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Characterization of a 14,15‐ Epoxyeicosatrienoic Acid Binding Site/Receptor with a New Agonist Ligand 14,15‐Epoxyeicosatrienoic‐Phenyliodosulfonylamide
Author(s) -
Yang Wenqi,
Holmes Blythe B,
Falck J. R.,
Campbell William B.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a670
Subject(s) - iberiotoxin , epoxyeicosatrienoic acid , chemistry , epoxygenase , agonist , hyperpolarization (physics) , epoxide hydrolase 2 , biophysics , extracellular , receptor , vascular smooth muscle , ligand (biochemistry) , medicine , pharmacology , biochemistry , stereochemistry , calcium , biology , enzyme , smooth muscle , nuclear magnetic resonance spectroscopy , arachidonic acid , organic chemistry
Epoxyeicosatrienoic acids (EETs) represent endothelium‐derived hyperpolarizing factor in many vascular beds and regulate vascular tone. EETs activate smooth muscle calcium‐activated K + channels hence cause hyperpolarization and relaxation of coronary arteries. However, whether EETs act through a membrane receptor is not known. Here, we developed a stable iodinated 14,15‐EET agonist, 14,15‐EET‐Phenyliodosulfonylamide (14,15‐EET‐PISA), to characterize the putative EET receptor. 14,15‐EET‐PISA induced concentration‐dependent relaxations of bovine coronary arteries, which was equipotent with 14,15‐EET. The relaxations to 14,15‐EET‐PISA were inhibited by the K + channel blocker iberiotoxin, the 14,15‐EET antagonist 14,15‐EEZE‐SI and increased extracellular K + . 14,15‐EET‐P 125 ISA binding to U937 cell membranes was time and concentration‐dependent. The specific binding reached equilibrium by 15 min at 4°C and remained unchanged at 30 min. With 50ug of protein, the estimated Kd of 14,15‐EET‐P 125 ISA was 33 nM. When 14,15‐EET‐P 125 ISA was incubated with myocardial or coronary arterial membranes, a 48kD protein was detected on SDS‐PAGE gels. The radiolabeling of the 48kD protein was displaced by unlabeled EETs in a concentration‐dependent manner. The order of potency was 11,12‐ > 14,15‐ = 5,6‐ > 8,9‐EET. These data suggest that 14,15‐EET may exert its effect through a membrane receptor.