data (triplicate mean ± SEM, n = three trials) of fluorescence monitored at λ_ex/em of 485/535 nm (gain = 75) after 1 h incubation at 37°C. Fluorescence values obtained with spermine/NO in the absence of XO (□) have been subtracted from the data acquired in the presence of XO (filled symbols). (B) ANA-1 murine macrophages (1 × 10⁶) stimulated to express iNOS as described in Methods. Arrow indicates time of XO addition to HX buffer to give O flux as indicated. (C) XO was added to HX buffer covering adherent MCF-7 cells loaded with DAF-diacetate as described in Methods to give the indicated O fluxes. Fluorescence values were obtained (as in A) 1 h after addition of spermine/NO to give the indicated NO fluxes.

Fig 3.

Modulators of NO + O reaction. (A) Spermine/NO (3 μM) was added to HX buffer containing DAF (1 μM) and XO to give O flux as indicated in either the presence (▴) or absence (□) of 25 mM NaHCO₃ with a 5% CO₂, 95% air atmosphere. Fluorescence was measured (λ_ex/em of 485/535 nm) after incubation at 37°C for 1 h. (B) DAF-triazole formation was monitored (as in A) in HX buffer containing DAF (1 μM) under conditions as indicated. (C) Spermine/NO (6 μM) was added to HX buffer containing DAF (1 μM) and XO to give O flux as indicated in the presence of SOD as indicated. Fluorescence was measured (as in A) after incubation at 37°C for 1 h.