CIS-4-METHYLSPHINGOSINE DECREASES SPHINGOLIPID BIOSYNTHESIS BY SPECIFICALLY INTERFERING WITH SERINE PALMITOYLTRANSFERASE ACTIVITY IN PRIMARY CULTURED NEURONS

The effect of six different structurally modified sphingosine analogue s on biosynthesis of sphingolipids was studied in primary cultured mur ine cerebellar neurons, Treatment of cells with cis-4-methylsphingosin e at micromolar levels resulted in a markedly decreased sphingolipid b iosynthesis, whereas the other compounds examined, trans-5-methylsphin gosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphing osine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis les s efficiently, The inhibition of sphingolipid biosynthesis by the vari ous compounds was paralleled by a decrease pf serine palmitoyltransfer ase activity in situ, For cis-4-methylsphingosine the inhibitory effec t on serine palmitoyltransferase activity was shown to be concentratio n- and time-dependent, Half-maximal reduction of enzyme activity occur red after 24 h of treatment with 10 mu M of the compound, The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid a!nd protein biosynthesis was not affected. Analysis of tile sphingoi d moiety of cellular sphingolipids suggests that the sphingosine analo gues listed above were subject to degradation rather than being utiliz ed as precursors for sphingolipid biosynthesis by cultured neurons, Ex cept of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine a nalogues the relative percentage of the respective intracellular l-pho sphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with th e unphosphorylated compounds, In contrast to the respective l-phosphat e derivatives of the other methyl-l,ranched sphingosine analogues exam ined, cis-4-methylsphingosine l-phosphate showed an intracellular accu mulation suggesting a delayed turnover rate in cultured murine neurons for this compound, These results suggest that the inhibitory effect o f the sphingosine analogues on serine palmitoyltransferase is mediated by their respective l-phosphate derivatives and that the pronounced e ffect of cis-4-methylsphingosine is caused by a high intracellular con centration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingo sine, in addition, caused drastic changes in cell morphology of primar y cerebellar neurons, which were not observed when these cells were tr eated with one of the other sphingosine analogues examined.