FLUID-PHASE ENDOCYTOSIS OF HORSERADISH-PEROXIDASE BY CEREBRAL ENDOTHELIAL-CELLS IN PRIMARY CULTURE - CHARACTERIZATION AND KINETIC-ANALYSIS

Endocytosis of horseradish peroxidase (HRP) was studied in primary cul tures of cerebral endothelial cells prepared from 2-week-old rats. The se cultures were considered ''endothelial-like'' on the basis of their ability to internalize acetylated low density lipoprotein. Cellular l ocalization of HRP protein was examined at the light and ultrastructur al levels and endocytosis of the protein was evaluated by a colorimetr ic assay. HRP was localized in discrete cytoplasmic granules by light microscopy. At the ultrastructural level these granules corresponded t o pleomorphic membrane-bound structures that were present throughout t he cytoplasm. The amount of internalized HRP was directly related to t he concentration of the protein in the medium, was not saturable at hi gh concentrations of HRP, and increased with time. Endocytosis proceed ed at 37 degrees C, but was abolished at 4 degrees C. In pulse-chase e xperiments, the quantity of internalized protein in the cells did not significantly change during the 2 hr chase period. Taken together, the se findings suggest that internalization of HRP occurs by fluid-phase endocytosis, a non-receptor-mediated process, and that the protein is stable within an intracellular compartment for at least several hours. (C) 1994 Wiley-Liss, Inc.