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• W2077845979 abstract "Mono-ADP-ribosyltransferase 2 (ART2) is found in mouse T cells and has mediated NAD-induced cell death (NICD) alongside the P2X7 pathway. We determined whether ART2 was expressed in mouse brain astrocytes and the possible function of the NAD-ART2-P2X7 pathway in astrocytes. Our results demonstrate that ART2 existed both in cultured mouse astrocytes and mouse brain slices. Exposure of astrocytes to the ART2 substrate, NAD, induced calcium elevation, which was blocked by ART2 and P2X7 inhibitors. ATP and NAD had an additive effect on calcium elevation. NICD in low-calcium conditions was blocked by ART2 and P2X7 inhibitors. The harmful effect of ATP on astrocytes was inhibited by P2X7 and ART2 inhibitors, meaning that endogenous NAD release may occur. Both NICD function and oxygen-glucose deprivation injury in mouse brain slices were also involved in the ART2-P2X7 pathway. Collectively, to our knowledge, our study provides the first evidence that ART2 exists in mouse brain astrocytes and NAD induces calcium elevation and astrocyte death by an ART2 and P2X7-mediated mechanism. The results suggest a novel approach for manipulating astrocyte death. Mono-ADP-ribosyltransferase 2 (ART2) is found in mouse T cells and has mediated NAD-induced cell death (NICD) alongside the P2X7 pathway. We determined whether ART2 was expressed in mouse brain astrocytes and the possible function of the NAD-ART2-P2X7 pathway in astrocytes. Our results demonstrate that ART2 existed both in cultured mouse astrocytes and mouse brain slices. Exposure of astrocytes to the ART2 substrate, NAD, induced calcium elevation, which was blocked by ART2 and P2X7 inhibitors. ATP and NAD had an additive effect on calcium elevation. NICD in low-calcium conditions was blocked by ART2 and P2X7 inhibitors. The harmful effect of ATP on astrocytes was inhibited by P2X7 and ART2 inhibitors, meaning that endogenous NAD release may occur. Both NICD function and oxygen-glucose deprivation injury in mouse brain slices were also involved in the ART2-P2X7 pathway. Collectively, to our knowledge, our study provides the first evidence that ART2 exists in mouse brain astrocytes and NAD induces calcium elevation and astrocyte death by an ART2 and P2X7-mediated mechanism. The results suggest a novel approach for manipulating astrocyte death. P2X7 belongs to the P2X family of ATP-gated ion channels expressed on different cell types, including astrocytes and T lymphocytes. P2X7 activation triggers calcium flux, the opening of a large nonselective membrane pore, and, ultimately, cell death by apoptosis.1Kim J.E. Ryu H.J. Yeo S.I. Kang T.C. P2X7 receptor differentially modulates astroglial apoptosis and clasmatodendrosis in the rat brain following status epilepticus.Hippocampus. 2011; 21: 1318-1333Crossref PubMed Scopus (45) Google Scholar P2X7 activation by ATP requires millimolar concentrations, whereas incubation of mouse T lymphocytes with micromolar NAD+ induces all these effects.2Haag F. Adriouch S. Braβ A. Jung C. Möller S. Scheuplein F. Bannas P. Seman M. Koch-Nolte F. Extracellular NAD and ATP: partners in immune cell modulation.Purinergic Signal. 2007; 3: 71-81Crossref PubMed Scopus (131) Google ScholarMammalian mono-ADP-ribosyltransferases (ARTs) constitute a family of ectoenzymes structurally related to bacterial toxins that catalyze the transfer of the ADP-ribose group from NAD+ onto amino acid residues of target proteins.3Ohlrogge W. Haag F. Löhler J. Seman M. Littman D.R. Killeen N. Koch-Nolte F. Generation and characterization of ecto-ADP-ribosyltransferase ART2.1/ART2.2-deficient mice.Mol Cell Biol. 2002; 22: 7535-7542Crossref PubMed Scopus (52) Google Scholar Previous reports showed that ART2 is expressed on the surface of most mature peripheral T lymphocytes in the mouse. Incubation with NAD+ leads to the rapid induction of ART-dependent T-cell death in vitro, a phenomenon that was termed NAD+-induced cell death (NICD).4Scheuplein F. Adriouch S. Glowacki G. Haag F. Seman M. Koch-Nolte F. Triggering of T-cell apoptosis by toxin-related ecto-ADP-ribosyltransferase ART2.Ann N Y Acad Sci. 2003; 1010: 296-299Crossref PubMed Scopus (11) Google Scholar NICD also results from the activation of the P2X7 ATP receptor, which is one target of murine ART2.5Seman M. Adriouch S. Scheuplein F. Krebs C. Freese D. Glowacki G. Deterre P. Haag F. Koch-Nolte F. NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor.Immunity. 2003; 19: 571-582Abstract Full Text Full Text PDF PubMed Scopus (265) Google ScholarAccumulating evidence has suggested that millimolar concentrations of ATP play critical roles in astrocyte death, via activation of P2X7 receptors. However, the extracellular ATP concentration in the normal brain is at micromolar levels.2Haag F. Adriouch S. Braβ A. Jung C. Möller S. Scheuplein F. Bannas P. Seman M. Koch-Nolte F. Extracellular NAD and ATP: partners in immune cell modulation.Purinergic Signal. 2007; 3: 71-81Crossref PubMed Scopus (131) Google Scholar It is warranted to investigate a novel pathway activated by low concentrations of NAD+, which may elucidate fundamental questions in biology.6Hong S. Schwarz N. Brass A. Seman M. Haag F. Koch-Nolte F. Schilling W.P. Dubyak G.R. Differential regulation of P2X7 receptor activation by extracellular nicotinamide adenine dinucleotide and ecto-ADP-ribosyltransferases in murine macrophages and T cells.J Immunol. 2009; 183: 578-592Crossref PubMed Scopus (47) Google Scholar The aims of the present study were to determine the existence of the ART2 enzyme in astrocytes, evaluate the effects of NAD+ and ATP on astrocytes, and determine whether NICD plays the same role in astrocyte death.Materials and MethodsMaterialsReagents were obtained from Sigma-Aldrich (St. Louis, MO), except where noted.Cell CulturesAstrocyte cultures were prepared from the cortices of 1-day-old Institute of Cancer Research mice (Simonsen Laboratories, Gilroy, CA) and plated into 24-well culture plates. The astrocytes were maintained in Eagle's minimal essential medium containing 5 mmol/L glucose and supplemented with 5% fetal bovine serum (Hyclone, Ogden, UT) and 2 mmol/L glutamine.7Zhu K. Swanson R.A. Ying W. NADH can enter into astrocytes and block poly(ADP-ribose) polymerase-1-mediated astrocyte death.Neuroreport. 2005; 16: 1209-1212Crossref PubMed Scopus (33) Google Scholar The cultures were used for experiments at 20 to 30 days in vitro.Experimental ProceduresExperiments were initiated by replacing the culture medium with balanced salt solution containing the following: KCl, 3.1 mmol/L; NaCl, 134 mmol/L; CaCl2, 1.2 mmol/L; MgSO4, 1.2 mmol/L; KH2PO4, 0.25 mmol/L; NaHCO3, 15.7 mmol/L; and glucose, 2 mmol/L. The pH was adjusted to 7.2 while the solution was equilibrated with 5% CO2 at 37°C. Osmolarity was verified at 290 to 310 mOsm with a vapor pressure osmometer (Wescor, Logan, UT). For low Ca2+ and Mg2+ balanced salt solution, both Ca2+ and Mg2+ concentrations were reduced to 0.1 mmol/L.RNA Extraction and Nested PCRRNA from astrocytes was extracted using an affinity resin column (Qiagen, Inc., Valencia, CA). cDNA was synthesized by random-primed RT reactions using random hexamer primers and 1 μg RNA in a 20-μL reaction volume. After completion of cDNA synthesis, one-tenth of the cDNA was used for subsequent PCR.The nested PCR was used to detect the ART2 expression in culture astrocytes. The two sets of primers of ART2 were primer A and primer B (sc-42733-PR) from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). This required two separate runs of PCR. The first PCR used 50-μL PCR medium containing 5 μL of 10 times PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 2 μL primer A, 2 μL cDNA, and 1 U TaqDNA, which underwent 35 cycles of PCR amplification under the following conditions: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds. The second PCR used 50 μL of PCR medium containing 5 μL of 10 times PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 2 μL primer B, 5 μL first PCR production, and 1 U TaqDNA, with 35 cycles of PCR amplification under the following conditions: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds. The size of the product of the first reaction was 1000 bp. The second pair of primers amplified a specific DNA fragment of 415 bp. The astrocyte RNA that was not subjected to the RT was used as the control group. PCR products were visualized on agarose gels stained with ethidium bromide.Cell Death DeterminationAstrocyte death was quantified in astrocyte monotypic cultures by measuring the lactate dehydrogenase (LDH) activity in cell lysates harvested 24 hours after drug exposure. The percentage cell death was calculated by normalizing the LDH values to those measured from control (wash only) culture wells.Slice PreparationMouse cortical slices were prepared from 14- to 22-day-old male Institute of Cancer Research mice. In brief, coronal sections were cut on a vibratome (Vibratome, Series 1000 Classic; St. Louis, MO) in an ice-chilled solution containing the following: sucrose, 200 mmol/L; piperazine-N,N′-bis(2-ethanesulphonate), 20 mmol/L; KCl, 2.5 mmol/L; NaH2PO4, 1.25 mmol/L; MgSO4, 10 mmol/L; CaCl2, 0.5 mmol/L; dextrose, 10 mmol/L, pH 7.35, adjusted with NaOH. Before the recording procedure, slices were kept submerged in artificial cerebrospinal fluid: NaCl, 125 mmol/L; KCl, 2.5 mmol/L; NaH2PO4, 1.25 mmol/L; NaHCO3, 24 mmol/L; MgSO4, 2 mmol/L; CaCl2, 2 mmol/L; dextrose, 10 mmol/L, pH adjusted to 7.35 by bubbling with a mixture of 95% O2 and 5% CO2.OGD in SlicesTo simulate hypoxic-hypoglycemic conditions, artificial cerebrospinal fluid was replaced with an identical solution lacking glucose (glucose was substituted with an equimolar concentration of sucrose) and equilibrated with 95% N2 and 5% CO2.ImmunohistochemistryAnimals were anesthetized with ether 24 hours after the last injection and perfused transcardially with PBS, followed by 4% paraformaldehyde (w/v) in PBS. Serial sections (7-μm thick) were cut from frozen tissue. Sections were dewaxed and hydrated in graded alcohols and PBS (10 mmol/L, pH 7.4), and immunostaining was visualized using fluorescent secondary antibody. In our study, ART2 antibody (Santa Cruz, Inc.) was used at a dilution of 1:400. Glial fibrillary acidic protein (GFAP) antibody (Dako, Glostrup, Denmark) was used at a dilution of 1:400. Alexa Fluor 488 (Molecular Probes, Carlsbad, CA) and Cy3 (Molecular Probes) were used as secondary antibodies to detect ART2 and GFAP immunoactivity, respectively. The controls run omitting the primary antibody showed no staining.Western Blot AnalysesCells were grown in six-well plates to 80% confluence and treated in serum-free minimal essential medium. The medium was then aspirated, and cells were rinsed twice with ice-cold PBS. Cells were then lysed and scraped loose into ice-cold lysis buffer (M-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, IL). After centrifugation at 14,000 × g for 10 minutes at 4°C, supernatants were boiled for 5 minutes in Laemmli sample buffer supplemented with 50 mmol/L dithiothreitol. Equal amounts of lysed and boiled protein (30 μg per well) were loaded and subjected to electrophoresis on 10% polyacrylamide gels (BioRad, Hercules, CA). Separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK), which were then blocked for 1 hour at room temperature with 5% nonfat dried milk (BioRad). Membranes were then incubated overnight at 4°C with the primary antibody. Concentrations were chosen according to the manufacturer's instructions. Secondary horseradish peroxidase–conjugated antibody was incubated for 1 hour at room temperature, and proteins were evaluated using an enhanced chemiluminescence kit (GE Healthcare).Measurement of Intracellular CalciumIntracellular calcium was measured from Fura-3 AM-loaded astrocytes using a spectrofluorimeter (Hitachi F-2000; Hitachi, Tokyo, Japan). Briefly, astrocytes (1 × 106 to 3 × 106 cells/mL) were incubated with 1 μmol/L Fura-2 AM for 45 minutes at room temperature in HEPES buffer (NaCl, 137 mmol/L; KCl, 2.7 mmol/L; CaCl2, 0.3 mmol/L; MgCl2, 1.0 mmol/L; NaH2PO4, 0.4 mmol/L; glucose, 5.6 mmol/L; HEPES, 10 mmol/L; and NaOH, to pH 7.4). Cells were then washed twice in HEPES buffer and resuspended in the same buffer. Cells were continuously stirred and sequentially excited at 488 nm for 1 second at room temperature. Emitted fluorescence was measured at 530 nm. The relative calcium intensity was calculated by the normal calcium quantification method. Relative ⁢Intensity=(F-F-base)/(F-base−B) where F is the measured fluorescence intensity of the calcium indicator, F-base is the fluorescence intensity of the calcium indicator in the cell before stimulation, and B is the background signal determined from the average of areas adjacent to the cell.Statistical AnalysisData were assessed by analysis of variance, followed by the Student-Newman-Keuls post hoc test. P < 0.05 was considered statistically significant. Results are presented as mean ± SEM of at least three independent experiments.ResultsExpression of ART2 in Cultured Astrocytes and Mouse Brain SlicesPrevious research reported that ART2 existed in T cells, but it was unknown if ART2 was expressed in astrocytes. Our RT-PCR (Figure 1A), immunocytochemistry (Figure 1B), and Western blot analysis (Figure 1C) results showed that ART2 mRNA and protein were expressed in cultured astrocytes, with T cells as a positive control. To confirm our PCR result, we sequenced the second PCR production. The sequence BLAST showed the identity with ADP-ribosyltransferase was 412 (99%) of 415. Immunostaining images for GFAP and ART2 (Figure 1, D–F) showed both markers merged well in the brain slice. Collectively, our results showed that cortical astrocytes in mouse brain expressed ART2.NAD Induces Astrocyte Calcium Elevation by the ART2 and P2X7 PathwayATP and NAD induced the flux additively on ATP and NAD induction (Figure 2, A–D). Calcium influx was induced by 100 μmol/L of either ATP or NAD, both in normal extracellular solution (ECS; Figure 2, A and B) and low-calcium ECS (Figure 2, C and D). The P2X7 antagonists, brilliant blue G (BBG) or suramin, or the ART2 inhibitor, nicotinamide, effectively blocked the NAD-induced calcium flux. After washing the inhibitors out, NAD induced calcium flux in the same astrocytes again (Figure 2, E and F). These results provided strong evidence that the ART2 and P2X7 pathway was involved in the NAD-induced calcium change in astrocytes. Treatment with glutamate was used as a control, and the inhibitors did not generally interfere with the calcium influx (data not shown).Figure 2NAD induces calcium flux in cultured astrocytes. A and B: Both 100 μmol/L ATP and 100 μmol/L NAD induce calcium elevation immediately after administration (arrow) in astrocytes incubated with Fluo-3 (5 μmol/L for 30 minutes). The effects of ATP plus NAD are additive in ECS with normal calcium. C and D: Both 100 μmol/L ATP and 100 μmol/L NAD induce calcium elevation immediately after administration (arrow) in astrocytes in low-calcium ECS. The effects of ATP plus NAD are additive in ECS with low calcium. E and F: Astrocytes are loaded with fluo-3 (5 μmol/L for 30 minutes). NAD at 100 μmol/L induces calcium elevation. After preincubation of astrocytes with the P2X7 antagonist BBG (100 μmol/L) or suramin (100 μmol/L) or the ART2 inhibitor nicotinamide (100 μmol/L for 10 minutes), calcium flux is not induced by 100 μmol/L NAD. On washing out the inhibitors, 100 μmol/L NAD induces calcium flux in the same astrocytes again. ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NAD Induces Astrocyte Death at a Lower Concentration than ATPIn our experiments with ECS containing a normal calcium concentration, both 5 mmol/L ATP (Figure 3A) and 500 μmol/L NAD (Figure 3B) induced astrocyte death. Compared with ATP, a lower dose of NAD induced astrocyte death. With low-calcium ECS, only 100 μmol/L ATP (Figure 3C) or 100 μmol/L NAD (Figure 3D) induced astrocyte death. In normal ECS, NAD and ATP needed higher doses to induce astrocyte death than in low-calcium ECS. There was a report showing that astrocytes exhibited P2X7 receptor channel opening even in the absence of exogenous ligands.8Koch-Nolte F. Adriouch S. Bannas P. Krebs C. Scheuplein F. Seman M. Haag F. ADP-ribosylation of membrane proteins: unveiling the secrets of a crucial regulatory mechanism in mammalian cells.Ann Med. 2006; 38: 188-199Crossref PubMed Scopus (35) Google Scholar Up-regulation of astrocyte P2X7R was observed in several pathological models, including the low-calcium environment and ATP stimulation. A low-calcium environment made astrocytes sensitive to the P2X7 agonist, ATP, or the ART2 agonist, NAD.9Seman M. Adriouch S. Haag F. Koch-Nolte F. Ecto-ADP-ribosyltransferases (ARTs): emerging actors in cell communication and signaling.Curr Med Chem. 2004; 11: 857-872Crossref PubMed Scopus (103) Google Scholar We also found that ATP plus NAD induced astrocyte death additively, both in normal (Figure 3E) and in low-calcium ECS (Figure 3F), suggesting that the ART2-P2X7 pathway was involved.Figure 3NAD induces astrocyte death at lower concentrations than ATP. A: ATP (5 mmol/L) induces astrocyte death in normal medium. Astrocytes are incubated in the presence of a different concentration of ATP for 24 hours. Twenty-four hours after washout of the drugs, cell death is assessed by LDH assay. B: NAD induces dose-dependent astrocyte death in normal medium. Astrocytes are treated with 50 μmol/L to 10 mmol/L NAD for 24 hours, and cell death is assessed by LDH assay after washout of the drugs. NAD (500 μmol/L) induces astrocyte death at a lower dosage than ATP. C: ATP induces astrocyte death in low-calcium ECS. The LDH release from the low-calcium group does not differ from that of the control group, meaning in our model the low-calcium ECS has no harmful effects on astrocytes. At least 100 μmol/L ATP induces astrocyte death in low-calcium ECS. D: Only 100 μmol/L NAD is needed to induce astrocyte death in low-calcium ECS. E: ATP and NAD induce astrocyte death in normal ECS. NAD (1 mmol/L) with ATP (1 mmol/L) show an additive effect. F: ATP and NAD induce astrocyte death in low-calcium ECS. NAD (1 mmol/L) plus ATP (1 mmol/L) show an additive effect. *P < 0.05, **P < 0.01. Data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NAD Induces Astrocyte Death via ART2 and P2X7From the previously described experimental results, we selected the low-calcium model as an ideal system to study the effects of NAD and ATP in astrocytes. In some pathological situations (eg, brain ischemia), there existed a low-calcium condition in the human brain. We found that the cytotoxic effect of ATP was inhibited by P2X7 and ART2 inhibitors (Figure 4A), suggesting that P2X7 was involved in the pathway and that endogenous NAD release occurred. We also found that exposure of astrocytes to the ART2 substrate, NAD, induced cell death, which was blocked by ART2 inhibitors (1–4 naphthoquinone or nicotinamide) and P2X7 inhibitors (periodate-oxidized ATP, BBG, or suramin) in low-calcium ECS (Figure 4B). This meant that the toxic effect of NAD was mediated by the ART2 and P2X7 pathway.Figure 4NAD induces astrocyte death via ART2 and P2X7. A: Astrocytes are incubated with 1 mmol/L ATP for 24 hours. Cotreatment with P2X7 inhibitors (oxATP, BBG, or suramin) or ART2 inhibitors (1,4-naphthoquinone or nicotinamide) decreases ATP-induced death in low-calcium ECS. B: Astrocytes are incubated with 1 mmol/L NAD for 24 hours. Cotreatment with P2X7 inhibitors (oxATP, BBG, or suramin) and ART2 inhibitors (1,4-naphthoquinone or nicotinamide) decreases NAD-induced astrocyte death in low-calcium ECS. C: Brain slices are incubated in the presence of 1 mmol/L NAD for 24 hours. Cotreatment with ART2 inhibitors [1,4-naphthoquinone, metaiodobenzylguanidine (MIBG), or nicotinamide] and the P2X7 inhibitor, oxATP, decreases NAD-induced brain slice injury. D: Brain slices are incubated with 4-hour oxygen-glucose deprivation and 24-hour recovery (OGD/R). Cotreatment with ART2 inhibitors (1,4-naphthoquinone and MIBG) and the P2X7 inhibitor, oxATP, decreases OGD-induced brain slice injury. *P < 0.05, **P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, we found that exposure of mouse brain slices to the ART substrate, NAD (Figure 4C), or oxygen-glucose deprivation (OGD; Figure 4D) induced injury in the slices. The LDH release was blocked by the inhibitor of cell surface ADP-ribosylation 1–4 naphthoquinone, or nicotinamide, a competitive inhibitor of NAD-dependent ADP-ribosylation, or by another ART2 inhibitor, metaiodobenzylguanidine, and the specific P2X7 inhibitor, oxATP. Because brain ischemia occurred with extracellular NAD release and OGD, the findings suggested that the ART2-P2X7 pathway in astrocytes may play an important role in brain ischemia and some other brain diseases.DiscussionOur previous study provided evidence that treatment with 100 μmol/L NAD decreases poly (ADP-ribose) polymerase-1–induced astrocyte death. Because NAD+ depletion is a key factor in poly (ADP-ribose) polymerase-1 cytotoxicity, NAD treatment could decrease poly (ADP-ribose) polymerase-1–induced cell death, at least partially by increasing the intracellular NAD+ levels. NAD treatment has a protective function in conditions of NAD depletion, such as oxidative stress.7Zhu K. Swanson R.A. Ying W. NADH can enter into astrocytes and block poly(ADP-ribose) polymerase-1-mediated astrocyte death.Neuroreport. 2005; 16: 1209-1212Crossref PubMed Scopus (33) Google Scholar Herein, our study showed that ART2 was expressed in murine astrocytes. We found that NAD is involved in triggering a change in astrocyte calcium and astrocyte death by the ART2 and P2X7 pathway. Our data showed a correlation between astrocyte calcium change and astrocyte death, but they do not establish the causality, although this causality might be proved by data on the role of calcium in NICD in other systems.2Haag F. Adriouch S. Braβ A. Jung C. Möller S. Scheuplein F. Bannas P. Seman M. Koch-Nolte F. Extracellular NAD and ATP: partners in immune cell modulation.Purinergic Signal. 2007; 3: 71-81Crossref PubMed Scopus (131) Google Scholar, 5Seman M. Adriouch S. Scheuplein F. Krebs C. Freese D. Glowacki G. Deterre P. Haag F. Koch-Nolte F. NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor.Immunity. 2003; 19: 571-582Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar Our results indicated that the ART2-P2X7 pathway existed in astrocytes, both in the pathological (cell death) and the physiological (calcium change) conditions.Near-millimolar concentrations of exogenous ATP were required to elicit P2X7 activation in vitro.8Koch-Nolte F. Adriouch S. Bannas P. Krebs C. Scheuplein F. Seman M. Haag F. ADP-ribosylation of membrane proteins: unveiling the secrets of a crucial regulatory mechanism in mammalian cells.Ann Med. 2006; 38: 188-199Crossref PubMed Scopus (35) Google Scholar In our experiments, we found that astrocyte death occurred at a low dose of NAD, but required a high dose of ATP. Under normal conditions, the concentrations of ATP and NAD in body fluids are lower than those required to induce P2X7-dependent death. The concentration of NAD is maintained between 0.1 and 0.5 μmol/L in the serum of untreated animals. It is, therefore, plausible that extracellular nucleotides are released from intracellular sources, where they are present in higher concentrations. Three situations may lead to the release of nucleotides from intracellular compartments: i) liberation of intracellular contents by dying cells, ii) exocytosis of nucleotide-containing granules, and iii) diffusion of these molecules toward the extracellular space across membrane channels.9Seman M. Adriouch S. Haag F. Koch-Nolte F. Ecto-ADP-ribosyltransferases (ARTs): emerging actors in cell communication and signaling.Curr Med Chem. 2004; 11: 857-872Crossref PubMed Scopus (103) Google Scholar Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and nonlytic mechanisms, and may serve as danger signals to alert astrocytes to respond to tissue damage.10Scheuplein F. Schwarz N. Adriouch S. Krebs C. Bannas P. Rissiek B. Seman M. Haag F. Koch-Nolte F. NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells.J Immunol. 2009; 182: 2898-2908Crossref PubMed Scopus (99) Google Scholar The intracellular NAD+ concentration is in the range of 1 mmol/L, whereas the plasma concentration is in the range of 0.1 μmol/L, lower than the Km of ARTs. Large amounts of ecto-NAD+ can be released during tissue injury as a consequence of cell lysis.11Adriouch S. Hubert S. Pechberty S. Koch-Nolte F. Haag F. Seman M. NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo.J Immunol. 2007; 179: 186-194Crossref PubMed Scopus (105) Google Scholar ATP or NAD+ also can be released by nonlytic processes under various physiological conditions, including hypoxia, ischemia, inflammation, and mechanical or chemical activation.12Hong S. Brass A. Seman M. Haag F. Koch-Nolte F. Dubyak G.R. Basal and inducible expression of the thiol-sensitive ART2.1 ecto-ADP-ribosyltransferase in myeloid and lymphoid leukocytes.Purinergic Signal. 2009; 5: 369-383Crossref PubMed Scopus (19) Google Scholar Hence, high local NAD+ concentrations may be reached in various physiological and pathophysiological situations, which would permit ADP-ribosylation of membrane proteins on ART-expressing neighboring cells.13Hong S. Brass A. Seman M. Haag F. Koch-Nolte F. Dubyak G.R. Lipopolysaccharide, IFN-gamma, and IFN-beta induce expression of the thiol-sensitive ART2.1 Ecto-ADP-ribosyltransferase in murine macrophages.J Immunol. 2007; 179: 6215-6227PubMed Google Scholar The NAD-dependent P2X7 activation provides a mechanism for signaling through P2X7 that requires only low extracellular concentrations of nucleotide, which may be more easily attainable in vivo than the high concentrations required for ATP-mediated signaling.14Nagasawa K. Escartin C. Swanson R.A. Astrocyte cultures exhibit P2X7 receptor channel opening in the absence of exogenous ligands.Glia. 2009; 57: 622-633Crossref PubMed Scopus (49) Google Scholar, 15Suadicani S.O. Brosnan C.F. Scemes E. P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling.J Neurosci. 2006; 26: 1378-1385Crossref PubMed Scopus (434) Google ScholarOur findings in this study have several biological implications. First, we reveal a novel finding of the presence of ART2 in astrocytes, which might be important for its roles in various physiological and pathophysiological situations, such as ischemia. Second, we had a finding regarding the roles of ART2 and P2X7 receptors in NICD and the calcium change within brain astrocytes: only micromolar concentrations of NAD are required, which represents an appealing alternative pathway for the activation of P2X7.16Adriouch S. Bannas P. Schwarz N. Fliegert R. Guse A.H. Seman M. Haag F. Koch-Nolte F. ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site.FASEB J. 2008; 22: 861-869Crossref PubMed Scopus (100) Google Scholar, 17Koch-Nolte F. Reyelt J. Schössow B. Schwarz N. Scheuplein F. Rothenburg S. Haag F. Alzogaray V. Cauerhff A. Goldbaum F.A. Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo.FASEB J. 2007; 21: 3490-3498Crossref PubMed Scopus (87) Google Scholar NAD-dependent activation of P2X7 purinergic signaling constitutes a novel mechanism for inducing astrocyte death.18Kawamura H. Aswad F. Minagawa M. Malone K. Kaslow H. Koch-Nolte F. Schott W.H. Leiter E.H. Dennert G. P2X7 receptor-dependent and -independent T cell death is induced by nicotinamide adenine dinucleotide.J Immunol. 2005; 174: 1971-1979Crossref PubMed Scopus (55) Google Scholar, 19Morrison A.R. Moss J. Stevens L.A. Evans J.E. Farrell C. Merithew E. Lambright D.G. Greiner D.L. Mordes J.P. Rossini A.A. Bortell R. ART2, a T cell surface mono-ADP-ribosyltransferase, generates extracellular poly(ADP-ribose).J Biol Chem. 2006; 281: 33363-33372Crossref PubMed Scopus (21) Google Scholar, 20Hubert S. Rissiek B. Klages K. Huehn J. Sparwasser T. Haag F. Koch-Nolte F. Boyer O. Seman M. Adriouch S. Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway.J Exp Med. 2010; 22: 2561-2568Crossref Scopus (135) Google Scholar Third, our findings suggest a novel approach to manipulating NAD+ levels for investigating its biological functions in ischemia. Future studies are necessary to determine whether our findings are applicable to in vivo ischemia models and other cell types under both in vitro and in vivo conditions. P2X7 belongs to the P2X family of ATP-gated ion channels expressed on different cell typ" @default.
• W2077845979 created "2016-06-24" @default.
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• W2077845979 date "2012-09-01" @default.
• W2077845979 modified "2023-10-07" @default.
• W2077845979 title "NAD Induces Astrocyte Calcium Flux and Cell Death by ART2 and P2X7 Pathway" @default.
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