Abstract
A current hypothesis states that tolerance to nitroglycerin (GTN) involves increased formation of superoxide (
). Studies showing that inhibitors of protein kinase C (PKC) prevent tolerance to GTN suggest the involvement of PKC activation, which can also increase 
. We examined the roles of 
, 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 
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, NG-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 
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.
Footnotes
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This work was supported by American Heart Association Grant SDG-0130154N (to G.A.) and National Institutes of Health Grants HBLI-70215 (to R.W.C.) and EYI-11766 and EYI-04618 (to R.B.C.).
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DOI: 10.1124/jpet.103.056119.
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ABBREVIATIONS: GTN, glyceryl trinitrate/nitroglycerin; PKC, protein kinase C;
, superoxide; ONOO-, peroxynitrite; CRC, concentration-response curve; BAEC, bovine aortic endothelial cells; ROS, reactive oxygen species; NO, nitric oxide; NOS, nitric-oxide synthase; PE, phenylephrine; pD2, negative log of the dose producing 50% of maximal response; BH4, tetrahydrobiopterin; BH2, dihydrobiopterin; l-NAME, NG-nitro-l-arginine methyl ester; M199, medium 199; SOD, superoxide dismutase; PMA, phorbol 12-myristate 13-acetate; eNOS, endothelial nitric-oxide synthase. - Received July 1, 2003.
- Accepted September 30, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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Roles of Superoxide, Peroxynitrite, and Protein Kinase C in the Development of Tolerance to Nitroglycerin
