RESUMO
OBJECTIVE: To investigate the effect of electroacupuncture (EA) at "Jiaji" (EX-B2) at different time points on the expression of OX-42 (a monoclonal antibody with specific expression of complement receptor-3 in spinal microglial cells) and purinergic receptor P2X4 (P2X4) in rats with chronic constriction injury (CCI) of the sciatic nerve, as well as the possible after-effect mechanism of EA analgesia in neuropathic pain. METHODS: Sprague-Dawley rats were randomly divided into blank group, model group, immediately after EA group, 0.5-hour after EA group, 1-hour after EA group, 2-hour after EA group, 4-hour after EA group, 12-hour after EA group, and 24-hour after EA group, with 6 rats in each group. The rats in the model group and the EA groups were used to establish a model of CCI-induced neuropathic pain, and those in the immediately after EA group and the 0.5, 1, 2, 4, 12 and 24 hours after EA groups were treated with EA at bilateral L3 and L5 "Jiaji" points for 20 min after 7 d of modeling. Samples were collected immediately and at 0.5, 1, 2, 4, 12, and 24 hours after EA, and for the rats in the blank group and the model group, samples were collected after fixation the rats for 20 min. Heat pain threshold was observed before and after intervention, and immunohistochemistry was used to measure the protein expression of OX-42 and P2X4 in the spinal cord lumbar enlargement. RESULTS: After 7 days of modeling (before intervention), compared with the blank group, the heat pain threshold had a significant reduction in the model group and the EA groups (P<0.01). Compared with the model group after intervention, the immediately after EA group and the 0.5, 1 and 2 hours after EA groups had a significant increase in heat pain threshold (P<0.05). Compared with the model group, immediately after EA, the 0.5, 1 and 2 hours after EA groups had a significant reduction in the protein expression of OX-42 (P<0.01), and immediately after EA, the 0.5 and 1 hour after EA groups had a significant reduction in the protein expression of P2X4 (P<0.01). CONCLUSION: EA at "Jiaji" points can significantly increase heat pain threshold and down-regulate the protein expression of OX-42 and P2X4 in the spinal cord of CCI rats. The analgesic effect can last for 2 h.
RESUMO
O receptor P2X4 (canal iônico controlado por adenosina-5'-trifosfato-ATP) está amplamente distribuído no sistema nervoso central e, após sua ativação, pode regular os níveis de cálcio intracelulares via permeação direta e por ativação de canais de cálcio voltagem-dependentes. Tem sido proposto que a atividade do receptor pode ser importante na plasticidade sináptica. Tendo em vista a importância do receptor P2X4, sobretudo na fisiologia do sistema nervoso central, é útil caracterizá-lo farmacologicamente e entender os mecanismos moleculares que regulam sua atividade. Examinamos o papel que resíduos específicos N- e C-terminais desempenham na atividade do receptor P2X4 humano, combinando técnicas de biologia molecular, bioquímica e patch-clamp em células de rim de embrião humano (células HEK-293T). Células HEK-293T expressando o receptor P2X4 wild-type apresentaram correntes iônicas, cujas amplitudes dependeram da concentração de ATP, fornecendo um valor de EC50 de 1,37 ± 0,21 µM. Os receptores mutantes E14A e D16A exibiram respostas ao ATP equiparáveis àquelas do receptor selvagem, ao passo que os mutantes Y15A e T17A não foram funcionais, apesar de serem expressos na membrana plasmática das células. A inibição de tirosina fosfatases por pervanadato diminuiu fortemente correntes induzidas por ATP. Subsequente análise de citometria de fluxo na presença de um anticorpo contra resíduos de fosfotirosina indicaram que, entre as células que expressam o receptor P2X4, a percentagem de células fosfo-tirosina-positivas é a mesma para os mutantes Y372A (86 ± 10%) e Y378A (79 ± 6.9%), mas substancialmente menor para os mutantes Y15A (35 ± 12%), Y367A (48 ± 6.4%) e Y372F (31 ± 1.7%), quando comparados com células que expressam o receptor wild-type (76 ± 5.6%). Resultados semelhantes foram obtidos quando quantificamos a expressão relativa de proteínas fosforiladas em resíduos de tirosina e expressamos através dos valores de intensidade de fluorescência média. Ensaios de western-blot revelaram que mesmo o mutante T17A é fosforilado em resíduos de treonina, sugerindo que o receptor P2X4 contém outros sítios de fosforilação. Entretanto, nenhum sinal de fosfotirosina foi detectado no receptor wild-type e nos mutantes, em que resíduos de tirosina foram substituídos por alanina ou fenilalanina. Não parece ser o resíduo Y15 o alvo de tal fosforilação, cabendo a ele um papel estrutural mais importante. Nossos dados também sugerem que a fosforilação em resíduos de tirosina de proteínas intermediárias regula a atividade do receptor P2X4
The human P2X4 receptor (ATP-gated ion channel) is widely distributed in the CNS and, after activation, participates in regulation of levels of intracellular calcium through direct permeation and activation of voltage-dependent calcium channels with well-defined functions including synaptic plasticity. Given the importance of the P2X4 receptor, especially in CNS physiology, we investigated the role that specific N- and C-termini residues play in human P2X4 receptor activity, by combining techniques of molecular biology, biochemistry and patch-clamping in human embryonic kidney cells (HEK-293T cells). HEK-293T cells expressing the wild-type P2X4 receptor showed ionic currents whose amplitudes depended on the ATP concentration, providing an EC50 value of 1.37 ± 0.21 mM. E14A and D16A receptor mutants exhibited responses to ATP comparable to those ones of wild-type receptor, whereas Y15A and T17A mutants were not functional, despite being expressed in the plasma membrane of cells. The inhibition of tyrosine phosphatases by pervanadate decreased strongly ATP-induced currents. Subsequent flow cytometry analysis in the presence of an antibody against phosphotyrosine residues indicated that, among the cells that express the P2X4 receptor, the percentage of phosphotyrosine-positive cells was the same for Y372A (86 ± 10%) and Y378A (79 ± 6.9%) mutants, however, substantially lower for Y15A (35 ± 12%), Y367A (48 ± 6.4%) and Y372F (31 ± 1.7%) mutants when compared with cells expressing the wild-type receptor (76 ± 5.6%). Similar results were obtained by quantifying the relative expression of phosphotyrosine proteins. Western blot assays revealed that even the T17A mutant was phosphorylated at threonine residues, suggesting that the human P2X4 receptor also contains further phosphorylation sites. However, no phosphotyrosine-antibody signal was detected in the wild-type receptor and mutants in which tyrosine residues were replaced by alanine or phenylalanine. The residue Y15 is supposedly not the target of such phosphorylation, despite its important structural role. However, the present work indicates that tyrosine phosphorylation of intermediate signaling proteins regulates P2X4 receptor activity