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

Abstract

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 m M ammonium chloride and 0.1 m M 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 <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 ~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.