Studies on the synthesis and transport of endogenous sphingomyelin in BHK-21 cells

The synthesis and intracellular transport of sphingomyelin in BHK-21 cells has been studied in the presence of brefeldin A (BFA) and monensin which are inhibitors of vesicular transport through the Golgi apparatus. Both drugs inhibit delivery of newly synthesised sphingomyelin to the cell surface, albeit by different mechanisms. BFA increases overall synthesis of sphingomyelin, but prevents vesicular transport of newly synthesised sphingomyelin to the trans Golgi network and the cell surface. Monensin seems to specifically inhibit synthesis of plasma membrane sphingomyelin by blocking vesicular transport of ceramide past the medial-Golgi. Ceramide can be produced at the plasma membrane by degradation of surface sphingomyelin by an extracellular sphingomyelinase. In untreated cells this ceramide is almost completely utilised for the resynthesis of plasma membrane sphingomyelin. Neither BFA nor monensin interfere with resynthesis of sphingomyelin from plasma membrane ceramide. However, resynthesis of sphingomyelin is largely prevented in mitotic and ATP-depleted cells in which endocytosis and vesicular transport are suppressed. These findings are conceptualised by a model which assumes in contrast to the prevailing orthodoxy, that the major site for the synthesis of plasma membrane sphingomyelin is not in the cis-lmedial-Go\gi, but distal to the trans-Golgi in an endosomal compartment on the plasma membrane recycling pathway. The depletion of plasma membrane sphingomyelin by either drug or sphingomyelinase treatment causes an increased esterification of cholesterol and, in the case of BFA, a decrease of cholesterol synthesis. These observations are consistent with the previous findings that cholesterol synthesis and esterification processes in cells are influenced by the plasma membrane content of sphingomyelin. It is suggested that the correct balance between plasma membrane cholesterol and sphingomyelin is created at the putative endosomal site of sphingomyelin synthesis after delivery of newly synthesised cholesterol to this site.