CERAMIDE AS A MODULATOR OF ENDOCYTOSIS

The effects of ceramide analogs on the uptake of markers for fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low density lipoprotein, LDL) endocytosis were studied in Chinese hamster fibroblasts. N-Hexanoyl-D-erythro-sphingosine (C-6-Cer) decreased the uptake of HRP in a dose-dependent manner. Internalization was inhibited >40% with 25 mu M C-6-Cer, relative to controls, and was apparent within 5 min. Internalization of HRP was also inhibited by other Cer analogs and by treatment with agents that raise levels of endogenous Cer (sphingomyelinase or the glycosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)), but not by N-hexanoyl-D-erythro-sphinganine (C-6-dihydro-Cer) or sphingosine. Internal of LDL was also inhibited by C-6-Cer in a concentration dependent manner, but was less pronounced than the effect on HRP internalization (10% versus 40% inhibition with 25 mu M C-6-Cer), suggesting that ceramide might affect fluid-phase and receptor-mediated endocytosis to different extents. C-6-Cer also slowed HRP and LDL transport from endosomes to lysosomes as studied by analysis of endocytic vesicles on Percoll density gradients and induced a redistribution of endocytic organelles as determined by fluorescence microscopy of intact cells using appropriate markers. This resulted in decreased degradation of I-125-Iabeled LDL in the presence of C-6-Cer. These results suggest that endogenous ceramide may modulate endocytosis.