P2X7 receptors mediate ischemic damage to oligodendrocytes

Brain ischemia leading to stroke is a major cause of disability in developed countries. Therapeutic strategies have most commonly focused on protecting neurons from ischemic damage. However, ischemic damage to white matter causes oligodendrocyte death, myelin disruption, and axon dysfunction, and it is partially mediated by glutamate excitotoxicity. We have previously demonstrated that oligodendrocytes express ionotropic purinergic receptors. The objective of this study was to investigate the role of purinergic signaling in white matter ischemia. We show that, in addition to glutamate, enhanced ATP signaling during ischemia is also deleterious to oligodendrocytes and myelin, and impairs white matter function. Thus, ischemic oligodendrocytes in culture display an inward current and cytosolic Ca²⁺ overload, which is partially mediated by P2X7 receptors. Indeed, oligodendrocytes release ATP after oxygen and glucose deprivation through the opening of pannexin hemichannels. Consistently, ischemia‐induced mitochondrial depolarization as well as oxidative stress culminating in cell death are partially reversed by P2X7 receptor antagonists, by the ATP degrading enzyme apyrase and by blockers of pannexin hemichannels. In turn, ischemic damage in isolated optic nerves, which share the properties of brain white matter, is greatly attenuated by all these drugs. Ultrastructural analysis and electrophysiological recordings demonstrated that P2X7 antagonists prevent ischemic damage to oligodendrocytes and myelin, and improved action potential recovery after ischemia. These data indicate that ATP released during ischemia and the subsequent activation of P2X7 receptor is critical to white matter demise during stroke and point to this receptor type as a therapeutic target to limit tissue damage in cerebrovascular diseases. © 2009 Wiley‐Liss, Inc.     

Neuron-specific distribution of P2X7 purinergic receptors in the monkey retina

Extracellular ATP is a signaling molecule, working through P2X purinoceptors in the nervous system. P2X7 is a major subtype of the purinoceptors in the brain, where it is expressed mostly in glia cells and considered to work as a trigger of cytolysis. In the rodent retina, however, P2X7 is expressed in several classes of neurons including ganglion cells. In the present study we identified cells immunopositive for P2X7 by double immunolabeling. Immunoreactivity for P2X7 was observed in the inner nuclear layer (INL), the inner plexiform layer (IPL), and the ganglion cell layer (GCL). In the INL, strongly immunopositive cells corresponded to the subpopulation of horizontal cells. In the IPL, fine processes were immunopositive. In the GCL, most of the ganglion cells showed P2X7 immunoreactivity. At the ultrastructural level, immunoreactivity was confirmed in the cytoplasm of ganglion cells. No P2X7 immunoreactivity was found in non-neural cells, i.e., Müller cells or microglia. The immunohistochemical distribution of other purinoceptor subtypes (P2X1, P2X2, and P2X4) was also examined in the monkey retina. Immunoreactivity for P2X1 was strongly detected in a band, in sublamina a of the IPL. The band existed at almost the same level as tyrosine hydroxylase immunoreactivity, but did not seem to actually overlap. P2X2 was not expressed in the retina, and P2X4 was only faintly expressed at the scleral margin of the INL. Because P2X7 in the primate retina is expressed exclusively in neurons, it may in this location be involved in neural transmission rather than in cytolysis, as found for glia cells.     

P2Y1 and P2X7 receptors induce calcium/calmodulin-dependent protein kinase II phosphorylation in cerebellar granule neurons

The activation of nucleotide receptors-- both ionotropic, P2X, and most of metabotropic, P2Y-- increases intracellular calcium concentration, resulting in calcium/calmodulin-dependent protein kinase II (CaMKII) activation. Stimulation of cerebellar granule neurons in culture-- with different P2X and P2Y agonists and their effect on CaMKII phosphorylation-- was studied using immunocytochemical and microfluorimetrical techniques. P2X agonist: 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP) and diadenosine pentaphosphate (Ap(5)A); and P2Y agonists: 2-(methylthyo)-adenosine diphosphate (2MeSADP) and uridine 5'-bisphosphate (UDP); tested induced a CaMKII phosphorylation but with a different immunostaining pattern in each group. Stimulation with 2MeSADP induced a Ca(2+) release from intracellular stores and a significant CaMKII phosphorylation in cell somas and neurites. This agrees with the subcellular distribution of P2Y(1). MRS 2179, a specific P2Y(1) inhibitor, antagonized the 2MeSADP effect. On the other hand, cerebellar granule neuron stimulation with BzATP, in Mg(2+)-free conditions, produced extracellular calcium entrance and, as a result, a significant increase in CaMKII phosphorylation mostly in fibres, which correspond with P2X(7) subdistribution. Immunocytochemical and microfluorimetrical experiments, using Zn(2+) and Brilliant Blue G (BBG), as a specific P2X(7) antagonist, confirmed that BzATP was acting through the P2X(7) receptor. These results indicate that P2Y(1) and P2X(7) produce a significant increase in CaMKII phosphorylation, but show important differences in subcellular distribution and in effect duration. P2X(7) activation in granule neurons is not associated with pore formation, according to the absence of YO-PRO-1 fluorescence. The abundant presence of P2X(7) at the synaptic structures suggests a relevant role played by this receptor in synaptic plasticity.     

Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors

Oxidative stress is the main cause of neuronal damage in traumatic brain injury, hypoxia/reperfusion injury, and neurodegenerative disorders. Although extracellular nucleosides, especially adenosine, are well known to protect against neuronal damage in such pathological conditions, the effects of these nucleosides or nucleotides on glial cell damage remain largely unknown. We report that ATP but not adenosine protects against the cell death of cultured astrocytes induced by hydrogen peroxide (H2O2). ATP ameliorated the H2O2-induced decrease in cell viability of astrocytes in an incubation time- and concentration-dependent fashion. Protection by ATP was inhibited by P2 receptor antagonists and was mimicked by P2Y1 receptor agonists but not by adenosine. The expressions of P2Y1 mRNAs and functional P2Y1 receptors in astrocytes were confirmed. Thus, ATP, acting on P2Y1 receptors in astrocytes, showed a protective action against H2O2. The astrocytic protection by the P2Y1 receptor agonist 2-methylthio-ADP was inhibited by an intracellular Ca2+ chelator and a blocker of phospholipase C, indicating the involvement of intracellular signals mediated by Gq/11-coupled P2Y1 receptors. The ATP-induced protection was inhibited by cycloheximide, a protein synthesis inhibitor, and it took more than 12 h for the onset of the protective action. In the DNA microarray analysis, ATP induced a dramatic upregulation of various oxidoreductase genes. Taken together, ATP acts on P2Y1 receptors coupled to Gq/11, resulting in the upregulation of oxidoreductase genes, leading to the protection of astrocytes against H2O2.     

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. GLIA 2013;61:1306–1319     

Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis

We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o-protein coupled P2Y(12) receptor (P2Y(12)R). Here we report discovering that the P2X(4) receptor (P2X(4)R) is also involved in ATP-induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y(12)R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3'-kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP-induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y(12)R antagonist. These results indicate that P2Y(12)R-mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X(4)R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X(4)R significantly inhibited it. Knockdown of the P2X(4) receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X(4)R as well as P2Y(12)R is involved in ATP-induced microglial chemotaxis.     

Purinergic signaling mediated by P2X7 receptors controls myelination in sciatic nerves

Adenosine‐5′‐triphosphate, the physiological ligand of P2X receptors, is an important factor in peripheral nerve development. P2X₇ receptor is expressed in Schwann cells (SCs), but the specific effects of P2X₇ purinergic signaling on peripheral nerve development, myelination, and function are largely unknown. In this study, sciatic nerves from P2X₇ knockout mice were analyzed for altered expression of myelin‐associated proteins and for alterations in nerve morphology. Immunohistochemical analyses revealed that, in the wild‐type peripheral nerves, the P2X₇ receptor was localized mainly in myelinating SCs, with only a few immunopositive nonmyelinating SCs. Complete absence of P2X₇ receptor protein was confirmed in the sciatic nerves of the knockout mice by Western blot and immunohistochemistry. Western blot analysis revealed that expression levels of the myelin proteins protein zero and myelin‐associated glycoprotein are reduced in P2X₇ knockout nerves. In accordance with the molecular results, transmission electron microscopy analyses revealed that P2X₇ knockout nerves possess significantly more unmyelinated axons, contained in a higher number of Remak bundles. The myelinating/nonmyelinating SC ratio was also decreased in knockout mice, and we found a significantly increased number of irregular fibers compared with control nerves. Nevertheless, the myelin thickness in the knockout was unaltered, suggesting a stronger role for P2X₇ in determining SC maturation than in myelin formation. In conclusion, we present morphological and molecular evidence of the importance of P2X₇ signaling in peripheral nerve maturation and in determining SC commitment to a myelinating phenotype. © 2014 Wiley Periodicals, Inc.     

The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes

Extracellular nucleotide di- and triphosphates such as ATP and ADP mediate their effects through purinergic P2 receptors belonging to either the metabotropic P2Y or the ionotropic P2X receptor family. The P2X7R is a unique member of the P2X family, which forms a pore in response to ligand stimulation, regulating cell permeability, cytokine release, and/or apoptosis. This receptor is also unique in that its affinity for the ligand benzoyl-benzoyl ATP (BzATP) is at least 10-fold greater than that of ATP. Primary human fetal astrocytes in culture express low-levels of P2X7R mRNA and protein, and BzATP induces only a slight influx in intracellular calcium [Ca2+]i, with little demonstrable effect on gene expression or pore formation in these cells. We now show that, following treatment with the proinflammatory cytokine IL-1beta, BzATP induces a robust rise in [Ca2+]i with agonist and antagonist profiles indicative of the P2X7R. IL-1beta also induced the formation of membrane pores as evidenced by the uptake of YO-PRO-1 (375 Da). Quantitative real-time PCR demonstrated transient upregulation of P2X7R mRNA in IL-1beta-treated cells, while FACS analysis indicated a similar upregulation of P2X7R protein at the cell membrane. In multiple sclerosis lesions, immunoreactivity for the P2X7R was demonstrated on reactive astrocytes in autopsy brain tissues. In turn, P2X7R stimulation increased the production of IL-1-induced nitric oxide synthase activity by astrocytes in culture. These studies suggest that signaling via the P2X7R may modulate the astrocytic response to inflammation in the human central nervous system.     

Astrocyte cultures exhibit P2X7 receptor channel opening in the absence of exogenous ligands

P2X7 receptors (P2X7Rs) gate the opening of large channels when activated by ATP or other ligands. P2X7Rs are expressed by astrocytes in culture and by reactive astrocytes in vivo, and astrocytes in culture have been shown to release glutamate and ATP in response to P2X7R activation. However, P2X7Rs are activated by ATP only at concentrations greater than 1 mM. The conditions under which astrocyte P2X7Rs would be activated in vivo are, thus, unclear. Here we show that astrocytes in culture exhibit basal P2X7R activity. Primary mouse astrocytes were found to take up the P2X7R permeant dyes YO-PRO-1 (YP) and propidium iodide in absence of any added ligands. By contrast, cultured rat astrocytes took up very little YP, consistent with their much lower level of P2X7R expression. The uptake by mouse astrocytes was inhibited by oxATP, suramin, KN-62 and brilliant blue G, and by siRNA knock-down of P2X7R. Astrocyte uptake of YP was also inhibited by phenol red at concentrations above 50 muM, suggesting that phenol red present in standard cell culture media may influence P2X7R channel activity. Treatment with apyrase, an enzyme that degrades extracellular ATP, partially decreased YP uptake in astrocytes. Conversely, exposure to the ectonucleotidase inhibitor ARL67156 enhanced YP uptake and astrocytes plated without contiguous neighboring astrocytes showed reduced basal YP uptake. These results suggest that the basal uptake of YP may be due to activation of P2X7R by release of ATP by astrocytes themselves into intercellular spaces.     

ATP stimulation of P2X(7) receptors activates three different ionic conductances on cultured mouse Schwann cells

Extracellular ATP, by acting on P2 purinergic receptors, is a potent mediator of cell-to-cell communication both within and between the nervous and the immune systems. We show here by patch-clamp recording, fluorescent dye uptake and immunocytochemistry that, in cultured mouse Schwann cells, ATP activates a P2X(7) receptor associated with three different ionic conductances. In control conditions, ATP activated an inward current (I(ATP)) with a low potency (EC(50), 7.2 mM). Replacing ATP either by the ATP analogue 2',3'-O-(4-benzoyl-4-benzoyl)-ATP (BzATP) or by the tetraacidic form ATP(4-) potentiated the inward current (ATP(4-) EC(50), 375 microM). ATP and BzATP currents were strongly reduced by periodate oxidized ATP (oATP), an antagonist of P2X(7) receptors. IATP was a mixed current composed of a nonselective cationic conductance, a cationic conductance selective for K(+) and an anionic conductance selective for Cl(-). The activation of the K(+) conductance was dependent on an influx of Ca(2+), and was blocked by charybdotoxin (ChTX) and tetraethylammonium (TEA), two potent antagonists of large conductance Ca(2+)-activated K(+) channels (BK channels). The activation of the Cl(-) conductance was insensitive to Ca(2+) but required the presence of K(+). Total removal of K(+) blocked both the Ca(2+)-activated K(+) conductance and the Cl(-) conductance, unveiling the P2X(7) nonselective cationic conductance. The P2X(7) receptor was localized by immunocytochemistry using a polyclonal antibody, anti-P2X(7), whilst its expression and functionality were both detected by the uptake of Lucifer Yellow. This receptor could regulate the synthesis and the release of cytokines by Schwann cells during pathophysiological events.     

Contribution of P2X7 receptors to adenosine uptake by cultured mouse astrocytes

Nucleotides and nucleosides play important roles by maintaining brain homeostasis, and their extracellular concentrations are mainly regulated by ectonucleotidases and nucleoside transporters expressed by astrocytes. Extracellularly applied NAD⁺ prevents astrocyte death caused by excessive activation of poly(ADP‐ribose) polymerase‐1, of which the molecular mechanism has not been fully elucidated. Recently, exogenous NAD⁺ was reported to enter astrocytes via the P2X7 receptor (P2X7R)‐associated channel/pore. In this study, we examined whether the intact form of NAD⁺ is incorporated into astrocytes. A large portion of extracellularly added NAD⁺ was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring‐labeled [¹⁴C]NAD⁺, but not nicotinamide moiety‐labeled [³H]NAD⁺, showed time‐ and temperature‐dependency, and was significantly enhanced on addition of apyrase, and was reduced by 8‐Br‐cADPR and ARL67156, inhibitors of CD38 and ectoapyrase, respectively, and P2X7R knockdown, suggesting that the detected uptake of [¹⁴C]NAD⁺ resulted from [¹⁴C]adenosine acting as a metabolite of [¹⁴C]NAD⁺. Pharmacological and genetic inhibition of P2X7R with brilliant blue G, KN‐62, oxATP, and siRNA transfection resulted in a decrease of [³H]adenosine uptake, and the uptake was also reduced by low concentration of carbenoxolone and pannexin1 selective peptide blocker ¹⁰panx. Taken together, these results indicate that exogenous NAD⁺ is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R‐associated channel/pore. © 2010 Wiley‐Liss, Inc.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

ATP binding cassette transporter ABC1 is required for the release of interleukin-1beta by P2X7-stimulated and lipopolysaccharide-primed mouse Schwann cells

Schwann cells are best known as myelinating glial cells of the peripheral nervous system, but they also participate actively in the sphere of immunity by producing pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). In a previous study, we demonstrated that posttranslational processing of IL-1beta by immune-challenged Schwann cells required the P2X7 receptor. Remarkably, the release of IL-1beta was not associated with cell death, indicating the involvement of an active mechanism. ATP binding cassette (ABC) transporters are known to transport leaderless secretory proteins, such as IL-1beta; therefore, we investigated whether such transporters were at work in Schwann cells. Mouse Schwann cells expressed ABC1 transporter mRNA and displayed the functional protein. Glybenclamide and diisothiocyanato-stilbene-disulfonic acid (DIDS), two blockers of chloride fluxes that drive the export activity of ABC1 transporters, inhibited IL-1beta release without altering its intracellular processing. Enhancing chloride efflux potentiated the release of IL-1beta, while decreasing it led to a strong reduction in its release. Because the stimulation of the P2X7 receptor also activates a chloride conductance, we investigated the possibility of a sole anionic pathway mobilized by the P2X7 receptor and ABC1. Glybenclamide and DIDS had no significant effects on the P2X7-activated chloride current suggesting therefore the existence of two different pathways. In summary, ABC1 transporters are required for the release of IL-1beta by mouse Schwann cells. Being associated together with chloride conductance, P2X7 receptors and ABC1 transporters delineate a subtle and complex regulation of IL-1beta production in mammalian Schwann cells. Furthermore, ABC1 transporters could be a target of therapeutic interest for regulating IL-1beta activity in neuroinflammation disorders.     

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors

ATP is the dominant messenger for astrocyte-to-astrocyte calcium-mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca(2+)](i)) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X(6) receptor, primary rat cortical astrocytes express all cloned ligand-gated P2X (i.e., P2X(1-5) and P2X(7)) and G-protein-coupled P2Y receptors (i.e., P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(12)). These cells also express the P2Y-like UDP-glucose receptor, which has been recently recognized as the P2Y(14) receptor. Single-cell image analysis showed that only some of these receptors are coupled to [Ca(2+)](i). While ATP induced rapid and transient [Ca(2+)](i) increases (counteracted by the P2 antagonists suramin, pyridoxal-phosphate-6-azophenyl-2'-4'-disulfonic acid and oxidized ATP), the P2X(1)/P2X(3) agonist alphabetameATP produced no changes. Conversely, the P2X(7) agonist BzATP markedly increased [Ca(2+)](i); the presence and function of the P2X(7) receptor was also confirmed by the formation of the P2X(7) pore. ADP and 2meSADP also produced [Ca(2+)](i) increases antagonized by the P2Y(1) antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar-nucleotides were also detected, which represents the first functional response reported for the putative P2Y(14) receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP-induced calcium rises are at least mediated by P2X(7) and P2Y(1) receptors; additional receptors (i.e., P2X(2), P2X(4), P2X(5), P2Y(2), P2Y(4), and P2Y(14)) may also contribute.     

Astrocyte–neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor‐mediated release of glutamate and reactive oxygen species

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole‐cell patch clamp recordings from SG spinal cord slices documented that in a low Ca²⁺/no Mg²⁺ (low X²⁺) external medium adenosine triphosphate (ATP)/dibenzoyl‐ATP, Bz‐ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X²⁺‐containing bath medium. The effect of Bz‐ATP was antagonized by the selective P2X7 receptor antagonist A‐438079. Neuronal, but not astrocytic Bz‐ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP‐5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H₂O₂ potentiated the effect of Bz‐ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP‐5 and CNQX almost abolished the effect of Bz‐ATP, H₂O₂ was inactive. A Bz‐ATP‐dependent and A‐438079‐antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7‐immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA‐ and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions. GLIA 2014;62:1671–1686     

Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura‐2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X_2,4,6 and P2Y_1,2 subtypes. P2X₁ and P2X₇ receptor immunoreactivity (IR) was found on thin axon‐like processes and presynaptic structures, respectively. Application of ATP caused a small concentration‐dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in most investigated neurons, whereas only about the half of these cells responded to 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (BzATP), ADPβS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, α,β‐meATP, UDP, and UDP‐glucose failed to produce any [Ca²⁺]_i signaling. The response to ATP itself was inhibited by pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), Reactive Blue 2, 2′‐deoxy‐N⁶‐methyl adenosine 3′,5′‐diphosphate (MRS2179), and suramin (300 μM) as well as by a cyclopiazonic acid‐induced depletion of intracellular Ca²⁺ stores. A Ca²⁺‐free external medium tended to decrease the ATP‐induced [Ca²⁺]_i transients, although this action did not reach statistical significance. Various blockers of voltage‐sensitive Ca²⁺ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(–)‐2‐amino‐5‐phosphonopentanoic acid (AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) decreased it. Cross‐desensitization experiments between ADPβS or UTP and ATP suggested that ATP acts on the one hand via P2Y_1,2 receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca²⁺ via store‐operated Ca²⁺ channels. Evidence for the presence of functional P2X receptors, in particular P2X₇, remains elusive. J. Comp. Neurol. 516:343–359, 2009. © 2009 Wiley‐Liss, Inc.     

Activation of P2X(7) receptors induced [(3)H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl(-)/HCO(3)(-)-dependent mechanism.

ATP is an important signaling molecule in the nervous system and it's signaling is mediated through the metabotropic P2Y and ionotropic P2X receptors. ATP is known to stimulate Ca(2+) influx and phospholipase D (PLD) activity in the type-2 astrocyte cell line, RBA-2; in this study, we show that the release of preloaded [(3)H]GABA from RBA-2 cells is mediated through the P2X(7) receptors. ATP and the ATP analogue 3'-O-(4-benoylbenoyl)-adenosine-5'-triphosphate (BzATP) both stimulated [(3)H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the P2X(7)-sensitive antagonist oxidized ATP (oATP), and high extracellular Mg(2+) all inhibited the ATP-stimulated [(3)H]GABA release. The ATP-stimulated [(3)H]GABA release was not affected neither by removing extracellular Na(+) nor by changes in the intracellular or extracellular Ca(2+) concentration. The GABA transporter inhibitors nipecotic acid and beta-alanine also had no effect. The ATP-stimulated [(3)H]GABA release was blocked, however, when media Cl(-) was replaced with gluconate and when extracellular HCO(3)(-) was removed. The Cl(-) channel/exchanger blockers 4,4'-diisothiocyanatostilbene-2',2'-disulfonic acid (DIDS) and 4-acetamido-4'- isothiocyanatostilbene-2',2'-disulfonic acids (SITS), but not diphenylamine-2-carboxylic acid (DPC) and furosemide, blocked the ATP-stimulated [(3)H]GABA release. The anionic selectivity of the process was F(-) > Cl(-) > Br(-) which is the same as that reported for volume-sensitive Cl(-) conductance. Treating cells with phorbol-12-myristate 13-acetate (PMA), forskolin, dibutyryl-cAMP, PD98059, neomycin, and D609 all inhibited the ATP-stimulated [(3)H]GABA release. We concluded that in RBA-2 cells, ATP stimulates [(3)H]GABA release through the P2X(7) receptors via a Cl(-)/HCO(3)(-)-dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD.     

Role of P2X4 receptors in synaptic strengthening in mouse CA1 hippocampal neurons

P2X4 receptors are calcium-permeable cation channels gated by extracellular ATP. They are found close to subsynaptic sites on hippocampal CA1 neurons. We compared features of synaptic strengthening between wild-type and P2X4 knockout mice (21-26 days old). Potentiation evoked by a tetanic presynaptic stimulus (100 Hz, 1 s) paired with postsynaptic depolarization was less in P2X4(-/-) mice than in wild-type mice (230 vs. 50% potentiation). Paired-pulse ratios and the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) were not different between wild-type and knockout mice. Prior hyperpolarization (ten 3 s pulses to -120 mV at 0.17 Hz) potentiated the amplitude of spontaneous EPSCs in wild-type mice, but not in P2X4(-/-) mice; this potentiation was not affected by nifedipine, but was abolished by 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) in the recording pipette. The amplitude of N-methyl-d-aspartate EPSCs (in 6-cyano-7-nitroquinoxaline-2,3-dione, 10 or 30 μm, at -100 mV) facilitated during 20 min recording in magnesium-free solution. In wild-type mice, this facilitation of the N-methyl-d-aspartate EPSC was reduced by about 50% by intracellular BAPTA (10 mM), ifenprodil (3 μm) or 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 μm). In P2X4(-/-) mice, the facilitation was much less, and was unaffected by intracellular BAPTA, ifenprodil (3 μm) or mitogen-activated protein (MAP) kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 μm). This suggests that the absence of P2X4 receptors limits the incorporation of NR2B subunits into synaptic N-methyl-d-aspartate receptors.     

Effect of tetramethylpyrazine on acute nociception mediated by signaling of P2X receptor activation in rat

Tetramethylpyrazine (TMP) has been used in traditional Chinese medicine as an analgesic for dysmenorrhea. In the present study, we try to investigate the effects of TMP on acute nociception mediated by P2X receptor activation of rat hindpaw and the membrane depolarization of rat dorsal root ganglion (DRG) neurons induced by P2X receptor agonists. The subcutaneous administration of TMP (0.1-10 mmol) into rat hindpaw in a dose-dependent manner decreased acute paw flinching responses mediated by adenosine 5'-triphosphate (ATP, 1000 nmol) or alpha,beta-methylene ATP (alpha,beta-meATP, 600 nmol). The subcutaneous administration of TMP (5 or 10 mmol) into rat hindpaw inhibited significantly the first phase of nociceptive behaviors induced by 5% formalin and attenuated slightly the second phase of nociceptive behaviors induced by 5% formalin. The subcutaneous administration of TMP (10 mmol) into rat hindpaw reduced the nociceptive responses induced by alpha,beta-meATP (200 nmol) co-injected with Prostaglandin E2 (PGE2), 5 micromol). The membrane depolarization induced by ATP (200 micromol) or alpha,beta-meATP (50 micromol) in DRG neurons was inhibited by TMP (300 micromol). The data suggest that the antinociceptive effect of TMP is involved in blocking the signaling of P2X3 receptor activation in rat.     

P2X4 receptor channels form large noncytolytic pores in resting and activated microglia

P2X4 ATP-gated cation channels have been shown to contribute to the microglial component of central sensitization, making their functional regulation a key element in chronic pain pathologies. Here we show that prolonged activation of native P2X4 receptor channels by ATP induces opening of a pore permeable to NMDG(+) and large fluorescent dyes in BV-2 microglial cells and primary murine microglia. This intrinsic pore formation mechanism is potentiated by LPS treatment, known to upregulate P2X4 expression in microglial cells and to mimic the microglial activation observed in neuropathic pain states. Sustained activation of the P2X7 channel subtype, also expressed in microglia, induces a pore formation that requires pannexin hemichannels and leads to plasma membrane blebbing and cytotoxicity. In contrast, P2X4 pore formation is unaffected by the pannexin blocker carbenoxolone, does not induce cytoskeletal rearrangements and does not lead to cell death. Furthermore, we show that P2X4 pore dilation is modulated by phosphoinositides (PIP(n) ) levels as it is inhibited by wortmannin, a blocker of PIP(n) synthesis, suggesting possible regulation by phospholipase C-coupled pathways. Nonlethal P2X4 pore dilation could play a role in neuropathic pain by allowing the flux of large organic molecules in microglia. Different outcomes of P2X4 and P2X7 membrane permeabilization point to subtype-specific microglial responses to ATP in normal and pathological neuro-immune crosstalks.