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Tashima, Toshihiko Murayama, John V. Priestley and Syunji HorieKenjiro Matsumoto, Emi Kurosawa, Hiroyuki Terui, Takuji Hosoya, Kimihitosensitivity in rectum and distal colon

andcapsaicin in mouse large intestine: high abundance Localization of TRPV1 and contractile effect of

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Localization of TRPV1 and contractile effect of capsaicin in mouse largeintestine: high abundance and sensitivity in rectum and distal colon

Kenjiro Matsumoto,1 Emi Kurosawa,2 Hiroyuki Terui,1 Takuji Hosoya,1 Kimihito Tashima,1

Toshihiko Murayama,2 John V. Priestley,3 and Syunji Horie1

1Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, Togane, Chiba;2Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba,Japan; and 3Neuroscience Centre, Institute of Cell and Molecular Science, Barts and The London School of Medicine andDentistry, Whitechapel, London, United Kingdom

Submitted 5 October 2008; accepted in final form 28 May 2009

Matsumoto K, Kurosawa E, Terui H, Hosoya T, Tashima K,Murayama T, Priestley JV, Horie S. Localization of TRPV1 andcontractile effect of capsaicin in mouse large intestine: high abun-dance and sensitivity in rectum and distal colon. Am J PhysiolGastrointest Liver Physiol 297: G348–G360, 2009. First publishedJune 4, 2009; doi:10.1152/ajpgi.90578.2008.—We investigated im-munohistochemical differences in the distribution of TRPV1 channelsand the contractile effects of capsaicin on smooth muscle in the mouserectum and distal, transverse, and proximal colon. In the immunohis-tochemical study, TRPV1 immunoreactivity was found in the mucosa,submucosal, and muscle layers and myenteric plexus. Large numbersof TRPV1-immunoreactive axons were observed in the rectum anddistal colon. In contrast, TRPV1-positive axons were sparsely distrib-uted in the transverse and proximal colon. The density of TRPV1-immunoreactive axons in the rectum and distal colon was much higherthan those in the transverse and proximal colon. Axons double labeledwith TRPV1 and protein gene product (PGP) 9.5 were detected in themyenteric plexus, but PGP 9.5-immunoreactive cell bodies did notcolocalize with TRPV1. In motor function studies, capsaicin induceda fast transient contraction, followed by a large long-lasting contrac-tion in the rectum and distal colon, whereas in the transverse andproximal colon only the transient contraction was observed. Thecapsaicin-induced transient contraction from the proximal colon to therectum was moderately inhibited by an NK1 or NK2 receptor antag-onist. The capsaicin-induced long-lasting contraction in the rectumand distal colon was markedly inhibited by an NK2 antagonist, but notby an NK1 antagonist. The present results suggest that TRPV1channels located on the rectum and distal colon play a major role inthe motor function in the large intestine.

vanilloid; immunohistochemistry; afferent nerve; substance P; neuro-kinin A

THE TRANSIENT RECEPTOR POTENTIAL vanilloid type 1 (TRPV1)channel, also referred to as the capsaicin receptor, is describedas a polymodal sensor of noxious heat (�43°C), low pH (pH �5.9), and inflammatory pain (12). In the gut, TRPV1 modulatesphysiological functions such as motility, secretion, circulation,and visceral nociception (8, 18, 19). Immunohistochemicalstudies have demonstrated the presence of TRPV1 channels inthe gastrointestinal tract in the mucosa, submucosal layer, andmuscular layer in the guinea pig, mouse, and rat gastrointesti-nal tract (23, 31, 37). Therefore, TRPV1 has become anattractive molecular target for pharmacological intervention ingastroenterological research.

Capsaicin, the main pungent constituent in red peppers ofthe genus Capsicum, stimulates TRPV1 channels, leading tothe activation of primary afferent neurons with unmyelinatedor thinly myelinated nerve fibers, the so-called capsaicin-sensitive afferent neurons. Capsaicin has been utilized bynumerous investigators to activate afferent fiber endings instudies of the visceral effects of TRPV1 in the gastrointestinaltract, and it has been established that capsaicin-sensitive pri-mary afferent neurons participate in the regulation of gastro-intestinal motility (8). For example, capsaicin induces contrac-tions in the guinea pig ileum (8) and relaxation in human smalland large intestines (7, 27, 28). Afferent nerves in the gut notonly send signals to the central nervous system but also providea local efferent-like effect by releasing neuropeptides. Capsa-icin-sensitive nerve fibers that contain neuropeptides includingtachykinins and calcitonin gene-related peptide have beenidentified in several mammalian species. In particular, tachy-kinins such as substance P and neurokinin A mediate theexcitatory effect of capsaicin on sensory neurons (8). Neuro-chemical and functional evidence indicates that tachykinins areexpressed in extrinsic and intrinsic primary afferent neurons(15, 21).

The gastrointestinal tract contains two types of primaryafferent neurons: intrinsic primary afferent neurons with cellbodies, processes, and synaptic connections in the gut wall, andextrinsic primary afferent neurons with their cell bodies innodose and jugular ganglia or in dorsal root ganglia (16).TRPV1 channels are found predominantly on the sensoryafferent nerve fibers implicated in pain transduction. TRPV1nerve fibers in the gut appear to be predominantly extrinsic inorigin (22, 31, 37), but their existence in intrinsic neurons inthe gastrointestinal tract was also reported (3).

The TRPV1 channel has become an attractive target fortreatment of colonic and rectal disorders such as irritable bowelsyndrome and inflammatory bowel disease (2, 13, 39). How-ever, most studies of the physiological roles of TRPV1 inmotor function in the intestine have been carried out mainlyusing isolated guinea pig ileum. The effects of capsaicin andthe distribution of TRPV1 channels in the lower gastrointesti-nal tract remain largely unstudied. In particular, regional andhence functional differences in TRPV1 among the rectum anddistal, transverse, and proximal colon have never been re-ported. In the present study, we investigated the immunohis-tochemical differences in the distribution of TRPV1 channelsand the contractile effect of capsaicin on smooth muscle in themouse rectum and distal, transverse, and proximal colon. Wealso investigated the contractile mechanism of capsaicin in

Address for reprint requests and other correspondence: K. Matsumoto, Labo-ratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai InternationalUniv., 1 Gumyo, Togane, Chiba 283-8555, Japan (e-mail: kenjiro@jiu.ac.jp).

Am J Physiol Gastrointest Liver Physiol 297: G348–G360, 2009.First published June 4, 2009; doi:10.1152/ajpgi.90578.2008.

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each segment of the lower gastrointestinal tract, with thespecial reference to the involvement of the tachykinin NK1 andNK2 receptors.

MATERIALS AND METHODS

Animals

Male ddY-strain mice (Japan SLC, Hamamatsu, Japan) 6–8 wk oldwere used. Animals were housed in a temperature-controlled room at24°C with lights on from 0700 to 1900 and had free access to food andwater. All experiments were performed in compliance with the “Guid-ing Principles for the Care and Use of Laboratory Animals” approvedby the Japanese Pharmacological Society and the guidelines approvedby the institutional Animal Care and Use Committee of Josai Inter-national University. The protocols used were approved by the com-mittee of Josai International University in accordance with the guide-lines of APS guiding principles in the care and use of animals. Thenumber of animals used was kept to the minimum necessary for ameaningful interpretation of the data, and animal discomfort was keptto the minimum.

Drugs

The drugs used in this study were acetylcholine chloride, tetrodo-toxin (TTX), capsaicin, and atropine sulfate (Wako Pure ChemicalIndustries, Tokyo, Japan), N-(4-tertiary butylphenyl)-4-(3-chloropyri-din-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC) (BiomolInternational, Boechout, Belgium), NG-nitro-L-arginine methyl ester(L-NAME), 1-(2-(5-fluoro-1H-indol-3-yl)ethyl)-4-methoxy-4-((phe-nylsulfinyl)methyl)piperidine (GR159897), substance P, neurokinin A(Sigma Chemical, St. Louis, MO), N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbony-1-L-prolyl]-N-methyl-N-phenylmethyl-3–2-(2-naphtyl)-L-alaninamide (FK888), and 3-methyl-2-phenyl-N-[(1S)-1-phenylpropyl]-4-quinolinecarboxamide (SB222200) (Tocris-Cookson, Bristol, UK).

Capsaicin was dissolved in ethanol prior to dilution in deionizedwater. FK888, GR159897, SB222200, BCTC, and neurokinin A weredissolved in DMSO prior to dilution in deionized water. Other drugswere dissolved in deionized water with no organic solvent or deter-gent. The final concentrations of DMSO and ethanol in the bath wereless than 0.2 and 0.1%, respectively. The vehicles used had nopharmacological effects on the tonus of preparations or the capsaicin-induced contraction.

Isolated lower gastrointestinal tract and measurement of tension.The preparation and measurement of contraction of segments of themouse rectum and distal, transverse, and proximal colon were per-formed as described previously (32). Each segment was removed andplaced in Krebs-Henseleit solution (in mM: 112.08 NaCl, 5.90 KCl,1.97 CaCl2, 1.18 MgCl2, 1.22 NaH2PO4, 25.00 NaHCO3, and 11.49glucose). A segment of each tissue, �1.0 cm length, was set up undera 0.7-g load in a 10-ml organ bath filled with Krebs-Henseleitsolution. The optimal resting tension was determined by preliminaryexperiments. When the resting tension was 0.7 g, the maximumreproducible contraction by capsaicin was obtained. The strips weremounted in the longitudinal direction, and longitudinal muscle tensionwas recorded. The bath was maintained at 37°C and continuouslybubbled with a mixture of 95% O2 and 5% CO2. Contraction wasrecorded by use of an isotonic transducer (TD-112S, Nihon Kohden,Tokyo, Japan), a balancing box (JD-112S, Nihon Kohden), and aPowerlab system (AD Instruments, Castle Hill, Australia). At the startof each experiment, the responsiveness to acetylcholine at 10 �M, asubmaximal concentration for acetylcholine-induced contraction, wasascertained to evaluate the contractile effects of the capsaicin, sub-stance P, and neurokinin A. After at least three stable contractionswere induced by 10 �M acetylcholine, the experiments wereperformed.

In the first series of experiments investigating the noncumulativeconcentration response to capsaicin, intestine segments were chal-lenged only once with an individual dose of capsaicin (10 nM–10�M) to avoid the influence of sensitization or desensitization on thecontraction (10, 14). To investigate the possibility of desensitizationto capsaicin in the contractile responses, the intestine preparationswere incubated for 3 min with capsaicin (first treatment), then washedthree times with the Krebs-Henseleit solution for 5 min each. Afterthat, capsaicin at the same dose as the first treatment was added(second treatment). Each response was expressed as a percentage ofthe maximum contraction induced by 10 �M acetylcholine (% ofacetylcholine contraction).

Mechanism of capsaicin-induced contraction. In studies of theeffects of TTX, atropine, the nitric oxide synthase inhibitor L-NAME,or TRPV1 antagonist BCTC on capsaicin-induced contraction, eachsegment was preincubated with 300 nM of TTX for 10 min, 1 �M ofatropine for 10 min, or 100 �M of L-NAME for 10 min, or 1 �M ofBCTC for 45 min. In studies of the effects of the NK1 receptorantagonist FK888 and NK2 receptor antagonist GR159897, eachsegment was preincubated with 10 �M of FK888 or 3 �M ofGR159897 for 20 min. The drug treatment protocols were previouslydescribed (4, 14). These drugs did not affect the baseline tension. Eachresponse was expressed as a percentage of the maximum contractioninduced by 10 �M acetylcholine (% of acetylcholine contraction).

Tissue preparation for immunohistochemistry. Segments of themouse rectum and distal, transverse, and proximal colon were re-moved, fixed by immersion in fresh 4% paraformaldehyde in 0.1 Mphosphate buffer for 2 h at 4°C, and washed three times withphosphate-buffered saline (PBS). They were cryoprotected overnightin 0.1 M phosphate buffer containing 20% sucrose. The tissues werefrozen in OCT (Sakura Finetek, Toyko, Japan) mounting medium, andsectioned on a cryostat (Leica Instruments, Nussloch, Germany) at athickness of 40 �m. The sections were thaw-mounted onto SuperfrostPlus slides (Matsunami Glass, Osaka, Japan).

Immunohistochemical study. The immunohistochemical proce-dures were performed as described by Horie et al. (23) and Watanabeet al. (38). Prior to staining, the slide-mounted sections were succes-sively incubated in 10% normal donkey serum containing 0.2% TritonX-100 and 0.1% sodium azide in PBS for 1 h, followed by PBScontaining 0.3% hydrogen peroxide for 30 min to quench endogenousperoxidase activity, and were then washed three times for 10 min eachwith PBS. In addition, the avidin and biotin sites of sections weresuccessively blocked using an avidin-biotin blocking kit (VectorLaboratories, Peterborough, UK), and the sections were then washedthree times for 10 min each in PBS again. Subsequently, sections wereincubated in rabbit polyclonal anti-TRPV1 antibody for 40 h at roomtemperature. Concentrations of the TRPV1 antibody (mouse TRPV1C-terminus; Neuromics, Minneapolis, MN) were 1:60,000 for rectum,1:40,000 for distal colon, and 1:20,000 for transverse and proximalcolon (Figs. 1, 4, and 5). To investigate the difference of the densityof TRPV1-immunoreactive nerve fibers in the rectum and distal,transverse, and proximal colon, two different anti-TRPV1 antibodies(1:60,000, Neuromics, and rat TRPV1 COOH-terminus, 1:1,000;Trans Genic, Kumamoto, Japan) were used as the primary antibody(Figs. 2 and 3). For immunoadsorption experiments, the antibody(1:60,000; Neuromics) was preincubated with 10 �M of the corre-sponding antigen peptide (Neuromics) fragment for 1 h at roomtemperature. After washes in PBS, the sections were incubated withbiotinylated donkey anti-rabbit immunoglobulin G (1:400; JacksonImmunoresearch Laboratories, West Grove, PA) for 90 min at roomtemperature. After further washes, the sections were incubated instreptavidin biotin-peroxidase complex (1:5; Vectastain Elite ABCkit, Vector Laboratories) for 1 h at room temperature, followed byfluorescein isothiocyanate (FITC) tyramide (1:75; TSA kit, Perkin-Elmer Life Sciences, Boston, MA) (1). In control experiments, theTRPV1 antibody was omitted from the staining procedures to verifythe specificity of the staining. No immunolabeling was observed in

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these controls. Double staining of the sections with TRPV1 antiserumcombined with a rabbit antiserum to the pan axonal marker proteingene product (PGP) 9.5 was also carried out. In the case of PGP 9.5,TRPV1 staining was carried out first by the TSA procedure describedabove with FITC as the label and then followed by PGP 9.5 staining(1:60,000; Biogenesis, Poole, UK) by the indirect labeling procedure.To visualize the PGP 9.5 labeling, sections were then incubated for4 h with donkey anti-rabbit secondary antibody linked to tetramethylrhodamine isothiocyanate (TRITC, 1:400; Jackson ImmunoresearchLaboratories). There was no cross-reactivity between TRPV1 andPGP 9.5 staining.

Microscopy and image analysis. The sections were viewed at �10magnification (Zeiss Plan-NeoFluar) via an inverted fluorescencemicroscope (Axioskop2 plus, Zeiss, Gottingen, Germany) equippedwith a filter for detection of fluorescein (FITC, 488 nm; TRITC; 543nm). Randomly chosen images were acquired via a charge-coupleddevice camera (AxioCam MRm, Zeiss), stored in a personal com-puter, and analyzed with Zeiss imaging software (Axiovision LEversion 4.6). To quantify TRPV1-positive axons, we measuredTRPV1-immunopositive areas in the image using the Automeasuremodule (AutoMeasure Plus, Zeiss). Random fields were selected foreach slide. Next, thresholds for the brightness that represents theTRPV1 immunoreactivity were selected by using the distribution ofbrightness information. The TRPV1-positive area was calculated bysubtracting a nonspecific signal area of the control image (i.e., notexposed to the antibody) from that of the experimental image (i.e.,antibody treated) on serial cryostat sections of rectum or distal,transverse, or proximal colon. In this manner, the absolute area ofTRPV1-positive axons could be determined for individual exper-

iments and calculated as follows: TRPV1-positive area (%) �[(TRPV1 positive area/total area of antibody-treated section) �(positive area of control section/total area of the control section)] �100 (%).

For observation of the double staining of TRPV1 and PGP 9.5 inthe submucosal layer and myenteric plexus, the sections were viewedat �20 magnification (Olympus UPlanSApo) via a confocal micro-scope (FV-1000, Olympus, Tokyo, Japan). Multiple images in Z-stacks were projected onto a single plane.

Statistical analysis. The data are expressed as means � SE.Statistical analyses were performed by the two-tailed Student’s t-testfor comparison of two groups, and by a one-way analysis of variancefollowed by a Bonferroni multiple-comparison test for comparison ofmore than two groups. A P value � 0.05 was considered statisticallysignificant.

RESULTS

Localization and quantification of TRPV1 immunoreactivityin isolated mouse lower gastrointestinal tract. NumerousTRPV1-immunoreactive nerve fibers were seen in the mucosa,submucosal layer, and myenteric plexus of the rectum anddistal colon (Fig. 1, A, B, and D). TRPV1-immunoreactivefibers were also observed in the muscle layer and aroundbundles of arterioles, venules, and lymphatic vessels in thesubmucosal layer (Fig. 1C). TRPV1-immunoreactive fiberswere not observed after preincubation of the antibody with theantigen peptide (Fig. 1E).

Fig. 1. Distribution of transient receptor potential va-nilloid type 1 (TRPV1) immunoreactivity in transversesection of mouse rectum (A, C, D, and E) and distalcolon (B). MU, mucosa; CM, circular muscle. Numer-ous TRPV1-immunoreactive nerve fibers are found inthe mucosa (arrowheads), submucosal layer (concavearrowheads), myenteric plexus (arrows), and mucosallayer (double arrows). TRPV1-immunoreactive fibersare also found around bundles of arterioles, venules, andlymphatic vessels in the submucosal layer (C). Pread-sorption of the primary antibody with the antigen pep-tide resulted in the loss of the signal (D and E). Scalebar is 100 �m.

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The difference in the distributions of TRPV1 in mouse largeintestine was examined by using serial cryostat sections of therectum and distal, transverse, and proximal colon under the sameimmunostaining condition (Fig. 2). To measure the amount ofTRPV1 immunoreactivity, two different anti-TRPV1 antibod-ies (Neuromics and Trans Genic) were used as the primaryantibody. In the rectum, numerous TRPV1-immunoreactivenerve fibers were found in the mucosa, submucosal layer, andmuscle layer and myenteric plexus compared with controltissues (Fig. 2, A and B). In the distal colon, the density ofTRPV1-positive nerve fibers was lower than in the rectum, butthey were clearly observed in the submucosal layer and my-enteric plexus (Fig. 2, D and E). In contrast, the TRPV1-positive axons were sparsely distributed in the transverse andproximal colon (Fig. 2, G, H, J, K). Thus similar results wereobtained from both experiments using two different anti-TRPV1 antibodies. In control experiments without the corre-sponding primary antibody, no immunolabeling was observed(Fig. 2, C, F, I, and L).

To better understand the difference between the amounts ofTRPV1-immunoreactive nerve fibers in the rectum and distal,transverse, and proximal colon, TRPV1-immunoreactive areaswere quantified by computerized binary image analysis (Fig.3). Figure 3 shows TRPV1-immunoreactive areas in all layersof the transverse section (Fig. 3A) and in the muscle layerincluding myenteric plexus (Fig. 3B) using a TRPV1 antibody

(Neuromics). The TRPV1-immunopositive area in the rectumwas the largest in the isolated mouse lower gastrointestinaltract. Both the rectum and distal colon contained a significantlyhigher density of TRPV1-immunoreactive nerve fibers than thetransverse and proximal colon. The density of TRPV1-positivenerve fibers was very low in the transverse and proximal colon.These results were further confirmed by an immunohistochem-ical experiment using another TRPV1 antibody (Trans Genic,Fig. 3, C and D).

Colocalization of TRPV1 with PGP 9.5. To investigate theprecise localization of TRPV1, we performed double-labelingexperiments with PGP 9.5, a pan axonal marker, to identifyneuronal cell bodies and nerve fibers using the fluorescencemicroscope (Fig. 4) and confocal microscope (Fig. 5). Abun-dant TRPV1 immunoreactivity was detected in the myentericand submucosal plexuses of each part of the lower gastroin-testinal tract (Fig. 4, A, D, G, and J). In all cases, theimmunoreactivity appeared axonlike in nature. TRPV1 stain-ing of ganglionic cells was not detected in either submucosal ormyenteric plexuses in the rectum. PGP 9.5 staining was widelydistributed within the mucosa, submucosa, and smooth musclelayer of each part of the lower gastrointestinal tract (Fig. 4, B,E, H, and K) and did not show a clear difference among theparts. Abundant PGP 9.5-immunoreactive fibers and bundleswere found in the myenteric plexus and submucosal layer, andTRPV1 immunoreactivity profiles in these areas showed dou-

Fig. 2. Comparison of immunoreactivities to 2 dif-ferent anti-TRPV1 antibodies in mouse rectum anddistal, transverse, and proximal colon. Left columns(A, D, G, J) show TRPV1 labeling with anti-TRPV1antibody (Neuromics). Middle columns (B, E, H, K)show TRPV1 labeling with anti-TRPV1 antibody(Trans Genic). Right columns (C, F, I, L) show thecontrol without the primary antibody. Scale bar is100 �m.

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ble labeling with PGP 9.5 (Fig. 4, C, F, I, L). Figure 5 showsthe high-power magnification confocal images of the submu-cosal layer and myenteric plexus of rectum. Many TRPV1/PGP 9.5 double-labeled axons were observed, but PGP 9.5-immunoreactive cell bodies did not colocalize with TRPV1.

Difference in capsaicin-induced contractions in isolatedmouse rectum, distal, transverse, and proximal colon. Next, weinvestigated the effect of the contraction induced by theTRPV1 agonist capsaicin on smooth muscle tension in theisolated mouse lower gastrointestinal tract. Figure 6 showstypical recordings and concentration-response curves of thecapsaicin-induced contraction in mouse rectum and distal,transverse, and proximal colon. In the rectum and distal colon,capsaicin induced fast transient contractions, followed byslowly developing long-lasting contractions that peaked within2–3 min after administration (Fig. 6, A and B). The contrac-tions induced by capsaicin were concentration dependent.However, only transient contractions were observed in thetransverse and proximal colon (Fig. 6, C and D). No statisticaldifferences were found between 1 �M capsaicin-induced tran-sient contractions in each part of the lower gastrointestinaltract. Preexposure of tissues to capsaicin caused a dose-depen-dent desensitization to the second application of capsaicin in allparts of the isolated lower gastrointestinal tract.

Effects of TTX, atropine, and TRPV1 antagonist BCTC oncapsaicin-induced contraction in isolated mouse lower gastro-intestinal tract. To clarify the mechanism underlying contrac-tile responses to TRPV1 activation by capsaicin in the mouserectum and distal, transverse, and proximal colon, we evalu-ated the effects of the neurotransmission blocker TTX, mus-

carinic receptor antagonist atropine, and TRPV1 antagonistBCTC on those contractile responses (Table 1). TTX abolishedtransient contractile responses to capsaicin from the rectum tothe proximal colon, compared with each control group. Cap-saicin-induced long-lasting contractions were significantly butpartially inhibited by TTX in the rectum and distal colon.Atropine abolished the transient contraction from the rectum tothe proximal colon. The long-lasting contractions were signif-icantly but partially inhibited by atropine in the rectum anddistal colon. The TRPV1 antagonist BCTC almost completelyinhibited both transient and long-lasting contractions in allparts of the isolated mouse lower gastrointestinal tract. BCTC(1 �M) did not affect the direct smooth muscle contractions inresponse to 10 �M of acetylcholine (data not shown), but thelong-lasting contraction was not abolished by BCTC. Then weperformed a preliminary experiment with another TRPV1 an-tagonist, iodoresiniferatoxin, but it also failed to abolish thecapsaicin-induced long-lasting contraction (data not shown).To investigate the capsaicin-induced relaxation in the mousecolon, inhibition by nitric oxide synthase was examined. Thedistal colon segments were preincubated with the nitric oxide-synthase inhibitor L-NAME (100 �M). L-NAME did not affect thecapsaicin-induced transient (control 33.1 � 3.4, L-NAME31.34 � 12.6; n � 3–6) or long-lasting contractions in the distalcolon (control 38.0 � 2.7, L-NAME 33.6 � 4.2; n � 3–6).

Effects of tachykinin-receptor antagonists on capsaicin-in-duced contraction in isolated mouse lower gastrointestinaltract. Tachykinins play an important role in the contractileeffect of capsaicin: they stimulate myenteric neurons andmediate the neurogenic excitatory effects of capsaicin. We

Fig. 3. Histogram showing positive areas (%)of TRPV1 fibers in all layers (A and C), and inthe muscle layer including the myentericplexus (B and D) of the rectum and distal,transverse, and proximal colon. Two kinds ofrabbit polyclonal anti-TRPV1 antibody (Neu-romics; A and C) and (Trans Genic; B andD) were used in each part of lower gastroin-testinal tract. Each value represents means �SE of data obtained from 4 mice. Signifi-cantly different from the data of the rectum byBonferroni correction: **P � 0.01. #Signifi-cantly different from the data of the distalcolon by Bonferroni correction: #P � 0.05,##P � 0.01.

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investigated the contractile effects of the tachykinins substanceP and neurokinin A on capsaicin-induced contraction in theisolated mouse lower gastrointestinal tract (Fig. 7). Preliminaryexperiments revealed that substance P (10 nM–1 �M) andneurokinin A (10 nM–3 �M) induced dose-dependent contrac-tions in the isolated mouse lower gastrointestinal tract. There-fore, we investigated the contractile response to substance Pand neurokinin A in the mouse rectum and distal, transverse,and proximal colon (Fig. 7, A–D). The concentrations ofsubstance P and neurokinin A were chosen to induce submaxi-mal contractions in each part. Substance P produced fasttransient contractions in all parts of the mouse isolated lower

gastrointestinal tract. Neurokinin A produced large long-last-ing contractions in the rectum and distal colon that peaked 2–3min after administration. In the transverse and proximal colon,neurokinin A induced fast transient contractions, followed bysmall long-lasting contractions. To determine the contributionof the cholinergic pathways, responses to substance P andneurokinin A were compared in the presence and absence ofthe muscarinic receptor antagonist atropine (Fig. 7, A–D).Atropine partially and significantly inhibited the substanceP-induced contractions, but no significant effect on the neuro-kinin A-induced contractions was observed in any part of themouse lower gastrointestinal tract.

Fig. 4. Colocalization of TRPV1-positive nerve fi-bers with protein gene product (PGP) 9.5 in mouserectum and distal, transverse, and proximal colon.Mouse lower gastrointestinal sections were doublelabeled with TRPV1 (green) and PGP 9.5 (red). TheTRPV1 and PGP 9.5 labelings are shown separately(A, D, G, J) and (B, E, H, K), respectively, andmerged (C, F, I, L). TRPV1 axons are sparse in thesmooth muscle layer but prominent in the myentericnerve plexus (arrows) and submucosal layer (con-cave arrowheads). In contrast, PGP 9.5 immunore-active axons are abundant in all regions. Arrows andconcave arrowheads indicate the colocalization ofTRPV1 immunoreactivity with PGP 9.5 immunore-activity. Scale bars are 100 �m.

Fig. 5. Confocal images showing TRPV1 and pro-tein gene product (PGP) 9.5 double labeling in thesubmucosal layer (A–C) and myenteric plexus(D–F) of the mouse rectum. Sections were doublelabeled with TRPV1 (green) and PGP 9.5 (red). TheTRPV1 and PGP 9.5 labelings are shown separatelyin (A and D) and (B and E), respectively, and merged(C and F). Arrowheads and arrows indicate thecolocalization of TRPV1/PGP 9.5 double-labeledaxons in the submucosal layer and myenteric plexus,respectively. Scale bar is 50 �m.

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Next, we evaluated the effects of the NK1 receptor antago-nist FK888 and the NK2 receptor antagonist GR159897 oncapsaicin-induced contractile responses (Fig. 8). Capsaicin-induced transient contractions from the rectum to the proximalcolon were significantly and moderately inhibited by FK888 orGR159897 compared with control responses (Fig. 8, A–D).The combined blockade of NK1 and NK2 receptors withFK888 and GR159897 markedly inhibited transient contractileresponses to capsaicin from the rectum to the proximal colon.The long-lasting contractions in the rectum and distal colon

were significantly and moderately inhibited by GR159897, butnot by FK888 (Fig. 8, A and B). Coadministration of FK888and GR159897 markedly inhibited long-lasting contractile re-sponses to capsaicin in the rectum and distal colon.

The substance P (100 nM)-induced contractions in the rec-tum and distal, transverse, and proximal colon were signifi-cantly blocked by 10 �M FK888 (rectum: control 59.2 � 7.9,FK888 8.4 � 1.7, P � 0.01; distal colon: control 66.6 � 11.6,FK888 18.2 � 3.8, P � 0.05; transverse colon: control 61.3 �11.3, FK888 20.5 � 3.4, P � 0.05; proximal colon: control

Fig. 6. Typical recordings and concentration-response curves of capsaicin-induced contrac-tion and desensitization in mouse rectum (A)and distal (B), transverse (C), and proximalcolon (D). Typical recordings show that capsa-icin induced a transient contraction, followedby a long-lasting contraction in the rectum anddistal colon. In the transverse and proximalcolon, only the transient contraction was ob-served. Data for each contraction are expressedas a percentage of the maximal contractioninduced by 10 �M acetylcholine (% of AChresponse) in each part of the lower gastrointes-tinal tract. Each value represents means � SEof data obtained from 3–7 mice. No statisticaldifferences were found on 1 �M capsaicin-induced transient contraction among each partof lower gastrointestinal tract.

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60.8 � 8.2, FK888 18.4 � 3.8%, P � 0.01; n � 5–8), but notsignificantly by 3 �M GR159897 (rectum: control 59.2 � 7.9,GR159897 49.9 � 8.5; distal colon: control 66.6 � 11.6,GR159897 61.9 � 12.6; transverse colon: control 61.3 �11.3, GR159897 53.8 � 10.4; proximal colon: control 60.8 �8.2, GR159897 62.4 � 10.8%; n � 5–8). The neurokinin A(300 nM or 1 �M)-induced contractions in the rectum anddistal, transverse, and proximal colon were significantlyblocked by 3 �M GR159897 (rectum: control 72.7 � 15.9,GR159897, 9.4 � 2.6, P � 0.05; distal colon: control 65.3 �16.5, GR159897 18.4 � 4.5, P � 0.05; transverse colon:control 46.0 � 8.5, GR159897 9.0 � 2.7, P � 0.01; proximalcolon: control 31.2 � 4.1, GR159897 6.8 � 1.6%, P � 0.01;n � 5–8), but not significantly by 10 �M FK888 (rectum:control 72.7 � 15.9, FK888, 81.1 � 13.8; distal colon: cont-rol 65.3 � 16.5, FK888 79.3 � 19.3; transverse colon: control46.0 � 8.5, FK888 47.7 � 18.7; proximal colon: control31.2 � 4.1, FK888 36.4 � 8.4%; n � 5–8). An NK3 receptorblockade partially inhibited the contraction induced by capsa-icin, suggesting the involvement of NK3 receptors in theresponse to capsaicin in mouse jejunum (14). We tried toclarify this by using the commercially available NK3 antago-nist SB222200 (30). SB222200 (30 �M) inhibited the neuro-kinin B-induced contraction, but it also markedly inhibited thesubstance P- and neurokinin A-induced contractions. There-fore, the involvement of NK3 receptors in capsaicin-inducedcontraction is unclear at this time (data not shown).

DISCUSSION

This study was designed to investigate differences in thedistribution of TRPV1 channels and the effects of capsaicin onsmooth muscle contraction using isolated mouse lower gastro-intestinal tract segments. The localization of TRPV1 channelsand their effects on motor function in the large intestine,especially site differences, remain to be elucidated. In thepresent study, we isolated the mouse rectum and distal, trans-verse, and proximal colon and examined the pharmacologicaland physiological differences among the parts by using immu-nohistochemical methods and tension recordings of capsaicin-induced contractions. The present results suggest that TRPV1

channels located on the rectum and distal colon play a majorrole in the motor function of the mouse lower gastrointestinaltract. Furthermore, neurokinin A was found to be the mainneurotransmitter in large long-lasting capsaicin-induced con-tractions in the mouse rectum and distal colon.

First, we mapped the localization and distribution of TRPV1in the mouse lower gastrointestinal tract using immunofluores-cence. To increase the sensitivity of immunodetection, wevisualized anti-TRPV1 antibodies using an amplificationmethod based on the catalyzed deposition of fluorescein-conjugated tyramide (1). In this study, we demonstrate TRPV1immunoreactivity in the mucosa, submucosal layer, musclelayer, and myenteric plexus of mouse lower gastrointestinaltract. In the mucosal layer of the rectum and distal colon,TRPV1-immunoreactive axons were observed running acrossand along the mucus glands. In the submucosal layer, TRPV1-immunoreactive fibers were found around bundles of arte-rioles, venules, and lymphatic vessels. In our previous study ofrat stomach, TRPV1 axons were observed running along gas-tric glands in the mucosa and around blood vessels in thesubmucosal layer, suggesting the role of TRPV1 nerve fibers ingastric acid and mucus secretion (22, 23). The gastrointestinalprotection produced by capsaicin-induced stimulation of sen-sory neurons is associated with a marked increase of mucosalblood flow in the small and large intestine of experimentalanimals (18). These findings suggest that the TRPV1 nervefibers of the mucosal layer are involved in mucus secretion andblood flow in the mouse large intestine. In the muscle layer,TRPV1-immunoreactive fibers were observed in the rectal sideof the mouse lower gastrointestinal tract.

Extrinsic primary afferent neurons have their cell bodies innodose and jugular ganglia or dorsal root ganglia. TRPV1-positive nerve fibers in the gut appear to originate predomi-nantly in spinal afferents, with very few in vagal afferents (37).Other investigators reported that TRPV1 immunoreactivitywas detected on both intrinsic and extrinsic neurons in the gut(3), and TRPV1 mRNA has also been reported in the mousecolon (25, 32). Ward et al. (37) reported that TRPV1 nervefibers form a network around myenteric nerve cell bodies in themouse, guinea pig, and rat gastric antrum. Consistent withprevious findings for TRPV1/PGP 9.5 double staining (23, 37)in the gastrointestinal tract, we observed TRPV1/PGP 9.5double-labeled axons in the myenteric plexus. However, PGP9.5-immunoreactive cell bodies were not colocalized withTRPV1 immunoreactivity. Pharmacological studies have shown thatthe population of capsaicin-sensitive gastrointestinal neurons iscomposed of extrinsic primary afferents, whereas intrinsicenteric neurons do not respond directly to capsaicin (8). Takentogether, the data imply that TRPV1-expressing nerve fibersobserved in the myenteric plexus are extrinsic primary affer-ents, but further studies are needed to determine the origin ofthe TRPV1 channels in the mouse large intestine.

The difference between the densities of TRPV-immunore-active nerve fibers in the rectum and distal, transverse, andproximal colon has not been studied under physiological con-ditions. In the present study, the TRPV1-immunoreactive areasof each segment were quantified by computerized binary imageanalysis using two anti-TRPV1 antibodies. Interestingly, highdensities of TRPV1-positive areas were detected in the rectumand distal colon, but not in the transverse and proximal colon.In contrast, clear differences in PGP 9.5 densities were not

Table 1. Effect of TTX, atropine, or TRPV1 antagonistBCTC on capsaicin-induced contraction in isolated mouserectum, distal colon, transverse colon, and proximal colon

Control TTX, 300 nM Atropine, 1 M BCTC, 1 M

RectumTransient contraction 33.2�5.5 1.6�1.6† 1.3�1.3† 3.1�0.6†Long-lasting

contraction 50.4�3.4 36.7�3.5* 35.8�5.4* 7.0�0.3†Distal colon

Transient contraction 33.1�3.4 0.0�0.0† 0.5�0.3† 3.7�1.2†Long-lasting

contraction 38.6�2.7 25.4�3.3* 14.3�4.6† 5.7�1.9†Transverse colon

Transient contraction 22.3�3.7 0.0�0.0† 3.7�2.2† 1.6�0.3†Proximal colon

Transient contraction 30.0�5.5 0.0�0.0† 6.4�4.2* 1.5�0.3†

Each value represents the mean � SE of 5–7 animals. TTX, tetrodotoxin;BCTC, N-(4-tertiary butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide. Values that were significantly different from those of thecontrol group: *P � 0.05, †P � 0.01.

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observed from the proximal colon to the rectum. Consistentwith the binary image analysis of the muscle layers includingthe myenteric plexus in each segment, the capsaicin-inducedcontraction patterns were different on the anal (rectum anddistal colon) and oral (transverse and proximal colon) sides ofthe mouse large intestine. These contraction patterns werecharacterized by transient and subsequent long-lasting contrac-tions in the rectum and distal colon and only transient contrac-tions in the transverse and proximal colon. Thus we speculatedthat TRPV1 channels located on the rectum and distal colonplay a major role in the motor function. On the basis of our invitro data, we speculated that capsaicin stimulates colonicmotility in the in vivo experiment. Indeed, it is reported thatintracolonic administration of capsaicin increases colonic mo-tility in a dose-dependent manner in conscious dogs (17).Therefore, the activation of TRPV1 channels is thought toresult in the promotion of motility in the colon.

TRPV1 channels located in the mucosa are considered toplay important roles in detecting visceral pain because TRPV1contributes to the perception of noxious mechanical stimuli andinflammatory stimuli in the mouse colon (24, 29). In thegastrointestinal tract, TRPV1 channels play both protective andnonprotective roles against inflammatory colitis (35). Holzer(20) suggested that TRPV1 channels involved in gastrointes-tinal protection may be differentiated pharmacologically fromTRPV1 channels mediating gastrointestinal inflammation. Azumaet al. (6) reported that the histological colitis score of the distalcolon was higher than that of the proximal colon in dextransodium sulfate-induced colitis. In this study, we clearly dem-onstrated that the TRPV1 channel density in the mucosal areais greater in the rectum and distal colon than in the transverseand proximal colon. Thus the abundant TRPV1 channel ex-pression may result in the induction of more pronounced colitisin the rectum and distal colon than in the transverse andproximal colon. However, further studies are needed to clarifythe roles of TRPV1 channels in colitis.

In the guinea pig esophagus, capsaicin induces a biphasiccontraction, i.e., immediate transient and late long-lasting con-tractions (9). A similar contraction pattern was observed in themouse rectum and distal colon in the present study. Thecontraction due to capsaicin is well studied in guinea pig ileum,and it is known that biologically active substances releasedfrom sensory nerves stimulate both smooth muscle and intrin-sic myenteric neurons (8). Acetylcholine released from intrin-sic cholinergic motor neurons is one of the final mediators ofthe capsaicin-induced contraction. Capsaicin-sensitive sensorynerves innervating the gastrointestinal tract are known to beTTX resistant (5, 11). In the guinea pig ileum, capsaicininduces TTX-sensitive acetylcholine release, suggesting thatthe TTX- and atropine-sensitive contractile effects of capsaicinare based on acetylcholine release from myenteric cholinergicneurons stimulated by neurotransmitters from sensory nerves(34). On the other hand, the TTX-insensitive contractile effect

of capsaicin is attributed to a direct activation of smoothmuscle stimulated by neurotransmitters from sensory nerveendings (14). In the present study, capsaicin-induced transientcontractions in the rectum to proximal colon were abolished byTTX and atropine. The long-lasting contractions in the rectumand distal colon were partially inhibited by TTX and atropine.The amplitudes of transient and long-lasting contractions werereduced by repeated administration of capsaicin, which is onepeculiar effect of TRPV1 activation. To study the involvementof TRPV1 in isolated gastrointestinal preparations, we usedBCTC as a selective TRPV1 antagonist (33). BCTC almostcompletely inhibited the response to capsaicin in all gastroin-testinal segments without affecting the direct smooth musclecontraction, as previously reported for isolated mouse jejunum(14). However, we observed that the long-lasting contractionwas not abolished even with adequate BCTC treatment. Next,we performed a preliminary experiment with another TRPV1antagonist, iodoresiniferatoxin, but iodoresiniferatoxin alsofailed to abolish the capsaicin-induced long-lasting contrac-tion. Therefore, capsaicin is thought to elicit the long-lastingcontraction partly via the TRPV1-independent pathway. DeMan et al. (14) also reported that TRPV1 antagonist iodores-iniferatoxin partially inhibited the contraction to capsaicin inmouse jejunum.

Benko et al. (10) reported that capsaicin induces nitricoxide-mediated relaxation in circular muscle-oriented prepara-tions isolated from human and mouse colon. In the presentstudy, we measured the longitudinal tension in longitudinallyoriented muscle preparations. Pretreatment with L-NAME didnot affect the capsaicin-induced contraction in the mouse distalcolon, suggesting that nitric oxide is not involved in thisresponse. Furthermore, in our preliminary experiments, wenever observed nitric oxide-mediated relaxation in colonic andrectal longitudinal muscle treated with capsaicin when theisolated segments were preincubated with the NK1 antagonistFK888, the NK2 antagonist GR159897, or atropine, unlike thereport of Benko et al. It is thought that the difference betweenBenko’s and our results arises from the preparations used.

Taken together, these results suggest that the capsaicin-induced transient contraction is mediated by intrinsic cholin-ergic activation, followed by the release of sensory neurotrans-mitters from the extrinsic sensory nerve. The long-lastingcontraction is mediated mainly by neurotransmitters releasedfrom extrinsic sensory nerve endings and partially by thecholinergic pathway in the mouse lower gastrointestinal tract.

It is well known that tachykinins released from capsaicin-sensitive primary afferents elicit contractions of smooth muscleby a local efferent action (8). The involvement of tachykininNK1 and NK2 receptors, which prefer substance P and neuro-kinin A, respectively, in the modulation of capsaicin-inducedcontractions has been shown in the guinea pig ileum. Thedistributions of substance P, neurokinin A, NK1 receptors, andNK2 receptors have been studied by immunohistochemical

Fig. 7. Typical recordings showing contractile response to substance P and neurokinin A in mouse rectum (A) and distal (B), transverse (C), and proximal colon(D). The concentrations of substance P and neurokinin A used in the rectum and distal, transverse, and proximal colon were submaximal concentrations that weredetermined by concentration response curves to substance P and neurokinin A in each segment. The effects of atropine (1 �M) on the contractile effects ofsubstance P and neurokinin A in mouse rectum to proximal colon. The concentrations of substance P and neurokinin A used in each segment were that sameas shown in each typical recording. Data are expressed as a percentage of the maximal contraction induced by 10 �M acetylcholine in each part of the lowergastrointestinal tract. Each value represents means � SE of data obtained from 5–8 mice. *Significantly different from those of the control by Student’s t-test(*P � 0.05, **P � 0.01).

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methods. Substance P and neurokinin A are expressed inextrinsic and intrinsic primary afferent neurons and myentericneurons (interneurons, excitatory motor neurons) (26). NK1

receptor immunoreactivity has been found in the myentericplexus and muscle layer (26, 36). The NK2 receptors are foundin longitudinal and circular muscle fibers of the mouse ileumand are thought to be expressed mainly on the smooth musclelayer (26, 31). To investigate the pattern of contraction inducedby the NK1 and NK2 receptor activation, we compared exoge-

nous substance P- and neurokinin A-induced contractions,respectively. We observed atropine-sensitive, fast transientcontractions induced by substance P in all parts of the isolatedmouse lower gastrointestinal tract. Neurokinin A producedlarge atropine-insensitive, long-lasting contractions in the rec-tum and distal colon. In the transverse and proximal colon,neurokinin A induced fast transient contractions, followed bysmall long-lasting contractions. The reactivities to neurokininA of the rectum and distal colon were higher than those of the

Fig. 8. Effects of NK1 antagonist FK888 (10�M), NK2 antagonist GR159897 (3 �M), andtheir combination on capsaicin-induced tran-sient and long-lasting contractions in mouserectum (A) and distal (B), transverse (C), andproximal colon (D). Data of each contractionare expressed as a percentage of the maximalcontraction induced by 10 �M acetylcholinein each part of the lower gastrointestinal tract.Each value represents means � SE of dataobtained from 5–8 mice. *Significantly dif-ferent from the control by Bonferroni test(*P � 0.05, **P � 0.01).

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transverse and proximal colon. It is suggested that NK1 andNK2 receptor activation induces a capsaicin-like contractileresponse in the mouse lower gastrointestinal tract.

Therefore, we next investigated the effects of the NK1

receptor and/or the NK2 receptor blockade on capsaicin-in-duced contractile responses. Our results show that the capsa-icin-induced transient contraction was significantly inhibitedby NK1 or NK2 receptor antagonists, and a combined blockadeof NK1 and NK2 receptors abolished transient contractileresponses to capsaicin, suggesting that NK1 and NK2 receptorscooperate in producing the transient contraction. On the otherhand, the long-lasting capsaicin-induced contraction was sig-nificantly inhibited by an NK2 antagonist but not by an NK1

antagonist, indicating that the release of neurokinin A fromsensory nerve endings is mainly responsible for the long-lasting contraction. Altogether, these results indicate that dif-ferent mechanisms are involved in the transient and long-lasting contractions induced by capsaicin in the mouse lowergastrointestinal tract, i.e., the intrinsic cholinergic and extrinsicsensory pathways are the main pathways of transient andlong-lasting contractions, respectively. Substance P and neu-rokinin A released from sensory nerves stimulate NK1 andNK2 receptors on parasympathetic ganglia, leading to acetyl-choline release from cholinergic nerve endings. This is theputative mechanism of capsaicin-induced transient contrac-tions in the rectum and proximal colon. In the long-lastingcontraction in the rectum and distal colon, the major finalmediator is the neurokinin A released from extrinsic sensorynerve endings, which activates NK2 receptors on the smoothmuscle cells. Our results suggest that TRPV1 channels locatedon the rectal and distal colon play a major role in the motorfunction of the large intestine. The differences between capsa-icin-induced contractions in the rectum, distal colon, transversecolon, and proximal colon segments may be attributed to thesedifferences in the TRPV1 density and neurokinin A reactivity.Although the present study provides significant informationon the mechanisms of capsaicin-induced contractions in themouse lower gastrointestinal tract, the localization of endoge-nous tachykinins and tachykinin receptors remains unknown.Colocalization studies on tachykinins and/or tachykinin recep-tors with TRPV1 channels may support the involvement oftachykinins, especially neurokinin A and NK2 receptors, in themechanism of TRPV1-mediated motor function of the rectumand distal colon.

The present results provide new information regarding thesite specificity of TRPV1 channels in the mouse lower gastro-intestinal tract. Our findings suggest that TRPV1 channelslocated on the rectum and distal colon play a major role in themotor function of the large intestine. Further studies of TRPV1channels in the rectum and distal colon may provide significantinformation about the role of TRPV1 in regulating a widevariety of functions in the large intestine.

ACKNOWLEDGMENTS

We thank Dr. Naoto Watanabe (Department of Respiratory and InfectiousDiseases, St. Marianna University School of Medicine; Laboratory of Phar-macology, Faculty of Pharmaceutical Sciences, Josai International University)for helpful advice and for discussions of the immunohistochemical study.

GRANTS

This work was supported in part by Grants-in-Aid for Scientific Researchfrom the Ministry of Education, Science, Sports, and Culture of Japan (nos.

19590156, 18790126, 18790127, and 20790075) and by the Uehara MemorialFoundation.

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