Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Fluid shear stress enhances the sphingosine 1-phosphate responses in cell-cell interactions between platelets and endothelial cells

Fluid shear stress modulates the functional responses of platelets and vascular cells, and plays an important role in the pathogenesis of vascular disorders, including atherosclerosis and restenosis. Since shear stress induces activation of platelets, which abundantly store sphingosine 1-phosphate (Sph-1-P), and upregulates the mRNA expression of S1P(1), the most important Sph-1-P receptor expressed on the endothelial cells, we examined the effects of shear stress on the Sph-1-P-related responses involving these cells. Shear stress was found to induce Sph-1-P release from the platelets in a shear intensity- and time-dependent manner. Inhibitors of protein kinase C suppressed this mechanical force-induced Sph-1-P release, suggesting involvement of this kinase. On the other hand, in vascular endothelial cells, shear stress increased S1P(1) protein expression, as revealed by flow-cytometric analysis, and the responsiveness to Sph-1-P, which was assessed by monitoring the intracellular Ca(2+) concentration. These results indicate that shear stress enhances the Sph-1-P responses in cell-cell interactions between platelets and endothelial cells.     

Sphingosine 1-phosphate formation and intracellular Ca2+ mobilization in human platelets: evaluation with sphingosine kinase inhibitors.

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.     

Sphingosine 1-phosphate inhibits migration of RBL-2H3 cells via S1P2: cross-talk between platelets and mast cells

To analyze the involvement in allergic reactions of platelets and sphingosine 1-phosphate (Sph-1-P), a lysophospholipid mediator released from activated platelets, the effects of Sph-1-P and a supernatant prepared from activated platelets on mast cell line RBL-2H3 were examined. Sph-1-P strongly inhibited the migration of both non-stimulated and fibronectin-stimulated RBL-2H3 cells, which was reversed by JTE-013, a specific antagonist of G protein-coupled Sph-1-P receptor S1P(2); S1P(2) was confirmed to be expressed in these cells. A similar anti-motility effect of Sph-1-P was observed in a phagokinetic assay. Consistent with these results, treatment of RBL-2H3 cells with Sph-1-P resulted in a rounded cell morphology, which was blocked by JTE-013. Under the present conditions, Sph-1-P failed to induce intracellular Ca(2+) mobilization or histamine degranulation, responses postulated to be elicited by intracellular Sph-1-P. Importantly, the Sph-1-P effect, i.e., the regulation of RBL-2H3 cell motility, was mimicked by the supernatant (both with and without boiling) prepared from activated platelets, and this effect of the supernatant was also blocked by JTE-013. Our results suggest that the motility of mast cells can be regulated by Sph-1-P and also platelets (which release Sph-1-P), via cell surface receptor S1P(2) (not through intracellular Sph-1-P actions, postulated previously in the same cells).     

Rho-mediated phosphorylation of focal adhesion kinase and myosin light chain in human endothelial cells stimulated with sphingosine 1-phosphate, a bioactive lysophospholipid released from activated platelets

Since sphingosine 1-phosphate (Sph-1-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions. In this study, we investigated the effects of Sph-1-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs). Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2. Sph-1-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs. The specific Rho inactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-1-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation. In contrast, Sph-1-P-induced intracellular Ca(2)(+) mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme. Our results suggest that Sph-1-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways.     

Plasma sphingosine 1-phosphate metabolism and analysis

The importance of sphingosine 1-phosphate (Sph-1-P) as an intercellular sphingolipid mediator has been established in various systems, and this is especially true in the areas of vascular biology and immunology. Blood platelets store Sph-1-P abundantly and release this bioactive lysophospholipid extracellularly upon stimulation, while vascular endothelial cells and smooth muscle cells respond dramatically to this platelet-derived bioactive lipid. Most of the responses elicited by extracellular Sph-1-P are believed to be mediated by G protein-coupled cell surface receptors, i.e., S1Ps. It is likely that regulation of Sph-1-P biological activity could be important for therapeutics, including but not limited to control of vascular disorders. Furthermore, elucidation of the mechanisms by which the levels of Sph-1-P in the blood are regulated seems important. Accordingly, the application of Sph-1-P analysis to laboratory medicine may be an important task in clinical medicine. In this review, Sph-1-P-related metabolism in the plasma will be summarized. Briefly, the levels and bioactivities of plasma Sph-1-P in vivo may be regulated by various factors, including Sph-1-P release from platelets (and red blood cells, based upon the recent reports), Sph-1-P distribution between albumin and lipoproteins, and S1P expression and lipid phosphate phosphatase activity on the cell surface. Then, application of Sph-1-P analysis to laboratory medicine will be discussed.     

Involvement of sphingosine 1-phosphate, a platelet-derived bioactive lipid, in contraction of mesangium cells

Platelet-derived mediators may play an important role in the development of renal diseases through interaction with glomerular mesangial cells (MCs), and we, in this study, examined the effect of sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, on the contraction of MCs. Sph-1-P was found to induce MC contraction through mediation of Rho kinase both in cell shape change and collagen gel contraction assays. The specific antagonist of the Sph-1-P receptor S1P(2) inhibited the response. Similar results were obtained when the supernatant from activated platelet suspensions were used instead of Sph-1-P. Our findings suggest that platelet-derived Sph-1-P may be involved in MC contraction via S1P(2) and that regulation of this receptor might be useful therapeutically.     

Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.     

G(i)-mediated Cas tyrosine phosphorylation in vascular endothelial cells stimulated with sphingosine 1-phosphate: possible involvement in cell motility enhancement in cooperation with Rho-mediated pathways

Since blood platelets release sphingosine 1-phosphate (Sph-1-P) upon activation, it is important to examine the effects of this bioactive lipid on vascular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions. In the present study, we examined Sph-1-P-stimulated signaling pathways related to human umbilical vein endothelial cell (HUVEC) motility, with a special emphasis on the cytoskeletal docking protein Crk-associated substrate (Cas). Sph-1-P stimulated tyrosine phosphorylation of Cas, which was inhibited by the G(i) inactivator pertussis toxin but not by the Rho inactivator C3 exoenzyme or the Rho kinase inhibitor Y-27632. Fyn constitutively associated with and phosphorylated Cas, suggesting that Cas tyrosine phosphorylation may be catalyzed by Fyn. Furthermore, upon HUVEC stimulation with Sph-1-P, Crk, through its SH2 domain, interacted with tyrosine-phosphorylated Cas, and the Cas-Crk complex translocated to the cell periphery (membrane ruffles), through mediation of G(i) (Fyn) but not Rho. In contrast, tyrosine phosphorylation of focal adhesion kinase, and formation of stress fibers and focal adhesion were mediated by Rho but not G(i) (Fyn). Finally, Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay using gold sol-coated plates and a Boyden's chamber assay, was markedly inhibited not only by pertussis toxin (or the Fyn kinase inhibitor PP2) but also by C3 exoenzyme (or Y-27632). In HUVECs stimulated with Sph-1-P, these data suggest the following: (i) cytoskeletal signalings may be separable into G(i)-mediated signaling pathways (involving Cas) and Rho-mediated ones (involving FAK), and (ii) coordinated signalings from both pathways are required for Sph-1-P-enhanced HUVEC motility. Since HUVECs reportedly express the Sph-1-P receptors EDG-1 (coupled with G(i)) and EDG-3 (coupled with G(13) and G(q)) and the EDG-3 antagonist suramin was found to block specifically Rho-mediated responses, it is likely that Cas-related responses following G(i) activation originate from EDG-1, whereas Rho-related responses originate from EDG-3.     

Sphingosine 1-phosphate-related metabolism in the blood vessel

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets and plays an important role in vascular biology. In this study, we investigated Sph-1-P-related metabolism in the blood vessel, mainly using radio-labeled Sph and Sph-1-P. Sph was metabolically stable in the plasma, while it was converted into Sph-1-P in the presence of activated platelets. When the mixture of Sph-1-P and plasma was fractionated on a gel-filtration column, all Sph-1-P co-eluted with protein fractions that coincide with lipoproteins and albumin by agarose gel electrophoresis. When evaluated by polyacrylamide gel electrophoresis, 7.2 +/- 3.8%, 53.3 +/- 6.4%, and 39.5 +/- 7.9% of the radioactivity of Sph-1-P added to plasma was recovered in the low-density lipoprotein (LDL), high-density lipoprotein (HDL), and albumin fractions, respectively. On the other hand, 5.2 +/- 3.2%, 38.4 +/- 5.5%, and 56.3 +/- 5.7% of the radioactivity of Sph-1-P converted from Sph in collagen-stimulated platelets and released into the plasma was recovered in the LDL, HDL, and albumin fractions, respectively. When Sph-1-P release from activated platelets was examined, a stronger response was observed in the presence of albumin than lipoproteins, suggesting efficient Sph-1-P extraction from platelets by albumin. Finally, Sph-1-P, which is stable in the plasma, was markedly degraded by an ectophosphatase activity in the presence of vascular endothelial cells or in whole blood. Although Sph-1-P is stable in the plasma, it is likely that the level of this bioactive lipid is dynamically controlled by various factors including release from platelets, distribution among plasma proteins, and degradation by ectophosphatase.     

Sphingosine 1-phosphate breakdown in platelets

We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.     

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.     

Roles for N-glycosylation in the dynamics of Edg-1/S1P1 in sphingosine 1-phosphate-stimulated cells

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid mediator released from activated platelets. To date, 5 seven-transmembrane-spanning receptors, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4 and Edg-8/S1P5, have been identified as specific Sph-1-P receptors. Our recent novel studies established that Edg-1/S1P1 is glycosylated in its N-terminal extracellular portion and further identified the specific glycosylation site as asparagine 30. We also demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. These studies revealed a possible regulatory role for the N-glycan on Edg-1/S1P1 in the dynamics of the receptor, such as its lateral and internal movements within the membrane, in ligand-stimulated mammalian cells. Published in 2004.     

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid.

An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.     

Selective non-lipid modulator of LPA5 activity in human platelets

Lysophosphatidic acid (LPA) is a potent activator of human platelets in vitro. Recently, the G protein-coupled receptor LPA5/GPR92 has been identified to be the relevant LPA receptor responsible for the activation of human platelets by LPA. In a high-throughput screening campaign we identified a diphenyl pyrazole carboxylic acid as a small-molecule inhibitor for LPA5. Confirmation for the specificity of this small molecule was achieved in human platelets as the relevant cellular in vitro model. We could confirm using antagonists for alternative LPA receptors that we identified in our work the first non-lipid, small-molecule inhibitor for LPA5/GPR92 specifically inhibiting LPA-mediated platelet activation in vitro.     

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.     

Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of human arterial smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.     

Regulation by sphingolipids of the fate of FRTL-5 cells

Sphingolipids, including ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1-P) have recently emerged as signal-transducing molecules. Functionally, a distinguishing characteristic of these lipids is their apparent participation in pro- or anti-proliferative cell regulation pathways. In this study, we examined the involvement of sphingolipids in the fate of FRTL-5 thyroid follicular cells. We first examined the effects of sphingolipids on FRTL-5 cell viability. Sph and Cer induced apoptosis, as revealed by fluorescence microscopy of TUNEL-positive fragmented nuclei and 180-300 bp DNA fragmentation on agarose gel electrophoresis while Sph-1-P was confirmed to prevent FRTL-5 cell apoptosis induced by deprivation of serum and TSH, possibly via cell surface receptors. We then analysed the metabolism of radiolabelled Sph and C(6)-Cer (a synthetic cell-permeable Cer) in FRTL-5 cells by thin layer chromatography, followed by autoradiography. Sph was mainly metabolized to Cer, and then to sphingomyelin, while Sph conversion into Sph-1-P was hardly detected. These changes were not affected by stimulation of the cells with TSH. Our results indicate the involvement of sphingolipid mediators in the fate of FRTL-5 thyroid cells.     

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.