Abstract
Epoxyeicosatrienoic acids (EETs) are endothelium-derived metabolites of arachidonic acid. They relax vascular smooth muscle by membrane hyperpolarization. These actions are inhibited by the EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE5ZE). We synthesized 20-125iodo-14,15-EE5ZE (20-125I-14,15-EE5ZE), a radiolabeled EET antagonist, and characterized its binding to cell membranes. 14,15-EET (10−9-10−5M) caused a concentration-related relaxation of the preconstricted bovine coronary artery and phosphorylation of p38 in U937 cells that were inhibited by 20-125I-14,15-EE5ZE. Specific 20-125I-14,15-EE5ZE binding to U937 cell membranes reached equilibrium within 5 min and remained unchanged for 30 min. The binding was saturable and reversible, and it exhibited KD and Bmax values of 1.11 ± 0.13 nM and 1.13 ± 0.04 pmol/mg protein, respectively. Guanosine 5′-O-(3-thio)triphosphate (10 μM) did not change the binding, indicating antagonist binding of the ligand. Various EETs and EET analogs (10−10-10−5M) competed for 20-125I-14,15-EE5ZE binding with an order of potency of 11,12-EET = 14,15-EET > 8,9-EET = 14,15-EE5ZE > 15-hydroxyeicosatetraenoic acid = 14,15-dihydroxyeicosatrienoic acid. 8,9-Dihydroxyeicosatrienoic acid and 11-hydroxyeicosatetraenoic acid did not compete for binding. The soluble and microsomal epoxide hydrolase inhibitors (1-cyclohexyl-3-dodecyl-urea, elaidamide, and 12-hydroxyl-elaidamide) and cytochrome P450 inhibitors (sulfaphenazole and proadifen) did not compete for the binding. However, two cytochrome P450 inhibitors, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) and miconazole competed for binding with Ki of 1558 and 315 nM, respectively. Miconazole and MS-PPOH, but not proadifen, inhibited 14,15-EET-induced relaxations. These findings define an EET antagonist's binding site and support the presence of an EET receptor. The inhibition of binding by some cytochrome P450 inhibitors suggests an alternative mechanism of action for these drugs and could lead to new drug candidates that target the EET binding sites.
Footnotes
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This work was supported by the National Institutes of Health National Heart, Lung and Blood Institute [Grant HL-51055]; the National Institutes of Health National Institute of General Medical Sciences [Grant GM-31278]; and the Robert A. Welch Foundation.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.157818
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. -
ABBREVIATIONS:
- EET
- epoxyeicosatrienoic acid
- 14,15-EE8ZE
- 14(S),15(R)-cis-epoxy-eicosa-8(Z)-enoic acid
- 14,15-EE5ZE
- 14(S),15(R)-cis-epoxyeicosa-5(Z)-enoic acid
- EH
- epoxide hydrolase
- 20-I-14,15-EE8ZE
- 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid
- OTs
- 20-tosyl
- 14,15-EET-mSA
- 14,15-Epoxyeicosatrienoyl-methylsulfonamide
- 15-HETE
- 15-hydroxyeicosatetraenoic acid
- 14,15-DHET
- 14,15-dihydroxyeicosatrienoic acid
- 14,15-DHE5ZE
- 14,15-dihydroxy-eicosa-5(Z)-enoic acid
- 8,9-DHET
- 8,9-dihydroxyeicosatrienoic acid
- 11-HETE
- 11-hydroxyeicosatetraenoic acid
- MS-PPOH
- N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- MAP
- mitogen-activated protein
- PPAR
- peroxisomal proliferator-activated receptor
- U46619
- 9–11-dideoxy-11α,9a-epoxymethano-prostaglandin F2α
- NS1619
- 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one
- BKCa
- large-conductance Ca2+ -activated K+ channel
- KATP
- ATP-sensitive K+ channel.
- Received June 22, 2009.
- Accepted September 16, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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