Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Abstract

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 microM digitonin. Blockade of VDAC with Koenig's polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 microM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.

Fig. 1

Digitonin-mediated release of cytosolic and mitochondrial enzymes from isolated rat hepatocytes. (A) Hepatocytes (2 × 10⁶ cells/ml) were incubated at room temperature in ICB and treated with different concentrations of digitonin for 10 min. A portion of digitonin-treated cells was taken to assess trypan blue labeling (TB), and the rest was sedimented by centrifugation at 14,000 rpm for 60 s for enzymatic analysis of the supernatants. Data are means from 6 experiments. (B) Hepatocytes (2 × 10⁶ cells/ml) treated with 0, 8 or 80 μM of digitonin for 10 min in ICB were centrifuged, and supernatants were subjected to Western blotting for cytochrome c, as described in Materials and methods. Lanes are: first lane (far left), molecular weight markers (MWM); second, third and fourth lanes, supernatants after 0, 8 and 80 μM digitonin; last lane (far right), 100 ng cytochrome c, (Cyt c). Representative Western blots are shown from three or more independent hepatocyte incubations yielding similar results.

Fig. 2

Effect of digitonin treatment of hepatocytes on mitochondrial membrane potential and respiration. (A) Cultured hepatocytes were loaded with TMRM to visualize mitochondria, as described in materials and methods. Red fluorescence of TMRM was imaged by laser scanning confocal microscopy. Shown are representative images of a hepatocyte before (Intact) and after consecutive treatment with 8 μM digitonin (+Digitonin), 5 mM succinate (+Succinate) and 50 μM 2,4-dinitrophenol (+DNP). As indicated by uptake of TMRM, mitochondria of intact hepatocytes partially depolarized after digitonin (+Digitonin). Succinate restored mitochondrial polarization (+Succinate), but nearly complete depolarization occurred after dinitrophenol (+DNP). Images are representative from three independent experiments. (B) Representative tracing of oxygen uptake by isolated hepatocytes incubated in ICB supplemented with 5 mM succinate after sequential addition of 50 μM dinitrophenol (DNP), 8 μM digitonin and 20 mM malonate. Inset shows respiration as a function of digitonin concentration. Data are means from six experiments. (C) Respiration of isolated hepatocytes in the absence (Intact) and presence of dinitrophenol (DNP) either with intact (None) or digitonin-permeabilized plasma membranes (Dig 8, 8 μM). Oligomycin and ATP were omitted from ICB for respiration experiments. Data are means from six experiments.

Fig. 3

Effects of Koenig's polyanion on DNP- and ADP-stimulated respiration in digitonin-permeabilized hepatocytes. Respiration of hepatocytes (2 × 10⁶ cells/ml) was measured in ICB supplemented with 5 mM succinate. (A) DNP-stimulated respiration was measured after sequential addition of 8 μM digitonin (Dig 8), 25 μg/ml KPA (25 KPA), and 80 μM digitonin (Dig 80). (B) ADP (500 μM)-stimulated respiration was measured after sequential addition of 8 μM digitonin (Dig 8), 25 μg/ml KPA (25 KPA) and 80 μM digitonin (Dig 80). Oligomycin and ATP were omitted from ICB for respiration experiments. Data are means ± SEM of at least six experiments. *p < 0.05 compared to Dig 8.

Fig. 4

Restoration of ethanol-inhibited respiration by high digitonin. (A) Respiration of untreated hepatocytes (2 × 10⁶ cells/ml) was measured in ICB (without oligomycin and ATP), supplemented with 5 mM succinate and 500 μM ADP after no addition (None) and after sequential addition of 8 μM and 80 μM digitonin (Dig 8 and Dig 80, respectively). (B) Respiration was measured as in A in hepatocytes exposed to 50 mM ethanol for 20 min. Data are means ± SEM of at least six experiments. *p < 0.05 compared to None; **p < 0.05 compared to Dig 8.

Fig. 5

Ethanol exposure limits accessibility to mitochondrial AK. Untreated and ethanol-treated hepatocytes (2 × 10⁶ cells/ml) were incubated with digitonin (8 μM) and centrifuged. AK activity was measured in the cytosol (A) and pellet (B), as described in Materials and methods. In (B) the pellet from low-digitonin treated, ethanol-treated hepatocytes was treated additionally with high digitonin (80 μM). Data are means ± SEM of at least six experiments. *p < 0.05 compared to None. **p < 0.05 compared to ethanol.

Fig. 6

Mechanical permeabilization and RhoDex entrapment in cultured hepatocytes. Cultured hepatocytes were loaded with green-fluorescing MTG and incubated in ICB (A) RhoDex (400 μM) was then added followed by mechanical perturbation using a micropipette. Afterwards, RhoDex penetrated the cytoplasm and nuclei (B) After 120 s, the medium was replaced with ICB containing both RhoDex and DIDS (30 μM), the latter a VDAC inhibitor to entrap RhoDex in the mitochondrial intermembrane space. After another 60 s, the hepatocytes were washed with ICB containing only DIDS to remove unbound RhoDex. After another 300 s, images of MTG and RhoDex fluorescence were collected to identify RhoDex remaining within mitochondria (C) Higher magnification (lower panels) shows retention of red-fluorescencing RhoDex (E) in MTG-labeled mitochondria (D) as evident in the overlay (F) White arrows identify strongly RhoDex-labeled structures that do not co-label with MTG.

Fig. 7

Decreased RhoDex entry into mitochondria after ethanol treatment. (A) Hepatocytes were pretreated with vehicle (c0ontrol), 50 mM ethanol, or 30 μM DIDS and subjected to the RhoDex entrapment protocol described in Fig. 6. Ethanol and DIDS pretreatment decreased retention of red-fluorescing RhoDex. (B) Mean intensities of red fluorescence associated with mitochondria are plotted for the treatment groups in A. Data are means ± SEM of 20 or more cells per group from five or more hepatocyte isolations. *p < 0.05 compared to control.