Panoply™ Human FFAR1 Over-expressing Stable Cell Line

Endothelial-derived epoxyeicosatrienoic acids (EETs) have diverse vascular activities mediated by G protein-coupled receptors. GPR40, or free fatty acid receptor-1 (FFAR-1), belongs to a family of receptors that use short- and long-chain fatty acids as ligands. Long-chain free fatty acids and EETs activate GPR40, prompting us to investigate the role of GPR40 in the activity of certain vascular EETs. 14,15-EETs, 11,12-EETs, arachidonic acid, and the GPR40 agonist GW9508 increase intracellular calcium concentrations in human HEK293 cells overexpressing GPR40. EETs with cis- and trans-epoxides exhibit similar activities, while replacement of the epoxy oxygen with thiirane sulfur reduces activity. 8,9-EET, 5,6-EET, and their epoxy hydrolysis products, 11,12- and 14,15-dihydroxyeicosatrienoic acid, are less active than 11,12-EET. The GPR40 antagonist GW1100 and siRNA-mediated silencing of GPR40 blocked the calcium elevation induced by EETs and GW9508. EETs are weak agonists of GPR120. GPR40 expression has been detected in human and bovine endothelial cells (ECs), smooth muscle cells, and arteries. 11,12-EET concentration-dependently relaxes pre-systolic coronary arteries; however, GW1100 does not alter this relaxation. In human endothelial cells, 11,12-EET increased microtubule-associated protein kinase (MAPK)-mediated ERK phosphorylation, connexin-43 (Cx43) phosphorylation and levels, and cyclooxygenase-2 (COX-2) expression, effects that were inhibited by both GW1100 and the MAPK inhibitor U0126. Furthermore, siRNA-mediated silencing of GPR40 reduced 11,12-EET-induced ERK phosphorylation. These results indicate that GPR40 is a low-affinity EET receptor in vascular cells and arteries.

The effects of GPR40 agonists and antagonists were assessed using a stable HEK293 cell line overexpressing human GPR40 and untransfected HEK293 cells by measuring [Ca2+]i as an indicator of GPR40 activity. 14,15-, 11,12-, 8,9-, and 5,6-EET stimulated increases in [Ca2+]i in GPR40 overexpressing HEK293 cells (Figure 1B). Although untransfected HEK293 cells may express low levels of GPR40 protein but no mRNA, this is clearly not functional. EETs did not alter [Ca2+]i in untransfected HEK293 cells (Figure 1A). Therefore, GPR40 overexpression is essential for EET activity. The [Ca2+]i increase induced by EETs was concentration-dependent. 11,12- and 14,15-EETs had similar activity and potency and were more potent than 8,9- and 5,6-EETs. 17,18-Epoxyeicosatetraenoic acid (17,18-EEQ), the epoxy form of the ω-3 fatty acid eicosapentaenoic acid, also elevated [Ca2+]i in GPR40-expressing cells; however, its activity was lower than that of the EETs. 11,12-EET (Figure 1D) and 14,15-EET (Figure 1F) caused transient increases in [Ca2+]i in GPR40 overexpressing HEK293 cells. Following this rapid increase, [Ca2+]i returned to baseline levels within the next 150 seconds. The transient height increased with increasing EET concentration, but the transient pattern was the same. Vehicle had no effect. Within the same concentration range, EETs did not induce transient changes in [Ca2+]i in untransfected HEK293 cells (Figure 1C and E).

Figure 1. Effect of EETs on [Ca2+]i in HEK293 cells and human GPR40 overexpressing HEK293 cells. (Park S K, et al., 2018)