Characterization of epoxyeicosatrienoic acid binding site in U937 membranes using a novel radiolabeled agonist, 20-¹²⁵I-14,15-epoxyeicosa-8(Z)- enoic acid

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.