RECEPTOR-MEDIATED ENDOCYTOSIS AND INTRACELLULAR TRAFFICKING OF INSULIN AND LOW-DENSITY-LIPOPROTEIN BY RETINAL VASCULAR ENDOTHELIAL-CELLS

Purpose. The authors investigated the receptor-mediated endocytosis (R ME) and intracellular trafficking of insulin and lo iv-density lipopro tein (LDL) in cultured retinal vascular endothelial cells (RVECs). Met hods. Low-density lipoprotein and insulin were conjugated to 10 nm col loidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degree s C and fixed after incubation times between 30 seconds and 1 hour. Co ntrol cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control ce lls were exposed to several concentrations of antiinsulin receptor ant ibody or a saturating solution of unconjugated insulin before incubati on with gold insulin. Results. 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 internaliz ation in coated vesicles. Gold was later visualized in uncoated cytopl asmic vesicles, corresponding to early endosomes and multivesicular bo dies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of en dosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both in sulin- and LDL-gold conjugates was seen to accumulate in large lysosom e-like compartments. However, a small but significant proportion of th e internalized ligands was transcytosed and released as discrete membr ane-associated quanta at the basal cell surface. The uptake of LDL gol d was greatly increased in highly vacuolated, late-passage RVECs. In c ontrols, anti-insulin receptor antibody and excess unconjugated insuli n caused up to 89% inhibition in gold-insulin binding and internalizat ion. Conclusion. These results illustrate the internalization and intr acellular trafficking by RVECs of insulin and LDL through highly effic ient RME, and they provide evidence for at least two possible fates fo r the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.