J. Neurochem. (2012) 122, 1118–1128. Abstract P2X7 receptor (P2X7R) is known to be a ‘death receptor’ in immune cells, but its functional expression in non‐immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP‐induced neuronal death in cultured cortical neurons. In P2X7R‐ and pannexin‐1‐expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R‐selective antagonists. ATP‐treated neurons exhibited Ca2+ entry and YO‐PRO‐1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin‐1 blocking with carbenoxolone, prevented the ATP‐induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP‐ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP‐treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP‐induced neuronal death. In ATP‐treated neurons, the cleavage of pro‐caspase‐3 was increased, and caspase inhibitors, Q‐VD‐OPh and Z‐DEVD‐FMK, inhibited the neuronal death. The cleavage of apoptosis‐inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation‐triggered Ca2+ entry and mitochondrial dysfunction played important roles in the ATP‐induced neuronal death.

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Nucleotides and nucleosides play important roles by maintaining brain homeostasis, and their extracellular concentrations are mainly regulated by ectonucleotidases and nucleoside transporters expressed by astrocytes. Extracellularly applied NAD(+) prevents astrocyte death caused by excessive activation of poly(ADP-ribose) polymerase-1, of which the molecular mechanism has not been fully elucidated. Recently, exogenous NAD(+) was reported to enter astrocytes via the P2X7 receptor (P2X7R)-associated channel/pore. In this study, we examined whether the intact form of NAD(+) is incorporated into astrocytes. A large portion of extracellularly added NAD(+) was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring-labeled [(14)C]NAD(+), but not nicotinamide moiety-labeled [(3)H]NAD(+), showed time- and temperature-dependency, and was significantly enhanced on addition of apyrase, and was reduced by 8-Br-cADPR and ARL67156, inhibitors of CD38 and ectoapyrase, respectively, and P2X7R knockdown, suggesting that the detected uptake of [(14)C]NAD(+) resulted from [(14)C]adenosine acting as a metabolite of [(14)C]NAD(+). Pharmacological and genetic inhibition of P2X7R with brilliant blue G, KN-62, oxATP, and siRNA transfection resulted in a decrease of [(3)H]adenosine uptake, and the uptake was also reduced by low concentration of carbenoxolone and pannexin1 selective peptide blocker (10)panx. Taken together, these results indicate that exogenous NAD(+) is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R-associated channel/pore.

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