Characterization of 14,15-Epoxyeicosatrienoyl-Sulfonamides as 14,15-Epoxyeicosatrienoic Acid Agonists: Use for Studies of Metabolism and Ligand Binding

摘要

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxy-genase 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 (BK_(Ca)) 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 BK_(Ca) channel blocker iberiotoxin (100 nM),the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoyl-methylsulfonamide (10 mu M),and abolished by 20 mM extracellular K+.14,15-EET-PISA is metabolized to 14,15-dihydroxyei-cosatrienoyl-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 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-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.