Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Biochemical and Biophysical Research Communications 405 (2011) 620–625

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39 Masataka Narukawa a,b,1, Chiaki Noga b, Yohei Ueno c, Tsutomu Sato a,b, Takumi Misaka c, Tatsuo Watanabe a,b,⇑ a b c

School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

a r t i c l e

i n f o

Article history: Received 20 January 2011 Available online 25 January 2011 Keywords: Green tea Catechin Bitter taste Bitter taste receptors hTAS2R39

a b s t r a c t Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as ( )-epicatechin (EC), ( )-epicatechin gallate (ECg), ( )-epigallocatechin (EGC), and ( )-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 lM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Green tea has long been a popular drink in East Asia. Many active ingredients, such as polyphenols, vitamins, and amino acids, are present in green tea [1]. It has been reported that green tea showed antimicrobial, immunostimulatory, anticarcinogenic, and anti-inflammatory capacities. It also exerts a protective effect against cardiovascular diseases [2]. Such functionalities mainly originate from catechins including green tea. ( )-Epicatechin (EC), ( )-epicatechin gallate (ECg), ( )-epigallocatechin (EGC), and ( )-epigallocatechin gallate (EGCg) are the major polyphenol components of green tea and are referred to as tea catechins. Recently, the pharmaceutical effects of catechins

Abbreviations: EC, ( )-epicatechin; ECg, ( )-epicatechin gallate; EGC, ( )epigallocatechin; EGCg, ( )-epigallocatechin gallate; hTAS2R, human TAS2R; 2+ [Ca ]i, intracellular calcium concentration; HEK, human embryonic kidney; PTC, phenylthiocarbamide. ⇑ Corresponding author at: School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Fax: +81 54 264 5550. E-mail address: [email protected] (T. Watanabe). 1 Present address: German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.079

have attracted attention. According to recent studies, the catechins possess antihypertensive [3], anti-oxidative [4,5], antiarteriosclerotic [6], anticarcinogenic [7,8], and hypocholesterolemic properties [9]. Although the polyphenols are key substances that influence the quality of certain foods [10,11], few studies have examined the sensory characteristics of tea catechins [12,13]. Because catechins generally have a strong bitter taste, we focused on bitter taste receptors as possible catechin receptors among the taste receptors [14]. Bitter compounds are detected by the TAS2R family, which consists of 25 members in humans and 34 members in the mouse [15–18]. Many agonists for these receptors have recently been characterized precisely [19]. On the other hand, catechins have a strong bitter taste, and certain taste bud cells isolated from mouse circumvallate papillae responded to the application of ECg [20]. These results suggest that catechins are recognized as tastants, and a bitter taste receptor that receives catechins actually exists in taste cells. However, the receptor for catechins has not been fully understood, and detailed information was not available, although there was a description that hTAS2R39 responded to EGCg [21]. Here, we examined the response of cultured cells expressing human bitter taste receptors (hTAS2Rs) to tea catechin to identify the receptor for them. Furthermore, we investigated whether

M. Narukawa et al. / Biochemical and Biophysical Research Communications 405 (2011) 620–625

bitterness of catechins can be evaluated using the bitter taste receptor by comparing a response of cell-based assay with human sensory evaluation. Our results suggest the participation of hTAS2R39 in the detection of tea catechins in humans, and also that of galloyl groups in the bitter taste intensities. 2. Materials and methods 2.1. Materials EC, ECg, EGC, and EGCg were provided by Mitsui Norin Co. Ltd. (Tokyo, Japan). All other reagents were of analytical grade and were obtained from standard suppliers. 2.2. Construction of expression plasmids for hTAS2Rs and the chimeric G protein To construct an expression plasmid for hTAS2Rs, DNA fragments encoding hTAS2R16 (NCBI refseq number: NM_016945), hTAS2R38 (NCBI refseq number: NM_176817), and hTAS2R39 (NCBI refseq number: NM_176881) were obtained from human genomic DNA (BD Clontech, Mountain View, CA, USA) by polymerase chain reaction (PCR) amplification. For hTAS2R38, point mutations were introduced to generate the high sensitive haplotype (hTAS2R38-PAV) [22] by PCR using mutation introducing primer sets. The fragments encoding hTAS2R proteins were tagged at the amino terminus with the sequence of the first 45 amino acids of rat somatostatin receptor type 3 (ssr3), and then subcloned into the EcoRI–NotI site of the pEAK10 expression vector (Edge Biosystems, Gaithersburg, MD). cDNA fragment encoding the chimeric Gprotein subunit Ga16gust44 were a kind gift provided by Dr. Ueda [23], and it was also subcloned into pEAK10 vector. 2.3. Measurement of intracellular calcium ([Ca2+]i) change To obtain a trace of [Ca2+]i change and a concentration–response curve, cellular responses were examined using a FlexStation™ II multimode fluorescent microplate reader (Molecular Devices, Sunnyvale, CA, USA). Human embryonic kidney 293T (HEK293T) cell was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. HEK293T cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (Sigma– Aldrich, Tokyo, Japan) supplemented with 10% fetal bovine serum (Thermo Scientific HyClone, Logan, UT, USA). Cells were seeded onto 35-mm dishes, and transiently transfected with the plasmids expressing each of ssr3-hTAS2R together with Ga16gust44 at a 4:1 ratio using Lipofectamine 2000 (Invitrogen). Transfected cells were transferred to a Cluster Plate (a 96-well flat bottom with lid, tissue culture treated, Corning Inc., Bedford, MA, USA) 6 h after transfection. The cells were incubated for an additional 20–24 h, then rinsed with assay buffer (10 mM HEPES, 5 mM KCl, 130 mM NaCl, 2 mM CaCl2
2H2O, 10 mM glucose, 1.2 mM MgCl2
6H2O at pH 7.4), loaded with 3 lM Fluo-4 AM (Molecular Probes, Eugene, OR, USA) diluted with the assay buffer, and incubated for an additional 30 min at 25 °C. The cells were then rinsed with the assay buffer and incubated in 180 lL of assay buffer for 10 min at 25 °C before the plate was loaded onto a FlexStation™ II for fluorescence detection. Fluorescence (excitation at 485 nm, emission at 525 nm, and cutoff at 515 nm) was monitored at 2-s intervals at 25 °C; 20 lL of assay buffer supplemented with 10 test compound solution was added at 30 or 150 s, respectively. The final concentrations of the test ligands were 10 mM salicin, 10 lM phenylthiocarbamide (PTC), and EC, ECg, EGC, and EGCg provided in a range between 1 and 300 lM. Because catechin concentrations above 300 lM give rise to a nonspecific calcium response, we did not use catechins

621

solution higher than 300 lM for the cell-based assay (data not shown). Because ATP increases [Ca2+]i via the endogenous P2Y receptor of HEK cells [24], 3 lM ATP was added to each well to elicit maximum fluorescence intensity. Data values for test compounds were expressed as a percent response to 3 lM ATP. Curve fitting and parameter estimation were carried out using Prism 4.0a software (Graph Pad Software, San Diego, CA, USA). To obtain fluorescent images, the transfected cells were seeded onto 96-well lumox multiwell (SARSTEDT AG & Co., Nümbrecht, Germany), and loaded with 5 lM Fura-2 AM (Invitrogen, Carlsbad, CA, USA). The fura-2 fluorescence intensities were measured with excitation at 340 and 380 nm followed by detection at 510 nm with a Lambda 10-3 computer-controlled filter changer (Sutter Instruments, San Rafael, CA, USA), a CoolSNAP HQ2 camera (Photometrics, Tucson, AZ, USA), and an IX-81 inverted fluorescence microscope (Olympus, Tokyo, Japan). The images were recorded at 4-s intervals and analyzed with MetaFluor software (Molecular Devices). The final concentrations of the test ligands were 300 lM salicin, 1 mM PTC, and 300 lM EC, ECg, EGC, and EGCg. 2.4. Sensory evaluation We recruited 11 volunteers of both sexes with a mean age of 23.7 (SD 3.7) years (mean (standard deviation)). All subjects had normal body mass, were non-smokers, and were in good physical health. All psychophysical tests were performed according to a protocol approved by the University of Shizuoka Ethics Committee. Taste intensity for catechins was assessed using a visual analog scale (VAS). The subjects were asked to spit out each solution after tasting and to rate the taste intensity by marking the appropriate position on a 100-mm VAS. The right and left ends of the scale represented ‘‘strong’’ and ‘‘weak’’ tastes, respectively. We prepared EC, ECg, EGC, and EGCg solutions at four different concentrations, 10, 30, 100, and 300 lM. For the taste test, the subjects were presented with plastic cups containing 30 mL of a sample solution at ambient temperature. For each taste test, the subjects were asked to thoroughly rinse their mouths with distilled water, sip the test solution, swirl it around in their mouths for several seconds to taste it, and then spit it out. The subjects rinsed their mouths with distilled water between the tasting of different samples. 3. Results The chemical structures of tea catechins tested were shown in Fig. 1. ECg and EGCg are characterized by having galloyl groups in its structure. In this study, we examined the response of cultured cells expressing each of human bitter taste receptor (hTAS2R) to tea catechins to identify the taste receptor for them. As in the publication that described the response of hTAS2R39 to EGCg, we also confirmed that a part of hTAS2R39-expressing HEK293T cells were found to respond to the application of 300 lM EGCg (Fig. 2A). In addition to EGCg, the clear response was also observed when 300 lM ECg was used for the application to hTAS2R39-expressing cells (Fig. 2A). The responses to these catechins were not observed when mock-transfected cells were used for the imaging experiments (Fig. 2A). The response of hTAS2R39expressing cells was also obtained by the cell-based assay using FlexStation™ II, and the intensities of cellular responses to 300 lM of ECg and EGCg were almost similar to each other (Fig. 2B). On the other hand, hTAS2R16- or hTAS2R38-expressing cells clearly and intensely responded to their cognate ligands, 300 lM salicin or 1 mM PTC, respectively, they didn’t responded to the application of 300 lM ECg or EGCg (Fig. 3A–C), indicating the specificity of hTAS2R39 to these tea catechins.

622

M. Narukawa et al. / Biochemical and Biophysical Research Communications 405 (2011) 620–625

OH

OH

OH

OH HO

HO

O

O

OH

OH

O

OH

OH

O

OH OH (–)-Epicatechin gallate (ECg)

(–)-Epicatechin (EC)

OH

OH

OH

OH HO

O

OH

HO

O

OH O

OH

OH

OH

OH

O

OH OH

(–)-Epigallocatechin (EGC)

(–)-Epigallocatechin gallate (EGCg)

Fig. 1. Chemical structures of ( )-epicatechin (EC), ( )-epigallocatechin (EGC), ( )epicatechin gallate (ECg), and ( )-epigallocatechin gallate (EGCg).

A

Next, we compared the responsiveness of hTAS2R39 to tea catechins listed in Fig. 1. When 300 lM of tea catechins were applied to the hTAS2R39-expressing cells, only a few responses were observed to EC or EGC, although clear responses were observed to ECg or EGCg (Fig. 4A). When we investigated the concentration–response relationship to tea catechins, it was clearly indicated that hTAS2R39-expressing cells responded to catechins in a concentration-dependent manner (Fig. 4B). EC50 of ECg, EGCg, EGC, and EC is 88.2, 181.6, 395.5, and 417.7 lM, respectively. At 300 lM ECg, EGCg, EGC, and EC, their relative responses to 3 lM ATP were 0.35 ± 0.05, 0.30 ± 0.02, 0.16 ± 0.03, and 0.13 ± 0.01, respectively. The strongest response to ECg was observed among the catechins tested. EGCg provided the next strongest response. The responses to EC and EGC were weaker than the responses to ECg and EGCg. To compare the result of the cell-based assay using the hTAS2R39-expressing cells with the taste intensity, we investigated the taste intensity of catechins by human sensory evaluation (Fig. 4C). At 300 lM catechin, the taste intensity of ECg was the strongest, followed by EGCg, EC, and EGC. Their taste intensities were 76.1 ± 7.4, 43.8 ± 8.4, 26.2 ± 8.2, and 21.3 ± 4.9, respectively. The taste intensities of EC and EGC are weaker than the intensities of ECg and EGCg. This result is similar to the result that obtained from the cell-based assay using hTAS2R39. Thus, the bitterness of catechins that humans recognize can be assessed by a cell-based assay using hTAS2R39-expressing cells.

ECg Mock

EGCg hTAS2R39

Mock

hTAS2R39

Ratio

1.25

Before

0.35

After

B

7000

ATP

Catechin

6000

ΔRFU

5000 4000 3000 2000

: ECg : EGCg

1000 0

0

50

100 Time (Sec)

150

200

Fig. 2. Responses of the cells expressing hTAS2R39 to catechins. (A) Representative ratiometric images of fura-2-loaded HEK293T cells coexpressing Ga16gust44 and hTAS2R39 after treatment with 300 lM ECg or EGCg. The top and bottom columns show the representative cell images obtained 2 and 30 s, respectively. The color scale indicates the F340/F380 fluorescence ratio as the pseudocolor. (B) Line traces obtained from the Flexstation™ II assay showing [Ca2+]i changes after treatment with 300 lM ECg and EGCg in HEK293T cells coexpressing hTAS2R39 and Ga16gust44.

623

M. Narukawa et al. / Biochemical and Biophysical Research Communications 405 (2011) 620–625

A

hTAS2R16

hTAS2R38

Ratio

1.25

Buffer

0.35

ECg

EGCg

Salicin / PTC

B

Taste substance

8000

ATP : PTC : ECg : EGCg

6000

4000

4000 2000

2000 0

Taste substance

8000

: Salicin : ECg : EGCg

ΔRFU

ΔRFU

6000

C

ATP

0 0

50

100 Time (sec)

150

200

0

50

100 Time (sec)

150

200

Fig. 3. Responses of the cells expressing hTAS2R16 or 38 to catechins. (A) Representative ratiometric images of fura-2-loaded HEK293T cells coexpressing Ga16gust44 and hTAS2R16 or 38. (B) Line traces obtained from the Flexstation™ II assay showing [Ca2+]i changes after treatment with 10 mM salicin, 300 lM ECg, or 300 lM EGCg in HEK293T cells coexpressing Ga16gust44 and hTAS2R16. (C) Line traces obtained from the Flexstation™ II assay showing [Ca2+]i changes after treatment with 10 lM PTC, 300 lM ECg, or 300 lM EGCg in HEK293T cells coexpressing Ga16gust44 and hTAS2R38.

4. Discussion Catechins are functional components in green tea and also influence on its quality [20]. It is well known that four kinds of catechins, such as EC, ECg, EGC, and EGCg, are major components of the polyphenols in green tea, and they are referred to as tea catechins. However, the taste receptor for these catechins had not been fully understood. To identify the taste receptor that receives catechins in the oral cavity of humans, we performed a cell-based assay using cultured cells expressing the human bitter taste receptor, and confirmed that catechins activated hTAS2R39 (Fig. 2), one of the human bitter taste receptor. What is important in this study is that there were clear differences in the ability of receptor activation among green tea catechins. hTAS2R39 showed the strongest response to ECg, followed by EGCg (Fig. 4). The responses to EC and EGC were weaker than those to ECg and EGCg (Fig. 4). Generally, bitter substances with high hydrophobicity tend to show the strong bitterness probably because of intense interactions with the bitter receptors [25]. ECg and EGCg are characterized by having galloyl groups in its chemical structures (Fig. 1). Our result strongly indicates that the

molecular hydrophobicity is increased by the addition of galloyl group. Log P values of tea catechins, which indicate the intensity of molecular hydrophobicity, are ECg +1.06, EGCg +0.39, EC +0.11, and EGC 0.50 [26]. This is the one of reason why ECg and EGCg induced stronger responses of hTAS2R39-expressing cells than EC and EGC did. In human sensory evaluation, the taste intensity for catechins was the strongest for ECg, followed by EGCg (Fig. 4C). Since the taste intensities of catechins without galloyl group (EC and EGC) were found to be weaker than those of ECg and EGCg, the participation of hTAS2R39 in the detection of tea catechins was strongly supposed. This is supported by the finding that hTAS2R16 and hTAS2R38 do not respond to catechins. The functional benefits of green tea have gained attention in the world, and global consumption of green tea has increased recently. Although catechins are the main components in green tea, they also provide an unpleasant flavor, which presumably hinders further consumption. It will be necessary to explore methods for reducing the unpleasant flavor. Among the catechins in green tea, EGCg is present at the highest level followed by ECg, EGC, and EC [27]. And, tea catechins is included at the range of 10–300 lM in green tea drink [27]. Therefore, to reduce the unpleasant flavor, it

624

M. Narukawa et al. / Biochemical and Biophysical Research Communications 405 (2011) 620–625

A

EGC

EC

EGCg

ECg

Ratio

1.25

Before

0.35

After

B

C

0.4

: ECg : EGCg : EC : EGC

0.3 0.2 0.1

: ECg : EGCg : EC : EGC

60

40

20

0.0 1

-0.1

100

80 Taste Intensity (mm)

Relative Response to 3 μM ATP

0.5

10

100

1000

Conc. ( μM)

0

1

10

100

1000

Conc. ( μM)

Fig. 4. Comparison of the responsiveness of hTAS2R39 to catechins with the human sensory evaluation. (A) Representative ratiometric images of the response to 300 lM EC, EGC, ECg, or EGCg in fura-2-loaded HEK293T cells coexpressing Ga16gust44 and hTAS2R39. (B) Dose–response relationships of 1–300 lM catechins on [Ca2+]i in HEK293T cells coexpressing hTAS2R39 and Ga16gust44. Data values for catechins were indicated as the percentage of response to 3 lM ATP (n = 7–15). (C) Change in taste intensity in response to 10, 30, 100, and 300 lM catechins in humans. The values represent the mean ± SE (n = 11).

is important to control the quantities of EGCg and ECg in green tea. Cell-based assay that employs hTAS2R39 is expected to be used for evaluation of the bitterness similar to the human taste sense. ECg and EGCg can then be determined to provide an objective measurement system to assess the bitterness of catechins. Acknowledgments We thank Dr. Keiko Abe (The University of Tokyo, Tokyo, Japan) for the technical advices and Dr. Takashi Ueda (Nagoya City University, Nagoya, Japan) for providing the Ga16gust44 chimera G protein. This work was supported by the Shizuoka Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST. References [1] H.N. Graham, Green tea composition, consumption, and polyphenol chemistry, Prev. Med. 21 (1992) 334–350. [2] T. Sato, G. Miyata, The nutraceutical benefit, part I: green tea, Nutrition 16 (2000) 315–317. [3] J.P. Henry, P. Stephens-Larson, Reduction of chronic psychosocial hypertension in mice by decaffeinated tea, Hypertension 6 (1984) 437–444. [4] Z.Y. Chen, P.T. Chan, Antioxidative activity of green tea catechins in canola oil, Chem. Phys. Lipids 82 (1996) 163–172. [5] A. Zhang, Q.Y. Zhu, Y.S. Luk, K.Y. Ho, K.P. Fung, Z.Y. Chen, Inhibitory effects of jasmine green tea epicatechin isomers on free radical-induced lysis of red blood cells, Life Sci. 61 (1997) 383–394. [6] M.G. Hertog, E.J. Feskens, P.C. Hollman, M.B. Katan, D. Kromhout, Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly study, Lancet 342 (1993) 1007–1011.

[7] S.T. Shi, Z.Y. Wang, T.J. Smith, J.Y. Hong, W.F. Chen, C.T. Ho, C.S. Yang, Effects of green tea and black tea on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation, DNA methylation, and lung tumorigenesis in A/J mice, Cancer Res. 54 (1994) 4641–4647. [8] Z.Y. Wang, M.T. Huang, Y.R. Lou, J.G. Xie, K.R. Reuhl, H.L. Newmark, C.T. Ho, C.S. Yang, A.H. Conney, Inhibitory effects of black tea, green tea, decaffeinated black tea, and decaffeinated green tea on ultraviolet B light-induced skin carcinogenesis in 7,12-dimethylbenz[a]anthracene-initiated SKH-1 mice, Cancer Res. 54 (1994) 3428–3435. [9] K. Imai, K. Nakachi, Cross sectional study of effects of drinking green tea on cardiovascular and liver diseases, Br. Med. J. 310 (1995) 693–696. [10] S.S. Noor-Soffalina, S. Jinap, S. Nazamid, S.A.H. Nazimah, Effect of polyphenol and pH on cocoa Maillard-related flavour precursors in a lipidic model system, Int. J. Food Sci. Tech. 44 (2009) 168–180. [11] K.B. Wang, J.Y. Ruan, Analysis of chemical components in green tea in relation with perceived quality, a case study with Longjing teas, Int. J. Food Sci. Tech. 44 (2009) 2476–2484. [12] S. Scharbert, T. Hofmann, Molecular definition of black tea taste by means of quantitative studies, taste reconstitution, and omission experiments, J. Agric. Food Chem. 53 (2005) 5377–5384. [13] D. Rossetti, J.H.H. Bongaerts, E. Wantling, J.R. Stokes, A.M. Williamson, Astringency of tea catechins: more than an oral lubrication tactile percept, Food Hydrocolloids 23 (2009) 1984–1992. [14] J. Chandrashekar, M.A. Hoon, N.J. Ryba, C.S. Zuker, The receptors and cells for mammalian taste, Nature 444 (2006) 288–294. [15] H. Matsunami, J.P. Montmayeur, L.B. Buck, A family of candidate taste receptors in human and mouse, Nature 404 (2000) 601–604. [16] E. Adler, M.A. Hoon, K.L. Mueller, J. Chandrashekar, N.J. Ryba, C.S. Zuker, A novel family of mammalian taste receptors, Cell 100 (2000) 693–702. [17] J. Chandrashekar, K.L. Mueller, M.A. Hoon, E. Adler, L. Feng, W. Guo, C.S. Zuker, N.J. Ryba, T2Rs function as bitter taste receptors, Cell 100 (2000) 703–711. [18] K.L. Mueller, M.A. Hoon, I. Erlenbach, J. Chandrashekar, C.S. Zuker, N.J. Ryba, The receptors and coding logic for bitter taste, Nature 434 (2005) 225–229. [19] W. Meyerhof, C. Batram, C. Kuhn, A. Brockhoff, E. Chudoba, B. Bufe, G. Appendino, M. Behrens, The molecular receptive ranges of human TAS2R bitter taste receptors, Chem. Senses 35 (2010) 157–170.

M. Narukawa et al. / Biochemical and Biophysical Research Communications 405 (2011) 620–625 [20] M. Narukawa, H. Kimata, C. Noga, T. Watanabe, Taste characterisation of green tea catechins, Int. J. Food Sci. Tech. 45 (2010) 1579–1585. [21] J.P. Slack, A. Brockhoff, C. Batram, S. Menzel, C. Sonnabend, S. Born, M.M. Galindo, S. Kohl, S. Thalmann, L. Ostopovici-Halip, C.T. Simons, I. Ungureanu, K. Duineveld, C.G. Bologa, M. Behrens, S. Furrer, T.I. Oprea, W. Meyerhof, Modulation of bitter taste perception by a small molecule hTAS2R antagonist, Curr. Biol. 20 (2010) 1104–1109. [22] B. Bufe, P.A. Breslin, C. Kuhn, D.R. Reed, C.D. Tharp, J.P. Slack, U.K. Kim, D. Drayna, W. Meyerhof, The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception, Curr. Biol. 15 (2005) 322–327. [23] T. Ueda, S. Ugawa, H. Yamamura, Y. Imaizumi, S. Shimada, Functional interaction between T2R taste receptors and G-protein alpha subunits expressed in taste receptor cells, J. Neurosci. 23 (2003) 7376–7380.

625

[24] R.S. Ostrom, C. Gregorian, P.A. Insel, Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways, J. Biol. Chem. 275 (2000) 11735–11739. [25] T. Kumazawa, M. Kashiwayanagi, K. Kurihara, Contribution of electrostatic and hydrophobic interactions of bitter substances with taste receptor membranes to generation of receptor potentials, Biochim. Biophys. Acta 888 (1986) 62–69. [26] A. Shoji, A. Yanagida, H. Shindo, Y. Shibusawa, Comparison of elution behavior of catechins in high-performance liquid chromatography with that on highspeed countercurrent chromatography, Bunseki Kagaku 53 (2004) 953–958. [27] Z.Y. Chen, Q.Y. Zhu, D. Tsang, Y. Huang, Degradation of green tea catechins in tea drinks, J. Agric. Food. Chem. 49 (2001) 477–482.