PT - JOURNAL ARTICLE AU - Elvira Salas AU - Luz María G. Carrasquero AU - Luis A. Olivos-Oré AU - Diego Bustillo AU - Antonio R. Artalejo AU - Maria Teresa Miras-Portugal AU - Esmerilda G. Delicado TI - Purinergic P2X7 Receptors Mediate Cell Death in Mouse Cerebellar Astrocytes in Culture AID - 10.1124/jpet.113.209452 DP - 2013 Dec 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 802--815 VI - 347 IP - 3 4099 - http://jpet.aspetjournals.org/content/347/3/802.short 4100 - http://jpet.aspetjournals.org/content/347/3/802.full SO - J Pharmacol Exp Ther2013 Dec 01; 347 AB - 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 3′-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 Mg2+, 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 Mg2+ 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 necrotic-type 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.