sphingosine-phosphorylcholine and sphingosyl-beta-glucoside

A deficiency of ordinary ceramides in the stratum corneum is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). We have proposed that the mechanism underlying that deficiency involves a novel sphingolipid metabolizing enzyme, termed sphingomyelin (SM) glucosylceramide (GCer) deacylase, which hydrolyzes SM or GCer at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) or glucosylsphingosine (GSP) instead of ceramide, leading to the ceramide deficiency in the AD skin. The enzymic characteristics observed showed a pH dependency of catalytic activity with a peak at pH 5.0 and a molecular weight of 40,000. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, GlcCDase, SMase and ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. Those enzymic characteristics of SM-GCer deacylase are completely distinct from ceramidase as well as the other known deacylases. Our enzymic measurements demonstrated that SM-GCer deacylase activity is enhanced more than 5-fold in involved stratum corneum, more than 3-fold in uninvolved stratum corneum and approximately 3-fold in the involved epidermis from patients with AD compared with healthy controls. Our findings suggest that the novel enzyme, SM-GCer deacylase, is expressed in situ at significant levels in the epidermis of AD patients. This results in the production of SPC and GSP, instead of ceramides, which leads in turn to the ceramide deficiency seen in the stratum corneum of those patients. It is likely that the biogenesis of SM-GCer deacylase may be critical to the pathogenesis of AD.

Topics: Amidohydrolases; Ceramides; Dermatitis, Atopic; Epidermis; Glucosylceramides; Humans; Phosphorylcholine; Psychosine; Sphingomyelins; Sphingosine

Gaucher disease (GD) is a rare autosomal recessive multisystemic lysosomal storage disorder presenting a marked phenotypic and genotypic variability. GD is caused by a deficiency in the glucocerebrosidase enzyme. The diagnosis of GD remains challenging because of the large clinical spectrum associated with the disease. Moreover, GD biomarkers are often not sensitive enough and can be subject to polymorphic variations. The main objective of this study was to perform a metabolomic study using an ultra-performance liquid chromatography system coupled to a time-of-flight mass spectrometer to identify novel GD biomarkers. Following the analysis of plasma samples from patients with GD, and age- and gender-matched control samples, supervised statistical analyses were used to find the best molecules to differentiate the two groups. Targeted biomarkers were structurally elucidated using accurate mass measurements and tandem mass spectrometry. This metabolomic study was successful in highlighting seven biomarkers associated with GD. Fragmentation tests revealed that these latter biomarkers were lyso-Gb

Topics: Adult; Aged; Biomarkers; Case-Control Studies; Chromatography, High Pressure Liquid; Early Diagnosis; Female; Gaucher Disease; Humans; Male; Mass Spectrometry; Metabolomics; Middle Aged; Phosphorylcholine; Prognosis; Psychosine; Sensitivity and Specificity; Sphingosine; Young Adult

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2-50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.

Topics: Adolescent; Adult; Aged; Biomarkers; Blood Specimen Collection; Case-Control Studies; Edetic Acid; Female; Heparin; Humans; Male; Niemann-Pick Disease, Type C; Phosphorylcholine; Psychosine; Reproducibility of Results; Retrospective Studies; Sphingosine; Tandem Mass Spectrometry; Young Adult

Ovarian cancer G-protein-coupled receptor 1 (OGR1), previously proposed as a receptor for sphingosylphosphorylcholine (SPC), has recently been identified as a proton-sensing or extracellular pH-responsive G-protein-coupled receptor stimulating inositol phosphate production, reflecting the activation of phospholipase C. In the present study, we found that acidic pH stimulated cAMP accumulation, reflecting the activation of adenylyl cyclase, in addition to inositol phosphate production in OGR1-expressing cells. The cAMP response was hardly affected by the inhibition of phospholipase C. SPC inhibited the acidification-induced actions in a pH-dependent manner, while no OGR1-dependent agonistic action of SPC was observed. Thus, the dose-response curves of the proton-induced actions were shifted to the right in the presence of SPC regardless of stereoisoform. The antagonistic property was also observed for psychosine and glucosylsphingosine. In conclusion, OGR1 stimulation may lead to the activation of adenylyl cyclase in addition to phospholipase C in response to extracellular acidification but not to SPC. However, SPC and related lysolipids antagonize the proton-induced and OGR1-mediated actions.

Topics: Adenylyl Cyclases; Animals; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Inositol Phosphates; Lysophosphatidylcholines; Phosphorylcholine; Psychosine; Receptors, G-Protein-Coupled; Second Messenger Systems; Sphingosine; Transfection; Type C Phospholipases

Sphingolipids contribute to modulation of two opposing cell processes, cell growth and apoptotic cell death; ceramide and sphingosine promote the latter and sphingosine-1-phosphate triggers the former. Thrombin, a pro-inflammatory protease that is regulated by the blood coagulation cascade, exerts similar effects depending on cell type. Here we report a new mechanism for cross-talk between sphingolipid metabolism and thrombin generation. Sphingosine and sphinganine, but not ceramide or sphingosine-1-phosphate, down-regulated thrombin generation on platelet surfaces (IC(50) = 2.4 and 1.4 microm for sphingosine and sphinganine, respectively) as well as in whole plasma clotting assays. Thrombin generation was also inhibited by glucosylsphingosine, lysosphingomyelin, phytosphingosine, and primary alkylamines with >10 carbons. Acylation of the amino group ablated anticoagulant activities. Factor Va was required for the anticoagulant property of sphingosine because prothrombin activation was inhibited by sphingosine, sphinganine, and stearylamine in the presence but not in the absence of factor Va. Sphingosine did not inhibit thrombin generation when Gla-domainless factor Xa was used in prothrombinase assays, whereas sphingosine inhibited activation of Gla-domainless prothrombin by factor Xa/factor Va in the absence of phospholipids (IC(50) = 0.49 microm). Fluorescence spectroscopy studies showed that sphingosine binds to fluorescein-labeled factor Xa and that this interaction required the Gla domain. These results imply that sphingosine disrupts interactions between factor Va and the Gla domain of factor Xa in the prothrombinase complex. Thus, certain sphingolipids may be bioactive lipid mediators of thrombin generation such that certain sphingolipid metabolites may modulate proteases that affect cell growth and death, blood coagulation, and inflammation.

Topics: Anticoagulants; Binding Sites; Blood Platelets; Carbon; Dose-Response Relationship, Drug; Down-Regulation; Factor Va; Factor Xa; Gene Expression Regulation; Humans; Inhibitory Concentration 50; Lipid Metabolism; Lipids; Micelles; Phosphorylcholine; Protein Binding; Protein Structure, Tertiary; Prothrombin; Psychosine; Sphingolipids; Sphingosine; Thrombin

To clarify the functional relevance of sphingomyelin (SM) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [(14)C]acetic anhydride to yield [(14)C-C(2)]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD.

Topics: Adolescent; Adult; Ceramides; Dermatitis, Atopic; Epidermis; Humans; Phosphorylcholine; Psychosine; Sphingosine; Up-Regulation

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.

Topics: Actins; Cell Division; Humans; Leukodystrophy, Globoid Cell; Phagocytosis; Phosphorylcholine; Psychosine; Sphingosine; Tumor Cells, Cultured; U937 Cells

Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their effects on the cardiovascular system in vivo in anaesthetized rats. The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1-100 microg kg(-1)) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood flows (RBF, MBF) using a pressure transducer and electromagnetic flow probes, respectively. Intravenous injection of SPP rapidly (within 30 s), transiently and dose-dependently reduced RBF (maximally -4.0+/-0.3 ml min(-1)) and MBF (maximally -1.4+/-0.2 ml min(-1)), without affecting mean arterial pressure or heart rate. Other sphingolipids had no significant effect. Intrarenal arterial SPP administration caused greater blood flow reductions (maximally -6.4+/-0.3 ml min(-1)) than systemic administration. Upon intrarenal administration, sphingosylphos- phorylcholine also lowered RBF (maximally -2.8+/-0.6 ml min(-1)), while the other sphingolipids remained without effect. Pretreatment with pertussis toxin (PTX, 10 microg kg(-1)) 3 days before the acute experiment abolished the SPP-induced reductions of RBF and MBF. These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any effects. The PTX-sensitivity strongly suggests that the effects of SPP on renal and mesenteric blood flow are mediated by receptors coupled to G(i)-type G-proteins.

Topics: Animals; Blood Pressure; Dose-Response Relationship, Drug; Heart Rate; Kidney; Lysophospholipids; Male; Mesentery; Pertussis Toxin; Phosphorylcholine; Psychosine; Rats; Rats, Wistar; Renal Circulation; Sphingosine; Splanchnic Circulation; Virulence Factors, Bordetella

Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G-protein-coupled receptors, some of which were cloned and designated as Edg-receptors. Sphingolipid-induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid-induced elevations in intracellular Ca(2+) concentration were measured in cultured rat aortic smooth muscle cells. SPPC and SPP (0.1-100 micromol l(-1)) caused concentration-dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC(50) 5.63+/-0.17 and E(max) 49+/-3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC(50) D-erythro-SPPC 5.69+/-0.08, L-threo-SPPC 5.31+/-0.06) and inhibited by pretreatment with pertussis toxin (E(max) from 44+/-5 to 19+/-4%), by chelation of extracellular Ca(2+) with EGTA and by nitrendipine (E(max) from 40+/-6 to 6+/-1 and 29+/-6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (E(max) from 87+/-3 to 70+/-4%). SPP and SPPC caused transient increases in intracellular Ca(2+) concentrations in rat aortic smooth muscle cells in a pertussis toxin-sensitive manner. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca(2+) concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin-sensitive G-proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

Topics: Animals; Calcium; Dose-Response Relationship, Drug; Egtazic Acid; In Vitro Techniques; Indomethacin; Lysophospholipids; Male; Mesenteric Arteries; Nitrendipine; Nitroarginine; Pertussis Toxin; Phosphorylcholine; Psychosine; Rats; Rats, Wistar; Renal Artery; Sphingosine; Vasoconstriction; Vasodilator Agents; Virulence Factors, Bordetella