Abstract
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.
Highlights
After2hinserum-free medium, 70% of the cells studies of neuroblastoma cell differentiation
Minana et al [16] reported that inhibition of protein kinase C (PKC) by the melium. These cells accumulated platelet-activating factor (PAF) with kinetics isoquinolinesulfonamide H-7 induced neurite outgrowth (NOG) in neuroblastoma similar to that ofNOG inducPAF was synthesized by cells. These findings suggest that PKC activation prevents the de novo pathway, as shown by the incorporatioonf NOG, while PKC inhibition stimulatesNOG
Since thrombin stimulates synthesis of platelet-activating factor (PAF; ~-O-alkyl-2-sn-acetylglycero-3-phosphocholine) in variouscell types, including platelets, endothelicaellls, and neutrophils [17], we examined whether thrombin stimulates PAF synthesis in NB-2acells and if PAF plays some role in modulating NOG
Summary
Laboratories (Merseyside, England);and Silica Gel F254 plates Effect of Thrombin and PKC Inhibitors or Activators on were from Merck. The cells were washed twice in serum-free medium, and test solutions were added as described in the figure legends. The PKCactivator PMA inhibited NOG induced by serum-free medium, with 100 ng/ml decreasing NOG to 27%.In contrast, the PKC inhibitor H-7 stimulated NOG in a dose-dependent fashion, with halfadded and NOG was scored by examining 200-300 cells/well from maximal response at -6 PM; 30 PM H-7 induced NOG to the randomlychosen fields. When NB-2a cells were treated for 2 h with thrombin in serum-free medium, NOG-positive cells were reduced to 24% (Fig. 2). The cells were scrapedfromcultureplates; lipids were extracted as described above and separated by thin-layer chromatographyon silica gel plates[20].Sections of theplates corresponding to PAF standardswere scraped and countedby liquid scintillation counting. Resuspended into 1 ml of buffer A, sonicated, and centrifugedfor 1 h of three independent experiments
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