van Blitterswijk W J, Hilkmann H, de Widt J, van der Bend R L
Division of Cellular Biochemistry, The Netherlands Cancer Institute (Antoni van Leeuwenhoek-Huis), Amsterdam.
J Biol Chem. 1991 Jun 5;266(16):10344-50.
Bradykinin (BK) and phorbol 12-myristate 13-acetate (PMA) both stimulate the hydrolysis of phosphatidylcholine (PC) in human fibroblasts, resulting in the formation of phosphatidic acid (PA) and diacylglycerol (DG) (Van Blitterswijk, W.J., Hilkmann, H., de Widt, J., and Van der Bend, R.L. (1990) J. Biol. Chem. 266, 10337-10343). Stimulation with BK resulted in the rapid and synchronous formation of [3H]choline and [3H]myristoyl-PA from the correspondingly prelabeled PC, indicative of phospholipase D (PLD) activity. In the presence of ethanol or n-butanol, transphosphatidylation by PLD resulted in the formation of [3H]phosphatidylethanol or - butanol, respectively, at the cost of PA and DG formation. This suggests that PC-derived DG is generated via a PLD/PA phosphohydrolase pathway. A more pronounced but delayed formation of these products was observed by PMA stimulation. The Ca2+ ionophore ionomycin also activated PLD and accelerated (synergized) the response to PMA. Both [3H] choline and [3H]phosphocholine were released into the extracellular medium in a time- and stimulus-dependent fashion, without apparent changes in the high intracellular levels of [3H]phosphocholine. The protein kinase C (PKC) inhibitors staurosporin and 1-O-hexadecyl-2-O-methylglycerol inhibited BK- and PMA-induced activation of PLD. Down-regulation of PKC by long-term pretreatment of cells with phorbol ester caused a dramatic drop in background [3H]choline levels, while subsequent stimulation with BK, ionomycin, or PMA failed to increase these levels and failed to induce transphosphatidylation. From these results we conclude that PLD activation is entirely mediated by (downstream of) PKC. Unexpectedly, however, BK stimulation of these PKC-depleted cells caused a marked generation of DG from PC within 15 s, which was not seen in BK-stimulated control cells, suggesting PC breakdown by a phospholipase C (PLCc). We conclude that cells stimulated with BK generate DG via both the PLCc and the PLD/PA hydrolase pathway, whereas PMA stimulates mainly the latter pathway. BK stimulation of normal cells leads to activation of PKC and, by consequence, to attenuation of the level of PLCc-generated DG and to stimulation of the PLD pathway, whereas the reverse occurs in PKC-down-regulated cells.
缓激肽（BK）和佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）均可刺激人成纤维细胞中磷脂酰胆碱（PC）的水解，导致磷脂酸（PA）和二酰甘油（DG）的形成（范·布利特维克，W.J.，希尔克曼，H.，德·维特，J.，以及范·德·本德，R.L.（1990年）《生物化学杂志》266卷，10337 - 10343页）。用BK刺激可使相应预先标记的PC迅速同步形成[3H]胆碱和[3H]肉豆蔻酰 - PA，这表明存在磷脂酶D（PLD）活性。在乙醇或正丁醇存在的情况下，PLD的转磷脂酰基作用分别导致形成[3H]磷脂酰乙醇或 - 丁醇，代价是PA和DG的形成减少。这表明PC衍生的DG是通过PLD/PA磷酸水解酶途径生成的。通过PMA刺激观察到这些产物的形成更为显著但延迟。钙离子载体离子霉素也激活PLD并加速（协同）对PMA的反应。[3H]胆碱和[3H]磷酸胆碱都以时间和刺激依赖性方式释放到细胞外培养基中，而细胞内高浓度的[3H]磷酸胆碱没有明显变化。蛋白激酶C（PKC）抑制剂星形孢菌素和1 - O - 十六烷基 - 2 - O - 甲基甘油抑制BK和PMA诱导的PLD激活。用佛波醇酯对细胞进行长期预处理导致PKC下调，使背景[3H]胆碱水平急剧下降，而随后用BK、离子霉素或PMA刺激未能提高这些水平，也未能诱导转磷脂酰基作用。从这些结果我们得出结论，PLD的激活完全由PKC介导（在PKC下游）。然而，出乎意料的是，对这些PKC缺失细胞进行BK刺激在15秒内导致PC显著生成DG，这在BK刺激的对照细胞中未观察到，提示存在磷脂酶C（PLCc）介导的PC分解。我们得出结论，用BK刺激的细胞通过PLCc和PLD/PA水解酶途径生成DG，而PMA主要刺激后一种途径。对正常细胞进行BK刺激导致PKC激活，结果是PLCc生成的DG水平降低，并刺激PLD途径，而在PKC下调的细胞中情况则相反。
