P2X7R参与炎性刺激大鼠硬脑膜三叉神经节中NLRP3炎性小体的激活

目的采用炎性刺激大鼠硬脑膜疼痛模型,探讨P2X7R是否参与偏头痛发作过程中三叉神经节内NLRP3炎性小体的激活。方法选择雄性SD大鼠42只,免疫印迹实验取27只随机分为9组,每组3只,分别为空白对照组、致炎剂3h组、致炎剂6h组、致炎剂1d组、致炎剂2d组、致炎剂2d对照组、致炎剂3d组、生理盐水对照组和亮蓝G治疗组。免疫荧光双重染色实验取15只随机分为3组,每组5只,分别为空白组、生理盐水组和致炎剂组。观察各组大鼠P2X7R和NLRP3在三叉神经节中的表达情况。结果免疫荧光双重染色结果显示,NLRP3蛋白主要表达于三叉神经元细胞,而P2X7R主要表达于卫星胶质细胞。致炎剂组NLRP3及P2X7R荧光强度明显高于空白组和生理盐水组。免疫印迹结果显示,各致炎剂组NLRP3、P2X7R蛋白表达明显高于空白对照组(P<0.05)。亮蓝G治疗组P2X7R和NLRP3蛋白表达明显低于致炎剂2d对照组和生理盐水对照组(P<0.05)。结论在炎性刺激大鼠硬脑膜模型中可以观察到三叉神经节内P2X7R和NLRP3炎性小体的激活。     

Ca2+/calmodulin-dependent protein kinase II potentiates ATP responses by promoting trafficking of P2X receptors

To elucidate the functional link between Ca(2+)/calmodulin protein kinase II (CaMKII) and P2X receptor activation, we studied the effects of electrical stimulation, such as occurs in injurious conditions, on P2X receptor-mediated ATP responses in primary sensory dorsal root ganglion neurons. We found that endogenously active CaMKII up-regulates basal P2X3 receptor activity in dorsal root ganglion neurons. Electrical stimulation causes prolonged increases in ATP currents that lasts up to approximately 45 min. In addition, the total and phosphorylated CaMKII are also up-regulated. The enhancement of ATP currents depends on Ca(2+) and calmodulin and is completely blocked by the CaMKII inhibitor, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine). Western analyses indicate that electrical stimulation enhances the expression of P2X3 receptors in the membrane and that the enhancement is blocked by the inhibitor. These results suggest that CaMKII up-regulated by electrical stimulation enhances ATP responses by promoting trafficking of P2X receptors to the membrane and may play a key role in the sensitization of P2X receptors under injurious conditions.     

Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells that initiate the immune response by activating T lymphocytes. DCs express plasma membrane receptors for extracellular nucleotides named P2 receptors (P2Rs). Stimulation of P2Rs in these cells is known to cause chemotaxis, cytokine release, and cell death and to modulate LPS-dependent differentiation. Here we show that stimulation of the P2X(7) receptor subtype (P2X(7)R) causes fast microvesicle shedding from DC plasma membrane. Vesicle release occurs from both immature and mature DCs; however, only vesicles from mature DCs, due to their previous exposure to LPS, contain IL-1beta. Microvesicles, whether from immature or mature DCs, also contain caspase-1 and -3 and cathepsin D. They also express the P2X(7)R in addition to other P2Rs and known markers of immune cells such as major histocompatibility complex II (MHC II) and CD39. Activation of the P2X(7)R by extracellular ATP causes IL-1beta release from the vesicle lumen. Previous studies demonstrated that high extracellular K(+) inhibits IL-1beta processing and release; here we show that high ionic strength reduces microvesicle shedding when compared with a low ionic strength medium but strongly increases microvesicle IL-1beta loading.     

P2X7 receptors control 2-arachidonoylglycerol production by microglial cells

Endogenous cannabinoid ligands (endocannabinoids) produced by neurons, astrocytes, and microglial cells activate cannabinoid receptors, the molecular target for marijuana's bioactive ingredient Delta(9)-tetrahydrocannabinol. The molecular mechanism underlying the production of the most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), is unclear. A prevalent hypothesis proposes that activation of metabotropic receptors coupled to the phosphatidylinositol-specific phospholipase C and diacylglycerol (DG) lipase pathway will systematically lead to increases in 2-AG production. Here, we show that ATP increases 2-AG production by cultured microglial cells in a phosphatidylinositol-specific phospholipase C and DG lipase-dependent manner. However, efficacious activation of metabotropic P2Y purinergic receptors coupled to phosphatidylinositol-specific phospholipase C does not increase 2-AG production. This suggests that ionotropic, and not metabotropic, purinergic receptors control 2-AG production at an unexpected enzymatic step of its metabolic pathway. We show that activation of P2X(7) ionotropic receptors, which are highly permeable to calcium, is necessary and sufficient to increase 2-AG production in microglial cells. We also show that the sustained rise in intracellular calcium induced by activation of P2X(7) receptors directly increases DG lipase activity while inhibiting the activity of monoacylglycerol lipase, the enzyme that degrades 2-AG. This inverse sensitivity of DG lipase and monoacylglycerol lipase to calcium constitutes an original and efficient modality for sustained accumulation of 2-AG. Because prolonged increases in 2-AG amounts in brain parenchyma are thought to orchestrate neuroinflammation, the enzymatic steps involved in 2-AG synthesis and degradation by microglial cells constitute appealing targets for therapy aimed at controlling exacerbated neuroinflammation.     

P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation.

Somatic sensation requires the conversion of physical stimuli into the depolarization of distal nerve endings. A single cRNA derived from sensory neurons renders Xenopus laevis oocytes mechanosensitive and is found to encode a P2Y1 purinergic receptor. P2Y1 mRNA is concentrated in large-fiber dorsal root ganglion neurons. In contrast, P2X3 mRNA is localized to small-fiber sensory neurons and produces less mechanosensitivity in oocytes. The frequency of touch-induced action potentials from frog sensory nerve fibers is increased by the presence of P2 receptor agonists at the peripheral nerve ending and is decreased by the presence of P2 antagonists. P2X-selective agents do not have these effects. The release of ATP into the extracellular space and the activation of peripheral P2Y1 receptors appear to participate in the generation of sensory action potentials by light touch.     

Extracellular adenosine 5'-triphosphate modulates interleukin-6 production by human thyrocytes through functional purinergic P2 receptors

We investigated the presence of P2 receptors (P2Rs) in human thyrocytes and their possible involvement in the modulation of cytokine release. P2Rs expression was assessed by RT-PCR and, when possible, by immunoblotting. Human primary thyrocytes express the mRNA for the following P2X and P2Y subtypes: P2X(3), P2X(5), P2X(6), P2X(7), and P2Y(1), P2Y(2), P2Y(4), and P2Y(11). Stimulation with extracellular nucleotides of fura-2-loaded thyrocytes triggered an intracellular Ca(2+) signal, suggesting expression of functional receptors. Thyrocytes spontaneously released the proinflammatory cytokine IL-6. The ATP-hydrolyzing enzyme apyrase reduced basal IL-6 release, whereas extracellular ATP dose-dependently increased IL-6 secretion. Uridine 5'-triphosphate was also an effective stimulus, whereas benzoyl-ATP was ineffective, suggesting a P2Y- rather than P2X-modulated response. Finally, TSH reduced both the intracellular Ca(2+) ([Ca(2+)](i)) rise and IL-6 release triggered by P2Rs stimulation. In conclusion, we provide functional, pharmacological, and biochemical evidence that human primary thyrocytes express P2YR and P2XR subtypes, coupled to increases in ([Ca(2+)](i)) and secretion of IL-6. P2R-dependent modulation of IL-6 release from human thyrocytes suggests a novel mechanism whereby an inflammatory and/or immune-mediated damage can be initiated and amplified in the thyroid.     

Ethanol sensitivity in ATP-gated P2X receptors is subunit dependent

BACKGROUND: P2X receptors are ligand-gated cation channels that are gated by synaptically released extracellular adenosine 5'-triphosphate (ATP). P2X receptors are inhibited by ethanol; however, few investigations have focused on ethanol's effects in P2X receptors. Recently, recombinant homomeric P2X4 receptors were reported to be sensitive to ethanol's inhibitory action, whereas recombinant P2X3 receptors were insensitive to ethanol. The two recombinant studies were conducted in different expression systems by using different techniques; therefore, questions remain. The present study tests the hypothesis that ethanol sensitivity in P2X receptors is subunit dependent. METHODS: The effects of ethanol (25-200 mM +/- ATP) on rat recombinant homomeric P2X2 and P2X4 receptors expressed in Xenopus oocytes were tested by using two-electrode voltage clamp techniques. RESULTS: Ethanol inhibited EC10 ATP-gated currents significantly less in P2X2 versus P2X4 receptors. A second study found that ethanol right-shifted the ATP concentration-response curve in P2X2 receptors, which significantly increased the EC50 for ATP without altering the Hill slope or maximal current response to ATP. These latter characteristics of ethanol action in P2X2 receptors agree with previous work in P2X4 receptors. There was no effect of ethanol when tested in the absence of ATP. CONCLUSION: The findings are the first to show (1) ethanol inhibition of ATP-activated currents on P2X2 receptors, (2) differences in ethanol sensitivity between homomeric P2X receptors when tested under matched conditions, and (3) evidence that suggests similar mechanisms of ethanol action for P2X2 and P2X4 receptors. These findings provide the first direct support for the hypothesis that ethanol sensitivity in P2X receptors is subunit dependent.     

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

P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22-92 nM) and was highly selective (IC50 >10 microM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 micromolkg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10-15 micromolkg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 micromolkg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.     

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X(2) receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X(2) receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X(2)-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X(2)-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X(2)-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X(2) receptor distribution on the seconds time scale.     

结肠扩张刺激对P2X4受体在IBS大鼠神经中枢中表达的影响

目的观察P2X4受体在结肠扩张刺激引起的IBS大鼠骶髓后连合核（DCN）和骶髓后角中表达的变化，为探讨IBS的神经作用机制提供理论依据。方法采用免疫组织荧光化学的方法，通过对P2X4受体和小胶质细胞的标志物OX42的荧光双标记，观察其在小胶质细胞和神经元上的反应；通过免疫电镜同步观察P2X4受体与小胶质细胞和神经元的相互关系。结果对照组大鼠的DCN和骶髓后角中几乎没有发现P2X4的表达。而在IBS大鼠组，P2X4在DCN和骶髓后角神经元和小胶质细胞中表达明显增高。电镜下，在对照组的DCN和骶髓后角，少数P2X4-LI产物表达在胶质细胞突起上，此突起可与神经元胞体、轴突、树突或另外的胶质细胞突起接触。而在IBS大鼠的DCN和骶髓后角中，P2X4在抑制性突触上的表达明显增加。有的表达在突触复合体上，并且在神经元上也有发现。然而P2X4阳性产物更多的表达在胶质细胞的突起上。结论P2X4是小胶质细胞活化的启动因素，可能是IBS内脏敏感性增高的重要因素。     

ATP gradients inhibit the migratory capacity of specific human dendritic cell types: implications for P2Y11 receptor signaling

Dendritic cells (DCs) are specialized antigen-presenting cells residing in tissues, from which they take up antigen. Activated DCs migrate through chemokine gradients from sites of inflammation to lymph nodes to stimulate T cells. At sites of inflammation, nucleotides, such as adenosine triphosphate (ATP), are released by activated or dying cells and can function as signaling molecules through P2 receptors (P2Rs). We investigated P2R expression in different DC populations and the effect of nucleotides on chemokine-directed migration. Exposure of monocyte-derived DCs (MoDCs) and CD1a+ dermal DCs to gradients of ATP inhibited their migratory capacity in a dose-dependent manner. Studies using P2R agonists and antagonists implicated signaling through the P2Y11R. On maturation, MoDCs down-regulated P2Y11R expression and were less sensitive to ATP-mediated inhibition of migration. In contrast, ATP did not inhibit the migration of CD1c+ peripheral blood (PB) DCs or interleukin-3 receptor-positive (IL-3R+) plasmacytoid DCs. Although all 4 DC populations expressed mRNA for P2Y11R, calcium-flux studies showed that blood DC types were unresponsive to P2Y11R agonists. In conclusion, DCs use distinct subtypes of P2R. The formation of ATP gradients at sites of inflammation may transiently inhibit the migration of local DCs, thus prolonging the time of antigen encounter. P2R inhibition may represent a new strategy to improve the migration of antigen-loaded DCs from the vaccination site to lymph nodes.     

P2 receptors in human heart: upregulation of P2X6 in patients undergoing heart transplantation, interaction with TNFalpha and potential role in myocardial cell death

ATP acts as a neurotransmitter via seven P2X receptor-channels for Na(+) and Ca(2+), and eight G-protein-coupled P2Y receptors. Despite evidence suggesting roles in human heart, the map of myocardial P2 receptors is incomplete, and their involvement in chronic heart failure (CHF) has never received adequate attention. In left myocardia from five to nine control and 5-12 CHF subjects undergoing heart transplantation, we analyzed the full repertoire of P2 receptors and of 10 "orphan" P2Y-like receptors. All known P2Y receptors (i.e. P2Y(1,2,4,6,11,12,13,14)) and two P2Y-like receptors (GPR91 and GPR17) were detected in all subjects. All known P2X(1-7) receptors were also detected; of these, only P2X(6) was upregulated in CHF, as confirmed by quantitative real time-PCR. The potential significance of this change was studied in primary cardiac fibroblasts freshly isolated from young pigs. Exposure of cardiac fibroblasts to ATP or its hydrolysis-resistant-analog benzoylATP induced apoptosis. TNFalpha (a cytokine implicated in CHF progression) exacerbated cell death. Similar effects were induced by ATP and TNFalpha in a murine cardiomyocytic cell line. In cardiac fibroblasts, TNFalpha inhibited the downregulation of P2X(6) mRNA associated to prolonged agonist exposure, suggesting that, by preventing ATP-induced P2X(6) desensitization, TNFalpha may abolish a defense mechanism meant at avoiding Ca(2+) overload and, ultimately, Ca(2+)-dependent cell death. This may provide a basis for P2X(6) upregulation in CHF. In conclusion, we provide the first characterization of P2 receptors in the human heart and suggest that the interaction between TNFalpha and the upregulated P2X(6) receptor may represent a novel pathogenic mechanism in CHF.     

Emerging roles for P2X1 receptors in platelet activation

The platelet surface membrane possesses three P2 receptors activated by extracellular adenosine nucleotides; one member of the ionotropic receptor family (P2X(1)) and two members of the G-protein-coupled receptor family (P2Y(1) and P2Y(12)). P2Y(1) and P2Y(12) receptors have firmly established roles in platelet activation during thrombosis and haemostasis, whereas the importance of the P2X(1) receptor has been more controversial. However, recent studies have demonstrated that P2X(1) receptors can generate significant functional platelet responses alone and in synergy with other receptor pathways. In addition, studies in transgenic animals indicate an important role for P2X(1) receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis. This review discusses the background behind discovery of P2X(1) receptors in platelets and their precursor cell, the megakaryocyte, and how signalling via these ion channels may participate in platelet activation.     

Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia

It has been generally assumed that the cell body (soma) of a neuron, which contains the nucleus, is mainly responsible for synthesis of macromolecules and has a limited role in cell-to-cell communication. Using sniffer patch recordings, we show here that electrical stimulation of dorsal root ganglion (DRG) neurons elicits robust vesicular ATP release from their somata. The rate of release events increases with the frequency of nerve stimulation; external Ca(2+) entry is required for the release. FM1-43 photoconversion analysis further reveals that small clear vesicles participate in exocytosis. In addition, the released ATP activates P2X7 receptors in satellite cells that enwrap each DRG neuron and triggers the communication between neuronal somata and glial cells. Blocking L-type Ca(2+) channels completely eliminates the neuron-glia communication. We further show that activation of P2X7 receptors can lead to the release of tumor necrosis factor-alpha (TNFalpha) from satellite cells. TNFalpha in turn potentiates the P2X3 receptor-mediated responses and increases the excitability of DRG neurons. This study provides strong evidence that somata of DRG neurons actively release transmitters and play a crucial role in bidirectional communication between neurons and surrounding satellite glial cells. These results also suggest that, contrary to the conventional view, neuronal somata have a significant role in cell-cell signaling.     

Research progress of P2X7 receptor in inflammatory bowel disease

Inflammatory bowel disease is a chronic nonspecific inflammatory disease of the intestine. Its pathogenesis is not yet fully understood. It may be related to heredity, environmental triggers, infection, immune dysfunction and other factors. Purinergic receptor (P2X7R) ligand-gated ion channel is closely related to inflammation and widely expressed in intestinal cells. Previous studies have shown that ATP/P2X7R signal is involved in the pathogenesis of intestinal inflammation, but its specific mechanism needs further study. This article reviews the research progress of P2X7 receptor in inflammatory bowel disease.     

Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling

The difficulty of conducting electrophysiologic recordings from the platelet has restricted investigations into the role of ion channels in thrombosis and hemostasis. We now demonstrate that the well-established synergy between P2Y(1) and P2Y(12) receptors during adenosine diphosphate (ADP)-dependent activation of the platelet alpha(IIb)beta(3) integrin also exists in murine marrow megakaryocytes, further supporting the progenitor cell as a bona fide model of platelet P2 receptor signaling. In patch clamp recordings, ADP (30 microM) stimulated a transient inward current at -70 mV, which was carried by Na(+) and Ca(2+) and was amplified by phenylarsine oxide, a potentiator of certain transient receptor potential (TRP) ion channels by phosphatidylinositol 4,5-bisphosphate depletion. This initial current decayed to a sustained phase, upon which repetitive transient inward cation currents with pre-dominantly P2X(1)-like kinetics were super-imposed. Abolishing P2X(1)-receptor activity prevented most of the repetitive currents, consistent with their activation by secreted adenosine triphosphate (ATP). Recordings in P2Y(1)-receptor-deficient megakaryocytes demonstrated an essential requirement of this receptor for activation of all ADP-evoked inward currents. However, P2Y(12) receptors, through the activation of PI3-kinase, played a synergistic role in both P2Y(1) and P2X(1)-receptor-dependent currents. Thus, direct stimulation of P2Y(1) and P2Y(12) receptors, together with autocrine P2X(1) activation, is responsible for the activation of nonselective cation currents by the platelet agonist ADP.     

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.