Glutathione depletion potentiates 12- O-tetradecanoyl phorbol-13-acetate(TPA)-induced inhibition of gap junctional intercellular communication in WB-F344 rat liver epithelial cells: relationship to intracellular oxidative stress

Abstract

Treatment of WB-F344 liver epithelial cells with buthionine sulfoximine (BSO, 100 /∗M) for 24 h caused a greater than 95% depletion in cellular glutathione (GSH) and potentiated the ability of 12- O-tetradecanoyl phorbol-13-acetate (TPA) to inhibit gap junctional intercellular communication (GJIC) between the cells (IC 50 shifted from 5 μM to 2 μM). Similarly, acute depletion of GSH by up to 30%, either with the thiol oxidant diamide or with BSO, also potentiated the inhibitory effect of the phorbol ester on GJIC. The treatment of the control cells with TPA caused a concomitant increase in the accumulation of oxidation products of 2′,7′-dichlorofluorescein (DCF), indicating elevated production of oxidants in the cells during the blockade of GJIC. The depletion of GSH over a 24 h period with BSO itself increased the flux of oxidants in the cells but did not inhibit GJIC. Treatment of these GSH-depleted cells with TPA caused an additive elevation in the accumulation of oxidised DCF metabolites. Direct application of H 2O 2 (25–200 μM) or benzoyl peroxide (25–150 μM) to the control cells for 60 min caused weak, dose-dependent inhibitions of gap junctional communication in these cells but these responses were accompanied by the induction of acute, sub-lethal cytotoxicity. The depletion of GSH from the cells did not potentiate these responses to the peroxides but did facilitate synergistic inhibition of gap junctional communication in response to both TPA and sub-toxic doses of either peroxide. The results of the above studies indicate that oxidants are produced in WB-F344 cells in response to TPA and that these function in a co-operative manner with other cellular responses to the phorbol ester in the inhibition of gap junctional communication. This may explain why priming the cells for the induction of oxidative stress by the depletion of GSH potentiates the inhibitory activity of TPA on gap junctional communication.