Green Tea Polyphenol Epigallocatechin Gallate Activates TRPA1 in an Intestinal Enteroendocrine Cell Line, STC-1

Mako Kurogi 1 Megumi Miyashita 1 Yuri Emoto 1 Yoshihiro Kubo 0 Osamu Saitoh 1 0 Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences , 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan 1 Department of Bio-Science, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology , 1266 Tamura-cho, Nagahama-shi, Shiga 526-0829, Japan A characteristic astringent taste is elicited by polyphenols. Among the polyphenols, catechins and their polymers are the most abundant polyphenols in wine and tea. A typical green tea polyphenol is epigallocatechin gallate (EGCG). Currently, the mechanism underlying the sensation of astringent taste is not well understood. We observed by calcium imaging that the mouse intestinal endocrine cell line STC-1 responds to the astringent compound, EGCG. Among major catechins of green tea, EGCG was most effective at eliciting a response in this cell line. This cellular response was not observed in HEK293T or 3T3 cells. Further analyses demonstrated that the 67-kDa laminin receptor, a known EGCG receptor, is not directly involved. The Ca2+ response to EGCG in STC-1 cells was decreased by inhibitors of the transient receptor potential A1 (TRPA1) channel. HEK293T cells transfected with the mouse TRPA1 (mTRPA1) cDNA showed a Ca2+ response upon application of EGCG, and their response properties were similar to those observed in STC-1 cells. These results indicate that an astringent compound, EGCG, activates the mTRPA1 in intestinal STC-1 cells. TRPA1 might play an important role in the astringency taste on the tongue. Introduction Tastants are detected mainly by taste receptor cells (TRCs) in taste buds on the tongue. Among the 5 basic taste stimuli, sweet, umami, and bitter taste are recognized by G protein coupled receptors (Chandrashekar et al. 2000, 2006; Nelson et al. 2001, 2002; Ishimaru 2009). As a candidate sour taste receptor, the heteromer of transient receptor potential (TRP) channels (PKD1L3 and PKD2L1) has been identified (Huang et al. 2006; Ishimaru et al. 2006). In the case of salty taste, epithelial Na+ channels have been identified as amiloride-sensitive salty receptors and are considered to play a role at least partly (Chandrashekar et al. 2006; Ishimaru 2009). In addition to the 5 basic taste stimuli, the pungent stimulation of hot peppers is also recognized in the mouth. This pungent taste is mainly mediated by TRPV1 receptors, which can be activated by capsaicin from pepper and are expressed in TRCs and sensory neurons in the oral cavity (Ishida et al. 2002). Furthermore, in beverages, such as tea, cider, and red wine as well as in several types of fruits, nuts, and chocolate, a characteristic astringent taste is elicited primarily by compounds known as polyphenols. Of these polyphenols, catechin, epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), and their polymers are most abundant in wine and tea. A typical green tea polyphenol is EGCG (Drewnowski and GomezCarneros 2000; Lesschaeve and Nobel 2005). Although recent reports demonstrated that a bitter taste receptor, hTAS2R39, is an oral sensor of EGCG (Slack et al. 2010; Narukawa et al. 2011), the mechanism underlying the sensation of astringent taste is not well understood. Green tea has been shown to have anticancer activity in many organs (Yang et al. 2006; Bettuzzi et al. 2006). Among constituents of green tea, EGCG is the major polyphenol and exhibits the greatest cancer-preventive effects (Chung et al. 1999; Saeki et al. 2000). Recently, Tachibana et al. (2004) have found that the 67-kDa laminin receptor (67LR) functions as a cell surface EGCG receptor inducing anticancer action. 67LR is a nonintegrin-type laminin receptor and expressed on a variety of tumor cells. Furthermore, EGCG has been shown to induce the disruption of actin fibers and the dephosphorylation of the myosin II regulatory light chain through the 67LR to inhibit the growth of cancer cells (Umeda et al. 2005). Because activation of 67LR with EGCG does not influence the intracellular Ca2+ level (Fujimura et al. 2006), it seems that the EGCG signaling using 67LR may not induce the astringent sensation in sensory terminals in the oral cavity. Other receptor molecule for EGCG must be present as an astringent sensor on the tongue. In addition to the gustatory system, chemosensory information perceived during the gastric and intestinal phases of digestion is important for the control of gastrointestinal (GI) function, such as the secretory activity of GI glands, the resorptive activity, motility and blood supply of the intestinal tract, and satiation (Dockray 2003). The enteroendocrine cells are specialized transducers of luminal factors. STC-1 cells were established in 1990 as a line of enteroendocrine cells (Rindi et al. 1990). A decade later, Wu et al. (2002) reported that STC-1 cells express T2R bitter taste receptors and respond to bitter taste substances. We also characterized the bitter taste responses of STC-1 cells (Masuho et al. 2005). Then, we recently investigated the cellular responses of intestinal STC-1 cells to compounds of 5 basic tastants using a calcium-imaging technique. Although this cell line was known to respond to bitter compounds, we found that compounds of 4 other basic tastants also stimulated STC-1 cells. When solutions containing glutamate, sucrose, HCl, or NaCl were applied, the intracellular Ca2+ concentration in STC-1 cells significantly increased. Therefore, we demonstrated that the GI system can sense all 5 of the basic taste stimuli and that it might contain a taste receptor signaling system similar to the oral taste system (Saitoh et al. 2007). The expression of T1R taste receptors in the gut cells has also been reported by Dyer et al. (2005) and Margolskee et al. (2007). Here, we investigated whether the intestinal STC-1 can respond to the astringent compound of green tea, EGCG, by the calcium-imaging technique. Interestingly, the results clearly indicated that STC-1 cells have a novel sensor for EGCG, which has not been described. When EGCG was applied to STC-1, a significant increase in the intracellular Ca2+ concentration occurred. This cellular response was not observed in HEK293T or 3T3 cells, both of which express 67LR. Using some channel blockers, we focused on members of the TRP channels and found that mouse TRPA1 (mTRPA1) is utilized in the EGCG-induced Ca2+ response in STC-1 cells. Then, we characterized the responding properties of heterologously expressed mTRPA1 to EGCG in HEK293T cells. Materials and methods ()-epigallocatechin-3-gallate (EGCG), ()-epicatechin (EC), ()-epicatechin gallate (ECG), ()-epigallocatechin (EGC), sodium L-glutamate (Glu-Na), menthol, capsaicin, and sodium saccharin were from Wako. Caffeine, ruthenium red (RR), and GdCl3 were from Sigma-Aldrich. AP-18 and HC-030031 were from Enzo Life Sciences. Denat (...truncated)