Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion.
The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 +/- 0.3 mmol/L and maximum velocity of 1.9 +/- 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A(2)C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A(2)C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion.
Coll
Olga
O
Liver Unit, Hospital Clínic i Provincial, Institut de Malalties Digestives, Instituto de Investigaciones Biomédicas August Pi i Sunyer, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.
Colell
Anna
A
García-Ruiz
Carmen
C
Kaplowitz
Neil
N
Fernández-Checa
J C
JC
D000428:Alcohol Drinking / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002784:Cholesterol / Q000494:pharmacology; D005978:Glutathione / Q000172:deficiency* / Q000378:metabolism; D006801:Humans; D008297:Male; D008560:Membrane Fluidity; D026901:Membrane Transport Proteins; D008930:Mitochondria, Liver / Q000378:metabolism*; D009865:Oocytes; D051381:Rats; D017207:Rats, Sprague-Dawley; D014982:Xenopus laevis
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C021723:oxoglutarate translocator; D002784:Cholesterol; D005978:Glutathione