Roles of Superoxide, Peroxynitrite, and Protein Kinase C in the Development of Tolerance to Nitroglycerin

A current hypothesis states that tolerance to nitroglycerin (GTN) involves increased formation of superoxide (O₂^.-). Studies showing that inhibitors of protein kinase C (PKC) prevent tolerance to GTN suggest the involvement of PKC activation, which can also increase O₂ ^.-. We examined the roles of O₂^.-, peroxynitrite (ONOO^-), and PKC activation in GTN tolerance. Pre-exposure of rat aortic rings to GTN (5 × 10^-4 M) for 2 h caused tolerance to the vasodilating effect of GTN, as evidenced by a substantial rightward shift of GTN concentration-relaxation curves. This shift was reduced by treatment of the rings with the antioxidants uric acid, vitamin C, or tempol or the PKC inhibitor chelerythrine. We also found that O ₂^.- generation via xanthine/xanthine oxidase in the bath induced tolerance to GTN. However, responses to nitroprusside were not affected. In vivo tolerance produced in rats by 3-day i.v. infusion of GTN was also almost completely prevented by coinfusion of tempol. In bovine aortic endothelial cells (EC), addition of GTN produced a marked increase in tyrosine nitrosylation, indicating increased ONOO^- formation. This action was blocked by prior treatment with uric acid, superoxide dismutase, N ^G-nitro-L-arginine methyl ester, or chelerythrine. We also demonstrated that GTN translocates the α-and εPKC isoforms in EC. However, PKCζ was not affected by GTN treatment. In conclusion, tolerance to GTN involves enhanced production of O₂^.- and ONOO ^- and activation of NO synthase. Furthermore, sustained activation of α- and εPKC isozymes in EC by GTN may play a role in development of tolerance.