Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.
Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.
Bladergroen
B A
BA
Department of Biochemistry and Cell Biology, Graduate School of Animal Health, Utrecht University, The Netherlands.
Bussière
M
M
Klein
W
W
Geelen
M J
MJ
Van Golde
L M
LM
Houweling
M
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology*; D002794:Choline / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019856:Ethanolamine / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D019308:Palmitic Acid / Q000378:metabolism; D010713:Phosphatidylcholines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D010714:Phosphatidylethanolamines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D051381:Rats
D002518:Ceramides; D004075:Diglycerides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D019308:Palmitic Acid; D019856:Ethanolamine; D002794:Choline