Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

概要

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.

著者

NAKAMURA Hiroyuki Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University
OKUMA Yasunobu Departmen of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University
Tada Eiko Laboratory Of Chemical Pharmacology Graduate School Of Pharmaceutical Sciences Chiba University
Saito Takeshi Department Of Forensic Medicine Tokai University School Of Medicine
Murayama Toshihiko Laboratory Of Chemical Pharamacology Graduate School Of Pharmaceutical Sciences Chiba University
Kaneko Masayuki Department Of Dental Radiology Health Sciences University Of Hokkaido
Toyomura Kaori Laboratory Of Chemical Pharmacology Graduate School Of Pharmaceutical Sciences Chiba University
Sasaki Hirotsune Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, J
Saito Takeshi Department Of Cardiovascular Surgery Sendai Open Hospital
Nakamura Hiroyuki Laboratory Of Chemical Pharmacology Graduate School Of Pharmaceutical Sciences Chiba University

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