BIDIRECTIONAL MEMBRANE-TRANSPORT OF INTACT GLUTATHIONE IN HEP G2 CELLS

Rat hepatocytes exhibit bidirectional carrier-mediated transport of re duced glutathione (GSH) across the plasma membrane. Transport of GSH h as 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 wer e incubated in buffer, no GSH appeared in the medium over 2 h. However , after pretreatment with acivicin to inhibit gamma-glutamyl transpept idase activity, GSH efflux was unmasked and measured 30 +/- 4 pmol . 1 0(6) cells-1 . min-1, which is comparable to rat hepatocytes. GSH effl ux was inhibited by sulfobromophthalein GSH adduct (BSP-GSH) and cysta thionine, agents that inhibit sinusoidal efflux in the rat, and was st imulated by adenosine 3',5'-cyclic monophosphate-dependent agents. GSH uptake was measured after cells were pretreated with acivicin and but hionine sulfoximine to prevent breakdown of GSH and resynthesis of GSH from precursors, respectively. In the presence of 4 muCi/ml of [S-35] GSH and 10 mM unlabeled GSH, GSH uptake was linear up to 45 min and di d not require Na+ or Cl-. GSH uptake exhibited saturability with a max imal velocity of 4.15 +/- 0.23 nmol . mg-1 . 30 min-1, a Michaelis con stant of 2.36 +/- 0.26 mM, and two interactive transport sites. BSP-GS H cis-inhibited GSH uptake in a dose-dependent manner with an inhibito ry constant of 0.46 +/- 0.05 mM. Inhibition by BSP-GSH (1 mM) of GSH u ptake was through a single inhibitor site and was overcome at >10 mM G SH, which is consistent with competitive inhibition. Similar to the ra t, 10 mM extracellular GSH trans-stimulated GSH efflux. These findings may be important in gaining better insights into GSH homeostasis in h uman liver cells.