title: 20-125Iodo-14,15-epoxyeicosa-5(Z)-enoic acid: a high-affinity radioligand used to characterize the epoxyeicosatrienoic acid antagonist binding site.
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-(125)iodo-14,15-EE5ZE (20-(125)I-14,15-EE5ZE), a radiolabeled EET antagonist, and characterized its binding to cell membranes. 14,15-EET (10(-9)-10(-5)M) caused a concentration-related relaxation of the preconstricted bovine coronary artery and phosphorylation of p38 in U937 cells that were inhibited by 20-(125)I-14,15-EE5ZE. Specific 20-(125)I-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 K(D) and B(max) values of 1.11 +/- 0.13 nM and 1.13 +/- 0.04 pmol/mg protein, respectively. Guanosine 5'-O-(3-thio)triphosphate (10 muM) did not change the binding, indicating antagonist binding of the ligand. Various EETs and EET analogs (10(-10)-10(-5)M) competed for 20-(125)I-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 K(i) 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.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Yuenmu
authors.initials: Y
authors.lastname: Chen
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: Venugopal R
authors.initials: VR
authors.lastname: Tuniki
authors.forename: William B
authors.initials: WB
authors.lastname: Campbell
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D004305:Dose-Response Relationship, Drug; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors; D004852:Epoxy Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D010766:Phosphorylation; D020298:U937 Cells; D014664:Vasodilation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: C545738:20-iodo-14,15-epoxyeicosa-5-enoic acid; D065607:Cytochrome P-450 Enzyme Inhibitors; D004852:Epoxy Compounds; D007457:Iodine Radioisotopes; D008024:Ligands; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; C067407:14,15-episulfide eicosatrienoic acid; D048051:p38 Mitogen-Activated Protein Kinases; D004851:Epoxide Hydrolases; D015126:8,11,14-Eicosatrienoic Acid