Modulation of neurite outgrowth in neuroblastoma cells by protein kinase C and platelet-activating factor.
Previous reports have shown that thrombin and activators of protein kinase C (PKC) inhibit neurite outgrowth (NOG) in neuroblastoma cells cultured in serum-free medium. Therefore, we tested the hypothesis that PKC activation mediates the effect of thrombin on NOG in murine neuroblastoma NB-2a cells. After 2 h in serum-free medium, 70% of the cells displayed neurites; addition of 300 ng/ml thrombin reduced NOG to 24% within 1 h. This inhibition was reduced after NB-2a cells were pretreated for 24 h with 200 nM phorbol dibutyrate down-regulate PKC. Thrombin and phorbol 12-myristate 13-acetate inhibited NOG in an additive way and the protein kinase inhibitors H-7, H-8, and HA1004 reversed the effect of thrombin on NOG with a rank order of activity consistent with PKC inhibition. Furthermore, PKC was translocated from the cytosol to a membrane-bound form 5 to 10 min after addition of thrombin. These findings indicate that thrombin inhibits NOG through a PKC-dependent pathway. Thrombin stimulates the synthesis of the phospholipid platelet-activating factor (PAF) in some cells. However, NOG was markedly stimulated when PAF or its analogue carbamyl-PAF were added to NB-2a cells in medium with serum. Furthermore, the PAF receptor antagonist SRI 63072 inhibited NOG in NB-2a cells in serum-free medium. These cells accumulated PAF with kinetics similar to that of NOG inducPAF was synthesized by the de novo pathway, as shown by the incorporation of [3H]choline. These findings suggest that PAF is a mediator of NOG in NB-2a cells. Thrombin neither stimulates nor inhibits PAF synthesis in these cells.
Herrick-Davis
K
K
Department of Biological Sciences, University at Albany, State University of New York 12222.
Camussi
G
G
Bussolino
F
F
Baglioni
C
C
D000818:Animals; D002455:Cell Division; D006801:Humans; D051379:Mice; D016501:Neurites / Q000502:physiology*; D009447:Neuroblastoma; D010972:Platelet Activating Factor / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000502:physiology; D014407:Tumor Cells, Cultured
D010972:Platelet Activating Factor; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate