Fibroblasts maintain a complete endocytic pathway in the presence of lysosomotropic amines

We have investigated the effects of the lysosomotropic amines, ammonium chloride and chloroquine, on the delivery of fluid-phase pinocytic tracers to lysosomes in Chinese hamster ovary (CHO) cells. In preliminary experiments, 15 mM ammonium chloride and 0.1 mM chloroquine were found to be sufficient to give maximal protection of endocytosed material from digestion in a lysosome. In the presence of either amine at these concentrations, the generation time of CHO cells was depressed by less than 30% even though selective depletion of lysosomal hydrolases was observed. For cells treated with either amine for 1 or 6 days the amount of horseradish peroxidase (HRP) internalized in a 1-h pulse was approximately 50-70% of that of control. By cell fractionation, cells treated with amine for 2 or 6 days were found to accumulate fluorescein-dextran or HRP in lysosomes. HRP accumulation in lysosomes in amine-treated cells was also observed by electron microscopy. Little exocytosis of lysosomal HRP into the media was observed under any condition. We conclude that in long-term amine-treated CHO cells endocytic vesicle traffic is maintained.