ATP, via activation of P2X3 receptors, has been highlighted as a key target in inflammatory hyperalgesia. Therefore, the aim of this study was to confirm whether the activation of P2X3 receptors in the gastrocnemius muscle of rats induces mechanical muscle hyperalgesia and, if so, to analyze the involvement of the classical inflammatory mediators (bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration) in this response. Intramuscular administration of the non-selective P2X3 receptor agonist α,β-meATP in the gastrocnemius muscle of rats induced mechanical muscle hyperalgesia, which, in turn, was prevented by the selective P2X3 and P2X2/3 receptors antagonist A-317491, the selective bradykinin B1-receptor antagonist Des-Arg9-[Leu8]-BK (DALBK), the cyclooxygenase inhibitor indomethacin, the β1- or β2-adrenoceptor antagonist atenolol and ICI 118,551, respectively. Also, the nonspecific selectin inhibitor fucoidan. α,β-meATP induced increases in the local concentration of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), which were reduced by bradykinin antagonist. Finally, α,β-meATP also induced neutrophil migration. Together, these findings suggest that α,β-meATP induced mechanical hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines release and neutrophil migration. It is also indicated that bradykinin is the key modulator of the mechanical muscle hyperalgesia induced by P2X3 receptors. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain.285 2433Ambalavanar, R., Moritani, M., Dessem, D., Trigeminal P2X3 receptor expression differs from dorsal root ganglion and is modulated by deep tissue inflammation (2005) Pain, 117, pp. 280-291Babenko, V.V., Graven-Nielsen, T., Svensson, P., Drewes, A.M., Jensen, T.S., Arendt-Nielsen, L., Experimental human muscle pain induced by intramuscular injections of bradykinin, serotonin, and substance P (1999) Eur J Pain (London, England), 3, pp. 93-102Barclay, J., Patel, S., Dorn, G., Wotherspoon, G., Moffatt, S., Eunson, L., Abdel'al, S., Ganju, P., Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain (2002) J Neurosci, 22, pp. 8139-8147Becq, F., CFTR channels and adenosine triphosphate release: the impossible rendezvous revisited in skeletal muscle (2010) J Physiol, 588, pp. 4605-4606Bjur, D., Danielson, P., Alfredson, H., Forsgren, S., Immunohistochemical and in situ hybridization observations favor a local catecholamine production in the human Achilles tendon (2008) Histol Histopathol, 23, pp. 197-208Boix, F., Roe, C., Rosenborg, L., Knardahl, S., Kinin peptides in human trapezius muscle during sustained isometric contraction and their relation to pain (1985) J Appl Physiol, 98, pp. 534-540Burnstock, G., Physiology and pathophysiology of purinergic neurotransmission (2007) Physiol Rev, 87, pp. 659-797Chen, C.C., Akopian, A.N., Sivilotti, L., Colquhoun, D., Burnstock, G., Wood, J.N., A P2X purinoceptor expressed by a subset of sensory neurons (1995) Nature, 377, pp. 428-431Christidis, N., Kopp, S., Ernberg, M., The effect on mechanical pain threshold over human muscles by oral administration of granisetron and diclofenac-sodium (2005) Pain, 113, pp. 265-270Cockayne, D.A., Dunn, P.M., Zhong, Y., Rong, W., Hamilton, S.G., Knight, G.E., Ruan, H.Z., Ford, A.P., P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP (2005) J Physiol, 567, pp. 621-639Cook, S.P., McCleskey, E.W., Cell damage excites nociceptors through release of cytosolic ATP (2002) Pain, 95, pp. 41-47Cunha, T.M., Verri, W.A., Schivo, I.R., Napimoga, M.H., Parada, C.A., Poole, S., Teixeira, M.M., Cunha, F.Q., Crucial role of neutrophils in the development of mechanical inflammatory hypernociception (2008) J leukoc Biol, 83, pp. 824-832Danielson, P., Alfredson, H., Forsgren, S., In situ hybridization studies confirming recent findings of the existence of a local nonneuronal catecholamine production in human patellar tendinosis (2007) Microsc Res Tech, 70, pp. 908-911Danielson, P., Alfredson, H., Forsgren, S., Studies on the importance of sympathetic innervation, adrenergic receptors, and a possible local catecholamine production in the development of patellar tendinopathy (tendinosis) in man (2007) Microsc Res Tech, 70, pp. 310-324Davis, A.J., Perkins, M.N., Induction of B1 receptors in vivo in a model of persistent inflammatory mechanical hyperalgesia in the rat (1994) Neuropharmacology, 33, pp. 127-133de Oliveira Fusaro, M.C., Pelegrini-da-Silva, A., Araldi, D., Parada, C.A., Tambeli, C.H., P2X3 and P2X2/3 receptors mediate mechanical hyperalgesia induced by bradykinin, but not by pro-inflammatory cytokines, PGE(2) or dopamine (2010) Eur J Pharmacol, 649, pp. 177-182Dessem, D., Ambalavanar, R., Evancho, M., Moutanni, A., Yallampalli, C., Bai, G., Eccentric muscle contraction and stretching evoke mechanical hyperalgesia and modulate CGRP and P2X(3) expression in a functionally relevant manner (2010) Pain, 149, pp. 284-295Ellrich, J., Fischer, A., Gilsbach, J.M., Makowska, A., Spangenberg, P., Inhibition of nitric oxide synthases prevents and reverses alpha, beta-meATP-induced neck muscle nociception in mice (2010) Cephalalgia, 30, pp. 1225-1232Ferrari, D., Chiozzi, P., Falzoni, S., Dal Susino, M., Melchiorri, L., Baricordi, O.R., Di Virgilio, F., Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages (1997) J Immunol, 159, pp. 1451-1458Ferreira, S.H., Lorenzetti, B.B., Cunha, F.Q., Poole, S., Bradykinin release of TNF-alpha plays a key role in the development of inflammatory hyperalgesia (1993) Agents Actions, 38 Spec No, pp. C7-C9Ferreira, S.H., Lorenzetti, B.B., Poole, S., Bradykinin initiates cytokine-mediated inflammatory hyperalgesia (1993) Br J Pharmacol, 110, pp. 1227-1231Fujii, Y., Ozaki, N., Taguchi, T., Mizumura, K., Furukawa, K., Sugiura, Y., TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness (2008) Pain, 140, pp. 292-304Gautam, M., Benson, C.J., Acid-sensing ion channels (ASICs) in mouse skeletal muscle afferents are heteromers composed of ASIC1a, ASIC2, and ASIC3 subunits (2013) FASEB J, 27, pp. 793-802Gerdle, B., Hilgenfeldt, U., Larsson, B., Kristiansen, J., Sogaard, K., Rosendal, L., Bradykinin and kallidin levels in the trapezius muscle in patients with work-related trapezius myalgia, in patients with whiplash associated pain, and in healthy controls - a microdialysis study of women (2008) Pain, 139, pp. 578-587Hamilton, S.G., Wade, A., McMahon, S.B., The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat (1999) Br J Pharmacol, 126, pp. 326-332Hanna, R.L., Kaufman, M.P., Activation of thin-fiber muscle afferents by a P2X agonist in cats (1985) J Appl Physiol, 96, pp. 1166-1169Hedenberg-Magnusson, B., Ernberg, M., Alstergren, P., Kopp, S., Pain mediation by prostaglandin E2 and leukotriene B4 in the human masseter muscle (2001) Acta Odontol Scand, 59, pp. 348-355Honore, P., Kage, K., Mikusa, J., Watt, A.T., Johnston, J.F., Wyatt, J.R., Faltynek, C.R., Lynch, K., Analgesic profile of intrathecal P2X(3) antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats (2002) Pain, 99, pp. 11-19Honore, P., Mikusa, J., Bianchi, B., McDonald, H., Cartmell, J., Faltynek, C., Jarvis, M.F., TNP-ATP, a potent P2X3 receptor antagonist, blocks acetic acid-induced abdominal constriction in mice: comparison with reference analgesics (2002) Pain, 96, pp. 99-105Jarvis, M.F., Burgard, E.C., McGaraughty, S., Honore, P., Lynch, K., Brennan, T.J., Subieta, A., Faltynek, C., A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat (2002) Proc Natl Acad Sci USA, 99, pp. 17179-17184Kaufman, M.P., Hayes, S.G., The exercise pressor reflex (2002) Clin Auton Res, 12, pp. 429-439Kennedy, C., Assis, T.S., Currie, A.J., Rowan, E.G., Crossing the pain barrier: P2 receptors as targets for novel analgesics (2003) J Physiol, 553, pp. 683-694Krismer, M., van Tulder, M., Strategies for prevention and management of musculoskeletal conditions. Low back pain (non-specific) (2007) Best Pract Res, 21, pp. 77-91Lewis, C., Neidhart, S., Holy, C., North, R.A., Buell, G., Surprenant, A., Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons (1995) Nature, 377, pp. 432-435Li, J., King, N.C., Sinoway, L.I., ATP concentrations and muscle tension increase linearly with muscle contraction (2003) J Appl Physiol (1985), 95, pp. 577-583Loram, L.C., Fuller, A., Fick, L.G., Cartmell, T., Poole, S., Mitchell, D., Cytokine profiles during carrageenan-induced inflammatory hyperalgesia in rat muscle and hind paw (2007) J Pain, 8, pp. 127-136Makowska, A., Panfil, C., Ellrich, J., ATP induces sustained facilitation of craniofacial nociception through P2X receptors on neck muscle nociceptors in mice (2006) Cephalalgia, 26, pp. 697-706Marceau, F., Hess, J.F., Bachvarov, D.R., The B1 receptors for kinins (1998) Pharmacol Rev, 50, pp. 357-386McGaraughty, S., Jarvis, M.F., Antinociceptive properties of a non-nucleotide P2X3/P2X2/3 receptor antagonist (2005) Drug News Perspect, 18, pp. 501-507McGaraughty, S., Wismer, C.T., Zhu, C.Z., Mikusa, J., Honore, P., Chu, K.L., Lee, C.H., Jarvis, M.F., Effects of A-317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration (2003) Br J Pharmacol, 140, pp. 1381-1388Mense, S., Algesic agents exciting muscle nociceptors (2009) Exp Brain Res, 196, pp. 89-100Noma, N., Shinoda, M., Honda, K., Kiyomoto, M., Dezawa, K., Nakaya, Y., Komiyama, O., Iwata, K., Interaction of IL-1beta and P2X(3) receptor in pathologic masseter muscle pain (2013) J Dent Res, 92, pp. 456-460Oliveira, M.C., Parada, C.A., Veiga, M.C., Rodrigues, L.R., Barros, S.P., Tambeli, C.H., Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain (2005) Eur J Pain (London, England), 9, pp. 87-93Oliveira, M.C., Pelegrini-da-Silva, A., Tambeli, C.H., Parada, C.A., Peripheral mechanisms underlying the essential role of P2X3,2/3 receptors in the development of inflammatory hyperalgesia (2009) Pain, 141, pp. 127-134Prado, F.C., Araldi, D., Vieira, A.S., Oliveira-Fusaro, M.C., Tambeli, C.H., Parada, C.A., Neuronal P2X3 receptor activation is essential to the hyperalgesia induced by prostaglandins and sympathomimetic amines released during inflammation (2013) Neuropharmacology, 67, pp. 252-258Randall, L.O., Selitto, J.J., A method for measurement of analgesic activity on inflamed tissue (1957) Arch Int Pharmacodynam Ther, 111, pp. 409-419Reinohl, J., Hoheisel, U., Unger, T., Mense, S., Adenosine triphosphate as a stimulant for nociceptive and non-nociceptive muscle group IV receptors in the rat (2003) Neurosci Lett, 338, pp. 25-28Reitz, M., Makowska, A., Ellrich, J., Excitatory and inhibitory purinergic control of neck muscle nociception in anaesthetized mice (2009) Cephalalgia, 29, pp. 58-67Ristic, D., Spangenberg, P., Ellrich, J., Inhibition of nNOS prevents and inhibition of iNOS reverses alpha, beta-meATP-induced facilitation of neck muscle nociception in mice (2010) Eur J Pharmacol, 647, pp. 55-61Rosland, J.H., The formalin test in mice: the influence of ambient temperature (1991) Pain, 45, pp. 211-216Safieh-Garabedian, B., Poole, S., Allchorne, A., Winter, J., Woolf, C.J., Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia (1995) Br J Pharmacol, 115, pp. 1265-1275Schafers, M., Sorkin, L.S., Sommer, C., Intramuscular injection of tumor necrosis factor-alpha induces muscle hyperalgesia in rats (2003) Pain, 104, pp. 579-588Shinoda, M., Kawashima, K., Ozaki, N., Asai, H., Nagamine, K., Sugiura, Y., P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain (2007) J Pain, 8, pp. 588-597Shinoda, M., Ozaki, N., Sugiura, Y., Involvement of ATP and its receptors on nociception in rat model of masseter muscle pain (2008) Pain, 134, pp. 148-157Sinoway, L.I., Li, J., A perspective on the muscle reflex: implications for congestive heart failure (1985) J Appl Physiol, 99, pp. 5-22Steranka, L.R., Manning, D.C., DeHaas, C.J., Ferkany, J.W., Borosky, S.A., Connor, J.R., Vavrek, R.J., Snyder, S.H., Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions (1988) Proc Natl Acad Sci USA, 85, pp. 3245-3249Stewart, L.C., Deslauriers, R., Kupriyanov, V.V., Relationships between cytosolic [ATP], [ATP]/[ADP] and ionic fluxes in the perfused rat heart: a 31P, 23Na and 87Rb NMR study (1994) J Mol Cell Cardiol, 26, pp. 1377-1392Takahashi, K., Taguchi, T., Itoh, K., Okada, K., Kawakita, K., Mizumura, K., Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes (2005) Somatosens Motor Res, 22, pp. 299-305Tegeder, L., Zimmermann, J., Meller, S.T., Geisslinger, G., Release of algesic substances in human experimental muscle pain (2002) Inflamm Res, 51, pp. 393-402Teixeira, J.M., de Oliveira-Fusaro, M.C., Parada, C.A., Tambeli, C.H., Peripheral P2X7 receptor-induced mechanical hyperalgesia is mediated by bradykinin (2014) Neuroscience, 277 C, pp. 163-173Torres-Chavez, K.E., Sanfins, J.M., Clemente-Napimoga, J.T., Pelegrini-Da-Silva, A., Parada, C.A., Fischer, L., Tambeli, C.H., Effect of gonadal steroid hormones on formalin-induced temporomandibular joint inflammation (2012) Eur J Pain (London, England), 16, pp. 204-216Waldron, J.B., Sawynok, J., Peripheral P2X receptors and nociception: interactions with biogenic amine systems (2004) Pain, 110, pp. 79-89Wang, C., Li, G.W., Huang, L.Y., Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons (2007) Mol Pain, 3, p. 22Wu, G., Whiteside, G.T., Lee, G., Nolan, S., Niosi, M., Pearson, M.S., Ilyin, V.I., A-317491, a selective P2X3/P2X2/3 receptor antagonist, reverses inflammatory mechanical hyperalgesia through action at peripheral receptors in rats (2004) Eur J Pharmacol, 504, pp. 45-53Zeisig, E., Ljung, B.O., Alfredson, H., Danielson, P., Immunohistochemical evidence of local production of catecholamines in cells of the muscle origins at the lateral and medial humeral epicondyles: of importance for the development of tennis and golfer's elbow? (2009) Br J Sports Med, 43, pp. 269-275Zimmermann, M., Ethical guidelines for investigations of experimental pain in conscious animals (1983) Pain, 16, pp. 109-110