20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide: Photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid receptor

Yuenmu Chen, J R Falck, Vijaya L. Manthati, Jawahar Lal Jat, William B. Campbell

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Abstract

Endothelium-derived epoxyeicosatrienoic acids (EETs) relax vascular smooth muscle by activating potassium channels and causing membrane hyperpolarization. Recent evidence suggests that EETs act via a membrane binding site or receptor. To further characterize this binding site or receptor, we synthesized 20-iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide (20-I-14,15-EE8ZE-APSA), an EET analogue with a photoactive azido group. 20-I-14,15-EE8ZE-APSA and 14,15-EET displaced 20-125I-14,15- epoxyeicosa-5(Z)-enoic acid binding to U937 cell membranes with Ki values of 3.60 and 2.73 nM, respectively. The EET analogue relaxed preconstricted bovine coronary arteries with an ED50 comparable to that of 14,15-EET. Using electrophoresis, 20-125I-14,15-EE8ZE-APSA labeled a single 47 kDa band in U937 cell membranes, smooth muscle and endothelial cells, and bovine coronary arteries. In U937 cell membranes, the 47 kDa radiolabeling was inhibited in a concentration-dependent manner by 8,9-EET, 11,12-EET, and 14,15-EET (IC50 values of 444, 11.7, and 8.28 nM, respectively). The structurally unrelated EET ligands miconazole, MS-PPOH, and ketoconazole also inhibited the 47 kDa labeling. In contrast, radiolabeling was not inhibited by 8,9-dihydroxyeicosatrienoic acid, 5-oxoeicosatetraenoic acid, a biologically inactive thiirane analogue of 14,15-EET, the opioid antagonist naloxone, the thromboxane mimetic U46619, or the cannabinoid antagonist AM251. Radiolabeling was not detected in membranes from HEK293T cells expressing 79 orphan receptors. These studies indicate that vascular smooth muscle, endothelial cells, and U937 cell membranes contain a high-affinity EET binding protein that may represent an EET receptor. This EET photoaffinity labeling method with a high signal-to-noise ratio may lead to new insights into the expression and regulation of the EET receptor.

Original languageEnglish (US)
Pages (from-to)3840-3848
Number of pages9
JournalBiochemistry
Volume50
Issue number18
DOIs
StatePublished - May 10 2011

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Cell membranes
U937 Cells
Labeling
Cell Membrane
Muscle
Endothelial cells
Membranes
Acids
Vascular Smooth Muscle
Binding Sites
Smooth Muscle Myocytes
Cannabinoid Receptor Antagonists
Miconazole
Coronary Vessels
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Endothelial Cells
Ketoconazole
Narcotic Antagonists
Thromboxanes
Potassium Channels

ASJC Scopus subject areas

  • Biochemistry

Cite this

20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide : Photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid receptor. / Chen, Yuenmu; Falck, J R; Manthati, Vijaya L.; Jat, Jawahar Lal; Campbell, William B.

In: Biochemistry, Vol. 50, No. 18, 10.05.2011, p. 3840-3848.

Research output: Contribution to journalArticle

Chen, Yuenmu ; Falck, J R ; Manthati, Vijaya L. ; Jat, Jawahar Lal ; Campbell, William B. / 20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide : Photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid receptor. In: Biochemistry. 2011 ; Vol. 50, No. 18. pp. 3840-3848.
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abstract = "Endothelium-derived epoxyeicosatrienoic acids (EETs) relax vascular smooth muscle by activating potassium channels and causing membrane hyperpolarization. Recent evidence suggests that EETs act via a membrane binding site or receptor. To further characterize this binding site or receptor, we synthesized 20-iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide (20-I-14,15-EE8ZE-APSA), an EET analogue with a photoactive azido group. 20-I-14,15-EE8ZE-APSA and 14,15-EET displaced 20-125I-14,15- epoxyeicosa-5(Z)-enoic acid binding to U937 cell membranes with Ki values of 3.60 and 2.73 nM, respectively. The EET analogue relaxed preconstricted bovine coronary arteries with an ED50 comparable to that of 14,15-EET. Using electrophoresis, 20-125I-14,15-EE8ZE-APSA labeled a single 47 kDa band in U937 cell membranes, smooth muscle and endothelial cells, and bovine coronary arteries. In U937 cell membranes, the 47 kDa radiolabeling was inhibited in a concentration-dependent manner by 8,9-EET, 11,12-EET, and 14,15-EET (IC50 values of 444, 11.7, and 8.28 nM, respectively). The structurally unrelated EET ligands miconazole, MS-PPOH, and ketoconazole also inhibited the 47 kDa labeling. In contrast, radiolabeling was not inhibited by 8,9-dihydroxyeicosatrienoic acid, 5-oxoeicosatetraenoic acid, a biologically inactive thiirane analogue of 14,15-EET, the opioid antagonist naloxone, the thromboxane mimetic U46619, or the cannabinoid antagonist AM251. Radiolabeling was not detected in membranes from HEK293T cells expressing 79 orphan receptors. These studies indicate that vascular smooth muscle, endothelial cells, and U937 cell membranes contain a high-affinity EET binding protein that may represent an EET receptor. This EET photoaffinity labeling method with a high signal-to-noise ratio may lead to new insights into the expression and regulation of the EET receptor.",
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