Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells
Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells
复制标题
DOI：
10.1016/s0041-0101(99)00110-5
复制
发表时间：
1999-11-01
期刊：
影响因子：
2.8
通讯作者：
Peterson, JW
中科院分区：
文献类型：
--
作者：
Saini, SS;Chopra, AK;Peterson, JW
用合成蜂毒肽攻击人单核细胞白血病细胞（U937），并通过薄层色谱（TLC）分析来自细胞（沉淀）和培养基（上清液）的花生四烯酸（AA）/酰化脂质。从这些数据中，蜂毒肽介导的激活/抑制U937细胞中的主要磷脂酶与孔形成，透化和细胞溶解，通过光学显微镜测定。此外，蜂毒肽对U937细胞的无细胞超声裂解物中酰基水解酶活性的影响进行了检查。在这里，我们报告，合成蜂毒素（1 μ M）引起的U937细胞在10-15分钟内的细胞溶解。细胞肥大（5分钟）和聚集（1分钟）之前的细胞溶解。这些脂质的TLC分析表明，总水平（细胞+培养基）的二酰基甘油（DAG），磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）下降，而花生四烯酸（AA）的持续增加（5-30分钟）。然而，磷脂酰乙醇（PEt）、磷脂酸（PA）和磷脂酰丝氨酸（PS）的水平在5-10分钟时短暂增加，在5分钟时达到最大值。（磷脂酶D的终产物，PLD）的水平比5- 10 ℃时AA的水平高约42倍。AA水平的增强似乎是由孔形成/细胞裂解期间释放到培养基中的各种酰基水解酶及其磷脂底物（游离/膜结合）的体外反应引起的。超声处理的细胞裂解物的孵育也增强了AA的释放，这减少后，加入蜂毒肽，表明蜂毒肽抑制这些酰基水解酶。DAG水平的持续降低表明磷脂酶C不受影响。因此，瞬时激活PLD蜂毒肽在开始的点的细胞溶解，建议PLD的作用蜂毒肽介导的膜破坏/细胞溶解的一个未知的信号转导机制。(C)1999 Elsevier Science Ltd.保留所有权利。
Human monocytic leukemia cells (U937) were challenged with synthetic melittin, and arachidonic acid (AA)/acylated lipids from both cells (pellet) and media (supernatant) were analyzed by thin layer chromatography (TLC). From these data, melittin-mediated activation/inhibition of major phospholipases in U937 cells was related to pore formation, permeabilization and cytolysis as determined by light microscopy. Also, the effect of melittin on acylhydrolase activity in the cell-free sonicated lysates of U937 cells was examined. Here we report that synthetic melittin (1 mu M) caused cytolysis of U937 cells within 10-15 min. Cellular hypertrophy (5 min) and aggregation (1 min) preceded cytolysis. TLC analysis of these lipids showed that total levels (cellular+medium) of diacylglycerol (DAG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) decreased, while that of arachidonic acid (AA) increased continuously (5-30 min). However, levels of phosphatidylethanol (PEt) phosphatidic acid (PA) and phosphatidylserine (PS) were increased transiently at 5-10 min being maximal at 5 min. Taken together, the combined levels of PEt and PA (an end product of phopholipase D, PLD) were about 42-fold higher than the level of AA at 5-10 min. Enhancement of AA levels appeared to result from in vitro reactions of various acylhydrolases and their phospholipid substrates (free/membrane bound) liberated into the medium during pore formation/cell lysis. Incubation of sonicated cell lysates also enhanced release of AA, which decreased upon addition of melittin, indicating that melittin inhibited these acylhydrolases. A consistent decrease in the level of DAG showed that phospholipase C was unaffected. Hence, transient activation of PLD by melittin at the point of initiation of cytolysis, suggested a role for PLD in melittin-mediated membrane disruption/cytolysis by an uncharacterized signal transduction mechanism. (C) 1999 Elsevier Science Ltd. All rights reserved.