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+/HCO3- 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+/H+ 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.
Carini
R
R
Department of Experimental Medicine and Oncology, University of Torino, Italy.
Bellomo
G
G
Benedetti
A
A
Fulceri
R
R
Gamberucci
A
A
Parola
M
M
Dianzani
M U
MU
Albano
E
E
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology; D016923:Cell Death; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology; D014812:Vitamin K / Q000494:pharmacology
D002352:Carrier Proteins; D027982:Sodium-Bicarbonate Symporters; D014812:Vitamin K; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase