The Thiol Sensitivity of Glutathione Transport in Sidedness-Sorted Basolateral Liver Plasma Membrane and in Oatp1-Expressing HeLa Cell Membrane

Abstract

Sinusoidal efflux of hepatic reduced glutathione (GSH) is a key step in interorgan GSH/cysteine homeostasis and extracellular detoxification. Rat organic anion transporter polypeptide1 (Oatp1) is known to transport GSH but several features of sinusoidal GSH uptake, such as electrogenic property and asymmetric effects of uncharged thiols (increased efflux, decreased uptake), either cannot be accounted for by Oatp1 or have not been studied. The asymmetric effect of thiols has only been studied in intact cells and not directly in membrane vesicles. To accomplish the latter, we studied GSH uptake in inside-out-(IO) and rightside-out-(RO) oriented basolateral plasma membrane vesicles (bLPM). We also studied the kinetics and effect of thiols on GSH transport by Oatp1 stably expressed in HeLa cells. GSH uptake was ϳ2-to 3-fold higher in IO than RO bLPM. Dithiothreitol-stimulated GSH uptake in IO but inhibited uptake in RO bLPM, demonstrating that thiols exert direct asymmetric side-specific effects on GSH transport. Uptake in IO and RO bLPM was sigmoid (K m ϳ13 mM) with a 2-fold higher capacity in IO compared with RO bLPM. In both IO and RO bLPM, a component with a high affinity but low capacity for GSH (K m ϳ100 M) was also present. Endogenous GSH transporter in HeLa cells was thiol-sensitive, electrogenic, and described by a single Michaelis-Menten component (K m ϳ15 mM). In contrast, GSH transport mediated by Oatp1 was insensitive to thiols and membrane potential, inhibited by cystine, and stimulated by an inward H ϩ gradient. These findings identify novel functional asymmetries in sinusoidal efflux and uptake of GSH and further clarify the role of Oatp1 in GSH transport.

Molecular and Cellular Physiology of Renal Organic Cation and Anion Transport

“…Proximal tubule cells contain a relatively large (5 mM) concentration of GSH (236) that could serve to support uphill accumulation of anions, consistent with a role for rOATP1 in reabsorption of selected substrates from the tubular filtrate. It has also been suggested that rOATP1 could mediate the electrogenic secretion of E17␤-D, a physiological metabolite of estradiol (194), and there is evidence that rOATP1 can support an electrogenic mode of activity (288).…”

Alcohol-induced oxidative stress

“…To date, experiments to demonstrate the synthesis of glutathione by isolated mitochondria have failed (Griffith and Meister, 1985;Reed, 1990). Therefore, it is assumed that the glutathione is synthesized in the cytosol and transported into the mitochondria to maintain a separate pool of glutathione (Mittur et al, 2002). Inhibition of the transport of GSH from the cytosol into mitochondria leads to depletion of the mitochondrial pool of GSH after ethanol intake (Colell et al, 1997).…”

Chronic Ethanol Consumption Results in Atypical Liver Injury in Copper/Zinc Superoxide Dismutase Deficient Mice

“…Glutathione is synthesized in the soluble fraction of the cytoplasm and transported into mitochondria, thereby maintaining 2 distinct intracellular pools of the antioxidant (Mittur et al, 2002). Reports have shown that ethanol administration not only depletes GSH levels by the generation of oxidants but also inhibits the mitochondrial GSH transporter by alteration of the mitochondrial membrane Fernandez-Checa et al, 1993Vina et al, 1980;Wheeler et al, 2003).…”