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 approximately 2- 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) approximately 13 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) approximately 100 microM) was also present. Endogenous GSH transporter in HeLa cells was thiol-sensitive, electrogenic, and described by a single Michaelis-Menten component (K(m) approximately 15 mM). In contrast, GSH transport mediated by Oatp1 was insensitive to thiols and membrane potential, inhibited by cystine, and stimulated by an inward H(+) gradient. These findings identify novel functional asymmetries in sinusoidal efflux and uptake of GSH and further clarify the role of Oatp1 in GSH transport.