Endocytosis, proteolysis, and exocytosis of exogenous proteins by cultured myotubes.
The endocytic activity of chick myotubes in cultures was investigated. Differentiated myotubes internalized the fluid-phase marker horseradish peroxidase in membrane-bound particles which typically displayed reaction product at the inner surface of the vesicle. Pulse-chase experiments demonstrated a rapid decrease in the number of horseradish peroxidase-containing vesicles and a redistribution from a uniform to a perinuclear pattern. Horseradish peroxidase uptake was extensively inhibited by incubation at 0-4 degrees C consistent with an endocytic mechanism. To further characterize the process, the fate of labeled protein was investigated. Following uptake [3H] hemoglobin A was extensively degraded (40-50%) to acid-soluble products within 10 h. Degradation displayed a biphasic pattern with a rapid early phase followed by a much slower second phase. The decreasing rate of proteolysis can be accounted for, in part, by a simultaneous exocytosis of a substantial fraction (25-30%) of acid-insoluble label from myotubes. The lysosomotropic agents methylamine, monensin, and chloroquine significantly inhibited (23-75%) proteolysis, indicating a lysosomal site of degradation. Part of the inhibitory effect results from an increase in exocytosis in the presence of these agents. Degradation of endocytosed [3H]hemoglobin A was not inhibited by insulin. In contrast degradation of endogenous myotube proteins was inhibited (40%) by insulin and blocked by methylamine. These results suggest that cultured myotubes possess a coupled endocytic/exocytic pathway for macromolecules and that a fraction of the internalized substrate is degraded by an insulin-insensitive lysosomal pathway.
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D004705:Endocytosis; D005089:Exocytosis; D006441:Hemoglobin A / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D008954:Models, Biological; D009132:Muscles / Q000166:cytology*; D011506:Proteins / Q000378:metabolism*
D007328:Insulin; D008744:Methylamines; D011506:Proteins; D006441:Hemoglobin A; C027451:methylamine; D006735:Horseradish Peroxidase