Phosphatidylcholine as the choline donor in sphingomyelin synthesis

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Abstract
Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [Methyl-14C]choline resulted in rapid accumulation of essentially all the label (≈95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 μM choline), loss of lable from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 μM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 μM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9–12 hr until it reached the specific radioactivity of PC. In the presence of 300 μM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion, we conclude that sphingomyelin synthesis in Novikoff cells circumvents the water-soluble choline pool and that phosphatidylcholine serves as the immediate choline source. All our data support the phosphatidylcholine:ceramide cholinephosphotransferase pathway of sphingomyelin synthesis.

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