Abstract
5-Hydroxytryptamine (5-HT; serotonin) is a potent vasoconstrictor and smooth muscle mitogen. Substances that produce similar responses also stimulate production of superoxide. We sought to determine whether 5-HT stimulates production of superoxide. 5-HT can be metabolized by cytochrome P450 to nitric oxide (NO), which scavenges superoxide. Thus, we hypothesized that inhibiting cytochrome P450 would potentiate 5-HT-induced contraction and reveal 5-HT-stimulated superoxide. In isolated tissue bath experiments using endotheliumintact rat aorta, the cytochrome P450 inhibitor ketoconazole (KTZ; 1–50 μM) caused a maximum 8-fold leftward shift in the 5-HT concentration-response curve that was not observed when aorta were stimulated with phenylephrine or KCl. 5-HT did not stimulate concentration-dependent increases in superoxide levels as measured by a lucigenin-enhanced chemiluminescent superoxide assay. KTZ (10 μM) did not reveal 5-HT-stimulated superoxide. The NO inhibitor Nω-nitro-l-arginine (l-NNA) (100 μM) with or without KTZ (10 μM) potentiated 5-HT-induced contraction independently of NADPH oxidase-derived superoxide but also did not reveal 5-HT-stimulated superoxide. Metabolism of 5-HT to NO depends on catalase, but the catalase inhibitor 3-amino-1,2,4-triazole (50 mM) attenuated 5-HT-induced contraction. Removal of endothelium did not alter the effects of KTZ on 5-HT-induced contraction, and, in endothelium-intact aorta, KTZ did not decrease acetylcholine-induced relaxation. Unlike KTZ, the cytochrome P450 inhibitors 1-aminobenzotriazole (0.5 mM) and clotrimazole (10 μM) did not potentiate 5-HT-induced contraction. Moreover, 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM), an epoxyeicosatrienoic acid antagonist, caused a small rightward shift in the 5-HT concentration-response curve. These data suggest KTZ acts by a potentially novel mechanism to potentiate 5-HT-induced contraction.
Footnotes
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This work was supported by the National Institutes of Health Grant HL081115 [(K0, SWW0), and National Institutes of Health Grant GM31278 (to J.R.F.)]. National meetings at which this work was orally presented: Ogden KK and Watts SW (2007) Ketoconazole potentiates 5-HT-induced contraction in rat aorta: is nitric oxide the culprit? FASEB J21:905.3.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.128454.
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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); ROS, reactive oxygen species; , superoxide; VSMC, vascular smooth muscle cell; EET, epoxyeicosatrienoic acid; 20-HETE, 20-hydroxyeicosatetraenoic acid; KTZ, ketoconazole; PE, phenylephrine; ACh, acetylcholine; l-NNA, Nω-nitro-l-arginine; 3-AT, 3-amino-1,2,4-triazole; ABT, 1-aminobenzotriazole; 14,15 EEZE, 14,15-epoxyeicosa-5(Z)-enoic acid; JKB, Jude's Kreb; DDC, diethyldithiocarbamic acid; NOS, nitric-oxide synthase.
- Received July 10, 2007.
- Accepted August 7, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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