PT  - JOURNAL ARTICLE
AU  - Wenqi Yang
AU  - Blythe B. Holmes
AU  - V. Raj Gopal
AU  - R. V. Krishna Kishore
AU  - Bhavani Sangras
AU  - Xiu-Yu Yi
AU  - J. R. Falck
AU  - William B. Campbell
TI  - Characterization of 14,15-Epoxyeicosatrienoyl-Sulfonamides as 14,15-Epoxyeicosatrienoic Acid Agonists: Use for Studies of Metabolism and Ligand Binding
AID  - 10.1124/jpet.107.119651
DP  - 2007 Jun 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 1023--1031
VI  - 321
IP  - 3
4099  - http://jpet.aspetjournals.org/content/321/3/1023.short
4100  - http://jpet.aspetjournals.org/content/321/3/1023.full
SO  - J Pharmacol Exp Ther2007 Jun 01; 321
AB  - 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 μM), 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-P125ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4°C and remained unchanged up to 30 min. The estimated Kd and Bmax 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-P125ISA 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. The American Society for Pharmacology and Experimental Therapeutics