Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: Effect of membrane physical properties andS-adenosyl-L-methionine

Abstract

ethanol were confined to the inner membrane; mitoplasts Ethanol intake depletes the mitochondrial pool of reduced from ethanol-fed rats showed features similar to those of intact glutathione (GSH) by impairing the transport of GSH from mitochondria such as impaired transport of GSH and incytosol into mitochondria. S-Adenosyl-L-methionine (SAM) creased order parameter. A different mitochondrial transsupplementation of ethanol-fed rats restores the mitochonporter, adenosine diphosphate (ADP)/ATP translocator, was drial pool of GSH. The purpose of the current study was to unaffected by ethanol feeding. Futhermore, fluidization of midetermine the effect of ethanol feeding on the kinetic parametochondria or mitoplasts from ethanol-fed rats by treatment ters of mitochondrial GSH transport, the fluidity of mitochonwith a fatty acid derivative restored their ability to transport dria, and the effect of SAM on these changes. Male Sprague-GSH to control levels. Thus, ethanol-induced impaired transDawley rats were fed ethanol-liquid diet for 4 weeks port of GSH into mitochondria is selective, mediated by desupplemented with either SAM or N-acetylcysteine (NAC).creased fluidity of the mitochondrial inner membrane, and SAM-supplementation of ethanol-fed rats restored the mitoprevented by SAM treatment. (HEPATOLOGY 1997;26:699-chondrial GSH pool but NAC administration did not. Kinetic 708.) studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanolThe pathogenesis of the alcohol-induced liver disease is feeding: lowering V max (0.22 and 1.6 in ethanol case vs. 0.44 not completely understood, despite intense research. Multiand 2.7 nmol/15 sec/mg protein in controls) for both low and ple factors have been implicated in the pathogenesis of the high affinity components with the latter showing an increased disease. 1-4 As a consequence of ethanol metabolism there are K m (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria important biochemical changes in the hepatocyte, including from SAM-supplemented ethanol-fed rats showed kinetic feacellular redox potential shift (a-nicotinamide adenine dinutures of GSH transport similar to control mitochondria. Detercleotide/a-dihydronicotinamide adenine dinucleotide demination of membrane fluidity revealed an increased order crease) and acetaldehyde production, a potent toxic intermeparameter in ethanol compared with control mitochondria, diate, which may mediate many of the toxic effects of ethanol. which was restricted to the polar head groups of the bilayer In addition, other factors such as auto-immune-induced inand was prevented by SAM but not NAC supplementation jury by antibodies to protein-acetaldehyde adducts, hemodyof ethanol-fed rats. The changes elicited in mitochondria by namic alterations of the hepatic blood supply along sinusoids, peroxidation of membrane lipids, and oxidative stress may contribute to the development of the disease. [5][6][7] It is well...

Cholesterol Impairs the Adenine Nucleotide Translocator-mediated Mitochondrial Permeability Transition through Altered Membrane Fluidity

“…Mitochondria from chronic alcohol-fed rat livers have decreased membrane fluidity because of altered mitochondrial lipid composition and cholesterol deposition (43, 60 -62); the DPH fluorescence anisotropy values reported in these membranes were similar to the range we observed in our present observations. Consistent with the current findings the adenine nucleotide exchange in mitochondria from chronic alcohol-fed rats was unaltered (43,63,64), confirming that this transport function of mitochondria is not regulated by membrane fluidity.…”

“…Preparation of Mitoplasts-Mitoplasts were prepared as previously described (43). Briefly, to a suspension of 20 mg of mitochondrial protein/ml in 220 mM mannitol, 70 mM sucrose, 2 mM HEPES, and 0.05% BSA, pH 7.4, an equal volume of digitonin (2.4 mg/ml) in the same buffer without BSA was added.…”

“…Fluidization of Mitochondria-Mitochondrial suspension (2 mg/ml) was incubated in 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 1 mM EDTA, with A 2 C (125 nmol/mg of protein) from a stock solution of 12 mg/ml as described in detail previously (43). The suspension was incubated at 37°C for 30 min and then washed twice by spinning at 9,500 ϫ g for 10 min.…”

“…Measurement of Fluorescence Anisotropy-Fluidity of mitochondrial membranes was evaluated by fluorescence anisotropy of mitochondria-bound dyes, TMA-DPH, DPH, or 9-(anthroyloxy)stearic acid, as described in detail previously (43). Briefly, TMA-DPH or 9-(anthroyloxy)stearic acid (final concentration 3 M) were injected into stirred mitochondrial suspensions (0.5 mg/ml) and the mixture was incubated for 30 min at 37°C, whereas DPH (20 mM in tetrahydrofuran) was diluted 100 times with 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 1 mM EDTA.…”

“…Briefly, TMA-DPH or 9-(anthroyloxy)stearic acid (final concentration 3 M) were injected into stirred mitochondrial suspensions (0.5 mg/ml) and the mixture was incubated for 30 min at 37°C, whereas DPH (20 mM in tetrahydrofuran) was diluted 100 times with 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 1 mM EDTA. Fluorescence polarization was measured in a Hitachi spectrofluorometer at wavelengths of 366 nm for excitation and 425 nm for emission for TMA-DPH or DPH, and 366 nm excitation and 440 nm emission wavelengths for 9-(anthroyloxy)stearic acid (43). The results are expressed as anisotropy units (r), where r ϭ (I 0 /I 90 )/(I 0 ϩ 2I 90 ).…”