Yu Q C, Marzella L
Department of Pathology, School of Medicine, University of Maryland, Baltimore.
Lab Invest. 1988 Jun;58(6):643-52.
The lysosomes of hepatocytes increase in numbers and size during acute cell injury in vivo. To elucidate the mechanism of this change, we have studied in vitro the response of the autophagic lysosomal system to several physiologic mediators of autophagy, and to agents known to induce injury and/or the accumulation of lysosomes in vivo. To this end, monolayer cultures of rat hepatocytes were labeled with [14C]leucine to measure hepatocyte protein degradation; ultrastructural analyses were carried out to measure the volume fraction of lysosomes in the hepatocytes. Dibutyryl cyclic AMP increased protein degradation in the hepatocytes either in the presence or absence of serum and insulin. Deprivation of serum and insulin also increased hepatocyte protein degradation. Morphometric analysis indicated parallel increases in the volume fraction of lysosomes in the hepatocytes. The calcium ionophore ionomycin (5 microM), in the presence of 1.3 mM extracellular calcium, increased protein degradation (but not the volume fraction of lysosomes), and this increase was abolished by the addition of ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. On the other hand, vasopressin (5 nM) caused an increase in protein degradation coupled with an increase in volume fraction of lysosomes. The microtubule depolymerizer vinblastine decreased protein degradation. The microtubule stabilizer taxol did not prevent the inhibitory effects caused by vinblastine. Parallel decreases in the lysosomal compartment were found in the hepatocytes exposed to vinblastine or taxol. Dimethylnitrosamine inhibited protein degradation as well as decreased the volume fraction of lysosomes. Finally, carbon tetrachloride also decreased protein degradation. These data indicate that in physiologic conditions, increases in numbers of hepatocyte lysosomes reflect increased sequestration and degradation of cytoplasmic proteins in response to changes in the levels of hormones, serum factors and nutrients as well as cyclic AMP. The induction of acute cell injury in vitro by calcium ionophore, microtubule active agents, and hepatotoxins inhibits lysosomal proteolysis and causes a decrease in the volume fraction of lysosomes. We conclude that the increase in lysosomal size and numbers occurring in acutely injured hepatocytes in vivo is induced primarily by altered levels of nutritional and hormonal regulators of lysosomal protein degradation.
在体内急性细胞损伤期间，肝细胞的溶酶体数量增加且体积增大。为阐明这种变化的机制，我们在体外研究了自噬溶酶体系统对几种自噬生理介质以及已知在体内诱导损伤和/或溶酶体积聚的药物的反应。为此，用[14C]亮氨酸标记大鼠肝细胞单层培养物以测量肝细胞蛋白质降解；进行超微结构分析以测量肝细胞中溶酶体的体积分数。二丁酰环磷腺苷在有或无血清和胰岛素的情况下均增加肝细胞蛋白质降解。血清和胰岛素缺乏也增加肝细胞蛋白质降解。形态计量分析表明肝细胞中溶酶体体积分数平行增加。在存在1.3 mM细胞外钙的情况下，钙离子载体离子霉素（5 microM）增加蛋白质降解（但不增加溶酶体体积分数），并且通过添加乙二醇双（β-氨基乙基醚）-N，N'-四乙酸可消除这种增加。另一方面，加压素（5 nM）导致蛋白质降解增加，同时溶酶体体积分数增加。微管解聚剂长春碱降低蛋白质降解。微管稳定剂紫杉醇不能阻止长春碱引起的抑制作用。在暴露于长春碱或紫杉醇的肝细胞中发现溶酶体区室平行减少。二甲基亚硝胺抑制蛋白质降解并降低溶酶体体积分数。最后，四氯化碳也降低蛋白质降解。这些数据表明，在生理条件下，肝细胞溶酶体数量增加反映了对激素、血清因子和营养物质以及环磷腺苷水平变化的反应，细胞质蛋白的隔离和降解增加。钙离子载体、微管活性剂和肝毒素在体外诱导急性细胞损伤会抑制溶酶体蛋白水解并导致溶酶体体积分数降低。我们得出结论，体内急性损伤肝细胞中溶酶体大小和数量的增加主要是由溶酶体蛋白降解的营养和激素调节因子水平改变引起的。
