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PubMed2002

Soluble Fas ligand activates the sphingomyelin pathway and induces apoptosis in luteal steroidogenic cells independently of stress-activated p38(MAPK).

Fas ligand (FasL) is implicated as a mediator of luteolysis. However, a gap exists in our understanding of the Fas-mediated signaling mechanisms that are involved in either the loss of progesterone production or the structural regression of the corpus luteum. In the present study we investigated the acute and chronic effects of FasL with respect to activation of cytokine/stress-induced signaling pathways and apoptosis in bovine steroidogenic cells. More specifically, we investigated soluble FasL (sFasL)-activated production of ceramide, a second messenger of the sphingomyelin pathway, and activation of p38(MAPK), a member of the MAPK family. sFasL activated the sphingomyelin pathway, as evidenced by a 2-fold increase (P < 0.05) in the production of ceramide. Pretreatment with imipramine (50 micro M), an inhibitor of acid sphingomyelinase activity, attenuated (75%; P < 0.05) sFasL-induced ceramide production, suggesting that the increase in ceramide was partially the result of acid sphingomyelinase-mediated hydrolysis of sphingomyelin. Treatment of luteal cells with sFasL or a cell-permeable ceramide analog (C8) for 24-48 h resulted in a significant increase (P < 0.05) in apoptosis. Western blot analysis revealed that sFasL had little effect on the activation of p38(MAPK) in primary bovine luteal steroidogenic cells. Furthermore, pretreatment with the p38(MAPK) inhibitor SB203580 failed (P > 0.05) to inhibit sFasL- or C8-induced death. Although sFasL did not alter basal progesterone levels detected in the culture medium, C8 caused a significant increase (P < 0.05) in progesterone concentrations within the medium. Collectively, these data suggest that the role of FasL in luteolysis may be to activate the stress-induced sphingomyelin pathway that, in turn, serves as a mediator of apoptosis.

Animals↗
PubMed1996

Interleukin-1 beta stimulates sphingomyelin hydrolysis in cultured granulosa cells: evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Dexamethasone-induced change in the sphingomyelin content of human polymorphonuclear leukocytes in vitro.

Human polymorphonuclear leukocytes were incubated with 8 x 10(-9) M dexamethasone for 2 h. A significant increase in the sphingomyelin content of the cells was found when 8 x 10(-8) M dexamethasone was used, with lesser changes being noted when the concentration was increased or decreased from this level. The increase in the phospholipid was blocked by the addition of cycloheximide to the incubation. Concurrent with the increase in sphingomyelin, an increase in sphingomyelinase was demonstrated. This increased enzymatic activity may have occurred secondary to the increase in substrate or may be involved in the reverse reaction, whereby phosphorylcholine and ceramide combine to form sphingomyelin. Other studies have demonstrated an effect of corticosteroids to suppress the production of superoxide anion and the importance of a membrane oxidase in the production of the anion. An action of dexamethasone to both suppress superoxide anion production and increase sphingomyelin content suggests a possible effect of the lipid changes upon the oxidase responsible for the production of the anion.

Cycloheximide↗
PubMed1985

Isolation of two forms of an activator protein for the enzymic sphingomyelin degradation from human Gaucher spleen.

Two activator proteins for sphingomyelin degradation were isolated from heat-treated extracts of human Gaucher spleen. The separation was based on the degree of affinity of the activators for ConA-Sepharose. Activator A1, which had affinity for ConA-Sepharose, was purified 1 430-fold, and activator A2, which had no affinity for ConA-Sepharose, 2 140-fold as compared with the original heat-treated extracts. The molecular masses of activator A1 and activator A2 were 6 000 and 3 500 Da, respectively, as determined by dodecyl sulfate electrophoresis, and approximately 5 000 Da as measured in the presence of 8M urea. The two activators had similar properties and a similar but not identical amino-acid composition. Both were shown to form a complex with sphingomyelin and stimulate the degradation of sphingomyelin by normal fibroblast homogenates and by an approximately 1 430-fold purified sphingomyelin phosphodiesterase ("acid sphingomyelinase") from normal human urine. This stimulation was greatly reduced after incubation with pronase E. The enzymic degradation of glucosylceramide and galactosylceramide was not affected by these activators.

Amino Acids↗
PubMed1980

A fluorometric determination of sphingomyelinase by use of fluorescent derivatives of sphingomyelin, and its application to diagnosis of Niemann-Pick disease.

We synthesized fluorescent derivatives of sphingomyelin (N-acyl-sphingosylphosphocholine) and used them as substrates for several sphingomyelinases. The following five fluorescent probes, each attached to the terminal carbon atom of the fatty acyl residue, were introduced into sphingomyelin: dansyl, pyrene, carbazole, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, and anthroic acid. We compared the rates at which the fluoro- and radiolabeled sphingomyelins were hydrolyzed. They were the same with the following three spingomyelinases: (a) a purified enzyme from Staphylococcus aureus; (b) at Triton X100-treated extract of human brain (assayed at pH 7.4 in the presence of Mg2+; and (c) aqueous extracts of brain lysosomes, skin fibroblasts, and amniotic cells, assayed at pH 5.0. Homogenates of skin fibroblasts of a patient with Niemann-Pick disease had practically no activity when assayed at pH 5, with fluorosphingomyelin as substrate. When fluorosphingomyelin was mixed in various proportions with natural sphingomyelin, enzymes from each of the three sources hydrolyzed the two substrates at equal rates. The fluorosphingomyelins can be used to estimate with great sensitivity the sphingomyelinase activity in extracts of tissues or cells, in tears, and probably in hair follicles, as well as diagnose Niemann-Pick disease, either pre- or post-natally

Amniotic Fluid↗
PubMed1982

[The enzymatic analysis of sphingomyelin in HDL (author's transl)].

A simple method is described for the enzymatic determination of sphingomyelin in the apolipoprotein B-free supernatants prepared by precipitation of blood sera with phosphotungstate/MgCl2. The analysis is based on the enzymatic hydrolysis of sphingomyelin, by sphingomyelinase from B. cereus, into phosphorylcholine and N-acylsphingosine, and subsequent hydrolysis of phosphorylcholine by alkaline phosphatase. The choline formed is determined by choline kinase in an optical test. The results from this method were in good agreement with those obtained by the conventional chemical sphingomyelin determination. Furthermore, there was a good correlation between the sphingomyelin concentrations obtained from the HDL fractions isolated by ultracentrifugation (1.063-1.21 kg/l) and those obtained from the apolipoprotein B free supernatants after phosphotungstate/mgCl2 precipitation of sera.

Bacillus cereus↗
PubMed1983

Enzymatic colorimetry of lecithin and sphingomyelin in aqueous solution.

A procedure for the enzymatic determination of lecithin and sphingomyelin in aqueous solution is described. The phospholipids are first dissolved in chloroform:methanol (2:1 by vol), the solvent is evaporated, and the residue is redissolved in an aqueous zwitterionic detergent solution. The enzymatic reaction sequences of both assays involve hydrolysis of the phospholipids to produce choline, which is then oxidized to betaine, thus generating hydrogen peroxide. The hydrogen peroxide is subsequently utilized in the enzymatic coupling of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, an intensely red color being formed. The presence of a non-reacting phospholipid enhances the hydrolysis of the reacting phospholipid. Thus we added lecithin to the sphingomyelin standards and sphingomyelin to the lecithin standards. This precise procedure may be applicable to determination of lecithin and sphingomyelin in amniotic fluid.

Alkaline Phosphatase↗
PubMed1994

Involvement of the sphingomyelin pathway in autocrine tumor necrosis factor signaling for human immunodeficiency virus production in chronically infected HL-60 cells.

Tumor necrosis factor (TNF) is a potent inducer of human immunodeficiency virus (HIV) proviral transcription and subsequent mature virus production. Recent investigations have shown that TNF can use a signal transduction pathway in HL-60 cells involving sphingomyelin hydrolysis to ceramide with subsequent stimulation of a ceramide-linked kinase. When sphingomyelinase was added exogenously to activate this cascade in HIV-1-infected HL-60 cells, it mimicked TNF-induced HIV production. Phospholipases A2, C, or D, which do not generate ceramide, had no effect; however, a synthetic ceramide analog added exogenously potently induced HIV production. In addition, anti-TNF antibodies blocked much of the effect of both sphingomyelinase and the synthetic ceramide analog on virus expression, suggesting that, although signaling is initiated through the sphingomyelin pathway, it is sustained by autocrine TNF synthesis. Thus, direct activation of the sphingomyelin pathway recapitulated the effect of TNF on both HIV and TNF production. These studies indicate that the sphingomyelin pathway is involved in TNF signaling for HIV production in chronically infected myeloid cells.

Antibodies, Monoclonal↗
PubMed1994

Quantification and comparison of some natural sphingomyelins by on-line high-performance liquid chromatography/discharge-assisted thermospray mass spectrometry.

Sphingomyelins obtained from bovine brain, chicken egg yolk and bovine milk fat were analysed. Separation was performed by reversed-phase high-performance liquid chromatography utilizing a binary solvent gradient consisting of n-butanol-water-1-propanol-isooctane. Detection was accomplished by light scattering and on-line discharge-assisted thermospray (plasmaspray) ionization mass spectrometry. The positive ion mass spectra exhibit prominent ions related to the amine base structure and fragments which can be utilized for identification of molecular species. The abundance of the fragments containing the fatty acid reflects relatively well the fatty acid composition of natural sphingomyelin mixtures. Bovine brain sphingomyelin has, for example, two distinctive major molecular species composed of C24:1 and C18:0 acids as indicated by the m/z 630.6 and m/z 548.5 ions, respectively. While egg yolk exhibits ions of m/z 520.6 which indicate the presence of C16:0, bovine milk sphingomyelin has several prominent ions of m/z 632.5, 618.6 and 604.7, reflecting the proportions of C24:0, C23:0 and C22:0, respectively.

Animals↗
PubMed1985

Fatty acids of sphingomyelin from amniotic fluid of normal and diabetic pregnancies.

Amniotic fluid collected from 14 normal and 11 diabetic patients was analyzed for phospholipids, and separated sphingomyelin and lecithin fractions were further studied for their acid composition by gas liquid chromatography. Notable differences in percent fatty acid composition of sphingomyelin were observed for palmitic (16:0; diabetic less than normal), oleic (18:1; diabetic greater than normal), behenic (22:0; diabetic less than normal) and arachidic acid (20:0; absent in diabetics) in the specimens studied. Notable differences were not observed in fatty acids from lecithin fraction. Fatty acid composition of sphingomyelin from amniotic fluid is similar to fatty acid distribution in sphingomyelin from serum and erythrocyte and suggests maternal origin of the lipid.

Amniotic Fluid↗
PubMed1998

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers.

To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state 31P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoyl-phosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, Tm, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 degrees C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 degrees C were found to be 6.1 +/- 0.8 mM-1 and 3.7 +/- 0.4 mM-1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, Ri < or = 5) mixed micelles are formed over the entire temperature range (20 degrees to 60 degrees C) for both kinds of lipids.

Animals↗
PubMed1977

Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus.

The cytolytic toxin from the sea anemone Stoichactis helianthus was inhibited up to 90--95% by suspensions of sphingomyelin but not by phosphatidylcholine or other membrane lipids. When the toxin was incubated with sphingomyelin and the mixture fractionated either by isoelectric focusing or Sephadex gel filtration, the residual hemolytic units migrated together with the lipid and not as free toxin. Incubation with phosphatidylcholine, however, did not shift the toxin peak in either type of column. A toxin-ferritin conjugate retaining hemolytic activity was observed by negative staining to bind to liposomes prepared with sphingomyelin but not with liposomes containing phosphatidylcholine. The results provide evidence that the membrane binding site of the toxin is sphingomyelin.

Animals↗
PubMed1980

2H- and 31P-NMR studies of cholesteryl palmitate in sphingomyelin dispersions.

Dispersions (50 wt% in water) of sphingomyelin/cholesteryl palmitate (95 : 5 mol%) have been studied by 2H- and 31P-NMR spectroscopy between 25 and 60 degrees C. The deuterated esters, cholesteryl palmitate-d31 and cholesteryl palmitate-16, 16, 16-d3, were used for 2H-NMR studies. Of the 5 mol% cholesteryl palmitate added, 1.5 mol% was found to incorporate in the sphingomyelin and this amount remained constant throughout the temperature range studied. The remainder of the cholesteryl palmitate was present as regions of solid. At temperatures above the gel-to-liquid crystalline phase transition of sphingomyelin the NMR spectra indicate that a fraction of the cholesteryl palmitate and sphingomyelin is undergoing rapid isotropic motions. This fraction, which increases with increasing temperature above the phase transition, is probably composed of small bilayer structures. When 50 mol% cholesterol (relative to sphingomyelin) was added to the sphingomyelin/cholesteryl palmitate dispersion, the isotropic component in the NMR spectral was no longer present, the gel-to-liquid crystalline phase transition was removed, and the incorporation of ester in the membrane decreased by more than an order of magnitude.

Animals↗
PubMed1991

Structure and thermotropic properties of hydrated N-stearoyl sphingomyelin bilayer membranes.

Hydrated multibilayers of N-stearoyl sphingomyelin were investigated as a function of hydration using differential scanning calorimetry (DSC) and X-ray diffraction. Anhydrous N-stearoyl sphingomyelin exhibits an endothermic transition at 75 degrees C (delta H = 3.8 kcal/mol); increasing hydration progressively lowers the transition temperature and increases the transition enthalpy, until limiting values (Tm = 45 degrees C, delta H = 6.7 kcal/mol) are observed for hydration values greater than 21.4% H2O. At low hydration levels, less than 20% H2O, an additional transition is observed at approx. 20 degrees C. X-ray diffraction studies at temperatures below (22 degrees C) and above (55 degrees C) the main endothermic transition confirm that the bilayer gel (sharp 4.2 A reflection)----bilayer liquid crystal (diffuse 4.5 A reflection) transition occurs at all hydration levels with limiting bilayer hydration occurring at approx. 31.5% H2O in the gel phase and at approx. 35% H2O in the liquid crystal phase. The thermotropic properties and metastability of this partial synthetic N-stearoyl sphingomyelin differ in some respects from that of the previously studied synthetic DL-erythro-N-stearoyl sphingomyelin (Estep, T.N., Calhoun, W.I., Barenholz, Y., Biltonen, R.L., Shipley, G.G. and Thompson, T.E. (1980) Biochemistry 19, 20-24), suggesting an influential role of the interfacial molecular conformation.

Calorimetry, Differential Scanning↗
PubMed1985

The synthesis of sphingomyelin in the Morris hepatomas 7777 and 5123D is restricted to the plasma membrane.

The site of sphingomyelin synthesis in the rapidly growing Morris hepatomas 7777 and 5123D was determined by incubating plasma membrane, mitochondrial and microsomal membrane fractions with vesicles of phosphatidyl[methyl-14C]choline in the presence of phosphatidylcholine transfer protein. In agreement with a previous study on rat liver (Voelker, D.R. and Kennedy, E.P., 1982, Biochemistry 21, 2753-2759) we have demonstrated that sphingomyelin synthesis in these hepatomas is restricted to the plasma membrane. The greatly elevated sphingomyelin content of mitochondria and microsomes (Hostetler, K.Y., Zenner, B.D. and Morris, H.P., 1979, Cancer Res. 39, 2978-2983) suggests that rapidly growing hepatomas, in contrast to liver, have an effective mechanism of intracellular sphingomyelin transfer.

Androgen-Binding Protein↗
PubMed1991

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Human peripheral lymph lipoproteins are enriched in sphingomyelin.

The concentrations of total choline-containing phospholipids and of sphingomyelin have been determined in peripheral lymph from six volunteers. The concentration in lymph of total phospholipids was 14.6 +/- 6.0 mg/dl and of sphingomyelin 6.1 +/- 2.7 mg/dl; the estimated concentration of phosphatidylcholine (by subtracting the concentration of sphingomyelin from that of total choline-containing lipoproteins) was 8.5 +/- 5.1 mg/dl. The concentration of free choline in lymph was less than 1 mg/dl. The ratio of the concentration in lymph to that in the subject's own plasma of sphingomyelin was 0.18 +/- 0.07 and that of (estimated) phosphatidylcholine 0.05 + 0.04; the difference between these two ratios was significant at P less than 0.002. The present data are compatible with the hypothesis that some of lipoprotein phosphatidylcholine is lost while crossing from plasma to lymph.

Adult↗
PubMed1992

Effects of sphingomyelin and phosphatidylcholine acyl chains on the clearance of triacylglycerol-rich lipoproteins from plasma. Studies with lipid emulsions in rats.

Series of lipid emulsions were prepared as physical models of lymph chylomicrons. The emulsion phospholipid was systematically varied with respect to sphingomyelin, in 0-100% mixtures with egg yolk phosphatidylcholine (EYPC). In other emulsions, the phospholipid was systematically varied with respect to dipalmitoylphosphatidylcholine (DPPC) in 0-100% mixtures with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). All emulsions contained unlabeled free cholesterol, radiolabeled triolein (TO) and radiolabeled cholesteryl oleate (CO). The emulsions were injected into conscious rats to measure the clearances of emulsion TO and CO and the capture of lipid radioactivity by selected organs. The emulsions containing EYPC or POPC were metabolized similarly to lymph chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of emulsion TO followed by hepatic uptake of the CO in the triglyceride-depleted emulsion remnants. Emulsions stabilized with either 1-oleoyl-2-stearoyl- or 1-stearoyl-2-oleoylphosphatidylcholine (OSPC or SOPC) were metabolized similarly. Increasing amounts of sphingomyelin in EYPC emulsions progressively slowed the removal of TO and CO labels from plasma. With 50% sphingomyelin clearance was very slow, while emulsion clearance was negligible with 100% sphingomyelin. Emulsions containing 20% of DPPC in POPC were metabolized similarly to 100% POPC, but 40% or more of DPPC progressively slowed the removal from plasma of both TO and CO. With 100% DPPC clearance was characterized by a rapid initial removal of about 30% of the injected material, followed by a second phase when removal was negligible, suggesting lack of hydrolysis of triacylglycerols by lipoprotein lipase. Changes in the apolipoproteins associated with the emulsions probably mediated the observed changes in clearance.

Animals↗