| Co-Investigator(Kenkyū-buntansha) |
TAKETOMI Tamotsu Shinshu University School of Medicine, Research Center for Aging and Adaptation,, 医学部, 教授 (30020704)
HARA Atsushi Shinshu University School of Medicine, Research Center for Aging and Adaptation,, 医学部, 講師 (70126697)
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| Research Abstract |
(1) Neurite outgrowth in murine neuroblastoma NS-20Y cells induced by serum deprivation was inhibited by shingosine (1-2muM) as well as by an inhibitor of glucosylceramide synthase (1-phenyl-2-decanoylamino-3-morpholino-3-propanol). The roles of protein kinases and calmodulin in regulating neurite outgrowth were investigated by testing the effect of several inhibitors on the neuritogenesis. W-7 (10muM), calmidazolium (0.3muM) and trifluoperazine (0.1muM), drugs reported to inhibit calmodulin-dependent events, reduced neurite outgrowth. On the other hand, H-7 and H-89 were ineffective, and genistein did not affect the number of cells bearing neurites. Activation of protein kinases, which are blocked by these inhibitors, does not appear to be essential to the extension and maintenance of neurites. KN-62 and KN-93 (inhibitors of Ca^<2+>/calmodulin-dependent protein kinase II) did not inhibit neurite outgrowth. These results suggest that a calmodulin-dependent process, other than the activ
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ation of Ca^<2+>/calmodulin-dependent protein kinase II,is involved in neuritogenesis of murine neuroblastoma NS-20Y cells in serum-free medium. The inhibitory effect of sphingosine on neurite outgrowth is most likely due to calmodulin inhibition.
(2) The effects of a series of sphingosine derivatives and an inhibitor of glucosylceramide synthase, PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol), on the cell cycle progression of murine neuroblastoma NS-20Y cells were investigated by flow cytometry. In the presence of PDMP (50muM,24h) the proportion of cells in the S phase was reduced, whereas that in the G0/G1 phase significantly increased. Incubation of cells with sphingosine, sphinganine, sphingosine-1-phosphate, short-chain ceramides (N-acethyl-or N-hexanoyl-sphingosine) or sphingosyl-phosphocholine did not affect the cell cycle. The DNA peaks characteristic of apoptosis were not observed. These results suggest that the inhibition of the proliferation of NS-20Y cells by PDMP is caused by its effect on cell cycle progression and is more likely due to the reduced levels of glycosphingolipids rather than the increased levels of ceramide or sphingosine. Less
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