Leitersdorf E, Stein O, Stein Y
Biochim Biophys Acta. 1984 Jul 6;794(2):261-8. doi: 10.1016/0005-2760(84)90154-1.
Rat hepatocytes isolated by collagenase perfusion were cultured for 48-72 h and examined for synthesis and secretion of hepatic triacylglycerol lipase activity. Low levels of enzyme activity found in the culture medium increased with time of incubation, and a 3-10-fold rise was encountered in the presence of optimal concentrations of heparin (5 U/ml). After interruption of enzyme synthesis by cycloheximide, plateauing of enzyme activity in the medium occurred, indicating that addition of heparin may not only stabilize but also enhance hepatic triacylglycerol lipase secretion. Synthesis and secretion of hepatic triacylglycerol lipase was not related to cell density, and enzyme secretion was encountered in subconfluent cultures. Release of enzyme activity into the medium was not sensitive to chlorpromazine, a lysosomal enzyme inhibitor, but was completely inhibited by treatment with tunicamycin, an inhibitor of glycosylation. As release of enzyme activity could be maintained for 12 h in the absence of serum, possible hormonal regulation was sought. Under the present experimental conditions, no modulation of hepatic triacylglycerol lipase was encountered by either gonadal or thyroid hormones. Addition of cyclic AMP to the culture medium resulted in a 30% decrease in enzyme activity. The dependence of hepatic triacylglycerol lipase secretion on the intactness of the Golgi apparatus and on vesicular transport was demonstrated by the treatment with monensin. The present results show that cultured rat hepatocytes provide a good model system by which the regulation of synthesis and secretion of hepatic triacylglycerol lipase can be studied.
通过胶原酶灌注分离的大鼠肝细胞培养48 - 72小时，并检测其肝三酰甘油脂肪酶活性的合成与分泌。在培养基中发现的低水平酶活性随孵育时间增加，在存在最佳浓度肝素（5 U/ml）的情况下酶活性升高了3 - 10倍。在用环己酰亚胺中断酶合成后，培养基中的酶活性趋于平稳，这表明添加肝素不仅可以稳定而且可以增强肝三酰甘油脂肪酶的分泌。肝三酰甘油脂肪酶的合成与分泌与细胞密度无关，在亚汇合培养物中也观察到了酶的分泌。酶活性释放到培养基中对溶酶体酶抑制剂氯丙嗪不敏感，但用糖基化抑制剂衣霉素处理可完全抑制。由于在无血清的情况下酶活性释放可维持12小时，因此研究了可能的激素调节。在当前实验条件下，性腺激素或甲状腺激素均未对肝三酰甘油脂肪酶产生调节作用。向培养基中添加环磷酸腺苷导致酶活性降低30%。莫能菌素处理证明了肝三酰甘油脂肪酶分泌对高尔基体完整性和囊泡运输的依赖性。目前的结果表明，培养的大鼠肝细胞提供了一个良好的模型系统，通过该系统可以研究肝三酰甘油脂肪酶合成与分泌的调节。
