Abstract
Extracellular adenosine 5′-triphosphate (ATP) is a damage-associated molecular pattern and contributes to inflammation associated diseases including cancer. Extracellular acidosis is a novel danger signal in the inflammatory sites, where it can modulate inflammation, immunity and tumor growth. Extracellular acidification was shown to inhibit P2X7-mediated channel currents, while it remains unknown how acidification and P2X7 together affect cellular responses. Here, we treated BV-2 microglial cells with ATP in a short period (<15 min) or a sustained acidified condition. For short acidification we compared the actions of neutralized ATP and acidic ATP in a condition with pH buffering. For sustained acidification, we treated cells with neutralized ATP in acidic medium or acidic ATP in medium without pH buffering. In the short acidified condition, neutralized ATP induced higher responses than acidic ATP to increase intracellular calcium and reactive oxygen species, decrease intracellular potassium and induce cell death. In contrast, these cellular responses and mitochondrial fission caused by neutralized ATP were enhanced by pH 6.0 and pH 4.5 media. P2X7 activation can also rapidly block mitochondrial ATP turnover and respiration capacity, both of which were mimicked by nigericin and enhanced by acidity. Taken together P2X7-mediated ionic fluxes and reactive oxygen species production are attenuated under short acidification, while sustained acidification itself can induce mitochondrial toxicity which deteriorates the mitochondrial function under P2X7 activation.
Highlights
Microglia are the resident immune cells in the central nervous system (CNS) [1]
Due to the rapid recovery of acidic pH of acidic adenosine 5′-triphosphate (ATP) (aATP)-containing medium to neutral condition in CO2-buffering incubator, we regarded the different responses of neutralized ATP (nATP) and aATP as the effect of short time acidification on P2X7 response
We found that pre-treatment of P2X7 selective antagonist A438079 (Figure 1A, 1B) and silencing P2X7 by siRNA approach (Figure 1C, 1D) can block the cell death induced by nATP and aATP, suggesting the involvement of P2X7 in extracellular ATP-induced cell death
Summary
Microglia are the resident immune cells in the central nervous system (CNS) [1]. In response to brain injury or immunological stimuli, these cells become activated and migrated to the site of injury and secrete numerous chemokines, reactive oxygen species (ROS), and pro-inflammatory cytokines. Microglia actively involve in the phagocytosis of apoptotic cells and microbes. Activation of P2X7 rapidly triggers Na+, K+ and Ca2+ ions across the plasma membrane, which is followed by increasing membrane permeability for larger organic cations and forming a large, non-selective pore allowing molecules with molecular weight up to 900 Da to enter the cells [5, 6]. The large pore formation initiates several cellular events, including activation www.impactjournals.com/oncotarget of the NLRP3 inflammasome, opening of pannexin 1 and connexin hemichannels, membrane blebbing, ROS production, loss of mitochondrial membrane potential, and eventually cellular death [7,8,9]. P2X7 is highly expressed in both macrophages and microglia [10], and is critical for protective innate immune responses during the early phases of microbial infection [11]. Pro-inflammatory and pro-apoptotic actions of P2X7 contribute to the chronic inflammation and pathogenesis of various diseases, including arthritis, inflammatory bowel disease, neurodegenerative diseases, chronic pain, mood disorders and cancers [12,13,14,15,16,17,18]
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