Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine

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gy 576 (2007) 143–150www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine

Angelica Penuelas a, Kimihito Tashima b,⁎, Shizuko Tsuchiya a, Kenjiro Matsumoto b,Tomonori Nakamura a, Syunji Horie b, Shingo Yano a

a Department of Molecular Pharmacology and Pharmacotherapeutics, Graduate School of Pharmaceutical Sciences,Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan

b Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo Togane, Chiba 283-8555, Japan

Received 9 March 2007; received in revised form 4 August 2007; accepted 13 August 2007Available online 17 August 2007

Abstract

TRPA1 is a member of the transient receptor potential (TRP) channel family expressed in sensory neurons. The present study focused on theeffects of TRPA1 activation on contractile responses in isolated mouse intestine preparations. The jejunum, ileum, and proximal and distal colonwere surgically isolated from male ddY mice. Intestinal motility was recorded as changes in isotonic tension. TRPA1, TRPM8, and TRPV1expressions were examined by reverse transcription-polymerase chain reaction (RT-PCR). A TRPA1 agonist allyl isothiocyanate (AITC) dose-dependently induced contractions in the proximal and distal colon, whereas in the jejunum and ileum, even 100 μM AITC caused very littlecontraction. Likewise, a TRPA1 and TRPM8 agonist icilin, a TRPA1 agonist allicin, and a TRPV1 agonist capsaicin induced contractions in thecolon. However, a TRPM8 agonist menthol induced long-lasting relaxation in the colon. Repeated exposure to AITC produced desensitization ofits own contraction in the colon. Moreover, contractions induced by AITC generate cross-desensitization with icilin and capsaicin. Tetrodotoxincompletely abolished AITC-induced contractions in the colon, whereas atropine significantly attenuated AITC-induced contractions in the distalcolon, but not in the proximal colon. Menthol-induced relaxation in the colon was not inhibited by tetrodotoxin and atropine. RT-PCR analysisrevealed the expression of TRPA1 and TRPV1, but not TRPM8, throughout the mouse intestine. These results suggest that TRPA1, but notTRPM8, are functionally expressed in the enteric nervous system throughout the mouse intestine on neurons that may also co-express TRPV1, yetthe contractile responses to TRPA1 activation differ depending on their location along the intestine.© 2007 Elsevier B.V. All rights reserved.

Keywords: Colon; Contraction; Allyl isothiocyanate; Capsaicin; TRPA1; TRPV1

1. Introduction

Capsaicin, the major pungent constituent of hot peppers, is anagonist of the vanilloid receptor (transient receptor potentialchannel-vanilloid subfamily member 1, TRPV1), which repre-sents a non-selective cation channel activated by noxious stimulisuch as heat, acidosis, and chemicals (Caterina et al., 2000; Jordtand Julius, 2002; Tominaga et al., 1998). Capsaicin excites asubset of primary sensory neurons with somata in dorsal rootganglia or trigeminal ganglia via TRPV1 (Calixto et al., 2005).In general, capsaicin-sensitive sensory neurons transmit noxiousinformation perceived as pain and itching to the central nervoussystem. TRPV1 immunoreactivity has been documented in

⁎ Corresponding author. Tel.: +81 475 53 4585; fax: +81 475 53 8811.E-mail address: [email protected] (K. Tashima).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.08.015

nerves within myenteric ganglia and interganglionic fiber tractsthroughout the gastrointestinal tract (Ward et al., 2003; Horieet al., 2004). Moreover, it has been shown that the capsaicin-sensitive sensory neurons participate in the regulation ofgastrointestinal motility in vitro (Takaki et al., 1989; Jin et al.,1990; Lefebvre et al., 1991; Horie et al., 2006).

Wasabi, horseradish, and mustard oil owe their pungency toisothiocyanate compounds such as allyl isothiocyanate (AITC).Topical application of mustard oil to the skin and mouth activatesthe underlying sensory nerve endings, thereby producing pain,which is similar to the pharmacological effects of capsaicin. Inaddition, mustard oil is thought to have beneficial effects in thegastrointestinal tract, including enhancement of appetite anddigestive function. However, there is no evidence concerning thefacilitating effects of mustard oil or allyl isothiocyanate ongastrointestinal functions such as motility.

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Recent studies have shown that some members of thetransient receptor potential (TRP) family respond not only to aunique range of temperatures but also to natural plant-derivedproducts including mustard oil and menthol, which is similar tocapsaicin (Calixto et al., 2005). TRPA1 is activated by noxiouscold, mustard oil, and allicin, a pungent natural compound ingarlic (Bandell et al., 2004; Jordt et al., 2004; Story et al., 2003).TRPM8 is activated by innocuous cold temperatures andmenthol, a mint-derived cooling compound (McKemy et al.,2002; Peier et al., 2002). Furthermore, it has reported thatTRPA1 is present in a sub-population of primary sensoryneurons expressing TRPV1 but not TRPM8 (Kobayashi et al.,2005; Story et al., 2003). Thus, it was suggested that TRPA1 isinvolved in regulating gastrointestinal motility through capsa-icin-sensitive sensory neurons. Indeed, in the rat urinarybladder, it has been shown that TRPA1 activation by allylisothiocyanate and TRPV1 activation by capsaicin both causecontraction (Andrade et al., 2006).

The present study was designed to investigate whetherTRPA1 is present in the mouse intestine, including the jejunum,ileum, and, proximal and distal colon, and if TRPA1 agonistsaffect gastrointestinal motility. The purposes were to (i)determine the mechanical effects induced by TRPA1 activationwith agonists in the longitudinal smooth muscle, (ii) character-ize the underlying mechanisms, and (iii) examine the expressionof TRPA1 in the mouse intestine.

2. Materials and methods

2.1. Animals

Male ddY-stain mice (Japan SLC, Hamamatsu, Japan)weighing 30–40 g were used. Animals were housed undercontrolled environmental conditions (temperature at 24±2 °Cand light on 7:00 am to 7:00 pm) and fed commercial mousechow MF (Oriental Yeast, Tokyo, Japan). Animal experimentswere performed in compliance with the “Guiding Principles forthe Care and Use of Laboratory Animals” approved by theJapanese Pharmacological Society and the guidelines approvedby the Ethical Committee on Animal Care and AnimalExperimentation of the Graduate School of PharmaceuticalSciences, Chiba University, Japan. The number of animals usedwas kept to the minimum necessary for a meaningfulinterpretation of the data. Animals were euthanized by cervicaldislocation before the isolation of tissues.

2.2. Isolated intestine preparations and measurement oftension

Whole segments of the mouse intestine, including thejejunum, ileum, and proximal and distal colon, were removedand placed in Krebs–Henseleit solution containing (in mM):112.1 NaCl, 5.9 KCl, 2.0 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25.0NaHCO3, and 11.5 glucose, at pH 7.4. A segment of each tissue,about 1.0–1.5 cm length, was set up under a 0.5 g load in a 10-ml organ bath filled with Krebs–Henseleit solution. The bathwas maintained at 37 °C and continuously bubbled with a

mixture of 95% O2 and 5% CO2. Contraction was recordedusing an isotonic transducer (NEC San-ei Instruments Ltd.,Tokyo Japan). At the start of each experiment, the maximumresponse to 1 μM acetylcholine, which was incubated for 3 min,was obtained from a segment of each tissue to evaluate thecontractile and relaxant effects of the tested agents, includingallyl isothiocyanate, icilin, allicin, and menthol. These agentswere added to the organ bath for 2 min before wash out.Because the second responses to some TRP channel activators,including allyl isothiocyanate, icilin, allicin, and capsaicin, weredesensitized easily in the same preparation (as shown in results),the concentration–response curves for allyl isothiocyanate,icilin, and allicin were obtained in other intestinal segmentsfrom different mice. In some experiments, preparations wereincubated for 5 min with antagonists or inhibitors, and then theresponse induced by TRP channel activators was measured inthe presence of the respective antagonists or inhibitors.

To investigate the possibility of cross-desensitization in thecontractile responses induced by allyl isothiocyanate, icilin, andcapsaicin, the intestine preparations were incubated for 3 minwith 100 μM allyl isothiocyanate or 3 μM capsaicin, thenwashed more than 3 times with the stimulant-free buffer,followed by incubation with the indicated agents for 2 min,which differed from that used for the first exposure, to measurethe contraction for the second time. The interval between thefirst exposure to the agents and the second exposure to theagents was about 15 min.

Each response was expressed as either a percentage of themaximum longitude contraction induced by 1 μM acetylcholine(% of ACh contraction) or relaxation induced by 100 μMpapaverine (% of papaverine relaxation).

2.3. Reverse transcription-polymerase chain reaction

Total RNA was isolated by using an RNeasy kit (Qiagen,CA, USA) according to the manufacturer's protocol. In brief,about 20–30 mg of freshly isolated mouse tissues, includingdorsal root ganglia, jejunum, ileum, and proximal and distalcolon, was immediately submerged in Buffer RLT (Qiagen),which inhibited RNase activation, and was homogenized byusing a Multi-beads shocker (Yasui Kikai, Osaka, Japan).Reverse transcription-polymerase chain reaction (RT-PCR) wasperformed by using a one-step RT-PCR Kit (Qiagen) and athermal cycler GeneAmp PCR System 9700 (Applied Biosys-tems, CA, USA) for 35 cycles (TRPA1, TRPV1, and β-actin) or40 cycles (TRPM8) under the following conditions: reversetranscription at 50 °C for 30 min, initial denaturation; 15 min at95 °C and then 1 min at 94 °C, followed by a 1-min annealingstep at 55 °C for TRPM8 or 56 °C for TRPA1, TRPV1, and β-actin and 1 min elongation at 72 °C. The primers sequenceswere 5′-ACA AGA AGTACC AAA CAT TGA CAC A-3′ and5′-TTA ACT GCG TTTAAG ACA AAATTC C-3′ for mouseTRPA1, 5′-CAA AAC ACC CAA CCT GGT CAT TTC-3′ and5′-CAC AGT GCG TGG TAA AAA GCG-3′ for mouseTRPM8, 5′-AGC GAG TTC AAA GAC CCA GA-3′ and 5′-TTC TCC ACCAAGAGGGTC AC-3′ for mouse TRPV1, and5′-GGC CCA GAG CAA GAG AGG TAT CC-3′ and 5′-ACG

Fig. 1. Representative recordings of the contractile responses to allylisothiocyanate (AITC) in the isolated mouse intestine. AITC (100 μM) wasapplied to the preparations of the proximal colon (A), distal colon (B), jejunum(C), and ileum (D). Results are expressed as a percentage of the contractileresponses induced by acetylcholine (ACh, 1 μM) in each tissue. Preparations ofthe distal colon were incubated with 1 μM acetylcholine (E). The initial and peakcontraction was expressed as a percentage of the maximal contraction inducedby 1 μM acetylcholine (% of ACh response). ▲: time when AITC or ACh wasadded. W: Wash-off. The recording is one example of 6 independentexperiments. (F) Concentration–response curve for AITC (10–100 μM) in theisolated mouse intestine, including jejunum (▲), ileum (●), proximal colon (■),and distal colon (◆). Results are expressed as a percentage of the contractileresponses induced by acetylcholine (ACh, 1 μM). Data represent means±S.E.M.from 4–7 experiments. Note that contractile responses were more pronounced inthe distal and proximal colon than that in jejunum and ileum.

Fig. 2. Concentration–response curves for icilin (3–30 μM, A) and allicin (1–10 μM, B) in the isolated mouse proximal colon (■) and distal colon (◆). Datarepresent means±S.E.M. from 3–4 experiments. Results are expressed as apercentage of contractile responses induced by acetylcholine (ACh, 1 μM). Notethat icilin and allicin also induced pronounced contractions in the isolated mouseproximal and distal colon.

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CAC GAT TTC CCT CTC AGC-3′ for β-actin. The PCRproducts were separated on 3% (wt/vol) agarose gel in Tris–acetate EDTA buffer, stained with ethidium bromide, andanalyzed by LAS 3000 (FUJIFILM, Tokyo, Japan).

2.4. Drugs

The following drugs were used: acetylcholine hydrochloride,atropine sulfate, ethidium bromide, and icilin were from Sigma-Aldrich (MO, USA). Allicin, allyl isothiocyanate, capsaicin,menthol, papaverine hydrochloride, absolute ethanol, dimethylsulfoxide, tetrodotoxin, and Tween 80 were from Wako PureChemical Industries (Tokyo, Japan). Allyl isothiocyanate wasdissolved in 0.5% Tween 80 in saline solution, icilin wasdissolved in DMSO, and menthol, allicin, and capsaicin weredissolved in absolute ethanol prior to dilution in deionizedwater, respectively. Other drugs were dissolved in deionizedwater with no organic solvent or detergent. The finalconcentrations of Tween 80, DMSO, and ethanol in the bath

were less than 0.06%, 0.1%, and 0.3%, respectively. Thevehicles used had no pharmacological effects on the tonus ofpreparations or the agonist-induced contraction.

2.5. Data analysis

Values are presented as means±S.E.M. for three or moreindependent experiments. The number in experiments (n) refersto the number of experimental animals used. Statistical analysisof data was done with Graph Pad Prism 4.0 (Hearne ScientificSoftware, IL, USA). The statistical significance of differencesbetween two groups was assessed using a two-tailed Student'st-test. Multiple comparisons against a single control groupwere made by one-way analysis of variance (ANOVA) withDunnett's multiple comparisons test. Pb0.05 was consideredstatistically significant.

3. Results

3.1. Contractions in response to TRPA1 agonists

Application of the TRPA1 agonist allyl isothiocyanate (10–100 μM) to mouse intestine preparations caused a concentra-tion-dependent contractile response in the mouse proximal anddistal colon, with maximum responses being 23.1±0.4% and32.7±1.8%, respectively, of that induced by 1 μM acetylcholine(Fig. 1E, F). The contractile responses to allyl isothiocyanate,however, were different in the proximal and distal colons(Fig. 1A, B). In the proximal colon, spontaneous contractions

Fig. 3. Representative recording of the relaxant responses to menthol (1 mM) inthe isolated mouse proximal (A) and distal colon (B). Concentration–responsecurves for menthol in the isolated mouse proximal colon and distal colon (C).Results are expressed as a percentage of relaxant responses induced bypapaverine (100 μM). Data represent means±S.E.M. from 4 experiments. ▲:time when menthol was added.

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were obviously enhanced when allyl isothiocyanate wasapplied, whereas in the distal colon the contraction was biphasicwith an initial, rapidly developing peak contraction followed bya long-lasting secondary contraction. In contrast, in the jejunumand ileum, contractile responses to allyl isothiocyanate even atthe maximal concentration (100 μM) were slight, maximum

Fig. 4. Desensitization to allyl isothiocyanate (AITC, 100 μM) in the isolated mouse pto AITC and capsaicin on the contractile responses to icilin and capsaicin in the is(100 μM), followed by the exposure of icilin (30 μM, B) or capsaicin (3 μM, D) infollowed by the exposure of AITC (100 μM) in the proximal and distal colon (C). Res1 μM). Data represent mean±S.E.M. from 4–5 experiments. ⁎Pb0.05 by Student's talone.

responses being 6.5±1.1% and 9.0±1.3%, respectively(Fig. 1C, D). To investigate the possibility that other TRPA1activators could also induce contractions on isolated intestinepreparations, tissues were stimulated with the TRPA1 andTRPM8 agonist icilin (3–30 μM). Icilin also induced aconcentration-dependent contraction in preparations of themouse proximal and distal colon, maximum responses being18.2±2.6% and 38.3±2.5%, respectively (Fig. 2A), whereas inpreparations of the mouse jejunum and ileum, weakercontractile responses to icilin were observed (data not shown).Like allyl isothiocyanate and icilin, the known TRPA1 andTRPV1 agonist allicin (1–10 μM) induced contractions in themouse proximal and distal colon. The maximal responses to10 μM allicin were 19.1±2.9 and 19.9±5.2, respectively, forthe proximal and distal colon (Fig. 2B).

Menthol, a TRPM8 agonist, produced slow and long-lastingrelaxations in the mouse proximal and distal colon, the maximumrelaxation responses being 40.8±1.8% and 46.8±2.9% in thepresence of 1 mM menthol, respectively of that induced by100 μM papaverine (Fig. 3A, B, C). These results excluded thepossibility that TRPM8 agonistic action is involved in icilin-induced contractions.

3.2. Self- and cross-desensitization to contractions in responseto TRPA1 or TRPV1 activation

Although no specific antagonist for TRPA1 has beendemonstrated to date, it has been shown that a typical featureof TRPV1 is its ability to desensitize rat trigeminal ganglionneurons in culture in the presence of the TRPV1 agonist

roximal and distal colon (A). Effect of desensitizing the preparations by exposureolated mouse proximal and distal colon. Desensitization was caused by AITCthe proximal and distal colon. Desensitization was caused by capsaicin (3 μM),ults are expressed as a percentage of contractile responses to acetylcholine (ACh,-test compared with the first response to AITC, or icilin, or capsaicin stimulation

Fig. 5. Effect of tetrodotoxin (TTX, 1 μM) or atropine (1 μM) on the contractile responses to allyl isothiocyanate (AITC, 30 and 100 μM) on the isolated mouseproximal and distal colon (A, B). Tetrodotoxin totally abolished the contractile responses to AITC (30 μM) in the isolated mouse proximal and distal colon, althoughtetrodotoxin by itself induced about 24 or 35%-increased basal tone in the isolated mouse proximal and distal colon, respectively. Interestingly, pretreatment withatropine (1 μM) did not affect contractile responses to AITC (100 μM) in the proximal colon, whereas atropine significantly decreased contractile responses to AITC(100 μM) in the distal colon. Effects of tetrodotoxin (1 μM) and atropine (1 μM) on the relaxant responses to menthol in the isolated mouse proximal and distal colon(C, D). Neither tetrodotoxin nor atropine affected relaxant responses to menthol (1 mM) in the isolated mouse proximal and distal colon. AITC-induced contractionsare expressed as a percentage of contractile responses to acetylcholine (ACh, 1 μM), whereas results for menthol-induced relaxations are expressed as a percentage ofrelaxant to papaverine (100 μM). Data are mean±S.E.M. of 3–4 experiments. ⁎Pb0.05 by Student's t-test compared with the control group.

Fig. 6. Expression of TRPA1, TRPM8 and TRPA1 mRNA in the mouseintestine, including the jejunum, ileum, proximal and distal colon by RT-PCR.Total RNAwas isolated from mouse dorsal root ganglia (DRG), jejunum, ileum,proximal and distal colon. The mRNA levels were analyzed by PCR (35–40cycles) using primers for TRPA1, TRPM8, TRPV1, and β-actin. Mouse DRGwas used as positive control samples for TRPA1, TRPM8, and TRPV1 mRNA.Predicted sizes of PCR products are TRPA1 (243 bp), TRPM8 (682 bp), TRPV1(233 bp), and β-actin (460 bp), respectively.

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capsaicin (Bartho et al., 1999; Jin et al., 1990; Szallasi andBlumberg, 1999). Indeed, it was observed that repeatedexposure of the isolated mouse proximal and distal colon tothe TRPV1 agonist capsaicin (3 μM) cause progressivedesensitization of its contractile responses under this experi-mental condition (data not shown). This observation led us tospeculate that allyl isothiocyanate will cause desensitization ifthe character of TRPA1 is very similar to that of TRPV1. Wefound that repeated exposure to the maximal concentration ofallyl isothiocyanate (100 μM) at 15 min intervals caused adesensitization of the contractile responses to thesecond application of allyl isothiocyanate in the mouse prox-imal and distal colon, the responses decreasing to 1.3±1.3%and 3.1±1.9% at the second exposure, which are signifi-cantly lower than the first exposure in the isolated mouseproximal (23.1±0.97%) and distal colon (32.7±4.9%), respec-tively (Fig. 4A). In addition, it has been shown that TRPA1 isexpressed in TRPV1-expressing sensory neurons, suggestingthat TRPA1 has properties similar to TRPV1. We investigatedwhether repeated exposure to allyl isothiocyanate leads to adiminished response to a subsequent addition of the TRPA1 andTRPM8 agonist icilin and the TRPV1 agonist capsaicin. In thisstudy, it was found that cross-desensitization mutually occurredamong various TRP agonists, including allyl isothiocyanate,icilin, and the TRPV1 agonist capsaicin, in the mouse proximaland distal colon. Contractile responses to 30 μM icilin werecompletely abolished in allyl isothiocyanate-desensitized pre-parations as compared with naive preparations (Fig. 4B),suggesting that both allyl isothiocyanate and icilin activated a

common receptor, TRPA1, in these preparations. Secondly,contractile responses to 100 μM allyl isothiocyanate werepartially attenuated in capsaicin-desensitized preparations of theisolated mouse proximal and distal colon, the reduction being60.7±14.2% and 64.5±10.3%, respectively (Fig. 4C). Theresponses to 3 μM capsaicin were also partially attenuated inallyl isothiocyanate-desensitized preparations of the iso-lated mouse proximal and distal colon, the reductions being45.7±16.1% and 85.6±7.6%, respectively (Fig. 4D), suggest-ing that allyl isothiocyanate and capsaicin activate sensoryneurons that co-express both TRPA1 and TRPV1.

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3.3. Pharmacological features of contractions in response toTRPA1 activation

To clarify the mechanism underlying contractile responses toTRPA1 activation with allyl isothiocyanate in the mouseproximal and distal colon, we evaluated the effects of the Na+

channel blocker tetrodotoxin and the muscarinic receptorantagonist atropine on those contractile responses. Tetrodotoxinalone at 1 μM enhanced the basal tone in the longitudi-nal muscle of the proximal and distal colon by 34.6±5.2% and23.5±4.5%, respectively. Tetrodotoxin completely abolishedcontractile responses to allyl isothiocyanate (30 μM) (Fig. 5A),suggesting that spontaneous contractions in both regions of thecolon are predominantly mediated by inhibitory enteric neuronsand that contractions induced by allyl isothiocyanate involvedneural mechanisms. In addition, pretreatment with 1 μMatropine significantly decreased, but did not abolish, thecontractile responses to allyl isothiocyanate (100 μM) in themouse distal colon, the inhibition being 68.7±6.6% (Fig. 5B).In the proximal colon, however, pretreatment with 1 μMatropine did not affect the contractile responses to allylisothiocyanate (100 μM). These results suggested that therelease of acetylcholine in the mouse distal colon partiallymediates allyl isothiocyanate-induced longitudinal musclecontraction, whereas in the mouse proximal colon atropine-insensitive neurons regulated contractile responses to allylisothiocyanate. On the other hand, pretreatment with neithertetrodotoxin nor atropine inhibited relaxant responses tomenthol (1 mM) in the proximal and distal colon (Fig. 5C, D).

3.4. mRNA expressions of TRPA1, TRPM8, and TRPV1

The expressions of TRPA1, TRPM8 and TRPV1 mRNAwere examined in the mouse intestine, including the jejunum,ileum, and proximal and distal colon. TRPA1 mRNA (predictedsize: 243 bp) was found in the mouse jejunum, ileum, andproximal and distal colon (Fig. 6). TRPM8 mRNA (predictedsize: 682 bp) was not detected throughout the mouse intestine,although TRPV1 mRNA (predicted size: 233 bp) was expressedas well as TRPA1 mRNA. TRPA1, TRPM8, and TRPV1mRNAwere observed in all positive controls, which were fromthe mouse dorsal root ganglia. The expression levels of β-actinmRNAwere unchanged among all tested samples. These resultssuggested that TRPA1 and TRPV1, but not TRPM8, areexpressed at mRNA levels throughout the mouse intestine.

4. Discussion

Allyl isothiocyanate, the pungent ingredient of mustard oil andwasabi, stimulates a subset of primary afferent neurons by directlyactivating the transient receptor potential channel TRPA1(Bandell et al., 2004; Bautista et al., 2006; Jordt et al., 2004).So far, it has been reported that allyl isothiocyanate induced aconcentration-dependent vasodilation in mesenteric arterialsegments (Bautista et al., 2005), and that allyl isothiocyanateelicited concentration-dependent contractions of isolated raturinary bladder (Andrade et al., 2006). However, whether or not

TRPA1 activation produces smooth muscle excitatory and/orinhibitory effects on gastrointestinal motility has not beenclarified. The present study demonstrated that allyl isothiocyanatepredominantly caused contractions in the mouse proximal anddistal colon. The responsiveness to allyl isothiocyanate was morepotent in the colon than that in the jejunum and ileum. In addition,we also demonstrated a marked difference in contraction patternsbetween in the proximal and distal colon, which werecharacterized by enhanced spontaneous tone in the proximalcolon and transient contractions in the distal colon.

We were intrigued by the possibility that TRPA1 activatorsother than allyl isothiocyanate might also induce coloncontractions. It has been shown that icilin, which is a coolingagent and potent activator of TRPM8, activates TRPA1 but notTRPV1, in CHO cells expressing TRPA1, TRPM8, and TRPV1(Story et al., 2003). Interestingly, here we showed that icilindose-dependently contracted the proximal and distal colon, thecontraction patterns observed for icilin being similar to thoseinduced by allyl isothiocyanate. Certainly, the TRPM8 agonisticaction of icilin may also be involved in those contractileresponses. To clarify that possibility, we found in the presentstudy that menthol, which is now well recognized as a TRPM8agonist (Andersson et al., 2004; Behrendt et al., 2004; Chuang etal., 2004; Peier et al., 2002; Voets et al., 2004), dose-dependentlycaused relaxation in the proximal and distal colon. In addition, itwas also found that the relaxant responses to menthol were notinhibited by pretreatment with tetrodotoxin, an inhibitor ofneurotransmission, and that the mRNA expression of TRPM8was undetected throughout the mouse intestine. Thus, theseresults indicate that the relaxant responses to menthol in themouse proximal and distal colon are mediated by a non-neuronalpathway and not by TRPM8 activation. Indeed, in agreementwith the present findings, it has been reported that menthol exertsCa2+ channel blocking properties, resulting in the intestinalsmooth muscle relaxation (Hawthorn et al., 1988). We could notclarify whether the contractions induced by icilin wereattributable to TRPM8 activation pharmacologically, becausementhol, by itself, elicited the inhibition of Ca2+ channels ratherthan TRPM8 activation under our experimental conditions.Further study will be required on this point in the future.

Based on in vitro studies, it has been suggested that allicin isthe active ingredient in garlic that causes a prickling sensationthrough activation of TRPA1 (Bautista et al., 2005, 2006). Thisstudy found that allicin dose-dependently induced contractionsin the mouse proximal and the distal colon, although thecontractions induced by allicin are smaller than those inducedby either allyl isothiocyanate or icilin. The potency may beweak because allicin is an unstable compound and is convertedeasily into the more-stable sulfide compounds, which have a farless potent action on TRPA1 (Macpherson et al., 2005).

Furthermore, Bandell et al. (2004) demonstrated thatcinnamaldehyde, the main component of cinnamon oil, is themost-specific TRPA1 agonist by using calcium imaging inTRPA1-expressed CHO cells and adult rat dorsal root ganglianeurons. Indeed, it was shown that cinnamaldehyde dose-dependently induced contractions in the isolated rat urinarybladder, but that these responses were 60% less efficacious

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when allyl isothiocyanate was applied (Andrade et al., 2006). Inthe preliminary experiments, however, we failed to observecontractions in response to cinnamaldehyde in the isolatedmouse proximal and distal colon, although it was found thatcinnamaldehyde produced long-lasting relaxations in thelongitudinal smooth muscle of these preparations (data notshown). Because a recent report has shown that cinnamalde-hyde induced endothelium-independent vasorelaxation throughthe blocking of Ca2+ channels (Yanaga et al., 2006), it ispossible that cinnamaldehyde induces smooth muscle relaxationthrough the blockade of Ca2+ channels. Therefore, all theseresults suggested that allyl isothiocyanate, icilin, and allicin acton TRPA1 to induce smooth muscle contractions in the mouseproximal and distal colon.

In contrast to menthol, the present study demonstrated thatallyl isothiocyanate-induced contractions exhibited a tetrodo-toxin-sensitive component in the mouse proximal and distalcolon, suggesting that these responses are mediated by someneurogenic processes. In addition, it was also found that thecontractile responses to allyl isothiocyanate were moderatelyinhibited by the muscarinic receptor antagonist atropine in themouse distal colon, suggesting that acetylcholine, as anexcitatory neurotransmitter, is mediated by and connected toTRPA1-expressed neurons in the mouse lower intestine. In themouse proximal colon, however, contractile responses to allylisothiocyanate were not affected by atropine, indicating that thecontractions were mediated by neurotransmitters other thanacetylcholine, i.e., possibly tachykinins such as substance P andneurokinin A. This idea is partly supported by a previous reportthat atropine was not capable of altering the allyl isothiocya-nate-induced contraction in rat urinary bladder, suggesting thatanother tetrodotoxin-sensitive component in the rat urinarybladder seems to be an axon reflex arrangement that involvestachykinins as neuropeptides (Andrade et al., 2006). In addition,it has been shown that atropine-insensitive contractions in theporcine ileum are abolished by the tachykinins neurokinin-1receptor antagonists (Schmidt and Hoist, 2002).

Due to a lack of specific TRPA1 channel blockers, only alimited pharmacological characterization has been conducted todate. Therefore, we focused on the self- and cross-desensitizationbetween agonists, because the tested TRPA1 antagonists in ourpreliminary study, gentamicin and camphor, which are reported toblock TRPA1 channels in vitro (Nagata et al., 2005; Macphersonet al., 2006), were unable to specifically block allyl isothiocya-nate-induced contractile responses in the isolated mouse intestinepreparations (data not shown). They were more effective ininhibiting capsaicin-induced contractions in the mouse proximaland distal colon, although ruthenium red,which is known to blockTRP channel family including TRPV1 and TRPA1 (Nagata et al.,2005), completely inhibited allyl isothiocyanate-induced con-tractions (data not shown).

One peculiar feature of capsaicin action is that prolonged orrepeated stimulation results in marked desensitization of theTRPV1 receptor (Maggi et al., 1987; Takaki et al., 1989; Lefebvreet al., 1991; Jin et al., 1990). It has been shown that allylisothiocyanate desensitized the current of HEK 293 cellsexpressing TRPA1 (Nagata et al., 2005). Recently, Andrade et al.

(2006) reported that capsaicin, allyl isothiocyanate, and cinnamal-dehyde produced desensitization of the urinary bladder contraction.In addition, the present study showed that allyl isothiocyanate-induced contractions in the mouse proximal and distal colon arefollowed by desensitization of sensory nerves, which becomeprogressively less responsive to stimulation. Moreover, we foundthat allyl isothiocyanate-desensitized preparations were almostcompletely unresponsive to icilin and only partially responsive tocapsaicin. It is possible that the repeated exposure to allylisothiocyanate desensitizes TRPA1 on sensory neurons and alsoreduces the stored neuropeptides in the nerve ending. The presentresults suggest that allyl isothiocyanate and icilin act on the samesubset of sensory neurons that express both TRPA1 and TRPV1,resulting in production of their contractile effects.

Interestingly, it has been reported that TRPA1 is expressedon dorsal root ganglia and trigeminal ganglia, which alsoexpress TRPV1, whereas TRPM8 is expressed independently ofTRPA1 and TRPV1 on dorsal root ganglia and trigeminalganglia (Bautista et al., 2005; Katsura et al., 2006; Kobayashiet al., 2005; McKemy, 2005; Story et al., 2003). In contrast,TRPA1 expression in peripheral tissues is still unknown,although Mustafa and Oriowo (2005) demonstrated mRNAexpression of TRPM8 in the rat gastric fundus by RT-PCRanalysis. We found in the present study that mRNA expressionsof TRPA1 and TRPV1, but not TRPM8, were presentthroughout the mouse intestine, including the jejunum, ileum,and proximal and distal colon. These findings strongly supportthe observation that allyl isothiocyanate, icilin, and allicin, butnot menthol, act on TRPA1 receptors to produce theircontractile effects.

Taking the present findings together, it is concluded thatTRPA1, but not TRPM8, is functionally expressed in the entericsensory neurons that may also be co-expressed with TRPV1throughout the mouse intestinal tract; however, the contractileresponses to TRPA1 activation differ according to where thesegments are located in the mouse intestine. The TRPA1-activated response is mediated by acetylcholine in the distalcolon, whereas it is elicited through an atropine-insensitivepathway in the proximal colon.

Acknowledgements

This work was supported by a Grant-in-Aid ScientificResearch from the Ministry of Education, Culture, Sports,Science and Technology, Japan (to S. Horie, K. Tashima, K.Matsumoto), and by a grant from the Hamaguchi Fundation forthe advancement of biochemistry. We are grateful to theresearch funds administered by the president of Josai Interna-tional University for supporting the present study.

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