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

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• 作者：Hiroto Okuda¹ ; Kentaro Nishida ; Youichirou Higashi² ; Kazuki Nagasawa ; ^{nagasawa@mb.kyoto-phu.ac.jp}
• 关键词：NAD+ ; Astrocyte ; Connexin hemichannel ; P2X7 receptor ; Poly(ADP-ribose) polymerase
• 刊名：Life Sciences
• 出版年：2013
• 出版时间：19 April, 2013
• 年：2013
• 卷：92
• 期：13
• 页码：808-814
• 全文大小：414 K

文摘

Aim

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.

Main methods

PARP activation was induced by treatment with N-methyl-N¡ä-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 findings

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 18¦Â-glycyrrhetinic acid, octanol and carbenoxolone inhibited the NAD⁺-mediated cell rescue of PARP-triggered cell death.

Significance

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.