Effects of brefeldin A on sphingomyelin transport and lipid synthesis in BHK21 cells.
1. Addition of brefeldin A (BFA) to BHK cells incubated for 4 h with [3H]acetate led to a 3-4-fold increase in incorporation of label into sphingomyelin, monoglucosylceramide and cholesterol ester compared with untreated controls. There was a similar increase in incorporation of [3H]choline into sphingomyelin. The level of cholesterol ester increased 3-fold when BFA was added to cells labelled to equilibrium with [3H]acetate, but no statistically significant changes in the levels of other lipids were seen. 2. BFA appeared to act by diverting incorporation of acetate into sphingolipids and cholesterol ester at the expense of phosphatidylcholine (decreased by up to 15%), cholesterol (decreased by 30-40%) and triacylglycerol (decreased by 35-50%). 3. Forskolin (100 microM) prevented the changes in labelling induced by 0.25 micrograms of BFA/ml, but in the presence of 1 micrograms of BFA/ml it had no effect on sphingomyelin and triacylglycerol labelling and only partly blocked the effects of BFA on labelling of cholesterol and cholesterol ester. 4. None of the labelled sphingomyelin was degraded in BFA-treated cells which were subsequently exposed to an extracellular sphingomyelinase, showing that all the newly synthesized sphingomyelin remained inside the cells. Determinations of phospholipid phosphorus in unlabelled cells confirmed that, in the presence of BFA, no newly synthesized sphingomyelin was able to reach the cell surface, supporting the idea that sphingomyelin normally depends on vesicular transport for its passage to the plasma membrane. 5. The results are consistent with the hypothesis that cholesterol synthesis and esterification processes in BHK cells are sensitive to the plasma-membrane deficit of sphingomyelin caused by BFA.
Kallen
K J
KJ
Department of Physiology, University College London, U.K.
Quinn
P
P
Allan
D
D
D000085:Acetates / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology*; D008055:Lipids / Q000096:biosynthesis*; D009183:Mycotoxins / Q000494:pharmacology*; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013109:Sphingomyelins / Q000096:biosynthesis / Q000378:metabolism*
D000085:Acetates; D003517:Cyclopentanes; D008055:Lipids; D009183:Mycotoxins; D013109:Sphingomyelins; D005576:Colforsin; D020126:Brefeldin A; D013108:Sphingomyelin Phosphodiesterase