JPET #157818 1 Title Page 20-Iodo-14,15-Epoxyeicosa-5Z-enoic Acid: a High Affinity Radioligand Used to Characterize the Epoxyeicosatrienoic Acid Antagonist Binding Site

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-EEZE). We synthesized 20iodo-14,15-EE5ZE (20-I-14,15-EE5ZE), a radiolabeled EET antagonist, and characterized its binding to cell membranes. 14,15-EET (10-10M) caused a concentration-related relaxation of the preconstricted bovine coronary artery and phosphorylation of p38 in U937 cells that were inhibited by 20-I-14,15-EE5ZE. Specific 20-I-14,15-EE5ZE binding to U937 cell membranes reached equilibrium within 5 min and remained unchanged for 30 min. The binding was saturable, reversible and exhibited KD and Bmax values of 1.11±0.13 nM and 1.72±0.059 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 M) competed for 20-I-14,15-EE5ZE binding with an order of potency of 11,12-EET=14,15EET>8,9-EET=14,15-EE5ZE>15-HETE=14,15-DHET. 8,9-DHET and 11-HETE did not compete for binding. The soluble and microsomal epoxide hydrolase inhibitors (1-cyclohexyl-3dodecyl-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 nM 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. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on September 17, 2009 as DOI: 10.1124/jpet.109.157818 at A PE T Jornals on Jne 0, 2017 jpet.asjournals.org D ow nladed from