Martin T W, Feldman D R, Michaelis K C
Department of Pathology, St. Louis University School of Medicine, MO 63104.
Biochim Biophys Acta. 1990 Jul 12;1053(2-3):162-72. doi: 10.1016/0167-4889(90)90009-3.
The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.
在预先用[甲基-3H]胆碱（[3H]胆碱）或[9,10-3H]肉豆蔻酸（[3H]肉豆蔻酸）标记的牛肺动脉内皮细胞中，研究了佛波醇肉豆蔻酸酯（PMA）刺激的磷脂酰胆碱（PC）降解机制。两种标记物都选择性地掺入到PC中，添加PMA会刺激预先用[3H]胆碱或[3H]肉豆蔻酸标记的细胞中PC的3H有相当程度的损失。在用[3H]胆碱预先标记的细胞中，PC中3H的损失与细胞内游离[3H]胆碱的快速增加相关。PMA刺激的细胞内[3H]胆碱的增加之前，任何其他3H标记的PC降解产物都没有增加。PMA不会刺激预先用[3H]胆碱标记的细胞中PC脱酰基产物的形成。在用[3H]胆碱预先标记的通透细胞中，PMA刺激[3H]胆碱的形成，但不刺激[3H]磷酸胆碱的形成。在用[3H]肉豆蔻酸预先标记的完整细胞中，PMA诱导的PC中3H的损失与[3H]磷脂酸（[3H]PA）和[3H]二酰甘油的形成相关。在乙醇存在下，PMA刺激[3H]磷脂酰乙醇（[3H]PEt）的形成，同时以[3H]PA为代价。[3H]PEt形成的时间进程与PMA刺激的细胞中细胞内[3H]胆碱形成的时间进程相似。综合这些数据支持这样一种观点，即PMA刺激的内皮细胞中PC的降解主要由磷脂酶D介导。在用不能与蛋白激酶C相互作用的佛波醇酯处理的细胞中未观察到通过磷脂酶D的PC分解。用PMA长期预处理细胞以下调蛋白激酶C以及用星形孢菌素预处理细胞可抑制佛波醇酯对磷脂酶D的激活。这些数据支持这样一种观点，即佛波醇酯对磷脂酶D的激活依赖于蛋白激酶C。
