Liu B, Nakashima S, Adachi T, Ito Y, Takano T, Shimizu T, Nozawa Y
Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan.
Biochem J. 1997 Oct 1;327 ( Pt 1)(Pt 1):239-44. doi: 10.1042/bj3270239.
The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCalpha, which might play an important role in the expression of c-fos and c-jun.
利用稳定表达野生型血小板活化因子受体（WT-H细胞）和缺失C末端胞质尾的截短型血小板活化因子受体（D-H细胞）的中国仓鼠卵巢细胞，研究了血小板活化因子（PAF）激活磷脂酶D（PLD）的机制和作用。用PAF处理D-H细胞导致肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）迅速形成，随后是持续超过10分钟的阶段。在这些细胞中，PAF诱导的PLD激活持续超过20分钟。相比之下，WT-H细胞中的PLD激活是短暂的。PAF刺激在两种类型的细胞中均导致1,2-二酰基甘油（DG）的双相形成。第一阶段迅速且短暂，与Ins(1,4,5)P3峰值一致。DG形成的第二个持续阶段被丁醇减弱，丁醇通过PLD的转磷脂酰基活性以磷脂酸（PA）为代价产生磷脂丁醇，还被普萘洛尔减弱，普萘洛尔是一种催化PA转化为DG的PA磷酸水解酶的选择性抑制剂。在WT-H细胞中，PAF刺激后20分钟时DG水平几乎恢复到基础水平，而在D-H细胞中，升高的DG水平持续超过20分钟。蛋白激酶Cα（PKCα）向膜的转位情况与DG形成相似。在WT-H细胞中，PKCα短暂地与膜结合，然后回到胞质溶胶中。然而，在D-H细胞中，PKCα迅速转位到膜上并在膜上停留超过20分钟。丁醇抑制了PKCα的这种持续转位。此外，WT-H细胞中PAF诱导的c-fos和c-jun的mRNA水平远低于D-H细胞中的水平。抑制DG形成的浓度的普萘洛尔和丁醇抑制了PAF诱导的c-fos和c-jun的mRNA表达。综上所述，D-H细胞中PLD的延长激活证实了磷脂酶C/PKC在PAF激活PLD中的主要作用。此外，此处获得的结果表明，持续的PLD激活反过来导致PKCα的慢性激活和膜转位，这可能在c-fos和c-jun的表达中起重要作用。
