GPR40 is a low-affinity epoxyeicosatrienoic acid receptor in vascular cells

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.

Keywords: connexin; cyclooxygenase (COX); endothelial cell; fatty acid; smooth muscle.

Figure 1.

Expression of GPR40 and GPR120 in human (h) and bovine (b) endothelial cells and smooth muscle cells and bovine coronary arteries. A, expression of human GPR40 measured by RT-PCR in human vascular cells. B, expression of bovine GPR40 measured by RT-PCR in bovine vascular cells and arteries. C, expression of GPR40 measured by immunoblotting in human and bovine vascular cells and bovine arteries. D, expression of human GPR120 measured by RT-PCR in human vascular cells. Nontransfected HEK293 cells (HEK), HEK293 cells stably expressing human GPR40 (HEK + GPR40), pancreatic cancer cells-1 (PanC-1), and HEK293 cells transiently expressing human GPR120 (S) (HEK + GPR120) were used as controls.

Figure 2.

Effect of EETs on [Ca²⁺]_i in HEK293 cells and HEK293 cells stably expressing human GPR40 (HEK293 + GPR40). A and B, effect of various concentrations of the EETs on [Ca²⁺]_i in HEK293 cells (A) and HEK293 + GPR40 cells (B) (data expressed in Δ relative fluorescence units (ΔRFU) per μg of protein). C and D, effect of 11,12-EET on [Ca²⁺]_i fluorescence over time in HEK293 cells (C) and HEK293 + GPR40 cells (D) (data shown in RFU × 10⁻³). E and F, effect of 14,15-EET on [Ca²⁺]_i fluorescence over time in HEK293 cells (E) and HEK293 + GPR40 cells (F) (data shown in RFU × 10⁻³). Each value represents the mean ± S.E. for n = 4. veh, vehicle.

Figure 3.

Effect of extracellular [Ca²⁺]_i on EET activity in HEK293 cells expressing human GPR40. A–C, comparison of the activity of various concentrations of EET regioisomers and GPR40 agonist GW9508 in the presence of 1.26 mm extracellular calcium (A), in the absence of extracellular calcium (B), and in the absence of extracellular calcium plus 50 μm EGTA (C). D, effect of 1 μm 11,12-EET on [Ca²⁺]_i with time in the presence of 1.26 mm extracellular Ca, in the absence of extracellular Ca, and in the absence of extracellular calcium plus 50 μm EGTA. Each value represents the mean ± S.E. for n = 4. veh, vehicle.

Figure 4.

Effect of EETs, DHETs, EET isomers, and EET analogs on [Ca²⁺]_i in HEK293 cells expressing human GPR40. A, comparison of the activity of 11,12-, 14,15-EETs, and -DHETs. B, comparison of 11,12- and 14,15-EET and their thiirane analogs. C, comparison of the cis- and trans-isomers of 11,12- and 14,15-EETs. D, effect of 11,12-EET in the absence and presence of 1 μm arachidonic acid and effect of arachidonic acid in the absence and presence of 0.1 μm 11,12-EET. Each value represents the mean ± S.E. for n = 4. veh, vehicle.

Figure 5.

Effect of the GPR40 antagonist GW1100 (10 μm) and GPR40 siRNA on the increase in [Ca²⁺]_i by 11,12-EET, 14,15-EET, and the GPR40 agonist GW9508 in human GPR40-expressing HEK293 cells. A, effect of GW1100 on the activity of GW9508. B, effect of GPR40 siRNA on the activity of GW9508. C, effect of GW1100 on the activity of EETs. D, effect of GPR40 siRNA on the activity of EETs. E and F, Western blotting (E) and densitometry (F) of GPR40 protein levels and knockdown in human GPR40-expressing HEK293 cells treated with control and GPR40 siRNA for 48 h. A and C, each value represents the mean ± S.E. for n = 4. B and D, each value represents the mean ± S.E. for n = 8. F, each value represents the mean ± S.E. for n = 4. * indicates p < 0.05, compared with control siRNA. veh, vehicle.

Figure 6.

Effects of the GPR40 agonist TAK-875 on [Ca²⁺]_i and EETs on [³H]TAK-875–specific binding in HEK293 + GPR40 cells. A, effect of the GPR40 agonist TAK-875 on [Ca²⁺]_i in the presence and absence of the GPR40 antagonist GW1100 (10 μm). B, effect of 11,12- and 14,15-EET on the specific binding of [³H]TAK-875 to HEK293 + GPR40 membranes. Each value represents the mean ± S.E. for n = 6–9. veh, vehicle.

Figure 7.

Effect of EETs and GW9508 on [Ca²⁺]_i in HEK293 cells expressing GPR120. Two splice variants for GPR120 were tested: short form (A) and long form (B). Each value represent the mean ± S.E. for n = 4. veh, vehicle.

Figure 8.

GPR40-mediated actions of the EETs. A, role of GPR40 in the increase in [Ca²⁺]_i in INS-1 832/13 insulinoma cells by EETs. Inset represents a GPR40 immunoblot comparing HEK293 + GPR40 and INS-1 832/13 cells. B, role of GPR40 on the relaxation of bovine coronary arteries by 11,12-EET. Arteries were precontracted with U46619. 11,12-EET was tested in the presence and absence of the GPR40 antagonist GW1100 (10 μm), the BK_Ca channel inhibitor iberiotoxin (100 nm), or the combination of both inhibitors. C and D, effect of 11,12-EET on whole-cell K⁺ currents in HUVECs: role of GPR40 and calcium. 11,12-EET (1 μm) increased whole-cell K⁺ currents that were inhibited by GW1100 (10 μm). Studies were repeated in the absence (C) and presence (D) of EDTA in the patch pipette. Each value represents the mean ± S.E. for the stated N values. veh, vehicle.

Figure 9.

Effect of 11,12-EET on ERK phosphorylation in HUVECs: role of GPR40. Cells were pretreated with vehicle, MEK inhibitor U0126 (1 μm) (A), or GPR40 antagonist GW1100 (10 μm) (B). 11,12-EET (1 μm) was then added and incubation continued for 10 min. C, GPR40 protein level and knockdown in HUVEC treated with control and GPR40 siRNA for 48 h. D, effect of GPR40 knockdown on 11,12-EET-stimulated phospho-p42/44 (P-ERK). Both phospho-p42/44 (P-ERK) and total p42/44 (ERK) were measured by immunoblotting. Each value represent mean ± S.D. of three independent experiments. ** and * indicate p < 0.001 and p < 0.05, respectively, compared with the untreated control. ## indicates p < 0.005, compared with 11,12-EET alone. § indicates p < 0.05, compared with control siRNA with 11,12-EET.

Figure 10.

Effect of 11,12-EET on Cx43 expression and phosphorylation in HUVECs: role of GPR40. Cells were pretreated with vehicle, MEK inhibitor U0126 (1 μm) (A), or GPR40 antagonist GW1100 (10 μm) (B) for 20 min. 11,12-EET (1 μm) was then added and incubation continued for 10 min. Cx43 was measured by immunoblotting, and β-actin was a loading control. Each value represent mean ± S.D. of three independent experiments. ** and * indicate p < 0.001 and p < 0.05, respectively, compared with the untreated control. ## and # indicate p < 0.005 and p < 0.05, respectively, compared with 11,12-EET alone.

Figure 11.

Effect of 11,12-EET on COX-2 expression in HUVECs: role of GPR40, MAPK, NF-κB, and soluble epoxide hydrolase. Cells were pretreated with vehicle, MEK inhibitor U0126 (1 μm) (A), or GPR40 antagonist GW1100 (10 μm) (B), NF-κB inhibitor andrographolide (Andro)(40 μm) (C), or sEH inhibitor EH1555 (1 μm) (D) for 20 min. 11,12-EET (1 μm) was then added and incubation continued for 24 h. COX-2 was measured by immunoblotting with β-actin as a loading control. Each value represent mean ± S.D. of three independent experiments. ** and * indicate p < 0.001 and p < 0.05, respectively, compared with the untreated control. # indicates p < 0.05, respectively, compared with 11,12-EET alone.