Plevin R, Cook S J, Palmer S, Wakelam M J
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Biochem J. 1991 Oct 15;279 ( Pt 2)(Pt 2):559-65. doi: 10.1042/bj2790559.
Platelet-derived growth factor (PDGF) stimulated sn-1,2-diacylglycerol (DAG) mass formation in Swiss 3T3 fibroblasts with a lag time of some 30 s. The response was biphasic, with the second phase being sustained over time. PDGF also stimulated the formation of Ins(1,4,5)P3 with a similar lag time to the DAG response, suggesting that DAG is derived from PtdIns(4,5)P2 hydrolysis at this time point. PDGF-stimulated phosphatidylcholine (PtdCho) hydrolysis in Swiss 3T3 fibroblasts, as measured by the formation of water-soluble choline metabolites and phosphatidylbutanol (PtdBut) accumulation, was by a phospholipase D (PLD)-catalysed pathway which was kinetically downstream of initial PtdIns(4,5)P2 hydrolysis. Accumulation of PtdBut increased up to 15 min, suggesting that PLD activity is not rapidly densitized in response to PDGF. The kinetics of PtdCho hydrolysis closely paralleled the second phase of DAG formation, strongly suggesting that during prolonged stimulation periods PtdCho is a major source of DAG in these cells. However, since PtdIns(4,5)P2 breakdown was also prolonged, PDGF-stimulated DAG may be derived from both phospholipids. Down-regulation of protein kinase C (PKC), by pre-treatment with phorbol 12-myristate 13-acetate, abolished both [3H]choline and [3H]PtdBut formation, suggesting that PLD-catalysed PtdCho hydrolysis may be dependent on PKC activation, supporting its dependence on prior PtdIns(4,5)P2 hydrolysis.
血小板衍生生长因子（PDGF）刺激瑞士3T3成纤维细胞中sn-1,2-二酰基甘油（DAG）的大量形成，延迟时间约为30秒。该反应呈双相性，第二阶段随时间持续。PDGF还刺激肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）的形成，其延迟时间与DAG反应相似，表明在此时间点DAG源自磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）的水解。通过水溶性胆碱代谢产物的形成和磷脂酰丁醇（PtdBut）积累来测量，PDGF刺激瑞士3T3成纤维细胞中的磷脂酰胆碱（PtdCho）水解，这是由磷脂酶D（PLD）催化的途径，在动力学上位于初始PtdIns(4,5)P2水解的下游。PtdBut的积累在15分钟内增加，表明PLD活性不会因PDGF而迅速脱敏。PtdCho水解的动力学与DAG形成的第二阶段密切平行，强烈表明在长时间刺激期间，PtdCho是这些细胞中DAG的主要来源。然而，由于PtdIns(4,5)P2的分解也延长，PDGF刺激的DAG可能源自这两种磷脂。用佛波醇12-肉豆蔻酸酯13-乙酸酯预处理下调蛋白激酶C（PKC），消除了[3H]胆碱和[3H]PtdBut的形成，表明PLD催化的PtdCho水解可能依赖于PKC激活，支持其对先前PtdIns(4,5)P2水解的依赖性。
