Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells

The uptake and transport of cholesterol-carrying low density lipoprotein (L DL) by the arterial wall is a continuous dynamic process, contributing to t he cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R), In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membran es in endothelial cells; (ii) the role of LDL receptors in the control of c holesterol homeostasis and (iii) the translocation of LDL receptor across t he EC, To this purpose bovine aortic EC were cultured on filters in a doubl e-chamber system, in Dulbecco's medium supplemented either with 10% fetal c alf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS), The cells w ere exposed for 3 h to [H-3]acetate (40 mu Ci) added to both compartments o f the cell culture inserts. The newly synthesized [H-3]cholesterol was dete cted by thin layer chromatography and quantified by liquid scintillation co unting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular path way of LDGR was examined by electron microscopy using a complex made of pro tein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor anti body (Au-PA-C7), To evaluate the distribution and the transport of LDL-R fr om one cell surface to the other, EC grown in LPDS were radioiodinated eith er on the apical or on the basolateral surface, incubated on the same surfa ce with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunopre cipitated with anti-LDL-R antibody or with mouse IgG (as control), The elut ed antigen-antibody complexes were precipitated with streptavidin-agarose b eads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the compl ex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lys osomes, irrespective of the cell surface exposed to the complex; (c) bioche mical assays indicated that upon ligand binding, the LDL-R were translocate d preferentially from the apical to the basal plasma membrane.