Activation of TRPV1 and TRPA1 by Black Pepper Components

90964 (178)

Biosci. Biotechnol. Biochem., 74 (5), 90964-1–5, 2010

Yukiko OKUMURA,1 Masataka NARUKAWA,1;2;3 Yusaku IWASAKI,1;2 Aiko ISHIKAWA,3 y Hisashi MATSUDA,4 Masayuki YOSHIKAWA,4 and Tatsuo WATANABE1;2;3;

1Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan 2Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan 3School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan 4Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan

Received December 25, 2009; Accepted January 26, 2010; Online Publication, May 7, 2010 [doi:10.1271/bbb.90964]

We searched in this study for novel agonists of V, member 1 (TRPV1).4,5) TRPV1 is also activated by transient receptor potential cation channel, subfamily capsaicin (CAP), a pungent component of hot pepper. It V, member 1 (TRPV1) and transient receptor potential is known that TRPV1 activation enhances the energy cation channel, subfamily A, member 1 (TRPA1) in metabolism.6) It is therefore possible that, like CAP, pepper, focusing attention on 19 compounds contained piperine can also enhance the energy metabolism via in black pepper. Almost all the compounds in HEK cells TRPV1, although the detailed mechanism for this is heterogeneously expressed TRPV1 or TRPA1, increas- unknown. Various piperine analogs have also been ingAdvance the intracellular Ca2þ concentration ([Ca View2þ] )ina 7,8) i identified in pepper. Since it has been reported that concentration-dependent manner. Among these, piper- CAP analogs activated TRPV1 as well as CAP,9,10) it is ine, isopiperine, isochavicine, piperanine, pipernonaline, also possible that piperine analogs could activate dehydropipernonaline, retrofractamide C, piperolein A, TRPV1. and piperolein B relatively strongly activated TRPV1. Many thermosensitive TRP receptors have recently The EC50 values of these compounds for TRPV1 were been cloned as TRPV1 homologs. Among these, the 0.6–128 M. Piperine, isopiperine, isochavicine, pipera- transient receptor potential cation channel, subfamily A, nine, piperolein A, piperolein B, and N-isobutyl- member 1 (TRPA1) is similar to TRPV1 in several (2E,4E)-tetradeca-2,4-diamide also relatively strongly respects: TRPA1 is coexpressed in TRPV1-expressing 11) activated TRPA1, the EC50 values of these compounds somatosensory neurons, and TRPA1 induces sensory for TRPA1 were 7.8–148 M. The Ca2þ responses of stimuli such as painProofs and pungency like TRPV1.12) Many these compounds for TRPV1 and TRPA1 were signifi- pungent components such as allyl isothiocyanate (AITC; cantly suppressed by co-applying each antagonist. We wasabi),13) cinnamaldehyde (cinnamon),14) hydroxy - identified in this study new transient receptor potential sanshool (zanthoxylum fruit),15) and miogadial and (TRP) agonists present in black pepper and found that miogatrial (myoga)16) have been reported to be TRPA1 piperine, isopiperine, isochavicine, piperanine, pipero- agonists. We therefore hypothesized that the pungent lein A, and piperolein B activated both TRPV1 and compounds contained in pepper could also activate TRPA1. TRPA1. We investigated in this study the activation of TRPV1 and TRPA1 by 19 components of pepper. Key words: pepper; piperine; transient receptor potential cation channel, subfamily V, member 1 Materials and Methods (TRPV1); transient receptor potential cation channel, subfamily A, member 1 (TRPA1); Materials. CAP, piperine, and capsazepine (CPZ) were purchased intracellular calcium concentration from Sigma (St. Luis, MO, USA). Allyl isothiocyanate (AITC) was obtained from Wako Pure Chem. Ind. (Osaka, Japan), and HC030031 was purchased from ChemBridge (San Diego, CA, USA). All other Pepper, Peperineer nigrum Linne, grows on a climb- chemicals were of guaranteed reagent grade. ing plant belonging to the Piperineerales family. The dried fruit of pepper is used as a spice throughout the Purification of the pepper components. We studied 19 components world. Traditional Chinese medicine classifies pepper of black pepper: piperine, isopiperine, isochavicine, piperanine, as a food that generates body heat. Pepper has such piperonal, methylpiperate, fragaramide, pipernonaline, dehydropiper- pharmacological effects as anti-oxidative,1) anti-proto- nonaline, piperlonguminine, retrofractamide A (retro A), retrofracta- zoan,2) and prokinetic.3) mide B (retro B), retrofractamide C (retro C), guineensine, brachystamide B, N-isobutyl-(2E,4E)-tetradeca-2,4-diamide (N-tetra), Piperine is a pungent component of pepper and has N-isobutyl-(2E,4E)-octadeca-2,4-diamide (N-octa), piperolein A, and been reported as acting on the capsaicin receptor, piperolein B. The structural formulae of the black pepper components transient receptor potential cation channel, subfamily are shown in Fig. 1. With the exception of piperine, these components

y To whom correspondence should be addressed. Fax: +81-54-264-5550; E-mail: [email protected] 2þ 2þ Abbreviations: AITC, allyl isothiocyanate; [Ca ]i, intracellular Ca concentration; CAP, capsaicin; CPZ, capsazepine; retro A, retrofractamide A; retro B, retrofractamide B; retro C, retrofractamide C; N-octa, N-isobutyl-(2E,4E)-octadeca-2,4-diamide; N-tetra, N-isobutyl-(2E,4E)-tetradeca- 2,4-diamide; TRP, transient receptor potential; TRPA1, transient receptor potential cation channel, subfamily A, member 1; TRPV1, transient receptor potential cation channel, subfamily V, member 1 90964-2 Y. OKUMURA et al.

Piperine Retrofractamide A (retro A) Piperanine

Piperolein A Retrofractamide B (retro B)

Pipernonailne Piperolein B Retrofractamide C (retro C)

Piperonal Dehydro pipernonailne Guineensine

Methyl piperate Brachystamide B Fragaramide

Isopiperine N-Isobutyl-(2E,4E)-tetradeca-2,4-diamide (N-Tetra)

AdvanceIsochavicine Piperlonguminine View N-Isobutyl-(2E,4E)-octadeca-2,4-diamide (N-Octa) Fig. 1. Chemical Structures of the 19 Pepper Components. were isolated and purified from black pepper (Gaban Co., Tokyo, inhibitory activity of the antagonists, 30 mM CPZ for TRPV1 or Japan) and the fruits of Piper chaba Hunter purchased in Thailand. HC030031 for TRPA1 was added to 100 mM of each pepper compound, Piperolein A and B were extracted with hexane from black pepper and except for N-isobutyl-(2E,4E)-octadeca-2,4-diamide; we used 30 mM of purified by silica gel and reversed-phase chromatography. The other this compound in DMSO since it did not dissolve in 100 mM. We used compounds were obtained from P. chaba. The dried fruits of P. chaba 0.01–100 mM piperine, isopiperine, isochavicine, piperanine and piper- were extracted with 80% aqueous acetone and partitioned between nonaline, 0.1–100 mM dehydropipernonaline, retro C, piperolein A and ethyl acetate and water, before the ethyl acetate fraction was purified piperolein B, and 0.1 nM–10 mM CAP to obtain the dose-response by silica gel and reversed-phase chromatography.8) curves for TRPV1. We used 0.03–100 mM piperine, isopiperine, isochavicine and piperanine,Proofs 0.1–100 mM piperolein A, piperolein B Cloning and expression of human TRPV1 and human TRPA1. and N-tetra, and 0.01–100 mM AITC for TRPA1. In some experiments, Human TRPV1 and TRPA1 cDNA were amplified by RT-PCR, using TRPV1 antagonist CPZ (30 mM) or TRPA1 antagonist HC030031 mRNA respectively obtained from human brain first-strand cDNA (30 mM) were added together with these compounds. Each test (Agilent Technologies, Santa Clara, CA, USA) and human WI38 cells. compound was prepared in DMSO and added to the loading solution Human TRPV1 cDNA was subcloned into pcDNA3 (Invitrogen, to a final DMSO concentration of 0.1% or 0.2%. A 5 mM amount of Carlsbad, CA, USA) and then transfected into HEK293T cells by using ionomycin was added to each well to elicit maximum fluorescence the SuperFect transfection reagent (Qiagen, Hilden, Germany). After intensity. The data values for the test compounds are expressed as the culturing in the presence of 750 mg/ml of G418, we obtained a stable percentage response to 5 mM ionomycin. Curves were fitted and HEK293T cell line that expressed human TRPV1. parameters estimated by using Prism 4.0a software (Graph Pad The expression of full-length human TRPA1 in stable HEK cells Software, San Diego, CA, USA). was induced by the tetracycline-inducible T-REx expression system from Invitrogen. The hTRPA1 cDNA was subcloned into pcDNA4/TO Results (Invitrogen) and then transfected into HEK T-REx cells by using the Lipofectamine 2000 reagent (Invitrogen). HEK T-REx cells that stably maintained the hTRPA1 gene were selected by using 500 mg/ml The activity of TRPV1 and TRPA1 by the 19 pepper of zeocin and 10 mg/ml of blasticidin, and were grown according to the components was compared to that by TRPV1 agonist manufacturer’s instructions. The following primers were used for CAP or TRPA1 agonist AITC. cloning: human TRPV1 forward primer, 50-GCAAGGATGAAGAA- GAAATGGA-30 and reverse primer, 50-TCACTTCTCCCCGGAAG- 0 0 Effect of the 19 pepper components on human TRPV1 CGC-3 ; human TRPA1 forward primer, 5 -TGGGTCAATGAAGTG- Figure 2A shows the calcium response induced by CAG-30 and reverse primer, 50-GAAGGTCTGAGGAGCTAAGGC-30. each of the 19 pepper components in TRPV1-expressing Measurement of the intracellular Ca2þ concentration. The intra- cells. Among these components, piperine, isopiperine, 2þ 2þ cellular Ca concentration ([Ca ]i) was measured by FlexStation isochavicine, piperanine, pipernonaline, dehydropiper- II (Molecular Devices, Sunnyvale, CA, USA). The cells were seeded nonaline, retro C, piperolein A, and piperolein B 2þ into 96-well plates 24 h before the assay. To obtain TRPA1-expressing strongly increased [Ca ]i in TRPV1-expressing HEK cells, 1 mg/ml of tetracycline was added to induce the expression HEK293T cells. A dose-response curve was drawn for of the TRPA1 protein. The cells were sub-cultured every week, the these compounds (Fig. 2B). Their EC50 values (Table 1) highest passage number being 50. The cells were loaded with 3 mM were 250 to 53000 times larger than that of representa- Fluo-4-AM (Molecular Probes, Eugene, OR, USA) for 1 h at 37 Cina tive TRPV1 agonist CAP (2.4 nM). The maximum loading buffer (5.37 mM KCl, 0.44 mM KH2PO4, 137 mM NaCl, 0.34 mM Na2HPO4.7H2O, 5.56 mMD-glucose, 20 mM HEPES, 1 mM activity of piperine, isopiperine, isochavicine, pipera- CaCl2, 0.1% BSA, and 250 mM probenecid at pH 7.4). To study the nine, dehydropipernonaline, piperolein A was nearly TRP Agonists in Black Pepper 90964-3

A 100 µM Sample only 100 µM Sample + 30 µM Capsazepine HEK293T ionomycin M µ M µ % Activity to 5 30

AdvanceB View ionomycin

M µ % Activity to 5

Concentration (Log M) Proofs Fig. 2. Activation of TRPV1 by the Different Pepper Components. Fluo-4 Ca2þ responses of HEK 293 cells expressing human TRPV1 are shown. The data value for each of these compounds is expressed as a percentage response to that of 5 mM ionomycin. A, Ca2þ responses to 19 different pepper components. Filled columns show TRPV1 activation by 100 mM of the components, except for N-isobutyl-(2E,4E)-octadeca-2,4-diamide when 30 mM was used. Striped columns indicate TRPV1 activation by these compounds with the addition of 30 mM CPZ. Unfilled columns indicate the response to each of these compounds of HEK293T not expressing TRPV1. Each column shows the mean SEM. and indicate p < 0:05 and p < 0:0005, respectively (unpaired t-test; n ¼ 8). B, Concentration-response curves for piperine, isopiperine, isochavicine, piperanine, pipernonaline, dehydropipernonaline, retro C, piperolein A, piperolein B, and CAP. Each point represents the mean SEM (n ¼ 4).

equal to that of CAP. The maximum response values Table 1. TRPV1 and TRPA1 Activation Potency of the Pepper Compounds were not calculated for piperononaline and retro C which were paticularly low when compared to that TRPV1 TRPA1 of CAP (60.3%). Adding TRPV1 antagonist CPZ 2þ EC50 Max EC50 Max signiﬁcantly decreased the Ca response by these (mM) (%) (mM) (%) compounds (Fig. 2A). In addition, these compounds 2þ Capsaicin 0.0024 60.3 — — hardly increased [Ca ]i in HEK293T cells not express- Allyl isothiocyanate — — 0.51 88.7 ing TRPV1 (Fig. 2A). These results indicate that the Piperine 0.6 57.7 29.7 77.0 black pepper components activated TRPV1. Isopiperine 5.6 64.9 32.6 84.8 Isochavicine 3.4 61.3 71.1 NC Eﬀect of the 19 pepper components on human TRPA1 Piperanine 6.0 61.3 148.3 NC Figure 3A shows the calcium response induced by Pipernonaline 128.0 NC —— Dehydropipernonaline 29.3 59.0 — — each of the 19 pepper components in TRPA1-expressing Retro C 119.0 NC —— cells. Among these components, piperine, isopiperine, Piperolein A 17.3 58.2 7.8 89.9 isochavicine, piperanine, piperolein A, piperolein B, and 2þ Piperolein B 19.1 39.9 11.1 70.4 N-tetra strongly increased [Ca ]i in TRPA1-expressing N-Tetra — — 19.2 56.4 HEK T-REx cells (Fig. 3A). The dose-response curve

Percent value to the response of 5 mM ionomycin for these compounds is shown in Fig. 3B. A higher NC, not calculated concentration of each of these compounds was required for action on TRPA1 than that on TRPV1. The EC50 values for these compounds were 16–297 times higher 90964-4 Y. OKUMURA et al.

A 100 µM Sample only 100 µM Sample + 30 µM HC-030031 HEK T-REx ionomycin M µ M µ % Activity to 5 30

AdvanceB View ionomycin M µ Proofs % Activity to 5

Concentration (Log M)

Fig. 3. Activation of TRPA1 by the Different Pepper Components. Fluo-4 Ca2þ responses of HEK T-REx cells expressing human TRPA1 are shown. Data values for these compounds are each expressed as a percentage response to that of 5 mM ionomycin. A, Ca2þ responses to 19 different pepper components. Filled columns show TRPA1 activation by 100 mM of the components, except for N-isobutyl-(2E,4E)-octadeca-2,4-diamide, when 30 mM was used. Striped columns indicate TRPA1 activation by these compounds with the addition of 30 mM HC030031. Unfilled columns indicate the responses to these compounds of HEK T- REx not expressing TRPA1. Each column shows the mean SEM. and indicate p < 0:05 and p < 0:0005, respectively (unpaired t-test; n ¼ 4). B, Concentration-response curves for piperine, isopiperine, isochavicine, piperanine, piperolein A, piperolein B, N-tetra, and AITC. Each point represents the mean SEM (n ¼ 4). than that of representative TRPA1 agonist AITC isopiperine, isochavicine, piperanine, piperolein A, and (0.5 mM). The relative maximum activity of piperine, piperolein B activated both TRPV1 and TRPA1. The isopiperine, piperolein A, piperolein B, and N-tetra to EC50 values show that the piperine analogs activated that of AITC was 0.867, 0.956, 1.01, 0.794, and 0.636, TRPV1 more strongly than TRPA1. However, none of respectively (Table 1). The maximum response was not components acted more strongly than CAP and AITC. calculated for isochavicine and piperanine. The addition The results also clarified that piperine activated both of TRPA1 antagonist HC-030031 (30 mM) significantly TRPV1 and TRPA1. decreased the Ca2þ response induced by these com- Six components containing the piperidine ring (piper- pounds (Fig. 3B). In addition, these compounds hardly ine, isopiperine, isochavicine, piperanine, piperolein A, 2þ increased [Ca ]i in HEK T-REx cells not expressing and piperolein B) activated both TRPV1 and TRPA1. TRPA1 (Fig. 3A). These results indicate that the black The piperidine ring might therefore be important for pepper components activated TRPA1. activating both of these receptors. N-tetra could strongly activate only TRPA1. Since the structure of N-tetra is Discussion similar to known TRPA1 agonist hydroxy--sanshool, we believe that, among all the TRP agonists in pepper, This study clarified that TRP agonists other than only N-tetra has a structure that can selectively activate piperine were present in pepper. Among them, piperine, TRPA1. TRP Agonists in Black Pepper 90964-5 TRPV1 agonist CAP is known to increase energy References metabolism by stimulating the sympathetic nervous system. 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