Natural ceramide is unable to escape the lysosome, in contrast to a fluorescent analogue

Since the generation upon cell stimulation of the second messenger ceramide has been reported to occur in an endosomal/lysosomal compartment, we investigated whether ceramide formed in the lysosomes can escape this compartment. The metabolic fate of radiolabelled ceramide produced by intralysosomal hydrolysis of LDL-associated [ceramide-³H]sphingomyelin or [stearoyl-1-¹⁴C]sulfatide was examined in fibroblasts from control individuals and a patient with inborn lysosomal ceramidase deficiency (Farber disease). The behavior of this radioactive ceramide was compared to that of a fluorescent (lissamine-rhodaminyl) ceramide analogue deriving from sulfatide degradation. While in Farber cells the natural, radiolabelled ceramide remained completely undegraded and accumulated in the lysosomes, the fluorescent derivative was rapidly converted to sphingomyelin. These findings strongly suggest that, in contrast to fluorescent derivatives, endogenous long-chain ceramide is unable to exit from lysosomes, therefore making the lysosomal ceramide unlikely to be a biomodulatory molecule.