Abstract
To determine whether receptor-mediated endocytosis occurs in vasopressin-responsive cells, we developed a model system using synthetic fluorescent-labeled vasopressin analogs and A10 (smooth muscle) and LLC-PK1 (kidney epithelial) cells in culture; these cell lines express V1 and V2 vasopressin cell surface receptor types, respectively. We used epifluorescence microscopy to examine the binding, internalization, and intracellular destination of [1-(2-mercapto)propionic acid,8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) and [1-(2-mercapto)propionic acid,8-lysine-N6-carboxyfluorescein]vasopressin (F-MLVP) in these cells. The rhodamine-labeled fluorescent vasopressin analog, R-MLVP, initially bound in a diffuse manner at the cell surface of both A10 and LLC-PK1 cells and could be displaced by excess unlabeled [8-arginine]vasopressin. After incubation at 37 degrees C, bound ligand rapidly aggregated into small clusters or patches, which were internalized in a manner consistent with receptor-mediated endocytosis. Subsequent processing of internalized ligand-receptor complexes appeared to differ between A10 and LLC-PK1 cells. In the case of LLC-PK1 cells, ligand was delivered to a tightly focused lysosome compartment in the perinuclear region of the cell, and receptor molecules were replenished at the cell surface. The lysosomal location of ligand was supported by the quenching of fluorescence in the internalized vesicles when F-MLVP was used as a fluorescent tracer. In the case of A10 cells, ligand became localized to a vesicular compartment and reappearance of receptor at the cell surface was limited. Our data are consistent with the occurrence of receptor-mediated endocytosis of vasopressin in cells with V1 and V2 receptors.