P2X(7) is a scavenger receptor for apoptotic cells in the absence of its ligand, extracellular ATP

Phagocytosis of apoptotic cells is essential during development and tissue remodeling. Our previous study has shown that the P2X(7) receptor regulates phagocytosis of nonopsonized particles and bacteria. In this study, we demonstrate that P2X(7) also mediates phagocytosis of apoptotic lymphocytes and neuronal cells by human monocyte-derived macrophages under serum-free conditions. ATP inhibited this process to a similar extent as observed with cytochalasin D. P2X(7)-transfected HEK-293 cells acquired the ability to phagocytose apoptotic lymphocytes. Injection of apoptotic thymocytes into the peritoneal cavity of wild-type mice resulted in their phagocytosis by macrophages, but injection of ATP prior to thymocytes markedly decreased this uptake. In contrast, ATP failed to inhibit phagocytosis of apoptotic thymocytes in vivo by P2X(7)-deficient peritoneal macrophages. The surface expression of P2X(7) on phagocytes increased significantly during phagocytosis of either beads or apoptotic cells. A peptide screen library containing 24 biotin-conjugated peptides mimicking the extracellular domain of P2X(7) was used to evaluate the binding profile to beads, bacteria, and apoptotic cells. One peptide showed binding to all particles and cell membrane lipids. Three other cysteine-containing peptides uniquely bound the surface of apoptotic cells but not viable cells, whereas substitution of alanine for cysteine abolished peptide binding. Several thiol-reactive compounds including N-acetyl-L-cysteine abolished phagocytosis of apoptotic SH-SY5Y cells by macrophages. These data suggest that the P2X(7) receptor in its unactivated state acts like a scavenger receptor, and its extracellular disulphide bonds play an important role in direct recognition and engulfment of apoptotic cells.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

The P2X<Subscript>7</Subscript> receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

Retinal ganglion cells process the visual signal and transmit it along their axons in the optic nerve to the brain. Molecular, immunohistochemical, and functional analyses indicate that the majority of retinal ganglion cells express the ionotropic P2X(7) receptor. Stimulation of the receptor can lead to a rise in intracellular calcium and cell death, although death does not involve the opening of a large diameter pore. Adenosine acting at A(3) receptors can attenuate the rise in calcium and death accompanying P2X(7) receptor activation, suggesting that dephosphorylation of ATP into adenosine is neuroprotective and that the balance of extracellular purines can influence neuronal survival. Increased intraocular pressure can lead to release of excessive extracellular ATP in the retina and damage ganglion cells by acting on P2X(7) receptors, implicating a role for the receptor in the loss of ganglion cell activity in glaucoma. In summary, the activation of P2X(7) receptors has both physiologic and pathophysiologic implications for ganglion cell function. These characteristics may also provide an insight into the contributions the P2X(7) receptor makes to neurons elsewhere.     

Lipid Raft Association and Cholesterol Sensitivity of P2X1-4 Receptors for ATP: CHIMERAS AND POINT MUTANTS IDENTIFY INTRACELLULAR AMINO-TERMINAL RESIDUES INVOLVED IN LIPID REGULATION OF P2X1 RECEPTORS*

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20–23 and 27–29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.     

Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor

Neural progenitor cells (NPCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, and have been used to treat several animal models of CNS disorders. In the present study, we show that the P2X7 purinergic receptor (P2X7R) is present on NPCs. In NPCs, P2X7R activation by the agonists extracellular ATP or benzoyl ATP triggers opening of a non-selective cationic channel. Prolonged activation of P2X7R with these nucleotides leads to caspase independent death of NPCs. P2X7R ligation induces NPC lysis/necrosis demonstrated by cell membrane disruption accompanied with loss of mitochondrial membrane potential. In most cells that express P2X7R, sustained stimulation with ATP leads to the formation of a non-selective pore allowing the entry of solutes up to 900 Da, which are reportedly involved in P2X7R-mediated cell lysis. Surprisingly, activation of P2X7R in NPCs causes cell death in the absence of pore formation. Our data support the notion that high levels of extracellular ATP in inflammatory CNS lesions may delay the successful graft of NPCs used to replace cells and repair CNS damage.     

Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels

P2X7 receptors are ATP-gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist, and the intrinsic channel pore dilates to allow the passage of fluorescent dyes. A 60-s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components. A second application of ATP evoked a response with a more rapid time to peak. This facilitation was reversed to initial levels following a 10-min agonist-free interval. A chimeric approach showed that replacement of the pre-TM1 amino-terminal region with the corresponding P2X2 receptor section (P2X7–2Nβ) gave responses that quickly reached a steady state and did not show facilitation. Subsequent point mutations of variant residues identified Asn-16 and Ser-23 as important contributors to the time course/facilitation. The P2X7 receptor is unique in having an intracellular carboxyl-terminal cysteine-rich region (Ccys). Deletion of this region removed the secondary slowly developing current, and, when expressed in HEK293 cells, ethidium bromide uptake was only ∼5% that of WT levels, indicating reduced large pore formation. Dye uptake was also reduced for the P2X7–2Nβ chimera. Surprisingly, combination of the chimera and the Ccys deletion (P2X7–2NβdelCcys) restored the current rise time and ethidium uptake to WT levels. These findings suggest that there is a coevolved interaction between the juxtatransmembrane amino and carboxyl termini in the regulation of P2X7 receptor gating.     

Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.     

The P2X7 receptor is a key modulator of aerobic glycolysis

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.     

Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function

Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.     

Functional characterization of a P2X receptor from Schistosoma mansoni

The cloning and characterization of a P2X receptor (schP2X) from the parasitic blood fluke Schistosoma mansoni provides the first example of a non-vertebrate ATP-gated ion channel. A number of functionally important amino acid residues conserved throughout vertebrate P2X receptors, including 10 extracellular cysteines, aromatic and positively charged residues involved in ATP recognition, and a consensus protein kinase C site in the amino-terminal tail, are also present in schP2X. Overall, the amino acid sequence identity of schP2X with human P2X(1-7) receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC(50) of 22.1 microM. 2',3'-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was a partial agonist (maximum response 75.4 +/- 4.4% that of ATP) with a higher potency (EC(50) of 3.6 microM) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid blocked schP2X responses to 100 microm ATP with IC(50) values of 9.6 and 0.5 microM, respectively. Ivermectin (10 microM) potentiated currents to both ATP and Bz-ATP by approximately 60% with a minimal effect on potency (EC(50) of 18.2 and 1.6 microM, respectively). The relative permeability of schP2X expressed in HEK293 cells to various cations was determined under bi-ionic conditions. schP2X has a relatively high calcium permeability (P(Ca)/P(Na) = 3.80 +/- 0.29) and an estimated minimum pore diameter similar to that of vertebrate P2X receptors. SchP2X provides a useful comparative model for the better understanding of human P2X receptor function and may also provide an alternative drug target for treatment of schistosomiasis.     

Extracellular nucleotides enhance agonist potency at the parathyroid hormone 1 receptor

Parathyroid hormone (PTH) activates the PTH/PTH-related peptide receptor (PTH1R) on osteoblasts and other target cells. Mechanical stimulation of cells, including osteoblasts, causes release of nucleotides such as ATP into the extracellular fluid. In addition to its role as an energy source, ATP serves as an agonist at P2 receptors and an allosteric regulator of many proteins. We investigated the effects of concentrations of extracellular ATP, comparable to those that activate low affinity P2X7 receptors, on PTH1R signaling. Cyclic AMP levels were monitored in real-time using a bioluminescence reporter and β-arrestin recruitment to PTH1R was followed using a complementation-based luminescence assay. ATP markedly enhanced cyclic AMP and β-arrestin signaling as well as downstream activation of CREB. CMP – a nucleotide that lacks a high energy bond and does not activate P2 receptors – mimicked this effect of ATP. Moreover, potentiation was not inhibited by P2 receptor antagonists, including a specific blocker of P2X7. Thus, nucleotide-induced potentiation of signaling pathways was independent of P2 receptor signaling. ATP and CMP reduced the concentration of PTH (1–34) required to produce a half-maximal cyclic AMP or β-arrestin response, with no evident change in maximal receptor activity. Increased potency was similarly apparent with PTH1R agonists PTH (1–14) and PTH-related peptide (1–34). These observations suggest that extracellular nucleotides increase agonist affinity, efficacy or both, and are consistent with modulation of signaling at the level of the receptor or a closely associated protein. Taken together, our findings establish that ATP enhances PTH1R signaling through a heretofore unrecognized allosteric mechanism.     

P2X7 receptor regulation of non‐classical secretion from immune effector cells

P2X7 receptors (P2X7R) are extracellular ATP‐gated ion channels expressed in the immune effector cells that carry out critical protective responses during the early phases of microbial infection or acute tissue trauma. P2X7R‐positive cells include monocytes, macrophages, dendritic cells and T cells. Given its presence in all host and pathogen cell types, ATP can be readily released into extracellular compartments at local sites of tissue damage and microbial invasion. Thus, extracellular ATP and its target receptors on host effector cells can be considered as additional elements of the innate immune system. In this regard, stimulation of P2X7R rapidly triggers a key step of the inflammatory response: induction of NLRP3/caspase‐1 inflammasome signalling complexes that drive the proteolytic maturation and secretion of the proinflammatory cytokines interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18). IL‐1β (and IL‐18) lacks a signal sequence for compartmentation within the Golgi and classical secretory vesicles and the proIL‐1β precursor accumulates within the cytosol following translation on free ribosomes. Thus, ATP‐induced accumulation of the mature IL‐1β cytokine within extracellular compartments requires non‐classical mechanisms of export from the cytosolic compartment. Five proposed mechanisms include: (i) exocytosis of secretory lysosomes that accumulate cytosolic IL‐1β via undefined protein transporters; (ii) release of membrane‐delimited microvesicles derived from plasma membrane blebs formed by evaginationsof the surface membrane that entrap cytosolic IL‐β; (iii) release of membrane‐delimited exosomes secondary to the exocytosis of multivesicular bodies formed by invaginations of recycling endosomes that entrap cytosolic IL‐β; (iv) exocytosis of autophagosomes or autophagolysosomes that accumulate cytosolic IL‐1β via entrapment during formation of the initial autophagic isolation membrane or omegasome and (v) direct release of cytosolic IL‐1β secondary to regulated cell death by pyroptosis or necroptosis. These mechanisms are not mutually exclusive and may represent engagement of parallel or intersecting membrane trafficking responses to P2X7R activation.     

Bi-functional effects of ATP/P2 receptor activation on tumor necrosis factor-alpha release in lipopolysaccharide-stimulated astrocytes

Neuroinflammation is associated with a variety of CNS pathologies. Levels of tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory cytokine, as well as extracellular ATP, are increased following various CNS insults. Here we report on the relationship between ATP/P2 purinergic receptor activation and lipopolysaccharide (LPS)-induced TNF-alpha release from primary cultures of rat cortical astrocytes. Using ELISA, we confirmed that treatment with LPS stimulated the release of TNF-alpha in a concentration and time dependent manner. ATP treatment alone had no effect on TNF-alpha release. LPS-induced TNF-alpha release was attenuated by 1 mm ATP, a concentration known to activate P2X7 receptors. Consistent with this, 3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a P2X7 receptor agonist, also attenuated LPS-induced TNF-alpha release. This reduction in TNF-alpha release was not due to loss of cell viability. Adenosine and 2-chloroadenosine were ineffective, suggesting that attenuation of LPS-induced TNF-alpha release by ATP was not due to ATP breakdown and subsequent activation of adenosine/P1 receptors. Interestingly, treatment of astrocyte cultures with 10 microm or 100 microm ATP potentiated TNF-alpha release induced by a submaximal concentration of LPS. UTP and 2methylthioADP (2-MeSADP), P2Y receptor agonists, also enhanced this LPS-induced TNF-alpha release. Our observations demonstrate opposing effects of ATP/P2 receptor activation on TNF-alpha release, i.e. P2X receptor activation attenuates, whereas P2Y receptor activation potentiates TNF-alpha release in LPS-stimulated astrocytes. These observations suggest a mechanism whereby astrocytes can sense the severity of damage in the CNS via ATP release from damaged cells and can modulate the TNF-alpha mediated inflammatory response depending on the extracellular ATP concentration and corresponding type of astrocyte ATP/P2 receptor activated.     

Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

Extracellular nucleotides, such as ATP, are released from cells and play roles in various physiological and pathological processes through activation of P2 receptors. Here, we show that autocrine signaling through release of ATP and activation of P2X7 receptor influences migration of human lung cancer cells. Release of ATP was induced by stimulation with TGF-β1, which is a potent inducer of cell migration, in human lung cancer H292 cells, but not in noncancerous BEAS-2B cells. Treatment of H292 cells with a specific antagonist of P2X7 receptor resulted in suppression of TGF-β1-induced migration. PC-9 human lung cancer cells released a large amount of ATP under standard cell culture conditions, and P2X7 receptor-dependent dye uptake was observed even in the absence of exogenous ligand, suggesting constitutive activation of P2X7 receptor in this cell line. PC-9 cells showed high motile activity, which was inhibited by treatment with ecto-nucleotidase and P2X7 receptor antagonists, whereas a P2X7 receptor agonist enhanced migration. PC-9 cells also harbor a constitutively active mutation in epidermal growth factor receptor (EGFR). Treatment with EGFR tyrosine kinase inhibitor AG1478 suppressed both cell migration and P2X7 receptor expression in PC-9 cells. Compared to control PC-9 cells, cells treated with P2X7 antagonist exhibited broadened lamellipodia around the cell periphery, while AG1478-treated cells lacked lamellipodia. These results indicate that P2X7-mediated signaling and EGFR signaling may regulate migration of PC-9 cells through distinct mechanisms. We propose that autocrine ATP-P2X7 signaling is involved in migration of human lung cancer cells through regulation of actin cytoskeleton rearrangement.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9411-x) contains supplementary material, which is available to authorized users.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

Increased proliferation rate of lymphoid cells transfected with the P2X(7) ATP receptor

Human leukocytes can express the P2X(7) purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X(7) cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X(7) transfectants is abolished by the P2X(7) receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X(7)-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.     

Characterization of ATP‐induced cell death in the GL261 mouse glioma

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP‐induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub‐population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP‐induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP‐induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP‐induced cell death and shed light on the importance of ATP‐induced cell death in glioma development. J. Cell. Biochem. 109: 983–991, 2010. © 2010 Wiley‐Liss, Inc.     

P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E₂ (PGE₂) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE₂ release and was attenuated by inhibition of either phospholipase A₂ or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.     

A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.     

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl⁻ channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50–300 μm) stimulated a 5–100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl⁻ channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.