The regulation of expression and function of glycoshingolipids on cell surfaces with an inhibitor of glucosylceramide synthesis.

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1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24h in the presence of 50muM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolilids in neurite outgrowth of murine neuroblastoma cells. Ceramide, sphingomyelin and sphingo-sine were found to accumulate in NS-20Y cells after treatment with threo-PDMP. Exogenous sphingosine inhibited neurite outgrowth and caused retraction of neurites. Neurite outgrowth from NS-20Y cells was inhibited by N,N-dimethyl-sphingosine (50% inhibition at -0.1 muM), sphingosine(-0.8 muM),N-hexanoyl-sphingosine (-1 muM), and N-acethylsphingosine(-10muM). A protein kinase inhibitor, H-7, did not affect the neurite outgrowth, suggesting the inhibitory effect of these sphingolipids is protein kinase C-independent.