TRPV1 receptor signaling mediates afferent nerve sensitization during colitis-induced motility disorders in rats

TRPV1 receptor signaling mediates afferent nerve sensitization duringcolitis-induced motility disorders in rats

H. U. De Schepper,1 J. G. De Man,1 N. E. Ruyssers,1 A. Deiteren,1 L. Van Nassauw,2 J.-P. Timmermans,2

W. Martinet,3 A. G. Herman,3 P. A. Pelckmans,1 and B. Y. De Winter1

1Division of Gastroenterology, Faculty of Medicine, 2Research Group Cell Biology and Histology, Department of VeterinarySciences, and 3Laboratory of Pharmacology, Faculty of Pharmacy, University of Antwerp, Antwerp, Belgium

Submitted 1 August 2007; accepted in final form 5 November 2007

De Schepper HU, De Man JG, Ruyssers NE, Deiteren A, VanNassauw L, Timmermans J-P, Martinet W, Herman AG, PelckmansPA, De Winter BY. TRPV1 receptor signaling mediates afferent nervesensitization during colitis-induced motility disorders in rats. Am JPhysiol Gastrointest Liver Physiol 294: G245–G253, 2008. First pub-lished November 8, 2007; doi:10.1152/ajpgi.00351.2007.—Rats withexperimental colitis suffer from impaired gastric emptying (GE). Wepreviously showed that this phenomenon involves afferent neuronswithin the pelvic nerve. In this study, we aimed to identify themediators involved in this afferent hyperactivation. Colitis wasinduced by trinitrobenzene sulfate (TNBS) instillation. We deter-mined GE, distal front, and geometric center (GC) of intestinaltransit 30 min after intragastric administration of a semiliquid Evans bluesolution. We evaluated the effects of the transient receptor potentialvanilloid type 1 (TRPV1) antagonists capsazepine (5–10 mg/kg) andN-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 1–10 mg/kg) and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (150 �g/kg). To determine TRPV1 receptor antagonist sensitivity, we exam-ined their effect on capsaicin-induced relaxations of isolated gastricfundus muscle strips. Immunocytochemical staining of TRPV1 andRT-PCR analysis of TRPV1 mRNA were performed in dorsal rootganglion (DRG) L6 –S1. TNBS-induced colitis reduced GE but hadno effect on intestinal motility. Capsazepine reduced GE in con-trols but had no effect in rats with colitis. At doses that had noeffects in controls, BCTC and CGRP-(8-37) significantly improvedcolitis-induced gastroparesis. Capsazepine inhibited capsaicin-induced relaxations by 35% whereas BCTC completely abolishedthem. TNBS-induced colitis increased TRPV1-like immunoreac-tivity and TRPV1 mRNA content in pelvic afferent neuronal cellbodies in DRG L6 –S1. In conclusion, distal colitis in rats impairsGE via sensitized pelvic afferent neurons. We provided pharma-cological, immunocytochemical, and molecular biological evi-dence that this sensitization is mediated by TRPV1 receptors andinvolves CGRP release.

gastric emptying; sensory nerve; pelvic nerve; CGRP

PATIENTS WITH INFLAMMATORY bowel disease (IBD) often sufferfrom disorders of gastrointestinal motility and sensitivity, im-posing a significant load on the patient’s quality of life (20).These alterations are known to appear both during inflamma-tory episodes and in periods of remission and can occur eitherat the site of inflammation or at a distance from this site (43).Especially concerning the latter situation, little is known aboutthe underlying pathophysiological mechanisms. There havebeen several studies documenting the effects of isolated exper-

imental colitis on small intestinal neuromuscular function, butthe in vivo consequences on gut transit were inconclusive (3, 5,29). McHugh et al. (37) reported that rats with trinitrobenzenesulfate (TNBS)-induced colitis suffer from a reduction ofgastric emptying but did not investigate the underlying mech-anisms. We recently confirmed that rats with experimentalacute colitis suffer from impaired gastric emptying in theabsence of local gastric inflammatory changes (13), a phenom-enon that has also been portrayed in human IBD patients withcolonic involvement (2, 22). The colitis-induced gastroparesisin rats was neuronally mediated and disappeared after sectionof the pelvic nerve, suggesting the involvement of an extrinsicreflex pathway activated by colonic inflammation (13). Be-cause colorectal distension in healthy rats also impairs gastricemptying (i.e., cologastric inhibitory reflex) (26), the colitis-induced gastroparesis can be interpreted as a sensitized state ofthis physiological reflex. This hypothesis was supported byc-Fos expression studies showing heightened activity in thepelvic nerve dorsal root ganglion (DRG) S1 in the absence ofa distension stimulus (13).

A large number of molecular targets have already beenidentified as possible mediators of extrinsic afferent nervesensitization (23). One of the most attractive candidates is thetransient receptor potential vanilloid type 1 (TRPV1) receptor,which occurs predominantly on extrinsic afferent nerve fibersin the rat gut (55). This polymodal cation channel receptor isbest known for its responsiveness to the spicy pungent capsa-icin and has been shown to act as an integrator of inflammatorystimuli and as a key player in the pathophysiology of nervesensitization. Importantly, the TRPV1 receptor has alreadybeen implicated in colonic mechanosensitivity and its modu-lation by inflammatory agents (30). In addition, TRPV1 recep-tor immunoreactivity was found to be increased in patientswith IBD (58). These data make TRPV1 a very likely mediatorfor colitis-induced changes of neuronal function.

The aim of this study was to determine whether peripheralTRPV1 receptors are involved in the colitis-induced sensitiza-tion of the afferent pelvic nerve fibers leading to impairedgastric emptying in the rat model of TNBS-induced colitis. Wealso investigated the effect of an antagonist of the CGRPreceptor on colitis-induced gastroparesis, because this neu-ropeptide is released by afferent nerves at the spinal dorsalhorn synapse. Moreover, it was shown to be involved in thegeneration of colonic hypersensitivity to distension (16).

Address for reprint requests and other correspondence: B. Y. De Winter,Laboratory of Gastroenterology, Faculty of Medicine, Univ. of Antwerp,Universiteitsplein 1, 2610 Antwerp (Belgium) (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Gastrointest Liver Physiol 294: G245–G253, 2008.First published November 8, 2007; doi:10.1152/ajpgi.00351.2007.

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MATERIALS AND METHODS

Animal model. Distal colitis was induced in male Wistar rats(200–225 g) according to published methods (13, 41). Rats werefasted for 24 h. After pentobarbital anesthesia (45 mg/kg ip) an enemaof 0.5 ml of saline (controls) or trinitrobenzene sulfate (7.5 mg TNBSdissolved in 50% ethanol) was administered in the colorectum. Allfurther experiments were performed 72 h after induction of colitis. Allprocedures were approved by the Committee for Medical Ethics andthe Use of Experimental Animals at the University of Antwerp.

Evaluation of colitis. After each experiment, the colorectum wasremoved and macroscopic mucosal damage was assessed by a standard-ized scoring system ranging from 0 to 10 (13, 40). Rats were included ifthe score was �5, indicating major signs of damage. Colonic tissuesamples were harvested for measurement of myeloperoxidase (MPO)content, a biochemical marker for the extent of inflammation. MPO wasquantified according to published methods (40).

In vivo measurements of gastrointestinal motility. A protocol wasadapted from De Winter et al. (14) as previously described (13). Briefly,rats were fasted for 48 h with free access to tap water containing 5%glucose. This prolonged fasting was necessary to assure that the stomachwas completely free of contents before administration of Evans blue. Onthe day of experiment, 1 ml of a semiliquid nonnutrient dye (Evans blue50 mg/ml dissolved in 0.5% methylcellulose) was instilled intragastri-cally. Thirty minutes later, rats were anesthetized and euthanized. Thestomach and small intestine were carefully removed. Intestinal transit wasmeasured from the pylorus to the most distal point of migration andexpressed as a percentage of the total length of the small intestine. Thesmall intestine was then divided into 10 segments of equal length. Thestomach (segment 1) and the intestinal segments (segments 2–11) wereput in 25 ml of 0.1 N NaOH, minced, and placed in an ultrasonic bath for1 h (Bransonic 2000). The resulting suspension was left at room temper-ature for 1 h, and 5 ml of the supernatant was then centrifuged at 1,356g for 20 min at 4°C. Samples were further diluted (1:5 for intestinalspecimens and 1:50 for the stomach), and absorbance (A) was read atwavelength 565 nm. Gastric emptying (GE) was calculated as %GE �[A(small intestine)/A(stomach�small intestine)] � 100. The geometriccenter (GC) of intestinal transit (38) was calculated both including(GCS�I) and omitting (GCI) the stomach segment, because the lattermethod gives a better idea about small intestinal peristaltic functionindependent of gastric emptying efficacy. These values were calcu-lated as GC � ¥[%A(segment) � (number of segment)]/100.

Protocols. Experiments were performed 72 h after induction ofcolitis. To study the involvement of TRPV1 receptors, we treated ratswith the most used TRPV1 receptor antagonist capsazepine (5, 10mg/kg ip, 1 h before instillation of Evans blue) (4) or its vehicle(78% saline, 20% DMSO, 1% ethanol, 1% Tween80). Alterna-tively, rats were treated with the novel, more specific TRPV1antagonist N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahy-dropyrazine-1(2H)carboxamide (BCTC 1, 5, 10 mg/kg ip, 1 h beforeinstillation of Evans blue) (53) or its vehicle (25% hydroxypropyl-�-cyclodextrin). The role of CGRP-mediated neurotransmission was as-sessed by treating the rats with the specific receptor antagonist CGRP-(8-37) (150 �g/kg ip, 1 h before instillation of Evans blue) or its vehicle(saline). The doses of capsazepine, BCTC, and CGRP-(8-37) we usedwere previously shown to reduce somatic or visceral hyperalgesia in rats(8, 16, 53). CGRP-(8-37) is a frequently used and well-validated antag-onist for the CGRP receptor and has been shown to abolish CGRP-induced responses both in vitro (19) and in vivo in rats (36). Capsazepineand BCTC are specific TRPV1 receptor antagonists that potently inhibitcapsaicin-induced responses in rats. Since capsazepine has documentedaspecific effects on ion channel dynamics, and since BCTC is arelatively novel compound, the effect of these antagonists was testedon capsaicin-induced relaxations of isolated rat gastric fundus musclestrips to prove their efficacy in rats (31).

The effects of each antagonist and vehicle on gastrointestinalmotility and contractility, on the macroscopic score of inflammation,

and on the MPO content of the colon were determined. The inflam-matory indexes were determined to exclude an acute modulation ofthe local inflammatory environment by the antagonists used.

In vitro measurements of gastrointestinal contractility. Rats werefasted for 24 h with free access to water, anesthetized with diethylether, and exsanguinated by cardiotomy. After laparotomy, the stom-ach was removed and immersed in ice-cold Krebs-Ringer solution(118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4,2.5 mM CaCl2, 25 mM NaHCO3, 0.026 mM CaEDTA, and 11.1 mMglucose). Muscle strips were prepared as previously described (11,12). After removal of the mucosa, gastric fundus muscle strips wereprepared in the longitudinal direction and mounted in an organ bath(volume 5 ml) filled with Krebs-Ringer solution (37°C, continuouslyaerated with a mixture of 95% O2-5% CO2). One end of the strip wasfixed and the other end was attached to a strain gauge transducer (ScaimeTransducers, Annemasse, France) for continuous recording of isometrictension. After an initial equilibration period of 30 min during which thestrips were washed every 10 min, the muscle strips were contracted with100 nM carbachol. After washout the strips were stretched, and when thebasal tone of the preparations was stabilized 100 nM carbachol was addedagain. This procedure was repeated until the carbachol-induced contrac-tion was maximal and an optimal length-tension relationship wasachieved (�0.5 g) (11, 12). Experiments were started after an additionalequilibration period of 60 min, during which the strips were washed withfresh Krebs-Ringer solution every 15 min. We investigated the relaxanteffect of capsaicin (1 �M) on a precontraction induced by carbachol (100nM), in the presence of capsazepine (1, 5 �M) or BCTC (10, 100 nM),or their respective vehicles. Relaxations were expressed as a percentageof the precontraction to carbachol (100 nM).

Tracing and immunocytochemistry. Rats were anesthetized withpentobarbital (60 mg/kg ip). A midline laparotomy was performed andthe colorectum was exposed over 2–3 cm. The tracer dye Fast blue (2%in 10% DMSO) was injected in the colonic wall at eight sites (fourinjections at two levels) under aseptic conditions. The animals wereallowed to recover for 14 days before induction of TNBS colitis. Threedays later the rats were anesthetized, a lumbar laminectomy was per-formed, and the DRG L6 and S1 were harvested. The specimens wereimmediately immersed in 4% paraformaldehyde in 0.1 M phosphatebuffer (pH 7.0) at room temperature. After a 10-min fixation period, theDRG were processed for cryosectioning according to published methods(9). To assess the presence of TRPV1 in spinal neurons, cryostatsections were immunocytochemically stained with a rabbit antibodydirected against TRPV1 (AB5370; CHEMICON International, Te-mecula, CA; diluted 1:400) and a Cy3-conjugated goat anti-rabbit IgG(Jackson Immunoresearch Laboratories, West Grove, PA; diluted1:4,000). Briefly, all incubations were performed at room temperature.Sections were immersed for 30 min in 0.1 M phosphate-bufferedsaline (PBS, pH 7.4) containing 0.05% thimerosal (PBS*), 5% normalhorse serum (NHS; Jackson Immunoresearch Laboratories), and 1%Triton X-100, prior to incubation for 18 h with the primary antibodydiluted in PBS* containing 5% NHS and 0.1% Triton X-100. After beingrinsed in 0.01 M PBS, they were incubated for 1 h with the secondaryantibody diluted in PBS* containing 1% NHS. After washing, thecryosections were mounted in Citifluor. For negative controls, the pri-mary antiserum was omitted. For quantification, the number of neuronsexpressing TRPV1 immunoreactivity and/or Fast blue tracing wascounted on 10 slides per ganglion, and results were expressed as apercentage of the total numbers of neurons identified or as the percentageof Fast blue positive neurons expressing TRPV1 immunoreactivity.

Real-time PCR. Rats were anesthetized with pentobarbital (60mg/kg ip) and a lumbar laminectomy was performed. For each rat, theDRG L6 and S1 were harvested bilaterally, embedded in optimalcutting temperature compound (OCT), and stored at �80°C. TheDRG were then sliced into 50 – 60 frozen sections (20 �m) thatwere immediately immersed in lysis buffer. Total RNA was iso-lated using the Absolutely RNA microprep kit (Stratagene; LaJolla, CA). A TaqMan gene expression assay was performed for

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TRPV1 (assay ID Rn00676880_m1; Applied Biosystems, FosterCity, CA) on an ABIPrism 7300 sequence detector system (AppliedBiosystems) in 25 �l reaction volumes containing One-step UniversalPCR Master Mix (Applied Biosystems). The parameters for polymer-ase chain reaction (PCR) amplification were 48°C for 30 min, 95°Cfor 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for1 min. All data were controlled for quantity of cDNA input byperforming measurements on the endogenous reference gene �-ac-tin (assay ID Rn00667869_m1; Applied Biosystems) and calcula-tion of comparative cycle thresholds [CT � CT(TRPV1) � CT(�-actin)]. Relative expression of mRNA species was then calculated as2�CT, CT � CT(TNBS) � CT(control).

Solutions and drugs. Carbachol, Evans blue, hexadecyltrimethylam-monium bromide, o-dianisidine dihydrochloride, and Tween 80 were allpurchased from Sigma-Aldrich, St. Louis, MO; BCTC was purchasedfrom Biomol International, Exeter, UK; capsazepine was purchased fromAscent Scientific, Weston-Super-Mare, UK; CGRP-(8-37) was obtainedfrom Sigma-Aldrich; hydrogen peroxide and diethyl ether were pur-chased from Merck, Darmstadt, Germany; capsaicin and TNBS wereobtained from Fluka, Neu Ulm, Germany; pentobarbital (Nembutal) waspurchased from Ceva, Brussels, Belgium; and Fast blue was obtainedfrom Polysciences Europe, Eppelheim, Germany.

Presentation of results and statistical analysis. Parametric valuesare shown as means � SE for n indicating the number of rats used. Forstatistical analysis, unpaired Student’s t-test or two-way ANOVA wasperformed. Post hoc testing was carried out by Student-Newman-Keuls analysis or Student’s t-test when appropriate. For nonparamet-ric data, results are presented as median with 25th and 75th percentile.Nonparametric analysis was performed via a Mann-Whitney U-test.

P 0.05 was considered statistically significant. Data were ana-lyzed via SPSS 11.5 software (SPSS, Chicago, IL) and GraphPadPrism 4.00 (GraphPad Software, San Diego, CA).

RESULTS

Effect of TNBS-induced colitis on gastrointestinal motility.Instillation of TNBS in the distal colon resulted in a high-gradeinflammatory response characterized macroscopically by mu-cosal thickening, ulceration, and necrosis. This was accompa-

nied by a pronounced increase in colonic MPO content from1.8 � 0.9 U/g in controls to 41.5 � 5.8 U/g in rats with TNBScolitis (P 0.001, n � 6). Distal experimental colitis signif-icantly reduced gastric emptying and the GCS�I in all vehicle-treated rats (Fig. 1, A and B) but had no effect on the front ofintestinal transit or on the GCI (Fig. 1, C and D).

Effect of capsazepine on colitis-induced motility changes.Capsazepine caused a significant but dose-independent de-crease of gastric emptying and the GCS�I in controls (P 0.05, Fig. 1, A and B). This inhibition was absent in rats withTNBS-induced colitis [not significant (NS), Fig. 1, A and B].Capsazepine had no significant effect on the GCI or the distal frontof intestinal transit in control or TNBS rats (Fig. 1, C and D).Capsazepine treatment did not modulate the magnitude of theinflammatory response, as evidenced by the macroscopic score ofinflammation and the MPO-content of the distal colon (Table 1).

Effect of BCTC on colitis-induced motility changes. Incontrols, BCTC caused a reduction of gastric emptying andthe GCS�I that was significant at 10 mg/kg (P 0.05,Fig. 2, A and B). In rats with TNBS-induced colitis, BCTC 5mg/kg significantly improved gastric emptying and the GCS�I

compared with vehicle treatment (P 0.05, Fig. 2, A and B).A lower dose had no effect whereas the effect of 10 mg/kg wascomparable to the effect of 5 mg/kg. BCTC had no effect onsmall intestinal motility (Fig. 2, C and D). BCTC did notinfluence the macroscopic score of inflammation or the MPOcontent of the distal colon (Table 1).

Effect of CGRP-(8-37) on colitis-induced motility changes.The CGRP antagonist CGRP-(8-37) did not significantlymodulate gastric emptying and the GCS�I in controls (NS,Fig. 3, A and B). Likewise, CGRP-(8-37) had no effect onthe GCI or the front of intestinal transit (Fig. 3, C and D). Inrats with TNBS-induced colitis, the antagonist significantlyimproved gastric emptying (P 0.05, Fig. 3A). This effectwas reflected in the GCS�I (Fig. 3B). CGRP-(8-37) had no

Fig. 1. Effects of capsazepine 5–10 mg/kg (CZP)on gastric emptying (GE; A), the geometric centerof transit including the stomach (GCS�I, B), thedistal front of intestinal transit (C), and the geo-metric center of transit omitting the stomach (GCI,D). Results are expressed as percentage gastricemptying (%GE), percentage intestinal transit(%transit), and segment number (GCS�I and GCI)and are shown as means � SE for n � 10. TNBS,trinitrobenzene sulfate. *P 0.05, significantlydifferent from vehicle-treated controls, 2-wayANOVA with Student-Newman-Keuls post hocanalysis.

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additional effect on the GCI nor on the front of intestinaltransit in rats with TNBS colitis (Fig. 3, C and D).

CGRP-(8-37) did not modulate the inflammatory responsesince controls and TNBS rats showed similar scores for mac-roscopic inflammation and colonic MPO content (Table 1).

Effect of TRPV1 receptor antagonists on capsaicin-inducedrelaxations. In gastric fundus muscle strips precontracted withcarbachol (100 nM), capsaicin (1 �M) induced a sharp relax-ation that gradually recovered (Fig. 4A). This relaxationshowed the tachyphylaxis typical for capsaicin-induced phe-nomena: a muscle strip that relaxed to capsaicin did notrespond a second time to the TRPV1 agonist. Capsazepinesignificantly decreased the relaxatory response to capsaicin atthe concentration of 5 �M (Fig. 4, B and D). BCTC dosedependently inhibited the relaxation to capsaicin and, at 100nM, almost completely abolished the relaxation to capsaicin(Fig. 4, C and E).

Effect of experimental colitis on TRPV1 receptor expressionin the DRG L6–S1. Two weeks after injection of Fast blue inthe colonic wall, 8.9 � 1.1% of neuronal cell bodies in DRGL6–S1 stained positive for Fast blue in controls and 10.1 �0.9% stained positive in rats with TNBS colitis (n � 5, NS).TRPV1 expression of these colon-derived DRG neurons was

significantly higher in rats with TNBS-induced colitis (53.4 �3.5%) compared with control rats (40.6 � 4.1%) (P 0.05,Fig. 5).

Effect of experimental colitis on TRPV1 receptor mRNAlevels in the DRG L6–S1. TNBS-induced colitis caused asignificant upregulation of TRPV1 receptor mRNA in thepelvic nerve DRG L6–S1, as evidenced by an increase com-pared with controls in its expression relative to the housekeep-ing gene �-actin (P 0.05, Fig. 6).

DISCUSSION

Patients with IBD such as Crohn’s disease have been shownto suffer from gastroparesis or reduced gastric emptying, lead-ing to symptoms like nausea, early satiety, and abdominaldiscomfort (2, 22).

We previously reported on the occurrence of gastric motorinhibition in rats with TNBS-induced colitis (13). We providedproof that the afferent branch of this reflex pathway is con-tained within the pelvic nerve, because its section resulted in arestoration of gastric motor function in TNBS-treated rats. Wehypothesized that this colitis-induced gastroparesis actuallyrepresents the sensitized state of an extrinsic reflex pathway

Table 1. Effect of CZP, BCTC, and CGRP-(8-37) or their respective vehicles on the MPO content and the macroscopicscore of inflammation of the distal colon in rats with TNBS-induced colitis

Vehicle CZP Vehicle BCTC Vehicle CGRP-(8-37)

MPO 47.3�5.6 53.7�7.2 40.5�50.9 38.7�6.3 37.3�5.9 41.5�4.7n � 6, NS n � 6, NS n � 6, NS

Macro 8.5 (7.5–10) 8 (6.5–9) 9 (8–10) 8 (6–8) 8 (8–10) 8 (6–9)n � 6, NS n � 6, NS n � 6, NS

MPO results are expressed as U/g tissue and presented as mean � SE. Macroscopic score (Macro) ranges from 0 to 10 and is presented as median with 25thand 75th percentile. CZP, capsazepine 10 mg/kg; BCTC, N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide 5 mg/kg;CGRP-(8-37), calcitonin gene-related peptide receptor antagonist 150 mg/kg; TNBS, trinitrobenzene sulfate; NS, not significantly different from vehicletreatment, unpaired Student’s t-test for MPO analysis or Mann-Whitney U-test for analysis of macroscopic score.

Fig. 2. Effects of N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)car-boxamide (BCTC; 1–10 mg/kg) on gastric emptying(A), the geometric center of transit including thestomach (B), the distal front of intestinal transit (C),and the geometric center of transit omitting thestomach (D). Results are expressed as percentagegastric emptying, percentage intestinal transit, andsegment number and are shown as means � SE forn � 10. *P 0.05, significantly different fromvehicle-treated controls, #P 0.05, significantlydifferent from vehicle- and BCTC (1 mg/kg)- treatedTNBS rats, 2-way ANOVA with Student-Newman-Keuls post hoc analysis.

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known as cologastric inhibition. Indeed, physiological inhibi-tion of gastric emptying in response to innocuous colorectaldistension occurs both in rats (26) and in humans (59) andprobably plays a role in the homeostasis of whole gut motorcoordination. In this study, we showed that sensitization ofpelvic afferent neurons is indeed involved in colitis-inducedgastroparesis and involves TRPV1 and CGRP.

We designed the present study to identify the neuropharma-cological mediators responsible for the sensitization processleading to colitis-induced gastroparesis. Of the key playersinvolved in sensory nerve sensitization, the vanilloid TRPV1receptor currently represents the most attractive target. This

polymodal cation channel receptor is present on the majority ofsensory nerve fibers in the rat colon (55) and is activated and/orsensitized by heat, protons, mechanical stimuli, and a numberof lipid derivatives such as LTB4 and 12-HPETE (21). A“true” specific endogenous agonist, however, remains uniden-tified. Likewise, the physiological role of TRPV1-mediatedsignaling is largely unknown, even though our immunocyto-chemical results clearly show that TRPV1 receptors are abun-dantly present in 40% of normal colonic afferent cell bodies inDRG L6–S1, comparable to other findings (7).

The lack of knowledge concerning the physiological roleof TRPV1 signaling is largely due to the lack of a TRPV1

Fig. 3. Effects of CGRP-(8-37) on gastric emptying(A), the geometric center of transit including thestomach (B), the distal front of intestinal transit (C),and the geometric center of transit omitting thestomach (D). Results are expressed as percentagegastric emptying, percentage intestinal transit, andsegment number and are shown as means � SE forn � 10. *P 0.05, significantly different fromvehicle-treated controls, #P 0.05, significantlydifferent from vehicle-treated TNBS rats, 2-wayANOVA with Student-Newman-Keuls post hocanalysis.

Fig. 4. Top: typical tracings of isolated mus-cle strips from the rat gastric fundus showingrelaxations to 1 �M capsaicin in strips treatedwith saline (A), capsazepine (CZP, 5 �M; B)and BCTC (100 nM; C). Arrow depicts addi-tion of capsaicin in the organ bath. W, wash-out of drugs from the organ bath. Bottom:mean effect of capsazepine (1–5 �M, n �7–9; D) and BCTC (10–100 nM, n � 7–9; E)on the relaxation to 1 �M capsaicin. Resultsare shown as means � SE. *P � 0.05, un-paired Student’s t-test.

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receptor antagonist with a good specificity profile and withpharmacokinetic properties that are optimized for in vivostudies. The most commonly used competitive antagonistfor in vivo use in rats is the synthetic capsaicin analogcapsazepine (4).

Capsazepine significantly inhibited gastric emptying and thegeometric center of gastrointestinal transit in controls, withoutaffecting intestinal transit or the GCI. In rats with TNBS colitis;however, it no longer significantly affected gastric emptying.This differential effect implies a modulation of vanilloid sig-naling under the influence of an acute and local inflammatoryprocess. The inhibition of gastric emptying by capsazepine incontrols could be due to a physiological role of TRPV1 in theregulation of gastric emptying, or it may be due to the aspecificactions attributed to this compound. Indeed, it is well knownthat capsazepine does not only inhibit TRPV1 receptor activa-

tion but equally affects neuronal calcium currents (17) andnicotinic acetylcholine receptors (33). Our in vitro data showthat even at a concentration of 5 �M capsazepine did notcompletely inhibit capsaicin-induced relaxations in the gastricfundus, raising additional questions about its sensitivity. There-fore, we also tested the effects of the competitive TRPV1receptor antagonist BCTC on colitis-induced motility disor-ders. BCTC has an optimized pharmacokinetic profile, com-bining favorable bioavailability with a relatively long half-lifeand improved specificity (46). Compared with capsazepine, ithas the additional advantage of blocking low-pH-inducedTRPV1 activation (53). This is very important, because localacidosis is a key phenomenon in tissue inflammation in generaland a powerful stimulus of TRPV1-mediated sensitization ofafferent nerve endings (27, 49). At the dose of 100 nM, BCTCcompletely inhibited capsaicin-induced relaxations of isolated

Fig. 5. Cryosections of Fast blue (FB)-traced dorsal root ganglion (DRG) (L6: A–F; S1: G–L) of control (A–C, G–I) and TNBS-treated (D–F, J–L) rats, revealingcolocalization (arrowheads C, F, I, L) between Fast blue-traced (A, D, G, J) and transient receptor potential vanilloid type 1 (TRPV1)-immunoreactive (B, E,H, K) neurons.

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muscle strips of the gastric fundus, confirming its sensitivityand affinity as a TRPV1 receptor antagonist. At a dose that didnot affect gastric emptying in controls, BCTC significantlyimproved gastroparesis in rats with TNBS-induced colitis. Thisobservation clearly highlights the modulatory role of TRPV1 insensitization related to colitis-induced gastroparesis. The high-est dose tested in this study significantly decreased gastricemptying in controls and likewise no longer improved it inTNBS rats. The mechanism by which high-dose BCTC selec-tively inhibits gastric emptying whereas it does not modulateintestinal motility was beyond the scope of this study and notinvestigated in further detail.

TRPV1 expression and function are heavily influenced by avariety of inflammatory mediators such as bradykinin (50),nerve growth factor (1), PGE2 (34), 5-HT (48), and ATP (51).The latter act through upregulation of protein kinases resultingin phosphorylation of TRPV1 and sensitization of the receptor.Recently, Jones et al. (30) demonstrated that colonic afferentfibers become resistant to the sensitizing effects of an inflam-matory soup in TRPV1 knockout mice, emphasizing the piv-otal role of vanilloid signaling in the immunomodulation ofvisceral sensory functions. Inflammation not only inducessensitization of receptor function but is also known to upregu-late TRPV1 receptor expression (57). Using immunocyto-chemical staining of traced pelvic afferent neuronal cell bodies(DRG L6–S1) we found that in our model colitis indeed causeda significant 1.3-fold increase of TRPV1 receptor expression,which is comparable with other reports (39). This upregulationwas also found when studying TRPV1 mRNA by RT-PCR.

Although the effects of immunomodulation on TRPV1 func-tion have been studied intensively, the in vivo consequences ofvisceral TRPV1 sensitization are less clear and largely derivedfrom somatic pain research (54), even though it was shown byChristianson et al. that TRPV1-positive colonic afferents aremore numerous than TRPV1-positive somatic afferents (6).Our combination of functional, immunocytochemical, and mo-lecular data clearly emphasizes the crucial role of TRPV1 inthe in vivo model of colitis-induced gastroparesis. Futurestudies will have to show whether BCTC effectively reduceselectrophysiological primary afferent neuron sensitization.

Administration of TRPV1 antagonists may modulate gutinflammation (28). Still, in our study inflammatory indexeswere not altered by capsazepine or BCTC 1 h after adminis-tration. It remains to be determined whether the participation ofTRPV1 in the inflammatory process may complicate the clin-

ical use of TRPV1 antagonists to treat inflammation-inducedmotility and sensitivity disorders.

Capsazepine and BCTC did not fully restore gastric empty-ing in colitis rats to control values. It is very likely that TRPV1is an important but not the only integrator of sensitizing stimulion afferent nerve terminals. Indeed, Wynn et al. (56) showedthat TNBS colitis-induced sensitization of pelvic afferent nerveactivity in an isolated colorectum setup was mediated by P2X3

receptors. Future studies will have to elucidate whether puri-nergic signaling is another important cofactor in colitis-induced gastroparesis, next to vanilloid signaling.

After peripheral stimulation of primary afferent neurons, thesensory signal is carried to the laminae I, II, V, and X of thespinal dorsal horn where the synapse with the second-orderneuron occurs and the information is either discarded or gatedto peripheral (autonomic) or central (thalamic) relay centers(24). It has been shown that CGRP plays an important modu-latory role as a neurotransmitter at this synapse. Indeed, nox-ious visceral and somatic stimulation increase both its synthe-sis (18) and release (25). CGRP will then increase proteinkinase activity in the postsynaptic neuron, thereby increasingneuronal excitability and NK1 receptor expression (47). Thisresults in windup of the second-order neuron, accounting for amore sustained central sensitization. Plourde et al. (44) re-ported that intrathecal administration of a CGRP antagonistreduced acetic acid-induced colonic visceral hypersensitivity.Systemic CGRP inhibition in our model had no significanteffect on gastric emptying in controls, in line with previousreports (36, 45). However, the CGRP antagonist significantlyrestored gastric emptying to normal levels in rats with TNBScolitis, suggesting that spinal sensitization of afferent nervefibers also accounts for colitis-induced gastroparesis. We can-not completely exclude a local gastric or colonic effect of theantagonist, since CGRP is also released from peripheral termi-nals of stimulated nerve fibers and may thus play a modulatoryrole in peripheral excitation of colonic afferents or in gastricmotility. Still, centrally administered CGRP induces a hyper-algesic state and intracisternal or intravenous application ofCGRP inhibit gastric emptying in a CGRP-(8-37)-sensitivemanner in rats, supporting our hypothesis (32, 35, 36).

An alternative explanation for colitis-induced gastroparesiscould be stress, which may have been present in our study asthe rats were fasted and which is known to inhibit gut motility(42). However, the pronounced effect of pelvic nerve section(13) and the beneficial effect of TRPV1 antagonists suggestthat a peripheral neural pathway involving afferent sensitiza-tion accounted for the gastroparesis in our study.

The motility alterations at distance of an inflammatoryregion we reported here bear resemblance to the panentericfield effect as described for postoperative and septic ileus andmay possibly involve similar pathophysiological mechanisms(10, 15, 52).

In conclusion, we showed that TRPV1 and CGRP receptors,biological mediators of sensitization, are involved in the patho-physiology of gastroparesis induced by TNBS colitis. Previousstudies revealed that the afferent arm of this pathological reflexphenomenon is contained within the pelvic nerve (13). In thisstudy we provide evidence that during colitis, both TRPV1receptor production and expression in the DRG of the afferentpelvic nerve are increased. This peripheral sensitization trig-gers a reflex pathway involving CGRP, leading to overt inhi-

Fig. 6. Effect of TNBS-induced colitis on the relative expression of TRPV1mRNA in the DRG L6–S1. *P 0.05, significantly different from controls,unpaired Student’s t-test (n � 5).

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bition of gastric emptying. Our data reveal that in colitis-induced gastroparesis we may have identified a novel in vivomodel for TRPV1-mediated visceral afferent nerve sensitiza-tion as an alternative for visceral hyperalgesia. In addition, ourresults indicate that TRPV1 and CGRP modulation may bebeneficial for IBD-associated motility disorders at a distancefrom the inflammatory site.

ACKNOWLEDGMENTS

H. De Schepper is an aspirant of the Fund for Scientific Research (FWO),Flanders. W. Martinet is a postdoctoral fellow of the FWO.

GRANTS

This work was supported financially by the Interuniversity Poles of Attrac-tion program P5/20 and by the FWO (Grant no. G.0200.05).

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 doi:10.1152/ajpgi.00351.2007 294:245-253, 2008. First published Nov 8, 2007;Am J Physiol Gastrointest Liver Physiol

Winter J.-P. Timmermans, W. Martinet, A. G. Herman, P. A. Pelckmans and B. Y. De H. U. De Schepper, J. G. De Man, N. E. Ruyssers, A. Deiteren, L. Van Nassauw,

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