Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase-Mediated Pathway
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- Tada Eiko
- Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan
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- Toyomura Kaori
- Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan
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- Nakamura Hiroyuki
- Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan
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- Sasaki Hirotsune
- Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan
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- Saito Takeshi
- Department of Health Science, Hokkaido University School of Medicine, Japan
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- Kaneko Masayuki
- Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Japan
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- Okuma Yasunobu
- Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Japan
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- Murayama Toshihiko
- Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan
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Abstract
Ceramide, a key molecule in the metabolism of sphingolipids, is converted by ceramidase to sphingosine, and phosphorylated by ceramide kinase to form ceramide-1-phosphate (C1P). In this study, we improved on a method of thin-layer chromatography using a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole–labeled C6-ceramide (NBD-ceramide) by adding another step for separation of extracted ceramide metabolites by lipophilicity, and determined levels of C1P, caproic acid, sphingomyelin, and glucosylceramide simultaneously. Also we found that 1) treatment of NBD-ceramide–labeled cells (human lung adenocarcinoma A549 cells and Chinese hamster ovary cells) with Na3VO4 increased the amount of NBD-C1P formed within 30 min, 2) the treatment increased production of NBD-caproic acid, a counterpart of sphingosine, by ceramidase within 2 h, 3) expression of ceramide kinase enhanced the Na3VO4-induced formation of NBD-C1P, and tyrosine kinase inhibitors (herbimycin and genistein) decreased the response, 4) the production of NBD-caproic acid in A549 cells was inhibited by genistein, and 5) the responses for 2 h after Na3VO4 treatment were accompanied by a decrease in the production of NBD-sphingomyelin, not a loss of NBD-ceramide. The improved thin-layer chromatography method was useful for the simultaneous determination of enzymatic activities for ceramide metabolism in cells.
Journal
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- Journal of Pharmacological Sciences
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Journal of Pharmacological Sciences 114 (4), 420-432, 2010
The Japanese Pharmacological Society
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Details 詳細情報について
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- CRID
- 1390001205180769920
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- NII Article ID
- 10029891066
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- NII Book ID
- AA11806667
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- ISSN
- 13478648
- 13478613
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- NDL BIB ID
- 10926454
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed