NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD(+) on: (1) the Ca(2+)-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca(2+)-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca(2+)-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca(2+)-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD(+) increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca(2+)-uptake and the free Ca(2+) concentration in the medium. NAD(H) suppressed the Ca(2+)-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca(2+)-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.