Internalization of vasopressin analogs in kidney and smooth muscle cells: Evidence for receptor-mediated endocytosis in cells with V₂ or V₁ receptors

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°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 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.