Alteration of Na⁺ homeostasis as a critical step in the development of irreversible hepatocyte injury after adenosine triphosphate depletion

The exposure of isolated hepatocytes to the redox-cycling quinone menadione caused an early loss of mitochondrial membrane potential, adenosine triphosphate (ATP) depletion, and decreased intracellular pH. These alterations were followed by an increase in intracellular Na⁺ and, ultimately, cell death. If HCO₃^- was omitted from the incubation buffer, or the hepatocytes were incubated in an acidic medium (pH 6.5) the accumulation of Na⁺ was markedly reduced. Inhibition of the Na⁺/H⁺ exchanger and of the Na⁺/HC0₃^- cotransporter by, respectively, amiloride and 4,4′-di-isothiocyano-2,2′-disulfonic acid stilbene (DIDS) suppressed the initial Na⁺ influx but did not prevent subsequent Na⁺ accumulation, because amiloride and DIDS inhibited the Na⁺/K⁺ pump. The omission of HCO₃^- from the extracellular medium or the incubation in acidic conditions also prevented menadione toxicity, without interfering with the loss of mitochondrial membrane potential and with ATP depletion. A similar protection was evident when hepatocytes were incubated with menadione in a medium without Na⁺. The preservation of adequate levels of ATP by supplementing hepatocytes with fructose allowed the initial Na⁺ load to be recovered and provided partial protection against menadione toxicity. These effects were suppressed if Na⁺/K⁺-ATPase was inhibited with ouabain. Taken together, these results indicated that the activation of the Na⁺/HCO₃^- cotransporter and of the Na⁺/K⁺ exchanger in response to the decrease of intracellular pH stimulated an enhanced influx of Na⁺. When the activity of the Na⁺/K⁺ pump was not able to control Na⁺ levels because of ATP depletion, such an uncontrolled Na⁺ influx precipitated irreversible injury and caused hepatocyte death.