Tyrosine nitration in blood vessels occurs with increasing nitric oxide concentration

1 Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O-2(.-)) and peroxynitrite (ONOO-) in rabbit aortic rings. 2 Following inhibition of endogenous superoxide dismutase (SOD), significant basal release of O-2(.-) was revealed (0.9 +/- 0.01 x 10(-12) mol min(-1) mg(-1) tissue). Generation of O-2(.-) 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 O-2(.-) chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15 x 10(3) 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 O-2(.-) 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 O-2(.-) 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 O-2(.-) resulting in the formation of ONOO-. Formation of ONOO- and nitration of tyrosine residues potentially could lead to vascular damage and might represent unexpected adverse effects of long-term nitrate therapy.