title: Endocytosis of native and cationized horseradish peroxidase by cultured mouse peritoneal macrophages. Variations in cell surface binding and intracellular traffic and effects of colchicine.
abstract: Thioglycollate-elicited mouse peritoneal macrophages were cultivated in vitro and endocytosis of native and cationized horseradish peroxidase was studied electron microscopically and biochemically. Native peroxidase (HRP) was ingested by fluid-phase endocytosis and accumulated in lysosomes. Cationized peroxidase (CHRP) bound diffusely to the macrophage surface in a saturable manner. It was then internalized via membrane folds and transferred not only to lysosomes but also to the Golgi complex, mainly those parts referred to as GERL and positive for acid phosphatase activity. Following initial uptake, surface staining for CHRP was lost, although the tracer remained present in the medium. This indicates that anionic binding sites were internalized together with the ligand and not immediately replaced. Accordingly, continued uptake of CHRP occurred at a rate similar to that for HRP. Exposure of the macrophages to cationized ferritin (CF) decreased their ability to bind CHRP. However, after 2 to 4 h in CF-free medium, the CHRP-binding ability returned and raised to 2 to 3 times higher values than in cells not exposed to CF. Treatment with cycloheximide at a concentration that effectively inhibits protein synthesis did not clearly affect this regeneration. These findings support the concept of recirculation of plasma membrane constituents. They further suggest that there exists an intracellular membrane pool which rapidly exchanges with the cell surface. Colchicine removed cytoplasmic microtubules, caused a characteristic disorganization of the Golgi complex, and inhibited uptake of both HRP and CHRP. Additionally, no transport of CHRP to the Golgi complex or GERL was observed in colchicine-treated cells. The regeneration of surface anions after exposure to CF was also delayed. Contrarily, lumicolchicine was without effect on cell morphology and uptake as well as intracellular transport of the tracers. Nevertheless, the effects of colchicine on endocytosis were not necessarily due to a direct role of microtubules. They could be secondary to a disturbed function of the Golgi complex or other organelles after collapse of the microtubular cytoskeleton.
mesh_terms: D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D006735:Horseradish Peroxidase / Q000276:immunology*; D008264:Macrophages / Q000276:immunology* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure; D010544:Peroxidases / Q000276:immunology*
chemical_list: D006735:Horseradish Peroxidase; D010544:Peroxidases; D003078:Colchicine