FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2153177211> ?p ?o ?g. }

• W2153177211 endingPage "13317" @default.
• W2153177211 startingPage "13309" @default.
• W2153177211 abstract "Primary rat microglia stimulated with either ATP or 2′- and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O2⨪). ATP and BzATP stimulate O2⨪ production through purinergic receptors, primarily the P2X7 receptor. O2⨪ 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 O2⨪production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O2⨪ 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 β-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576). Primary rat microglia stimulated with either ATP or 2′- and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O2⨪). ATP and BzATP stimulate O2⨪ production through purinergic receptors, primarily the P2X7 receptor. O2⨪ 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 O2⨪production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O2⨪ 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 β-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576). Alzheimer's disease 4-(2-aminoethyl)-benzenesulfonyl fluoride 2′- and 3′-O-(4-benzoylbenzoyl)-ATP diphenyleneiodonium chloride extracellular signal-regulated protein kinase hydrogen peroxide interferon-γ lipopolysaccharide mitogen-activated protein kinase superoxide oxidized ATP phosphatidylinositol 3-kinase phorbol 12-myristate 13-acetate pyridoxal-5-phosphate-6-azophenyl-2′4-disulfonic acid reactive oxygen intermediates tumor necrosis factor-α 1,4-piperazinediethanesulfonic acid Hanks' balanced salt solution nitro blue tetrazolium glial fibrillary acidic protein amyloid precursor protein Activated microglia have been observed in patients suffering from both acute (stroke) and chronic (Alzheimer's disease) neurological disorders (1Cagnin 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. Cuzner M.L. Lancet. 2001; 358: 461-467Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar, 2Pappata S. Levasseur M. Gunn R.N. Myers R. Crouzel C. Syrota A. Jones T. Kreutzberg G.W. Banati R.B. Neurology. 2000; 55: 1052-1054Crossref PubMed Scopus (217) Google Scholar). 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 (3Akiyama 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. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3733) Google Scholar). Reactive oxygen intermediates (ROIs), one of several pro-inflammatory substances released by microglia (4Bianca V.D. Dusi S. Bianchini E. Dal Pra I. Rossi F. J. Biol. Chem. 1999; 274: 15493-15499Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), 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 (3Akiyama 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. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3733) Google Scholar). 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 (5Cunha R.A. Ribeiro J.A. Life Sci. 2000; 68: 119-137Crossref PubMed Scopus (177) Google Scholar). ATP evokes a variety of biological responses in microglia (6Honda S. Sasaki Y. Ohsawa K. Imai Y. Nakamura Y. Inoue K. Kohsaka S. J. Neurosci. 2001; 21: 1975-1982Crossref PubMed Google Scholar, 7Sanz J.M. Di Virgilio F. J. Immunol. 2000; 164: 4893-4898Crossref PubMed Scopus (232) Google Scholar, 8Hide I. Tanaka M. Inoue A. Nakajima K. Kohsaka S. Inoue K. Nakata Y. J. Neurochem. 2000; 75: 965-972Crossref PubMed Scopus (381) Google Scholar, 9Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Collo G. Buell G. Di Virgilio F. Neuropharmacology. 1997; 36: 1295-1301Crossref PubMed Scopus (244) Google Scholar). The effects of ATP are mediated through interactions with the P2 purinoceptors, broadly classified into P2Y metabotropic and P2X ionotropic receptors (10Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (625) Google Scholar). The P2Y receptors are G protein-coupled and P2X receptors are ligand-gated ion channels (10Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (625) Google Scholar). 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 (11Kettenmann H. Banati R. Walz W. Glia. 1993; 7: 93-101Crossref PubMed Scopus (138) Google Scholar, 12Norenberg W. Langosch J.M. Gebicke-Haerter P.J. Illes P. Br. J. Pharmacol. 1994; 111: 942-950Crossref PubMed Scopus (93) Google Scholar, 13Visentin S. Renzi M. Frank C. Greco A. Levi G. J. Physiol. (Lond.). 1999; 519: 723-736Crossref Scopus (86) Google Scholar). 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 (9Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Collo G. Buell G. Di Virgilio F. Neuropharmacology. 1997; 36: 1295-1301Crossref PubMed Scopus (244) Google Scholar, 14Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (243) Google Scholar). 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 (15Nuttle L.C. el-Moatassim C. Dubyak G.R. Mol. Pharmacol. 1993; 44: 93-101PubMed Google Scholar, 16Ferrari D. Villalba M. Chiozzi P. Falzoni S. Ricciardi-Castagnoli P. Di Virgilio F. J. Immunol. 1996; 156: 1531-1539PubMed Google Scholar). Oxidized ATP (oATP) is a specific antagonist of P2X7 that binds irreversibly to the receptor and prevents its activation by ATP (17Murgia M. Hanau S. Pizzo P. Rippa M. Di Virgilio F. J. Biol. Chem. 1993; 268: 8199-8203Abstract Full Text PDF PubMed Google Scholar). In this study, these pharmacological tools were used to determine the purinergic receptors involved in O2⨪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. Reagents not specified otherwise were obtained from Sigma. PD98059, SB203580, LY294002, and DPI were obtained fromBiomol (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). 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. Primary cortical cell cultures were prepared from embryos of timed pregnant Sprague-Dawley rats at E14 (18Parvathenani L.K. Calandra V. Roberts S.B. Posmantur R. Neuroreport. 2000; 11: 2293-2297Crossref PubMed Scopus (13) Google Scholar). 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 mml-glutamine. Neuronal cells constituted 90–95% of the total cells and were used on day 10 for experiments. Neutrophils were isolated from peripheral blood of healthy human donors as reported previously (19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Superoxide (O2⨪) was measured indirectly through the detection of hydrogen peroxide (H2O2) by the method of Mohantyet al. (20Mohanty J.G. Jaffe J.S. Schulman E.S. Raible D.G. J. Immunol. Methods. 1997; 202: 133-141Crossref PubMed Scopus (421) Google Scholar). O2⨪ production was measured in initial experiments by O2⨪-dependent superoxide dismutase-sensitive reduction of ferricytochrome c (21Johnston Jr., R.B. Methods Enzymol. 1984; 105: 365-369Crossref PubMed Scopus (90) Google Scholar). However, microglia released very little O2⨪, 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 (20Mohanty J.G. Jaffe J.S. Schulman E.S. Raible D.G. J. Immunol. Methods. 1997; 202: 133-141Crossref PubMed Scopus (421) Google Scholar). 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. The conversion of nitro blue tetrazolium (NBT) to formazan was used to detect the intracellular generation of O2⨪ (21Johnston Jr., R.B. Methods Enzymol. 1984; 105: 365-369Crossref PubMed Scopus (90) Google Scholar, 22Patterson C. Ruef J. Madamanchi N.R. Barry-Lane P. Hu Z. Horaist C. Ballinger C.A. Brasier A.R. Bode C. Runge M.S. J. Biol. Chem. 1999; 274: 19814-19822Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). 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 O2⨪generation. 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). Relativef/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. Fractionation of microglia was performed according to the method of Zhao et al. (23Zhao X. Bey E.A. Wientjes F.B. Cathcart M.K. J. Biol. Chem. 2002; 277: 25385-25392Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 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 μmphenylmethylsulfonyl 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 mml-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 TNFα with LPS and ROIs with BzATP (data not shown). Immunoblotting was performed as described previously (18Parvathenani L.K. Calandra V. Roberts S.B. Posmantur R. Neuroreport. 2000; 11: 2293-2297Crossref PubMed Scopus (13) Google Scholar). 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. Primary rat microglia (1 × 105) in Neurobasal Medium containing B-27, 1% penicillin/streptomycin, and 10 mml-glutamine were seeded into a 48-well plate containing 1 × 105primary 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. Supernatants were assayed for TNFα using OPtEIA Rat TNFα kit (Pharmingen). 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. 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% H2O2for 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-β 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. 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. Student's t test was performed to determine group differences (p < 0.05). Primary rat microglia stimulated with ATP and BzATP rapidly generate ROIs, superoxide (O2⨪) 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 H2O2production 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 H2O2produced (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. The conversion of Amplex Red to highly fluorescent resorufin in the presence of H2O2 is an indirect measure of O2⨪ generation. Hence the production of O2⨪ was confirmed by a more direct but less sensitive method. The inhibition of reduction of ferricytochrome c by O2⨪-dependent superoxide dismutase was used to detect the generation of O2⨪. As shown in Fig. 1D, microglia treated with BzATP showed a significant increase in superoxide dismutase-inhibitable reduction of ferricytochromec compared with untreated microglia. O2⨪ production by neutrophils treated with BzATP was examined as a control. Similar increases in O2⨪ 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 O2⨪, 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 O2⨪. Because microglia produce little O2⨪, the conversion of Amplex Red to highly fluorescent resorufin, a more sensitive but indirect indicator of O2⨪production (20Mohanty J.G. Jaffe J.S. Schulman E.S. Raible D.G. J. Immunol. Methods. 1997; 202: 133-141Crossref PubMed Scopus (421) Google Scholar), was used as the choice reagent in the remaining experiments. 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+]ichange was lower than required for maximal H2O2production. 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) (24Toescu E.C. Moller T. Kettenmann H. Verkhratsky A. Neuroscience. 1998; 86: 925-935Crossref PubMed Scopus (85) Google Scholar, 25Wang X. Kim S.U. van Breemen C. McLarnon J.G. Cell Calcium. 2000; 27: 205-212Crossref PubMed Scopus (46) Google Scholar). 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 H2O2production 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. 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 (26Jiang L.H. Mackenzie A.B. North R.A. Surprenant A. Mol. Pharmacol. 2000; 58: 82-88Crossref PubMed Scopus (351) Google Scholar). Brilliant Blue G (500 nm) inhibited BzATP (250 μm)-induced H2O2 production by more than 80% (Fig. 4A). 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 P2X7is the primary receptor stimulated by BzATP to generate H2O2. 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 (27Stolk J. Hiltermann T.J. Dijkman J.H. Verhoeven A.J. Am. J. Respir. Cell Mol. Biol. 1994; 11: 95-102Crossref PubMed Scopus (588) Google Scholar, 28Diatchuk V. Lotan O. Koshkin V. Wikstroem P. Pick E. J. Biol. Chem. 1997; 272: 13292-13301Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). 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 c" @default.
• W2153177211 created "2016-06-24" @default.
• W2153177211 creator A5021929537 @default.
• W2153177211 creator A5025797635 @default.
• W2153177211 creator A5049553015 @default.
• W2153177211 creator A5061766900 @default.
• W2153177211 creator A5069914515 @default.
• W2153177211 creator A5072260939 @default.
• W2153177211 date "2003-04-01" @default.
• W2153177211 modified "2023-10-18" @default.
• W2153177211 title "P2X7 Mediates Superoxide Production in Primary Microglia and Is Up-regulated in a Transgenic Mouse Model of Alzheimer's Disease" @default.
• W2153177211 cites W1532386517 @default.
• W2153177211 cites W1541085684 @default.
• W2153177211 cites W1571485493 @default.
• W2153177211 cites W1581364974 @default.
• W2153177211 cites W1608870321 @default.
• W2153177211 cites W1668344921 @default.
• W2153177211 cites W1718629648 @default.
• W2153177211 cites W1751283081 @default.
• W2153177211 cites W1830416824 @default.
• W2153177211 cites W1886167013 @default.
• W2153177211 cites W1897243328 @default.
• W2153177211 cites W1947551305 @default.
• W2153177211 cites W1964175388 @default.
• W2153177211 cites W1967199758 @default.
• W2153177211 cites W1975199357 @default.
• W2153177211 cites W2001210466 @default.
• W2153177211 cites W2010166433 @default.
• W2153177211 cites W2013561452 @default.
• W2153177211 cites W2021185580 @default.
• W2153177211 cites W2022507354 @default.
• W2153177211 cites W2024296781 @default.
• W2153177211 cites W2029446823 @default.
• W2153177211 cites W2032505238 @default.
• W2153177211 cites W2038370680 @default.
• W2153177211 cites W2041086215 @default.
• W2153177211 cites W2048825417 @default.
• W2153177211 cites W2050361888 @default.
• W2153177211 cites W2051148897 @default.
• W2153177211 cites W2055662392 @default.
• W2153177211 cites W2056461411 @default.
• W2153177211 cites W2063359028 @default.
• W2153177211 cites W2083095157 @default.
• W2153177211 cites W2095829875 @default.
• W2153177211 cites W2101955048 @default.
• W2153177211 cites W2116784956 @default.
• W2153177211 cites W2119780153 @default.
• W2153177211 cites W2134030137 @default.
• W2153177211 cites W2135089333 @default.
• W2153177211 cites W2135482947 @default.
• W2153177211 cites W2144042798 @default.
• W2153177211 cites W2146506131 @default.
• W2153177211 cites W2149954869 @default.
• W2153177211 cites W2157660452 @default.
• W2153177211 cites W2159027833 @default.
• W2153177211 cites W2255446000 @default.
• W2153177211 cites W2414939366 @default.
• W2153177211 cites W4313324522 @default.
• W2153177211 cites W4322697597 @default.
• W2153177211 cites W93284168 @default.
• W2153177211 doi "https://doi.org/10.1074/jbc.m209478200" @default.
• W2153177211 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12551918" @default.
• W2153177211 hasPublicationYear "2003" @default.
• W2153177211 type Work @default.
• W2153177211 sameAs 2153177211 @default.
• W2153177211 citedByCount "436" @default.
• W2153177211 countsByYear W21531772112012 @default.
• W2153177211 countsByYear W21531772112013 @default.
• W2153177211 countsByYear W21531772112014 @default.
• W2153177211 countsByYear W21531772112015 @default.
• W2153177211 countsByYear W21531772112016 @default.
• W2153177211 countsByYear W21531772112017 @default.
• W2153177211 countsByYear W21531772112018 @default.
• W2153177211 countsByYear W21531772112019 @default.
• W2153177211 countsByYear W21531772112020 @default.
• W2153177211 countsByYear W21531772112021 @default.
• W2153177211 countsByYear W21531772112022 @default.
• W2153177211 countsByYear W21531772112023 @default.
• W2153177211 crossrefType "journal-article" @default.
• W2153177211 hasAuthorship W2153177211A5021929537 @default.
• W2153177211 hasAuthorship W2153177211A5025797635 @default.
• W2153177211 hasAuthorship W2153177211A5049553015 @default.
• W2153177211 hasAuthorship W2153177211A5061766900 @default.
• W2153177211 hasAuthorship W2153177211A5069914515 @default.
• W2153177211 hasAuthorship W2153177211A5072260939 @default.
• W2153177211 hasBestOaLocation W21531772111 @default.
• W2153177211 hasConcept C102230213 @default.
• W2153177211 hasConcept C104317684 @default.
• W2153177211 hasConcept C121332964 @default.
• W2153177211 hasConcept C1276947 @default.
• W2153177211 hasConcept C141035611 @default.
• W2153177211 hasConcept C142724271 @default.
• W2153177211 hasConcept C181199279 @default.
• W2153177211 hasConcept C185592680 @default.
• W2153177211 hasConcept C203014093 @default.
• W2153177211 hasConcept C2776914184 @default.
• W2153177211 hasConcept C2777977315 @default.
• W2153177211 hasConcept C2779134260 @default.

• next