Abstract
The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 microM of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except 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 analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.
Highlights
Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons
Mass Measurements of Free Sphingoid Bases and Sphingolipids— After treatment of cells with the respective sphingosine analogue, mass measurements of free sphingoid bases were conducted by HPLC as Effect of Different Sphingosine Analogues on Sphingolipid Biosynthesis—Primary cultured murine cerebellar neurons were treated with 10 M cis-4-methylsphingosine, cis-5-methylsphingosine, cis-sphingosine, trans-4-methylsphingosine, and trans-5-methylsphingosine as well as 1-deoxysphingosine, respectively
That trans-5-methylsphingosine treatment caused a band pattern comigrating with sphingomyelin which in this experiment was quite different compared with the other sphingosine analogues
Summary
Labeling and Isolation of Cellular Sphingolipids and Phospholipids—After 4 days in culture, murine cerebellar neurons were incubated with sphingoid bases added as complexes with bovine serum albumin to the culture medium containing 0.3% heat-inactivated horse serum. The reaction mixture in a total volume of 80 l contained 0.1 M Tris buffered with sodium acetate at pH 7.4, 0.5 mM dithiothreitol, 100 M stearoyl-CoA, 50 M of the labeled sphinganine (0.5 Ci), which was previously sonicated for 2 min at 0 °C in the buffer solution, and 120 g of cell protein. After incubation for 15 min at 37 °C, the lipids were extracted, separated by TLC (chloroform/ methanol/water, 80:10:1, v/v), and the radiolabeled ceramide was determined by liquid scintillation counting as described above. After an additional 24 h, cells were harvested, sphingolipids were isolated and evaluated as described under “Experimental Procedures.” Alternatively SPT activity in the cell homogenate was determined after a 24-h incubation with the respective sphingosine analogue as described under “Experimental Procedures.” Results are means from duplicates of four different experiments. 100% serine incorporated equals 100,000 Ϯ 18,000 cpm/mg of protein, while 100% of SPT activity corresponds to 900 Ϯ 120 pmol ϫ hϪ1 ϫ mgϪ1
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