Effects of carbon tetrachloride on calcium homeostasis. A critical reconsideration.
The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.
Dipartimento di Medicina ed Oncologia Sperimentale, Universita' di Torino, Italy.
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Albano
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Carini
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Parola
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Bellomo
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Goria-Gatti
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Poli
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Dianzani
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002251:Carbon Tetrachloride / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006706:Homeostasis / Q000187:drug effects*; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects; D010762:Phosphorylase a / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D002251:Carbon Tetrachloride; D010762:Phosphorylase a; D002118:Calcium