Characterization of 14,15-epoxyeicosatrienoyl-sulfonamides as 14,15-epoxyeicosatrienoic acid agonists: Use for studies of metabolism and ligand binding

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BKCa) channels to cause hyperpolarization and relaxation. We developed a series of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA) as tools to characterize 14,15-EET metabolism and binding. Agonist activities of these analogs were characterized in precontraced bovine coronary arterial rings. All three analogs induced concentration-dependent relaxation and were equipotent with 14,15-EET. Relaxations to these analogs were inhibited by the BKCa channel blocker iberiotoxin (100 nM), the 14,15-EET antagonist 14,15- epoxyeicosa-5(Z)-enoylmethylsulfonamide (10 mu M), and abolished by 20 mM extracellular K+. 14,15-EET-PISA is metabolized to 14,15-dihydroxyeicosatrienoyl-PISA by soluble epoxide hydrolase in bovine coronary arteries and U937 cells but not U937 cell membrane fractions. 14,15-EET-P(125)ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4 degrees C and remained unchanged up to 30 min. The estimated K-d and B-max were 148.3 +/- 36.4 nM and 3.3 +/- 0.5 pmol/mg protein, respectively. These data suggest that 14,15-EET- PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full 14,15-EET agonists. 14,15-EET-P(125)ISA is a new radiolabeled tool to study EET metabolism and binding. Our results also provide preliminary evidence that EETs exert their biological effect through a membrane binding site/receptor.