P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

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-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.

Lack of the P2X7 receptor protects against AMD-like defects and microparticle accumulation in a chronic oxidative stress-induced mouse model of AMD

“…4). Activation of the P2X 7 R is involved in the generation of reactive oxygen species (ROS) in microglia [10,21]. Here we show that concurrent knockout of this receptor blocked CML accumulation and resulted in lower 3-NT immunofluorescence induced by Sod1 KO, indicating the P2X 7 R is also involved in the generation of ROS in retina/RPE/choroid.…”

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

“…The generation of reactive oxygen species (ROS) after ATP activation of P2X7 is now well established in macrophages (Lenertz et al, 2009;Moore and MacKenzie, 2009), microglia (Apolloni et al, 2013;Bartlett et al, 2013), submandibular gland cells (Seil et al, 2008), and erythroid cells (Wang and Sluyter, 2013). Activation of NADPH oxidase 2 by P2X7 was a common feature found in all cell types studied, although there is evidence that ATP, possibly via P2X7 activation, can also enhance mitochondrial reactive oxygen species (Nakahira et al, 2011).…”

The P2X7 receptor is not essential for development of imiquimod-induced psoriasis-like inflammation in mice

“…+ uptake assay P2X7 pore formation was quantified by measuring ATPinduced ethidium + uptake as described [24]. Briefly, J774 cells in NaCl medium (145 mM NaCl, 5 mM KCl, 5 mM glucose and 10 mM HEPES, pH 7.5) were pre-incubated in the absence or presence of BBG or A-804598 (as indicated) for 15 min at 37°C.…”