Role of TRPV1 in nociception and edema induced by monosodium urate crystals in rats

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PAIN 152 (2011) 1777–1788

www.elsevier.com/locate/pain

Role of TRPV1 in nociception and edema induced by monosodium urate crystals in rats Carin Hoffmeister a, Gabriela Trevisan b, Mateus Fortes Rossato c, Sara Marchesan de Oliveira c, Marcus Vinícius Gomez d, Juliano Ferreira a,b,c,d,⇑ a

Programa de Pós-graduação em Farmacologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, RS, Brazil Departmento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS, Brazil Programa de Pós-graduação em Ciências Biológicas, Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS, Brazil d Programa de Pós-graduação em Farmacologia Bioquímica e Molecular, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil b c

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e

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Article history: Received 15 December 2009 Received in revised form 25 February 2011 Accepted 21 March 2011

Keywords: Pain Gout Mast cells Capsaicin Tryptase

a b s t r a c t Gout is characterized by the deposition of monosodium urate (MSU) crystals. Despite being one of the most painful forms of arthritis, gout and the mechanisms responsible for its acute attacks are poorly understood. In the present study, we found that MSU caused dose-related nociception (ED50 [ie, the necessary dose of MSU to elicit 50% of the response relative to the control value] = 0.04 [95% confidence interval 0.01–0.11] mg/paw) and edema (ED50 = 0.08 [95% confidence interval 0.04–0.16] mg/paw) when injected into the hind paw of rats. Treatment with the selective TRPV1 receptor (also known as capsaicin receptor and vanilloid receptor-1) antagonists SB366791 or AMG9810 largely prevented nociceptive and edematogenic responses to MSU. Moreover, the desensitization of capsaicin-sensitive afferent fibers as well as pretreatment with the tachykinin NK1 receptor antagonist RP 67580 also significantly prevented MSU-induced nociception and edema. Once MSU was found to induce mast cell stimulation, we investigated the participation of these cells on MSU effects. Prior degranulation of mast cells by repeated treatment with the compound 48/80 decreased MSU-induced nociception and edema or histamine and serotonin levels in the injected tissue. Moreover, pretreatment with the mast cell membrane stabilizer cromolyn effectively prevented nociceptive and edematogenic responses to MSU. MSU induced a release of histamine, serotonin, and tryptase in the injected tissue, confirming mast cell degranulation. Furthermore, the antagonism of histaminergic H1 and serotoninergic receptors decreased the edema, but not the nociception of MSU. Finally, the prevention of the tryptase activity was capable of largely reducing both MSU-induced nociception and edema. Collectively, the present findings demonstrate that MSU produces nociceptive and edematogenic responses mediated by TRPV1 receptor activation and mast cell degranulation. Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction Gout, a medical condition described over 2500 years ago, is still considered one of the most painful acute conditions that afflicts human beings [4,45]. It took until 1962 to demonstrate that injecting monosodium urate (MSU) crystals into joints caused pain similar to that observed in gout [11]. It is now generally accepted that the deposition of microcrystalline MSU in synovial fluid initiates an attack of gout [9].

⇑ Corresponding author at: Departamento de Química, Universidade Federal de Santa Maria, Avenida Roraima 1000, Camobi, CEP 97105-900, Santa Maria, RS, Brazil. Tel.: +55 55 3220 8053; fax: +55 55 3220 8031. E-mail address: [email protected] (J. Ferreira).

Several studies have demonstrated that MSU induces leukocyte infiltration and activation [9,13,15,28,30]. Moreover, it has frequently been suggested that gout-associated pain is due to leukocytes phagocytosing MSU and subsequently releasing pronociceptive mediators that stimulate nociceptors [9,11]. Although these events may be important in maintaining gout-associated pain, it has also been demonstrated that urate crystals alone can cause joint pain without leukocyte infiltration [18]. Therefore, additional events besides leukocyte activation likely contribute to gout-associated pain induction; the additional mechanisms involved in MSU-induced nociception are not yet known. An acute gout attack classically presents as an initial warm sensation followed by excruciating burning pain [11,50]. Interestingly, the TRPV1 receptor (also known as capsaicin receptor and vanilloid receptor-1), a type of transient receptor potential (TRP)

0304-3959/$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2011.03.025

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ion channel, appears to be responsible for noxious heat sensation [5]. TRPV1 is expressed in a subset of small-diameter primary afferent sensory fibers and is activated by several noxious stimuli, such as heat, protons, some lipid mediators, and the active ingredient of chili peppers (capsaicin). The stimulation of TRPV1 causes a neurogenic nociception caused by the depolarization and subsequent neuropeptide release by nociceptors, the degranulation of mast cells, and also by plasma extravasation [2,4,41]. Moreover, repeated applications of capsaicin result in long-lasting analgesic effects [5]. Of note, chili pepper extracts have been used medicinally for gout treatment [48]. We speculated that because TRPV1 is involved in the detection of several harmful stimuli (especially noxious heat), and gout-related pain is described as a burning sensation, it was possible that this pain was related to the activation of the TRPV1 receptor. Therefore, the present study investigated the role of leukocytes, mast cells, and TRPV1 in the nociceptive and edematogenic response to MSU crystals. 2. Materials and methods 2.1. Animals Adult male Wistar rats weighing 200–300 g were used in all experiments. All animals were housed in a room maintained at a constant temperature of 22 ± 1 °C under a 12-h light/dark cycle with food and water available ad libitum. Animals were acclimatized to the laboratory for at least 2 h before testing. The number of animals and the nociceptive stimuli used were the minimum necessary to demonstrate the consistent effects of drug treatments. All experiments were performed in accordance with the ethical guidelines established for investigations of experimental pain in conscious animals [52] and were approved by the university ethics committee (process number: 23081.003640/2009-61). 2.2. Drugs Synthetic MSU crystals were prepared as previously described [39]. Briefly, 4 g of uric acid (Vetec, Rio de Janeiro, Brazil) was dissolved and heated in 800 mL of H2O, adjusted to pH 8.9 with NaOH (9 mL, 0.5 N) at 60 °C, cooled overnight in a cold room, and then washed and dried. Needle-like crystals were recovered and suspended in phosphate-buffered saline (PBS; K2HPO4 10.71 mM, NaH2PO4 6.78 mM, NaCl 120.4 mM, pH 7.4). Polarized light microscopic examination confirmed that the crystals were rod-shaped and varied in length (12 ± 2 lm). The preparation was endotoxinfree as determined by an amebocyte cell lysate assay (Sigma, St Louis, MO, USA). Capsaicin was purchased from Sigma, and the stock solution was prepared in 90% ethanol and 10% Tween 80. Ruthenium red, methysergide, sodium cromoglycate (cromolyn), histamine, substance P, ninhydrin, serotonin, RP 67580, a1-acid glycoprotein, and resiniferatoxin were purchased from Sigma and dissolved in PBS. In addition, o-phthaldialdehyde, p-nitrophenil-2-acetamide-b-D-glicopyranoside (NAG), 5-(N,N-diethylamino)-pentyl-3,4,5-trimethoxybenzoate, hexadecyltrimethylammonium bromide, and N-p-Tosyl-GlyPro-Arg-p-nitroanilide were purchased from Sigma. Soybean trypsin inhibitor (SBTI) was purchased from Fluka Chemical Corp. (Milwaukee, WI, USA), and promethazine was purchased from Aventis (São Paolo, Brazil); both were dissolved in PBS. Radiolabeled [3H] -resiniferatoxin was purchased from PerkinElmer (Waltham, MA, USA). 2.3. Injection of urate crystals To observe the possible nociceptive and edematogenic effects produced by MSU, 100 lL of MSU suspension (between 0.015

and 2 mg/paw) was administered subcutaneously (s.c.) under the plantar surface of the right hind paw of unanesthetized animals. Separate groups of animals received s.c. injection of vehicle alone (PBS). Signs of stimuli-independent nociception (spontaneous nociception) and edema were observed over time. 2.4. MSU-induced spontaneous nociception and edema Animals were individually placed in transparent glass chambers and allowed to adapt to their surroundings for 20 min before being injected as previously described [12]. The duration of flinch responses during the 30 min (in 5-min increments) following injection were recorded and used to determine the level of nociception. The edema formation was assessed as an increase in paw thickness measured by a digital caliper at several time points (30, 60, 120, and 180 min) after the s.c. injection of MSU or vehicle alone. The results were expressed as the difference between the basal value and the test value at each time observed [32]. To investigate the mechanism of MSU-induced nociception and edema, several antagonists and inhibitors were also subcutaneously injected with MSU (0.25 mg/paw). The treatment time and drug doses were based on data from previous literature as well as pilot experiments using positive controls (data not shown). Behavioral testing was performed in a blinded manner with respect to drug administration. 2.5. TPRV1 and NK1 receptor involvement in nociceptive and edematogenic responses induced by MSU To investigate the possible involvement of TRP receptors in MSU-induced spontaneous pain, the selective TRPV1 receptor antagonists SB366791 (0.1–100 nmol/paw) and AMG9810 (30 pmol/paw), were co-injected subcutaneously with MSU (0.25 mg/paw). Nociception was observed and recorded as described above. As a positive control, we evaluated the effects of SB366791 on nociception and edema caused by capsaicin (0.01 or 1 nmol/paw, respectively). To confirm the effects of locally injected drugs, we pretreated (for 20 min) separate groups of animals with intraperitoneal SB366791 (0.5 mg/kg). Additionally, separate groups of animals received an s.c. injection of vehicle (PBS) with a neutral (7.4) or acidic pH (6.4, acidified with 1 M HCl), either alone or with a low dose of MSU (0.015 mg/paw) before nociception was measured. The involvement of the tachykinin NK1 receptor was also investigated. To elucidate its participation in MSU-induced nociception and edema, the NK1 receptor antagonist RP 67580 (20 nmol/paw) was co-injected subcutaneously with MSU (0.25 mg/paw) before nociception and edema were observed. As a positive control, we also evaluated the effects of RP 67580 on nociception and edema caused by substance P (0.1 nmol/paw). 2.6. Role of capsaicin-sensitive afferent fibers in MSU-induced nociceptive and edematogenic responses To further explore the role of capsaicin-sensitive fibers in MSU-induced nociceptive and edematogenic effects, animals were desensitized to perineural capsaicin as previously described [35]. First, animals were anesthetized with ketamine (90 mg/kg) and xylazine (3 mg/kg), and an incision was made over the hip joint to expose the sciatic nerve. Next, 10 lL of 2% capsaicin or vehicle (10% ethanol, 10% Tween 80, and 80% PBS) was injected into the nerve sheath with a microsyringe. After 7 days, animals were submitted to an s.c. injection of MSU (0.25 mg/ paw), capsaicin (for a positive control; 0.01 nmol/paw), or PBS (100 lL/paw).

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2.7. The role of mast cells in MSU-induced nociception and edema To further investigate the participation of mast cells in MSU-induced nociception and edema, the right hind paws of rats were pretreated (15 min before injection of MSU) with the mast cell stabilizer sodium cromoglycate (2 lmol/paw) [38]. In another experiment, mast cells were degranulated by pretreatment with compound 48/80 for 5 consecutive days (at 1, 3, 10, 10, and 10 lg/paw) [2]. An hour after the last treatment, rats were injected with MSU (0.25 mg/paw) or compound 48/80 (used as a positive control; 10 lg/paw). To evaluate the participation of histamine and serotonin in MSU-induced nociception and edema, animals were co-administered MSU (0.25 mg/paw) and the H1 receptor antagonist promethazine (120 nmol/paw) or the 5-HT receptor antagonist methysergide (10 nmol/paw). In separate groups of animals, histamine (250 lg/paw or 20 lmol/paw) and serotonin (10 lg/paw or 60 nmol/paw) were used as positive controls for promethazine and methysergide, respectively [17]. As a negative control, we also evaluated the effects of SB366791 (10 nmol/paw) on nociception and edema caused by serotonin (60 nmol/paw). To investigate the participation of tryptase, groups of rats were pretreated with the nonselective tryptase inhibitor SBTI (100 lg/ paw) or the selective tryptase inhibitor gabexate (0.1 nmol/paw) 10 min before MSU injection [22]. Nociception and edema were then evaluated as described above. 2.8. Measurement of histamine or serotonin levels and tryptase activity To confirm mast cell degranulation by repeated compound 48/ 80 treatment, separate groups of rats were killed by cervical dislocation 24 h after the final compound injection. Histamine and serotonin levels were measured in the injected tissue homogenates. Histamine content was evaluated as previously described [42]. Paw skin samples were homogenized in PBS (50 mM, pH 7.4) with 1 mM metronidazole (a histamine methyl transferase inhibitor used to reduce histamine degradation) and centrifuged at 12,000g at 4 °C for 10 min. The resulting supernatants were used to evaluate the histamine content. Briefly, 150 lL of 1 M NaOH was added to 400 lL of supernatant and incubated with 40 lL of 1% o-phthaldialdehyde. Then, 75 lL of HCl (3 M, pH 10.4) was added to the reaction. The solution was excited at 360 nm, and the subsequent fluorescence was read at 450 nm in a fluorescence photometer. Serotonin content was evaluated as previously described [47]. Paw tissue samples were homogenized in 2 mL of perchloric acid (0.4 M) and centrifuged at 900g for 10 min. Then, 300 lL of supernatant was combined with 0.125 mL of borate buffer (0.5 mM, pH 10, NaCl saturated) and 3 mL of 1-butanol. The mixture was stirred for 10 min. The organic phase was then separated and incubated with 0.350 mL of phosphate buffer (0.05 M, pH 7.4) and 3 mL of n-heptane. The mixture was mixed for 2 min, and the aqueous phase (0.5 mL) was incubated with 0.5 mL of ninhydrin (0.24%) and 0.5 mL of phosphate buffer (100 mM, pH 7.0). The reaction was incubated at 100 °C for 10 min and allowed to rest at room temperature in the dark for 5 h. The solution was excited at 380 nm, and the subsequent fluorescence was read at 500 nm with a fluorescence photometer. To verify whether or not MSU crystals were capable of promoting mast cell degranulation, separate groups of rats were killed by cervical dislocation 10 min after paw s.c. injection of MSU or vehicle alone. The injected paw was coaxially perfused as previously described [12]. A double polyethylene tube was inserted into the subcutaneous space of the paw, and 500 lL of PBS was perfused at a rate of 200 lL/min 1 through the inner tube, after which the

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perfusate was collected through the outer tube. To assess histamine and serotonin levels, paws were perfused with PBS plus metronidazole or PBS alone, respectively, and their levels were measured as described above. To assess tryptase release after MSU treatment, the paw was perfused with 10 mM tris(hydroxymethyl)aminomethane (Tris; pH 6.1) containing 2 M NaCl 10 min after MSU injection. Then, 50 lL of perfusate was combined with 50 lL of 500 g/mL N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide in Tris buffer (60 mM, pH 7.8) containing 0.4% dimethyl sulfoxide and 30 lg/mL heparin at 37 °C for 1 h. The released nitroaniline was measured colorimetrically at 420 nm [46]. 2.9. The role of cyclooxygenase in MSU-induced nociception and edema and measurement of prostaglandin E2 levels To investigate the participation of cyclooxygenase metabolite in MSU-induced nociception and edema, the right hind paws of the rats were pretreated (15 min before injection of 0.25 mg/paw MSU) with cyclooxygenase inhibitor (100 lg/paw) [27]. Then, nociception and edema were evaluated as described above. To verify whether or not MSU crystals were capable of promoting prostanoid production, separate groups of rats were killed by cervical dislocation 10 min after s.c. paw injection of MSU or vehicle alone. The injected paw was coaxially perfused with PBS as described above, and the exudates were collected and stored at 70 °C until further analyzed. Prostaglandin E2 levels were evaluated using an enzyme-linked immunosorbent assay kit according to the manufacturer’s recommendations (Cayman Chemical Company, Ann Arbor, MI, USA). 2.10. Determination of neutrophil and macrophage infiltration To evaluate the possible cellular infiltration induced by MSU, myeloperoxidase (MPO) and N-acetyl-b,D-glucaminidase (NAGase) activities were used as indexes of neutrophil and macrophage accumulation, respectively [26]. The plantar surface of the hind paw was harvested at 10, 60, or 480 min after the s.c. injection of MSU crystals (0.25 mg/paw) or vehicle. Samples were homogenized in sodium acetate buffer (80 mM, pH 5.5) containing 0.5% hexadecyltrimethylammonium bromide and held at 4 °C. Just before the assay, the tissue homogenates were centrifuged at 20,000g for 20 min and the supernatants were collected. To determine MPO activity, 10 lL of supernatant was mixed with 100 lL of sodium acetate buffer (80 mM, pH 5.5) and 10 lL of 5-(N,N-diethylamino)-pentyl-3,4,5-trimethoxybenzoate. The solution was incubated for 3 min at 37 °C and stopped on ice by the addition of 30 lL acetic acid. The solution was analyzed with a spectrophotometer at 630 nm. Alternatively, to determine NAGase activity, 25 lL of supernatant was mixed with sodium citrate buffer (50 mM, pH 4.5) and 25 lL of p-nitrofenil-2-acetamide-bD-glicopiranoside (NAG; 2.25 nM). The solution was incubated for 60 min at 37 °C, stopped on ice by the addition of 100 lL glycine buffer (0.2 lM, pH 10.4), and analyzed on a spectrophotometer at 405 nm. A Fisher Biotech Microkinetics BT 2000 (Fisher Scientific, Pittsburgh, PA, USA) microplate reader was utilized for these experiments, and values were expressed as optical densities corrected by tissue weight (in mg). 2.11. [3H]-Resiniferatoxin binding assay Binding assays were performed as previously described [44]. To obtain membranes for these studies, rat spinal cords were removed and disrupted with the aid of a tissue homogenizer in ice-cold buffer A (pH 7.4) that contained 5 mM KCl, 5.8 mM NaCl, 2 mM MgCl2, 0.75 mM CaCl2, 12 mM glucose, 137 mM sucrose, and 10 mM HEPES. The homogenate was firstly centrifuged for 10 min at

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1000g at 4 °C. The low-speed pellets were discarded, and the supernatants were further centrifuged for 30 min at 35,000g at 4 °C. The resulting high-speed pellets, re-suspended in buffer A, were stored at 70 °C until assayed. Binding assays were conducted in duplicate with a final volume of 500 lL buffer A supplemented with 0.25 mg/mL bovine serum albumin, membranes (100 lg/protein), and 50 pM [3H]-resiniferatoxin. To measure nonspecific binding, 100 nM nonradioactive [3H]-resiniferatoxin was included in some tubes. Assay mixtures were set up on ice, and the binding reaction was initiated by transferring the tubes to 37 °C. Mixtures were incubated for 60 min, and reactions were terminated by cooling them on ice. Then, 100 g of bovine a1-acid glycoprotein was added to each tube to reduce nonspecific binding. Finally, the free (and bound) [3H]-resiniferatoxin membranes were separated by centrifugation for 15 min at 20,000g at 4 °C. The pellet was quantified with a scintillation counter. Specific binding was calculated as the difference between the total and nonspecific binding. 2.12. Statistical analysis All values are expressed as mean ± SEM, except for ED50 values (ie, the necessary dose of MSU to elicit 50% of the response relative to the control value) and ID50 values (ie, the necessary dose to elicit 50% of the preventative response relative to the control value), which are reported as geometric means accompanied by their respective 95% confidence limits. The percentages of prevention are reported as the mean ± SEM and calculated with the maximum developed responses obtained after injection of MSU when compared to vehicle-treated animals. The statistical significance between groups was assessed by the Student t-test, in addition

to 1- and 2-way analysis of variance (ANOVA) when appropriate. Post hoc tests (Student–Newman-Keuls’ for 1-way or Bonferroni for 2-way ANOVA) were conducted when appropriate. P values lower than 0.05 (P < 0.05) were considered to be significant. The ED50 and ID50 values were determined by nonlinear regression analysis with a sigmoid dose–response equation using GraphPad Software version 4.0 (GraphPad Software Inc, La Jolla, CA, USA).

3. Results 3.1. MSU crystal-induced nociception and edema The s.c. injection of MSU crystals (0.25 mg/paw) caused a slow onset of spontaneous nociception that was not observed in the initial 5 min after its administration. Instead, nociception reached its maximum at between 5 and 10 min and then disappeared altogether 10 min after injection (Fig. 1A). The same treatment also induced edema formation that began at 30 min and reached its peak effect at 1 h (Fig. 1C). Based on these findings, further experiments of nociceptive and edematogenic responses were measured between 10 min and 1 h after injection, respectively. The nociceptive and edematogenic effects produced by MSU (0.015–2 mg/paw) were dose-dependent (Fig. 1B and D), with an ED50 of 0.042 (95% confidence interval [CI] 0.015–0.118) mg/paw or 0.089 (95% CI 0.046–0.170) mg/paw, respectively. The maximal nociceptive and edematogenic effects were observed as 36 ± 7 seconds and 1 ± 0.1 mm, respectively. Thus, an MSU dose of 0.25 mg/paw was chosen for further experiments to induce nociception and edema to avoid supramaximal stimulation and unnecessary animal discomfort.

Fig. 1. Monosodium urate (MSU) crystal-induced nociception and edema. Time courses (A and B) and dose–responses (C and D) for nociception and edema caused by subcutaneous injection of MSU (A and C). Each point on the curve represents the mean ± SEM of 7–10 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01, oneway analysis of variance followed by Student–Newman–Keuls’ test.

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3.2. Participation of TRPV1 receptors, NK1 receptors, and capsaicin-sensitive sensory fibers in MSU-induced nociception and edema The co-administration of the selective TRPV1 receptor antagonist SB366791 (0.1–100 nmol/paw) reduced MSU-induced nociception and edema (Fig. 2A and B), with calculated ID50 values of

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0.34 (95% CI 0.07–4.22) nmol/paw and 0.07 (95% CI 0.001–5.8) mg/paw. Additionally, co-injection of SB366791 prevented 90 ± 6% and 100% of MSU-induced nociception and edema, respectively. Similarly to MSU, s.c. injection of the TRPV1 receptor agonist capsaicin (0.01 or 1 nmol/paw) into rat paws also produced nociceptive and edematogenic actions, both abolished by SB366791 (10 nmol/paw) (Table 1). Demonstrating its selectivity, SB366791

Fig. 2. Role of TRPV1 and NK1 receptors on monosodium urate (MSU)-induced nociception and edema. (A and B) Effects of co-treatment with the TRPV1 receptor antagonist SB366791 (10 nmol/paw) on the nociceptive (A) and edematogenic (B) effects induced by subcutaneous (s.c.) injection of MSU (0.25 mg/paw). (C and D) Effects of intraperitoneal pretreatment with the TRPV1 receptor antagonist SB366791 (0.5 mg/kg) on the nociceptive (C) and edematogenic (D) effects induced by s.c. injection of MSU (0.25 mg/paw). (E and F) Effects of co-treatment with the TRPV1 receptor antagonist AMG9810 (30 pmol/paw) on the nociceptive (E) and edematogenic (F) effects induced by s.c. injection of MSU (0.25 mg/paw). (G and H) Effects of co-treatment with the NK1 receptor antagonist RP 67580 (20 nmol/paw) on the nociceptive (G) and edematogenic (H) effects induced by s.c. injection of MSU (0.25 mg/paw). Each column represents the mean ± SEM of 8 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01 in comparison to vehicle, and #P < 0.05, ##P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman–Keuls’ test. (I and J) Nociceptive effects caused by the injection of a low dose of MSU (0.015 mg/paw) in acidified phosphate-buffered saline (vehicle, pH 6.5, I) and with a low dose of capsaicin (Cap, 10 pmol/paw, J). Each column represents the mean ± SEM of 6 rats. The asterisk denotes significance levels. ⁄P < 0.05 denotes comparison to MSU in neutral pH (7.4; I) or with capsaicin vehicle (Veh; J), one-way analysis of variance followed by Student–Newman–Keuls’ test.

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Table 1 Controls for the pharmacological treatments. Treatment

Nociceptive response (s)

D Paw thickness (mm)

Vehicle (100 lL/paw) Capsaicin (0.1 or 1 mol/paw)+ +SB 366791 (10 nmol/paw) Vehicle (100 lL/paw) Serotonin (60 nmol/paw) +SB366791 (10 nmol/paw) Vehicle (100 lL/paw) Serotonin (60 nmol/paw) +Methysergide (10 nmol/paw) Vehicle (100 lL/paw) Histamine (20 lmol/paw) +Prometazine (120 nmol/paw) Vehicle (100 lL/paw) Substance P (0.1 nmol/paw) +RP 67580 (20 nmol/paw) Compound 48/80 (10 lg/paw) in vehicle pretreated animals Compound 48/80 (10 lg/paw) in 48/80 pretreated animals Capsaicin (0.1 or 1 mol/paw) + in perineural vehicle pretreated animals Capsaicin (0.1 or 1 mol/paw) in perineural capsaicin (2%) pretreated animals

3±1 31 ± 3# 12 ± 2* 3±2 16 ± 4## 17 ± 4 5±1 26 ± 7## 2 ± 6** 4±1 21 ± 6## 6 ± 2* 4±1 62 ± 6## 19 ± 6** 57 ± 9##

0.3 ± 0.07 0.6 ± 0.1# 0.3 ± 0.04* 0.4 ± 0.1 2.9 ± 0.1## 2.8 ± 0.2* 0.4 ± 0.04 2.0 ± 0.2## 0.3 ± 0.1** 0.4 ± 0.03 1.5 ± 0.2## 0.6 ± 0.2** 0.3 ± 0.05 1.0 ± 0.1## 0.5 ± 0.04** 3.5 ± 0.2##

14 ± 4**

2.0 ± 0.3**

34 ± 7

0.7 ± 0.04

8 ± 1**

0.4 ± 0.08*

Data are presented as mean ± SEM. * P < 0.05. ** P < 0.01, compared with vehicle (#) or nociceptive/edematogenic substances (*) group; one-way analysis of variance followed by Student–Newman–Keuls’ test or Student t-test. + The doses of 0.1 and 1 nmol/paw were used for evaluate nociception or edema, respectively.

altered neither nociception nor edema induced by serotonin (60 nmol/paw) (Table 1). Besides being effective when injected locally (into the paw), SB366761 was also capable of reducing MSUinduced nociception (inhibition levels of 73 ± 5%) and edema (inhibition levels of 46 ± 11%) when administered systemically (0.5 mg/ kg, intraperitoneally) (Fig. 2C and D). Confirming the results obtained with SB366791, co-administration of the selective TRPV1 receptor antagonist AMG9810 (30 pmol/paw) also significantly reduced MSU-induced nociception (inhibition levels of 78 ± 12%) and edema (inhibition levels of 96 ± 20%) (Fig. 2E and F). Furthermore, high concentrations of urate (75–750 lM) did not alter the specific binding of [3H]-resiniferatoxin to TRPV1 (specific binding of 105.7 ± 5.7%, 108.1 ± 13.5%, 99.5 ± 0.2%, and 46.7 ± 0.3% for vehicle, 75 lM urate, 750 lM urate, and 10 lM capsaicin, respectively). The results shown in Fig. 2(I and J) suggest that MSU sensitized TRPV1 to agonist activation. The s.c. injection of a low dose of MSU (0.015 mg/paw), low dose of capsaicin (10 pmol/paw), or acidified PBS (pH 6.5) did not cause nociception when compared with the neutral PBS-treated animals. However, acidified PBS or capsaicin low-dose administration in conjunction with a low dose of MSU resulted in a significant nociceptive response (Fig. 2I and J). The NK1 receptor antagonist RP 67580 (20 nmol/paw) partially prevented MSU-induced nociceptive (55 ± 9%) and edematogenic (48 ± 6%) responses (Fig. 2G and H, respectively). The same treatment with RP 67580 prevented nociception (84 ± 11%) and edema (95 ± 1%) caused by the NK1 receptor agonist substance P (0.1 nmol/paw) (Table 1). To investigate the participation of capsaicin-sensitive sensory fibers in the MSU-induced edema and nociception, animals were subjected to perineural capsaicin desensitization. This process prevented nociception induced by capsaicin (0.01 nmol/paw) in 87 ± 3% of animals (Table 1) and MSU (0.25 mg/paw) in 88 ± 4% of animals (Fig. 3A). Similarly, desensitization prevented edema induced by capsaicin (0.01 nmol/paw) in 84 ± 7% of animals (Table 1) and by MSU (0.25 mg/paw) in 50 ± 13% of animals (Fig. 3B).

Fig. 3. Role of capsaicin-sensitive sensory fibers on monosodium urate (MSU)induced nociception and edema. Perineural pretreatment with capsaicin (2%, 7 days prior to treatment) on the nociceptive (A) and edematogenic (B) effects inducible by subcutaneous injection of MSU (0.25 mg/paw). Each column represents the mean ± SEM of 6 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01 in comparison to vehicle, and #P < 0.05, ##P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman–Keuls’ test.

3.3. Mast cell participation in MSU nociception and edema Mast cell degranulation caused by compound 48/80 over 4 consecutive days resulted in a significant prevention of nociception (71 ± 12%) and edema (50 ± 18%) caused by MSU (Fig. 4C and D, respectively). After degranulation of mast cells treated with compound 48/80, we observed a decrease in the histamine and serotonin content of injected tissues (reduction of 85 ± 3% and 41 ± 7%, respectively) (Fig. 5A and B). Similarly, pretreatment with the mast cell membrane-stabilizer cromolyn (2 lmol/paw) prevented MSU-induced (0.25 mg/paw) nociception and edema (inhibitions of 58 ± 9% and 67 ± 5%, respectively) (Fig. 4A and B). Additionally, we observed that MSU treatment (0.25 mg/paw) caused the release of histamine, serotonin, and tryptase when compared with vehicle-treated animals (increases of 138 ± 34%, 72 ± 17%, and 31 ± 7% in relation to control animals, respectively) (Fig. 5C, D, and F). The role of histamine or serotonin in the MSU response was confirmed by the administration of histaminergic and serotoninergic antagonists. The co-administration of the H1 receptor antagonist promethazine (120 nmol/paw) partially prevented the edematogenic effect induced by MSU crystals by 71 ± 10% (Fig. 6B), but failed to reduce the nociceptive response (Fig. 6A). On the other hand, promethazine was able to abolish both nociception and edema caused by histamine (20 lmol/paw) (Table 1). Likewise, the 5-HT receptor antagonist methysergide (10 nmol/ paw) partially prevented edema (67 ± 7%), but not MSU-induced

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Fig. 4. Previous mast cell degranulation reduces monosodium urate (MSU)-induced nociception and edema. Effects of compound 48/80 pretreatment (1, 3, 10, 10 lg/paw for 4 consecutive days, respectively) on nociceptive (A) and edematogenic (B) effects inducible by subcutaneous injection of MSU (0.25 mg/paw) measured on the fifth day of the experiment. Tissue levels of histamine (C) and serotonin (D) on day 5. Each column represents the mean ± SEM of 6–8 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄ P < 0.01 in comparison to vehicle, and #P < 0.05, ##P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman–Keuls’ test (A and B) or Student t-test (C and D).

Fig. 5. Mast cell degranulation-mediated monosodium urate (MSU)-induced nociception and edema. (A and B) Effects of pretreatment with sodium cromoglycate (cromolyn, 2 lmol/paw) on MSU-induced nociception (A) and edema (B). (C and D) Levels of histamine (C) and serotonin (D) in paw perfusates 10 min after MSU injection. Each column represents the mean ± SEM of 6 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01 in comparison to vehicle, and #P < 0.05, ##P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman–Keuls’ test (A and B) or Student t-test (C and D).

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Fig. 6. Role of receptors in histamine and serotonin during monosodium urate (MSU)-induced nociception and edema. Effects of subcutaneous co-treatment with the H1receptor antagonist promethazine (120 nmol/paw; A and B) and the 5-HT-receptor antagonist methysergide (10 nmol/paw; C and D) on MSU-induced nociception (A and B) and edema (C and D). Each column represents the mean ± SEM of 6 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01 in comparison to vehicle, and #P < 0.05, ## P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman–Keuls’ test.

nociception (Fig. 6C and D). Additionally, methysergide abolished nociception and edema caused by serotonin (60 nmol/paw) (Table 1). Finally, the role of tryptase, a serine peptidase released by mast cells, in MSU-induced responses was investigated. The preadministration of the nonselective serine peptidase inhibitor SBTI (100 lg/ mL) significantly prevented MSU-induced nociception (53 ± 9%) and edema (30 ± 10%) (Fig. 7A and B). Moreover, the preadministration of the selective tryptase inhibitor gabexate mesilate (0.1 nmol/paw) largely prevented both nociceptive (87 ± 5%) and edematogenic (61 ± 9%) responses to MSU (Fig. 7C and D, respectively). 3.4. Cyclooxygenase metabolite participation in MSU nociception and edema Pretreatment with the cyclooxygenase inhibitor indomethacin (100 lg/paw) partially prevented nociception and edema (inhibitions of 82 ± 8% and 49 ± 8%, respectively), caused by MSU treatment (0.25 mg/paw) (Fig. 8A and B). Additionally, we observed that MSU (0.25 mg/paw) stimulated prostaglandin E2 production when compared to vehicle-treated animals (increase of 223 ± 70% in relation to control animals) (Fig. 8C). 3.5. Evaluation of leukocyte infiltration by MSU Infiltration of leukocytes was measured by the activity of marker enzymes for neutrophils (MPO) and macrophages (NAGase) in

injected tissues. MSU crystals (0.25 mg/paw) induced neutrophil and macrophage infiltration after 240 min but had no effect during 10 to 60 min post injection (Table 2). 4. Discussion First, we characterized the nociceptive response induced by MSU crystals because we did not find previous literature assessing nociception after s.c. injection of MSU into rat paws. The flinching behavior induced by MSU appeared slowly; it was not observed in the initial 5 min after its administration. This delayed pattern of response suggests that MSU did not directly activate nociceptors, but rather mediated its effects through the activation of other cells present in the injected tissue that likely stimulate the peripheral terminals of afferent fibers. In fact, in contrast to MSU, direct stimulators of nociceptors, such as capsaicin, substance P, and serotonin, induced numerous flinching responses in the first minute after their s.c. injection into rodent paws [16,40] (results not shown). Moreover, we observed that the flinching behavior seemed to be independent of MSU-induced edema. This finding was expected because edema development depends on several previous events, such as arteriolar vasodilatation, venous constriction, and plasma extravasation [15]. Accordingly, we detected significant plasma extravasation 5 and 10 min after MSU injection (increases of 120 ± 37% and 79 ± 18% compared to vehicle when assessed by Evans blue staining) (results not shown). Thus, MSU-induced delayed flinching behavior seems to be dependent on the contribution of several inflammatory mediators released by resident cells

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Fig. 7. Participation of tryptase in monosodium urate (MSU)-induced nociception and edema. (A and B) Effects of co-treatment with the nonselective tryptase inhibitor SBTI (10 lg/paw) on MSU-induced nociception (A) and edema (B). (C and D) Effects of co-treatment with the selective tryptase inhibitor gabexate (0.1 nmol/paw) on MSU-induced nociception (C) and edema (D). (E) Tryptase activity in paw perfusates 10 min after MSU injection. Each column represents the mean ± SEM of 6 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01 in comparison to vehicle, and #P < 0.05, ##P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman– Keuls’ test (A–D) or Student’s t-test (E).

and derived from plasma (see the discussion below). Besides flinching behavior, we observed that MSU-injected (but not vehicle-injected) rats presented more long-lasting, low-magnitude nociceptive behaviors (for at least 4 h) characterized by shaking, lifting, and paw favoring (results not shown). These findings are in accordance with the clinic, where acute gouty arthritis is associated with pain over a 6-h period [23], with the peak of spontaneous pain after MSU injection in human subjects occurring at 4 h [11]. Finally, we observed that MSU produced a significant leukocyte infiltration just 4 h after its injection. Thus, despite the idea that gout-associated pain is only due to leukocyte infiltration and activation by MSU [9,11], we suggest that some resident tissue cells, such as mast cells (see below), may also contribute to pain generation during gout attacks. Additionally, urate can also cause joint pain in the absence of apparent leukocyte infiltration [18].

In acute gout attacks, an initial warm sensation is followed by an excruciating burning pain [11,50]. TRPV1 seems to be important in the detection of noxious heat because TRPV1 agonists induce (while TRPV1 antagonists reverse) burning pain in humans [3]. Furthermore, extracts of chili peppers have been used medicinally as a remedy for gout. In fact, continuous topical treatment with the active compound of chili pepper, capsaicin, has been shown to be beneficial in patients with some types of arthritis [29,48]. Therefore, we investigated the participation of TRPV1 in MSU-induced nociception and edema. We found that treatments with selective TRPV1 antagonists are effective in reducing MSU-induced nociceptive and edematogenic responses. Because our binding assay demonstrated that urate does not directly bind to the TRPV1 receptor (at least in the vanilloid site), the action of MSU on the receptor seems to be indirect, possibly by sensitizing TRPV1. The TRPV1

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Fig. 8. Role of cyclooxygenase on monosodium urate (MSU)-induced nociception and edema. (A and B) Effects of pretreatment with indomethacin (100 lg/paw) on MSU nociception (A) and edema (B). (C). Levels of prostaglandin E2 in paw perfusates 10 min after MSU injection. Each column represents the mean ± SEM of 6–8 rats. The asterisks denote significance levels. ⁄P < 0.05, ⁄⁄P < 0.01 in comparison to vehicle, and #P < 0.05, ##P < 0.01 in comparison to MSU, one-way analysis of variance followed by Student–Newman–Keuls’ test (A and B) or Student t-test (C).

sensitization can be demonstrated by the fact that low-dose MSU did not cause nociception by itself in neutral pH conditions, while it consistently induced a nociceptive response in low pH conditions or when combined with a low dose of capsaicin. Furthermore, we also observed that MSU-injected animals presented long-lasting (up to 4 h) heat hyperalgesia (data not shown), an effect that is related to TRPV1 stimulation in inflammatory settings [6]. These results clearly demonstrate a critical role of TRPV1 in the nociceptive and edematogenic effects elicited by MSU. TRPV1 receptors are highly expressed in sub-types of primary afferent fibers, almost exclusively in peptidergic C fibers, but also in a small number of Ad fibers [24]. Thus, capsaicin treatment can desensitize peptidergic C fibers to capsaicin itself or to other stimuli that utilize these fibers to mediate their actions [19,37]. Demonstrating that MSU depends on capsaicin-sensitive TRPV1expressing fibers to exert its action, perineural desensitization prevents both algesic and edematogenic effects produced by capsaicin or MSU. Our results are in accordance with data showing that different capsaicin desensitization protocols can reduce the nociceptive and edematogenic responses induced by MSU [24,25,37]. Moreover, some studies have demonstrated that the stimulation of C fibers, but not Ad fibers, is related to MSU-induced nociception [14]. TRPV1 activation by the agonist capsaicin induces substance P release from C-primary-afferent fibers [16]. Accordingly, we have determined that the NK1 receptor antagonist RP 67580 decreased MSU-induced nociception and edema. In agreement, the injection of MSU into domestic chicken ankle joints produced a depletion of substance P from C fibers in the synovial and subsynovial tissues [27]. Thus, these results suggest that substance P released from capsaicin-sensitive fibers is involved in nociceptive and edematogenic responses. Substance P is a major inflammatory mediator that produces, among other events, mast cell degranulation, a recognized component of the arthritic inflammatory process [36]. Moreover, it has been reported that MSU has the ability to degranulate rat peritoneal mast cells [15]. Activation of both human and rodent mast cells leads to the release of several proinflammatory mediators [31]. We demonstrated that the nociceptive and edematogenic response caused by MSU was significantly prevented by both mast cell membrane stabilization and prior degranulation, suggesting that gout-related pain and inflammation results from the activation of mediators released by these cells. Accordingly, mast cells are found in large numbers in the inflamed human rheumatoid synovium [51]. Histamine and serotonin are important substances present in mast cells that are released after their degranulation. To further investigate the role of these mediators after MSU treatment, we tested the effects of H1 and 5-HT antagonists on MSU-induced nociception and edema, measuring the levels of histamine and serotonin in the perfusates of the injected tissue. Promethazine (an H1 antagonist) and methysergide (not selective for any subtype

Table 2 Infiltration of neutrophil and macrophages in injected paw tissue. Time after injection (min)

10 60 480

MPO (OD/tissue mg)a

NAGase (OD/tissue mg)a

Vehicle (100 lL/paw)

MSU (0.25 mg/paw)

Vehicle (100 lL/paw)

MSU (0.25 mg/paw)

1.0 ± 0.1 2.0 ± 0.6 5.0 ± 0.4

1.0 ± 0.1 3.0 ± 0.9 7.0 ± 0.6*

18.0 ± 0.1 14.0 ± 0.2 16.0 ± 1.4

15.0 ± 2.3 14.0 ± 1.7 29.0 ± 3.2*

MPO, myeloperoxidase; NAGase, N-acetyl-b,D-glucaminidase. The asterisks denote the significance levels. * P < 0.05, compared with vehicle group (Student t-test). a Data are presented as mean of optical density (OD) ± SEM.

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of 5-HT receptors, but still antagonizes the 5-HT2C,7,5,2B,6 receptors) prevented MSU-induced edema, but not nociception. These results are not unexpected because both serotonin and histamine are much more potent inductors of edema than nociception in rats [7,17]. Accordingly, the minimum doses at which s.c. histamine and serotonin cause nociception in rats were 10 and 1 lg/paw, respectively [17]. Therefore, the amounts of histamine and serotonin released in the perfusate of MSU-injected paws were not enough to cause nociception (equivalent to 0.001 lg/paw histamine and 0.06 lg/paw serotonin). Moreover, our data confirm previous studies that have demonstrated the role of histamine and serotonin in MSU-induced edema [49]. The increase in both mediators might be responsible for the development of edema, but not nociceptive behavior. In addition to histamine and serotonin, mast cells may also release tryptase, a trypsin-like serine protease that is the most abundant mediator stored in rat mast cell granules [33]. We observed that both the nonselective and the selective prevention of tryptase prevented MSU-induced nociception and edema. Tryptase is responsible for the activation of proteinase-activated receptor-2 (PAR-2) [33,34], which is present in primary sensory neurons [43] and involved in inflammatory responses and nociception [20]. Therefore, our results indicate a possible role for the PAR-2 receptor in MSU-induced nociception and edema. Interestingly, sensitization of TRPV1 receptor by the PAR-2 receptor has been previously demonstrated [1,8,20]. Because nonsteroidal anti-inflammatory drugs are used to treat pain and inflammation in gouty patients [45], we also investigated the role of cyclooxygenase metabolites in MSU responses. We observed that the cyclooxygenase inhibitor indomethacin reduced MSU-induced nociception and edema in rats. Moreover, we observed that MSU increased prostaglandin E2 levels in injected tissue. Of note, prostaglandin E2 (released from resident tissue cells such as mast cells) acting on prostanoid E receptor (EP) receptors may sensitize TRPV1, thereby enhancing TRPV1 responses [23]. This mechanism could contribute to the induction of gout-associated pain and inflammation. As MSU cannot gate the TRPV1 receptor, the mediators generated by MSU treatment (such as prostaglandin E2 and tryptase) should not only sensitize TRPV1, but also stimulate (over several hours) the channel to prolong TRPV1-mediated nociceptive behaviors. Accordingly, the peripheral activation of either PAR-2 or EP receptors are capable of sensitizing and directly exciting nociceptors in vitro, as well as inducing spontaneous nociceptive behaviors in vivo [10,17,21,33]. Furthermore, we observed that MSU produced a significant increase in the skin temperature of the injected paw (from 26.16 ± 0.14 °C before treatment to 29.78 ± 0.80 °C, 28.15 ± 0.46 °C, and 27.80 ± 0.37 °C after 5, 10, and 30 min following MSU injection, respectively) (P < 0.05, one-way ANOVA). In conjunction with other stimuli, the increase of tissue temperature might contribute to TRPV1 stimulation because the sensitization of TRPV1 produced by PAR-2 or EP activation may decrease the temperature threshold to below 30 °C [8,35]. Thus, the increased temperature of the MSU-injected tissue could contribute to directly gate the TRPV1 receptor in the presence of inflammatory mediators. In summary, the TRPV1 receptor is very likely involved in MSU-induced pain and edema, probably through the release of mediators that stimulate this receptor. Thus, TRPV1 may be a potential target for the development of new therapies for acute gout.

Conflict of interest statement The authors declare no conflicts of interest.

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Acknowledgments This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Dr. José Antônio Trindade Borges da Costa and Mariana Saibit, a student from the Laboratório iVic (Grupo de Imageamento e Visão Computacional), for their crystal measurements and support. We also acknowledge fellowships from CNPq.

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