Neuroprotection of exercise: P2X4R and P2X7R regulate BDNF actions

The neurotrophin brain-derived neurotrophic factor (BDNF), which acts as a transducer, is responsible for improving cerebral stroke, neuropathic pain, and depression. Exercise can alter extracellular nucleotide levels and purinergic receptors in central nervous system (CNS) structures. This inevitably activates or inhibits the expression of BDNF via purinergic receptors, particularly the P2X receptor (P2XR), to alleviate pathological progression. In addition, the significant involvement of sensitive P2X4R in mediating increased BDNF and p38-MAPK for intracerebral hemorrhage and pain hypersensitivity has been reported. Moreover, archetypal P2X7R blockade induces mouse antidepressant-like behavior and analgesia by BDNF release. This review summarizes BDNF-mediated neural effects via purinergic receptors, speculates that P2X4R and P2X7R could be priming molecules in exercise-mediated changes in BDNF, and provides strategies for the protective mechanism of exercise in neurogenic disease.     

Silencing of essential genes by RNA interference in Haemonchus contortus

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.     

Regulation of the P2X7R by microRNA-216b in human breast cancer

Breast cancer is the most common cancer in women around the world. However, the molecular mechanisms underlying breast cancer pathogenesis are only partially understood. Here, in this study, we found that P2X7R was up-regulated and miR-216b was down-regulated in breast cancer cell lines and tissues. Using bioinformatic analysis and 3′UTR luciferase reporter assay, we determined P2X7R can be directly targeted by miR-216b, which can down-regulate endogenous P2X7R mRNA and protein levels. Ectopic expression of miR-216b mimics leads to inhibited cell growth and apoptosis, while blocking expression of the miR-216b results in increased cell proliferation. Furthermore, our findings demonstrate that knockdown of P2X7R promotes apoptosis in breast cancer cells through down-regulating Bcl-2 and increasing the cleavage caspase-3 protein level. Finally, we confirmed that down-regulation of miR-216b in breast cancer is inversely associated with P2X7R expression level. Together, these findings establish miR-216b as a novel regulator of P2X7R and a potential therapeutic target for breast cancer.     

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R^−/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [³H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R^−/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [³H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.     

P2X7R: pivotal player in sepsis-induced liver damage

Purinergic Signalling -     

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.     

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2’,3’-O-(2,4,6-trinitrophenyl)-adenosine 5’-triphosphate), a nonspecific P2X1–7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4–L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.     

Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia

In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.     

Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5′-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.     

Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages

Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1β production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1β. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy.     

用慢病毒转染RNAi技术沉默胆固醇7α羟化酶的基因表达，降低肝细胞中胆汁酸的分泌

为了降低生物人工肝(bioartificial liver system)中肝细胞胆汁酸的分泌,构建了胆固醇7α羟化酶慢病毒RNA干涉载体,并转染人肝脏细胞(L-02).根据绿色荧光蛋白的表达评估转染效率后进行流式分选,获得高表达慢病毒干涉载体的细胞,并以野生型L-02细胞和仅转染pSicoR空载体的L-02细胞作对照,观察肝细胞胆固醇7α羟化酶的表达以及培养上清中总胆汁酸含量.利用半定量PCR、实时荧光定量PCR及Western-blot等实验方法检测了转染细胞中基因的干涉效果,结果显示:与对照组相比,在mRNA水平,转染慢病毒siRNA载体的L-02细胞,其胆固醇7α羟化酶基因的表达量仅为野生型L-02细胞表达量的31.2%,为转染pSicoR空载体的L-02细胞的34.1%,干涉效率分别为68.8%和65.9%,均具有显著差异(P＜0.05);Western-blot结果显示胆固醇7α羟化酶在蛋白质水平表达也明显受到抑制,表明转染慢病毒siRNA下调了肝细胞中胆固醇7α羟化酶基因的表达,减少了胆汁酸的分泌.以上研究结果表明,利用RNAi技术可以获得低表达胆固醇7α羟化酶基因的肝细胞,并有效降低肝细胞中胆汁酸的分泌,为临床上生物人工肝的构建及应用奠定基础.     

Ezrin/Radixin/Moesin Are Required for the Purinergic P2X7 Receptor (P2X7R)-dependent Processing of the Amyloid Precursor Protein

The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.     

Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7

The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1β and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.     

A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.     

Activation of P2X7R and downstream effects in bleomycin treated lung epithelial cells

Changes in intracellular calcium concentration [Ca(2+)](i) are believed to influence the proliferation and differentiation of airway epithelial cells both in vivo and in vitro. In the present study, using mouse alveolar epithelial E10 cells, we demonstrated that the treatment of lung epithelial cells with BLM resulted in elevated intracellular Ca(2+) levels. BLM further increased P2rx7 mRNA expression and P2X7R protein levels, paralleled by increased PKC-β1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-β1 from the cytoplasm to the membrane. The expression of PKC-β1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-β1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-β1 in Cav-1 containing lipid raft fractions. The comparison of lung tissues from wild-type and P2rx7(-/-) mice revealed decreased protein and mRNA levels of PKC-β1 and CaM as well as decreased immunoreactivity for PKC-β1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-β1. These data suggest that the effect of P2X7R on expression of PKC-β1 detected in alveolar epithelial cells is also functioning in the animal model. Immunohistochemical evaluation of fibrotic lungs derived from a BLM-induced mouse model revealed a strong increase in PKC-β1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-β1. We predict that increased Ca(2+) concentration stimulates PKC-β1, whereas the prerequisite for activating PKC-β1 after P2X7R increase remained to be determined. Our findings suggest that PKC-β1 is important in the pathogenesis of pulmonary fibrosis.     

Increased P2X7R expression in atrial cardiomyocytes of caveolin-1 deficient mice

It has recently been shown in epithelial cells that the ATP-gated ion channel P2X7R is in part, associated with caveolae and colocalized with caveolin-1. In the present study of the mouse heart, we show for the first time, using immunohistochemistry and cryoimmunoelectron microscopy, that P2X7R is expressed in atrial cardiomyocytes and in cardiac microvascular endothelial cells, but not in the ventricle cardiomyocytes. Furthermore, biochemical data indicate the presence of two forms of P2X7R, the classical glycosylated 80 kDa isoform and a protein with the molecular weight of 56 kDa, in both cardiomyocytes and endothelial cells of the mouse heart. The functionality of both proteins in heart cells is still unclear. In cardiac tissue homogenates derived from caveolin-1 deficient mice (cav-1 ^−/−), an increase of the P2Xrx7 mRNA and P2X7R protein (80 kDa) was found, particularly in atrial samples. In addition, P2rx7 ^−/− mice showed enhanced protein levels of caveolin-1 in their atrial tissues. Although the details of cellular mechanisms that underlie the relationship between caveolin-1 and P2X7R in atrial cardiomyocytes and the electrophysiological consequences of the increased P2X7R expression in atrial cells of cav-1 ^−/− mice remain to be elucidated, the cardiomyopathy detectable in cav-1 ^−/− mice is possibly related to a disturbed crosstalk between P2X7R and caveolin-1 in different heart cell populations.     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.