(A and C) are the representative Ca2+ traces in HCT116 cells and HT-29 cells, respectively. and 1D). Furthermore, LA induced the co-localization of FFA4 and -arrestin in both cell types, implying the recruitment of -arrestin by FFA4 to cell membranes (Physique 1C and 1D). Thus, FFA4 expression and its activation by LA were observed in both cell lines at the RNA and protein levels. Similarly, EPA and DHA induced the recruitment of -arrestin in both cell lines (Physique 1C and 1D). However, the response to DHA was weaker than that to LA or EPA (Physique 1C and ?and1D1D). Open in a separate window Physique 1 FFA4 expression in human colonic epithelial cells. (A) RT-PCR analyses of FFA1 and FFA4 expression in HCT116 and PT2977 HT-29 human colon epithelial cells. GAPDH was used as the loading control. (B) RT-PCR analyses of long- and short-form FFA4 expression p21-Rac1 in HCT116 and HT-29 human colon epithelial cells. The long- and short-form FFA4 were used as positive controls. (C and D) Fluorescence confocal microscopy of HCT116 and HT-29 cells, respectively. FFA4 and -arrestin 2 protein expression was detected with specific antibodies 30 min after exposing the cells to LA, EPA, or DHA (50 mol/L). Due to the two splicing variants of FFA4 in the colon, specific primers for short- and long-form FFA4 were used. As shown in Physique 1B, HCT116 cells expressed only long-form FFA4, whereas HT-29 cells expressed both forms. Intracellular Ca2+ increase by -3 PUFAs in human colonic epithelial cells Because FFA4 couples to G proteins and increases [Ca2+]i in FFA4-overexpressing cells, this Ca2+ response was measured in colonic epithelial cells endogenously expressing FFA4. As shown Physique 2A, the -3 PUFAs, LA (C18:3) and EPA (C20:5) increased [Ca2+]i in a concentration-dependent manner in HCT116 cells, whereas DHA (C22:6) did not. The -6 fatty, linoleic (C18:2) and -linoleic (C18:3) acids increased [Ca2+]i (Physique 2A), whereas monounsaturated oleic acid (C18:1) and saturated fatty acids, such as stearic (C18:0), palmitic (C16:0), myristic (C14:0), and lauric (C12:0) acid, did not increase [Ca2+]i in HCT116 cells (Physique 2B). Open in a separate window Physique 2 Concentration-response curves of different fatty acids in inducing [Ca2+]i increase in HCT116 cells (A, B) and HT-29 cells (C, D). Long-chain fatty acids with 18 or more carbons were used in (A, C), whereas fatty acids with less than 18 carbons were used in (B, D). Ca2+ responses are shown versus the digitonin-induced maximum response. PT2977 These observations suggested that there is an optimum structure for 18C20 carbon atoms with more than two double bonds and that the location of the unsaturation does not influence endogenous FFA4 activation in colonic cells. TUG-891, an FFA4-specific agonist, increased [Ca2+]i in HCT116 cells (Physique 2A)24,26. Although this agonist has been reported PT2977 to be more potent than LA in several cell systems24,26, it was less potent than other natural PUFAs in HCT116 cells (Physique 2A). We also examined the Ca2+ response in HT-29 cells. As shown in Physique 2C and 2D, the -3 PUFAs, LA, EPA, linoleic acid, and -linoleic acid increased [Ca2+]i PT2977 in a concentration-dependent manner, whereas DHA, stearic acid, palmitic acid, myristic acid, and lauric acid did not. Therefore, the optimum structure for the Ca2+ response in HT-29 cells was the same as that in HCT116 cells, PT2977 that is, 18C20 carbon atoms and more than 2 double bonds. Further, the Ca2+ response to TUG-891 in HT-29 cells was weaker than that to LA (Physique 2C), which was in agreement with the results for HCT116 cells. Taken together, these obtaining suggest that these results are common for human colonic epithelial cells. Effects of edelfosine, EGTA, and PTX on LA-induced Ca2+ response Because LA was the best ligand in terms of inducing Ca2+ response in HCT116 and HT-29 cells and FFA4-overexpresssing systems, LA was used in the subsequent studies. To elucidate the mechanism responsible for Ca2+ signaling, cells were treated with.