RESUMO
Previous studies have demonstrated that acute colonic inflammation leads to an increase in dorsal root ganglia (DRG) neuronal excitability. However, the signaling elements implicated in this hyperexcitability have yet to be fully unraveled. Extracellular adenosine 5'-triphosphate (ATP) is a well-recognized sensory signaling molecule that enhances the nociceptive response after inflammation through activation of P2X3 receptors, which are expressed mainly by peripheral sensory neurons. The aim of this study is to continue investigating how P2X3 affects neuronal hypersensitivity in an acute colitis animal model. To achieve this, DNBS (Dinitrobenzene sulfonic acid; 200 mg/kg) was intrarectally administered to C57BL/6 mice, and inflammation severity was assessed according to the following parameters: weight loss, macroscopic and microscopic scores. Perforated patch clamp technique was used to evaluate neuronal excitability via measuring changes in rheobase and action potential firing in T8-L1 DRG neurons. A-317491, a well-established potent and selective P2X3 receptor antagonist, served to dissect their contribution to recorded responses. Protein expression of P2X3 receptors in DRG was evaluated by western blotting and immunofluorescence. Four days post-DNBS administration, colons were processed for histological analyses of ulceration, crypt morphology, goblet cell density, and immune cell infiltration. DRG neurons from DNBS-treated mice were significantly more excitable compared with controls; these changes correlated with increased P2X3 receptor expression. Furthermore, TNF-α mRNA expression was also significantly higher in inflamed colons compared to controls. Incubation of control DRG neurons with TNF-α resulted in similar cell hyperexcitability as measured in DNBS-derived neurons. The selective P2X3 receptor antagonist, A-317491, blocked the TNF-α-induced effect. These results support the hypothesis that TNF-α enhances colon-innervating DRG neuron excitability via modulation of P2X3 receptor activity.
Assuntos
Colite , Gânglios Espinais , Trifosfato de Adenosina , Animais , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X3 , Células Receptoras Sensoriais , Fator de Necrose Tumoral alfaRESUMO
Cannabinoid system is a potential target for pain control. Cannabinoid receptor 1 (CB1) activation play a role in the analgesic effect of cannabinoids once it is expressed in primary afferent neurons. This study investigates whether the anti-hyperalgesic effect of CB1 receptor activation involves P2X3 receptor in primary afferent neurons. Mechanical hyperalgesia was evaluated by electronic von Frey test. Cannabinoid effect was evaluated using anandamide or ACEA, a non-selective or a selective CB1 receptor agonists, respectively; AM251, a CB1 receptor antagonist, and antisense ODN for CB1 receptor. Calcium imaging assay was performed to evaluated α,ß-meATP-responsive cultured DRG neurons pretreated with ACEA. Anandamide or ACEA administered in peripheral tissue reduced the carrageenan-induced mechanical hyperalgesia. The reduction in the carrageenan-induced hyperalgesia induced by ACEA was completely reversed by administration of AM251 as well as by the intrathecal treatment with antisense ODN for CB1 receptor. Also, ACEA reduced the mechanical hyperalgesia induced by bradykinin and by α,ß-meATP, a P2X3 receptor non-selective agonist, but not by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and chemokine-induced chemoattractant-1 (CINC-1). Finally, CB1 receptors are co-localized with P2X3 receptors in DRG small-diameter neurons and the treatment with ACEA reduced the number of α,ß-meATP-responsive cultured DRG neurons. Our data suggest that the analgesic effect of CB1 receptor activation is mediated by a negative modulation of the P2X3 receptor in the primary afferent neurons.
Assuntos
Hiperalgesia/metabolismo , Hiperalgesia/patologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Animais , Bradicinina/farmacologia , Carragenina/farmacologia , Tamanho Celular , Citocinas/metabolismo , Gânglios Espinais/patologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Neurônios Aferentes/patologia , Oligodesoxirribonucleotídeos Antissenso/genética , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/genéticaRESUMO
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,ß-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 ß1- or ß2-adrenoceptor antagonist atenolol and ICI 118,551, respectively. Also, the nonspecific selectin inhibitor fucoidan. α,ß-meATP induced increases in the local concentration of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß), which were reduced by bradykinin antagonist. Finally, α,ß-meATP also induced neutrophil migration. Together, these findings suggest that α,ß-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.