Sensitivities of Two Zebrafish TRPA1 Paralogs to Chemical and Thermal Stimuli Analyzed in Heterologous Expression Systems.

Transient receptor potential A1 (TRPA1) is the only member of the mouse, chick, and frog TRPA family, whereas 2 paralogs (zTRPA1a and zTRPA1b) are present in zebrafish. We herein investigated functional differences in the 2 zebrafish TRPA1s. HEK293T cells were used as heterologous expression systems, and the sensitivities of these cells to 4 chemical irritants (allyl isothiocyanate [AITC], caffeine, auto-oxidized epigallocatechin gallate [EGCG], and hydrogen peroxide [H2O2]) were compared with Ca(2+) imaging techniques. Sensitivities to the activators for AITC, oxidized EGCG, and H2O2 were higher in cells expressing zTRPA1a than in those expressing zTRPA1b, whereas caffeine appeared to activate both cells equally. We also characterized the thermal sensitivity of Xenopus oocytes expressing each TRPA1 electrophysiologically using a 2-electrode voltage clamp. Although endogenous currents induced by a cold stimulation were observed in control oocytes in some batches, oocytes expressing zTRPA1b showed significantly stronger cold- and heat-induced responses. However, significant thermal activation was not observed in oocytes expressing zTRPA1a. The results obtained using in vitro expression systems suggest that zTRPA1a is specialized for chemical sensing, whereas zTRPA1b responds to thermal stimuli. Furthermore, characterization of the chimeric molecule of TRPA1a and 1b revealed the importance of the N-terminal region in chemical and thermal sensing by zTRPA1s.

Auto-oxidation products of epigallocatechin gallate activate TRPA1 and TRPV1 in sensory neurons.

The sensation of astringency is elicited by catechins and their polymers in wine and tea. It has been considered that catechins in green tea are unstable and auto-oxidized to induce more astringent taste. Here, we examined how mammalian transient receptor potential V1 (TRPV1) and TRPA1, which are nociceptive sensors, are activated by green tea catechins during the auto-oxidation process. Neither TRPV1 nor TRPA1 could be activated by any of the freshly prepared catechin. When one of the major catechin, epigallocatechin gallate (EGCG), was preincubated for 3h in Hank's balanced salt solution, it significantly activated both TRP channels expressed in HEK293 cells. Even after incubation, other catechins showed much less effects. Results suggest that only oxidative products of EGCG activate both TRPV1 and TRPA1. Dorsal root ganglion (DRG) sensory neurons were also activated by the incubated EGCG through TRPV1 and TRPA1 channels. Liquid chromatography-mass spectrometry revealed that theasinensins A and D are formed during incubation of EGCG. We found that purified theasinensin A activates both TRPV1 and TRPA1, and that it stimulates DRG neurons through TRPV1 and TRPA1 channels. Results suggested a possibility that TRPV1 and TRPA1 channels are involved in the sense of astringent taste of green tea.

Green tea polyphenol epigallocatechin gallate activates TRPA1 in an intestinal enteroendocrine cell line, STC-1.

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 Ca(2+) 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 Ca(2+) 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.

The diversity in sensitivity of TRPA1 and TRPV1 of various animals to polyphenols.

The perception of tastes is sensed by the receptors that stimulate sensory cells. We previously reported that TRPA1 and TRPV1 channels expressed in the oral cavity of mammals, are activated by the auto-oxidized product of epigallocatechin gallate (oxiEGCG), a major astringent catechin in green tea. Here, we investigated and compared the sensitivity of TRPA1 and TRPV1 from various animals to astringent polyphenols. We selected three polyphenols, oxiEGCG, tannic acid and myricetin. HEK293T cells expressing TRPA1 or TRPV1 from mammal, bird, reptile, amphibian, and fish, were analyzed for their activation by the Ca2+-imaging. We found the apparent diversity in the polyphenol-sensitivity among various animals. Mammalian TRPs showed relatively higher sensitivity to polyphenols, and especially, human TRPA1 and TRPV1 could be activated by all of three polyphenols at 20 µM. Reptile TRP channels, however, were insensitive to any polyphenols examined. Moreover, the polyphenol-sensitivity of zebrafish TRPA1 and TRPV1 was quite different from that of medaka TRP channels. Since many polyphenols are present in plants and the sensing of polyphenols using TRP channels in the oral cavity might cause astringent taste, the observed diversity of the polyphenol-sensitivity of TRP channels might be involved in the divergence in the food habit of various animals.

Chemical sensing properties of Takifugu TRPA1.

Transient receptor potential ankyrin 1 (TRPA1) is one of the main sensors for noxious stimuli in animals. Recent studies on the cloning and characterization of TRPA1 channels from several organisms showed the functional diversity of TRPA1 in sensing chemicals and temperature. Nociceptive receptors have been suggested to play important roles in adaptation to the environment by and survival strategies of animals; therefore, the sensitivity of various vertebrate TRPA1s needs to be examined in more detail. Here, we focused on fish TRPA1s and investigated the chemical sensing properties of pufferfish (Takifugu) TRPA1 (pfTRPA1). We determined how mammalian TRPA1 ligands activated pfTRPA1 using a Ca-imaging technique. The results obtained indicated that the sensitivity of pfTRPA1 to known TRPA1 ligands was lower than that of mammalian TRPA1s, except for the response ability to allyl isothiocyanate. We also investigated the effects of tannic acid, a type of polyphenol, by measuring ionic currents in Xenopus oocytes in a two-electrode voltage clamp. Although mouse TRPA1 was inhibited by tannic acid, pfTRPA1 channels were enhanced by the treatment with tannic acid. Taken together, these results suggest that pfTRPA1 is not a simple sensor with a lower sensitivity to chemical stimulation, but is actually a specialized sensor with unique properties.

Effect of five taste ligands on the release of CCK from an enteroendocrine cell line, STC-1.

Here, we investigated which taste ligand induces the CCK (cholecystokinin) release from intestinal STC-1 cells. We first developed a new assay to measure the release of CCK. The expression vector for CCK type A receptor (CCKAR) was permanently introduced into HEK293T cells and a cell line was established (CCKAR/HEK). Then, STC-1 cells were treated with taste ligands and the incubated buffer of STC-1 cells containing released CCK was applied to CCKAR/HEK cells.Since CCKAR couples to Gq-signaling, the CCK-induced receptor activation can be monitored by the method of Ca2+-imaging. Therefore, when CCK is released from STC-1 cells to culture medium with taste stimulation, Ca2+ activation of CCKAR/HEK should be observed. Among five different taste ligands (saccharin, Na-glutamate, NaCl, denatonium benzoate, HCl), only denatonium benzoate and HCl induced the release of CCK in STC-1 cells. Thus, we found that only specific taste ligands induce the CCK release, and that other three taste ligands cannot induce the release of CCK despite of their ability to elevate the intracellular Ca2+ level in STC-1 cells.

Effects of spinophilin on the function of RGS8 regulating signals from M2 and M3-mAChRs.

We found that a scaffold protein, spinophilin (SPL), can interact with M2 and M3-muscarinic acetylcholine receptors (mAChRs). As SPL can also bind to RGS8 by using the different region of SPL, we investigated the effects of SPL on the function of RGS8 regulating signals from M2 and M3 receptors. M2 receptor-mediated Gi-signaling was studied by monitoring G-protein-coupled inwardly rectifying K+ channels, and M3 receptor-mediated Gq-signaling was monitored by the increase of Ca2+-activated Cl(-) current. The expression of SPL could enhance the regulatory function of RGS8 on the M3-mAChR, but the acceleration function of RGS8 on the M2-mediated signaling could not be enhanced by SPL. Results showed that the recruitment of RGS8 to the receptor differentially affects the function of RGS8 among receptors.