ABSTRACT
The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-induced molecular changes during the differentiation process. The involvement of sphingomyelin (SM) was studied using the fluorescent analogue of ceramide, i.e. N-lissamine rhodaminyl-(12-aminododecanoyl) D-erythro-sphingosine (C12-LRh-Cer). This fluorescent analogue is metabolically active and can be used to follow the biosynthesis of SM in intact cells. NGF induces a 4-fold increase of fluorescent SM content in PC12 cells, when loaded with C12-LRh-Cer. Treatment of PC12 cells with actinomycin D or cycloheximide completely abolishes the NGF-induced elevation of SM. Inhibition of p140(trkA) receptor by AG-879 prevents extracellular signal-regulated kinase 1/2 phosphorylation and suppresses the increase of SM. Inhibition of protein kinase C (PKC), protein kinase A (PKA) and phosphatidylinositol 3-kinase does not have any effect on NGF-induced C12-LRh-SM accumulation. On the other hand, activation of PKA or PKC with simultaneous treatment with NGF has a synergistic effect on increase of SM content. The NGF-induced SM increase in PC12 cells is an effect promoted by other differentiating agents like dibutyryl cyclic AMP or fibroblast growth factor-2 but not by a mitogenic agent like epidermal growth factor.
Subject(s)
Nerve Growth Factor/metabolism , Sphingomyelins/biosynthesis , Animals , Cyclic AMP-Dependent Protein Kinases/metabolism , Cycloheximide/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , PC12 Cells , Phosphorylation , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Rats , Receptor, trkA/metabolism , Tyrphostins/pharmacologyABSTRACT
GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been administered to cultured Madin-Darby canine kidney (MDCK) cells for different pulse times (0.5-24 h), and its metabolic fate was followed. The fluorescent GM2, asialo-GM2, asialo-GM1 and ceramide were the only detectable metabolites. The complete absence of fluorescent GM3 is consistent with the presence in these cells of a sialidase working on GM1 and GM2 gangliosides. After treatment of the cells with chloroquine the fluorescent GM1 remained essentially undegraded, indicating a catabolic processing at lysosomal level.
