Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides

Phospholipids and sphingolipids are important precursors of lipid-derived s econd 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 sphingoli pid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (Ptd Etn) synthesis in Rat-2 fibroblasts. Addition of short-chain Ch-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pat hways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 m u M C-6-ceramide caused an 83% and a 56% decrease in incorporation of radio labelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exp osure of the cells for longer time-periods (greater than or equal to 16 h) to 50 mu M C-6-ceramide resulted in apoptosis. The structural analogue dihy dro-Ch-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-cholin e and CDP-ethanolamine under the influence of Ch-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotra nsferase activities in membrane fractions from C-6-ceramide-treated cells w ere reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol fr om radiolabelled palmitate were observed. It was concluded that C-6-ceramid e affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethano laminephosphotransferase.