Diallyl sulfides in garlic activate both TRPA1 and TRPV1

Biochemical and Biophysical Research Communications 382 (2009) 545–548

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Diallyl sulfides in garlic activate both TRPA1 and TRPV1 Kanako Koizumi a, Yusaku Iwasaki a,b, Masataka Narukawa a,b, Yuji Iitsuka c, Tomomi Fukao c, Taiichiro Seki c, Toyohiko Ariga c, Tatsuo Watanabe a,b,* a

Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c Department of Applied Life Sciences, Nihon University Graduate School of Bioresource Sciences, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b

a r t i c l e

i n f o

Article history: Received 25 February 2009 Available online 17 March 2009

Keywords: Garlic Diallyl sulfide Diallyl disulfide Diallyl trisulfide TRPV1 TRPA1 Intracellular calcium concentration

a b s t r a c t We searched for novel agonists of TRP receptors especially for TRPA1 and TRPV1 in foods. We focused attention on garlic compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). In TRPA1 or TRPV1 heterogeneously expressed CHO cells, all of those compounds increased [Ca2+]i in concentration-dependent manner. The EC50 values of DADS and DATS were similar to that of allyl isothiocyanate (AITC) and that of DAS was 170-fold larger than that of AITC. Maximum responses of these sulfides were equal to that of AITC. The EC50 values of these compounds for TRPV1 were around 100 lM against that of capsaicin (CAP), 25.6 nM and maximum responses of garlic compounds were half to that of CAP. The Ca2+ responses were significantly suppressed by co-application of antagonist. We conclude that DAS, DADS, and DATS are agonist of both TRPA1 and TRPV1 but with high affinity for TRPA1. Ó 2009 Elsevier Inc. All rights reserved.

Garlic is a perennial herbaceous plant belonged to Liliaceae. It is classified into foods having body-warming nature in traditional Chinese medicine. Garlic is made up of 65% of water, 30% of carbohydrates, and 5% of other components, mainly consisting sulfurcontaining compounds [1]. Sulfur-containing compounds show various pharmacological effects such as anti-virus [2], anti-oxidative [3], anti-cancer [4], immunoactive [5], anti-protozoan [6] ones. Diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are volatile compounds produced when garlic bulb is cut and have been reported to inhibit carcinogenesis [7–9]. Of these, DATS exerts cancer inhibiting effect by modifying cysteine residues in b-tubulin of microtubule then blocking tubulin polymerization, resulting in interruption of cell cycle at M phase [9]. Allyl isothiocyanate (AITC) and cinnamaldehyde (CNA) are pungent compounds in Wasabi and cinnamon, respectively. Both compounds cause pungency by activating TRPA1 (transient receptor potential cation channel, subfamily A, member 1) expressed in sensory nerve endings [10,11]. Allicin, a pungent compound of garlic,

Abbreviations: TRP, transient receptor potential; TRPA1, transient receptor potential cation channel, subfamily A, member 1; TRPV1, transient receptor potential cation channel, subfamily V, member 1; DAS, diallyl sulfide; DADS, diallyl disulfide; DATS, diallyl trisulfide; AITC, allyl isothiocyanate; CAP, capsaicin; CNA, cinnamaldehyde; CPZ, capsazepine; [Ca2+]i, intracellular Ca2+ concentration. * Corresponding author. Address: Graduate School of Nutritional and Environmental 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). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.066

also activates TRPA1 strongly [12]. Recently, AITC and CNA have been shown to activate TRPA1 by covalently binding to cysteine residue of TRPA1 in cell membrane [13]. Because DATS binds to b-tubulin of microtubule, DATS is expected to activate TRPA1. Therefore, we investigated effects of DATS and its structural analogs DADS and DATS on TRPA1 activation. On the other hand, TRPA1 is known to co-expressed with TRPV1 (transient receptor potential cation channel, subfamily V, member 1) in sensory nerve endings [14]. TRPV1-activating food components have been found mainly from foods of warm or hot nature such as hot pepper, black pepper, garlic, ginger, and sansho in traditional Chinese medicine [15]. Hydroxy a-sanshool has been reported to act both TRPV1 and TRPA1 [16,17], and there is a possibility that these foods components may also stimulate both ion channels. Accordingly, we studied effects of DAS, DADS, and DATS on TRPV1 activation together with TRPA1 activity. Materials and methods Materials. Capsaicin (CAP) and capsazepine (CPZ) were purchased from Sigma (St. Louis, MO, USA). Allyl isothiocyanate (AITC) was purchased from Wako Pure Chem. Ind. (Osaka, Japan). HC030031 was purchased from ChemBridge (San Diego, CA, USA). All other chemicals were of guaranteed reagent grade. Purification of DAS, DADS, and DATS. Structural formula of DAS, DADS, and DATS, was shown in Fig. 1. DAS (97% purity) was purchased from LKT Lab. Inc. (St. Paul, MN, USA) and used without

K. Koizumi et al. / Biochemical and Biophysical Research Communications 382 (2009) 545–548

diallyl sulfide (DAS) S S diallyl disulfide (DADS) S

S S

diallyl trisulfide (DATS) Fig. 1. Chemical structures of DAS, DADS, and DATS.

further purification. DADS was obtained from Tokyo Chemical Ind. (Tokyo, Japan) purified by high-performance liquid chromatography (HPLC, Alliance 2695, Waters Co., Milford, MA, USA) on an Inertsil ODS-3 column (6  250 mm, GL Science, Tokyo, Japan). DATS was synthesized by the method of Milligan et al. [18] and purified by HPLC. Purity of DADS and DADS was higher than 99%. Cloning and expression of rat TRPV1 and human TRPA1. Human TRPA1 and rat TRPV1 cDNA were amplified by RT-PCR, using mRNA obtained from human WI38 cells and rat C6 glioma cells, respectively. Stable CHO cell lines expressing full length human TRPA1, was generated using tetracycline inducible T-RExTM expression system from Invitrogen (Carlsbad, CA, USA). hTRPA1 cDNA was subcloned into pcDNA4/TO (Invitrogen), and then transfected into CHO T-RExTM cells by using the Lipofectamine 2000 reagent (Invitrogen). CHO T-RExTM cells that stably maintained hTRPA1 gene were selected in the presence of 500 lg/ml zeocin and 10 lg/ml blastidin and grown according to the manufacturer’s instructions. Rat TRPV1 cDNA was subcloned into pcDNA3 (Invitrogen, CA, USA), and then transfected into CHO cells by using the SuperFect transfection reagent (Quiagen, Hilden, Germany). After culturing in the presence of 750 lg/ml G418, we obtained stable CHO cell line, which express rat TRPV1. The following primers were used for cloning: human TRPA1 forward primer: 50 -tgggtcaatgaagtg cag-30 ; reverse primer: 50 -gaaggtctgaggagctaaggc-30 , rat TRPV1 forward primer: 50 -atccaagcttgaaaggatggaacaacggg-30 ; reverse primer: 50 -tatctctagattatttctcccctgggacc-30 . Measurement of intracellular Ca2+ concentration. Intracellular 2+ concentration, [Ca2+]i was measured by Flex StationTM II Ca (Molecular Devices, Sunnyvale, CA, USA). Twenty four hours before assay, cells were seeded in 96-well plate. In regard to TRPA1 expressing CHO cells, 1 lg/ml tetracycline was added to induce the expression of the TRPA1 protein. Cells were sub-cultured every week and the highest passage number used was 50. Their cells were loaded with 3 lM Fluo4-AM (Molecular Probes, Eugene, OR, USA) for 1 h at 37 °C in a loading buffer (5.37 mM KCl, 0.44 mM KH2PO4, 137 mM NaCl, 0.34 mM Na2HPO47H2O, 5.56 mM D-glucose, 20 mM HEPES, 1 mM CaCl2, 0.1% BSA, and 250 mM probenecid at pH 7.4). For measurements of dose–response curves for TRPA1 1–1000 lM DAS, 0.1–300 lM DADS, 0.001–300 lM DATS, and 0.01–100 lM AITC were employed. In the case of TRPV1, 1–100 lM DAS, DADS, and DATS and 0.1 nM–1 lM CAP were used. In some experiments, TRPV1 antagonist CPZ or TRPA1 antagonist HC030031 was added with these compounds. In the case of inhibition by HC030031, 30 lM HC030031 was added to 300, 1000 lM DAS, 10, 30 lM DADS, 1, 3 lM DATS, and 10, 100 lM AITC. When inhibition by CPZ is measured, 10 lM CPZ was added to 100, 300 lM DAS, 30, 100 lM DADS, 30 lM DATS, and 0.1, 10 lM CAP. Because turbid occurred when 10 lM CPZ was added to 100 lM DATS, 3 lM CPZ was added to 100 lM DATS. The test compounds were prepared in DMSO and add to the loading solution (final DMSO conc. 0.1% or 0.2%). Five micromolar ionomycin was

added to each well to elicit maximum fluorescence intensity. Data values for test compounds were expressed as a percent response to 5 lM ionomycin. Curve fitting and parameter estimation were carried out using Prism 4.0a software (Graph Pad Software, San Diego, CA, USA). Results The activities of DAS, DADS, and DATS to TRPA1 and TRPV1 were compared with the activity of TRPA1 agonist AITC or TRPV1 agonist CAP. Action of DADS, DADS, and DATS on human TRPA1 DAS, DADS, and DATS increased [Ca2+]i in TRPA1-expressing CHO T-RExTM cells in concentration-dependent manner (Fig. 2A). EC50 of DAS, DADS, and DATS is 254, 7.55, and 0.49 lM, respectively (Table 1). In comparison with the EC50 1.47 lM of representative TRPA1 agonist AITC, DATS acts on TRPA1 by lower concentration. Maximum activity of DAS, DADS, and DATS is 90.8%, 76.6%, and 83.6%, respectively (Table 1). By addition of TRPA1 antagonist HC-030031 (30 lM), Ca2+ response by these compounds significantly decreased (Fig. 3B). In addition, these compounds hardly increased [Ca2+]i in CHO T-RExTM cells which did not express TRPA1 (data not shown). These results indicated that DAS, DADS, and DATS activate TRPA1.

A

B

100 M Ionomycin

S

% Activity to 5

546

***

: sample only : sample + HC030031

***

*

*** ***

75 ***

***

300 1000

10

***

50

25

0 10

100

AITC

DAS

30

DADS

1

30 (

M)

DATS 2+

Fig. 2. TRPA1 is activated by DAS, DADS, and DATS. Fluo-4 Ca responses of CHO T-RExTM cells expressing human TRPA1. Data values for these compounds were expressed as a percent response to 5 lM ionomycin. (A) DAS, DADS, DATS, and AITC induced concentration–response curves. Each point represents the mean ± SEM. n = 7–8. (B) DAS, DADS, and DATS induced Ca2+ responses were inhibited by TRPA1 antagonist 30 lM HC030031. White columns indicate TRPA1 activities by DAS (300, 1000 lM), DADS (10, 30 lM), and DATS (1, 3 lM). Black columns indicate TRPA1 activities by these compounds with HC030031. Each column is the mean ± SEM. * and *** indicates p < 0.05 and 0.0005, respectively (unpaired t-test). n = 8.

K. Koizumi et al. / Biochemical and Biophysical Research Communications 382 (2009) 545–548

DAS

DADS

DATS

AITC

addition, these compounds hardly increased [Ca2+]i in CHO cells which did not express TRPV1 (data not shown). These results indicated that DAS, DADS, and DATS activate TRPV1.

254 90.8

7.55 76.6

0.49 83.6

1.47 79.8

Discussion

Table 1 TRPA1 activation potencies of DAS, DADS, DATS, and AITC.

EC50 (lM) Maximum response (%)

Number of experiments are 7–8. Maximum responses were normalized to 5 lM ionomycin induced response. EC50 is expressed as the mean value.

A

M Ionomycin

100

% Activity to 5

B

***

***

: sample only : sample + CPZ

75

50

***

*** *** **

25

*

***

0 0.1

1

100

CAP

300

DAS

30

100

DADS

30

100 (

M)

DATS

This study clearly showed that DAS, DADS, and DATS not only activate TRPA1 but also TRPV1 and these compounds have higher affinity and larger maximum response for TRPA1 than TRPV1. Further, both TRPA1 and TRPV1 activation by DAS, DADS, and DATS were inhibited by specific antagonists of these receptors and no activation by these compounds were found in parent cells, indicating that non-specific activation of these receptors did not occur. Many of TRPA1 agonists are known to activate this ion channel by covalent binding to cysteine residues at inner cellular domain of human TRPA1 [9]. DATS also modifies cysteine residue of b-tubulin. Therefore, TRPA1 activation by DATS is thought to be caused by modification of cysteine and activations by DAS and DADS may also be produced by the same mechanism. Oi et al. reported that of sulfur-containing compounds in garlic DADS and DATS enhance energy metabolism [19]. One of TRPV1 agonists, capsaicin is known to increase energy metabolism through stimulation of sympathetic nervous system. Through this, capsaicin induces adrenaline secretion and secreted adrenaline acts on b-receptor in liver and white adipose tissues (WAT), then decomposition of glycogen of liver and triglyceride of WAT occurs, resulted in energy metabolism enhancement [20]. Recently, we showed that TRPA1 agonists such as allyl isothiocyanate and cinnamaldehyde induce adrenaline secretion in anesthetized rats [21]. DADS and DATS also stimulated adrenaline secretion in rats [19]. On the contrary, DAS did not induce significant catecholamine secretion [19]. This is consistent with this study: DAS was less potent agonist on EC50 values of both TRPA1 and TRPV1 than DADS and DATS. Acknowledgment

Fig. 3. TRPV1 is activated by DAS, DADS, and DATS. Fluo-4 Ca2+ responses of CHO cells expressing rat TRPV1. Data values for these compounds were expressed as a percent response to 5 lM ionomycin. (A) DAS, DADS, DATS, and CAP induced concentration–response curves. Each point represents the mean ± SEM. n = 6–8. (B) DAS, DADS, and DATS induced Ca2+ responses were inhibited by TRPV1 antagonist CPZ (100 lM DATS: 3 lM CPZ, other compounds: 10 lM CPZ). White columns indicate TRPV1 activities by DAS (100, 300 lM), DADS (30, 100 lM), and DATS (30, 100 lM). Black columns indicate TRPV1 activities by these compounds with CPZ. Each column is the mean ± SEM. *, ** and *** indicates p < 0.05, 0.005, and 0.0005, respectively (unpaired t-test). n = 6–18.

Action of DADS, DADS, and DATS on rat TRPV1 DAS, DADS, and DATS increased [Ca2+]i in TRPV1-expressing CHO cells in concentration-dependent manner (Fig. 3A). EC50 of DAS, DADS, and DATS was 151, 36.7, and 43.7 lM, respectively (Table 2). In comparison with the EC50 (1.47 lM) of representative TRPV1 agonist CAP, their EC50 were considerably high. Maximum activity of DAS, DADS, and DATS were about 1/2 in comparison with that of CAP. By addition of TRPV1 antagonist CPZ, Ca2+ response by these compounds significantly decreased (Fig. 3B). In

Table 2 TRPV1 activation potencies of DAS, DADS, DATS, and CAP.

EC50 (lM) Maximum response (%)

547

DAS

DADS

DATS

CAP

151 48.6

36.7 35.9

43.7 44.4

0.0256 86.2

Number of experiments are 6–8. Maximum responses were normalized to 5 lM ionomycin induced response. EC50 is expressed as the mean value.

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