Purinergic P2X7 Receptors Mediate Cell Death in Mouse Cerebellar Astrocytes in Culture

Abstract

The brain distribution and functional role of glial P2X7 receptors are broader and more complex than initially anticipated. We characterized P2X7 receptors from cerebellar astrocytes at the molecular, immunocytochemical, biophysical, and cell physiologic levels. Mouse cerebellar astrocytes in culture express mRNA coding for P2X7 receptors, which is translated into P2X7 receptor protein as proven by Western blot analysis and immunocytochemistry. Fura-2 imaging showed cytosolic calcium responses to ATP and the synthetic analog 39-O-(4-benzoyl)benzoyl-ATP (BzATP) exhibited two components, namely an initial transient and metabotropic component followed by a sustained one that depended on extracellular calcium. This latter component, which was absent in astrocytes from P2X7 receptor knockout mice (P2X7 KO), was modulated by extracellular Mg 21, and was sensitive to Brilliant Blue G (BBG) and 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079) antagonism. BzATP also elicited inwardly directed nondesensitizing whole-cell ionic currents that were reduced by extracellular Mg 21 and P2X7 antagonists (BBG and calmidazolium). In contrast to that previously reported in rat cerebellar astrocytes, sustained BzATP application induced a gradual increase in membrane permeability to large cations, such as N-methyl-D-glucamine and 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]diiodide, which ultimately led to the death of mouse astrocytes. Cerebellar astrocyte cell death was prevented by BBG but not by calmidazolium, removal of extracellular calcium, or treatment with the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, thus suggesting a necrotictype mechanism of cell death. Since this cellular response was not observed in astrocytes from P2X7 KO mice, this study suggests that stimulation of P2X7 receptor may convey a cell death signal to cerebellar astrocytes in a species-specific manner.

Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists

“…As such, inhibition of the P2X7R offers protection against ATP-mediated cell death in a number of cells expressing the P2X7R [12], [30], [36]. Therefore, C23, C40 and C60 compounds were finally tested for their effectiveness in preventing ATP-induced cell death in hP2X7R-expressing HEK293 cells to provide further supporting evidence for their action on the hP2X7R.…”

Extracellular ATP and glutamate drive pyruvate production and energy demand to regulate mitochondrial respiration in astrocytes

“…shows that 1 mM ATP, which activates ionotropic purinergic receptors of astrocytes(Salas et al, 2013), causes a larger increase in[Ca 2+ ] I than that produced by 100 μM ATP. The [Ca 2+ ] I signal decreases in absence of external Ca 2+ (Figure 2b).…”

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

“…S16C), which has been shown to inhibit rat P2X7-mediated cation flux but not dye uptake (22), and to inhibit mouse P2X7-mediated cation flux but not cell death (23). We also found that calmidazolium blocked Caspase-1 activation by the cation-specific inflammasome agonist nigericin in these cells (fig.…”