title: Characterization of epoxyeicosatrienoic acid binding site in U937 membranes using a novel radiolabeled agonist, 20-125i-14,15-epoxyeicosa-8(Z)-enoic acid.
abstract: Epoxyeicosatrienoic acids (EETs) are important regulators of vascular tone and homeostasis. Whether they initiate signaling through membrane receptors is unclear. We developed 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), a radiolabeled EET agonist, to characterize EET binding to membranes of U937 cells. 20-I-14,15-EE8ZE stimulated cAMP production in U937 cells with similar potency, but it decreased efficacy compared with 11,12-EET. Maximum cAMP production increased 4.2-fold, with an EC(50) value of 9 muM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC(50) value. Both 20-I-14,15-EE8ZE agonist activities were blocked by the EET antagonist 14,15-epoxyeicosa-5(Z)enoic acid (14,15-EE5ZE). Specific 20-(125)I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4 degrees C. The binding was saturable, reversible, and exhibited K(D) and B(max) values of 11.8 +/- 1.1 nM and 5.8 +/- 0.2 pmol/mg protein, respectively. Pretreatment of the membranes with guanosine 5'-O-(3-thio)triphosphate reduced the B(max) in a concentration-related manner. 20-(125)I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE approximately 14,15-EET >> 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-(125)I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
authors.forename: Wenqi
authors.initials: W
authors.lastname: Yang
authors.forename: Venugopal Raju
authors.initials: VR
authors.lastname: Tuniki
authors.forename: Siddam
authors.initials: S
authors.lastname: Anjaiah
authors.forename: J R
authors.initials: JR
authors.lastname: Falck
authors.forename: Cecilia J
authors.initials: CJ
authors.lastname: Hillard
authors.forename: William B
authors.initials: WB
authors.lastname: Campbell
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry* / Q000378:metabolism*; D020298:U937 Cells
chemical_list: D001095:Arachidonic Acids; D007457:Iodine Radioisotopes