Pacini L, Limatola C, Frati L, Luly P, Spinedi A
Department of Biology, University of Rome Tor Vergata, Italy.
Biochem J. 1993 Jan 1;289 ( Pt 1)(Pt 1):269-75. doi: 10.1042/bj2890269.
Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.
毒蕈碱对人神经母细胞瘤细胞系SK-N-BE(2)的刺激引发磷酸肌醇和磷脂酰胆碱（PtdCho）的水解，并使二酰基甘油（DG）含量迅速且持续升高。加入卡巴胆碱（CCh）后，磷脂酶C（PLC）立即催化磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）裂解，随后磷酸肌醇水解持续至少5分钟。通过长期给予[3H]胆碱对PtdCho进行广泛标记后，细胞刺激导致[³H]磷酸胆碱（PCho）向细胞外培养基中的释放增强；相对于加入CCh延迟15秒出现的[³H]PCho释放增强，在刺激的第一分钟内尤为明显，且被证明是由PtdCho特异性PLC激活引起的。事实上，当细胞短期暴露于[³H]胆碱以广泛标记细胞内PCho库而非PtdCho时，刺激并未导致[³H]PCho向培养基中的释放增强。在用[³H]肉豆蔻酸预标记并在1%（v/v）乙醇存在下刺激的细胞中，[³H]磷脂酰乙醇的积累证明了PtdCho特异性磷脂酶D（PLD）的激活；然而，这一代谢途径被证明是细胞刺激过程中导致磷脂酸（PtdOH）生成的次要途径，未观察到PtdCho特异性PLD和PtdOH磷酸水解酶的顺序作用产生DG。对用[³H]肉豆蔻酸和[¹⁴C]花生四烯酸进行双重标记的细胞的研究表明，在刺激的15秒内，DG唯一来源于PtdIns(4,5)P2，而在随后的时间里，PtdCho是主要来源。有证据表明，磷酸肌醇衍生的DG快速且选择性地转化为PtdOH可能在决定上述来源的DG的时间积累模式中起重要作用。
