ALTERATION OF NA-TRIPHOSPHATE DEPLETION( HOMEOSTASIS AS A CRITICAL STEP IN THE DEVELOPMENT OF IRREVERSIBLE HEPATOCYTE INJURY AFTER ADENOSINE)

The exposure of isolated hepatocytes to the redox-cycling quinone mena dione caused an early loss of mitochondrial membrane potential, adenos ine triphosphate (ATP) depletion, and decreased intracellular pH. Thes e alterations were followed by an increase in intracellular Na+ and, u ltimately, cell death. If HCO3- was omitted from the incubation buffer , or the hepatocytes were incubated in an acidic medium (pH 6.5) the a ccumulation of Na+ was markedly reduced. Inhibition of the Na+/H+ exch anger and of the Na+/HCO3- cotransporter by, respectively, amiloride a nd 4,4'-di-isothiocyano-2,2'-disulfonic acid stilbene (DIDS) suppresse d the initial Na+ influx but did not prevent subsequent Na+ accumulati on, because amiloride and DIDS inhibited the Na+/K+ pump, The omission of HCO3- from the extracellular medium or the incubation in acidic co nditions also prevented menadione toxicity, without interfering with t he loss of mitochondrial membrane potential and with ATP depletion. A similar protection was evident when hepatocytes were incubated with me nadione in a medium without Na+. The preservation of adequate levels o f ATP by supplementing hepatocytes with fructose allowed the initial N a+ load to be recovered and provided partial protection against menadi one toxicity. These effects were suppressed if Na+/K+-ATPase was inhib ited with ouabain, Taken together, these results indicated that the ac tivation of the Na+/HCO3- cotransporter and of the Na+/H+ exchanger in response to the decrease of intracellular pH stimulated an enhanced i nflux of Na+. When the activity of the Na+/K+ pump was not able to con trol Na+ levels because of ATP depletion, such an uncontrolled Na+ inf lux precipitated irreversible injury and caused hepatocyte death.