Nerve growth factor induces sphingomyelin accumulation in pheochromocytoma cells.
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.
Piccinotti
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Unit of Biochemistry, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Via Valsabbina 19, 25123, Brescia, Italy. piccinot@med.unibs.it
Benaglia
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G
Bresciani
R
R
Zizioli
D
D
Presta
M
M
Preti
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A
Marchesini
S
S
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020932:Nerve Growth Factor / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D020917:Receptor, trkA / Q000378:metabolism; D013109:Sphingomyelins / Q000096:biosynthesis*; D020032:Tyrphostins / Q000494:pharmacology
C081020:AG-879; D004791:Enzyme Inhibitors; D013109:Sphingomyelins; D020032:Tyrphostins; D020932:Nerve Growth Factor; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D020917:Receptor, trkA; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases