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

Wenqi Yang, Blythe B. Holmes, V. Raj Gopal, R. V Krishna Kishore, Bhavani Sangras, Xiu Yu Yi, J R Falck, William B. Campbell

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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-EETBSA), 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)- enoyl-methylsulfonamide (10 μM), and abolished by 20 mM extra-cellular 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.

Original languageEnglish (US)
Pages (from-to)1023-1031
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
StatePublished - Jun 1 2007


ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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