Vascular Peroxynitrite Formation during Organic Nitrate Tolerance1

Abstract

Nitroglycerin (NTG) is an important cardiovascular agent, but tolerance during continuous administration limits its clinical utility. Increased vascular superoxide production may mediate nitrate tolerance via a reduction in nitric oxide availability. Because superoxide anion and nitric oxide react avidly 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, and that protein nitration participates in tolerance development. Preincubation of rat thoracic aorta segments with NTG (22 μM, EC₉₅ for 30 min) caused a significant shift in NTG relaxation response (EC₅₀; 6.7 ± 1.7 versus 0.50 ± 0.13 μM, NTG versus vehicle,p < .05). After functional evaluations, tissues were fixed in formalin for immunohistochemistry and digital image analysis. NTG-induced vascular tolerance was associated with increased immunoprevalence of 3-nitrotyrosine (3NT, stable biomarker of protein nitration; 11.41 ± 2.48 versus 0.04 ± 0.02% positive pixels, NTG versus vehicle, p < .05). Staining was observed throughout vascular smooth muscle layers. Addition of 500 μM free tyrosine to the preincubation medium did not alter tolerance development (NTG EC₅₀ 6.5 ± 3.0 μM) but abolished 3NT immunoprevalence (0.16 ± 0.10%). No significant relationship between NTG potency and 3NT immunoprevalence was observed. These data support the hypothesis that protein nitration occurs during nitrate vascular tolerance, however, it apparently does not mediate this phenomenon.