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Am J Physiol. 1993 Dec;265(6 Pt 1):G1128-34.

Bidirectional membrane transport of intact glutathione in Hep G2 cells.

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1
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033.

Abstract

Rat hepatocytes exhibit bidirectional carrier-mediated transport of reduced glutathione (GSH) across the plasma membrane. Transport of GSH has not been well characterized in human-derived cells. We examined Hep G2 cells as a possible human liver model for GSH homeostasis. Hep G2 cell GSH averaged 25.9 +/- 1.4 nmol/10(6) cells. When Hep G2 cells were incubated in buffer, no GSH appeared in the medium over 2 h. However, after pretreatment with acivicin to inhibit gamma-glutamyl transpeptidase activity, GSH efflux was unmasked and measured 30 +/- 4 pmol x 10(6) cells-1 x min-1, which is comparable to rat hepatocytes. GSH efflux was inhibited by sulfobromophthalein GSH adduct (BSP-GSH) and cystathionine, agents that inhibit sinusoidal efflux in the rat, and was stimulated by adenosine 3',5'-cyclic monophosphate-dependent agents. GSH uptake was measured after cells were pretreated with acivicin and buthionine sulfoximine to prevent breakdown of GSH and resynthesis of GSH from precursors, respectively. In the presence of 4 microCi/ml of [35S]GSH and 10 mM unlabeled GSH, GSH uptake was linear up to 45 min and did not require Na+ or Cl-. GSH uptake exhibited saturability with a maximal velocity of 4.15 +/- 0.23 nmol.mg-1 x 30 min-1, a Michaelis constant of 2.36 +/- 0.26 mM, and two interactive transport sites. BSP-GSH cis-inhibited GSH uptake in a dose-dependent manner with an inhibitory constant of 0.46 +/- 0.05 mM. Inhibition by BSP-GSH (1 mM) of GSH uptake was through a single inhibitor site and was overcome at > 10 mM GSH, which is consistent with competitive inhibition. Similar to the rat, 10 mM extracellular GSH trans-stimulated GSH efflux. These findings may be important in gaining better insights into GSH homeostasis in human liver cells.

PMID:
7904127
DOI:
10.1152/ajpgi.1993.265.6.G1128
[Indexed for MEDLINE]

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