Differences among cell types in NAD+ compartmentalization: A comparison of neurons, astrocytes, and cardiac myocytes

Activation of the nuclear enzyme poly(ADP‐ribose)‐1 leads to the death of neurons and other types of cells by a mechanism involving NAD+ depletion and mitochondrial permeability transition. It has been proposed that the mitochondrial permeability transition (MPT) is required for NAD+ to be released from mitochondria and subsequently consumed by PARP‐1. In the present study we used the MPT inhibitor cyclosporine‐A (CsA) to preserve mitochondrial NAD+ pools during PARP‐1 activation and thereby provide an estimate of mitochondrial NAD+ pool size in different cell types. Rat cardiac myocytes, mouse cardiac myocytes, mouse cortical neurons, and mouse cortical astrocytes were incubated with the genotoxin N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) in order to activate PARP‐1. In all four cell types MNNG caused a reduction in total NAD+ content that was blocked by the PARP inhibitor 3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinolinone. Inhibition of the mitochondrial permeability transition with cyclosporine‐A (CsA) prevented PARP‐1‐induced NAD+ depletion to a varying degree in the four cell types tested. CsA preserved 83.5% ± 5.2% of total cellular NAD+ in rat cardiac myocytes, 85.7% ± 8.9% in mouse cardiac myocytes, 55.9% ± 12.9% in mouse neurons, and 22.4% ± 7.3% in mouse astrocytes. CsA preserved nearly 100% of NAD+ content in mitochondria isolated from these cells. These results confirm that it is the cytosolic NAD+ pool that is consumed by PARP‐1 and that the mitochondrial NAD+ pool is consumed only after MPT permits mitochondrial NAD+ to exit into the cytosol. These results also suggest large differences in the mitochondrial and cytosolic compartmentalization of NAD+ in these cell types. © 2007 Wiley‐Liss, Inc.