[The role of the voltage-dependent anion channels in the outer membrane of mitochondria in the regulation of cellular metabolism]

Abstract

The role of voltage-dependent anion channels (VDAC/porins) of the mitochondrial outer membrane in the regulation of cell metabolism is assessed using an experimental model of ethanol toxicity in cultured hepatocytes. It is demonstrated that ethanol inhibits the phosphorylating and the uncoupled mitochondrial respiration, decreases the accessibility of mitochondrial adenylate kinase in the intermembrane space, and suppresses ureagenic respiration in the cells. Treatment with digitonin at high concentrations (>80 μM)—which creates pores in the mitochondrial outer membrane, allowing bypass of closed VDAC—restores all the processes suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial function because of closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space. Our studies also reveal the role of VDAC in the regulation of liver-specific intracellular processes such as ureagenesis. The data obtained can be used in development of pharmaceuticals that would prevent VDAC closure in mitochondria of ethanol-oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.

Fig. 1

Release of cytoplasmic and mitochondrial marker enzymes (a) and cytochrome c (b) from hepatocytes (2 × 10⁶ cells/mL) upon 10-min digitonin treatment. (a) TB, trypan blue; LDH, lactate dehydrogenase; AK, adenylate kinase; (b) 1, mol. wt markers; supernatants after treatment with 2 – 0; 3 – 8; 4 – 80 μM digitonin; 5 – 10 ng commercial cyt c.

Fig. 2

Effects of digitonin, substrate and uncoupler on (a–d) mitochondrial membrane potential (TMRM fluorescence) and (e, f) oxygen consumption by hepatocytes with 5 mM succinate. Here and further, data of at least six experiments.

Fig. 3

Effects of Koenig’s polyanion (KPA) and digitonin on (a) uncoupled and (b) phosphorylating respiration of hepatocytes.

Fig. 4

Effect of digitonin on phosphorylating respiration (5 mM succinate, 500 μM ADP) of (a) control and ethanol-exposed (100 mM, 60 min) hepatocytes.

Fig. 5

Effects of ethanol and digitonin on adenylate kinase activity in the (a) cytosol and (b) cell-bound fractions.

Fig. 6

Suppression by ethanol of the respiration of urea-synthesizing hepatocytes. (a) Specified concentrations of ethanol were added together with ureagenic substrates (3 mM NH₄Cl, 5 mM L-ornithine, 5 mM L-lactate) at the 15th minute. (b) Dependence of the respiration rate on ethanol concentration.