Glycine protects against hepatocyte killing by KCN or hypoxia by preventing intracellular Na+ overload in the rat

Glycine has been shown to prevent hepatocyte death induced by anoxia and by several toxic agents, However, the mechanisms responsible for such a cytoprotective effect have not yet been entirely clarified. We have previously shown that an uncontrolled increase in intracellular Na+ is critical for hepatocyte killing induced by adenosine triphosphate (ATP) depletion, We herein report that protection by glycine (2 mmol/L) against cytotoxicity induced in isolated rat hepatocyte by potassium cyanide (KCN) or hypoxia was associated with the prevention of cytosolic Na+ accumulation. The addition of the Na+ ionophore, monensin, abolished the effects of glycine on both Na+ increase and cytotoxicity. Pretreating hepatocytes with the glycine-receptor antagonist, strychnine (1 mmol/L), similarly prevented Na+ overload and cell killing. Glycine at high concentrations and strychnine are known to block Cl- channels in many cell types. Consistently, we have observed that glycine and strychnine prevented the increase of intracellular Cl- levels caused by hypoxia or KCN, Incubation of hepatocytes in a Cl--free medium, obtained by substituting chloride with membrane-impermeable gluconate, significantly reduced Naf accumulation and cell killing triggered by hypoxia or KCN. Both these effects were abolished by the addition of monensin. The cytoprotective action exerted by hepatocyte incubation in the Cl--free medium was, however, lost when membrane-permeable nitrate, which allowed Na+ accumulation, was used instead to replace chloride. Altogether, these results indicate that glycine inhibition of Cl- conductance protects against hepatocyte killing induced by KCN and hypoxia by interfering with intracellular Na+ accumulation triggered by ATP depletion.