Cellular cholesterol effect mediated by cyclodextrins - Demonstration of kinetic pools and mechanism of efflux
Cellular cholesterol effect mediated by cyclodextrins - Demonstration of kinetic pools and mechanism of efflux
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DOI：
10.1074/jbc.271.27.16026
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发表时间：
1996-07-05
影响因子：
4.8
通讯作者：
Rothblat, GH
中科院分区：
文献类型：
--
作者：
Yancey, PG;Rodriqueza, WV;Rothblat, GH
在这项研究中，我们比较了 2-羟丙基-β-环糊精和盘状高密度脂蛋白 (HDL) 颗粒的细胞胆固醇流出动力学，以探讨控制环糊精介导的快速流出速率的机制。通过摇动单层生长的细胞来增强胆固醇流出速率，并通过将细胞置于悬浮液中来进一步增强胆固醇流出速率，以达到最大流出速率。流出程度取决于环糊精浓度，并且在浓度> 50 mM时观察到最大流出速率。对于几种细胞类型，观察到细胞胆固醇流出的双指数动力学，表明存在两个胆固醇动力学池：快速池（半时（t（1/2））类似于19-23秒）和慢池，t（1/2）为15-30分钟。还用模型膜观察到两个不同的胆固醇动力学池（大的单层含胆固醇囊泡），这意味着细胞池位于质膜中。通过将细胞与与增加的胆固醇水平复合的环糊精溶液一起孵育，可以改变细胞胆固醇含量。动力池的数量不受细胞胆固醇含量升高的影响，但快速池的大小增加。在耗尽胆固醇快速池的细胞后，该池在40分钟恢复期后完全恢复。比较了环糊精介导的胆固醇从细胞和模型膜流出的温度依赖性；活化能分别为7 kcal/mol和2 kcal/mol。用apo-HDL-磷脂受体颗粒观察到的等效活化能为20 kcal/mol，环糊精分子似乎比磷脂受体更有效，因为从膜表面解吸的胆固醇分子可以直接扩散到环糊精分子的疏水核心中，而不必在被受体隔离之前完全解吸到水相中。
In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-beta-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates, The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t(1/2)) similar to 19-23 s) and a slow pool with t(1/2) of 15-30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane, Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased, After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrin-mediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol, It seems that cyclodextrin molecules are substantially more efficient than phospholipid accepters, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.