NAD(+) influx through connexin hemichannels prevents poly(ADP-ribose) polymerase-mediated astrocyte death

Cell 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.

PARP 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.

NAD(+), 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.

These 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.