Vascular peroxynitrite formation during organic nitrate tolerance

Nitroglycerin (NTG) is an important cardiovascular agent, but tolerance dur ing continuous administration limits its clinical utility. Increased vascul ar superoxide production may mediate nitrate tolerance via a reduction in n itric oxide availability. Because superoxide anion and nitric oxide react a vidly to form peroxynitrite, an aggressive cellular toxicant that nitrates protein tyrosine residues, we tested the hypotheses that protein nitration, indicative of peroxynitrite formation, occurs during vascular tolerance, a nd that protein nitration participates in tolerance development. Preincubat ion of rat thoracic aorta segments with NTG (22 mu M, EC95 for 30 min) caus ed a significant shift in NTG relaxation response (EC50; 6.7 +/- 1.7 versus 0.50 +/- 0.13 mu M, NTG versus vehicle, p<.05). After functional evaluatio ns, tissues were fixed in formalin for immunohistochemistry and digital ima ge analysis. NTG-induced vascular tolerance was associated with increased i mmunoprevalence of 3-nitrotyrosine (3NT, stable biomarker of protein nitrat ion; 11.41 +/- 2.48 versus 0.04 +/- 0.02% positive pixels, NTG versus vehic le, p<.05). Staining was observed throughout vascular smooth muscle layers. Addition of 500 mu M free tyrosine to the preincubation medium did not alt er tolerance development (NTG EC50 6.5 +/- 3.0 mu M) but abolished 3NT immu noprevalence (0.16 +/- 0.10%). No significant relationship between NTG pote ncy and 3NT immunoprevalence was observed. These data support the hypothesi s that protein nitration occurs during nitrate vascular tolerance, however, it apparently does not mediate this phenomenon.