Härmälä A S, Pörn M I, Mattjus P, Slotte J P
Department of Biochemistry and Pharmacy, BioCity, Abo Akademi University, Turku, Finland.
Biochim Biophys Acta. 1994 Mar 24;1211(3):317-25. doi: 10.1016/0005-2760(94)90156-2.
The compound U1866A (3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one) has been shown to inhibit the cellular transfer of low-density lipoprotein-derived cholesterol from lysosomes to plasma membranes (Liscum and Faust (1989) J. Biol. Chem. 264, 11796-806). We have in this study examined the effects of U18666A on cholesterol translocation from plasma membranes to intracellular membranes. Translocation of plasma membrane cholesterol was induced by degradation of plasma membrane sphingomyelin. The sphingomyelinase-induced activation of the acyl-CoA cholesterol acyl transferase (ACAT) reaction was completely inhibited in a dose-dependent manner by U18666A, both in cultured human skin fibroblasts and baby hamster kidney cells. Half-maximal inhibition (within 60 min) was obtained with 0.5-1 microgram/ml of U18666A. A time-course study indicated that the onset of inhibition was rapid (within 10-15 min), and reversible if U18666A was removed from the incubation mixture. Using a cholesterol oxidase assay, we observed that the extent of plasma membrane cholesterol translocation in sphingomyelinase-treated HSF cells was significantly lowered in the presence of U18666A (at 3 micrograms/ml). The effect of U18666A on cholesterol translocation was also fully reversible when the drug was withdrawn. In mouse Leydig tumor cells, labeled to constant specific activity with [3H]cholesterol, the compound U18666A inhibited in a dose-dependent manner the cyclic AMP-stimulated secretion of [3H]steroid hormones. The effects seen with compound U18666A appeared to be specific for this molecule, since another hydrophobic amine, imipramine, did not in our experiments affect cholesterol translocation or ACAT activation. Since different cell types display sensitivity to U18666A in various intracellular cholesterol transfer processes, they appear to have a common U18666A-sensitive regulatory mechanism.
化合物U1866A（3β-[2-(二乙氨基)乙氧基]雄甾-5-烯-17-酮）已被证明可抑制低密度脂蛋白衍生胆固醇从溶酶体向质膜的细胞转运（Liscum和Faust，(1989)《生物化学杂志》264, 11796 - 11806）。在本研究中，我们检测了U18666A对胆固醇从质膜向细胞内膜转运的影响。质膜鞘磷脂的降解诱导了质膜胆固醇的转运。在培养的人皮肤成纤维细胞和幼仓鼠肾细胞中，U18666A以剂量依赖的方式完全抑制了鞘磷脂酶诱导的酰基辅酶A胆固醇酰基转移酶（ACAT）反应的激活。在0.5 - 1微克/毫升的U18666A作用下，60分钟内达到半数最大抑制。时间进程研究表明，抑制作用起效迅速（10 - 15分钟内），如果从孵育混合物中去除U18666A，抑制作用是可逆的。使用胆固醇氧化酶测定法，我们观察到在U18666A（3微克/毫升）存在的情况下，鞘磷脂酶处理的人皮肤成纤维细胞中质膜胆固醇转运的程度显著降低。当撤去该药物时，U18666A对胆固醇转运的影响也是完全可逆的。在用[3H]胆固醇标记至恒定比活性的小鼠睾丸间质细胞瘤细胞中，化合物U18666A以剂量依赖的方式抑制了环磷酸腺苷刺激的[3H]类固醇激素分泌。化合物U18666A的作用似乎对该分子具有特异性，因为在我们的实验中，另一种疏水性胺丙咪嗪并未影响胆固醇转运或ACAT激活。由于不同细胞类型在各种细胞内胆固醇转运过程中对U18666A表现出敏感性，它们似乎具有共同的U18666A敏感调节机制。
