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PubMed2002

Sphingosine 1-phosphate signaling in atherosclerosis and vascular biology.

PURPOSE OF REVIEW: Sphingosine 1-phosphate is a novel lipid mediator which exerts various actions on endothelial cells and vascular smooth muscle cells. In this review, we discuss the latest findings about the molecule in vascular biology. RECENT FINDINGS: It has been demonstrated that most sphingosine 1-phosphate-induced actions are mediated by the Edg-family of its receptors. Sphingosine 1-phosphate stimulates the migration and proliferation of endothelial cells and is cytoprotective towards them. The involvement of phosphoinositide 3-kinase and nitric oxide in sphingosine 1-phosphate downstream signaling in endothelial cells was recently reported, as was the enhancement of endothelial barrier integrity induced by the molecule. Sphingosine 1-phosphate inhibits migration of vascular smooth muscle cells and this inhibition was reported to be mediated by inhibition of Rac. Sphingosine 1-phosphate is concentrated in the lipoprotein fraction in plasma, and high-density lipoprotein exerted endothelial cytoprotection through its component of this molecule. SUMMARY: Sphingosine 1-phosphate might play a critical role in the development of atherosclerosis.

Animals↗
PubMed2004

Early activation of sphingosine kinase in mast cells and recruitment to FcepsilonRI are mediated by its interaction with Lyn kinase.

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca(2+), released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-gamma and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcepsilonRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcepsilonRI triggering, enhanced sphingosine kinase activity was associated with FcepsilonRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn(-/)(-) mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcepsilonRI proximal event.

Animals↗
PubMed2004

Interaction of cholesterol with sphingosine: physicochemical characterization and impact on intestinal absorption.

Molecular associations between sphingomyelin and cholesterol provide a molecular basis for the colocalization of these lipids in plasma membrane microdomains (lipid rafts) and for the inhibitory effect of sphingomyelin on the intestinal absorption of cholesterol. Using surface pressure measurements at the air-water interface, we showed that sphingosine, the common sphingoid backbone of most sphingolipids, formed condensed lipid complexes with cholesterol. Structure-activity relationship studies with long-chain analogs of sphingosine, together with molecular mechanics simulations, were consistent with a specific interaction between sphingosine and the alpha face of cholesterol. The uptake of micellar cholesterol and the effect of sphingosine on cholesterol absorption were studied with two human model intestinal epithelial cell lines, Caco-2 and HT-29-D4. Real-time PCR quantifications of the putative cholesterol transporter Niemann-Pick C1 like 1 (NPC1L1) mRNA revealed that, in these cell lines, the activity of cholesterol transport correlated with the level of NPC1L1 expression. In both cell lines, sphingosine induced a dose-dependent decrease of cholesterol absorption. Yet the effect of sphingosine was more dramatic in Caco-2 cells, which also displayed the highest expression of NPC1L1 mRNA. Altogether, these data suggested that sphingosine interacts specifically with cholesterol and inhibits the intestinal NPC1L1-dependent transport of micellar cholesterol.

Binding Sites↗
PubMed2006

Sphingosine increases the permeability of model and cell membranes.

Sphingosine, at 5-15 mol % total lipids, remarkably increases the permeability to aqueous solutes of liposomal and erythrocyte ghost membranes. The increased permeability cannot be interpreted in terms of leakage occurring at the early stages of a putative membrane solubilization by sphingosine, nor is it due to a sphingosine-induced generation of nonlamellar structures, or flip-flop lipid movement. Instead, sphingosine stabilizes (rigidifies) gel domains in membranes, raising their melting temperatures and increasing the transition cooperativity. Structural defects originating during the lateral phase separation of the "more rigid" and "less rigid" domains are likely sites for the leakage of aqueous solutes to the extravesicular medium. The presence of coexisting domains in the plasma membrane makes it a target for sphingosine permeabilization. The sphingosine-induced increase in rigidity and breakdown of the plasma membrane permeability barrier could be responsible for some of the physiological effects of sphingosine.

Calorimetry, Differential Scanning↗
PubMed1996

Sphingosine stimulates calcium mobilization and modulates calcium signals evoked by thapsigargin in glioma C6 cells.

The effect of sphingosine on intracellular calcium signalling in glioma C6 cells was studied with Fura-2 video imaging technique. Sphingosine had a direct effect on changes in cytosolic Ca2+ concentration only when applied at high concentration of 100 microM, causing the cytosolic Ca2+ level to rise. However, at a much lower concentration of 15 microM sphingosine diminished calcium responses triggered by thapsigargin (a specific inhibitor of calcium pump in the endoplasmic reticulum) and ionomycin (calcium ionophore). Since responses to thapsigargin and ionomycin were blocked in Ca(2+)-free medium, we postulate that sphingosine is acting on the intracellular calcium stores. Additionally, sphingosine (at 15 microM and 100 microM) markedly decreases thapsigargin-induced sustained elevation in cytosolic Ca2+ concentration, indicating its inhibitory effect on thapsigargin-evoked Ca2+ influx. Sphingosine is a known inhibitor of protein kinase C and the involvement of this enzyme is postulated in the modulatory effects of sphingosine on intracellular calcium dynamics.

Animals↗
PubMed1999

Exogenous sphingosine 1-phosphate and sphingosylphosphorylcholine do not stimulate phospholipase D in C6 glioma cells.

In the present study we investigate the effect of exogenous sphingosine, sphingosine 1-phosphate and sphingosylphosphorylcholine on phospholipase D (PLD) activity in glioma C6 cells. The cells were prelabeled with [1-14C]palmitic acid and PLD-mediated synthesis of [14C]phosphatidylethanol was measured. Sphingosine 1-phosphate and sphingosylphosphorylcholine did not stimulate [14C]phosphatidylethanol formation either at low (0.1-10 microM) or high (25-100 microM) concentrations. On the other hand, sphingosine at concentrations of 100-250 microM strongly stimulated PLD activity as compared to the effect of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), known as a PLD activator. The effect of TPA on PLD is linked to the activation of protein kinase C. The present study also shows that sphingosine additively enhances TPA-mediated PLD activity. This is in contrast to the postulated role of sphingosine as a protein kinase C inhibitor. These results demonstrate that in glioma C6 cells sphingosine not only affects PLD independently of its effect on protein kinase C, but also is unable to block TPA-mediated PLD activity.

Brain Neoplasms↗
PubMed2004

Inhibition of skin sphingosine synthesis: enhanced percutaneous permeation of 5-fluorouracil.

The present study was designed to investigate the role of skin sphingosine inhibition in enhancing the transcutaneous permeation of 5-fluorouracil (5-FU), a hydrophilic drug, across rat skin. Ethanol-perturbation significantly reduced the sphingosine content as compared to that in normal skin until 24 h of perturbation (p < 0.05). To maintain the low content of sphingosine for a longer time, beta-chloroalanine (beta-CA), a selective inhibitor of serine palmitoyl transferase, was used for inhibiting the sphingosine synthesis in viable skin. Application of beta-CA (600 microg or 1200 microg/7 cm2) to viable skin perturbed with ethanol significantly reduced the sphingosine content until 48 h as compared to that in perturbed viable skin (p < 0.05). However, the sphingosine content in viable skin perturbed with ethanol and treated with lower doses (200 or 400 microg/7 cm2) of beta-CA, returned significantly close to that in ethanol-perturbed viable skin at 36 h (p < 0.05). Skin sphingosine synthesis inhibition efficacy of 1200 microg beta-CA was insignificantly different to that of 600 microg dose of beta-CA at 36 h or 48 h (p < 0.05). The systemic delivery of percutaneously applied 5-FU across ethanol-perturbed rat skin treated with either 600 microg or 1200 microg beta-CA was significantly greater as compared to that obtained after oral administration or after application of lower percutaneous doses of beta-CA (p < 0.05). Higher Cmax, MRT, AUC and maintenance of effective plasma concentration of 5-FU for 46 h was achieved by a single topical application of a formulation containing 5-FU and 600 microg beta-CA to ethanol-perturbed skin.

Administration, Oral↗
PubMed1990

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts.

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase.

We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine actions in addition to the well-characterized protein kinase C. Effects of sphingosine and lysophingolipids were studied on the activity of purified cytidylyltransferase prepared by the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M.E., and Feldman, D.A. (1986) J. Biol. Chem. 261, 5104-5110). The sphingolipids were tested as components of egg phosphatidylcholine (PC) vesicles, 25 mol% sphingosine inhibited the CT activity by about 50%. The inhibition of CT by sphingosine and lysosphingolipids was reversible. Sphingosine was found to be a reversible inhibitor of CT with respect to the activating lipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and fatty acid:phosphatidylcholine vesicles. Egg PC vesicles containing sphingosine, psychosine (galactosylsphingosine), glucopsychosine (glucosylsphingosine), and lysosphingomyelin (sphingosylphosphorylcholine) suppressed the activation by PC/oleic acid vesicles, whereas the parent sphingolipids did not. Egg PC vesicles containing oleylamine and hexadecyltrimethylamine inhibited CT activity, whereas egg PC-octylamine vesicles did not alter the enzyme activity. This indicates the importance of an amino group and long alkyl chain. LysoPC, a known detergent, did not inhibit the enzyme activity under the same assay conditions in which sphingosine inhibited. These results are the first report of a lipid inhibitor of purified CT.

Animals↗
PubMed1987

Sphingosine inhibition of agonist-dependent secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction pathways.

Sphingosine is a potent inhibitor of [3H]phorbol dibutyrate binding and protein kinase C activity in vitro and in human platelets (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. (1986) J. Biol. Chem. 261, 12604-12609). Preincubation of platelets with sphingosine resulted in the inhibition of platelet secretion and second phase aggregation in response to ADP, gamma-thrombin, collagen, arachidonic acid, and platelet activating factor. Sphingosine did not affect the initial shape change of platelets or the first phase of aggregation in response to these agonists. Ristocetin-induced platelet agglutination was not affected by sphingosine. Sphingosine inhibition of secondary aggregation (secretion and second phase aggregation) was overcome by phorbol dibutyrate and by the cell-permeable protein kinase C activator, dioctanoylglycerol. Furthermore, platelet secretion and irreversible aggregation were induced by protein kinase C activators in platelets that had been "primed" to undergo initial shape change and first phase aggregation by low concentrations of agonists. These results suggest that protein kinase C activation is a necessary component in the signal transducing pathways that lead to platelet activation. Higher concentrations of agonists, however, induced irreversible aggregation and partial secretion in the presence of sphingosine, suggesting the existence of protein kinase C-independent pathways for platelet activation. These results demonstrate the utility of sphingosine as a pharmacologic tool in probing the role of protein kinase C in signal transduction.

Adenosine Diphosphate↗
PubMed1995

[Correlation of cytotoxic activity of mutant forms of tumor necrosis factor alpha with changes in the level of free sphingosine in murine liver].

Sphingosine, the product of enzymatic degradation of sphingomyelin, displays a high cytotoxic activity and is accumulated in animal organs under the action of the tumour necrosis factor (TNF alpha). To elucidate the role of sphingosine in the realization of TNF cytotoxicity, TNF mutants were obtained which differed in their cytotoxic action on L929 cells. The wild strain of TNF and the mutant having a deletion in position 67-71 displayed the highest toxicity and sharply stimulated sphingosine accumulation in mouse hepatocytes. A moderate increase in the sphingosine content was induced by mutants with point and double mutations in positions E127Q, I155L and V150I displaying a much lower toxicity in comparison with the wild strain. The toxic, mutagenic and antimutagenic activities of sphingosine were investigated. Despite the high degree of cytotoxicity, sphingosine did not display any mutagenic activity but had a pronounced antimutagenic effect on E. coli cells. The role of phospholipid enzymatic degradation products in activation of sphingomyelin cycle enzymes stimulating of sphingosine accumulation in animal cells under the action of TNF alpha is discussed.

Amino Acid Sequence↗
PubMed1996

Effects of sphingomyelinase and sphingosine on arterial vasomotor regulation.

The sphingomyelin pathway is an important signal transduction system regulating various cellular functions. However, little is known about the effect of sphingomyelin metabolites on vasomotor function. We examined the vascular effects of sphingomyelin, sphingosine, and sphingomyelinase (SPMase) in vitro. In pig coronary rings precontracted with prostaglandin F2 alpha, sphingosine and SPMase evoked initial contraction and subsequent gradual relaxation; however, sphingomyelin did not influence the tone. The initial contractions in response to either SPMase (40 microU/ml to 0.4 U/ml) or sphingosine (0.5-10 microM) treatment were abolished in rings denuded of endothelium. This initial contraction in response to sphingosine treatment was significantly attenuated by a cyclo-oxygenase inhibitor indomethacin, but not altered by either a nitric oxide synthase inhibitor, N omega-monomethyl-L-arginine (L-NMMA), a protein kinase C (PKC) inhibitor staurosporine, or superoxide dismutase (SOD, 100 U/ml). Incubation of coronary rings with sphingosine (10 microM) or SPMase (0.4 U/ml) for 120 min significantly attenuated subsequent endothelium-dependent relaxation in response to thrombin and A23187, but did not affect endothelium-independent relaxation in response to sodium nitroprusside. In contrast, sphingomyelin (10 microM) did not alter the endothelium-dependent relaxation. In conclusion, in the sphingomyelin pathway, sphingosine induces vasoconstriction in coronary arteries that seems to be mediated by the release of cyclooxygenase-sensitive vasoconstrictor prostanoids from the endothelium. Sphingosine also induced endothelial dysfunction characterized by impaired endothelium-dependent relaxation. Thus, the sphingomyelin pathway may be an important regulator of vascular function.

Animals↗
PubMed1998

Functions of sphingosine in cell proliferation and death.

Interest in the biological functions of sphingosine, the metabolic product of sphingolipids, increased dramatically during the last few years. Sphingosine was found to be an exogenous inhibitor of protein kinase C and of many cell functions which depend on this enzyme including cell proliferation, differentiation, and programmed death. Sphingosine also activates some other protein kinases and regulates a variety of enzymes which are involved in the transmission of cell signals. Sphingosine mobilizes Ca2+ from intracellular stores and controls the specific Ca2(+)-channel. Sphingosine influences the synthesis of DNA and interacts with DNA in competition for the binding sites with histones, some enzymes, and transcriptional factors. Sphingosine is suggested to be a second messenger in the transmission of cell proliferation and apoptosis signals. The possible use of sphingosine in combined treatment of various diseases as a synergist of many drugs is considered.

Animals↗
PubMed1999

Activation of caspase-3-like proteases in apoptosis induced by sphingosine and other long-chain bases in Hep3B hepatoma cells.

Sphingosine and other long-chain bases (including sphinganine, dimethylsphingosine and stearylamine), but not octylamine (a short-chain analogue of sphinganine), induced apoptosis in Hep3B hepatoma cells. Because both D- and L-erythrosphingosine and stearylamine exert potent apoptotic effects on Hep3B cells, it is possible that these long-chain bases may activate apoptosis by inhibiting protein kinase C (PKC) activity. However, pretreatment with the PKC activator PMA could not rescue cells from apoptosis triggered by long-chain bases. Therefore the involvement of PKC in this apoptotic process requires further characterization. We also investigated whether these long-chain bases might be metabolized into ceramide in order to elicit their apoptotic action. We found that long-chain bases acted independently of ceramide in the induction of apoptosis, since addition of fumonisin B1, a fungal agent which effectively inhibits ceramide synthesis from sphingosine, did not protect against apoptosis. Additionally, we found that sphingosine-induced apoptosis was accompanied by activation of caspases. The functional role of caspases in this apoptotic process was examined by using specific caspase inhibitors. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, which exhibits a broad specificity for caspase-family proteases, effectively blocked sphingosine-induced apoptosis. Furthermore, our results indicate that caspase-3-like proteases, but not caspase-1, are activated during apoptosis triggered by sphingosine. Enhancement of caspase-3-like activity and cleavage of poly(ADP-ribose) polymerase, an in vivo substrate for caspase-3, was clearly demonstrated in sphingosine-treated Hep3B cells. Considered together, these results suggest that caspase-3-like proteases participate in apoptotic cell death induced by sphingosine.

Amino Acid Chloromethyl Ketones↗
PubMed1976

C20-sphingosine as a determining factor in aggregation of gangliosides.

Aggregation properties of gangliosides, the major synaptic membrane glycosphingolipids of mammalian brain, may prevent their segregation during membrane assembly and promote a uniform membrane matrix with minimum maintenance energy. The sphingosine residues of bovine brain gangliosides show an increase in C20-sphingosine corresponding with an increase in sialic acid. Concentrations of C20-sphingosine varied from 37% for the monosialoganglioside to 64% for the trisialoganglioside, the remainder being C18-sphingosine. Ultracentrifugal analysis showed that changes in sialic acid content and in C20-sphingosine content individually affect micellar size. Increases in sialic acid content decreased micellar size from 225 for the monosialoganglioside to 120 monomersper micelle for the trisialoganglioside. Monosialogangliosides enzymatically prepared from oligosialohomologues with a higher C20-sphingosine content gave evidence for a considerable effect of C20sphingosine upon the free energy of the aggregate form; the number of monomers per micelle increased from 225 for the natural monosialoganglioside to 280 for monosialoganglioside derived from trisialoganglioside. The similar aggregation energies of the major synaptic membrane gangliosides apparently result from a metabolic balancing of increased C20-sphingosine with increased sialic acid content.

Animals↗
PubMed1998

Chemoenzymatic Synthesis of All Four Stereoisomers of Sphingosine from Chlorobenzene: Glycosphingolipid Precursors(1)(a).

Advantageous use of homochiral cyclohexadiene-cis-1,2-diol 2, available by means of biocatalytic oxidation of chlorobenzene with toluene dioxygenase, has enabled the synthesis of all four enantiomerically pure C(18)-sphingosines 1. The four requisite diastereomers of azido alcohols 4a-d were accessed by regioselective opening of epoxides 7 and 8 with either azide or halide ions. The configuration of C4 and C5 in azides 4 defines the stereochemistry of the incipient sphingosine chain, liberated from 4 by the oxidative cleavage of the C1-C6 olefin. For L-threo-sphingosine (1b), lactol 20b generated by this cleavage was converted by periodate oxidation to azido deoxy L-threose 22b, which gave 1b upon Wittig olefination and reduction. Similarly, D-erythro-sphingosine (1a) and L-erythro-sphingosine (1c) were generated from 4a,c, respectively. The last sphingosine (1d) was synthesized from the silyl-protected azido alcohol 29d. Subsequent transformations provided silyl-protected azido deoxy D-threose 32d, which upon Wittig olefination and reduction gave D-threo-sphingosine (1d). Experimental and spectral data are provided for all new compounds.

Journal Article↗
PubMed2000

Characterization of a novel sphingosine 1-phosphate receptor, Edg-8.

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [(35)S]guanosine 5'-(3-O-thio)triphosphate binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.

Amino Acid Sequence↗
PubMed2005

A rapid radioassay for sphingosine kinase.

A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.

Animals↗