Activation of ceramidase and ceramide kinase by vanadate via a tyrosine kinase-mediated pathway.
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 Na(3)VO(4) 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 Na(3)VO(4)-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 Na(3)VO(4) 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.
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Eiko
E
Tada
Kaori
K
Toyomura
Hiroyuki
H
Nakamura
Hirotsune
H
Sasaki
Takeshi
T
Saito
Masayuki
M
Kaneko
Yasunobu
Y
Okuma
Toshihiko
T
Murayama
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002478:Cells, Cultured; D055572:Ceramidases / Q000502:physiology*; D002518:Ceramides / Q000032:analysis / Q000378:metabolism*; D002855:Chromatography, Thin Layer / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D013110:Sphingosine / Q000032:analysis*; D013268:Stimulation, Chemical; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology*
D002518:Ceramides; C065576:ceramide 1-phosphate; D014638:Vanadates; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D011505:Protein-Tyrosine Kinases; D055572:Ceramidases; D013110:Sphingosine