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3 receptors mediate mechanical hyperalgesia induced by bradykinin, but not by pro-inflammatory cytokines, PGE2 or dopamine
European Journal of Pharmacology 649 (2010) 177–182
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
P2X3 and P2X2/3 receptors mediate mechanical hyperalgesia induced by bradykinin, but not by pro-inflammatory cytokines, PGE2 or dopamine Maria Cláudia Gonçalves de Oliveira Fusaro a,c, Adriana Pelegrini-da-Silva a, Dionéia Araldi b, Carlos Amílcar Parada b, Cláudia Herrera Tambeli a,⁎ a b c
Department of Physiological Sciences, Piracicaba Dental School, State University of Campinas—UNICAMP, Limeira Av, 901 Zip Code: 13414-900 Piracicaba, São Paulo, Brazil Department of Physiology and Biophysics, Institute of Biology, State University of Campinas—UNICAMP, Monteiro Lobato 255, Zip Code: 13083-862 Campinas, São Paulo, Brazil Faculty of Applied Sciences, State University of Campinas—UNICAMP, Pedro Zaccaria 1300, Zip Code: 13484-350, Limeira, São Paulo, Brazil.
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Article history: Received 19 March 2010 Received in revised form 11 August 2010 Accepted 14 September 2010 Available online 21 September 2010 Keywords: P2X3 receptor ATP Inflammation Hyperalgesia Neutrophil Cytokine
a b s t r a c t Activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP is essential to the development of inflammatory hyperalgesia. We have previously demonstrated that this essential role of P2X3 and P2X2/3 receptors in the development of mechanical hyperalgesia induced by the inflammatory agent carrageenan is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of tumor necrosis factor alpha (TNF-α) and by a direct sensitization of the primary afferent nociceptors. Therefore, in this study we asked whether activation of P2X3 and P2X2/3 receptors contribute to the mechanical hyperalgesia induced by the inflammatory mediators involved in carrageenan-induced mechanical hyperalgesia, such as bradykinin, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL1β), interleukin-6 (IL-6), chemokine-induced chemoattractant-1 (CINC-1), prostaglandin E2 (PGE2) and dopamine. Co-administration of the non-selective P2X3 receptor antagonist TNP-ATP or the selective P2X3 and P2X2/3 receptor antagonist A-317491 with bradykinin, but not with TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine, prevented in a dose-dependent manner the mechanical hyperalgesia. We also verified whether the activation of P2X3 and P2X2/3 receptors by endogenous ATP contributes to bradykinin-induced mechanical hyperalgesia via neutrophil migration and/or cytokine release. Co-administration of TNP-ATP or A-317491 did not affect either neutrophil migration or the increased concentration of TNF-α, IL-1β, IL-6 and CINC-1 induced by bradykinin. These findings demonstrate that the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia by a mechanism that does not depend on neutrophil migration or cytokines release. © 2010 Published by Elsevier B.V.
1. Introduction ATP released from damaged or inflamed tissues plays an important role in the development of inflammatory hyperalgesia by activating P2X3 receptors, which are expressed on primary afferent neurons, preferentially on nociceptive C-fibers (Chen et al., 1995; Lewis et al., 1995). Recent reports using inflammatory pain models, such as administration of Complete Freund Adjuvant (Honore et al., 2002; Jarvis et al., 2002; McGaraughty et al., 2005; McGaraughty et al., 2003; Wu et al., 2004), formalin (Honore et al., 2002; McGaraughty et al., 2005; Souslova et al., 2000) or carrageenan (Oliveira et al., 2009) in the rat's hind paw indicate that the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia. ⁎ Corresponding author. Tel.: + 55 19 2106 5305; fax: + 55 19 2106 5212. E-mail addresses: [email protected] (M.C.G. de Oliveira Fusaro), [email protected] (A. Pelegrini-da-Silva), [email protected] (D. Araldi), [email protected] (C.A. Parada), [email protected] (C.H. Tambeli). 0014-2999/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ejphar.2010.09.037
We have recently demonstrated that activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP not only contributes but it is essential to the development of the mechanical hyperalgesia induced by carrageenan in the subcutaneous tissue of rat's hind paw. Furthermore, we showed that this essential role of P2X3 and P2X2/3 receptors is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of tumor necrosis factor alpha (TNF-α) and by a direct sensitization of the primary afferent nociceptors (Oliveira et al., 2009). In addition to ATP, carrageenan induces mechanical hyperalgesia by releasing other inflammatory mediators. For example, carrageenan induces the release of bradykinin, which in turn, triggers the subsequent release of pro-inflammatory cytokines, such as TNF-α, interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8) (Ferreira et al., 1993a,b). These cytokines induce the synthesis of final inflammatory mediators such as prostaglandins and sympathetic amines that directly sensitize the primary afferent nociceptors (Gold et al., 1996; Rush and Waxman, 2004). Furthermore, these cytokines also induce neutrophil migration (Bombini et al., 2004; Oliveira et al., 2008; Ramos et al., 2003)
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that contributes to the development of inflammatory hyperalgesia (Jain et al., 2001; Oliveira et al., 2007; Tambeli et al., 2006). However, it is not known whether the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP contributes to the mechanical hyperalgesia induced by the inflammatory mediators involved in carrageenan-induced mechanical hyperalgesia and, if so, which mechanisms underlie the contribution of peripheral P2X3 and P2X2/3 receptors to the hyperalgesic response induced by these inflammatory mediators. Therefore, the aims of this study were to investigate, for the first time, whether the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP contributes to the mechanical hyperalgesia induced by the inflammatory mediators bradykinin, TNF-α, IL-1β, IL-6, CINC-1 (rat IL-8 related chemokine), PGE2 and dopamine and, if so, whether this contribution is mediated by an indirect sensitization of the primary afferent nociceptors. 2. Material and methods 2.1. Drugs and doses The following drugs were used: the non-selective P2X3 receptor antagonist, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP; 9.6, 48 and 240 μg/paw; Oliveira et al., 2009); the selective P2X3 and P2X2/3 receptor antagonist, 5-([(3-Phenoxybenzyl) [(1 S)1,2,3,4-tetrahydro-1naphthalenyl] [amino] carbonyl)-1,2,4-benzenetricarboxylic acid (A-317491; 6.0, 20 and 60 μg/paw; Oliveira et al., 2009); bradykinin (0.15, 0.5 and 1.5 μg/paw; Ferreira et al., 1993a), prostaglandin E2 (0.05, 0.075, 0.1 and 0.2 μg/paw; Cunha et al., 2008), dopamine (1.0, 3.0, 10 and 30 μg/paw; Cunha et al., 2008) and the nonspecific selectin inhibitor fucoidan (25 mg/kg, i.v.; Cunha et al., 2008) were obtained from Sigma Chemicals (St. Louis, Missouri, USA). Tumor necrosis factor alpha (TNF-α, 0.25 and 0.8 pg/paw; Ferreira et al., 1993a), interleukin-1 beta (IL-1β, 0.01, 0.05 and 0.15 pg/paw; Cunha et al., 1992), interleukin-6 (IL-6, 0.03, 0.06, 0.1 and 0.3 ng/paw; Cunha et al., 1992) and chemokine-induced chemoattractant-1 (CINC-1, 0.1, 0.3, 1.0 and 3.0 pg/paw; Cunha et al., 1991) were obtained from R&D Systems (Minneapolis, USA). All drugs were dissolved in phosphatebuffered saline (PBS, Sigma Chemicals, St. Louis, Missouri, USA). 2.2. Subjects Male albino Wistar rats weighing 200–350 g were used. Experiments were conducted in accordance with the guidelines of the Committee for Research and Ethical Issues of IASP on using laboratory animals (Zimmermann, 1983) and the guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. All animal experimental procedures and protocols were approved by the Committee on Animal Research of the State University of Campinas—Unicamp. Animals were housed in plastic cages with soft bedding (five/cage) on a 12:12 light cycle (lights on at 06:00 A.M.) with food and water available ad libitum. They were maintained in a temperature-controlled room (±23 °C) for 1 h habituation period prior to the test. 2.3. Subcutaneous injections Drugs or their vehicle were subcutaneously injected in the dorsum of the rat's hind paw by tenting the skin and puncturing it with a 30gauge needle prior to injecting the test agent, as previously described
(Oliveira et al., 2007). The needle was connected to a catheter of polyethylene and also to a Hamilton syringe (50 μl). The animals were briefly restrained and the total volume administered in the paw was 50 μl. 2.4. Mechanical paw-withdrawal nociceptive threshold test Testing sessions took place during light phase (between 09:00 A. M. and 5:00 P.M.) in a quiet room maintained at 23 °C (Rosland, 1991). The Randall–Selitto nociceptive paw-withdrawal flexion reflex test (Randall and Selitto, 1957) was performed using an Ugo-Basile analgesymeter (Stoelting, Chicago, IL, USA), which applies a linearly increasing mechanical force to the dorsum of the rat's hind paw (Oliveira et al., 2007). The nociceptive threshold was defined as the force in grams, with which the rat withdrew its paw. The baseline paw-withdrawal threshold was defined as the mean of three tests performed at 5-min intervals before test agents were injected. Mechanical hyperalgesia was quantified as the change in mechanical nociceptive threshold calculated by subtracting the mean of three mechanical nociceptive threshold measurements taken after injection of the test agent from the mean of the three baseline measurements. The mechanical hyperalgesia was quantified 180 min after the administration of bradykinin, TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine (Cunha et al., 1991, 1992; Ferreira et al., 1993b). 2.5. Measurement of myeloperoxidase activity (MPO) The neutrophil migration to the site of the inflammatory mediator administration in the skin of rat's hind paw was evaluated by the myeloperoxidase (MPO) kinetic-colorimetric assay as previously described (Bradley et al., 1982). Approximately 0.5 cm2 of cutaneous tissue was harvested 180 min after the subcutaneous administration of inflammatory mediator. The samples were homogenized in pH 4.7 buffer (0.1 M NaCl, 0.02 M NaPO4, 1.015 M NaEDTA) and centrifugated at 3000 rpm for 15 min. The pellet was subjected to hypotonic lyses (1.5 ml of 0.2% NaCl solution followed 30 s later by addition of an equal volume of a solution containing NaCl 1.6% and glucose 5%). After further centrifugation, the pellet was resuspended in 0.05 M NaPO4 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB). After that, the pellet was snap-frozen in liquid nitrogen three times, it was centrifuged at 10,000 rpm for 15 min and it was re-homogenized. Myeloperoxidase activity in the resuspended pellet was assayed by measuring the change in optical density at 450 nm using tetramethylbenzidine and H2O2 (0.5 mM). Results were calculated by comparing the optical density of hind paw tissue supernatant with a standard curve of neutrophil (N95% purity) numbers. The results were presented as number of neutrophils × 106/mg tissue. All procedures were repeated two times to guarantee the accuracy of the results. 2.6. ELISA procedure An adaptation of ELISA (Safieh-Garabedian et al., 1995) was used. The subcutaneous tissue of the dorsum of the rat's hind paw was collected 180 min after the subcutaneous administration of the inflammatory mediator or its vehicle (0.9% NaCl). These tissues were weighed and homogenized in the same weight/volume proportion in a solution of phosphate-buffered saline (PBS) containing 0.4 M NaCl, 0.05% Tween 20, 0.5% bovine serum albumin (BSA),
Fig. 1. Effect of P2X3 and P2X2/3 receptor antagonists on the mechanical hyperalgesia induced by inflammatory mediators. Bradykinin (0.5 and 1.5 μg/paw, A), TNF-α (0.8 pg/paw, C), IL-1β (0.05 and 0.15 pg/paw, D), IL-6 (0.06, 0.1 and 0.3 ng/paw, E), CINC-1 (1.0 and 3.0 pg/paw, F), PGE2 (0.075, 0.1 and 0.2 μg/paw, G) or dopamine (3.0, 10 and 30 μg/paw, H) induced a dose-related mechanical hyperalgesia at 180 min after their administration. Co-administration TNP-ATP (240 μg/paw) or A-317491 (20 and 60 μg/paw) significantly prevented in a dose-dependent manner the mechanical hyperalgesia induced by bradykinin (1.5 μg/paw, B), but not by TNF-α (0.8 pg/paw, C), IL-1β (1.5 pg/paw, D), IL-6 (0.1 ng/ paw, E), CINC-1 (1.0 pg/paw, F), PGE2 (0.1 μg/paw, G) or dopamine (10 μg/paw, H) at 180 min after their administration. The administration of the highest dose of TNP-ATP or A317491 in the contralateral paw (c.t.) did not affect the bradykinin-induced mechanical hyperalgesia. The symbol “*” indicates responses significantly higher than that induced by PBS (P b 0.05, Tukey test). The symbol “#” indicates responses significantly lower than that induced by bradykinin plus PBS (P b 0.05, Tukey test).
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0.1 mM phenyl-methyl-sulfonyl fluoride, 0.1 mM benzotonic chloride, 10 mM EDTA, and 20 Kl/ml aprotinin (Sigma, USA). The samples were centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatants were stored at −70 °C for later use to evaluate the protein levels of TNF-α, IL-1β, IL-6 and CINC-1 in the subcutaneous tissue of rat's hind paw. The cytokines were quantified by the following kits: TNF-α–Rat TNF-alpha/TNFSF1A Quantikine ELISA Kit (R&D Systems, catalog number RTA00, sensitivity less than 5.0 pg/ml); IL-1β–Rat IL-1 beta/ IL-1F2 Quantikine ELISA Kit (R&D Systems, catalog number RLB00, sensitivity less than 5.0 pg/ml), IL-6 (R&D Systems, catalog number R6000B, sensitivity less than 14 pg/ml) and CINC-1–Rat CINC-1 Quantikine ELISA Kit (R&D Systems, catalog number RCN100, sensitivity less than 1.1 pg/ml). All procedures followed the instructions of the manufacturer R&D Systems. All procedures were repeated three times to guarantee the accuracy of the results.
bradykinin-induced MPO activity (Fig. 2, P N 0.05, Tukey test) suggesting that P2X3 and P2X2/3 receptors do not contribute to bradykinin-induced neutrophils migration. 3.3. Effect of the P2X3 and P2X2/3 receptor antagonists on bradykinininduced local increase in cytokines concentration Bradykinin (1.5 μg/paw) significantly increased (P b 0.05, T test) the local concentration of TNF-α (Fig. 3A), IL-1β (Fig. 3B), IL-6 (Fig. 3C) and CINC-1 (Fig. 3D). Co-administration of TNP-ATP (240 μg/ paw) or A-317491 (60 μg/paw) with bradykinin did not affect (P N 0.05, Tukey test) the concentration of TNF-α (Fig. 3A), IL-1β (Fig. 3B), IL-6 (Fig. 3C) and CINC-1 (Fig. 3D). These findings suggest that P2X3 and P2X2/3 do not contribute to bradykinin-induced endogenous release of pro-inflammatory cytokines.
2.7. Statistical analysis 4. Discussion To determine if there were significant differences (P b 0.05) between treatment groups, one-way ANOVA or t-test was performed. If there was a significant between-subjects main effect of treatment group following one-way ANOVA, post-hoc contrasts, using the Tukey test, were performed to determine the basis of the significant difference. Data are expressed in figures by the decrease in pawwithdrawal threshold and presented as means ± S.E.M. 3. Results 3.1. Effect of P2X3 and P2X2/3 receptor antagonists on the mechanical hyperalgesia induced by bradykinin, TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine Bradykinin (0.5 and 1.5 μg/paw, Fig. 1A), TNF-α (0.8 pg/paw, Fig. 1C), IL-1β (0.05 and 0.15 pg/paw, Fig. 1D), IL-6 (0.06, 0.1 and 0.3 ng/paw, Fig. 1E), CINC-1 (1.0 and 3.0 pg/paw, Fig. 1F), PGE2 (0.075, 0.1 and 0.2 μg/paw, Fig. 1G) or dopamine (3.0, 10 and 30 μg/paw, Fig. 1H) induced a dose-related mechanical hyperalgesia measured 180 min after administration in the subcutaneous tissue of rat's hind paw (P b 0.05, Tukey test). To verify whether the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates the mechanical hyperalgesia induced by these inflammatory mediators, the nonselective P2X3 receptor antagonist TNP-ATP or the selective P2X3 and P2X2/3 receptor antagonist A-317491 was co-administered with each one of them. Co-administration of the non-selective P2X3 receptor antagonist TNP-ATP (240 μg/paw) or the selective P2X3 and P2X2/3 receptor antagonist A-317491 (20 and 60 μg/paw) prevented (P b 0.05, Tukey test) the mechanical hyperalgesia induced by bradykinin (1.5 μg/paw, Fig. 1B). The administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) on the contralateral rat's hind paw did not affect bradykinin-induced mechanical hyperalgesia (P N 0.05, T test, Fig. 1B), ruling out a systemic effect. Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) did not significantly reduce (P N 0.05, Tukey test) the mechanical hyperalgesia induced by TNF-α (0.8 pg/paw, Fig. 1C), IL-1β (0.15 pg/paw, Fig. 1D), IL-6 (0.1 ng/paw, Fig. 1E), CINC-1 (1.0 pg/paw, Fig. 1F), PGE2 (0.1 μg/ paw, Fig. 1G) or dopamine (10 μg/paw, Fig. 1H). These findings suggest that P2X3 and P2X2/3 receptors mediate the mechanical hyperalgesia induced by bradykinin but not by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine. 3.2. Effect of P2X3 and P2X2/3 receptor antagonists on bradykinininduced neutrophils migration Bradykinin (1.5 μg/paw) significantly increased the neutrophil migration (Fig. 2, P b 0.05, T test). Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) with bradykinin did not affect
In this study we demonstrated that the selective P2X3 and P2X2/3 receptor antagonist A-317491 (McGaraughty et al., 2003) or the nonselective P2X3 receptor antagonist TNP-ATP (Jarvis et al., 2001) prevented the mechanical hyperalgesia induced by bradykinin, but not by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine. Although the activation of spinal P2X3 and P2X2/3 receptors contributes to the inflammatory hyperalgesia in the rat hind paw (McGaraughty et al., 2003), the contralateral administrations of P2X3 and P2X2/3 receptor antagonists had no effect confirming that only local P2X3 and P2X2/3 receptors of the peripheral tissue were targeted. These findings indicate that bradykinin in the subcutaneous tissue, as previously demonstrated in cultured cells (Chopra et al., 2005; Zhao et al., 2007), induces the endogenous release of ATP, which in turn, activates P2X3 and P2X2/3 receptors. The activation of peripheral P2X3 and P2X2/3 receptor by endogenous ATP not only contributes but it is essential to the development of the mechanical hyperalgesia induced by bradykinin in the subcutaneous tissue. Considering that bradykinin is an inflammatory mediator released at the early phase of the inflammatory hyperalgesia (Ferreira et al., 1993a,b), the activation of P2X3 and P2X2/3 receptors by endogenous ATP may play a role in the beginning of the development of the inflammatory hyperalgesia. Similarly, we have recently demonstrated that the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP is essential to the development, but not to the maintenance of the hyperalgesic response induced by carrageenan. Specifically, TNP-ATP blocks carrageenan-induced mechanical hyperalgesia when it is co-administered with carrageenan, but
Fig. 2. Effect of P2X3 and P2X2/3 receptor antagonists on bradykinin-induced neutrophil migration. Bradykinin (1.5 μg/paw) induced a significant increase in the MPO activity measured 180 min after its administration. Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) did not affect the MPO activity induced by bradykinin. The symbol “*” indicates a response significantly higher than that induced by PBS (P b 0.05, t-test).
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Fig. 3. Effect of P2X3 and P2X2/3 receptor antagonists on bradykinin-induced local increase in cytokine concentration. Bradykinin (1.5 μg/paw) induced a significant increase in the local concentration of TNF-α (A), IL-1β (B), IL-6 (C) and CINC-1 (D) measured 180 min after its administration. Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/ paw) did not affect the bradykinin-induced local increase in TNF-α (A), IL-1β (B), IL-6 (C) and CINC-1 (D) concentrations. The symbol “*” indicates responses significantly higher than that induced by PBS (P b 0.05, t-test).
not when it is administered 60, 120 or 180 min after the carrageenan administration (Oliveira et al., 2009). Bradykinin has been reported to induce hyperalgesia by two distinct pathways that ultimately result in the local production of prostaglandins and in the local release of sympathetic amines (Ferreira et al., 1993a,b), which directly sensitize the primary afferent nociceptor (Gold et al., 1996; Rush and Waxman, 2004). Therefore, the prevention of bradykinin-induced mechanical hyperalgesia by the co-administration of TNP-ATP or A-317491 indicates that the activation of the P2X3 and P2X2/3 receptors is crucial to bradykinin induction of prostaglandinand sympathetic amine-mediated sensitization of the primary afferent nociceptor. Like bradykinin-induced mechanical hyperalgesia, carrageenan-induced mechanical hyperalgesia, which is also mediated by prostaglandins and sympathetic amines (Cunha et al., 1991, 1992; Ferreira et al., 1993a), was prevented by P2X3 and P2X2/3 receptor antagonists (Oliveira et al., 2009). It has been proposed that bradykinin triggers the synthesis of prostaglandins and sympathetic amines through the release of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and CINC-1 (Ferreira et al., 1993a,b) to induce inflammatory hyperalgesia. However, the lack of effect of P2X3 and P2X2/3 receptor antagonists on the endogenous release of TNF-α, IL-1β, IL-6 and CINC-1 induced by bradykinin indicates that the mechanism by which the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia is not dependent on cytokines release. Furthermore, the mechanical hyperalgesia induced by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine was not affected by P2X3 and P2X2/3 receptor antagonists. It has been recently demonstrated that neutrophil migration is essential to the development of inflammatory pain in the subcutane-
ous tissue of the rat's hind paw (Cunha et al., 2008). However, the coadministration of A-317491 or TNP-ATP with bradykinin did not affect the neutrophil migration induced by bradykinin. This finding indicates that the mechanism by which the activation of P2X3 and P2x2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia does not depend on neutrophil migration. Considering that the mRNA distribution of the P2X3 receptors in the peripheral tissues is almost exclusively expressed on primary afferent neurons (Chen et al., 1995; Collo et al., 1996; Kennedy and Leff, 1995; Lewis et al., 1995), endogenous ATP may contribute to bradykinin-induced mechanical hyperalgesia by a direct action on P2X3 receptors expressed on primary afferent nociceptors. Studies that support this suggestion demonstrate that treatment with oligonucleotide antisense against P2X3 receptors significantly reduce the inflammatory hyperalgesia induced by Complete Adjuvant Freund (Barclay et al., 2002; Honore et al., 2002), carrageenan (Oliveira et al., 2009) or nerve injury (Barclay et al., 2002; Honore et al., 2002). Recent studies have demonstrated that the action of TNF-α on primary afferent nociceptors increase their susceptibility to the development of inflammatory hyperalgesia induced by PGE2 (Parada et al., 2003a,b). Therefore, a similar sensitizing effect induced by the activation of neuronal P2X3 by endogenous ATP might underlie bradykinin-induced mechanical hyperalgesia. 5. Conclusion In summary, we demonstrated that the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates the mechanical hyperalgesia induced by bradykinin but not by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine. The mechanism by which the activation of
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
P2X3 and P2X2/3 receptors mediate mechanical hyperalgesia induced by bradykinin, but not by pro-inflammatory cytokines, PGE2 or dopamine Maria Cláudia Gonçalves de Oliveira Fusaro a,c, Adriana Pelegrini-da-Silva a, Dionéia Araldi b, Carlos Amílcar Parada b, Cláudia Herrera Tambeli a,⁎ a b c
Department of Physiological Sciences, Piracicaba Dental School, State University of Campinas—UNICAMP, Limeira Av, 901 Zip Code: 13414-900 Piracicaba, São Paulo, Brazil Department of Physiology and Biophysics, Institute of Biology, State University of Campinas—UNICAMP, Monteiro Lobato 255, Zip Code: 13083-862 Campinas, São Paulo, Brazil Faculty of Applied Sciences, State University of Campinas—UNICAMP, Pedro Zaccaria 1300, Zip Code: 13484-350, Limeira, São Paulo, Brazil.
a r t i c l e
i n f o
Article history: Received 19 March 2010 Received in revised form 11 August 2010 Accepted 14 September 2010 Available online 21 September 2010 Keywords: P2X3 receptor ATP Inflammation Hyperalgesia Neutrophil Cytokine
a b s t r a c t Activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP is essential to the development of inflammatory hyperalgesia. We have previously demonstrated that this essential role of P2X3 and P2X2/3 receptors in the development of mechanical hyperalgesia induced by the inflammatory agent carrageenan is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of tumor necrosis factor alpha (TNF-α) and by a direct sensitization of the primary afferent nociceptors. Therefore, in this study we asked whether activation of P2X3 and P2X2/3 receptors contribute to the mechanical hyperalgesia induced by the inflammatory mediators involved in carrageenan-induced mechanical hyperalgesia, such as bradykinin, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL1β), interleukin-6 (IL-6), chemokine-induced chemoattractant-1 (CINC-1), prostaglandin E2 (PGE2) and dopamine. Co-administration of the non-selective P2X3 receptor antagonist TNP-ATP or the selective P2X3 and P2X2/3 receptor antagonist A-317491 with bradykinin, but not with TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine, prevented in a dose-dependent manner the mechanical hyperalgesia. We also verified whether the activation of P2X3 and P2X2/3 receptors by endogenous ATP contributes to bradykinin-induced mechanical hyperalgesia via neutrophil migration and/or cytokine release. Co-administration of TNP-ATP or A-317491 did not affect either neutrophil migration or the increased concentration of TNF-α, IL-1β, IL-6 and CINC-1 induced by bradykinin. These findings demonstrate that the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia by a mechanism that does not depend on neutrophil migration or cytokines release. © 2010 Published by Elsevier B.V.
1. Introduction ATP released from damaged or inflamed tissues plays an important role in the development of inflammatory hyperalgesia by activating P2X3 receptors, which are expressed on primary afferent neurons, preferentially on nociceptive C-fibers (Chen et al., 1995; Lewis et al., 1995). Recent reports using inflammatory pain models, such as administration of Complete Freund Adjuvant (Honore et al., 2002; Jarvis et al., 2002; McGaraughty et al., 2005; McGaraughty et al., 2003; Wu et al., 2004), formalin (Honore et al., 2002; McGaraughty et al., 2005; Souslova et al., 2000) or carrageenan (Oliveira et al., 2009) in the rat's hind paw indicate that the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia. ⁎ Corresponding author. Tel.: + 55 19 2106 5305; fax: + 55 19 2106 5212. E-mail addresses: [email protected] (M.C.G. de Oliveira Fusaro), [email protected] (A. Pelegrini-da-Silva), [email protected] (D. Araldi), [email protected] (C.A. Parada), [email protected] (C.H. Tambeli). 0014-2999/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ejphar.2010.09.037
We have recently demonstrated that activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP not only contributes but it is essential to the development of the mechanical hyperalgesia induced by carrageenan in the subcutaneous tissue of rat's hind paw. Furthermore, we showed that this essential role of P2X3 and P2X2/3 receptors is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of tumor necrosis factor alpha (TNF-α) and by a direct sensitization of the primary afferent nociceptors (Oliveira et al., 2009). In addition to ATP, carrageenan induces mechanical hyperalgesia by releasing other inflammatory mediators. For example, carrageenan induces the release of bradykinin, which in turn, triggers the subsequent release of pro-inflammatory cytokines, such as TNF-α, interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8) (Ferreira et al., 1993a,b). These cytokines induce the synthesis of final inflammatory mediators such as prostaglandins and sympathetic amines that directly sensitize the primary afferent nociceptors (Gold et al., 1996; Rush and Waxman, 2004). Furthermore, these cytokines also induce neutrophil migration (Bombini et al., 2004; Oliveira et al., 2008; Ramos et al., 2003)
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that contributes to the development of inflammatory hyperalgesia (Jain et al., 2001; Oliveira et al., 2007; Tambeli et al., 2006). However, it is not known whether the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP contributes to the mechanical hyperalgesia induced by the inflammatory mediators involved in carrageenan-induced mechanical hyperalgesia and, if so, which mechanisms underlie the contribution of peripheral P2X3 and P2X2/3 receptors to the hyperalgesic response induced by these inflammatory mediators. Therefore, the aims of this study were to investigate, for the first time, whether the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP contributes to the mechanical hyperalgesia induced by the inflammatory mediators bradykinin, TNF-α, IL-1β, IL-6, CINC-1 (rat IL-8 related chemokine), PGE2 and dopamine and, if so, whether this contribution is mediated by an indirect sensitization of the primary afferent nociceptors. 2. Material and methods 2.1. Drugs and doses The following drugs were used: the non-selective P2X3 receptor antagonist, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP; 9.6, 48 and 240 μg/paw; Oliveira et al., 2009); the selective P2X3 and P2X2/3 receptor antagonist, 5-([(3-Phenoxybenzyl) [(1 S)1,2,3,4-tetrahydro-1naphthalenyl] [amino] carbonyl)-1,2,4-benzenetricarboxylic acid (A-317491; 6.0, 20 and 60 μg/paw; Oliveira et al., 2009); bradykinin (0.15, 0.5 and 1.5 μg/paw; Ferreira et al., 1993a), prostaglandin E2 (0.05, 0.075, 0.1 and 0.2 μg/paw; Cunha et al., 2008), dopamine (1.0, 3.0, 10 and 30 μg/paw; Cunha et al., 2008) and the nonspecific selectin inhibitor fucoidan (25 mg/kg, i.v.; Cunha et al., 2008) were obtained from Sigma Chemicals (St. Louis, Missouri, USA). Tumor necrosis factor alpha (TNF-α, 0.25 and 0.8 pg/paw; Ferreira et al., 1993a), interleukin-1 beta (IL-1β, 0.01, 0.05 and 0.15 pg/paw; Cunha et al., 1992), interleukin-6 (IL-6, 0.03, 0.06, 0.1 and 0.3 ng/paw; Cunha et al., 1992) and chemokine-induced chemoattractant-1 (CINC-1, 0.1, 0.3, 1.0 and 3.0 pg/paw; Cunha et al., 1991) were obtained from R&D Systems (Minneapolis, USA). All drugs were dissolved in phosphatebuffered saline (PBS, Sigma Chemicals, St. Louis, Missouri, USA). 2.2. Subjects Male albino Wistar rats weighing 200–350 g were used. Experiments were conducted in accordance with the guidelines of the Committee for Research and Ethical Issues of IASP on using laboratory animals (Zimmermann, 1983) and the guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. All animal experimental procedures and protocols were approved by the Committee on Animal Research of the State University of Campinas—Unicamp. Animals were housed in plastic cages with soft bedding (five/cage) on a 12:12 light cycle (lights on at 06:00 A.M.) with food and water available ad libitum. They were maintained in a temperature-controlled room (±23 °C) for 1 h habituation period prior to the test. 2.3. Subcutaneous injections Drugs or their vehicle were subcutaneously injected in the dorsum of the rat's hind paw by tenting the skin and puncturing it with a 30gauge needle prior to injecting the test agent, as previously described
(Oliveira et al., 2007). The needle was connected to a catheter of polyethylene and also to a Hamilton syringe (50 μl). The animals were briefly restrained and the total volume administered in the paw was 50 μl. 2.4. Mechanical paw-withdrawal nociceptive threshold test Testing sessions took place during light phase (between 09:00 A. M. and 5:00 P.M.) in a quiet room maintained at 23 °C (Rosland, 1991). The Randall–Selitto nociceptive paw-withdrawal flexion reflex test (Randall and Selitto, 1957) was performed using an Ugo-Basile analgesymeter (Stoelting, Chicago, IL, USA), which applies a linearly increasing mechanical force to the dorsum of the rat's hind paw (Oliveira et al., 2007). The nociceptive threshold was defined as the force in grams, with which the rat withdrew its paw. The baseline paw-withdrawal threshold was defined as the mean of three tests performed at 5-min intervals before test agents were injected. Mechanical hyperalgesia was quantified as the change in mechanical nociceptive threshold calculated by subtracting the mean of three mechanical nociceptive threshold measurements taken after injection of the test agent from the mean of the three baseline measurements. The mechanical hyperalgesia was quantified 180 min after the administration of bradykinin, TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine (Cunha et al., 1991, 1992; Ferreira et al., 1993b). 2.5. Measurement of myeloperoxidase activity (MPO) The neutrophil migration to the site of the inflammatory mediator administration in the skin of rat's hind paw was evaluated by the myeloperoxidase (MPO) kinetic-colorimetric assay as previously described (Bradley et al., 1982). Approximately 0.5 cm2 of cutaneous tissue was harvested 180 min after the subcutaneous administration of inflammatory mediator. The samples were homogenized in pH 4.7 buffer (0.1 M NaCl, 0.02 M NaPO4, 1.015 M NaEDTA) and centrifugated at 3000 rpm for 15 min. The pellet was subjected to hypotonic lyses (1.5 ml of 0.2% NaCl solution followed 30 s later by addition of an equal volume of a solution containing NaCl 1.6% and glucose 5%). After further centrifugation, the pellet was resuspended in 0.05 M NaPO4 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB). After that, the pellet was snap-frozen in liquid nitrogen three times, it was centrifuged at 10,000 rpm for 15 min and it was re-homogenized. Myeloperoxidase activity in the resuspended pellet was assayed by measuring the change in optical density at 450 nm using tetramethylbenzidine and H2O2 (0.5 mM). Results were calculated by comparing the optical density of hind paw tissue supernatant with a standard curve of neutrophil (N95% purity) numbers. The results were presented as number of neutrophils × 106/mg tissue. All procedures were repeated two times to guarantee the accuracy of the results. 2.6. ELISA procedure An adaptation of ELISA (Safieh-Garabedian et al., 1995) was used. The subcutaneous tissue of the dorsum of the rat's hind paw was collected 180 min after the subcutaneous administration of the inflammatory mediator or its vehicle (0.9% NaCl). These tissues were weighed and homogenized in the same weight/volume proportion in a solution of phosphate-buffered saline (PBS) containing 0.4 M NaCl, 0.05% Tween 20, 0.5% bovine serum albumin (BSA),
Fig. 1. Effect of P2X3 and P2X2/3 receptor antagonists on the mechanical hyperalgesia induced by inflammatory mediators. Bradykinin (0.5 and 1.5 μg/paw, A), TNF-α (0.8 pg/paw, C), IL-1β (0.05 and 0.15 pg/paw, D), IL-6 (0.06, 0.1 and 0.3 ng/paw, E), CINC-1 (1.0 and 3.0 pg/paw, F), PGE2 (0.075, 0.1 and 0.2 μg/paw, G) or dopamine (3.0, 10 and 30 μg/paw, H) induced a dose-related mechanical hyperalgesia at 180 min after their administration. Co-administration TNP-ATP (240 μg/paw) or A-317491 (20 and 60 μg/paw) significantly prevented in a dose-dependent manner the mechanical hyperalgesia induced by bradykinin (1.5 μg/paw, B), but not by TNF-α (0.8 pg/paw, C), IL-1β (1.5 pg/paw, D), IL-6 (0.1 ng/ paw, E), CINC-1 (1.0 pg/paw, F), PGE2 (0.1 μg/paw, G) or dopamine (10 μg/paw, H) at 180 min after their administration. The administration of the highest dose of TNP-ATP or A317491 in the contralateral paw (c.t.) did not affect the bradykinin-induced mechanical hyperalgesia. The symbol “*” indicates responses significantly higher than that induced by PBS (P b 0.05, Tukey test). The symbol “#” indicates responses significantly lower than that induced by bradykinin plus PBS (P b 0.05, Tukey test).
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0.1 mM phenyl-methyl-sulfonyl fluoride, 0.1 mM benzotonic chloride, 10 mM EDTA, and 20 Kl/ml aprotinin (Sigma, USA). The samples were centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatants were stored at −70 °C for later use to evaluate the protein levels of TNF-α, IL-1β, IL-6 and CINC-1 in the subcutaneous tissue of rat's hind paw. The cytokines were quantified by the following kits: TNF-α–Rat TNF-alpha/TNFSF1A Quantikine ELISA Kit (R&D Systems, catalog number RTA00, sensitivity less than 5.0 pg/ml); IL-1β–Rat IL-1 beta/ IL-1F2 Quantikine ELISA Kit (R&D Systems, catalog number RLB00, sensitivity less than 5.0 pg/ml), IL-6 (R&D Systems, catalog number R6000B, sensitivity less than 14 pg/ml) and CINC-1–Rat CINC-1 Quantikine ELISA Kit (R&D Systems, catalog number RCN100, sensitivity less than 1.1 pg/ml). All procedures followed the instructions of the manufacturer R&D Systems. All procedures were repeated three times to guarantee the accuracy of the results.
bradykinin-induced MPO activity (Fig. 2, P N 0.05, Tukey test) suggesting that P2X3 and P2X2/3 receptors do not contribute to bradykinin-induced neutrophils migration. 3.3. Effect of the P2X3 and P2X2/3 receptor antagonists on bradykinininduced local increase in cytokines concentration Bradykinin (1.5 μg/paw) significantly increased (P b 0.05, T test) the local concentration of TNF-α (Fig. 3A), IL-1β (Fig. 3B), IL-6 (Fig. 3C) and CINC-1 (Fig. 3D). Co-administration of TNP-ATP (240 μg/ paw) or A-317491 (60 μg/paw) with bradykinin did not affect (P N 0.05, Tukey test) the concentration of TNF-α (Fig. 3A), IL-1β (Fig. 3B), IL-6 (Fig. 3C) and CINC-1 (Fig. 3D). These findings suggest that P2X3 and P2X2/3 do not contribute to bradykinin-induced endogenous release of pro-inflammatory cytokines.
2.7. Statistical analysis 4. Discussion To determine if there were significant differences (P b 0.05) between treatment groups, one-way ANOVA or t-test was performed. If there was a significant between-subjects main effect of treatment group following one-way ANOVA, post-hoc contrasts, using the Tukey test, were performed to determine the basis of the significant difference. Data are expressed in figures by the decrease in pawwithdrawal threshold and presented as means ± S.E.M. 3. Results 3.1. Effect of P2X3 and P2X2/3 receptor antagonists on the mechanical hyperalgesia induced by bradykinin, TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine Bradykinin (0.5 and 1.5 μg/paw, Fig. 1A), TNF-α (0.8 pg/paw, Fig. 1C), IL-1β (0.05 and 0.15 pg/paw, Fig. 1D), IL-6 (0.06, 0.1 and 0.3 ng/paw, Fig. 1E), CINC-1 (1.0 and 3.0 pg/paw, Fig. 1F), PGE2 (0.075, 0.1 and 0.2 μg/paw, Fig. 1G) or dopamine (3.0, 10 and 30 μg/paw, Fig. 1H) induced a dose-related mechanical hyperalgesia measured 180 min after administration in the subcutaneous tissue of rat's hind paw (P b 0.05, Tukey test). To verify whether the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates the mechanical hyperalgesia induced by these inflammatory mediators, the nonselective P2X3 receptor antagonist TNP-ATP or the selective P2X3 and P2X2/3 receptor antagonist A-317491 was co-administered with each one of them. Co-administration of the non-selective P2X3 receptor antagonist TNP-ATP (240 μg/paw) or the selective P2X3 and P2X2/3 receptor antagonist A-317491 (20 and 60 μg/paw) prevented (P b 0.05, Tukey test) the mechanical hyperalgesia induced by bradykinin (1.5 μg/paw, Fig. 1B). The administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) on the contralateral rat's hind paw did not affect bradykinin-induced mechanical hyperalgesia (P N 0.05, T test, Fig. 1B), ruling out a systemic effect. Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) did not significantly reduce (P N 0.05, Tukey test) the mechanical hyperalgesia induced by TNF-α (0.8 pg/paw, Fig. 1C), IL-1β (0.15 pg/paw, Fig. 1D), IL-6 (0.1 ng/paw, Fig. 1E), CINC-1 (1.0 pg/paw, Fig. 1F), PGE2 (0.1 μg/ paw, Fig. 1G) or dopamine (10 μg/paw, Fig. 1H). These findings suggest that P2X3 and P2X2/3 receptors mediate the mechanical hyperalgesia induced by bradykinin but not by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine. 3.2. Effect of P2X3 and P2X2/3 receptor antagonists on bradykinininduced neutrophils migration Bradykinin (1.5 μg/paw) significantly increased the neutrophil migration (Fig. 2, P b 0.05, T test). Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) with bradykinin did not affect
In this study we demonstrated that the selective P2X3 and P2X2/3 receptor antagonist A-317491 (McGaraughty et al., 2003) or the nonselective P2X3 receptor antagonist TNP-ATP (Jarvis et al., 2001) prevented the mechanical hyperalgesia induced by bradykinin, but not by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine. Although the activation of spinal P2X3 and P2X2/3 receptors contributes to the inflammatory hyperalgesia in the rat hind paw (McGaraughty et al., 2003), the contralateral administrations of P2X3 and P2X2/3 receptor antagonists had no effect confirming that only local P2X3 and P2X2/3 receptors of the peripheral tissue were targeted. These findings indicate that bradykinin in the subcutaneous tissue, as previously demonstrated in cultured cells (Chopra et al., 2005; Zhao et al., 2007), induces the endogenous release of ATP, which in turn, activates P2X3 and P2X2/3 receptors. The activation of peripheral P2X3 and P2X2/3 receptor by endogenous ATP not only contributes but it is essential to the development of the mechanical hyperalgesia induced by bradykinin in the subcutaneous tissue. Considering that bradykinin is an inflammatory mediator released at the early phase of the inflammatory hyperalgesia (Ferreira et al., 1993a,b), the activation of P2X3 and P2X2/3 receptors by endogenous ATP may play a role in the beginning of the development of the inflammatory hyperalgesia. Similarly, we have recently demonstrated that the activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP is essential to the development, but not to the maintenance of the hyperalgesic response induced by carrageenan. Specifically, TNP-ATP blocks carrageenan-induced mechanical hyperalgesia when it is co-administered with carrageenan, but
Fig. 2. Effect of P2X3 and P2X2/3 receptor antagonists on bradykinin-induced neutrophil migration. Bradykinin (1.5 μg/paw) induced a significant increase in the MPO activity measured 180 min after its administration. Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/paw) did not affect the MPO activity induced by bradykinin. The symbol “*” indicates a response significantly higher than that induced by PBS (P b 0.05, t-test).
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Fig. 3. Effect of P2X3 and P2X2/3 receptor antagonists on bradykinin-induced local increase in cytokine concentration. Bradykinin (1.5 μg/paw) induced a significant increase in the local concentration of TNF-α (A), IL-1β (B), IL-6 (C) and CINC-1 (D) measured 180 min after its administration. Co-administration of TNP-ATP (240 μg/paw) or A-317491 (60 μg/ paw) did not affect the bradykinin-induced local increase in TNF-α (A), IL-1β (B), IL-6 (C) and CINC-1 (D) concentrations. The symbol “*” indicates responses significantly higher than that induced by PBS (P b 0.05, t-test).
not when it is administered 60, 120 or 180 min after the carrageenan administration (Oliveira et al., 2009). Bradykinin has been reported to induce hyperalgesia by two distinct pathways that ultimately result in the local production of prostaglandins and in the local release of sympathetic amines (Ferreira et al., 1993a,b), which directly sensitize the primary afferent nociceptor (Gold et al., 1996; Rush and Waxman, 2004). Therefore, the prevention of bradykinin-induced mechanical hyperalgesia by the co-administration of TNP-ATP or A-317491 indicates that the activation of the P2X3 and P2X2/3 receptors is crucial to bradykinin induction of prostaglandinand sympathetic amine-mediated sensitization of the primary afferent nociceptor. Like bradykinin-induced mechanical hyperalgesia, carrageenan-induced mechanical hyperalgesia, which is also mediated by prostaglandins and sympathetic amines (Cunha et al., 1991, 1992; Ferreira et al., 1993a), was prevented by P2X3 and P2X2/3 receptor antagonists (Oliveira et al., 2009). It has been proposed that bradykinin triggers the synthesis of prostaglandins and sympathetic amines through the release of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and CINC-1 (Ferreira et al., 1993a,b) to induce inflammatory hyperalgesia. However, the lack of effect of P2X3 and P2X2/3 receptor antagonists on the endogenous release of TNF-α, IL-1β, IL-6 and CINC-1 induced by bradykinin indicates that the mechanism by which the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia is not dependent on cytokines release. Furthermore, the mechanical hyperalgesia induced by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine was not affected by P2X3 and P2X2/3 receptor antagonists. It has been recently demonstrated that neutrophil migration is essential to the development of inflammatory pain in the subcutane-
ous tissue of the rat's hind paw (Cunha et al., 2008). However, the coadministration of A-317491 or TNP-ATP with bradykinin did not affect the neutrophil migration induced by bradykinin. This finding indicates that the mechanism by which the activation of P2X3 and P2x2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia does not depend on neutrophil migration. Considering that the mRNA distribution of the P2X3 receptors in the peripheral tissues is almost exclusively expressed on primary afferent neurons (Chen et al., 1995; Collo et al., 1996; Kennedy and Leff, 1995; Lewis et al., 1995), endogenous ATP may contribute to bradykinin-induced mechanical hyperalgesia by a direct action on P2X3 receptors expressed on primary afferent nociceptors. Studies that support this suggestion demonstrate that treatment with oligonucleotide antisense against P2X3 receptors significantly reduce the inflammatory hyperalgesia induced by Complete Adjuvant Freund (Barclay et al., 2002; Honore et al., 2002), carrageenan (Oliveira et al., 2009) or nerve injury (Barclay et al., 2002; Honore et al., 2002). Recent studies have demonstrated that the action of TNF-α on primary afferent nociceptors increase their susceptibility to the development of inflammatory hyperalgesia induced by PGE2 (Parada et al., 2003a,b). Therefore, a similar sensitizing effect induced by the activation of neuronal P2X3 by endogenous ATP might underlie bradykinin-induced mechanical hyperalgesia. 5. Conclusion In summary, we demonstrated that the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates the mechanical hyperalgesia induced by bradykinin but not by TNF-α, IL-1β, IL-6, CINC-1, PGE2 or dopamine. The mechanism by which the activation of
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