Cook G A, Gattone V H, Evan A P, Harris R A
Biochim Biophys Acta. 1983 Dec 19;763(4):356-67. doi: 10.1016/0167-4889(83)90097-6.
The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.
通过扫描电子显微镜和透射电子显微镜研究了伴随洋地黄皂苷诱导分离的肝细胞释放酶和代谢产物的结构变化。在初始阶段，以胞质标记酶乳酸脱氢酶的完全释放为特征，质膜立即受损，随后内质网迅速受到广泛损伤。然而，细胞形状得以维持，线粒体和细胞核通过细胞骨架紧密相连。线粒体最初保持完整，而胞质的电子密度降低，核染色质更加分散。中间阶段的特征是腺苷酸激酶和大部分葡萄糖-6-磷酸酶（分别为线粒体膜间隙和内质网的标记酶）完全释放。线粒体外膜破裂，但线粒体保持其正常的基质电子密度。在最后阶段，以柠檬酸合酶从线粒体基质空间开始释放为特征，线粒体肿胀，只有细胞核、线粒体内外膜和细胞骨架能够清晰分辨。尽管在初始阶段后电子显微镜照片中难以轻易辨别质膜，但质膜标记酶5'-核苷酸酶仍与经洋地黄皂苷处理的肝细胞相关。乙酰辅酶A羧化酶的释放比乳酸脱氢酶慢得多，表明其释放受到一些严重限制。乙酰辅酶A羧化酶的释放与葡萄糖-6-磷酸酶的释放密切平行。因此，将肝细胞可控地暴露于洋地黄皂苷会导致可溶性、分隔的细胞成分和一些膜结合成分的顺序释放，但线粒体膜、细胞骨架和核骨架即使在长期洋地黄皂苷处理下仍能存活。
