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.
Highlights
Nicotinamide adenine dinucleotide (NAD(H)) plays a pivotal role in the regulation of a huge number of cellular processes
We investigated the effects of external NAD(H) on the maximum Ca2+-retention capacity (CRC) of mitochondria isolated from various organs, the swelling, and contraction of mitochondria in incubation media containing solutes transported and not transported by carriers of the inner mitochondrial membrane (IMM), and the Ca2+-dependent dissipation of the mitochondrial membrane potential (∆Ψm) and the release of 620-Da fluorescent dye from individual mitochondria of hepatocytes permeabilized with digitonin
The release of Ca2+ from mitochondria can occur much earlier than the complete opening of mitochondrial permeability transition pore (mPTP) and entry into the matrix of low-molecular-weight solutes (K+, Cl−, sucrose, mannitol, etc.), which cause the swelling of mitochondria [26,27,28,29]
Summary
Nicotinamide adenine dinucleotide (NAD(H)) plays a pivotal role in the regulation of a huge number of cellular processes. It was shown that NADH at millimolar concentrations reversed the opening of mPTP and increased the capability of mitochondria to accumulate and retain Ca2+, acting on an unidentified regulatory site in the mitochondrial matrix [8,9,10,11]. We have found that NAD(H) added at physiological concentrations to the incubation medium (cytosolic side of mitochondrial membranes) potently suppresses the opening of mPTP in the mitochondria of differentiated cells and enhances the protective effect of adenine nucleotides and cyclosporin A (CsA) [23]. According to the earlier data of Haworth and Hunter, it was exogenous NAD(H) that caused the closure of the opened mPTP by acting on the allosteric regulatory site in the matrix [8,9]. The data obtained unambiguously indicate that the site of the regulation of mPTP by cytosolic NAD(H) is located either at the OMM, or the IMS, or the outer surface of the IMM
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