Myriocin, an inhibitor of serine palmitoyl transferase, impairs the uptake of transferrin and low-density lipoprotein in mammalian cells.

The role of sphingolipids in clathrin-mediated endocytosis is only poorly understood in mammalian cells. Thus the relationship between sphingolipid de novo synthesis and clathrin-mediated endocytosis of transferrin were studied in L929 fibroblasts and two other cell lines. Endocytosis was measured using live cell imaging with fluorescent transferrin or (125)I-transferrin. Lipids were primarily measured using electrospray ionization tandem mass spectrometry. At physiological temperature, transferrin uptake was significantly decreased by the inhibitor of serine palmitoyl transferase myriocin. Myriocin inhibited also the uptake of low-density lipoproteins. The endocytosis inhibition by myriocin could be released by the addition of sphingoid base and by the protein phosphorylation effectors phorbol-12-myristate, 13-acetate (PMA) and okadaic acid. Myriocin influenced not only sphingolipids but also the glycerophospholipid profile. The study of phosphatidylcholine species shows adaptations to more saturated, alkylated and longer fatty acid moieties. The reported results imply that in mammalian cells, at 37°C, sphingolipid de novo synthesis is required for clathrin-mediated endocytosis.

2n-fatty acids from phosphatidylcholine label sphingolipids--a novel role of phospholipase A2?

In order to find out whether there is a phospholipase A2 (PLA2)-mediated link between glycerophospholipids and sphingolipids, L929 cells were labeled with 1n-palmitoyl-2n-[1-14C]palmitoyl phosphatidylcholine for 16-18 h or 90 min. After labeling for 16-18 h, 14C-sphingomyelin (SM), 14C-ceramide and 14C-sphingosine were demonstrated on autoradiograms of thin layer chromatograms of untreated or mildly hydrolyzed lipid extracts in different chromatographic systems. Strong hydrolysis of labeled SM proved that both possible moieties of SM, sphingosine and acyl moiety, had been labeled. The identity of SM and its enzymatic degradation product, ceramide, was verified by mass spectrometry. The label in SM-derived ceramide was demonstrated on an autoradiogram after thin layer chromatography. The inhibitor of (dihydro)ceramide synthase fumonisin B1 suppressed the label in sphingolipids significantly during 16-18 h (ceramide and SM), as well as during 90-min labeling (SM). The presence of inhibitors of PLA2 (bromoenol lactone, aristolochic acid and quinacrine dihydrochloride) diminished the label in SM significantly during the 90-min labeling. These results demonstrate a close metabolic relationship between glycerophospholipids and sphingolipids and give evidence for a novel role of PLA2.

Cycloserine and threo-dihydrosphingosine inhibit TNF-alpha-induced cytotoxicity: evidence for the importance of de novo ceramide synthesis in TNF-alpha signaling.

Measuring the cell death induced by tumor necrosis factor (TNF-alpha) in L929 cells, we discovered for the first time that L-cycloserine, an established inhibitor of serine palmitoyltransferase, as well as DL-threo-dihydrosphingosine (threo-DHS, threo-sphinganine) significantly protected against TNF-alpha-induced cytotoxicity. Under the same conditions sphingosine and DL-erythro-dihydrosphingosine (erythro-DHS) did not change TNF-alpha-induced cytotoxicity, thus underlining the specificity of threo-DHS. In serine-labeled cells, newly (de novo) synthetized labeled ceramide was significantly diminished by threo-DHS alone or together with TNF-alpha, which makes the (dihydro) ceramide synthase the likely target of threo-DHS. These results suggest the decisive role of ceramide de novo synthesis in TNF signaling.

[14C]serine from phosphatidylserine labels ceramide and sphingomyelin in L929 cells: evidence for a new metabolic relationship between glycerophospholipids and sphingolipids.

After incubation of L929 cells with [14C]serine and various effectors an inverse correlation between label in ceramide and phosphatidylserine (PS) was displayed. This surprising behavior of the two metabolites prompted us to check whether serine of PS could be a source for ceramide synthesis. We therefore incubated L929 cells for 30 min in serum-free medium with L-phosphatidyl-L-[3-14C]serine in the presence or in the absence of cycloserine, an established inhibitor of serine palmitoyltransferase. During this short period L-phosphatidyl-L-[3-14C]serine labeled ceramide and this label was suppressed by cycloserine. Then L929 cells were grown for 16-18 h in the presence of L-phosphatidyl-L-[3-14C]serine. After this period the label was seen in sphingomyelin. Labeling of ceramide and sphingomyelin by serine from PS provides evidence for a new metabolic relationship between glycerophospholipids and sphingolipids.