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CARDIOVASCULAR

Characterization of Epoxyeicosatrienoic Acid Binding Site in U937 Membranes Using a Novel Radiolabeled Agonist, 20-¹²⁵I-14,15-Epoxyeicosa-8(Z)-Enoic Acid

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin (W.Y., C.J.H., W.B.C.); and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (V.R.T., S.A., J.R.F.)

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 EC₅₀ value of 9 µM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC₅₀ 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 20-¹²⁵I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4°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. 20-¹²⁵I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE 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, 20-¹²⁵I-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.

Address correspondence to: Dr. William B. Campbell, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: wbcamp{at}mcw.edu

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