Hatch G M, Tsukitani Y, Vance D E
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Biochim Biophys Acta. 1991 Jan 4;1081(1):25-32. doi: 10.1016/0005-2760(91)90245-d.
There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.
有证据表明，肝细胞中磷脂酰胆碱（PC）的生物合成受磷酸化-去磷酸化机制调控。但其中涉及的磷酸酶尚未明确。因此，我们研究了强效蛋白磷酸酶抑制剂冈田酸对分离的大鼠肝细胞悬浮培养物中通过CDP-胆碱途径进行的PC生物合成的影响。在连续脉冲标记实验中，冈田酸使[Me-3H]胆碱摄取量降低了15%（P<0.05）。处理120分钟后，PC的标记量降低了46%（P<0.05），而磷酸胆碱的标记量相应增加了20%（P<0.05）。用[Me-3H]胆碱脉冲处理细胞30分钟，随后在不存在或存在冈田酸的情况下用胆碱追踪长达120分钟。在冈田酸处理的肝细胞中，追踪120分钟后，磷酸胆碱的标记量增加了86%（P<0.05），PC的标记量降低了29%（P<0.05）。PC中标记量的减少在磷酸胆碱部分中得到了定量解释。用冈田酸和CPT-cAMP共同孵育肝细胞，对PC标记的抑制作用并未产生叠加效应。用冈田酸处理后，胆碱激酶和胆碱磷酸转移酶的活性未发生改变。用洋地黄皂苷孵育肝细胞以释放胞质成分。在冈田酸处理的肝细胞中，细胞空壳膜胞苷酰转移酶（CT）活性降低了37%（P<0.005），同时释放到胞质中的胞苷酰转移酶活性相应增加了33%（P<0.05）。我们推测，在分离的大鼠肝细胞中，CT活性和PC生物合成受蛋白磷酸酶活性调控。
