AimCell death induced by excessive activation of poly(ADP-ribose) polymerase (PARP) is inhibited by administration of NAD+ extracellularly, but its preventive mechanism remains unclear. Here we investigated the involvement of NAD+ and/or its metabolites, adenosine and nicotinamide, in the rescue of PARP-mediated astrocyte death by NAD+ and explored the pathway through which intact NAD+ could enter cells. Main methodsPARP activation was induced by treatment with N-methyl-N′-nitro-N-nitrosoguanidine, a DNA-alkylating agent. The cellular NAD+ content was determined by an enzymatic recycling assay, and cell viability was determined by measuring intracellular LDH activity. Key findingsNAD+, but not adenosine and nicotinamide, could restore the cellular NAD+ levels decreased by PARP activation. Pharmacological inhibition of the uptake of adenosine and nicotinamide had no effect on the prevention of PARP-triggered cell death by NAD+, suggesting that unmetabolized NAD+ remaining in the extracellular milieu might prevent PARP-mediated NAD+ consumption and cell death. The increase in the cellular NAD+ level caused by NAD+ administration to PARP-activated cells was significantly inhibited by a connexin hemichannel blocker, carbenoxolone, but not by P2X7 receptor inhibition with selective antagonists and siRNA, or pannexin-selective blockers. Finally, pharmacological blockade of connexin hemichannels with 18β-glycyrrhetinic acid, octanol and carbenoxolone inhibited the NAD+-mediated cell rescue of PARP-triggered cell death. SignificanceThese findings suggested that intact NAD+ could get into astrocytes through connexin hemichannels, and that this process should play a key role in NAD+-mediated prevention of PARP-triggered astrocyte death.