Abstract
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 Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-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 Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-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 Ca2+-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.