A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

The P2X7 receptor is widely recognized to mediate the proinflammatory effects of extracellular ATP. However this receptor in the absence of ATP may have a function unrelated to inflammation. Our data show that P2X7 expressed on the surface of monocyte/macrophages or on epithelial HEK-293 cells greatly augments the engulfment of latex beads and live and heat-killed bacteria by effector phagocyte in the absence of ATP and serum. The expression of P2X7 on the effector also confers the ability to phagocytose apoptotic target cells and an accumulation of P2X7 can be seen at the attachment point to the target. Activation of the P2X7 receptor by ATP causes a slow dissociation (over 10–15 min) of nonmuscle myosin from the P2X7 membrane complex and abolishes further P2X7-mediated phagocytosis of these targets. The recent crystal structure of the homologous zebrafish P2X4 receptor shows an exposed “nose” of the ectodomain (residues 115–162) which contains three of the five disulfide bonds conserved in all P2X receptors. Three short biotin-labeled peptides mimicking sequence of this exposed region bound to apoptotic target cells but not to either viable cells or to other target particles. All three peptides contained one or two cysteine residues and their replacement by alanine abolished peptide binding. These data implicate thiol-disulfide exchange reactions in the initial tethering of apoptotic cells to macrophage and establish P2X7 as one of the scavenger receptors involved in the recognition and removal of apoptotic cells in the absence of extracellular ATP and serum.     

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.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

P2X7 receptor signaling promotes inflammation in renal parenchymal cells suffering from ischemia-reperfusion injury

Extracellular adenosine triphosphate (ATP) and its receptor, P2X7 receptor (P2X7R), are playing an important role in the pathological process of renal ischemia-reperfusion injury, but their underlying mechanism remains unclear. Also, the effects of tubular epithelium-expressed P2X7 receptor on ischemia acute kidney injury is still unknown. The aim of this study is to clarify if this mechanism involves the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the renal tubular epithelial cells. In our research, we used male C57BL/6 wild type and P2X7R (−/−) mice, cultured human proximal tubular epithelial cells, and kidneys from acute kidney injury patients. Mice underwent for unilateral nephrectomy combined with the lateral renal pedicle clamping. Cultured cells were subjected to hypoxia/reoxygenation or ATP. Apyrase and A438079 were used to block the extracellular ATP/P2X7 receptor pathway. We also constructed radiation-induced bone marrow (BM) chimeras by using P2X7R (−/−) mice and P2X7R (+/+) wild-type mice. P2X7 receptor deficiency protected from renal ischemia-reperfusion injury and attenuated the formation of NLRP3 inflammasome. By using BM chimeras, we found a partial reduction of serum creatinine and less histological impairment in group wild-type BM to P2X7R (−/−) recipient, compared with group wild-type BM to wild-type recipient. In renal tubular epithelial cells, hypoxia/reoxygenation induced ATP release and extracellular ATP depletion reduced the expression of active IL-1β. ATP activated the NLRP3 inflammasome in renal tubular epithelial cells, which were blunted by transient silence of P2X7 receptor, as well as by P2X7 receptor blocking with A438079. In human samples, we found that patients with Stage 3 AKI had higher levels of P2X7 receptor expression than patients with Stage 1 or Stage 2. Extracellular ATP/P2X7 receptor axis blocking may protect renal tubular epithelial cells from ischemia-reperfusion injury through the regulation of NLRP3 inflammasome.Subject terms: Membrane proteins, Mechanisms of disease, Acute kidney injury     

Characterization of P2 receptors in thymic epithelial cells.

The presence of P2 receptors was investigated in three distinct preparations of murine thymic epithelial cells (TEC): 2BH4 murine cell line, IT45-R1 rat cell line, and a primary murine cell derived from the Nurse cell lympho-epithelial complex. In all preparations, application of ATP to the extracellular milieu triggered intracellular calcium signals indicating the presence of P2 receptor(s) in these cells. After an initial peak of calcium concentration, a plateau phase that could last more than 10 min was frequently observed. Ion replacement and channel blockage experiments indicated that the initial peak was associated with the release of calcium from intracellular stores, while the plateau phase was associated with an influx from the extracellular medium. ATP and UTP induced similar calcium signals, suggesting the presence of P2Y2 receptors in all three cell types. The murine 2BH4 cells also expressed P2X7/P2Z receptor, since under exposure to millimolar concentrations of ATP, a continuous rise in intracellular calcium concentration was observed and their plasma membranes became permeabilized to the fluorescent dyes Lucifer yellow and ethidium bromide. In addition, this permeabilization phenomenon was blocked by the P2Z-specific antagonist, oxidized ATP. RT-PCR assays confirmed the presence of mRNAs for the P2Y2 molecule in all TEC, while mRNA for the P2X7 molecule was detected only in 2BH4 cells. Our data indicate that P2Y2 purinergic receptors are widely expressed by thymic epithelial cells, whereas the expression of the P2X7 receptor appears to be more restricted, raising the possibility that its expression is related only to a particular epithelial microenvironment within/the thymus.     

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

Tear hyperosmolarity is a key event in dry eye. In this work, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined. High-performance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.     

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.     

人白血病细胞系P2X7受体的表达及其介导的细胞内钙浓度变化

采用半定量RT-PCR和流式细胞术,在基因和蛋白水平研究了白血病细胞系U937、HL60和Ramos细胞P2X7受体的表达。荧光染料Fura-2／AM负载后,用荧光分光光度计测定P2X7受体激动剂三磷酸腺苷(adenosine 5′-triphosphate,ATP)和苯甲酰苯甲酸ATP(2′,3′-O-(4-benzoyl)benzoyl-ATP,BzATP)刺激前后细胞内钙离子浓度的变化,以确认其功能。结果表明：U937和HL60细胞系表达P2X7受体的mRNA和蛋白,Ramos不表达;在激动剂的刺激下,可引发U937和HL60细胞胞内钙浓度的显著升高,但对Ramos没有作用。当去除胞外钙离子时,ATP和BzATP刺激均不能引起U937和HL60细胞胞内钙离子浓度的升高。提示U937和HL60细胞表达P2X7受体的基因和功能蛋白,Ramos细胞则不表达该受体。     

ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b⁺/Gr-1⁺ cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1⁺ population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.     

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.     

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.     

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.     

Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus

The binding of extracellular ATP to the P2X(7) receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1beta secretion and eventual death. The latter three effects are unique to the P2X(7) receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X(7) receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X(7) receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X(7) was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X(7). The P2X(7) receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X(7) receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X(7) C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X(7) receptor activation.     

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.     

Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells

The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

Lung epithelial cells release ATP during ozone exposure: signaling for cell survival

The common air pollutant ozone causes acute toxicity to human airways. In primary and transformed epithelial cells from all levels of human or rat airways, ozone levels relevant to air pollution (50-200 ppb) increased extracellular [ATP] within 7-30 min. A human bronchial epithelial cell line (16HBE14o(-)) that forms electrically resistant polarized monolayers had up to 10-fold greater apical than basolateral surface extracellular [ATP] within 7 min of ozone exposure. Increased extracellular [ATP] appeared due to ATP secretion or release because (1) inhibition of ectonucleotidase (cell surface enzyme(s) which degrade ATP) by ozone did not occur until >120 min of ozone exposure and (2) brefeldin A, a secretory inhibitor, eliminated elevation of extracellular [ATP] without affecting intracellular ATP. Extracellular ATP protected against ozone toxicity in a P2Y receptor-dependent manner as (1) removal of ATP and adenosine by apyrase and adenosine deaminase, respectively, potentiated ozone toxicity, (2) extracellular supplementation with ATP, a poorly hydrolyzable ATP analog ATPgammaS, or UTP inhibited apoptotic and necrotic ozone-mediated cell death, and (3) ATP-mediated protection was eliminated by P2 and P2Y receptor inhibitors suramin and Cibacron blue (reactive blue 2), respectively. The decline in glucose uptake caused by prolonged ozone exposure was prevented by supplemental extracellular ATP, an effect blocked by suramin. Further, Akt and ERK phosphorylation resulted from exposure to supplemental extracellular ATP. Thus, extracellularly released ATP signals to prevent ozone-induced death and supplementation with ATP or its analogs can augment protection, at least in part via Akt and /or ERK signaling pathways and their metabolic effects.     

Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia

Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-alpha secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-alpha release, resulting from TNF-alpha mRNA expression in rat cultured brain microglia. The TNF-alpha release was maximally elicited by 1 mM ATP and also induced by a P2X(7) receptor-selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, suggesting the involvement of P2X(7) receptor. ATP-induced TNF-alpha release was Ca(2+)-dependent, and a sustained Ca(2+) influx correlated with the TNF-alpha release in ATP-stimulated microglia. ATP-induced TNF-alpha release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca(2+). These results indicate that extracellular ATP triggers TNF-alpha release in rat microglia via a P2 receptor, likely to be the P2X(7) subtype, by a mechanism that is dependent on both the sustained Ca(2+) influx and ERK/p38 cascade, regulated independently of Ca(2+) influx.