European Journal of Pharmacology 714 (2013) 332–344

Contents lists available at ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia Mechanisms involved in abdominal nociception induced by either TRPV1 or TRPA1 stimulation of rat peritoneum

Gabriela Trevisan a, Mateus F. Rossato a, Carin Hoffmeister b, Sara M. Oliveira a, Cássia R. Silva a, Filipe C. Matheus c, Gláucia C. Mello d, Edson Antunes d, Rui D.S. Prediger c, Juliano Ferreira a,b,c,n a Graduate Program in Biological Sciences, Toxicological Biochemistry, Department of Chemistry, Center of Natural and Exact Sciences, Federal University of Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil b Graduate Program in Pharmacology, Department of Physiology and Pharmacology, Center of Health Sciences, Federal University of Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil c Graduate Program in Pharmacology, Department of Pharmacology, Center of Biological Sciences, Federal University of Santa Catarina (UFSC), Florianópolis, SC 88049-900, Brazil d Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, Campinas. SP 13084-971, Brazil article info abstract

Article history: Abdominal pain is a frequent symptom of peritoneal cavity irritation, but little is known about the role of Received 22 January 2013 the receptors for irritant substances, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 Received in revised form (TRPA1), in this painful condition. Thus, we investigated the abdominal nociception caused by peritoneal 21 May 2013 stimulation with TRPV1 (capsaicin) and TRPA1 (allyl isothiocyanate, AITC) agonists and their mechanisms Accepted 12 July 2013 in rats. The intraperitoneal (i.p.) injection of either capsaicin or AITC (0.03–10 mg/kg) induced short-term Available online 1 August 2013 (up to 20 min) and dose-dependent abdominal nociception, and also produced c-fos expression in spinal Keywords: afferents of the dorsal horn. TRPV1 antagonism prevented (9474% inhibition) nociception induced by Capsaicin capsaicin but not by AITC. In contrast, the TRPA1 antagonism almost abolished AITC-induced nociception Allyl isothiocyanate (9572% inhibition) without altering the capsaicin response. Moreover, nociception induced by either Substance p capsaicin or AITC was reduced by the desensitisation of TRPV1-positive sensory ﬁbres with resinifer- Mast cells 7 7 Resiniferatoxin atoxin (73 18 and 76 15% inhibitions, respectively) and by the NK1 receptor antagonist aprepitant Pain (5675 and 5378% inhibitions, respectively). Likewise, the i.p. injections of capsaicin or AITC increased the content of substance P in the peritoneal ﬂuid. Nevertheless, neither the mast cell membrane

stabiliser cromoglycate, nor the H1 antagonist promethazine, nor depletion of peritoneal macrophages affected abdominal nociception induced either by capsaicin or AITC. Accordingly, neither capsaicin nor AITC increased the histamine content in the peritoneal fluid or provoked peritoneal mast cell degranulation in vitro. Collectively, our findings suggest that TRPV1 and TRPA1 stimulation in the peritoneum produces abdominal nociception that is mediated by sensory fibres activation. & 2013 Elsevier B.V. All rights reserved.

1. Introduction TRPV1 and TRPA1 are usually co-expressed in a subset of small diameter sensory ﬁbres together with the neuropeptide substance Transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 P(Bautista et al., 2005; Story et al., 2003). It has been well (TRPA1) are non-selective cation channels involved in the periph- described that activation of TRPV1 and TRPA1 in the sensory eral detection of several painful stimuli (Andrade et al., 2012; Jara- neurons releases neuropeptides and transmits painful stimuli to Oseguera et al., 2008; Moran et al., 2011). An important feature of the central nervous system, which causes pain (Cortright and both channels is that they are activated by irritant substances, Szallasi, 2009; Geppetti et al., 2008). In fact, the receptors TRPV1 such as capsaicin (the “hot” component of chilli peppers), which and TRPA1 have been implicated in painful processes observed in activates TRPV1, and allyl isothiocyanate (AITC, the major compo- somatic (such as skin and joints) and visceral (such as intestine, nent of mustard oil), which stimulates TRPA1 (Moran et al., 2011). pancreas and urinary bladder) organs (Akbar et al., 2008; Andrade et al., 2012; Lapointe and Altier, 2011; Moran et al., 2011; Schwartz et al., 2011). In addition to sensory neuron activation and neuro-

n peptide release, mast cell stimulation and histamine secretion Corresponding author at: Department of Chemistry, Federal University of Santa have been implicated in nociception caused by triggering TRPA1 Maria, Avenida Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. Tel.: +55 55 3220 8053; fax: +55 55 3220 8978. and TRPV1 in the skin and viscera (Andrade et al., 2008; Futamura E-mail address: [email protected] (J. Ferreira). et al., 2009; Inoue et al., 1993; Massaad et al., 2004).

The peritoneum is the largest and most complex serous mem- 2.3. Evaluation of capsaicin- and AITC-induced abdominal brane of the body and consists of two layers: the parietal peritoneum nociception in rats lines the abdomino-pelvic cavity and the visceral peritoneum reflects over the external surface of the viscera (Tanaka et al., 2002). Diseases Animals were placed individually in chambers (transparent involving the peritoneum are frequently encountered in medical glass boxes) and were allowed to adapt for 20 min before the practices (Elsayes et al., 2006). The irritation or inflammation of the algogen injection. Abdominal nociception was elicited by the peritoneum by chemical substances or microorganisms usually intraperitoneal (i.p.) administration of either capsaicin or AITC. induces abdominal pain, which is a common complaint in all settings The control animals received the same volume of the vehicle of medical practice (Flasar and Goldberg, 2006; Mactier et al., 1998). (10 mL/kg, 0.95% ethanol and 0.05% Tween 80 in PBS). Abdominal The peritoneal cavity contains both visceral and somatic sensory nociception was qualitatively evaluated using a scale from 0 to innervations and has a large number of resident cells, such as mast 3 points for each 10-min interval, as previously described with cells (Anaf et al., 2006; Flasar and Goldberg, 2006; Lantéri-Minet some modifications (Schmauss and Yaksh, 1984). The abdominal et al., 1993). nociceptive score was assigned as follows: 0¼normal body posi- In addition, both TRPV1 and TRPA1 are expressed in somatic tion of the rat and normal exploratory behaviour, 1¼leaning and visceral sensory neurons, as well as in mast cells (Biro et al., posture favouring the left or right body side, 2¼stretching of the 1998; Malin et al., 2011; Prasad et al., 2008; Stander et al., 2004; hindlimbs, dorsoflexion of the hind paws, and body stretched and Weller et al., 2011), and are important receptors for chemical flat on the bottom, frequently with the pelvis rotated sideward, irritants (Moran et al., 2011). However, the ability of such receptors 3¼contraction of the abdominal muscles followed by a stretching to induce abdominal pain after peritoneum stimulation and the of the body and extension of the hind limbs (writhing response). mechanisms involved are poorly understood. The goal of this Abdominal nociception was also quantitatively measured by the study was to investigate whether abdominal nociception is amount of time an animal presented a nociceptive score ≥1 timed induced by the activation of either TRPV1 or TRPA1 and some of in 10-min blocks (Schmauss and Yaksh, 1984). the mechanisms involved in this response. Initially, we observed the abdominal nociception elicited by the i.p. administration of capsaicin (0.1 mg/kg) or AITC (3 mg/kg) at 10-min intervals over a total time of 30 min. Afterwards, a dose– 2. Material and methods response curve for the abdominal nociception induced by the i.p. administration of capsaicin (0.03–0.3 mg/kg) or AITC (1–10 mg/kg) 2.1. Animals was carried out. For this experiment, the abdominal nociception time and score were observed for 10 min after the algogen injection. Adult male Wistar rats (200–250 g) bred in our animal house To observe a possible co-participation of the TRPV1 and TRPA1 were used in all of the experiments. The animals were housed in receptors in the abdominal nociception induced by capsaicin or AITC, groups of 5 to a cage in a controlled temperature environment we have co-injected these substances. Then, we have co-administered maintained at 2271 1C with a 12-h light/dark cycle (lights on from the capsaicin (0.01 mg/kg, i.p.) and AITC (1 mg/kg, i.p.) in doses that 6:00 a.m. to 6 p.m.) and fed standard lab chow and tap water ad did not induce nociception previously. In addition, we have also co- libitum. The animals were acclimated to the experimental room for at injected the TRPV1 (SB-366791, 0.25 mg/kg, i.p.) and TRPA1 (camphor, least 2 h before the experiments. Each animal was used only once. 0.25 mg/kg, s.c.) antagonists 30 min before the administration of All of the experiments were carried out according to the current capsaicin (0.01 mg/kg, i.p.) plus AITC (3 mg/kg, i.p.) or its vehicles. guidelines for the care of laboratory animals and the ethical guidelines for the investigation of experimental pain in conscious animals (Zimmermann, 1983). The number of animals and intensity of noxi- 2.4. Fos immunohistochemistry and quantification ous stimuli used were the minimum necessary to demonstrate consistent effects of the drug treatments. All of the protocols were Fosimmunohistochemistrywasperformedaspreviouslydescribed approved by the Ethics Committee of the Federal University of Santa (Bonaz et al., 1994, 2000). Rats were sacrificed 10 min after i.p. Maria (CIETEA, protocol number: 029/2012). injection of vehicle, capsaicin (0.1 mg/kg, i.p.) or AITC (3 mg/kg, i.p.). Animals were transcardially perfused with 0.1 M phosphate 2.2. Drugs buffer (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M PBS. Spinal cords segments were rapidly removed, postfixed for Capsaicin, allyl isothiocyanate (AITC), 4′-chloro-3-methoxycin- 24 h at the same fixative, and subsequently cryoprotected over- namanilide (SB-366791), sodium cromoglycate, compound 48/80, night in 30% sucrose in 0.1 M PBS. Frozen coronal sections (40 mm) o-phthaldialdehyde, resiniferatoxin (RTX), metronidazole, HC- of the spinal cord (segments thoracic or cervical) were cut on a 030031, and histamine dihydrochloride were purchased from cryostat (Leica 1850, Germany) and processed for Fos-IR. Free- Sigma (Sigma, St Louis, MO, USA). Camphor was purchased from floating sections were incubated for 16–18 h at 4 1C with the VETEC (Rio de Janeiro, Brazil). Promethazine was obtained from primary antibody (Fos AB-5 rabbit polyclonal antibody, Santa Cruz Cristália (São Paulo, Brazil). Aprepitant (MK-869) was extracted Biotechnology, Santa Cruz, CA, USA; dilution 1:1000 in PBS 0.02 M, from commercially available capsules (Emends, Merck, USA), and containing 0.5% Triton X-100 and 10% normal goat serum) and its identity and purity (greater than 98%) were confirmed by then with a biotinylated secondary antibody (goat anti-rabbit, nuclear resonance methods. The stock solutions of capsaicin and Dako, USA; dilution 1: 250) for 2 h at room temperature. Sections AITC were prepared in 90% ethanol and 10% Tween 80. Resinifer- were finally processed for avidin-biotin-peroxidase using diami- atoxin was diluted in 10% ethanol and 10% Tween 80 in phosphate- nobenzidine as a chromogen (Sigma, St Louis, MO, USA), then buffered saline (PBS). Camphor and SB-366791 were suspended in mounted on gelatin-coated slides, dehydrated, cleared in xylene, 1% Tween 80 and 1% DMSO in PBS. Sodium cromoglycate, and cover-slipped. compound 48/80, promethazine and aprepitant were diluted in The presence of Fos immunoreactivity (Fos-IR) was detected by PBS for injection. The PBS had the following composition: 137 mM optic microscopy as a brown-black reaction product in cell nuclei. NaCl and 10 mM sodium phosphate buffer (pH 7.4). The final Fos positive cells were counted with the software Image J at the concentrations of ethanol and Tween 80 did not exceed 1% and thoraco-lumbar (T2-L2) and cervical (C1-C5) levels of the spinal also did not produce any effect on their own. cord on 5 consecutive sections, in the dorsal horn of the gray 334 G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344 matter. Results were expressed by the mean number of Fos-IR- responses were observed individually for 10 min, as described cells per dorsal horn. above. Control animals were not submitted to peritoneal macrophage depletion, but were anesthetized as described above. We 2.5. Role of sensory fibres, TRPV1, TRPA1 and NK1 in abdominal detected that peritoneal lavage decreased (about 90%) the number nociception evoked by capsaicin or AITC in rats of resident macrophages (data not shown), in accordance with previous studies (Ribeiro et al., 2000). To assess the possible contribution of the TRPV1, TRPA1 and NK1 in the nociceptive responses induced by capsaicin (0.1 mg/kg) and 2.7. Determination of the histamine and substance P contents in the AITC (3 mg/kg), the animals were treated with SB-366791 (a selective peritoneal cavity TRPV1 antagonist, 0.25 mg/kg, i.p. or s.c.), camphor (a non-selective TRPA1 antagonist, 0.25 mg/kg, s.c.), HC-030031 (a selective TRPA1 To observe if the i.p. injections of either capsaicin or AITC were antagonist,30mg/kg,i.p.),aprepitant(atachykininNK1receptor capable of inducing the degranulation of mast cells, the histamine antagonist, 2.5 mg/kg, i.p.) or the respective vehicles 30 min before content in the peritoneal fluid was determined, as previously the algogen injection. Then, abdominal nociception was observed described (Oliveira et al., 2011). The animals received an i.p. admin- individually for 10 min after the i.p. administration of capsaicin istration of capsaicin (0.1 mg/kg, i.p.), AITC (3 mg/kg, i.p.) or the (0.1 mg/kg), AITC (3 mg/kg) or the vehicle, as described above. The vehicle (10 mL/kg, i.p., 0.95% ethanol and 0.05% Tween 80 in PBS). dose of the antagonists and their treatment time were based on After 10 min, the animals were euthanised using a carbon dioxide previous data described in the literature as well as those determined chamber, and their peritoneal cavities were washed with 3 mL of PBS in pilot experiments (Kopczynska, 2008; Millan et al., 2010). containing 1 mM metronidazole (a histamine methyl transferase In order to investigate the effect of TRPV1-positive sensory inhibitor used to reduce histamine degradation). Aliquots of the fibres on nociception induced by capsaicin or AITC, we used a peritoneal fluids were centrifuged at 12,000 g and 4 1Cfor10min, desensitisation protocol employing resiniferatoxin, as described and the resulting supernatants were used to evaluate the histamine previously (Steiner et al., 2007). The animals were pre-treated content. Next, 150 μL of NaOH (1 M) was added to 400 μLofthe with a single i.p. injection of resiniferatoxin (200 mg/kg) or the supernatant and incubated with 40 μL of 1% o-phthaldialdehyde for vehicle (10% ethanol and 10% Tween 80 in PBS, 1 mL/kg) under 4min.Next,75μL of HCl (3 M) was added to stop the reaction and anaesthesia induced by a mixture of ketamine and xylazine allow for the development of fluorescence. The histamine contents (90 mg/kg and 3 mg/kg, respectively, i.p.). The animals were tested were determined spectrofluorometrically with an excitation wave- 7 days after treatment with resiniferatoxin or the vehicle. length of 360 nm and an emission wavelength of 450 nm using a To assess whether a complete desensitisation of the TRPV1- fluorescence photometer. The histamine release was expressed as the positive fibreshadoccurredaftertreatmentwithRTX,theanimals content of histamine (in μg) per mL of peritoneal fluid, as compared were submitted to an eye-wiping test, as previously described (Ikeda to a standard curve of histamine. et al., 2001; Jakab et al., 2005). For this experiment, 20 mLof10mg/mL The substance P levels were also measured from an aliquot of capsaicin solution was administeredintotheeye,andthenumberof the peritoneal fluid supernatant obtained 10 min after the i.p. wiping movements that occurred in a 1-min time interval was injection of capsaicin, AITC, or the vehicle using a commercially counted. The animals that wiped their eyes no more than 5 times available enzyme-linked immunosorbent assay (ELISA) according were considered to be desensitised by the RTX treatment. Subse- to the manufacturer′s instructions (Cayman Chemicals Company, quently, the animals received an i.p. injection of capsaicin (0.1 mg/ Ann Arbor, USA). For this experiment, the animals received an i.p. kg), AITC (3 mg/kg) or the vehicle, and their nociceptive responses administration of capsaicin (0.1 mg/kg, i.p.), AITC (3 mg/kg, i.p.) or were observed individually for 10 min, as described above. the vehicle (10 mL/kg, i.p., 0.95% ethanol and 0.05% Tween 80 in PBS). After 10 min, the animals were euthanised using a carbon 2.6. Assessment of the participation of resident peritoneal mast cells dioxide chamber, and their peritoneal cavities were washed with and macrophages in capsaicin- and AITC-elicited abdominal 3 mL of PBS. Aliquots of the peritoneal fluids were centrifuged at nociception in rats 12,000 g and 4 1C for 10 min, and the resulting supernatants were used to evaluate the content of substance P, which was The role of resident peritoneal mast cells on capsaicin- and expressed as the SP (in pg) content per mL of peritoneal fluid, as AITC-induced abdominal nociception in rats was investigated by compared to a standard curve of substance P. using the mast cell membrane stabiliser cromoglycate and the histaminergic H1 receptor antagonist promethazine. Cromoglycate 2.8. Mast cell degranulation in vitro (80 mg/kg, i.p.), promethazine (20 mg/kg, i.p.) were administered 1 or 0.5 h, respectively, before the i.p. injection of capsaicin Mast cells from the peritoneal cavities of the rats were purified on (0.1 mg/kg), AITC (3 mg/kg), the mast cell degranulator compound a Percoll gradient, as described previously (Desouza et al., 2009). 48/80 (0.75 mg/kg, used as a positive control) or the vehicle Briefly, the rats were exsanguinated under halothane anaesthesia, (10 mL/kg, i.p.). The nociceptive response was observed individu- and 10 mL of Krebs-Ringer phosphate solution (pH 7.4) was injected ally for 10 min, as described above. The dose of the drugs and their into their intraperitoneal cavities. The Krebs-Ringer solution had the treatment time were based on previous data described in the following composition (mM): NaCl (150), KCl (6.1), Na2HPO4 (10), literature as well as those determined in pilot experiments of our MgSO4 (1.5), CaCl2 (42.9), and glucose (5.6). The abdomen was group using positive controls (Ohta et al., 2003). carefully massaged, and the peritoneal fluid was withdrawn, placed To observe the effect of resident peritoneal macrophages in the in polypropylene centrifuge tubes and centrifuged at 300 g for nociceptive effect of capsaicin and AITC, we have used the protocol 5minat41C. The resulting cell pellet (of which mast cells comprise of peritoneal macrophage depletion as the method described 10%) was gently resuspended in a small volume of Krebs-Ringer before (Ribeiro et al., 2000; Kamei et al., 2010). In order to deplete phosphate solution, layered over the isotonic Percoll gradient and left the macrophage content in the peritoneal cavity, rats were i.p. at room temperature for 10 min prior to centrifugation (150 g for injected with PBS (10 mL) under isoflurane anesthesia and the 25 min at 4 1C). The gradient zone containing the mast cells was peritoneal cavity was washed three times. Immediately after the removed and washed twice in Krebs-Ringer phosphate solution. procedure, animals were injected with capsaicin (0.1 mg/kg, i.p.), The purity of the cells in the final preparation was 90–95%, and AITC (3 mg/kg, i.p.), or vehicle (10 mL/kg, i.p.) and their nociceptive their viability (as assessed by 0.1% Trypan blue dye exclusion) was G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344 335 approximately 95%. Aliquots of the mast cell suspension (0.5 mL (10 mL/kg, 0.95% ethanol and 0.05% Tween 80 in PBS). Consecu- containing 4 105 cells/mL) were warmed to 37 1Cfor10min. tively, rats were given a standard charcoal meal (5% charcoal and Capsaicin (0.001–10 mM), AITC (1–1000 mM), the vehicle (0.01% 20% Arabic gum, 2 mL) by gavage. Ten minutes after administra- ethanol in Krebs-Ringer phosphate solution), or compound 48/80 tion of the charcoal meal, the animals were euthanized and their (1 μg/mL, used as a positive control) was added to the suspension stomachs and small intestines were removed. The length of the (final volume of 1 mL). After 15 min of incubation, the reaction was intestine (from the pyloric sphincter to the ileo-caecal junctions) quenched by placing the test tubes in ice-cold water. The cells were and the distance travelled by the charcoal meal were measured. then centrifuged (300 g for 10 min at 4 1C), and the supernatant The propulsive activity of the gut was evaluated by determining was removed for histamine determination. Then, Krebs-Ringer the percentage of gastrointestinal travelled charcoal, using the phosphate solution (1 mL) was added to the pellet, which was boiled following equation: travelled (%)¼100 (distance travelled/total at 100 1C for 10 min to release the residual histamine. Then, 150 μLof gut length). NaOH (1 M) was added to 400 μL of the supernatant or the resuspended pellet and incubated with 40 μL of 1% o-phthaldialdehyde for 4 min. Next, 75 μL of HCl (3 M) was added to stop the reaction 2.10. Myeloperoxidase activity assessment and allow for the development of fluorescence. The histamine contents were determined spectrofluorometrically with an excitation Myeloperoxidase (MPO) activity was determined as described wavelength of 360 nm and an emission wavelength of 450 nm using before (Sauzem et al., 2009). Animals were i.p. injected with a fluorescence photometer, as previously described (Oliveira et al., capsaicin (0.1 mg/kg), AITC (3 mg/kg), or vehicle (10 mL/kg, 0.95% 2011). The histamine release was expressed as a percentage of the ethanol and 0.05% Tween 80 in PBS). Ten minutes after, samples of total cellular content of the amine. intestine were used to analyze the MPO activity. Samples were homogenized in 0.3 mL of sodium acetate buffer (80 mM, pH 5.5) plus 0.5% hexadecyltrimethylammonium bromide (HTAB), and 2.9. Gastrointestinal transit centrifuged (10,000 g)at41C for 20 min; supernatants were used for the assay. Then, 10 μL of supernatant was added to 200 mL We have also observed if capsaicin (0.1 mg/kg) or AITC (3 mg/ of sodium acetate buffer (80 mM, pH 5.5) and 10 mL of 5-(N,N- kg) i.p. administration alters the normal gastrointestinal transit it diethylamino)-pentyl-3,4,5-trimethoxybenzoate. After an incuba- was analysed, as described previously (Holtman et al., 2010). tion period of 3 min at 37 1C, the reaction was stopped on ice Rats were housed in cages without food for 18 h. Then, animals by the addition of 30 mL acetic acid. The visible absorbance were treated with capsaicin (0.1 mg/kg), AITC (3 mg/kg), or vehicle was analyzed on a spectrophotometer at 610 nm, a Fisher Biotech

Fig. 1. Intraperitoneal injection of the TRPV1 agonist capsaicin induced a short-term abdominal nociceptive response. ((A) and (B)) Time-course of the abdominal nociception elicited by the i.p. injection of capsaicin in rats. The animals received an i.p. injection of capsaicin (CPS, 0.1 mg/kg) or the vehicle (Veh, 0.95% ethanol and 0.05% Tween in PBS, 10 mL/ kg), and then the nociception time (A) and nociception score (B) were observed for 30 min. ((C) and (D)) Dose–response curve of capsaicin-induced abdominal nociception in rats. Different doses of capsaicin (0.03–0.3 mg/kg) or the vehicle (0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg) were administered by i.p. injection. The nociceptive time (C) and nociceptive score (D) were observed for 10 min. Each column represents the mean7S.E.M. ((A) and (C)) or the median7interquartile range ((B) and (C)) of ﬁve to six rats. The asterisks denote the signiﬁcance levels: nPo0.05, nnPo0.01, and nnnPo0.001 in comparison to the vehicle-treated group. Student′s “t” test (B); Student′s “t” test followed by the Mann–Whitney test (B); one-way ANOVA followed by Bonferroni′s post-hoc test (C) or the Kruskal Wallis test followed by Dunn′s test (D). 336 G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344

Microkinetics BT 2000 (Fisher Scientiﬁc, Pittsburgh, PA, USA) micro- some modiﬁcations (An et al., 2011). The plasma protein extravasa- plate reader. Values were expressed as optical density corrected by tion was observed after 10 min of the injection of capsaicin (0.1 mg/ mg of tissue. kg), AITC (3 mg/kg), or vehicle (10 mL/kg, 0.95% ethanol and 0.05% Tween 80 in PBS). Animals were anesthetized with sodium pento- 2.11. Measurement of plasma protein extravasation barbital (100 mg/kg, i.p.) and the Evans blue (50 mg/mL in saline solution, NaCl 0.9%) was injected through the tail vein (1 mL/kg, i.v.). The vascular permeability induced by the i.p. injection of capsai- Then, ear or meningeal samples were removed and immersed in N, cin or AITC was determined according to previously described with N-dimethylformamide at 55 1C for 24 h. Afterwards, the extract of

Fig. 2. Intraperitoneal injection of the TRPA1 agonist allyl isothiocyanate caused short-term abdominal nociception. ((A) and (B)) Time-course of the abdominal nociception provoked by i.p. injection of AITC in rats. The animals received an i.p. injection of AITC (3 mg/kg) or the vehicle (Veh, 0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg), and then the nociception time (A) and nociception score (B) were observed for 30 min. ((C) and (D)) Dose–response curve of AITC-elicited abdominal nociception in rats. Different doses of AITC (1–10 mg/kg) or the vehicle (0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg) were administered by i.p. injection and the nociception time (C) and nociception score (D) were observed for 10 min. The co-injection of sub-effective doses of capsaicin plus AITC induced nociception, and these responses were reduced by the co-administration of TRPV1 and TRPA1 antagonists. The selective TRPV1 antagonist SB-366791 (0.25 mg/kg, s.c.) plus the non-selective TRPA1 antagonist camphor (0.25 mg/kg, s.c.) administered systemically 30 min before the i.p. injection of capsaicin (CPS) plus AITC (1 mg/kg, i.p.) diminished the nociceptive time (E) and score (F). Each column represents the mean7S.E.M. ((A), (C), and (E)) or the median7interquartile range ((B), (C), and (F)) of five to six rats. The asterisks denote the significance levels: nPo0.05, nnPo0.01, and nnnPo0.001 in comparison to the vehicle-treated group, and ##Po0.01 in comparison to the capsaicin plus AITC-treated groups (pre-treated with vehicle); Student “t” test (A); Student “t” test followed by the Mann–Whitney test (B); one-way ANOVA followed by Bonferroni′s post-hoc test ((C) and (E)) or the Kruskal Wallis test followed by Dunn′s test ((D) and (F)). G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344 337 each sample was centrifuged at (5000 g,10minat201C). The experiment in relation to the control values. P values less than 0.05 Evans blue content in supernatants was quantified by reading the (Po0.05) were considered significant. optical density at 620 nm (measured in a Plate reader, Bioteck, USA). The content of Evans blue was compared with a standard curve, and 3. Results plasma extravasation was expressed as mmol of Evans blue extracted per mg of tissue. 3.1. Characterisation of abdominal nociception induced by i.p. injections of capsaicin and AITC 2.12. Statistical analysis The i.p. injection of the TRPV1 agonist capsaicin (0.1 mg/kg) The results were expressed as the mean7S.E.M for the nocicep- induced a short-term nociceptive response, observed by the nociception time and mediator concentration and the median followed by tion time and score, that peaked during the first 10 min and the interquartile range for the nociception scores. The ED50 values disappeared within 20–30 min after administration (Fig. 1AandB). were reported as the geometric means accompanied by their The capsaicin-induced nociceptive response was dose-dependent respective 95% confidence limits. Parametric data were analysed by with calculated values of ED50 and Emax of 0.083 (0.067–0.102) mg/ one-way analysis of variance (ANOVA) followed by Bonferroni′spost- kg and 563720 s and 0.124 (0.094–0.163) mg/kg and 100% for the hoc test or by the Student′s “t test when appropriate. Non-parametric nociception time and score, respectively (Fig. 1CandD). data were analysed either by the Student′s “t” test followed by the Similar to the results obtained using capsaicin, i.p. injection of Mann–Whitney test or the Kruskal–Wallis test followed by Dunn′s the TRPA1 agonist AITC (3 mg/kg) was also capable of inducing test. The ED50 values were determined by non-linear regression short-term nociception, peaking at 10 and lasting for a maximum analyses with a sigmoid dose-–response equation using GraphPad of 20 min (Fig. 2A and B). The calculated ED50 and Emax values were Software 5.0 (GraphPad Software, San Diego, CA, USA). The Emax 2.3 (1.4–3.9) mg/kg and 514754 s or 3.1 (2.1–4.7) mg/kg and 100% (maximum effect for the TRPV1 and TRPA1 agonists) was described for nociception time and score, respectively (Fig. 2C and D). in seconds for the nociception time response or in a percentage Moreover, we have observed that the co-injection of sub-effective for the maximal nociceptive score (3). The percentages of inhibition doses of capsaicin (0.03 mg/kg, i.p.) plus AITC (1 mg/kg, i.p.) induced are reported as the mean7S.E.M. obtained in each individual nociception (indicated as nociceptive time and nociceptive score).

Fig. 3. Capsaicin or AITC i.p. injection induced c-fos expression in thoraco-lumbar level spinal cord lumbar sections. (A) Photomicrographs of transverse sections of the thoraco-lumbar spinal cord and (B) pooled data representative of the action of i.p. injection of capsaicin (CPS, 0.1 mg/kg) or AITC (3 mg/mg) on the expression of c-fos-like immunoreactivity (LI) in rats 10 min after administration (5 animals for each treatment). The arrows indicate the c-fos immunoreactive cells. The representative photomicrograph of each group is an example one section (T2-L2) of the dorsal horn. Each column represents the mean7S.E.M. of of Fos-IR-cells per dorsal horn; n≥5 slices per condition in T2-L2 segments; nnnPo0.001 in comparison to the vehicle (Veh)-treated group one-way ANOVA followed by Bonferroni′s post-hoc test. 338 G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344

These responses were reduced by the administration of the combi- To avoid supra-maximal stimulation and unnecessary animal nation of the TRPV1 (SB-366791, 0.25 mg/kg, s.c.) plus the TRPA1 discomfort, we selected the doses of 0.1 mg/kg for capsaicin and (camphor, 0.25 mg/kg, s.c.) antagonists (9778 or 100% inhibition, for 3 mg/kg for AITC with an observational time of 10 min for the next nociceptive time and score, respectively) (Fig. 2EandF). experiments. Confirming the behavioral findings and indicating neuronal activation, capsaicin or AITC i.p. injection caused an increase in the 3.2. Role of TRPV1, TRPA1 and positive sensory fibres in abdominal c-Fos immunoreactivity (Fos-IR) of the dorsal horn cells in the nociception elicited by capsaicin and AITC thoraco-lumbar level of the spinal cord, when compared with vehicle i.p. injection (Fig. 3A and B). On the other hand, capsaicin, The nociception induced by capsaicin (0.1 mg/kg) was almost AITC, or vehicle i.p. treatment did not induce Fos-IR of cells in the abolished by i.p. pre-treatment with the selective TRPV1 receptor cervical level of the spinal cord (Suppl. Fig. 1A and B). antagonist SB-366791 (0.25 mg/kg i.p.; 9474 and 100% inhibition,

Fig. 4. The effect of TRPA1 and TRPV1 antagonists on agonist-induced nociception. The selective TRPV1 antagonist SB-366791 administered systemically diminished the nociceptive time (A) and score (B) elicited by capsaicin (CPS) i.p. injection. SB-366791 (0.25 mg/kg, i.p.) or the vehicle (Veh, 1% Tween 80 and 1% DMSO in PBS, 1 mL/kg) was injected 30 min before the i.p. administration of capsaicin (0.1 mg/kg), AITC (3 mg/kg) or the vehicle (0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). The non-selective (camphor) or the selective (HC-030031) TRPA1 antagonists were able to reduce the nociceptive time ((C) and (E)) and score ((D) and (F)) provoked by AITC i.p. administration. Camphor (0.25 mg/kg, s.c.), HC-030031 (30 mg/kg, i.p.), or the vehicle (1% Tween 80, 1% DMSO in PBS, 1 mL/kg) was injected 30 min before the i.p. injection of capsaicin (0.1 mg/kg), AITC (3 mg/kg) or the vehicle (0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). Afterwards, the nociception time ((A), (C), and (E)) and nociception score ((B), (D), and (F)) were observed for 10 min. Each column represents the mean7S.E.M. ((A), (C), and (E)) or the median7interquartile range ((B), (D), and (F)) of five rats. The asterisks denote the significance levels: nPo0.05 and nnnPo0.001 in comparison to the vehicle-treated group (pre-treated with vehicle), and #Po0.05 and ###Po0.001 in comparison to the capsaicin- or AITC-treated groups (pre-treated with vehicle); one-way ANOVA followed by Bonferroni′s post-hoc test ((A), (C), and (E)) or the Kruskal Wallis test followed by Dunn′s test ((B), (D), and (F)). G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344 339 for the nociception time and score, respectively; Fig. 4A and B). nociception time and score, respectively (Fig. 4E and F). Moreover, However, the response induced by AITC (3 mg/kg) was not pre-treatment with camphor (0.25 mg/kg) or HC-030031 (30 mg/ prevented by pre-treatment with SB-366791 (Fig. 4A and B). The kg, i.p.) did not affect capsaicin-induced abdominal nociception antagonism effect of SB-366791 was not related to a neutralizing (Fig. 4C–F). effect because we have also injected SB-366791 by subcutaneous To confirm the ablation of TRPV1- and TRPA1-positive fibres, route (0.25 mg/kg, s.c.) and it was also able to reduce capsaicin in a wiping movements induced by the administration of capsaicin similar way that was observed to the i.p. injection of this TRPV1 (10 mg/mL, 20 mL) into the eye were counted. The rats pre-treated antagonist (9873 and 100% inhibition, for the nociception time with RTX (200 mg/kg, i.p., 7 days prior) showed an almost com- and score, respectively) (data not shown). plete reduction in nociception induced by capsaicin when com- Abdominal nociception caused by AITC (3 mg/kg) was mark- pared with the vehicle-treated animals (12.070.8 and 3.070.3 edly reduced by the non-selective TRPA1 antagonist camphor wiping movements, for the vehicle- and RTX-treated animals, (0.25 mg/kg, s.c.), with inhibitions of 9572 and 67% for the respectively; Po0.001, Student′s “t” test). nociception time and score, respectively (Fig. 4C and D). A similar In addition, the desensitisation of TRPV1-positive sensory fibres reduction was observed for the selective TRPA1 antagonist (HC- caused by pre-treatment with RTX largely reduced capsaicin- 030031, 30 mg/kg, i.p.), with inhibitions of 9675 and 100% for the (0.1 mg/kg) and AITC-elicited (3 mg/kg) abdominal nociception with inhibitions of 73718 and 76715% for the nociception time, respectively, and 100% for the nociceptive score for both treatments (Fig. 5A and B).

3.3. Participation of substance P and the tachykinin NK1 receptor in abdominal nociception induced by TRPV1 and TRPA1 agonists

To determine the participation of substance P and its NK1 receptor in nociceptive behaviours induced by either capsaicin or AITC, we first used the selective tachykinin NK1 receptor antagonist aprepitant (MK-869). Pre-treatment with aprepitant (2.5 mg/ kg, i.p.) partially, but significantly, reduced the nociception time and score induced by capsaicin (5675 and 50% inhibition, respectively) and AITC (5378 and 50% inhibition, respectively; Fig. 6A and B). Further, we measured the substance P content in the peritoneal fluid 10 min after i.p. injection of capsaicin (0.1 mg/kg), AITC (3 mg/kg) or the vehicle (10 mL/kg, i.p.; 0.95% ethanol and 0.05% Tween 80 in PBS; Fig. 5C). The concentration of substance P in the peritoneal fluid of the vehicle-treated animals was below the detection limit of the high-sensitivity EIA kit (3.9 pg/mL). How- ever, treatment with capsaicin and AITC significantly elevated the concentration of substance P in the peritoneal fluid (1273 and 12 72 pg/mL, respectively; Fig. 6C).

3.4. Role of resident peritoneal mast cells and macrophages in abdominal nociception induced by TRPV1 and TRPA1 agonists

Afterwards, we evaluated the possible participation of mast cells in nociception elicited by capsaicin and AITC. Pre-treatment with the mast cell membrane stabiliser cromoglycate (80 mg/kg, i.p.) did not affect the nociceptive responses elicited by capsaicin (0.1 mg/kg) or AITC (3 mg/kg) (Fig. 7A and B). Nevertheless, abdominal nociception trigged by the mast cell degranulator compound 48/80 was almost abolished by cromoglycate (inhibitions of 9075 and 100%, for the nociception time and score,

respectively). Similarly, pre-treatment with the H1 receptor antagonist promethazine did not affect nociception induced by capsaicin or AITC, but markedly reduced nociception induced by Fig. 5. The desensitisation of capsaicin-positive fibres decreased the nociceptive compound 48/80 (inhibitions of 9176 and 100% for the nocicep- time (A) and score (B) elicited by capsaicin (CPS) and AITC i.p. injections. The tion time and score, respectively; Fig. 7C and D). animals were pre-treated with an i.p. injection of resiniferatoxin (RTX, 200 mg/kg) In addition, neither the i.p. administration of capsaicin (0.1 mg/ or the vehicle (Veh, 10% ethanol and 10% Tween 80 in PBS, 1 mL/kg), and after kg) nor AITC (3 mg/kg) was capable of altering the histamine 7 days were administered an i.p. injection of capsaicin (0.1 mg/kg), AITC (3 mg/kg) fl or the vehicle (0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). Afterwards, the concentration in the peritoneal uid when compared with the nociception time (A) and nociception score (B) were observed for 10 min. Each vehicle-treated animals, while the i.p. injection of compound 48/ column represents the mean7S.E.M. (A) or the median7interquartile range (B) of 80 (0.75 mg/kg) significantly increased the histamine content of n nnn four rats. The asterisks denote the significance levels: Po0.05 and Po0.001 in the peritoneal fluid (2.6-fold, when compared to vehicle; Fig. 8A). comparison to the vehicle-treated group (pre-treated with vehicle), and #Po0.05 Accordingly, incubation of the isolated peritoneal mast cells and ###Po0.001 in comparison to the capsaicin- or AITC-treated groups (pre- – m – m treated with RTX); one-way ANOVA followed by Bonferroni′s post-hoc test (A) or with capsaicin (0.001 10 M) and AITC (1 1000 M) in vitro did the Kruskal Wallis test followed by Dunn′s test (B). not induce the release of histamine (Fig. 8B and C). In contrast, 340 G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344

Fig. 6. The selective tachykinin NK1 receptor antagonist (MK-869) reduced the nociceptive time (A) and score (B) elicited by capsaicin (CPS) and AITC i.p. injections. The animals were pre-treated with an i.p. injection of MK-869 (2.5 mg/kg) or the vehicle (PBS, 1 mL/kg), and after 30 min received an i.p. injection of capsaicin (0.1 mg/kg), AITC (3 mg/kg) or the vehicle (Veh, 0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). Afterwards, the nociception time (A) and nociception score (B) were observed for 10 min. (C) The i.p. injection of either capsaicin or AITC induced the release of substance P (SP) in the peritoneal cavity. The SP content was evaluated in the peritoneal fluid 10 min after the i.p. administration of capsaicin (0.1 mg/kg), AITC (3 mg/kg) or the vehicle (0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). Each column represents the mean7S. E.M. ((A) and (C)) or the median7interquartile range (B) of five rats (N.D., not detectable). The asterisks denote the significance levels: nPo0.05 and nnnPo0.001 in comparison to the vehicle-treated group (pre-treated with vehicle), and #Po0.05 and ###Po0.001 in comparison to the capsaicin- or AITC-treated groups (pre-treated with vehicle); one-way ANOVA followed by Bonferroni′s post-hoc test ((A) and (C)) or the Kruskal Wallis test followed by Dunn′s test (B). compound 48/80 (positive control) caused a release of 8573% of capsaicin (0.1 mg/kg, i.p.) or AITC (3 mg/kg, i.p.) i.p. injection did not the total histamine content of the mast cells in vitro. alter gastrointestinal transit or induced MPO activity when Moreover, we observed that peritoneal macrophage depletion observed 10 min after administration (Suppl. Fig. 2A and B). TRPA1 by repeated peritoneal lavage was not able to decrease the and TRPV1 agonists did not produce systemic effects thus the nociceptive time (452754 and 456723 s, for depleted or control i.p. injection of capsaicin (0.1 mg/kg, i.p.) or AITC (3 mg/kg, i.p.) animals, respectively) and score (2 (2–3) and 2 (2–2.75) score was not able to induce plasma extravasation in peripheral and medians (25–75 percentiles), for depleted or control animals, central tissues, such as ear and meningeal samples, after 10 min of respectively) induced by capsaicin administration (0.1 mg/kg, i. i.p. administration (Suppl. Fig. 3A and B). p.). In addition, AITC (3 mg/kg, i.p.) induced nociceptive time (423763 and 372735 s, for depleted or control animals, respectively) or score (2 (1–3) and 2 (1.25–2.75) score medians (25–75 4. Discussion percentiles) for depleted or control animals, respectively) were unaltered by the macrophage depletion protocol. Vehicle i.p. Irritation of the peritoneum by chemical substances induces injection in control or depleted animals did not induce nociception abdominal pain, which is a common complaint in many areas of (data not shown). medical practice. However, little is known about the role of TRPV1 and TRPA1 in this painful condition. In the present study, we focused 3.5. Effect of capsaicin or AITC i.p. injection on gastrointestinal on the mechanisms involved in abdominal pain induced by TRPV1 transit, gut inflammation or plasma extravasation and TRPA1 agonists in rats. We observed that an injection of either capsaicin or AITC into the rat peritoneum induces nociception Demonstrating that TRPA1 and TRPV1 agonists did not alter mediated by TRPV1 and TRPA1 receptor activation. Furthermore, gastrointestinal function or produced gastrointestinal inflammation, stimulation of TRPV1-positive sensory fibres triggers the release of G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344 341

Fig. 7. Capsaicin- and AITC-induced nociceptive responses are independent of mast cell activation. Cromoglycate (a mast cell membrane stabiliser) and the H1 histaminergic receptor antagonist (promethazine) were not able to reduce the nociceptive time ((A) and (C)) and score ((B) and (D)) elicited by the i.p. injection of either capsaicin (CPS) or AITC. The animals were pre-treated with an i.p. injection of promethazine (20 mg/kg) or cromoglycate (80 mg/kg) and after 30 or 60 min, respectively, received an i.p. injection of capsaicin (0.1 mg/kg), AITC (3 mg/kg), compound 48/80 (0.75 mg/kg, positive control) or the vehicle (Veh, 0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). Afterwards, the nociception time ((A) and (C)) and nociception score ((B) and (D)) were observed for 10 min. Each column represents the mean7S.E.M. ((A) and (C)) or the median7interquartile range ((B) and (D)) of five rats. The asterisks denote the significance levels: nPo0.05 and nnnPo0.001 in comparison to the vehicle-treated group (pre-treated with vehicle), and #Po0.05 and ###Po0.001 in comparison to the compound-48/80-treated group (pre-treated with the vehicle); one-way ANOVA followed by Bonferroni′s post-hoc test ((A) and (C)) or the Kruskal Wallis test followed by Dunn′s test ((B) and (D)). substance P and, in turn, the activation of the tachykinin NK1 recep- not alter gut function or induce gut inflammation, however, no tor but not by mast cell degranulation. systemic inflammatory effects were observed. Together, these We first observed that intraperitoneal injections of capsaicin or results indicated that the nociceptive effect induced by capsaicin AITC are able to induce short-term abdominal nociception in a or AITC i.p. injection was caused by a local action of agonists on dose-dependent manner and with similar efficacy. Analysis of the peritoneum sensory fibers.

Emax values showed that both agonists were very effective, reach- In the present study, capsaicin and AITC were selective in ing the maximal score on the abdominal nociception scale (3) and inducing pain through TRPV1 and TRPA1 stimulation because the presenting nociception over nearly the entire observation period abdominal nociception induced by these agonists was fully abolished (600 s). Similarly, it has been demonstrated that abdominal pain by pre-treatment with either TRPV1 or TRPA1 antagonists, respec- induced by peritoneal irritation in humans is very severe and tively. A recent study reported that some aversive and nociceptive short-lived (Mactier et al., 1998). Although capsaicin and AITC actions resulting from high concentrations of AITC (41mM)inmice were equiefficacious, the TRPV1 agonist was approximately 25- are mediated by TRPV1 (Everaerts et al., 2011). In our study, fold more potent than the TRPA1 agonist. This result is consistent AITCseemstobeselectiveforTRPA1becausetheselectiveTRPV1 with previous studies showing that capsaicin is more potent than antagonist SB366791 was not capable of altering AITC-induced allyl isothiocyanate in inducing depolarisation of somatic and nociception in a dose where it fully prevented capsaicin-induced visceral nociceptors and pain/nociception in the skin and viscera abdominal nociception. One may also argue that camphor is a TRPV1 (Andrade et al., 2006, 2008; Brozmanova et al., 2012; Weller et al., agonist at high concentrations (Xu et al., 2005). However, this activity 2011). Confirming our behavioral findings, we observed that i.p. was not observed in our experimental setting because camphor did capsaicin and AITC were able to increase the Fos-IR in dorsal horn not induce nociception alone or potentiate capsaicin- or AITC- cells of the spinal cord at the thoraco-lumbar level, but not at induced nociception, as expected for a TRPV1 agonist. Moreover, cervical level. These findings are in accordance with the literature the selective TRPA1 antagonist, HC-030031, was also able to reduce that demonstrates that i.p. injection of the irritant substance acetic the nociception induced by AITC and not capsaicin effects. These acid caused nociception and Fos-IR mainly at the thoraco-lumbar results confirm the data obtained with the poorly selective TRPA1 levels of the spinal cord (where there are the spinal terminations antagonist camphor. of afferent fibers that innervates the peritoneum), but not at It was previously demonstrated that TRPV1 activation may cross- cervical level (indicating a selective stimulation of the peritoneal sensitise TRPA1 or vice versa (Akopian, 2011). However, it is unlikely afferent fibers) (Lantéri-Minet et al., 1993; Sinniger et al., 2005). In that cross-sensitisation occurred in the abdominal pain induced by addition, we have observed that capsaicin or AITC i.p. injection did either the TRPV1 or TRPA1 agonists because selective antagonism of 342 G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344

Fig. 8. Evaluation of the histamine release induced by capsaicin and AITC in vivo (A) and in vitro ((B) and (C)). (A) The histamine levels were evaluated in the peritoneal ﬂuid 10 min after the i.p. administration of capsaicin (CPS, 0.1 mg/kg), AITC (3 mg/kg), compound 48/80 (0.75 mg/kg, positive control), or the vehicle (Veh, 0.95% ethanol and 0.05% Tween in PBS, 10 mL/kg). ((B) and (C)) The histamine released from the isolated rat pleural mast cells was also measured in the presence of capsaicin (0.001–10 mM), AITC (1–1000 mM), compound 48/80 (1 mg/mL, positive control) or the vehicle (0.01% ethanol in Krebs-Ringer phosphate solution). Each column represents the mean7S.E.M. of four rats or four samples. The asterisks denote the signiﬁcance levels: nPo0.05 and nnnPo0.001 in comparison to the vehicle-treated group; one-way ANOVA followed by Bonferroni′s post-hoc test.

both of the receptors did not alter the nociceptive response of either treatment with RTX reduces the expression of both TRPV1 and AITC or capsaicin. However, cross-sensitisation may occur with non- TRPA1 (Pecze et al., 2009). Accordingly, we observed that systemic selective irritants. The intraperitoneal injection of acetic acid is one of treatment with RTX significantly reduced both capsaicin- and the oldest and most studied models of abdominal pain in rodents AITC-elicited abdominal nociception. Similar nociception results (Porreca et al., 1987). Nociception caused by acetic acid is reduced were found with the subcutaneous injection of either capsaicin or by both TRPV1 and TRPA1 antagonists and also by ablation of AITC into the paw skin of mice and rats (Andrade et al., 2008; TRPV1-positive fibres in rodents (Ikeda et al., 2001; Pereira et al., Massaad et al., 2004). Collectively, our data clearly showed the 2012). Moreover, acetic-acid-induced abdominal pain is mediated by essential role of primary sensory afferent fibres expressing TRPV1 TRPV1-positive fibres, mast cells, and macrophages in rodents (Ikeda and TRPA1 in the capsaicin- and AITC-induced abdominal noci- et al., 2001; Ribeiro et al., 2000). Because TRPV1 and TRPA1 are ceptive responses in rats. expressed in somatic and visceral sensory neurons as well as in mast An important feature of TRPV1- and TRPA1-positive fibres is the cells (Biro et al., 1998; Malin et al., 2011; Prasad et al., 2008; Stander presence of neuropeptide substance P (Bautista et al., 2005; Story et al., 2004; Weller et al., 2011), we further investigated the role et al., 2003). Furthermore, the release of substance P is a critical of mast cells and sensory fibres in the abdominal pain caused by mechanism that is associated with nociception provoked by the s.c. capsaicin and AITC. administrations of capsaicin and AITC of experimental animals and The peritoneal cavity contains both visceral and somatic sen- humans (Anand and Bley, 2011; Andrade et al., 2008; Banvolgyi et al., sory innervations (Flasar and Goldberg, 2006; Lantéri-Minet et al., 2004; Massaad et al., 2004; Santos and Calixto, 1997). Similarly to the

1993). It has been well recognised that systemic treatment with skin, pre-treatment with a selective NK1 antagonist largely reduced TRPV1 agonists in rodents cause a discerning degeneration of abdominal nociception induced by intraperitoneally injected capsai- TRPV1-expressing primary sensory fibres, mainly peptidergic C cin and AITC. Our results also indicate that the NK1 receptor is fibres (Ferreira et al., 2004; Hsieh et al., 2008; Kopczynska, 2008). activated by substance P released from sensory neurons, because we Because TRPA1 and TRPV1 are co-expressed in sensory neurons, also observed that the administration of either capsaicin or AITC G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344 343 induced an increase of substance P in the peritoneal fluid at the dose Acknowledgments and time that caused nociception. In addition to pain, substance P may also cause neurogenic This study was supported by the Conselho Nacional de Desen- inflammation by inducing plasma extravasation, as well as activat- volvimento Científico (CNPq), the Programa de Apoio aos Núcleos ing resident immune cells such as mast cells (Geppetti et al., 2008; de Excelência (PRONEX), the Fundação de Amparo à Pesquisa do Richardson and Vasko, 2002). Of note, we detected that the Estado do Rio Grande do Sul (FAPERGS) and the Coordenação de intraperitoneal injection of capsaicin or AITC produced plasma Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) (Brazil). extravasation in the rat peritoneum (data not shown). Moreover, it We also acknowledge fellowships from CNPq and CAPES. has been described that substance P is capable of inducing mast cell degranulation, provoking the release of histamine (Ansel et al., 1993; Ogawa et al., 1999; Okada et al., 1999; Suzuki et al., 1995, Appendix A. Supporting information 1999). However, neither the mast cell membrane stabiliser cro- moglicate nor the H1 receptor antagonist promethazine were able Supplementary data associated with this article can be found in to reduce the abdominal nociceptive responses induced by either theonlineversionathttp://dx.doi.org/10.1016/j.ejphar.2013.07.029. capsaicin or AITC. In addition, we failed in detecting an increase in the histamine content in the peritoneal fluid of rats after intraperitoneal injections of capsaicin and AITC. Accordingly, the concentration of substance P was markedly elevated in the peritoneal References fluid after the capsaicin and AITC injections, but the levels were several orders of magnitude lower than that necessary to cause Akbar, A., Yiangou, Y., Facer, P., Walters, J.R., Anand, P., Ghosh, S., 2008. Increased histamine release in rat peritoneal mast cells in vitro (Ogawa et al., capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syn- 1999; Suzuki et al., 1995). drome and their correlation with abdominal pain. Gut 57, 923–929. Akopian, A.N., 2011. Regulation of nociceptive transmission at the periphery via Because TRPA1 and TRPV1 are expressed in mast cells (Biro TRPA1–TRPV1 interactions. Current Pharmaceutical Biotechnology 12, 89–94. et al., 1998; Prasad et al., 2008; Stander et al., 2004), we further An, J.X., He, Y., Qian, X.Y., Wu, J.P., Xie, Y.K., Guo, Q.L., Williams, J.P., Cope, D.K., 2011. confirmed the potential ability of their agonists in directly indu- A new animal model of trigeminal neuralgia produced by administration of cing mast cell degranulation. Moreover, the ability of capsaicin in cobra venom to the infraorbital nerve in the rat. Anesthesia & Analgesia 113, 652–656. inducing mast cell degranulation in vitro is still controversial and Anaf, V., Chapron, C., El Nakadi, I., De Moor, V., Simonart, T., Noel, J.C., 2006. Pain, was investigated only in cell lines and in bone-marrow-derived mast cells, and nerves in peritoneal, ovarian, and deep infiltrating endome- – mast cells (Biro et al., 1998; Giudice et al., 2007). Currently, the triosis. Fertility and Sterility 86, 1336 1343. Anand, P., Bley, K., 2011. Topical capsaicin for pain management: therapeutic direct activation of TRPV1 and TRPA1 in the mature peritoneal potential and mechanisms of action of the new high-concentration capsaicin mast cells of rats has not been documented. In accordance with 8% patch. British Journal of Anaesthesia 107, 490–502. our in vivo results, capsaicin and AITC failed to provoke histamine Andrade, E.L., Ferreira, J., Andre, E., Calixto, J.B., 2006. Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder. Biochemical release by the peritoneal mast cell of rats in vitro in concentrations Pharmacology 72, 104–114. that were efficacious in the activation sensory neurons (Caterina Andrade, E.L., Luiz, A.P., Ferreira, J., Calixto, J.B., 2008. Pronociceptive response et al., 2000; Jordt et al., 2004). Thus, TRPA1 and TRPV1 agonists do elicited by TRPA1 receptor activation in mice. Neuroscience 152, 511–520. Andrade, E.L., Meotti, F.C., Calixto, J.B., 2012. TRPA1 antagonists as potential not seem to induce a direct degranulation of mature peritoneal analgesic drugs. Pharmacology & Therapeutics 133, 189–204. mast cells. We next asked whether peritoneal mast cell degranula- Ansel, J.C., Brown, J.R., Payan, D.G., Brown, M.A., 1993. Substance P selectively tion is able to cause pain and histamine release. The intraperito- activates TNF-alpha gene expression in murine mast cells. The Journal of Immunology 150, 4478–4485. neal injection of a mast cell degranulator (compound 48/80) Banvolgyi, A., Pozsgai, G., Brain, S.D., Helyes, Z.S., Szolcsanyi, J., Ghosh, M., caused abdominal nociception and increased the peritoneal hista- Melegh, B., Pinter, E., 2004. Mustard oil induces a transient receptor potential mine concentration, which was fully prevented by cromoglycate vanilloid 1 receptor-independent neurogenic inflammation and a non-neuro- fl – and promethazine. This finding demonstrates clearly that perito- genic cellular in ammatory component in mice. Neuroscience 125, 449 459. Bautista, D.M., Movahed, P., Hinman, A., Axelsson, H.E., Sterner, O., Hogestatt, E.D., neal mast cell activation is able to cause abdominal nociception Julius, D., Jordt, S.E., Zygmunt, P.M., 2005. Pungent products from garlic activate through histamine release, but this mechanism is not implicated in the sensory ion channel TRPA1. Proceedings of the National Academy of – the nociceptive responses to selective TRPV1 and TRPA1 agonists. Sciences of the United States of America 102, 12248 12252. Biro, T., Maurer, M., Modarres, S., Lewin, N.E., Brodie, C., Acs, G., Acs, P., Paus, R., We have also observed that resident peritoneal macrophages were Blumberg, P.M., 1998. Characterization of functional vanilloid receptors not involved in the abdominal nociceptive responses induced by expressed by mast cells. Blood 91, 1332–1340. capsaicin or AITC i.p. injection. Bonaz, B., Plourde, V., Tache, Y., 1994. Abdominal surgery induces fosimmunor- eactivity in the rat brain. Journal of Comparative Neurology 349, 212–222. The TRPV1 and TRPA1 receptors have been highlighted as Bonaz, B., Rivière, P.J., Sinniger, V., Pascaud, X., Junien, J.L., Fournet, J., Feuerstein, C., important sensors of noxious stimulus in different pain models, 2000. Fedotozine, a kappa-opioid agonist, prevents spinal and supra-spinal Fos including visceral and pancreatic pain (Schwartz et al., 2011; expression induced by a noxious visceral stimulus in the rat. Neurogastroen- terology and Motility 12, 135–147. Moran et al., 2011). However, in this study we have proposed to Brozmanova, M., Mazurova, L., Ru, F., Tatar, M., Kollarik, M., 2012. Comparison of observe the participation of these channels in abdominal pain, TRPA1-versus TRPV1-mediated cough in guinea pigs. European Journal of which is an important feature in clinical settings (Flasar and Pharmacology 689, 211–218. Caterina, M.J., Leffler, A., Malmberg, A.B., Martin, W.J., Trafton, J., Petersen-Zeitz, K.R., Goldberg, 2006; Mactier et al., 1998). Until now, TRPV1 and TRPA1 Koltzenburg, M., Basbaum, A.I., Julius, D., 2000. Impaired nociception and pain antagonists are still not used in the clinical practice, but different sensation in mice lacking the capsaicin receptor. Science 288, 306–313. evidences have pointed to a possible use of these substances for Cortright, D.N., Szallasi, A., 2009. TRP channels and pain. Current Pharmaceutical – pain treatment (Moran et al., 2011). Collectively, our findings Design 15, 1736 1749. Desouza, I.A., Camargo, E.A., Mariano, N.S., Optiz-Neto, J.B., Resende, J.S., Mello, G.C., suggest that TRPV1 and TRPA1 stimulation in the peritoneum Costa, S.K., De Nucci, G., Antunes, E., 2009. Role of sensory innervation in the rat produces abdominal nociception that is mediated by sensory fibres pulmonary neutrophil recruitment induced by staphylococcal enterotoxins – and substance P but not by mast cells and histamine or macro- type A and B. European Journal of Pharmacology 613, 128 134. Elsayes, K.M., Staveteig, P.T., Narra, V.R., Leyendecker, J.R., Lewis Jr., J.S., Brown, J.J., phages. Because irritant substances, such as some peritoneal 2006. MRI of the peritoneum: spectrum of abnormalities. AJR American Journal dialysis solutions and chemotherapy drugs, may produce pain of Roentgenology 186, 1368–1379. when intraperitoneally injected and lead to the activation of Everaerts, W., Gees, M., Alpizar, Y.A., Farre, R., Leten, C., Apetrei, A., Dewachter, I., fi van Leuven, F., Vennekens, R., De Ridder, D., Nilius, B., Voets, T., Talavera, K., TRPV1 and TRPA1 receptors, our results may bene t the under- 2011. The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects standing and treatment of abdominal pain. of mustard oil. Current Biology 21, 316–321. 344 G. Trevisan et al. / European Journal of Pharmacology 714 (2013) 332–344

Ferreira, J., da Silva, G.L., Calixto, J.B., 2004. Contribution of vanilloid receptors to the Okada, T., Hirayama, Y., Kishi, S., Miyayasu, K., Hiroi, J., Fujii, T., 1999. Functional overt nociception induced by B2 kinin receptor activation in mice. British neurokinin NK-1 receptor expression in rat peritoneal mast cells. Inflammation Journal of Pharmacology 141, 787–794. Research 48, 274–279. Flasar, M.H., Goldberg, E., 2006. Acute abdominal pain. Medical Clinics of North Oliveira, S.M., Drewes, C.C., Silva, C.R., Trevisan, G., Boschen, S.L., Moreira, C.G., de America 90, 481–503. Almeida Cabrini, D., Da Cunha, C., Ferreira, J., 2011. Involvement of mast cells in Futamura, M., Goto, S., Kimura, R., Kimoto, I., Miyake, M., Ito, K., Sakamoto, T., 2009. a mouse model of postoperative pain. European Journal of Pharmacology 672, Differential effects of topically applied formalin and aromatic compounds on 88–95. neurogenic-mediated microvascular leakage in rat skin. Toxicology 255, Pecze, L., Pelsoczi, P., Kecskes, M., Winter, Z., Papp, A., Kaszas, K., Letoha, T., + 100–106. Vizler, C., Olah, Z., 2009. Resiniferatoxin mediated ablation of TRPV1 neurons – Geppetti, P., Nassini, R., Materazzi, S., Benemei, S., 2008. The concept of neurogenic removes TRPA1 as well. Canadian Journal of Neurological Sciences 36, 234 241. inflammation. BJU International 101 (3), 2–6. Pereira, L.M., Lima-Junior, R.C., Bem, A.X., Teixeira, C.G., Grassi, L.S., Medeiros, R.P., Giudice, E.D., Rinaldi, L., Passarotto, M., Facchinetti, F., D′Arrigo, A., Guiotto, A., Marques-Neto, R.D., Callado, R.B., Aragao, K.S., Wong, D.V., Vale, M.L., Brito, G.A., Carbonare, M.D., Battistin, L., Leon, A., 2007. Cannabidiol, unlike synthetic Ribeiro, R.A., 2012. Blockade of TRPA1 with HC-030031 attenuates visceral fl cannabinoids, triggers activation of RBL-2H3 mast cells. Journal of Leukocyte nociception by a mechanism independent of in ammatory resident cells, nitric – Biology 81, 1512–1522. oxide and the opioid system. European Journal of Pain 2012, 1532 2149. Holtman Jr., J.R., Crooks, P.A., Johnson-Hardy, J., Wala, E.P., 2010. Antinociceptive Porreca, F., Mosberg, H.I., Omnaas, J.R., Burks, T.F., Cowan, A., 1987. Supraspinal and spinal potency of selective opioid agonists in the mouse writhing test. Journal effects and toxicity of morphine-6-O-sulfate sodium salt in rat models of pain. of Pharmacology and Experimental Therapeutics 240, 890–894. European Journal of Pharmacology 648, 87–94. Prasad, P., Yanagihara, A.A., Small-Howard, A.L., Turner, H., Stokes, A.J., 2008. Hsieh, Y.L., Chiang, H., Tseng, T.J., Hsieh, S.T., 2008. Enhancement of cutaneous nerve Secretogranin III directs secretory vesicle biogenesis in mast cells in a manner regeneration by 4-methylcatechol in resiniferatoxin-induced neuropathy. Jour- dependent upon interaction with chromogranin A. The Journal of Immunology nal of Neuropathology & Experimental Neurology 67, 93–104. 181, 5024–5034. Ikeda, Y., Ueno, A., Naraba, H., Oh-ishi, S., 2001. Involvement of vanilloid receptor Ribeiro, R.A., Vale, M.L., Thomazzi, S.M., Paschoalato, A.B., Poole, S., Ferreira, S.H., VR1 and prostanoids in the acid-induced writhing responses of mice. Life Cunha, F.Q., 2000. Involvement of resident macrophages and mast cells in the – Science 69, 2911 2919. writhing nociceptive response induced by zymosan and acetic acid in mice. fi Inoue, H., Nagata, N., Koshihara, Y., 1993. Pro le of capsaicin-induced mouse ear European Journal of Pharmacology 387, 111–118. fl oedema as neurogenic in ammatory model: comparison with arachidonic acid- Richardson, J.D., Vasko, M.R., 2002. Cellular mechanisms of neurogenic inflamma- – induced ear oedema. British Journal of Pharmacology 110, 1614 1620. tion. Journal of Pharmacology and Experimental Therapeutics 302, 839–845. Jakab, B., Helyes, Z., Varga, A., Bolcskei, K., Szabo, A., Sandor, K., Elekes, K., Borzsei, Santos, A.R., Calixto, J.B., 1997. Further evidence for the involvement of tachykinin R., Keszthelyi, D., Pinter, E., Petho, G., Nemeth, J., Szolcsanyi, J., 2005. Pharma- receptor subtypes in formalin and capsaicin models of pain in mice. Neuropep- cological characterization of the TRPV1 receptor antagonist JYL1421 (SC0030) tides 31, 381–389. in vitro and in vivo in the rat. European Journal of Pharmacology 517, 35–44. Sauzem, P.D., Sant′Anna, Gda.S., Machado, P., Duarte, M.M., Ferreira, J., Mello, C.F., Jara-Oseguera, A., Simon, S.A., Rosenbaum, T., 2008. TRPV1: on the road to pain Beck, P., Bonacorso, H.G., Zanatta, N., Martins, M.A., Rubin, M.A., 2009. Effect of relief. Current Molecular Pharmacology 1, 255–269. 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles on chronic inflammatory pain Jordt, S.E., Bautista, D.M., Chuang, H.H., McKemy, D.D., Zygmunt, P.M., Hogestatt, E. model in rats. European Journal of Pharmacology 616, 91–100. D., Meng, I.D., Julius, D., 2004. Mustard oils and cannabinoids excite sensory Schmauss, C., Yaksh, T.L., 1984. In vivo studies on spinal opiate receptor systems nerve fibres through the TRP channel ANKTM1. Nature 427, 260–265. mediating antinociception. II. Pharmacological profiles suggesting a differential Kamei, K., Yasuda, T., Ueda, T., Qiang, F., Takeyama, Y., Shiozaki, H., 2010. Role of association of mu, delta and kappa receptors with visceral chemical and triggering receptor expressed on myeloid cells-1 in experimental severe acute cutaneous thermal stimuli in the rat. Journal of Pharmacology and Experi- pancreatitis. Journal of Hepato-Biliary-Pancreatic Sciences 17, 305–312. mental Therapeutics 228, 1–12. Kopczynska, B., 2008. Role of VR1 and CB1 receptors in modelling of cardio- Schwartz, E.S., Christianson, J.A., Chen, X., La, J.H., Davis, B.M., Albers, K.M., respiratory response to arvanil, an endocannabinoid and vanilloid hybrid, in Gebhart, G.F., 2011. Synergistic role of TRPV1 and TRPA1 in pancreatic pain rats. Life Science 83, 85–91. and inflammation. Gastroenterology 140 (1283-1291), e1281–e1282. Lantéri-Minet, M., Isnardon, P., de Pommery, J., Menetrey, D., 1993. Spinal and Sinniger, V., Mouchet, P., Bonaz, B., 2005. Effect of nor-trimebutine on neuronal hindbrain structures involved in visceroception and visceronociception as revealed activation induced by a noxious stimulus or an acute colonic inflammation in by the expression of Fos, Jun and Krox-24 proteins. Neuroscience 55, 737–753. the rat. Life Science 77, 2927–2941. Lapointe, T.K., Altier, C., 2011. The role of TRPA1 in visceral inflammation and pain. Stander, S., Moormann, C., Schumacher, M., Buddenkotte, J., Artuc, M., Shpacovitch, Channels (Austin) 5, 525–529. V., Brzoska, T., Lippert, U., Henz, B.M., Luger, T.A., Metze, D., Steinhoff, M., 2004. Mactier, R.A., Sprosen, T.S., Gokal, R., Williams, P.F., Lindbergh, M., Naik, R.B., Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, Wrege, U., Grontoft, K.C., Larsson, R., Berglund, J., Tranaeus, A.P., Faict, D., 1998. mast cells, and epithelial cells of appendage structures. Experimental Derma- – Bicarbonate and bicarbonate/lactate peritoneal dialysis solutions for the treat- tology 13, 129 139. ment of infusion pain. Kidney International 53, 1061–1067. Steiner, A.A., Turek, V.F., Almeida, M.C., Burmeister, J.J., Oliveira, D.L., Roberts, J.L., Malin, S., Molliver, D., Christianson, J.A., Schwartz, E.S., Cornuet, P., Albers, K.M., Bannon, A.W., Norman, M.H., Louis, J.C., Treanor, J.J., Gavva, N.R., Romanovsky, Davis, B.M., 2011. TRPV1 and TRPA1 function and modulation are target tissue A.A., 2007. Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense dependent. The Journal of Neuroscience 31, 10516–10528. effectors. Journal of Neuroscience 27, 7459–7468. Massaad, C.A., Safieh-Garabedian, B., Poole, S., Atweh, S.F., Jabbur, S.J., Saade, N.E., Story, G.M., Peier, A.M., Reeve, A.J., Eid, S.R., Mosbacher, J., Hricik, T.R., Earley, T.J., 2004. Involvement of substance P, CGRP and histamine in the hyperalgesia and Hergarden, A.C., Andersson, D.A., Hwang, S.W., McIntyre, P., Jegla, T., Bevan, S., cytokine upregulation induced by intraplantar injection of capsaicin in rats. Patapoutian, A., 2003. ANKTM1, a TRP-like channel expressed in nociceptive Journal of Neuroimmunology 153, 171–182. neurons, is activated by cold temperatures. Cell 112, 819–829. Millan, M.J., Dekeyne, A., Gobert, A., Mannoury la Cour, C., Brocco, M., Rivet, J.M., Di Suzuki, H., Miura, S., Liu, Y.Y., Tsuchiya, M., Ishii, H., 1995. Substance P induces Cara, B., Lejeune, F., Cremers, T.I., Flik, G., de Jong, T.R., Olivier, B., de Nanteuil, G., degranulation of mast cells and leukocyte adhesion to venular endothelium. 2010. S41744, a dual neurokinin (NK)1 receptor antagonist and serotonin (5- Peptides 16, 1447–1452. HT) reuptake inhibitor with potential antidepressant properties: a comparison Suzuki, R., Furuno, T., McKay, D.M., Wolvers, D., Teshima, R., Nakanishi, M., to aprepitant (MK869) and paroxetine. European Neuropsychopharmacology Bienenstock, J., 1999. Direct neurite-mast cell communication in vitro occurs – 20, 599 621. via the neuropeptide substance P. The Journal of Immunology 163, 2410–2415. Moran, M.M., McAlexander, M.A., Biro, T., Szallasi, A., 2011. Transient receptor Tanaka, K., Matsugami, T., Chiba, T., 2002. The origin of sensory innervation of the potential channels as therapeutic targets. Nature Reviews Drug Discovery 10, peritoneum in the rat. Anatomy and Embryology (Berlin) 205, 307–313. 601–620. Weller, K., Reeh, P.W., Sauer, S.K., 2011. TRPV1, TRPA1, and CB1 in the isolated vagus Ogawa, K., Nabe, T., Yamamura, H., Kohno, S., 1999. Nanomolar concentrations of nerve—axonal chemosensitivity and control of neuropeptide release. Neuro- neuropeptides induce histamine release from peritoneal mast cells of a peptides 45, 391–400. substrain of Wistar rats. European Journal of Pharmacology 374, 285–291. Xu, H., Blair, N.T., Clapham, D.E., 2005. Camphor activates and strongly desensitizes Ohta, Y., Kobayashi, T., Inui, K., Yoshino, J., Nakazawa, S., 2003. Protective effect of the transient receptor potential vanilloid subtype 1 channel in a vanilloid- teprenone against acute gastric mucosal lesions induced by compound 48/80, independent mechanism. Journal of Neuroscience 25, 8924–8937. a mast cell degranulator, in rats. Journal of Pharmacological Sciences 93, Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain 337–346. in conscious animals. Pain 16, 109–110.