P2X7 Mediates Superoxide Production in Primary Microglia and Is Up-regulated in a Transgenic Mouse Model of Alzheimer's Disease*

Lav K. ParvathenaniDagger, Svetlana Tertyshnikova, Corinne R. Greco, Susan B. Roberts, Barbara Robertson, and Rand Posmantur

From the Neuroscience Drug Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb Co., Wallingford, Connecticut 06492

Received for publication, September 16, 2002, and in revised form, January 23, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Primary rat microglia stimulated with either ATP or 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>). ATP and BzATP stimulate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production through purinergic receptors, primarily the P2X7 receptor. O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is produced through the activation of the NADPH oxidase. Although both p42/44 MAPK and p38 MAPK were activated rapidly in cells stimulated with BzATP, only pharmacological inhibition of p38 MAPK attenuated O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production to a greater extent than an inhibitor of p38 MAPK. Both ATP and BzATP stimulated microglia-induced cortical cell death indicating this pathway may contribute to neurodegeneration. Consistent with this hypothesis, P2X7 receptor was specifically up-regulated around beta -amyloid plaques in a mouse model of Alzheimer's disease (Tg2576).

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activated microglia have been observed in patients suffering from both acute (stroke) and chronic (Alzheimer's disease) neurological disorders (1, 2). Microglia are believed to contribute to the progression of Alzheimer's disease (AD)1 because these cells can release pro-inflammatory substances known to induce neurotoxicity (3). Reactive oxygen intermediates (ROIs), one of several pro-inflammatory substances released by microglia (4), are likely to play a very important role in AD because hallmark modifications of ROI damage such as lipid peroxidation and nitrotyrosine conjugates are characteristic of post-mortem AD brains (3). Hence, pro-inflammatory stimuli that promote microglial ROI production might contribute to the pathogenesis of AD.

ATP is an important messenger in the brain and can be released from cells by both lytic and non-lytic mechanisms (5). ATP evokes a variety of biological responses in microglia (6-9). The effects of ATP are mediated through interactions with the P2 purinoceptors, broadly classified into P2Y metabotropic and P2X ionotropic receptors (10). The P2Y receptors are G protein-coupled and P2X receptors are ligand-gated ion channels (10). Whereas the P2Y receptors are responsible for Ca2+ release predominantly from intracellular stores, P2X receptors are responsible for Ca2+ influx from extracellular sources.

Microglia possess both P2Y and P2X receptors (11-13). The P2X7 receptor is highly expressed by cells of the macrophage lineage, such as dendritic cells, alveolar macrophages, and microglia. Activation of the P2X7 receptor is unique in triggering the formation of large nonselective membrane pores, permeable to molecules up to 900 Da which ultimately results in death of the cell (9, 14). ATP and ATP analogs have been used to characterize the role of P2 receptors in microglial activation. Micromolar concentrations of ATP are required to activate the P2Y receptors, whereas millimolar (1-5 mM) concentrations of ATP are required to activate the P2X receptors. The ATP analog BzATP is a selective agonist at the P2X receptor and does not bind P2Y receptors (15, 16). Oxidized ATP (oATP) is a specific antagonist of P2X7 that binds irreversibly to the receptor and prevents its activation by ATP (17). In this study, these pharmacological tools were used to determine the purinergic receptors involved in O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in microglia.

The P2X7 receptor plays a role in the generation of superoxide in microglia. Our studies elucidate a putative signal transduction pathway that mediates this response. These studies also demonstrate that BzATP- and ATP-activated microglia can mediate neurotoxicity. Finally, a distinct alteration was detected in the staining pattern for P2X7 receptor in a transgenic mouse model of AD, suggesting that P2X7 receptor activation could play a contributing role in AD.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- Reagents not specified otherwise were obtained from Sigma. PD98059, SB203580, LY294002, and DPI were obtained from Biomol (Plymouth Meeting, PA). p38 MAPK and p42/44 MAPK phospho-antibody kits were obtained from New England Biolabs (Beverly, MA). P2X7 and p67phox antibodies were obtained from Pharmingen. Anti-CD45 was purchased from Serotec (Oxford, UK). Amplex red kit and Fluo-4 were from Molecular Probes (Eugene, OR). Hematoxylin kit obtained from Shandon, Inc. (Pittsburgh, PA). The Tg2576 transgenic mice overexpressing mutant APP (K670N,M671L) and control mice were purchased from the Mayo Clinic (Jacksonville, FL).

Isolation of Microglia-- Rat microglia were prepared from 2-day-old Sprague-Dawley rat pups. The rat cortices were separated from meninges and minced, triturated, and centrifuged (200 × g for 10 min) to remove dead cells. The pellet was resuspended in media and triturated, and two brains were transferred to a 175-mm2 flask containing medium and incubated at 37 °C, 95% relative humidity in a 5% CO2 atmosphere. The medium was changed after 3-4 days and twice a week thereafter. Microglia were isolated on day 10 by shaking the flasks on an orbital shaker (VWR Scientific) at 125 rpm for 15 min. The supernatant was passed through a sterile nylon mesh (20 µM) (VWR Scientific), and cells were collected by centrifugation (200 × g for 10 min) and used the same day. The purity of the cultures was 98-100% as determined by immunostaining with ED-40 antibody.

Isolation of Cortical Neurons-- Primary cortical cell cultures were prepared from embryos of timed pregnant Sprague-Dawley rats at E14 (18). Briefly, the cortex triturated in DNase/Protease dissociation buffer was centrifuged and resuspended in PC-1 SF medium (BioWhittaker). The cells (2 × 105/ml) were plated onto poly-L-ornithine-coated 24-well plates, and 4 days later the media were replaced with Neurobasal Medium containing B-27 supplement (Invitrogen), 1% penicillin/streptomycin, and 10 mM L-glutamine. Neuronal cells constituted 90-95% of the total cells and were used on day 10 for experiments.

Isolation of Neutrophils-- Neutrophils were isolated from peripheral blood of healthy human donors as reported previously (19).

Measurement of Superoxide Production-- Superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>) was measured indirectly through the detection of hydrogen peroxide (H2O2) by the method of Mohanty et al. (20). O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production was measured in initial experiments by O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>-dependent superoxide dismutase-sensitive reduction of ferricytochrome c (21). However, microglia released very little O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, and this procedure required a large number of cells. In subsequent experiments the more sensitive method of H2O2 detection using conversion of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly fluorescent resorufin in the presence of horseradish peroxidase was followed (20). Briefly, 5 × 105/ml microglia in Hanks' balanced salt solution (HBSS) were preincubated with the inhibitors for either 2 (oATP, PPADS) or 1 h (SB203580, PD98059, or LY294002, Brilliant Blue G) or 30 min (DPI, AEBSF, apocyanin) in a 96-well plate. The various stimuli were directly added into the plate containing cells in the presence of 0.2 units/ml horseradish peroxidase, 50 µM Amplex Red, and the change in fluorescence was measured at 590 nm after excitation at 544 nm every 2.5 min using a fluorometric plate reader (Fluostar, BMG Labtechnologies, Durham, NC). The H2O2 released was calculated as picomoles of H2O2, 1 × 105 cells using a standard curve generated using known amounts of H2O2.

Intracellular Measurement of Superoxide Production-- The conversion of nitro blue tetrazolium (NBT) to formazan was used to detect the intracellular generation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (21, 22). Briefly, NBT at a final concentration of 1 mg/ml was added to wells containing cells. After treatment, the cells were applied to a glass slide using a Cytospin III (Shandon Southern, Sewickley, PA) and counterstained with safranin. The number of purple granules of formazan was counted microscopically to give a qualitative measure O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation.

Measurement of Calcium-- Isolated rat microglia were plated onto 384-well plates (Falcon, part 353961) at ~50% confluence. Cells were loaded with Fluo-4,AM (5 µM) in HBSS containing 10 mM HEPES (pH 7.4) for 1 h before the experiment at room temperature and washed with the buffer. ATP and BzATP were used to stimulate the [Ca2+]i signal. In the experiments where oATP and PPADS were used, cells were pretreated with the inhibitors for 2 h at 37 °C and then loaded with Fluo-4,AM. The fluorescent signal from ~104 cells per well was measured using a fluorometric plate reader (FLIPR, Molecular Devices). Fluo-4 was excited at 488 nm, and fluorescence was measured at 510 nm in a time-resolved mode (1-Hz frequency). Relative f/f0 intensity (in counts/ms) was used as an indication of [Ca2+]i signal. Data acquisition and preliminary analysis were done using FLIPR software (Molecular Devices). All calcium measurements were done at room temperature.

Membrane Translocation of p67phox-- Fractionation of microglia was performed according to the method of Zhao et al. (23). Briefly, microglia (14 × 106 cells/ml) in HBSS were treated with or without 500 µM BzATP for 5-10 min. Cells were centrifuged, and the cell pellet was resuspended in 0.5 ml of relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 10 mM PIPES (pH 7.3), 500 µM phenylmethylsulfonyl fluoride, and 1:100 dilution of protease inhibitor mixture), sonicated (3 × 10 s, 4 °C using a microprobe sonicator), and centrifuged (500 × g for 10 min) to remove nuclei and unbroken cells. The post-nuclear lysates were then ultracentrifuged (100,000 × g for 60 min, 4 °C), and the resulting supernatant was designated the cytosolic fraction. The membrane/particulate pellet was resuspended in 200 µl of relaxation buffer containing 1% Triton X-100. Protein concentration was estimated using the Bio-Rad DC Protein Assay, and 25 µg of protein (for both the cytosolic and membrane/particulate fraction) was loaded onto a gel.

A spontaneously occurring rat microglial cell line was used for the above experiment because we were unable to generate sufficient numbers of primary microglia required for this experiment. The spontaneously occurring rat microglial cell line was isolated from primary rat microglia growing in LADMAC-conditioned media (ATCC, Manassas, VA). The cells were propagated in media (Dulbecco's modified Eagle's medium, 1% penicillin/streptomycin, 10 mM L-glutamine, 0.1 mM nonessential amino acids, 10% fetal bovine serum) containing 20% LADMAC-conditioned media. The cells were positive for ED-1, a microglial marker. The microglial cells responded to both LPS and BzATP as demonstrated by the generation of TNFalpha with LPS and ROIs with BzATP (data not shown).

Immunoblotting-- Immunoblotting was performed as described previously (18). Briefly 25 µg of protein was fractionated on a 10% SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and blocked in 5% milk/Tris-buffered saline containing 0.1% Tween 20 for 2 h. The membrane were washed and incubated overnight with antibodies specific for phospho-p42/44 MAPK (Thr-202/Tyr-204), phospho-p38 MAPK (Thr-180/Tyr-182) diluted 1:1000 in TBST containing 5% bovine serum albumin. Membranes were washed with TBST and incubated with an horseradish peroxidase-conjugated secondary antibody (1:2000) for 2 h. The membrane was washed extensively, and bands were detected using LumiGLO. The membranes were stripped using RESTORE Western blot stripping buffer (Pierce), washed several times, and blocked for 1 h. Membranes were incubated with antibodies specific for either unphosphorylated p42/44 MAPK or p38 MAPK diluted 1:1000 in blocking buffer. The next day membranes were incubated with the secondary antibody and visualized using LumiGLO.

The P2X7 (1:500), p67phox (1:500), and actin (1:750) antibodies were used according to the manufacturer's recommendation. In some experiments a P2X7 control peptide corresponding to amino acid 576-595 of rat P2X7 (the immunogen used to generate the antibody) was utilized to determine specificity of the bands. The P2X7 antibody was preincubated with the control peptide at a 1:1 dilution (v/v) for 1 h at room temperature prior to the addition to the membrane.

Neurotoxicity Assay-- Primary rat microglia (1 × 105) in Neurobasal Medium containing B-27, 1% penicillin/streptomycin, and 10 mM L-glutamine were seeded into a 48-well plate containing 1 × 105 primary cortical neurons. The cells were allowed to settle for 2 h prior to the addition of stimuli. After a 72-h incubation, the supernatant was assayed for lactate dehydrogenase (LDH). Microglia and cortical cells were also independently cultured for 72 h in the presence of stimuli, and LDH released from microglia alone ± stimuli were subtracted out from the values obtained from the combination of microglia and cortical neurons. The LDH was measured with a commercial kit obtained from Promega (WI). In one experiment a WST-1 cell survival assay was performed on the cells remaining in the well with a commercial kit obtained from Roche Diagnostics. WST-1 is a modified 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium assay. The WST-1 assay enables colorimetric measurement of cell viability based on the cleavage of tetrazolium salts by mitochondrial dehydrogenase in viable cells.

TNFalpha Enzyme-linked Immunosorbent Assay-- Supernatants were assayed for TNFalpha using OPtEIA Rat TNFalpha kit (Pharmingen).

Nitrite Assay-- Nitrite assay was performed in a 96-well plate using modified Griess Reagent. In brief, 100 µl of Griess Reagent was added to 100 µl of supernatant in a 96-well plate. Samples were read at 540 nm, and values were calculated against a sodium nitrite standard curve.

Tissue Processing and Immunohistochemistry-- The mice were sedated, perfused with 4% paraformaldehyde and decapitated. The brains (2-year Tg2576 mice and aged-matched controls) were removed and fixed in 4% paraformaldehyde. The brains were dehydrated in graded alcohol solutions followed by Histoclear and embedded in paraffin. Longitudinal serial sections were cut at 6-µm thickness.

The sections were deparaffinized in Histoclear, rehydrated through a series of graded alcohols, washed in deionized water, and incubated in 1:5 methanol/water solution containing 3% H2O2 for 30 min to quench endogenous peroxidase activity. The slides were rinsed in deionized water for 5 min followed by blocking in 5% normal goat serum in phosphate-buffered saline containing 0.01% Triton-X-100 for 1 h. Sections were incubated with primary antibody (Pan A-beta 1:1000 (QCB), 4G8 1:1000 (Signet), P2X7 1:100 (Pharmingen), CD45 1:200 (Serotec), or GFAP 1:1000 (Chemicon)) in 1% normal goat serum in phosphate-buffered saline overnight at 4 °C. Immunohistochemistry was completed with appropriate biotinylated secondary antibody (1:500) in 2% normal goat serum/phosphate-buffered saline followed by avidin-biotin complex and visualized by diaminobenzidine development (Vector Laboratories). Primary or secondary antibodies were omitted from some sections to serve as negative controls. After the enzyme substrate (Vector Laboratories) was added, the manufacturer's protocol was followed. The slides were washed in water and counter-stained with Shandon-Lippshaw hematoxylin stain for 2 min at room temperature. Sections were washed in water for 1 min, incubated for 10 s in 50% ethanol/ H2O + 1% HCl to remove residual hematoxylin, and then washed in water for 1 min. The slides were then dehydrated in a series of alcohol washes and sealed with a coverslip using DPX mounting medium.

Immunofluorescence was used to detect dual antigen labeling. Tissue sections were deparaffinized via a series of xylenes and alcohols. Sections were blocked in 10% donkey serum for 1 h and then incubated in primary antibodies using 1% serum in Tris-buffered saline overnight at 4 °C. Primary antibodies (1:1000) specific for GFAP and P2X7 were pooled for dual immunofluorescence. Slides were then incubated in pooled secondary antibodies using donkey anti-mouse Cy-2 (1:100) and donkey anti-rabbit Cy-3 (1:400) in 2% normal donkey serum in Tris-buffered saline for 1 h in the dark. Slides were mounted and coverslip sealed with Vectashield (Vector Laboratories, Burlingame, CA). Images were captured using a Zeiss Axiovert S100TV microscope with the Zeiss KS400 imaging system.

Generation of Hippocampal Lysates-- Hippocampi from Tg2576 mice and age-matched controls (19 months, 1 male and 2 female) were excised and snap-frozen in liquid nitrogen and stored at -80 °C. Tissues were homogenized for 20 s on ice in TNE buffer (50 mM Tris, 150 mM NaCl) at 20% weight/volume using a Polytron homogenizer. Samples were then diluted 1:1 in TNE buffer containing 2% SDS, 1% Nonidet P-40, and 1% deoxycholate, and sonicated (2 × 15 s, 4 °C). Protein concentration was estimated using the Bio-Rad DC Protein Assay, and 25 µg of protein from each hippocampus was loaded onto a gel.

Statistics-- Student's t test was performed to determine group differences (p < 0.05).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Reactive Oxygen Intermediates by Microglia-- Primary rat microglia stimulated with ATP and BzATP rapidly generate ROIs, superoxide (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>) in particular, measured indirectly as hydrogen peroxide (H2O2). Fig. 1 shows time- and dose-response curves of H2O2 generated by microglia stimulated with ATP or BzATP. The response to ATP was maximal at 1 mM. The production of H2O2 continued slowly for at least 90 min after stimulation. The H2O2 production by microglia stimulated with BzATP was rapid and peaked by about 30 min. The maximal stimulus was ~250 µM. The magnitude of the response was higher in cells treated with BzATP compared with ATP. The total amount of H2O2 produced (picomoles) with stimulation by ATP and BzATP was lower than that generated by 10 ng/ml phorbol 12-myristate 13-acetate (PMA). However at early time points (5 min) 250 µM BzATP generated 9-10-fold more H2O2 than PMA (Fig. 1C). These results demonstrate there is a distinct difference in both the magnitude and duration of H2O2 production depending on the stimulus.


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Fig. 1.   Effect of ATP and BzATP on H2O2 production in microglia. Primary rat microglia (1 × 105) were plated onto a 96-well plate in HBSS. The H2O2 production in microglia stimulated with various concentrations of ATP (A), various concentrations of BzATP (B), or various stimuli (C) was determined every 5 min for 90 min. The data are the means of quadruplicate samples repeated three times. O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in the presence and absence of superoxide dismutase (SOD) was determined in 2 × 105 microglia (D) or 1 × 105 neutrophils (E) stimulated with BzATP (100-250 µM) or PMA (20 ng/ml) for 30 min. The data are the mean ± S.D. of triplicate samples repeated at least twice.

The conversion of Amplex Red to highly fluorescent resorufin in the presence of H2O2 is an indirect measure of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation. Hence the production of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> was confirmed by a more direct but less sensitive method. The inhibition of reduction of ferricytochrome c by O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>-dependent superoxide dismutase was used to detect the generation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. As shown in Fig. 1D, microglia treated with BzATP showed a significant increase in superoxide dismutase-inhibitable reduction of ferricytochrome c compared with untreated microglia. O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by neutrophils treated with BzATP was examined as a control. Similar increases in O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> were observed when neutrophils were stimulated with BzATP suggesting that BzATP activates a similar cascade in neutrophils (Fig. 1E). To determine whether BzATP generated any intracellular O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, the reduction of NBT in neutrophils treated with BzATP was examined. In neutrophils treated with BzATP or PMA but not control cells, formations of purple granules of formazan were visible microscopically indicating that NBT was being reduced to formazan (data not shown). These results confirm that cells stimulated with BzATP generate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. Because microglia produce little O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, the conversion of Amplex Red to highly fluorescent resorufin, a more sensitive but indirect indicator of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production (20), was used as the choice reagent in the remaining experiments.

Effect of ATP and BzATP on Calcium-- Treatment of microglia with ATP or BzATP resulted in a very rapid increase in the level of intracellular calcium (Fig. 2). ATP stimulated a transient increase of intracellular calcium (Fig. 2A). BzATP caused a sustained increase in the level of intracellular free calcium ([Ca2+]i) that was maintained for more than 6 min (Fig. 2B). The concentration of ATP or BzATP required for maximal [Ca2+]i change was lower than required for maximal H2O2 production. Maximal [Ca2+]i changes were stimulated by 30-100 µM of both ATP and BzATP, whereas maximal ROI production required 1 mM ATP and 250µM BzATP (Figs. 1 and 2). To determine whether ATP and BzATP were mobilizing intracellular or extracellular sources of Ca2+ or both, additional experiments were carried out in Ca2+-free media containing 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Ca2+ chelator). In the absence of extracellular Ca2+, the BzATP response was completely blocked indicating that BzATP was mobilizing only extracellular Ca2+ (Fig. 2C). However, with ATP, the initial transient peak was reduced by about 75%, suggesting that ATP mobilizes both intracellular (via inositol 1,4,5-trisphosphate-induced Ca2+ release) and extracellular sources of Ca2+ (possibly via capacitative Ca2+ influx) (24, 25).


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Fig. 2.   Intracellular free Ca2+ changes in cells treated with ATP and BzATP in the presence and absence of extracellular Ca2+. Primary rat microglia (1 × 104) were plated onto a 384-well plate and stimulated 10 s after beginning the experiment with various concentrations of ATP in HBSS (A), various concentrations of BzATP in HBSS (B), 100 µM ATP or 100 µM BzATP in HBSS without Ca2+ or Mg2+ containing 0.5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (C). The response was measured using a fluorescence plate reader. The black lines represent responses in the presence and the gray lines represent responses in the absence of Ca2+/Mg2+ ions. The data are the means of quadruplicate samples repeated at least twice.

Effect of Extracellular Calcium on ROI Production-- Because ATP appeared to stimulate Ca2+ release from intracellular stores and Ca2+ influx from extracellular sources, whereas BzATP appeared to stimulate only Ca2+ influx from extracellular sources, the effect of removal of extracellular Ca2+ on H2O2 production was examined. Both ATP- and BzATP-stimulated H2O2 production was blocked to below control levels in the absence of extracellular Ca2+ (Fig. 3). These results suggest that despite the differences in Ca2+ mobilization, both ATP and BzATP required only extracellular Ca2+ to generate H2O2.


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Fig. 3.   Effect of EGTA on ATP- and BzATP-induced H2O2 production. Primary rat microglia (1 × 105) were plated onto a 96-well plate in either HBSS or HBSS without Ca2+ and Mg2+ containing 500 µM EGTA and stimulated with various concentrations of ATP or BzATP. The data are the means ± S.E. of two experiments each done in quadruplicate.

Receptors Involved in the Generation of ROI-- The ability of BzATP, an agonist of P2X receptors, to stimulate H2O2 production and the requirement of extracellular Ca2+ for this response suggest P2X receptors mediate the production of H2O2 in microglia. To determine whether the production of H2O2 was mediated through the P2X7 receptor, two selective inhibitors of P2X7, PPADS and oATP, were tested. Both PPADS and oATP blocked H2O2 production by BzATP treatment (Fig. 4A) suggesting that BzATP activates H2O2 production primarily through the P2X7 receptor. Further support for the role of P2X7 in H2O2 production was obtained by treating cells with Brilliant Blue G, a potent and highly selective inhibitor of P2X7, at nanomolar concentrations (26). Brilliant Blue G (500 nM) inhibited BzATP (250 µM)-induced H2O2 production by more than 80% (Fig. 4A).


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Fig. 4.   Effect of P2X selective inhibitor oATP and PPADS on BzATP-induced H2O2 production and calcium changes. Primary rat microglia (1 × 105) were plated and pretreated with oATP or PPADS for 2 h or Brilliant Blue-G (BB) for 1 h. H2O2 production (A) or Ca2+ changes (B) were measured in cells treated with BzATP. The data are the means ± S.E. of five (PPADS, oATP) or three (Brilliant Blue-G (BB)) experiments each done in quadruplicate. The data are presented as a percent of H2O2 produced by microglia stimulated with 250 µM BzATP for 30 min. * indicates p < 0.05 by Student's t test.

To determine whether oATP and PPADS affected Ca2+ responses similarly, Ca2+ changes were measured in cells pretreated with oATP and PPADS in the presence or absence of BzATP. oATP (100 µM) inhibited BzATP-induced Ca2+ flux to near control levels (Fig. 4B). Similar results were obtained with PPADS (Fig. 4B). These results suggest that P2X7 is the primary receptor stimulated by BzATP to generate H2O2.

Source of ROI-- Several sources can contribute to the production of ROI. These include the classical NADPH oxidase, the mitochondrial respiratory chain, and microsomal enzymes. Pharmacological inhibitors of the NADPH oxidase were used to determine whether P2X7 receptor activates NADPH oxidase. Three selective inhibitors with different mechanisms of action, diphenyleneiodonium chloride (DPI), apocyanin, and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) were used (27, 28). As shown in Fig. 5A, all three inhibitors completely inhibited BzATP-induced H2O2 release from microglia. To confirm the activation of NADPH oxidase by BzATP in microglia, a functional change in NADPH oxidase was examined. A critical step in the activation of the NADPH oxidase is the translocation of p67phox from the cytosol to the membrane. In BzATP-stimulated microglia, p67phox, which is primarily cytosolic, rapidly translocated to the particulate/membrane fraction (Fig. 5B). These results suggest that BzATP stimulates the release of ROI in microglia via the activation of the NADPH oxidase.


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Fig. 5.   NADPH oxidase is activated by BzATP. A, primary rat microglia (1 × 105) were plated and pretreated with AEBSF, apocyanin, or DPI for 30 min, and H2O2 production was measured in cells stimulated with 250 µM BzATP. The data are the means ± S.D. of quadruplicate samples repeated three times. * indicates p < 0.05 by Student's t test. B, microglia were left untreated or activated with 500 µM BzATP for 5-10 min. The cells were then collected and fractionated as described under "Materials and Methods." p67phox was detected using a polyclonal antibody.

Signal Transduction Cascade Involved in the Generation of H2O2-- The production of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>/H2O2 is stimulated through several different signal transduction pathways. Two kinases implicated in the activation of the NADPH oxidase are p42/44 ERK and p38 MAPK (29-31). Both p42/44 ERK and p38 MAPK were rapidly activated in cells stimulated with 250 µM BzATP (Fig. 6). However, pretreatment with SB203580 (a selective p38 MAPK inhibitor) but not PD98059 (a selective p42/44 MAPK inhibitor) attenuated H2O2 production in cells stimulated with BzATP (Fig. 6). Nonetheless, PD98059 did attenuate BzATP-induced TNFalpha release suggesting that p42/44 MAPK was involved in cytokine signaling but not in the generation of H2O2 (data not shown). LY294002, a selective phosphatidylinositol 3-kinase inhibitor (PI3-K) also significantly inhibited H2O2 stimulated by BzATP. LY294002 at 50 and 10 µM inhibited H2O2 production by 74.7 ± 2 (n = 3) and 47 ± 5% (n = 3), respectively. The inhibitors used were not toxic to the cells at the concentrations used. These results suggest that whereas both p38 MAPK and p42/44 ERK are rapidly activated in microglia stimulated with BzATP, only the inhibition of p38 MAPK attenuates H2O2 production. Furthermore, PI3-K may play a more important role in the release of H2O2 than p38 MAPK because the PI3-K inhibitor blocks H2O2 release to a greater extent than the p38 MAPK inhibitor.


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Fig. 6.   The signal transduction cascade activated by BzATP in the generation of H2O2. A, primary rat microglia (1 × 105) were plated and pretreated with SB203580, PD98059, or LY294002 for 1 h, and then H2O2 production was measured in cells stimulated with 250 µM BzATP. The data represent the means ± S.E. of three experiments each done in quadruplicate. The data are presented as a percent of H2O2 produced by microglia stimulated with 250 µM BzATP for 30 min. * indicates p < 0.05 by Student's t test. Lysates of cells stimulated with 250 µM BzATP or 100 ng/ml of LPS for various periods were probed with antibodies against phosphorylated p38 or unphosphorylated p38 MAPK antibodies (B) against phosphorylated p42/44 MAPK or unphosphorylated p42/44 MAPK (C).

Microglia Stimulated with ATP or BzATP Are Neurotoxic-- To determine whether activation of microglia with ATP and BzATP is neurotoxic, a co-culture system using highly purified primary rat cortical neurons and primary rat microglia was employed. Stimulation of microglia with LPS resulted in massive production of TNFalpha and nitric oxide but no H2O2 release (Figs. 1 and 7, B and C). LPS did not stimulate LDH release from microglia/cortical neurons co-cultures (Fig. 7A). Conversely, ATP or BzATP stimulated very little TNFalpha and nitric oxide production but induced neurotoxicity at 72 h (Fig. 7). There was no significant neurotoxicity up to 48 h post-stimulation with either ATP or BzATP. The amount of neurotoxicity was much greater with BzATP compared with ATP, which is consistent with the observation that BzATP is a more potent stimulus compared with ATP in the generation of H2O2. The values shown in Fig. 7A represent LDH released from both microglia and cortical cells minus LDH released from microglia alone. A WST-1 cell survival assay was used to confirm that the LDH release is a measure of neuronal toxicity. Neither ATP nor BzATP had a significant neurotoxic effect on cortical neurons (Fig. 7A). Results comparing LPS-treated co-cultures to ATP/BzATP-treated co-cultures show that factors other than TNFalpha or nitric oxide are contributing to toxicity in our system.


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Fig. 7.   Toxicity of microglia stimulated with ATP or BzATP toward primary rat cortical neurons. A, primary rat microglia were co-cultured with primary rat cortical neurons without any stimuli or in the presence of 100 ng/ml LPS, 1 mM ATP, or 250 µM BzATP. The viability of cortical neurons after 72 h of incubation was assessed by LDH release into the supernatant. The data represent the mean ± S.E. of three separate experiments each done in triplicate expressed as a percentage of LDH released into the supernatant compared with control (LDH released from the co-culture of microglia and cortical neurons in the absence of any stimuli). * indicates p < 0.05 by Student's t test. B and C, microglia were treated with LPS (100 ng/ml), BzATP (500 µM), and ATP (3 mM) for various times. Media were assayed for TNFalpha release by enzyme-linked immunosorbent assay (B) and nitrite release by modified Greiss reagent (C). The data represents the mean ± S.D. of triplicate samples repeated at least three times.

Up-regulation of P2X7 Receptor in a Transgenic Mouse Model of Alzheimer's Disease-- By having demonstrated that ATP- and BzATP-stimulated microglia release H2O2 and kill cortical neurons in vitro, we examined the brains of a transgenic mouse model of AD (Tg2576 carrying a APP(K670N,M671L) double mutation) (32) to determine whether the mechanism is important in vivo. In 24-month-old transgenic mouse brains but not in aged-matched control brains, plaques were evident in the hippocampus and surrounding outer cortical region when stained with two different antibodies for Abeta (Fig. 8, A, B, and E; data not shown). P2X7 immunostaining gave a ring-like pattern around plaques only in transgenic mice suggesting that cells staining for P2X7 were surrounding the plaques (Fig. 8, C, D, and F). This staining was not evident in the absence of the primary antibody indicating that the staining was not due to nonspecific binding of the secondary antibody to the plaques. There was some basal staining with the P2X7 antibody in both control and transgenic animals suggesting low levels of P2X7 are expressed in the brain.


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Fig. 8.   P2X7 expression and localization in APP(K670N,M671L) transgenic and age-matched control mice. Two-year-old Tg2576 (B and D-F) and age-matched control mice (A and C) were stained for beta -amyloid with a pan-Abeta antibody (A, B, and E) and P2X7 with a polyclonal anti-P2X7 antibody (C, D, and F). Magnification bar = 300 µm for A-D and 50 µm for E-F. G, hippocampal lysates from three 19-month-old Tg2576 mice or age-matched controls were probed with an antibody against P2X7. The specificity of the P2X7 band was confirmed by the use of a blocking peptide as described under "Materials and Methods." The blot was then stripped and reprobed with an antibody against actin.

To determine whether the increased staining of P2X7 in the transgenic mice was due to increased expression of P2X7 receptor, lysates from the hippocampi (region with higher concentration of plaques) of three 19-month Tg2576 mice and age-matched controls were separated by SDS-PAGE, electrophoretically transferred, and probed with a polyclonal antibody specific for P2X7 (Fig. 8G). The signal for the 55-kDa isoform was higher in the transgenic mice compared with age-matched controls. Preincubation of the P2X7 antibody with the P2X7 peptide (amino acid 576-595) resulted in the disappearance of the P2X7 band and had no effect on the actin band, demonstrating the specificity of the antibody (Fig. 8G).

To determine the identity of cells expressing P2X7 around the plaques, serial sections were stained with markers selective for either pan-Abeta (Fig. 9A), P2X7 (Fig. 9, B and E), microglia (CD45 (Fig. 9C)), astrocytes (GFAP (Fig. 9F)), or P2X7 and GFAP (Fig. 9D). Microglial and astrocytic staining were seen surrounding plaques in the transgenic animals (Fig. 9, C and F, respectively). It could not be determined if P2X7 immunoreactivity was restricted specifically to either microglia or astrocytes; however, very little co-localization was detected by dual immunofluorescence with anti-GFAP and anti-P2X7 (assessed by the number of dual labeled yellow cells (Fig. 9D)). These results show that P2X7 is up-regulated in Tg2576 mice around amyloid plaques and is regionally localized with activated microglia and astrocytes.


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Fig. 9.   P2X7 immunoreactivity is associated with GFAP- and CD45-positive cells around plaques in APP(K670N,M671L) transgenic mice. Two-year-old Tg2576 mice (A-F) were stained with pan-Abeta antibody (A), P2X7 (B and E), CD45 (C), GFAP (F), or GFAP + P2X7 (D). Magnification bar = 50 µm. Photomicrographs A-C were obtained using immunohistochemistry using DAB substrate; D-F utilizing dual immunofluorescence (Cy-2-GFAP (green)/Cy-3-P2X7 (red)). Dual labeled cells are represented as yellow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The P2X7 receptor has been implicated in the activation of transcription factors, apoptosis, and in the release of pro-inflammatory substances like TNFalpha and interleukin-1beta in microglia (8, 9, 14, 33). In this report we demonstrate that P2X7 is the primary receptor involved in H2O2 production in primary rat microglia stimulated with ATP or BzATP. The P2X-selective agonist BzATP was a more potent stimulus than ATP, a P2Y/P2X agonist. Functionally, the activation of microglia with ATP or BzATP induced cell death in primary cortical neurons. In vivo there was a striking association of P2X7 receptor-positive cells around plaques in a transgenic mouse model of Alzheimer's disease.

No detailed study has profiled the expression of P2Y and P2X receptors in microglia, but microglial expression of both P2X and P2Y is supported by electrophysiological studies (13, 34). Our results demonstrating Ca2+ changes induced by ATP or BzATP also support the existence of functional P2X and P2Y receptors on primary rat microglia.

Several lines of evidence point to P2X receptors and P2X7, in particular, as the primary receptor involved in the generation of H2O2 in ATP- or BzATP-stimulated microglia. Stimulation of H2O2 production by P2X-selective agonist BzATP (35, 36) provides the first line of evidence for the involvement of P2X receptors. P2X receptors mobilize only extracellular Ca2+, consequently the experiments that demonstrate BzATP mobilizes only extracellular Ca2+ provide further evidence for the involvement of this receptor. The inability of BzATP to stimulate H2O2 production in the absence of extracellular Ca2+ provides a direct link between Ca2+ mobilization and H2O2 production. The inhibition of H2O2 production and Ca2+ influx by P2X-selective antagonist PPADS, P2X7-selective antagonist oATP (17), and P2X7-selective inhibitor Brilliant Blue-G in BzATP-stimulated cells provide additional lines of evidence for the involvement of P2X7 receptors. The contribution of other purinergic receptors cannot be excluded, and can be evaluated only by measuring H2O2 production in microglia lacking P2X7 receptors.

The levels of ATP required to stimulate the P2X7 receptor in vitro suggests that the low concentrations of ATP found in the extracellular milieu of the brain would not be sufficient for microglia to induce neurotoxicity. However, recent reports (6) suggest that low concentrations of ATP can act as a chemoattractant for microglia directing them to a region of injury. Furthermore, ATP released from activated astrocytes has been shown recently (37) to activate microglia. Finally, a recent report (38) has documented the significance, the source, and needed high concentrations of ATP to activate P2X7 receptors in the brain, as well as the role of ATP in neurodegeneration.

Endogenous production of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is a biochemical process that requires tight regulation. NADPH oxidase, a known generator of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, is regulated by an intricate signal transduction cascade within cell types such as neutrophils (39). Dysregulation of the oxidase leads to the damage of surrounding tissue and results in inflammatory conditions. Both p42/44 ERK and p38 MAPK play an important role in the regulation of various proteins of NADPH oxidase complex in neutrophils (29-31). However, little is known about the regulation of the NADPH oxidase in microglia. Although both p42/44 ERK and p38 MAPK were activated very rapidly in microglia stimulated with BzATP, only inhibition of p38 MAPK attenuated the release of H2O2. This suggests that there are subtle differences in the regulation of the NADPH oxidase complex in neutrophils compared with microglia. Other kinases like PI3-K promote NADPH oxidase activity through the PX domain of p47phox and p40phox (40). Inhibition of H2O2 levels by the PI3-K inhibitor, LY294002, suggests that PI3-K plays a similar yet more important role in the assembly of the oxidase complex in microglia compared with p38 MAPK.

Microglia activated by certain stimuli can induce cell death in cortical neurons. Although the neurotoxicity is attributed to various individual secretory products like TNFalpha , nitric oxide, and ROIs, toxicity is likely due to a combination of factors (41-43). The inability of LPS-activated microglia to induce cell death in cortical neurons despite large increases in TNFalpha and inducible nitric-oxide synthase shows that these products do not solely induce cell death in our microglia/cortical neuron co-culture system. Published reports support this observation. Klegeris et al. (44, 45) demonstrated that LPS-stimulated microglia are not neurotoxic but in combination with IFNgamma induces neurotoxicity. IFNgamma can prime microglia to generate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in the absence and presence of TNFalpha suggesting that TNFalpha could be indirectly inducing cell death by generating O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (46, 47). Moreover, both IFNgamma /LPS or IFNgamma /TNFalpha can up-regulate P2X7 expression in THP-1 cells and monocytes (48, 49). Given that ROIs can directly induce neurotoxicity, it will be important to determine the exact mechanism by which ATP and BzATP are inducing cortical cell death. The link between LPS, IFNgamma , and P2X7 expression has yet to be examined in detail in microglia.

Currently there are no reports on the expression profile of P2X7 in Alzheimer's disease. Ours is the first paper demonstrating a remarkable difference in the staining pattern for P2X7 in brain slices of a transgenic (Tg2576) mice model. This intense staining for P2X7 around plaques can be the result of up-regulation of the P2X7 receptor and/or aggregation of glia around plaques. The fact that P2X7 message and receptor is up-regulated in monocytes treated with LPS/IFNgamma or LPS/TNFalpha (48, 50) raises the possibility that P2X7 indeed can be up-regulated in the mouse model of AD. The increased P2X7 immunoreactivity in immunoblots comparing Tg2576 hippocampi lysates to age-matched control hippocampi lysates and the presence of P2X7-immunopositive cells around plaques supports this theory. However, the identity of the P2X7 immunopositive cells around plaques is still not clear, even though activated microglia and astrocytes are found in the same vicinity. Although a correlation has been established between increased P2X7 immunoreactivity and amyloid plaques, the question of cause and effect is beyond the scope of this paper. Because P2X7 knock out mice have been made, it will be interesting to see if microglia from these mice generate H2O2 in response to BzATP or if a P2X7 knock out mouse crossed with a Tg2576 mouse would have any alteration in plaque deposition. It is possible that receptor antagonists of P2X7 could have therapeutic utility in treatment of AD by regulating pathologically activated microglia.

    ACKNOWLEDGEMENTS

We thank Melissa Dodson for providing primary cortical neurons, Donna Barten for providing Tg2576 and age-matched mouse brains, Kristen Renzi for preparing the paraffin sections, and Alexander Vinitsky for critical review of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: CuraGen Corp., 322 East Main St., Branford, CT 06405. Tel.: 203-871-4432; E-mail: bparvathenani@curagen.com.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M209478200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; BzATP, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP; DPI, diphenyleneiodonium chloride; ERK, extracellular signal-regulated protein kinase; H2O2, hydrogen peroxide; IFNgamma , interferon-gamma ; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, superoxide; oATP, oxidized ATP; PI3-K, phosphatidylinositol 3-kinase; PMA, phorbol 12-myristate 13-acetate; PPADS, pyridoxal-5-phosphate-6-azophenyl-2'4-disulfonic acid; ROI, reactive oxygen intermediates; TNFalpha , tumor necrosis factor-alpha ; PIPES, 1,4-piperazinediethanesulfonic acid; HBSS, Hanks' balanced salt solution; NBT, nitro blue tetrazolium; GFAP, glial fibrillary acidic protein; APP, amyloid precursor protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Cagnin, A., Brooks, D. J., Kennedy, A. M., Gunn, R. N., Myers, R., Turkheimer, F. E., Jones, T., Banati, R. B., Pappata, S., Levasseur, M., Crouzel, C., Syrota, A., Kreutzberg, G. W., Newcombe, J., Turkheimer, F., Heppner, F., Price, G., Wegner, F., Giovannoni, G., Miller, D. H., Perkin, G. D., Smith, T., Hewson, A. K., Bydder, G., and Cuzner, M. L. (2001) Lancet 358, 461-467[CrossRef][Medline] [Order article via Infotrieve]
2. Pappata, S., Levasseur, M., Gunn, R. N., Myers, R., Crouzel, C., Syrota, A., Jones, T., Kreutzberg, G. W., and Banati, R. B. (2000) Neurology 55, 1052-1054[Abstract/Free Full Text]
3. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R., Eikelenboom, P., Emmerling, M., Fiebich, B. L., Finch, C. E., Frautschy, S., Griffin, W. S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I. R., McGeer, P. L., O'Banion, M. K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F. L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G., and Wyss-Coray, T. (2000) Neurobiol. Aging 21, 383-421[CrossRef][Medline] [Order article via Infotrieve]
4. Bianca, V. D., Dusi, S., Bianchini, E., Dal Pra, I., and Rossi, F. (1999) J. Biol. Chem. 274, 15493-15499[Abstract/Free Full Text]
5. Cunha, R. A., and Ribeiro, J. A. (2000) Life Sci. 68, 119-137[CrossRef][Medline] [Order article via Infotrieve]
6. Honda, S., Sasaki, Y., Ohsawa, K., Imai, Y., Nakamura, Y., Inoue, K., and Kohsaka, S. (2001) J. Neurosci. 21, 1975-1982[Abstract/Free Full Text]
7. Sanz, J. M., and Di Virgilio, F. (2000) J. Immunol. 164, 4893-4898[Abstract/Free Full Text]
8. Hide, I., Tanaka, M., Inoue, A., Nakajima, K., Kohsaka, S., Inoue, K., and Nakata, Y. (2000) J. Neurochem. 75, 965-972[CrossRef][Medline] [Order article via Infotrieve]
9. Ferrari, D., Chiozzi, P., Falzoni, S., Dal Susino, M., Collo, G., Buell, G., and Di Virgilio, F. (1997) Neuropharmacology 36, 1295-1301[CrossRef][Medline] [Order article via Infotrieve]
10. Di Virgilio, F., Chiozzi, P., Ferrari, D., Falzoni, S., Sanz, J. M., Morelli, A., Torboli, M., Bolognesi, G., and Baricordi, O. R. (2001) Blood 97, 587-600[Abstract/Free Full Text]
11. Kettenmann, H., Banati, R., and Walz, W. (1993) Glia 7, 93-101[Medline] [Order article via Infotrieve]
12. Norenberg, W., Langosch, J. M., Gebicke-Haerter, P. J., and Illes, P. (1994) Br. J. Pharmacol. 111, 942-950[Abstract]
13. Visentin, S., Renzi, M., Frank, C., Greco, A., and Levi, G. (1999) J. Physiol. (Lond.) 519, 723-736[Abstract/Free Full Text]
14. Ferrari, D., Los, M., Bauer, M. K., Vandenabeele, P., Wesselborg, S., and Schulze-Osthoff, K. (1999) FEBS Lett. 447, 71-75[CrossRef][Medline] [Order article via Infotrieve]
15. Nuttle, L. C., el-Moatassim, C., and Dubyak, G. R. (1993) Mol. Pharmacol. 44, 93-101[Abstract]
16. Ferrari, D., Villalba, M., Chiozzi, P., Falzoni, S., Ricciardi-Castagnoli, P., and Di Virgilio, F. (1996) J. Immunol. 156, 1531-1539[Abstract]
17. Murgia, M., Hanau, S., Pizzo, P., Rippa, M., and Di Virgilio, F. (1993) J. Biol. Chem. 268, 8199-8203[Abstract/Free Full Text]
18. Parvathenani, L. K., Calandra, V., Roberts, S. B., and Posmantur, R. (2000) Neuroreport 11, 2293-2297[Medline] [Order article via Infotrieve]
19. Parvathenani, L. K., Buescher, E. S., Chacon-Cruz, E., and Beebe, S. J. (1998) J. Biol. Chem. 273, 6736-6743[Abstract/Free Full Text]
20. Mohanty, J. G., Jaffe, J. S., Schulman, E. S., and Raible, D. G. (1997) J. Immunol. Methods 202, 133-141[CrossRef][Medline] [Order article via Infotrieve]
21. Johnston, R. B., Jr. (1984) Methods Enzymol. 105, 365-369[Medline] [Order article via Infotrieve]
22. Patterson, C., Ruef, J., Madamanchi, N. R., Barry-Lane, P., Hu, Z., Horaist, C., Ballinger, C. A., Brasier, A. R., Bode, C., and Runge, M. S. (1999) J. Biol. Chem. 274, 19814-19822[Abstract/Free Full Text]
23. Zhao, X., Bey, E. A., Wientjes, F. B., and Cathcart, M. K. (2002) J. Biol. Chem. 277, 25385-25392[Abstract/Free Full Text]
24. Toescu, E. C., Moller, T., Kettenmann, H., and Verkhratsky, A. (1998) Neuroscience 86, 925-935[CrossRef][Medline] [Order article via Infotrieve]
25. Wang, X., Kim, S. U., van Breemen, C., and McLarnon, J. G. (2000) Cell Calcium 27, 205-212[CrossRef][Medline] [Order article via Infotrieve]
26. Jiang, L. H., Mackenzie, A. B., North, R. A., and Surprenant, A. (2000) Mol. Pharmacol. 58, 82-88[Abstract/Free Full Text]
27. Stolk, J., Hiltermann, T. J., Dijkman, J. H., and Verhoeven, A. J. (1994) Am. J. Respir. Cell Mol. Biol. 11, 95-102[Abstract]
28. Diatchuk, V., Lotan, O., Koshkin, V., Wikstroem, P., and Pick, E. (1997) J. Biol. Chem. 272, 13292-13301[Abstract/Free Full Text]
29. Rane, M. J., Carrithers, S. L., Arthur, J. M., Klein, J. B., and McLeish, K. R. (1997) J. Immunol. 159, 5070-5078[Abstract]
30. Lal, A. S., Clifton, A. D., Rouse, J., Segal, A. W., and Cohen, P. (1999) Biochem. Biophys. Res. Commun. 259, 465-470[CrossRef][Medline] [Order article via Infotrieve]
31. Dewas, C., Fay, M., Gougerot-Pocidalo, M. A., and El-Benna, J. (2000) J. Immunol. 165, 5238-5244[Abstract/Free Full Text]
32. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F., and Cole, G. (1996) Science 274, 99-102[Abstract/Free Full Text]
33. Ferrari, D., Wesselborg, S., Bauer, M. K., and Schulze-Osthoff, K. (1997) J. Cell Biol. 139, 1635-1643[Abstract/Free Full Text]
34. Norenberg, W., Cordes, A., Blohbaum, G., Frohlich, R., and Illes, P. (1997) Br. J. Pharmacol. 121, 1087-1098[Abstract]
35. el-Moatassim, C., and Dubyak, G. R. (1992) J. Biol. Chem. 267, 23664-23673[Abstract/Free Full Text]
36. Nuttle, L. C., and Dubyak, G. R. (1994) J. Biol. Chem. 269, 13988-13996[Abstract/Free Full Text]
37. Verderio, C., and Matteoli, M. (2001) J. Immunol. 166, 6383-6391[Abstract/Free Full Text]
38. Le Feuvre, R., Brough, D., and Rothwell, N. (2002) Eur. J. Pharmacol. 447, 261-269[CrossRef][Medline] [Order article via Infotrieve]
39. Bokoch, G. M. (1995) Blood 86, 1649-1660[Free Full Text]
40. Ellson, C. D., Gobert-Gosse, S., Anderson, K. E., Davidson, K., Erdjument-Bromage, H., Tempst, P., Thuring, J. W., Cooper, M. A., Lim, Z. Y., Holmes, A. B., Gaffney, P. R., Coadwell, J., Chilvers, E. R., Hawkins, P. T., Stephens, L. R., Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E., Cantley, L. C., and Yaffe, M. B. (2001) Nat. Cell Biol. 3, 679-682[CrossRef][Medline] [Order article via Infotrieve]
41. Combs, C. K., Karlo, J. C., Kao, S. C., and Landreth, G. E. (2001) J. Neurosci. 21, 1179-1188[Abstract/Free Full Text]
42. Bal-Price, A., and Brown, G. C. (2001) J. Neurosci. 21, 6480-6491[Abstract/Free Full Text]
43. McDonald, D. R., Brunden, K. R., and Landreth, G. E. (1997) J. Neurosci. 17, 2284-2294[Abstract/Free Full Text]
44. Klegeris, A., Walker, D. G., and McGeer, P. L. (1999) Neuropharmacology 38, 1017-1025[CrossRef][Medline] [Order article via Infotrieve]
45. Klegeris, A., McGeer, P. L., and Walker, D. G. (2000) J. Leukocyte Biol. 67, 127-133[Abstract]
46. Janabi, N., Chabrier, S., and Tardieu, M. (1996) J. Immunol. 157, 2129-2135[Abstract]
47. Hu, S., Sheng, W. S., Peterson, P. K., and Chao, C. C. (1995) Glia 13, 45-50[Medline] [Order article via Infotrieve]
48. Humphreys, B. D., and Dubyak, G. R. (1998) J. Leukocyte Biol. 64, 265-273[Abstract]
49. Humphreys, B. D., and Dubyak, G. R. (1996) J. Immunol. 157, 5627-5637[Abstract]
50. Buell, G., Chessell, I. P., Michel, A. D., Collo, G., Salazzo, M., Herren, S., Gretener, D., Grahames, C., Kaur, R., Kosco-Vilbois, M. H., and Humphrey, P. P. (1998) Blood 92, 3521-3528[Abstract/Free Full Text]


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