P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia
The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the mu...
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| Format: | Article |
| Language: | English |
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Wiley
2013-01-01
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| Series: | Mediators of Inflammation |
| Online Access: | http://dx.doi.org/10.1155/2013/271813 |
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| author | Rachael Bartlett Justin J. Yerbury Ronald Sluyter |
| author_facet | Rachael Bartlett Justin J. Yerbury Ronald Sluyter |
| author_sort | Rachael Bartlett |
| collection | DOAJ |
| description | The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′-triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium+ or YO-PRO-12+ uptake into both cell lines. ATP induced ethidium+ uptake into EOC13 cells in a concentration-dependent manner, with an EC50 of ~130 μM. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75–100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia. |
| format | Article |
| id | doaj-art-98c97882bc8843fe996931556621e080 |
| institution | Kabale University |
| issn | 0962-9351 1466-1861 |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Wiley |
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| series | Mediators of Inflammation |
| spelling | doaj-art-98c97882bc8843fe996931556621e0802025-02-03T01:11:07ZengWileyMediators of Inflammation0962-93511466-18612013-01-01201310.1155/2013/271813271813P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 MicrogliaRachael Bartlett0Justin J. Yerbury1Ronald Sluyter2School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, AustraliaSchool of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, AustraliaSchool of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, AustraliaThe P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′-triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium+ or YO-PRO-12+ uptake into both cell lines. ATP induced ethidium+ uptake into EOC13 cells in a concentration-dependent manner, with an EC50 of ~130 μM. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75–100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.http://dx.doi.org/10.1155/2013/271813 |
| spellingShingle | Rachael Bartlett Justin J. Yerbury Ronald Sluyter P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia Mediators of Inflammation |
| title | P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia |
| title_full | P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia |
| title_fullStr | P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia |
| title_full_unstemmed | P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia |
| title_short | P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia |
| title_sort | p2x7 receptor activation induces reactive oxygen species formation and cell death in murine eoc13 microglia |
| url | http://dx.doi.org/10.1155/2013/271813 |
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