P2X7 receptor activation induces reactive oxygen species formation and cell death in murine EOC13 microglia
Rachael Bartlett1, Justin J Yerbury, Ronald Sluyter
1School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
Abstract:
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-1(2+) uptake into both cell lines. ATP induced ethidium(+) uptake into EOC13 cells in a concentration-dependent manner, with an EC(50) 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 Ca(2+) 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.
Insights
Activation of the P2X7 receptor in microglia triggers organic cation uptake, reactive oxygen species (ROS) generation, and cell death. These findings highlight P2X7 receptor
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- The P2X7 purinergic receptor is a critical ion channel found on immune cells, including microglia.
- Microglia play a vital role in central nervous system immunity and disease.
Purpose of the Study:
- To investigate the functional consequences of P2X7 receptor activation in the murine microglial EOC13 cell line.
- To determine if P2X7 activation leads to organic cation uptake, reactive oxygen species (ROS) formation, and cell death.
Main Methods:
- Utilized RT-PCR, immunoblotting, and immunolabelling to confirm P2X7 expression in EOC13 cells.
- Employed cytofluorometric assays to measure cation uptake, ROS production, and cell death markers (Annexin-V, 7AAD).
- Tested the effects of P2X7 agonists (ATP) and antagonists, as well as an ROS scavenger (N-acetyl-L-cysteine).
Main Results:
- P2X7 receptor agonists (ATP) induced concentration-dependent ethidium(+) uptake in EOC13 cells, inhibited by specific antagonists.
- P2X7 activation led to ROS formation independent of Ca(2+) influx and K(+) efflux.
- P2X7 activation resulted in EOC13 cell death, which was attenuated by an ROS scavenger, indicating ROS mediation.
Conclusions:
- P2X7 receptor activation in microglia (EOC13 cell line) triggers significant organic cation influx.
- The study demonstrates that P2X7 activation induces ROS formation and subsequent cell death in microglia.
- Reactive oxygen species play a crucial role in mediating P2X7-induced microglial cell death.
