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.