Rapid Internalization and Intracellular Metabolic Processing of Exogenous Ganglioside by Cerebellar Granule Cells Differentiated in Culture

Ganglioside GMl, tritiated at the level of the long chain base (sphingosine) ([Sph‐3H]GMl), sialic acid (N‐ace‐tylneuraminic acid) ([NeuAc‐3H]GMl), or terminal galactose ([Gal‐3H]GMl) was supplied to cerebellar granule cells differentiated in vitro, and its metabolic processing was followed with pulse time. Using [Sph‐3H]GM 1 and [NeuAc‐3H]GMl the formation of radioactive compounds of catabolic origin (GM2, GM3, lactosylceramide, glucosylceramide, and ceramide) started being detectable at 10–15 min of pulse, whereas compounds of biosynthetic origin (GD1a, GD1b, GT1b, O‐acetylated GT1b, sphingomyelin, and sialoglycoprotein) appeared after 15–30 min of pulse. Using [Gal‐3H]GM1 two radioactive substances were formed, GD1 a and GT1b, with the former (produced by direct sialosylation of GM1) appearing after 30 min of pulse and the latter (formed by bio‐synthetic recycling of released galactose) appearing after 2 h. The radioactivity linked to all metabolites increased with increasing pulse time until 4 h. The percentage of GM1 taken up and subjected to metabolic processing was found to increase from 1.8% after 10 min of pulse to 12.5% after 4 h. Cerebellar granule cells were able to release enzymes of lysosomal origin, β‐D‐N‐acetylhexosaminidase and β‐D‐galac‐tosidase, into the culture medium, with the release being markedly decreased by the absence in the medium of fetal calf serum, a condition that was used for studying exogenous GM1 uptake and metabolization. However, these enzymes exerted no activity at the pH of the culture medium, and no radioactive gangliosides, besides GM1, were detected in the culture medium during pulse. Addition of 50 μM chloroquine to granule cells during the pulse experiments with radioactive GM1 completely blocked the metabolic processing of exogenous GM1 with the exception of direct sialosylation, a result indicating the key role played by lysosomes in the process. The overall results show that cerebellar granule cells take up, internalize, and metabolize exogenous GM1 rapidly, with the lysosomes being involved immediately in degradation, and the biosynthetic apparatuses somewhat later in recycling metabolic intermediates. The hypothesis can be proposed that the ganglioside present in the membrane domain subjected to internalization is rapidly turned over.