Stitt A W, Anderson H R, Gardiner T A, Bailie J R, Archer D B
Department of Ophthalmology, Queen's University of Belfast, Ireland.
Invest Ophthalmol Vis Sci. 1994 Aug;35(9):3384-92.
The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs).
Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin.
Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization.
These results illustrate the internalization and intracellular trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.
作者研究了培养的视网膜血管内皮细胞（RVECs）中胰岛素和低密度脂蛋白（LDL）的受体介导的内吞作用（RME）及细胞内运输。
将低密度脂蛋白和胰岛素与10纳米胶体金偶联，然后在4℃下将这些配体添加到培养的牛RVECs中20分钟。然后将培养物升温至37℃，并在30秒至1小时的孵育时间后固定。对照细胞在与配体相似的时间和浓度下与未偶联的金胶体孵育。另外的对照细胞在与金胰岛素孵育之前，先暴露于几种浓度的抗胰岛素受体抗体或未偶联胰岛素的饱和溶液中。
使用透射电子显微镜，在RME的各个阶段均观察到胰岛素金和LDL金。胰岛素金颗粒首先被观察到与顶端质膜（PM）结合，然后聚集在网格蛋白包被的小窝中并在内化到包被小泡中。金随后在未包被的细胞质小泡中可见，对应于早期内体和多泡体（MVBs）或晚期内体。在几个实例中，MVBs的限制膜的局部区域似乎被包被，这是以前未描述过的内体膜的特征。在顶端PM处进行RME并通过内体系统后，胰岛素和LDL金偶联物的大部分都积聚在大的溶酶体样区室中。然而，一小部分但相当数量的内化配体被转胞吞并作为离散的膜相关量子在基底细胞表面释放。在高度空泡化的晚期传代RVECs中，LDL金的摄取大大增加。在对照中，抗胰岛素受体抗体和过量的未偶联胰岛素导致金胰岛素结合和内化受到高达89%的抑制。
这些结果说明了RVECs通过高效的RME对胰岛素和LDL的内化及细胞内运输，并且它们为内吞配体的至少两种可能命运提供了证据。本研究概述了重要大分子可能穿过内血视网膜屏障的途径。
