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 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 KD and Bmax 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 Bmax 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.
Footnotes
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This study was supported by National Institutes of Health Grants HL-51055, HL-83297, and GM-31278 and the Robert A. Welch Foundation.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.129577.
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ABBREVIATIONS: EET, epoxyeicosatrienoic acid; P450, cytochrome P450; BKCa, large conductance, calcium-activated potassium; 14,15-EE8ZE, 14(S),15(R)-cis-epoxy-eicosa-8Z-enoic acid; 14,15-EE5ZE,14,15-epoxyeicosa-5(Z)-enoic acid; 14,15-DHET, 14,15-dihydroxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; sEH, soluble epoxide hydrolase; 20-I-14,15-EE8ZE, 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid; OTs, 20-tosyl; HPLC, high-performance liquid chromatography; TBSTM, Tris-buffered saline containing 0.1% Tween 20 and 5% milk; 15-HETE, 15-hydroxyeicosatetraenoic acid; Ro 20,1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; AUDA, adamantyl dodecanoic acid urea; IBTX, iberiotoxin; GTPγS, guanosine 5′-O-(3-thio)triphosphate; GPCR, G protein-coupled receptor; U46619, 9–11-dideoxy-11α, 9a-epoxymethano-prostaglandin F2a.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received August 1, 2007.
- Accepted December 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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