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PubMed1996

Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis.

We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8-Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8-Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8-Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8-Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer.

Apoptosis↗
PubMed2005

Endothelial nitric oxide synthase activation by tumor necrosis factor alpha through neutral sphingomyelinase 2, sphingosine kinase 1, and sphingosine 1 phosphate receptors: a novel pathway relevant to the pathophysiology of endothelium.

OBJECTIVE: Tumor necrosis factor alpha (TNF-alpha), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown. METHODS AND RESULTS: In human endothelial cells, we found that eNOS activation by TNF-alpha is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and sphingosine-kinase-1 (SK1) and generation of sphingosine-1-phosphate (Sph1P). Inhibition of N-SMase2 inhibited Sph1P formation, whereas inhibition of SK1 did not affect N-SMase2 activation by TNF-alpha. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P1 and S1P3, either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-alpha via S1P receptors, activated by Sph1P generated through N-SMase2 and SK1 activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-alpha. CONCLUSIONS: This study establishes a previously undescribed link among TNF-alpha, Sph1P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-alpha.

Calcium↗
PubMed2005

Mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets.

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.

Acyltransferases↗
PubMed1975

Specific tritium labeling of cerebrosides at the 3-positions of erythro-sphingosine and threo-sphingosine.

Cerebrosides containing either threo- or erythro-[3-3-H]sphingosine were synthesized by a new procedure. Glucopyranosyl or galactopyranosyl ceramides were converted to their 3-keto derivatives with 2,3-dichloro-5,6-dicyanobenzoquinone and reduced with 3-H-labeled sodium borohydride. The resulting tritiated cerebrosides, which contained erythro- and threo-sphingosines in the ratio of 84:16, were deacylated with butanol-KOH, and the erythro- and threo-psychosines were separated by silica gel column chromatography and reacylated with lignoceroyl chloride.

Animals↗
PubMed2006

Inhibition of interleukin-4 production in activated T cells via the downregulation of AP-1/NF-AT activation by N-lauroyl-D-erythro-sphingosine and N-lauroyl-D-erythro-C20-sphingosine.

Allergic diseases are hypersensitivity disorders that are associated with the generation of specific immunoglobulin E (IgE) in response to environmental allergens. Interleukin (IL)-4, which is primarily produced by the CD4(+) T cells, is an important stimulus for the switching of the antibody isotype to IgE in both mice and humans. In a previous study, we demonstrated that ceramide derivatives coupled with a lauroyl group exerted strong inhibitory effects on IL-4 production in T cells. In this study, we attempted to characterize the mechanisms underlying the inhibition of IL-4 production in T cells. Two ceramide derivatives, N-lauroyl-D-erythro-sphingosine and N-lauroyl-D-erythro-C(20)-sphingosine (hereafter abbreviated as LES and LECS, respectively), were shown to significantly inhibit the production of IL-4 in both primary CD4(+) T cells and EL4 T thymoma cells in a dose-dependent manner. LES and LECS also inhibited the activity of the IL-4 gene promoter in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells that had been transfected with an IL-4 promoter construct deleted of a P4 site harboring the AP-1 and NF-AT binding sites. Furthermore, LES and LECS inhibited the DNA binding activities of both AP-1 and NF-AT transcription factors. In addition, LES and LECS were demonstrated to suppress PMA-stimulated PKC activity, although they exerted no significant effects on the protein levels of the conventional PKCs. These results indicate that the ceramides, LES and LECS, may inhibit the production of IL-4 in the activated T cells, via the downregulation of AP-1/NF-AT activation and PKC activity.

Animals↗
PubMed2005

Sphingosine kinases, sphingosine-1-phosphate and sphingolipidomics.

It has become abundantly clear over the past decade that sphingolipids and their metabolites are key signaling molecules. Ceramide, the backbone of all sphingolipids, predominantly inhibits cell growth and induces apoptosis, while its metabolite, sphingosine-1-phosphate promotes growth and survival. Given the interconvertibility of these two opposing signaling molecules, it is essential that any study that examines the effects of one also look at the other. The newly available technology of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) is increasingly being applied for this purpose, as it can quickly identify and measure many different sphingolipids simultaneously. An added benefit of LC-MS/MS is that it is several orders of magnitude more sensitive than enzymatic methods or more traditional chromatographic techniques, allowing smaller sample sizes and increased throughput. Here, we briefly discuss the importance of LC-MS/MS for measuring sphingolipid metabolites and some future directions researchers may take given the increasingly accessibility to this technology.

Animals↗
PubMed1999

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.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes.

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes.

Animals↗
PubMed1989

Enzymatic synthesis of N,N-dimethyl-sphingosine: demonstration of the sphingosine: N-methyltransferase in mouse brain.

Based on our recent finding that N,N-dimethyl-D-erythro-sphingenine strongly inhibits protein kinase C (PK-C) whereas D-erythro-sphingenine produces only weak inhibition, we have studied the presence of N-methyltransferase responsible for conversion of sphingosine to its N,N-dimethyl derivative. The enzyme activity was detected in crude mouse brain tissue homogenate but was hardly detectable in liver homogenate, in which N-methylation of phosphatidylethanolamine (PE) is predominant.

Animals↗
PubMed2000

Analysis of sphingomyelin, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate by tandem mass spectrometry.

Free sphingoid bases such as sphingosine, sphinganine, and the respective phosphorylated bases, as well as the complex sphingolipids ceramides, glucosylceramide, and sphingomyelin, all dissociate to form structurally distinctive product ions. For sphingomyelin these ions are characteristic of their phosphorylcholine headgroup and are observed at m/z 184. The other sphingolipids dissociate to form carbocations characteristic of their sphingoid base. For common mammalian sphingoid bases such as d18:1 or d18:0 these product ions are detected at m/z 264 or 266, respectively. However, changes in the sphingoid base chain length, degree of unsaturation, or other modifications may correspondingly result in a shift in m/z of [figure: see text] this product ion. Additionally, the kinetics that govern the formation of these product ions is affected by the presence of a delta 4 double bond. Thus, internal standards for each type of sphingoid base are required for quantitative data. Structurally distinctive product ions, when used with either precursor ion or constant neutral loss scans allow highly specific and sensitive methods for sphingolipid analysis. They serve to greatly reduce background chemical noise, and enhance detection of sphingolipids at very low concentrations. This occurs by allowing only those ions that dissociate to yield a specific product ion or neutral loss to be passed to the detector. Additionally, these scans reveal the exact combinations of headgroup, sphingoid base, and fatty acid in a complex mixture by mass. The free sphingoid bases and Cer readily decompose in the ion source, whereas GlcCer and SM do not. Finally, each individual sphingolipid species fragmented optimally at a different collision energy, precluding the use of either precursor ion or neutral loss scans for quantitation. Multiple reaction monitoring (MRM) experiments directly address the issues regarding accurate quantitation of sphingolipids that precursor ion and neutral loss scans cannot. In these experiments both ionization and dissociation parameters are optimized for each individual species. By detecting only specific precursor and product ion pairs instead of scanning wide m/z ranges maximum sensitivity is attained. Furthermore, relative ion abundance data are not biased with regard to instrumental parameters. At this point simple loop injections can be used with the MRM scanning methods developed to observe changes in sphingolipid type and quantity in crude extracts on a class-by-class basis. This, however, is labor intensive requiring multiple injections and multiple runs for each class in order to obtain a complete picture of all sphingolipids present. As an alternative [figure: see text] to loop injections, HPLC-MS/MS methods are being developed. In these methods sphingolipids are eluted by class, thus, each individually optimized MRM method can be used at specific times in an LC run. This provides a highly sensitive and accurate quantitation as well as a complete picture of all sphingolipids in a single run (Fig. 6). Additionally, this methodology is amenable to automation and can be used for high-throughput screening of multiple samples.

Ceramides↗
PubMed1997

Extracellular sphingosine 1-phosphate stimulates formation of ethanolamine from phosphatidylethanolamine: modulation of sphingosine 1-phosphate-induced mitogenesis by ethanolamine.

In this work, we determined the effects of sphingosine 1-phosphate (S1P) on phospholipase D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn), and evaluated the effects of the water-soluble product ethanolamine on S1P-induced DNA synthesis in NIH 3T3 cells. In [14C]ethanolamine-labelled cells, S1P (0.5-5 microM) stimulated PLD-mediated hydrolysis of PtdEtn 1.5-2.1-fold. Down-regulation of protein kinase C by chronic (24 h) treatment of cells with 300 nM PMA, or pretreatments (10 min) with the cell-permeant calcium chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N, N',N'-tetra-acetic acid tetra-acetoxymethyl ester led to the inhibition of S1P-induced PtdEtn hydrolysis. S1P alone was a weak inducer of DNA synthesis, but its effects were enhanced by phosphocholine (PCho), insulin, ATP or PMA. Ethanolamine (5-100 microM) did not modify the mitogenic effect of S1P alone, whereas at 50-100 microM concentrations it actually enhanced the mitogenic effect of PCho via a mitogen-activated protein (MAP) kinase-independent mechanism. In contrast, 5-20 microM concentrations of ethanolamine, which correspond to normal blood ethanolamine levels in humans, strongly inhibited DNA synthesis induced by S1P plus PCho via a MAP kinase-dependent mechanism; importantly, less or no inhibition was observed with 50-100 microM concentrations of ethanolamine. At 5-50 microM concentrations, ethanolamine also inhibited the synergistic mitogenic effects of both S1P plus insulin (22-27% inhibition) and PCho plus ATP (45-73% inhibition) but not those of S1P plus PMA or S1P plus ATP. The results indicate that S1P stimulates PLD-mediated hydrolysis of PtdEtn by a mechanism that may involve a regulatory protein kinase C isoform. Increased formation of ethanolamine by PLD-mediated PtdEtn hydrolysis or by other means may be required for maximal stimulation of DNA synthesis by S1P in the presence of insulin, and particularly PCho.

3T3 Cells↗
PubMed1998

Quantification of sphingosine derivatives in human platelets: inducible formation of free sphingosine.

To elucidate the physiologic role of sphingolipid-derived products as signaling molecules, we analyzed the levels of endogenous sphingosine (Sph) derivatives in human platelets. When the platelets were stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate, neither ceramide formation nor sphingomyelin hydrolysis was observed, which suggests that the sphingomyelin cycle may not be an essential part of the signaling pathway under these conditions. In contrast, Sph was found to increase in platelets upon stimulation. The level of Sph 1-phosphate, which is formed from Sph by the action of Sph kinase, was not affected under our conditions. Although it has been established that Sph inhibits protein kinase C, which regulates the functional responses of the platelets, Sph levels which exert an inhibitory effect on protein kinase C cannot be attained under physiological conditions (without exogenous Sph). Considering the stimulation of the synthesis of Sph by the physiological agonist thrombin, we speculate that Sph is a signaling molecule of physiological importance in platelets, but protein kinase C may not be its target.

Adult↗