P2X4 receptor exacerbates ischemic AKI and induces renal proximal tubular NLRP3 inflammasome signaling.

We tested the hypothesis that the P2X4 purinergic receptor (P2X4) exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation after ischemia and reperfusion (IR). Supporting this, P2X4-deficient (KO) mice were protected against ischemic AKI with significantly attenuated renal tubular necrosis, inflammation, and apoptosis when compared to P2X4 wild-type (WT) mice subjected to renal IR. Furthermore, WT mice treated with P2X4 allosteric agonist ivermectin had exacerbated renal IR injury whereas P2X4 WT mice treated with a selective P2X4 antagonist (5-BDBD) were protected against ischemic AKI. Mechanistically, induction of kidney NLRP3 inflammasome signaling after renal IR was significantly attenuated in P2X4 KO mice. A P2 agonist ATPγS increased NLRP3 inflammasome signaling (NLRP3 and caspase 1 induction and IL-1ß processing) in isolated renal proximal tubule cells from WT mice whereas these increases were absent in renal proximal tubules isolated from P2X4 KO mice. Moreover, 5-BDBD attenuated ATPγS induced NLRP3 inflammasome induction in renal proximal tubules from WT mice. Finally, P2X4 agonist ivermectin induced NLRP3 inflammasome and pro-inflammatory cytokines in cultured human proximal tubule cells. Taken together, our studies suggest that renal proximal tubular P2X4 activation exacerbates ischemic AKI and promotes NLRP3 inflammasome signaling.

Microglia P2X4R-BDNF signalling contributes to central sensitization in a recurrent nitroglycerin-induced chronic migraine model.

BACKGROUND: According to our previous study, microglia P2X4 receptors (P2X4Rs) play a pivotal role in the central sensitization of chronic migraine (CM). However, the molecular mechanism that underlies the crosstalk between microglia P2X4Rs and neurons of the trigeminal nucleus caudalis (TNC) is not fully understood. Therefore, the aim of this study is to examine the exact P2X4Rs signalling pathway in the development of central sensitization in a CM animal model. METHODS: We used an animal model with recurrent intermittent administration of nitroglycerin (NTG), which closely mimics CM. NTG-induced basal mechanical and thermal hypersensitivity were evaluated using a von Frey filament test and an increasing-temperature hot plate apparatus (IITC). We detected P2X4Rs, brain-derived neurotrophic factor (BDNF) and phosphorylated p38 mitogen-activated protein kinase (p-p38-MAPK) expression profiles in the TNC. We investigated the effects of a P2X4R inhibitor (5-BDBD) and an agonist (IVM) on NTG-induced hyperalgesia and neurochemical changes as well as on the expression of p-p38-MAPK and BDNF. We also detected the effects of a tropomyosin-related kinase B (TrkB) inhibitor (ANA-12) on the CM animal model in vivo. Then, we evaluated the effect of 5-BDBD and SB203580 (a p38-MAPK inhibitors) on the release and synthesis of BDNF in BV2 microglia cells treated with 50 µM adenosine triphosphate (ATP). RESULTS: Chronic intermittent administration of NTG resulted in chronic mechanical and thermal hyperalgesia, accompanied by the upregulation of P2X4Rs and BDNF expression. 5-BDBD or ANA-12 prevented hyperalgesia induced by NTG, which was associated with a significant inhibition of the NTG-induced increase in phosphorylated extracellular regulated protein kinases (p-ERK) and calcitonin gene related peptide (CGRP) release in the TNC. Repeated administration of IVM produced sustained hyperalgesia and significantly increased the levels of p-ERK and CGRP release in the TNC. Activating P2X4Rs with ATP triggered BDNF release and increased BDNF synthesis in BV2 microglia, and these results were then reduced by 5-BDBD or SB203580. CONCLUSIONS: Our results indicated that the P2X4R contributes to the central sensitization of CM by releasing BDNF and promoting TNC neuronal hyper-excitability. Blocking microglia P2X4R-BDNF signalling may have an effect on the prevention of migraine chronification.

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

Ivermectin and its target molecules: shared and unique modulation mechanisms of ion channels and receptors by ivermectin.

Ivermectin (IVM) is an antiparasitic drug that is used worldwide and rescues hundreds of millions of people from onchocerciasis and lymphatic filariasis. It was discovered by Satoshi Omura and William C. Campbell, to whom the 2015 Nobel Prize in Physiology or Medicine was awarded. It kills parasites by activating glutamate-gated Cl- channels, and it also targets several ligand-gated ion channels and receptors, including Cys-loop receptors, P2X4 receptors and fernesoid X receptors. Recently, we found that IVM also activates a novel target, the G-protein-gated inwardly rectifying K+ channel, and also identified the structural determinant for the activation. In this review, we aim to provide an update and summary of recent progress in the identification of IVM targets, as well as their modulation mechanisms, through molecular structures, chimeras and site-directed mutagenesis, and molecular docking and modelling studies.

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.

The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

Chronic lead exposure enhances the sympathoexcitatory response associated with P2X4 receptor in rat stellate ganglia.

Chronic lead exposure causes peripheral sympathetic nerve stimulation, including increased blood pressure and heart rate. Purinergic receptors are involved in the sympathoexcitatory response induced by myocardial ischemia injury. However, whether P2X4 receptor participates in sympathoexcitatory response induced by chronic lead exposure and the possible mechanisms are still unknown. The aim of this study was to explore the change of the sympathoexcitatory response induced by chronic lead exposure via the P2X4 receptor in the stellate ganglion (SG). Rats were given lead acetate through drinking water freely at doses of 0 g/L (control group), 0.5 g/L (low lead group), and 2 g/L (high lead group) for 1 year. Our results demonstrated that lead exposure caused autonomic nervous dysfunction, including blood pressure and heart rate increased and heart rate variability (HRV) decreased. Western blotting results indicated that after lead exposure, the protein expression levels in the SG of P2X4 receptor, IL-1ß and Cx43 were up-regulated, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was activated. Real-time PCR results showed that the mRNA expression of P2X4 receptor in the SG was higher in lead exposure group than that in the control group. Double-labeled immunofluorescence results showed that P2X4 receptor was co-expressed with glutamine synthetase (GS), the marker of satellite glial cells (SGCs). These changes were positively correlated with the dose of lead exposure. The up-regulated expression of P2X4 receptor in SGCs of the SG maybe enhance the sympathoexcitatory response induced by chronic lead exposure.

Deletion of the P2X4 receptor is neuroprotective acutely, but induces a depressive phenotype during recovery from ischemic stroke.

INTRODUCTION: Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. METHODS: We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30days after occlusion) effects of receptor deletion. RESULTS: Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. CONCLUSIONS: P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization.

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes.

Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V-ATPase generated voltage gradient as the driving force.

KEY POINTS: SLC17A9 proteins function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation. P2X4 receptors act as lysosomal ion channels activated by luminal ATP. SLC17A9-mediated ATP transport across the lysosomal membrane is suppressed by Bafilomycin A1, the V-ATPase inhibitor. SLC17A9 mainly uses voltage gradient but not pH gradient generated by the V-ATPase as the driving force to transport ATP into the lysosome to activate P2X4. ABSTRACT: The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9-mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V-ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9.

Role of purinergic P2X4 receptors in regulating striatal dopamine homeostasis and dependent behaviors.

Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of ion channels regulated by ATP. We recently demonstrated that P2X4R knockout (KO) mice exhibited deficits in sensorimotor gating, social interaction, and ethanol drinking behavior. Dopamine (DA) dysfunction may underlie these behavioral changes, but there is no direct evidence for P2X4Rs' role in DA neurotransmission. To test this hypothesis, we measured markers of DA function and dependent behaviors in P2X4R KO mice. P2X4R KO mice exhibited altered density of pre-synaptic markers including tyrosine hydroxylase, dopamine transporter; post-synaptic markers including dopamine receptors and phosphorylation of downstream targets including dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa and cyclic-AMP-response element binding protein in different parts of the striatum. Ivermectin, an allosteric modulator of P2X4Rs, significantly affected dopamine and cyclic AMP regulated phosphoprotein of 32 kDa and extracellular regulated kinase1/2 phosphorylation in the striatum. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Using the 6-hydroxydopamine model of DA depletion, P2X4R KO mice exhibited an attenuated levodopa (L-DOPA)-induced motor behavior, whereas ivermectin enhanced this behavior. Collectively, these findings identified an important role for P2X4Rs in maintaining DA homeostasis and illustrate how this association is important for CNS functions including motor control and sensorimotor gating. We propose that P2X4 receptors (P2X4Rs) regulate dopamine (DA) homeostasis and associated behaviors. Pre-synaptic and post-synaptic DA markers were significantly altered in the dorsal and ventral striatum of P2X4R KO mice, implicating altered DA neurotransmission. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Ivermectin (IVM), a positive modulator of P2X4Rs, enhanced levodopa (L-DOPA)-induced motor behavior. These studies highlight potential interactions between P2X4Rs and DA system.

Microglia-derived purines modulate mossy fibre synaptic transmission and plasticity through P2X4 and A1 receptors.

Recent data have provided evidence that microglia, the brain-resident macrophage-like cells, modulate neuronal activity in both physiological and pathophysiological conditions, and microglia are therefore now recognized as synaptic partners. Among different neuromodulators, purines, which are produced and released by microglia, have emerged as promising candidates to mediate interactions between microglia and synapses. The cellular effects of purines are mediated through a large family of receptors for adenosine and for ATP (P2 receptors). These receptors are present at brain synapses, but it is unknown whether they can respond to microglia-derived purines to modulate synaptic transmission and plasticity. Here, we used a simple model of adding immune-challenged microglia to mouse hippocampal slices to investigate their impact on synaptic transmission and plasticity at hippocampal mossy fibre (MF) synapses onto CA3 pyramidal neurons. MF-CA3 synapses show prominent forms of presynaptic plasticity that are involved in the encoding and retrieval of memory. We demonstrate that microglia-derived ATP differentially modulates synaptic transmission and short-term plasticity at MF-CA3 synapses by acting, respectively, on presynaptic P2X4 receptors and on adenosine A1 receptors after conversion of extracellular ATP to adenosine. We also report that P2X4 receptors are densely located in the mossy fibre tract in the dentate gyrus-CA3 circuitry. In conclusion, this study reveals an interplay between microglia-derived purines and MF-CA3 synapses, and highlights microglia as potent modulators of presynaptic plasticity.

Relative Role of Akt, ERK and CREB in Alcohol-Induced Microglia P2X4R Receptor Expression.

AIMS: Previously we have demonstrated altered microglia P2X4R expression in response to alcohol and pharmacological blockade with a selective P2X4R antagonist can reverse the action, suggesting that P2X4R play a role in mediating alcohol-induced effects on microglia. In the present study, we investigated the underlying signaling mediators, which may play a role in modulating P2X4R expression in microglia cells in response to alcohol. METHODS: Embryonic stem cell-derived microglia (ESdM) cells were used to investigate the potential mechanisms involved in the regulation of P2X4R in response to alcohol. Selective P2X4R antagonist and kinase inhibitors were used to further corroborate the signal transduction pathway through which alcohol modulates P2X4R expression in microglia. RESULTS: Alcohol (100 mM) suppressed phosphorylated AKT and ERK cascades in native ESdM cells. This alcohol-induced suppression was confirmed to be P2X4R-dependent through the use of a selective P2X4R antagonist and knockdown of P2XR4 by siRNA. Alcohol increased transcriptional activity of CREB. P2X4R antagonist blocked alcohol-induced effects on CREB, suggesting a P2X4R-mediated effect. CONCLUSION: These findings provide important clues to the underlying mechanism of purinoceptors in alcohol-induced microglia immune suppression.

Involvement of Purinergic P2X4 Receptors in Alcohol Intake of High-Alcohol-Drinking (HAD) Rats.

BACKGROUND: The P2X4 receptor (P2X4R) is thought to be involved in regulating alcohol-consuming behaviors, and ethanol (EtOH) has been reported to inhibit P2X4Rs. Ivermectin is an antiparasitic agent that acts as a positive allosteric modulator of the P2X4R. This study examined the effects of systemically and centrally administered ivermectin on alcohol drinking of replicate lines of high-alcohol-drinking (HAD-1/HAD-2) rats, and the effects of lentiviral-delivered short-hairpin RNAs (shRNAs) targeting P2rx4 on EtOH intake of female HAD-2 rats. METHODS: For the first experiment, adult male HAD-1 and HAD-2 rats were given 24-hour free-choice access to 15% EtOH versus water. Dose-response effects of ivermectin (1.5 to 7.5 mg/kg, intraperitoneally [i.p.]) on EtOH intake were determined; the effects of ivermectin were then examined for 2% w/v sucrose intake over 5 consecutive days. In the second experiment, female HAD-2 rats were trained to consume 15% EtOH under 2-hour limited access conditions, and dose-response effects of intracerebroventricular (ICV) administration of ivermectin (0.5 to 2.0 µg) were determined over 5 consecutive days. The third experiment determined the effects of microinfusion of a lentivirus expressing P2rx4 shRNAs into the posterior ventral tegmental area (VTA) on 24-hour EtOH free-choice drinking of female HAD-2 rats. RESULTS: The highest i.p. dose of ivermectin reduced alcohol drinking (30 to 45%) in both rat lines, but did not alter sucrose intake. HAD-2 rats appeared to be more sensitive than HAD-1 rats to the effects of ivermectin. ICV administration of ivermectin reduced 2-hour limited access intake (~35%) of female HAD-2 rats; knockdown of P2rx4 expression in the posterior VTA reduced 24-hour free-choice EtOH intake (~20%). CONCLUSIONS: Overall, the results of this study support a role for P2X4Rs within the mesolimbic system in mediating alcohol-drinking behavior.

Exaggerated renal fibrosis in P2X4 receptor-deficient mice following unilateral ureteric obstruction.

BACKGROUND: The ATP-sensitive P2X7 receptor (P2X7R) has been shown to contribute to renal injury in nephrotoxic nephritis, a rodent model of acute glomerulonephritis, and in unilateral ureteric obstruction (UUO), a rodent model of chronic interstitial inflammation and fibrosis. Renal tubular cells, endothelial cells and macrophages also express the closely related P2X4 receptor (P2X4R), which is chromosomally co-located with P2X7R and has 40% homology; it is also pro-inflammatory and has been shown to interact with P2X7R to modulate its pro-apoptotic and pro-inflammatory effects. Therefore, we chose to explore the function of P2X4R in the UUO model of renal injury using knockout mice. We hypothesized that UUO-induced tubulointerstitial damage and fibrosis would also be attenuated in P2X4R(-/-) mice. METHOD: P2X4R(-/-) and wild-type (WT) mice were subjected to either UUO or sham operation. Kidney samples taken on Days 7 and 14 were evaluated for renal inflammation and fibrosis, and expression of pro-fibrotic factors. RESULTS: To our surprise, the obstructed kidney in P2X4R(-/-) mice showed more severe renal injury, more collagen deposition (picrosirius red staining, increase of 53%; P < 0.05) and more type I collagen staining (increase of 107%; P < 0.01), as well as increased mRNA for TGF-ß (increase of 102%, P < 0.0005) and CTGF (increase of 157%; P < 0.05) by Day 14, compared with the UUO WT mice. CONCLUSION: These findings showed that lack of P2X4R expression leads to increased renal fibrosis, and increased expression of TGF-ß and CTGF in the UUO model.

Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).

P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.

Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes.

Ca(2+) is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca(2+) concentration ([Ca(2+)])(c) leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca(2+) channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca(2+) entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca(2+) entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca(2+) channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X(4) receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X(4) receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X(4). Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca(2+) influx via vesicular Ca(2+) channels regulates fusion pore expansion and vesicle content release.

P2X4 receptors influence inflammasome activation after spinal cord injury.

P2X(4) and P2X(7) are the predominant purinergic P2X receptor subtypes expressed on immune and neural cells. These receptor subtypes traffic between intracellular compartments and the plasma membrane and form protein interactions with each other to regulate ATP-dependent signaling. Our recent studies have shown that P2X(7) receptors in neurons and astrocytes activate NLRP1 inflammasomes, but whether P2X(4) receptors regulate inflammasome signaling is essentially unknown. Here, we demonstrate that P2X(4) receptors are expressed in neurons of the spinal cord. We provide direct evidence that spinal cord injury (SCI) induces an innate inflammatory response that leads to increased caspase-l cleavage and production of IL-1ß but not IL-18. Consistent with these findings, P2X(4) knock-out mice showed impaired inflammasome signaling in the cord, resulting in decreased levels of IL-1ß and reduced infiltration of neutrophils and monocyte-derived M1 macrophages, resulting in significant tissue sparing and improvement in functional outcomes. These results indicate that P2X(4) receptors influence inflammasome signaling involving caspase-1 activation and IL-1ß processing in neurons after SCI. P2X(4) might thus represent a potential therapeutic target to limit inflammatory responses associated with SCI and neurodegenerative disorders.

Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus.

Within the central nervous system, functions of the ATP-gated receptor-channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)-induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R-deficient mice, behavioral responses during KA-induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage-dependent potassium channels were impaired in P2X4R-deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity-dependent degenerative processes, the CA1 area was partially protected from SE-induced neuronal death in P2X4R-deficient mice compared with wild-type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation.

P2X7 receptor-stimulation causes fever via PGE2 and IL-1ß release.

Prostaglandins (PGs) are important lipid mediators involved in the development of inflammatory associated pain and fever. PGE2 is a well-established endogenous pyrogen activated by proinflammatory cytokine interleukin (IL)-1ß. P2X7 receptors (P2X7Rs) expressed by inflammatory cells are stimulated by the danger signal extracellular ATP to activate the inflammasome and release IL-1ß. Here we show that P2X7R activation is required for the release of PGE2 and other autacoids independent of inflammasome activation, with an ATP EC(50) for PGE2 and IL-1ß release of 1.58 and 1.23 mM, respectively. Furthermore, lack of P2X7R or specific antagonism of P2X7R decreased the febrile response in mice triggered after intraperitoneal LPS or IL-1ß inoculation. Accordingly, LPS inoculation caused intraperitoneal ATP accumulation. Therefore, P2X7R antagonists emerge as novel therapeutics for the treatment for acute inflammation, pain and fever, with wider anti-inflammatory activity than currently used cyclooxygenase inhibitors.-Barberà-Cremades, M., Baroja-Mazo, A., Gomez, A. I., Machado, F., Di Virgilio, F., Pelegrín, P. P2X7 receptor-stimulation causes fever via PGE2 and IL-1ß release.

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin.

The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.