Abstract
Endothelium-derived epoxyeicosatrienoic acids (EETs) have numerous vascular activities mediated by G protein-coupled receptors. Long-chain free fatty acids and EETs activate GPR40, prompting us to investigate the role of GPR40 in some vascular EET activities. 14,15-EET, 11,12-EET, arachidonic acid, and the GPR40 agonist GW9508 increase intracellular calcium concentrations in human GPR40-overexpressing HEK293 cells (EC50 = 0.58 ± 0.08 μm, 0.91 ± 0.08 μm, 3.9 ± 0.06 μm, and 19 ± 0.37 nm, respectively). EETs with cis- and trans-epoxides had similar activities, whereas substitution of a thiirane sulfur for the epoxide oxygen decreased the activities. 8,9-EET, 5,6-EET, and the epoxide hydrolysis products 11,12- and 14,15-dihydroxyeicosatrienoic acids were less active than 11,12-EET. The GPR40 antagonist GW1100 and siRNA-mediated GPR40 silencing blocked the EET- and GW9508-induced calcium increases. EETs are weak GPR120 agonists. GPR40 expression was detected in human and bovine endothelial cells (ECs), smooth muscle cells, and arteries. 11,12-EET concentration-dependently relaxed preconstricted coronary arteries; however, these relaxations were not altered by GW1100. In human ECs, 11,12-EET increased MAP kinase (MAPK)-mediated ERK phosphorylation, phosphorylation and levels of connexin-43 (Cx43), and expression of cyclooxygenase-2 (COX-2), all of which were inhibited by GW1100 and the MAPK inhibitor U0126. Moreover, siRNA-mediated GPR40 silencing decreased 11,12-EET-induced ERK phosphorylation. These results indicated that GPR40 is a low-affinity EET receptor in vascular cells and arteries. We conclude that epoxidation of arachidonic acid to EETs enhances GPR40 agonist activity and that 11,12-EET stimulation of GPR40 increases Cx43 and COX-2 expression in ECs via ERK phosphorylation.
Highlights
Endothelium-derived epoxyeicosatrienoic acids (EETs) have numerous vascular activities mediated by G protein– coupled receptors
Because EETs affect endothelial cells (ECs) and smooth muscle cells (SMCs), we determined the expression of GPR40 in human and bovine vascular cells
Receptors for the other EET regioisomers have not been characterized. This high-affinity GPCR explains 14,15-EET actions occurring in nanomolar concentrations, but not those requiring micromolar concentrations
Summary
Endothelium-derived epoxyeicosatrienoic acids (EETs) have numerous vascular activities mediated by G protein– coupled receptors. EET isomers inhibit platelet adhesion to the endothelium, and 11,12- and 8,9-EETs inhibit inflammation by decreasing leukocyte adhesion to the endothelium [18, 19] Some of these actions occur with nanomolar concentrations of the EETs (BKCa channel activation, relaxation, tPA release, and decreasing leukocyte adhesion), whereas other activities require micromolar concentrations (inhibition of smooth muscle cell growth and aromatase expression, COX-2 expression, gap junction communication, stimulation of endothelial growth, and inhibition of platelet adhesion) [4]. These differences in EET concentrations for particular activities and differences in activities of EET regioisomers suggest that multiple mechanisms and/or receptors are involved. The transient receptor potential vanilloid-1 (TRPV1) channel has been proposed to represent the vascular EET receptor; there is no evidence for an EETbinding site on this channel [30]
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