Extracellular acidosis delays onset of cell death in ATP-depleted hepatocytes.
A fluorometric assay using propidium iodide total fluorescence was utilized to quantitate cell viability in hepatocyte suspensions continuously. For viable hepatocytes exposed to KCN, fluorescence was linearly proportional to lactate dehydrogenase release and to nuclear labeling by propidium iodide. In KCN-treated hepatocytes, iodoacetate eliminated the protective effect of the fed state and fructose against the onset of cell death. A model of cell death with KCN and iodoacetate was developed to mimic the ATP depletion of anoxia. This "chemical hypoxia" was used to investigate the role of pH in cell death. At pH 7.4, cell viability decreased to 10% after 120 min, whereas at pH 5.5-7.0, cell viability was 65-85%, nearly the same as normoxic cells. During chemical hypoxia under acidotic conditions, a return of pH to 7.4 resulted in a rapid acceleration of cell killing, a "pH paradox." Inhibition of Na+-H+ exchange increased survival, whereas promoting exchange of intracellular Cl- for extracellular HCO3- potentiated cell killing. Monensin, a Na+-H+ ionophore, potentiated cell killing at pH 7.4 but not at pH 6.2. The results show that extracellular acidosis markedly protects against cell killing after ATP depletion, an effect that appears mediated through cytoplasmic acidification.
Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina, Chapel Hill 27599.
G J
GJ
Gores
A L
AL
Nieminen
K E
KE
Fleishman
T L
TL
Dawson
B
B
Herman
J J
JJ
Lemasters
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005215:Fasting; D005632:Fructose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008985:Monensin / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D005632:Fructose; D000255:Adenosine Triphosphate; D008985:Monensin; D007770:L-Lactate Dehydrogenase