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首页> 外文期刊>Biochemistry >CHOLESTEROL REDISTRIBUTION WITHIN HUMAN PLATELET PLASMA MEMBRANE - EVIDENCE FOR A STIMULUS-DEPENDENT EVENT
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CHOLESTEROL REDISTRIBUTION WITHIN HUMAN PLATELET PLASMA MEMBRANE - EVIDENCE FOR A STIMULUS-DEPENDENT EVENT

机译：人血小板血浆膜中胆固醇的重新分配-刺激依赖事件的证据

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摘要

The fluorescent analog NBD-phosphatidylethanolamine and the analogs of cholesterol NBD-cholesterol and cholestatrienol were used to study the distribution of these lipids within the plasma membrane bilayer of human platelets. The probes were incorporated into platelets using phosphatidylcholine donor vesicles. The distribution of NBD lipid and of cholestatrienol in the platelet plasma membrane bilayer was followed by quenching with dithionite and TNBS, respectively. The t(1/2) of cholestatrienol incorporation into platelet membranes was 39 min, and approximately 65% of the probe was quenched by addition of TNBS. When platelets were exposed to collagen or to ADP, a portion of the probe became inaccessible to quenching. Within 2 min of stimulation by collagen (10 mu g/mL), the percentage of cholestatrienol fluorescence quenched by TNBS decreased to 45%. The fluorescent probe was not found to be associated either with the intracellular membranes or in the extracellular media after collagen stimulation. Similar data were obtained with NBD-cholesterol, but the decrease in accessibility of this probe to quenching was considerably slower. The redistribution of endogenous membrane cholesterol was also measured using cholesterol oxidase. Exposure of platelets to collagen decreased the accessibility of endogenous membrane cholesterol to enzymatic oxidation with cholesterol oxidase. Taken together, the foregoing observations are consistent with the stimulus-dependent translocation of cholesterol out of the outer monolayer. Coincident with the redistribution of cholesterol is the reciprocal movement of NBD-phosphatidylethanolamine into the outer monolayer. In the presence of the chaotropic agents urea and guanidine HCl, the movement of cholestatrienol upon collagen stimulation was prevented, but the redistribution of NBD-phosphatidylethanol amine was still detected. We propose that cholesterol translocates to the inner platelet monolayer following collagen stimulation, but the possibility that the sterol moves laterally within the outer membrane monolayer cannot be rigorously excluded.
机译：使用荧光类似物NBD-磷脂酰乙醇胺和胆固醇NBD-胆固醇和胆甾烯醇的类似物来研究这些脂质在人血小板质膜双层中的分布。使用磷脂酰胆碱供体囊泡将探针掺入血小板中。在血小板质膜双层中分布NBD脂质和胆甾烯三醇后，分别用连二亚硫酸盐和TNBS淬灭。胆甾烯醇掺入血小板膜的t（1/2）为39分钟，通过添加TNBS淬灭约65％的探针。当血小板暴露于胶原蛋白或ADP时，探针的一部分变得难以淬灭。在胶原蛋白（10μg / mL）刺激下的2分钟内，TNBS猝灭的胆甾烯醇荧光的百分比降低至45％。在胶原蛋白刺激后，未发现荧光探针与细胞内膜或细胞外介质相关。使用NBD-胆固醇获得了相似的数据，但是该探针淬灭的可及性下降明显较慢。还使用胆固醇氧化酶测量内源膜胆固醇的重新分布。血小板暴露于胶原蛋白会降低内源性膜胆固醇与胆固醇氧化酶进行酶促氧化的可及性。综上所述，上述观察结果与胆固醇依赖刺激的外单层转运有关。与胆固醇的重新分布相吻合的是NBD-磷脂酰乙醇胺向外单层的往复运动。在离液剂尿素和盐酸胍的存在下，胶原蛋白刺激下胆甾烯醇的运动被阻止，但仍检测到NBD-磷脂酰乙醇胺的重新分布。我们建议胆固醇刺激胶原蛋白后胆固醇转移到内部血小板单层，但是不能严格排除固醇在外部膜单层内横向移动的可能性。

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