Synthesis and transport of different sphingomyelin species in rat Sertoli cells

Cellular phospholipids of Sertoli cells from immature rats were labeled wit h [C-14]-choline. Two sphingomyelin bands (SM1 and SM2) were identified by TLC. The incorporation of [C-14]-choline over a 45 h period of incubation d emonstrated that there are differences in labeling kinetics between SM1 and SM2. The subcellular location of SM1 and SM2 was investigated by accessibi lity to bacterial sphingomyelinase. The results showed the existence of two SM pools in Sertoli cells, but an equal cellular distribution of SM1 and S M2. SM2 is characterized by a relatively high content of unsaturated fatty acids. The inhibition of vesicular flow by monensin determines a decrease o f about 60-70% in incorporation into SM1 and SM2, suggesting the existence of at least two sites of sphingomyelin synthesis. Pulse-chase and time-cour se experiments indicated a phosphatidylcholine --> SM precursor product rel ationship and differences in kinetic properties between SM1 and SM2. Resynt hesis experiments showed that monensin had only a partial inhibitory effect on SM1 resynthesis, and a second sphingomyelinase treatment demonstrated t hat the resynthesized fraction reached the outer leaflet of the plasma memb rane. The 60-70% inhibition of SM synthesis by monensin showed that the tra ns-Golgi cisternae and the trans-Golgi network are the most likely sites of bulk SM synthesis, and that about 15% of SM was synthesized in the cis/med ial Golgi apparatus. Additionally the results indicated that plasma membran e SM synthase activity could be the site of about 15% of SM synthesis in Se rtoli cells.