Reaven E, Tsai L, Maffe B, Azhar S
Geriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Palo Alto, CA 94304.
Cell Mol Biol Res. 1993;39(3):275-88.
The aim of this study with rat hepatocytes was to describe the effect of okadaic acid (OKA) (a potent and specific inhibitor of protein phosphatases) on the biosynthesis, processing and/or secretion of various lipid and protein molecules. Gel radioautograms indicated that low concentrations of okadaic acid (100 nM) induced hyperphosphorylation of a number of hepatocyte phosphoserine/threonine residues in the Mr range of 35-220 kDa. The effects of okadaic acid on the morphology of the hepatocytes was time and dose-dependent; early changes included cell rounding, loss of typical Golgi staining of beta COP, and fragmentation of the Golgi compartment at the EM level. General hepatocyte cell functions such as protein synthesis, lactate dehydrogenase activity, and ATP levels were unchanged with 100 nM okadaic acid as were all hepatocyte functions carried out in the endoplasmic reticulum of these cells. As such, incubation with okadaic acid did not alter the biosynthesis of phosphatidylcholine (from labeled choline), or very low density lipoproteins (VLDL) from labeled fatty acids or glycerol. Likewise, the biosynthesis of various endoplasmic reticulum synthesized proteins (transferrin, albumin, apolipoprotein E, and HMG CoA Reductase) continued normally in the presence of okadaic acid. However, incubation with okadaic acid led to major changes in all hepatocyte functions normally carried out in the Golgi compartment; i.e., the incorporation of labeled ceramide into sphingomyelin was profoundly reduced, as was the Golgi-required packaging and secretion of various proteins synthesized in the endoplasmic reticulum. These findings point to the Golgi compartment as an specific target for okadaic acid and suggest that one or more okadaic acid-sensitive phosphoproteins may be involved in maintaining its normal structure and function.
本项以大鼠肝细胞为研究对象的实验旨在描述冈田酸（一种有效的蛋白磷酸酶特异性抑制剂）对多种脂质和蛋白质分子的生物合成、加工及/或分泌的影响。凝胶放射自显影片显示，低浓度的冈田酸（100 nM）可诱导多种肝细胞磷酸丝氨酸/苏氨酸残基在分子量范围为35 - 220 kDa内发生过度磷酸化。冈田酸对肝细胞形态的影响具有时间和剂量依赖性；早期变化包括细胞变圆、β - COP的典型高尔基体染色消失以及在电子显微镜水平下高尔基体区室的碎片化。一般的肝细胞功能，如蛋白质合成、乳酸脱氢酶活性和ATP水平，在100 nM冈田酸作用下未发生改变，这些细胞内质网中进行的所有肝细胞功能也未改变。因此，与冈田酸孵育不会改变磷脂酰胆碱（由标记胆碱合成）的生物合成，也不会改变由标记脂肪酸或甘油合成极低密度脂蛋白（VLDL）的过程。同样，在冈田酸存在的情况下，各种内质网合成蛋白（转铁蛋白、白蛋白、载脂蛋白E和HMG CoA还原酶）的生物合成仍正常进行。然而，与冈田酸孵育会导致通常在高尔基体区室中进行的所有肝细胞功能发生重大变化；即，标记神经酰胺掺入鞘磷脂的过程被显著降低，内质网中合成的各种蛋白质所需的高尔基体包装和分泌过程也是如此。这些发现表明高尔基体区室是冈田酸的一个特定作用靶点，并提示一种或多种对冈田酸敏感的磷蛋白可能参与维持其正常结构和功能。
