Tyrosine nitration in blood vessels occurs with increasing nitric oxide concentration

Abstract

1. Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O2-) and peroxynitrite (ONOO-) in rabbit aortic rings. 2. Following inhibition of endogenous superoxide dismutase (SOD), significant basal release of O2- was revealed (0.9 ± 0.01 x 10-12 mol min-1 mg-1 tissue). Generation of O2- - increased in a concentration-dependent manner in response to NADH or NADPH (EC50= 2.34 ± 1.18 x 10-4 and 6.21 ± 1.79 x 10-3 M respectively, n = 4). NADH-stimulated O2- chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15 x 103 U ml-1). 3. incubation of aortic rings with S-nitrosoglutathione (GSNO; 1 x 10-5 - 3 x 10-3 M) or sodium nitroprusside (SNP; 1 x 10-8 - 1 x 10-3 M), resulted in a concentration-dependent quenching of O2-1 chemiluminescence which was proportional to NO release. 4. ONOO- formation was assessed indirectly by determining protein tyrosine nitration in rabbit aorta using a specific antibody against nitrotyrosine. Basally and in the presence of NADH, a single band was detected. Incubation of aortic rings with either GSNO (1 x 10-3 M) alone or GSNO with NADH resulted in the appearance of additional nitrotyrosine bands. Incubation of serum albumin with GSNO alone did not cause nitrotyrosine formation. In contrast, incubation with 3-morpholinosydonomine (SIN-1; 1 x 10-3 M, 10 min), resulted in marked nitration of albumin which was reduced by oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol, a O2- generator, also resulted in protein nitration. 5. Addition of exogenous NO results in nitrotyrosine formation in rabbit aortic rings. Nitrotyrosine formation is likely to result from the reaction of exogenous NO and basal endogenous O2- resulting in the formation of ONOO-. Formation of ONOO- and nitration of tyrosine residues polentially could lead to vascular damage and might represent unexpected adverse effects of long-term nitrate therapy.