Albano E, Carini R, Parola M, Bellomo G, Goria-Gatti L, Poli G, Dianzani M U
Dipartimento di Medicina ed Oncologia Sperimentale, Universita' di Torino, Italy.
Biochem Pharmacol. 1989 Aug 15;38(16):2719-25. doi: 10.1016/0006-2952(89)90559-5.
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.
将分离的大鼠肝细胞与0.172 mM四氯化碳一起孵育，会导致线粒体和线粒体外部分的钙含量迅速下降。然而，通过磷酸化酶α的激活或喹啉-2荧光测量，细胞内储存中Ca2+的释放与细胞质Ca2+水平的增加无关。仅在四氯化碳浓度高于0.172 mM时，才观察到肝细胞游离钙的快速升高。磷酸化酶α缺乏激活不是由于四氯化碳对该酶的抑制，因为在经四氯化碳处理的肝细胞中，磷酸化酶活性可被胰高血糖素、丁酰-cAMP或添加A23187诱导的细胞钙增加所刺激。在四氯化碳中毒的早期阶段，当线粒体和线粒体外钙池已经降低时，质膜的Ca2+依赖性ATP酶仅受到轻微影响。这导致得出结论，从细胞内细胞器释放的钙可以足够量地从细胞中挤出，以防止细胞质水平的增加。在与四氯化碳孵育的第二个小时内，观察到肝细胞游离钙升高，同时出现乳酸脱氢酶泄漏和质膜起泡。向培养基中添加乙二醇双（2-氨基乙基醚）四乙酸可防止细胞质Ca2+增加和起泡，这表明它们是钙流入细胞的结果。然而，乙二醇双（2-氨基乙基醚）四乙酸以及添加钙依赖性磷脂酶A2或非溶酶体蛋白酶的抑制剂均不能防止细胞死亡。这些结果表明，四氯化碳在分离的肝细胞中诱导的钙分布改变不是毒性作用的主要原因，尽管它们不排除细胞质Ca2+的持续升高可能有助于细胞损伤的进展。
