SPHINGOMYELIN SYNTHASE, A POTENTIAL REGULATOR OF INTRACELLULAR LEVELSOF CERAMIDE AND DIACYLGLYCEROL DURING SV40 TRANSFORMATION - DOES SPHINGOMYELIN SYNTHASE ACCOUNT FOR THE PUTATIVE, PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE-C

Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite direct ions, the levels of ceramide and diacylglycerol. in this study SMS act ivity was investigated in normal and SV40-transformed human lung fibro blasts (WI38). The addition of [H-3]C-2-ceramide to cells resulted in a time-dependent formation of [H-3]C-2-SM. At 24 h after treatment, no rmal WI38 cells cleared 17% of [H-3]C-2-ceramide producing [H-3]C-2-SM , which accounted for 13% of total radioactivity. On the other hand, S V40-transformed cells cleared 45% of [H-3]C-2-ceramide and produced C- 2-SM, which accounted for 24% of total radioactivity. This enhanced pr oduction of C-2-SM was also supported by an increase in the total SMS activity of cells (measured in vitro), such that SV40-transformed cell s had SMS activity of 222 pmol/mg of protein/h, whereas wild type cell s had 78 pmol/mg of protein/h of activity. Additional studies aimed at examining the SMS activity directed at ceramide produced in the plasm a membrane. Treatment of cells with exogenous bacterial sphingomyelina se (SMase) for 25 min resulted in cleavage of 90-95% of total SM and t he concomitant generation of ceramide, After bacterial SMase treatment , wild type WI38 cells cleared ceramide very slowly (19.2 pmol of cera mide/nmol of phosholipid P-i after 6 h of incubation) and hardly regen erated slay SM. On the other hand, SV40-transformed cells cleared cera mide much faster (41.1 pmol/nmol of P-i after 6 h of incubation) and r egenerated approximately 80% of the original SM. These results show th at the enhanced SMS activity of transformed cells is particularly pron ounced when ceramide is produced in the plasma membrane. Finally, seve ral observations led us to consider the relationship of SMS to the ''p utative'' phosphatidylcholine-specific phospholipase C (PC-PLC), We, t herefore, tested the effects of D609, a pur-ported PC-PLC-specific inh ibitor on the activity of SMS, D609 inhibited SMS activity in vitro. I n addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC ( 10-50 mu g/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attribu table to SMS.