RT Journal Article SR Electronic T1 20-125Iodo-14,15-Epoxyeicosa-5(Z)-enoic Acid: a High-Affinity Radioligand Used to Characterize the Epoxyeicosatrienoic Acid Antagonist Binding Site JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 1137 OP 1145 DO 10.1124/jpet.109.157818 VO 331 IS 3 A1 Chen, Yuenmu A1 Falck, John R. A1 Tuniki, Venugopal R. A1 Campbell, William B. YR 2009 UL http://jpet.aspetjournals.org/content/331/3/1137.abstract AB 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.© 2009 by The American Society for Pharmacology and Experimental Therapeutics