Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped.
Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped.
复制标题
包埋 Fura 2 的脂质体中的离子霉素和磷脂酸介导的跨膜钙运动。
DOI：
10.1021/bi00415a040
复制
发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Weissmann,G
中科院分区：
文献类型：
--
作者：
Blau,L;Weissmann,G
Yeshiva大学化学系，纽约列克星敦大道245号，纽约，纽约10016；纽约大学医学中心风湿病学系，纽约第一大道550号，纽约，纽约10016；摘要：一种新的脂质体方法允许研究钙在多层囊泡（MLV）双层上的运动，该多层囊泡包裹了钙依赖的荧光指示剂Fura 2。离子霉素介导的钙在磷脂酰胆碱(PC)/磷酸二酯（DCP）的MLV上的转运，比例为9∶1，服从简单的一级动力学，因为初始速率与离子霉素或钙浓度的对数对数图斜率约为1。由于Ca在细胞的刺激-反应偶联过程中以Ca依赖的方式易位，这些细胞从多磷酸肌苷中形成二酰基甘油（DAG）和磷脂酸（PA），因此我们比较了PA和DAG的作用。PA和DAG预掺入PC/DCP囊泡中，其中微量的离子霉素提供跨膜电位（由于Ca2+/H+交换）。与含有dcp或dag的MLV相比，在鸡蛋卵磷脂PA、二聚乙二醇-PA和双棕榈酰-PA存在时，观察到Ca运动显著增加。脂质体中的dag如1 -油基-2-乙酰甘油或1,2 -二油基甘油可降低钙易位率。Ca内流到含PA的MLV中取决于双层PA的摩尔百分比；钙内流的复杂动力学与非双层态的形成是相容的。将胆固醇掺入脂质体可能通过压缩双分子层抑制MLV对钙的初始吸收速率。在pH为7.4的脂质体中，钙内流随外部介质pH从6.9增加到7.9而增加。结果不仅表明，跨膜pH梯度和电离层的电离程度影响钙在脂质双分子层上的转运速率，而且还表明，在模型系统中，PA而不是DAG促进钙转运，其速率受双分子层的脂质组成和悬浮液的pH的影响。胞质游离Ca水平（[Ca];） 1在介导细胞对细胞外信号的反应中起关键作用（Berridge & Irvine, 1984; Kikkawa & Nishizuka, 1986; Spat et al., 1986）。这些反应可能是由配体诱导的肌醇磷脂水解引起的（Hokin & Hokin, 1953; Nishizuka, 1984a; Berridge, 1984），随后[Ca]升高；(米歇尔,1975)。缺乏磷脂
Department of Chemistry, Yeshiva University, 245 Lexington Avenue, New York, New York 10016, and Division of Rheumatology, Department of Medicine, New York UniversityMedical Center, 550 First Avenue, New York, New York 10016 Received October 14, 1987; Revised Manuscript Received March 24, 1988 abstract: A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9: 1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which traceamounts of ionomycin provided transmembrane potential (dueto Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP-or DAG-containing MLV. DAGs such as l-oleoyl-2-acetoylglycerol or 1, 2-dioleoylglycerol in liposomesdecreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external mediumfrom 6.9 to 7.9 in liposomes with an internal pHof 7.4. The results not only indicatethat transmembrane pH gradients and the extent of ionization of the ionophore affect rates of Ca translocation across lipid bilayers but also demonstrate that in model systems PA but not DAG promotes Ca translocation, the rate of which is altered by lipid composition of the bilayer and the pH of the suspension.The levels of free cytoplasmic Ca ([Ca];) 1play a critical role in mediating cell responses to extracellular signals (Berridge & Irvine, 1984; Kikkawa & Nishizuka, 1986; Spat et al., 1986). These responses are probably initiated by ligand-in-duced hydrolysis of inositol phospholipids (Hokin & Hokin, 1953; Nishizuka, 1984a; Berridge, 1984) followed by a rise in the [Ca];(Michell, 1975). In absence of phospholipid