Interference of P2X4 receptor expression in tumor-associated macrophages suppresses migration and invasion of glioma cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells.@*METHODS@#C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment.@*RESULTS@#The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05).@*CONCLUSION@#Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.

Dexmedetomidine attenuates P2X4 and NLRP3 expression in the spine of rats with diabetic neuropathic pain

Abstract Purpose: To evaluate the effects of Dexmedetomidine (Dex) on spinal pathology and inflammatory factor in a rat model of Diabetic neuropathic pain (DNP). Methods: The rats were divided into 3 groups (eight in each group): normal group (N group), diabetic neuropathic pain model group (DNP group), and DNP model with dexmedetomidine (Dex group). The rat model of diabetes was established with intraperitoneal streptozotocin (STZ) injections. Nerve cell ultrastructure was evaluated with transmission electron microscopy (TEM). The mechanical withdrawal threshold (MWT) and motor nerve conduction velocity (MNCV) tests documented that DNP rat model was characterized by a decreased pain threshold and nerve conduction velocity. Results: Dex restored the phenotype of neurocytes, reduced the extent of demyelination and improved MWT and MNCV of DNP-treated rats (P=0.01, P=0.038, respectively). The expression of three pain-and inflammation-associated factors (P2X4, NLRP3, and IL-IP) was significantly upregulated at the protein level in DNP rats, and this change was reversed by Dex administration (P=0.0022, P=0.0092, P=0.0028, respectively). Conclusion: The P2X4/NLRP3 signaling pathway is implicated in the development and presence of DNP in vivo, and Dex protects from this disorder.

Role of spinal P2X4 receptor in remifentanil-induced postoperative hyperalgesia / 中南大学学报(医学版)

To explore the role of P2X4 receptor in opioid-induced hyperalgesia (OIH).
 Methods: A total of 30 Sprague-Dawley (SD) male rats were randomly divided into 5 groups: a saline (N0) group, a remifentanil at 0.5 μg/(kg.min) (R1) group, a remifentanil at 1.0 μg/(kg.min) (R2) group, a remifentanil at 1.5 μg/(kg.min) (R3) group, and a remifentanil at 5.0 μg/(kg.min) (R4) group. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at follow time points to optimize the dosages: the day before treatment (T1), 30 min after tail intravenous catheterization (T2), and 30 min (T3), 1 h (T4), 2 h (T5), 24 h (T6) after withdrawal from remifentanil. Then, the rats were randomly divided into 2 groups: a saline group (N group), a remifentanil at 1.0 μg/(kg.min) group (R group). The PWMT and PWTL were measured at follow time points: T1, T2, and T4. The lumbar enlargement of spine was selected at 1 h after withdrawal from remifentanil, and the expression of P2X4 receptor mRNA and protein was examined in OIH. Additional male rats were selected and randomly divided into 2 groups: a plantar incision surgery followed by saline treatment group (I+N group), a plantar incision surgery followed by remifentanil treatment group (I+R group). The PWMT and PWTL were measured at follow time points: T1, T2, T3, T4, T5, T6, 48 h (T7) and 72 h (T8) after withdrawal from remifentanil. The lumbar enlargement of spine was selected at 1 h after withdrawal from remifentanil, the expression of P2X4 receptor mRNA and protein was examined by PCR and Western blotting, and the microglial activation in spine 1 h after withdrawal from remifentanil were assessed by immunofluorescence.
 Results: The pain thresholds including PWMT and PWTL in different groups were as follows: R4 group

Trigeminal purinergic P2X4 receptor involved in experimental occlusal interference-induced hyperalgesia in rat masseter muscle / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To explore the expression of purinergic p2X4 receptor (P2X4R) in trigeminal ganglion of rats after occlusal interference. Investigation of peripheral receptor mechanism of occlusal interference-induced masticatory muscle pain will aid the development of drug intervention against this condition.</p><p><b>METHODS</b>Experimental occlusal interference was established by application of 0.4 mm metal crown to the upper right first molar of male Sprague-Dawley rats. Real-time PCR assay was used to investigate P2X4R mRNA level in trigeminal ganglion in rats with occlusal interference for 3, 7, 10 and 14 days and in control rats without occlusal interference (n=5 in each). Retrograde labelling combining immunofluorescence was performed to evaluate the percentage of P2X4R-positive cells in masseter afferent neurons (n=5 in each group). Graded concentrations of P2XR antagonist TNP-ATP (0.1, 10, 125, 250, 500 μmol/L) or saline (n=5 in each group) was administrated in right masseter and the mechanical sensitivity of bilateral masseters was measured before occlusal interference application, before the injection, and 30 min as well as 60 min after the injection.</p><p><b>RESULTS</b>Compared with control rats (P2X4R mRNA: right side: 1.00±0.26, left side: 0.94± 0.21; percentage of P2X4R-positive masseter afferents: right side: [64.3±6.3]%, left side: [67.7±5.8]%), the level of P2X4R mRNA in bilateral trigeminal ganglia (right side: 5.98±3.56; left side: 5.06±2.88) of rats with occlusal interference for 7 days up-regulated (P<0.01) and the percentage of P2X4R-positive masseter afferent neurons(right side: [81.7±1.5]%; left side: [82.9±2.3]%) increased (P<0.05). Local administration of 10, 125, 250, 500 μmol/L TNP-ATP increased the mechanical withdrawal threshold in masseter 30 min after injection, compared with those before injection (P<0.05).</p><p><b>CONCLUSIONS</b>Increased expression of trigeminal P2X4R involves in the development of occlusal interference-induced masseter hyperalgesia.</p>

Participation of Central P2X7 Receptors in CFA-induced Inflammatory Pain in the Orofacial Area of Rats

We investigated the role of central P2X receptors in inflammatory pain transmission in the orofacial area in rats. Experiments were carried out using male Sprague-Dawley rats weighing 230-280g. Complete Freund's adjuvant (CFA, 40 microL) was applied subcutaneously to the vibrissa pad to produce inflammatory pain. The intracisternal administration of iso-PPADS tetrasodium salt, a non-selective P2X receptor antagonist, A317491 sodium salt hydrate, a P2X2/3 receptor antagonist, 5-BDBD, a P2X4 receptor antagonist, or A438079 hydrochloride, a P2X7 receptor antagonist, was performed 5 days after CFA injection. Subcutaneous injections of CFA produced increases in thermal hypersensitivity. Intracisternal injections of iso-PPADS (25 microg) or A438079 (25 or 50 microg) produced significant anti-hyperalgesic effects against thermal stimuli compared to the vehicle group. A317491 or 5-BDBD did not affect the head withdrawal latency times in rats showing an inflammatory response. Subcutaneous injections of CFA resulted in the up-regulation of OX-42, a microglia marker, and GFAP, an astrocyte marker, in the medullary dorsal horn. The intracisternal administration of A438079 reduced the numbers of activated microglia and astrocytes in the medullary dorsal horn. These results suggest that a blockade of the central P2X7 receptor produces antinociceptive effects, mediated by inhibition of glial cell function in the medullary dorsal horn. These data also indicate that central P2X7 receptors are potential targets for future therapeutic approaches to inflammatory pain in the orofacial area.

Identificação de resíduos de treonina e tirosina importantes na regulação da atividade do receptor P2X4 humano através de mutagênese sítio-dirigida / Identification of threonine and tyrosine residues important for human P2X4 receptor activity by site-directed mutagenesis

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

Mg(2+) inhibits ATP-activated current mediated by rat P2X4 receptors expressed in Xenopus oocytes / 生理学报

To investigate the modulation of Mg(2+) on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg(2+) on ATP-activated currents (I(ATP)) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg(2+) on I(ATP) were as follows: (1) In oocytes expressing P2X4 receptors, Mg(2+) with concentration ranging from 0.5-10 mmol/L inhibited the amplitude of I(ATP) in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC(50)) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg(2+) (1 mmol/L) shifted the dose-response curve for I(ATP) right-downward without changing the EC(50), but reduced the maximal current (E(max)) by (42.0 ± 2.1)%. (3) After being preincubated with Mg(2+) for 80 s, the inhibitory effect of the Mg(2+) on I(ATP) reached the maximum. (4) The inhibition of Mg(2+) on I(ATP) was independent of membrane potential from -120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of I(ATP) was increased almost one-fold, and Mg(2+) with concentration ranging from 0.5-10 mmol/L did not affect the I(ATP) significantly. The results suggest that Mg(2+) inhibits I(ATP) mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg(2+) might be realized by acting on the site Asp280 of the P2X4 receptors.

Characteristic and effect of cadmium on ATP-activated currents mediated by P2X4 receptors / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the characteristic and effect of cadmium on ATP-activated currents (I(ATP)) mediated by P2X4 purinoceptors.</p><p><b>METHODS</b>Transcribe cDNA coding for the rat P2X4 receptor to cRNA in vitro. Inject the cRNA to oocytes of an xenopus laevis using the microinjection technique. Reveal the effect of cadmium on I(ATP) mediated by P2X4 receptor using the two-electrode whole-cell voltage clamp technique.</p><p><b>RESULTS</b>(1) Within a certain concentration range, cadmium was found to reversibly magnify I(ATP) mediated by P2X4 receptors expressed in oocytes of an xenopus. When the concentration of cadmium reached 30 micromol/L, the increase of I(ATP) was the most significant. I(ATP) turned to decrease when the concentration of cadmium was more than 30 micromol/L; (2) The concentration-response curve was shifted to left by applying cadmium at 10 micromol/L; the EC50 was reduced from (17.1 +/- 1.5) micromol/L to (9.8 +/- 1.8) micromol/L (n = 6, P < 0.01) and the Hill coefficient was increased from 1.14 +/- 0.13 to 1.57 +/- 0.36; (3) The effect of cadmium on I(ATP) showed no dependence on membrane voltage; (4) The magnifying effect on I(ATP) reached maximum when preincubating cadmium for 120 seconds.</p><p><b>CONCLUSION</b>The increase I(ATP) by cadmium is reversible, concentration-dependent, time-dependent, and voltage-independent. One reason of this augment effect could be the allosteric modulation on P2X4 receptors.</p>