Glutathione and glutathione conjugate efflux from cultured liver cells

Abstract

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C1 and the gamma-glutamyl transpeptidase containing, tumorigenic ARL-16T2, has been assessed under basal condition and during chronic treatment with 75 and 150 microM ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines. Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 microM EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of gamma-glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T2 cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T2 cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased intracellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.

Induction of mitochondrial fusion by cysteine‐alkylators ethacrynic acid and N‐ethylmaleimide

“…This was expected and in keeping with its known function as a GSH precursor (Moldeus et al, 1986). It is likely that NAC is acting to either detoxify EA from the cell by formation of GSH‐conjugates through GSTs to be removed by the multidrug resistance pump (Meredith, 1991; Zaman et al, 1996) or by directly reversing one or more EA‐alkylated cysteine residues responsible for its effects. Treatment with 200 μM dEM alone for 2 h resulted in a similar depletion of cellular GSH as that seen with 60 μg/ml (∼200 μM) EA.…”