Abstract
Reactive oxygen species elicit vascular effects ranging from acute dilatation because of hydrogen peroxide-mediated opening of K+ channels to contraction arising from superoxide-dependent inactivation of endothelium-derived nitric oxide. Given that NADPH oxidases are major sources of superoxide in the vascular wall, this study examined the effects of exogenous NADPH, a substrate of these enzymes, on superoxide generation and isometric tone in mouse isolated aortic rings. NADPH caused concentration-dependent increases in superoxide generation (measured by lucigenin-enhanced chemiluminescence) and vascular tone (isometric tension recordings). However, surprisingly, whereas oxidized NADP+ was unable to support superoxide production, it was equally as effective as reduced NADPH at stimulating vasocontraction. In addition, an NADPH oxidase inhibitor, diphenyleneiodonium, markedly attenuated NADPH-induced superoxide production, yet had no effect on vasocontractions to NADPH. In contrast, a broad specificity P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, as well as the P2X1 selective antagonist, NF023, markedly attenuated both endothelium-dependent and -independent vasocontractions to NADPH, as did the P2X-desensitizing agent α,β-methylene-ATP. Importantly, α,β-methylene-ATP had no effect on superoxide production induced by NADPH. In conclusion, these findings suggest little role for NADPH oxidase-derived superoxide in the contractile effects of NADPH in the mouse aorta. Rather, NADPH seems to act as an agonist at two distinct P2X receptor populations; one located on the endothelium and the other on smooth muscle layer, both of which ultimately lead to contraction.
Footnotes
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This work was supported by National Health and Medical Research Council (NHMRC) of Australia Grant 300013 and National Heart Foundation of Australia Grant G00M0612. G.R.D. is supported by a Monash Category I Fellowship, whereas fellowships from the NHMRC provided support for C.G.S. (350327) and G.J.D. (400303).
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doi:10.1124/jpet.105.096610.
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ABBREVIATIONS: ROS, reactive oxygen species; DETCA, diethyldithiocarbamic acid; SOD, superoxide dismutase; l-NAME, NG-nitro-l-arginine methyl ester; NF023, 8,8′-[carbonylbis (imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid); PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid; Ach, acetylcholine chloride; U46619, 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2a; M40403, a manganese(II)-bis(cyclohexylpyridine)-substituted macrocyclic superoxide dismutase mimetic; Umax, maximum contraction to 0.3 μM U46619.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received October 8, 2005.
- Accepted January 4, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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