Ceramide Molecules Research Articles

Interleukin-1β Induces Chronic Activation and de Novo Synthesis of Neutral Ceramidase in Renal Mesangial Cells

The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163-166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1beta treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1beta and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novo synthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions.

Superoxide Potently Induces Ceramide Formation in Glomerular Endothelial Cells

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2–30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.

Regulation of Cyclin-Dependent Kinase 2 Activity by Ceramide

Cyclin-dependent kinases have been implicated in the inactivation of retinoblastoma (Rb) protein and cell cycle progression. Recent studies have demonstrated that the lipid molecule ceramide is able to induce Rb hypophosphorylation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C6-Ceramide induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblasts. Employing immunoprecipitation kinase assays, we found that ceramide specifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C6-dihydroceramide was not effective. Ceramide did not directly inhibit CDK2 in vitro but caused activation of p21, a major class of CDK-inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both protein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 in vitro. Further, calyculin A and okadaic acid, both potent protein phosphatase inhibitors, together almost completely reversed the effects of ceramide on CDK2 inhibition. Taken together, these results demonstrate a dual mechanism by which ceramide inhibits the cell cycle. Ceramide causes an increase in p21 association with CDK2 and through activation of protein phosphatases selectively regulates CDK2. These events may lead to activation of Rb protein and subsequent cell cycle arrest.

Enhancement of FcγR- and CR3-Mediated Neutrophil Phagocytosis by Cerebrosides

There is increasing evidence that the ligation of adhesion molecules such as L-selectin can activate phagocytes to their full inflammatory potential. Sulfatide has been established as ligand for L-selectin and shown to trigger intracellular signals in human neutrophils. However, it remains unclear whether the ligation of L-selectin with sulfatide affects neutrophil phagocytosis. We studied the effects of sulfatide upon FcγR- and CR3-mediated human neutrophil phagocytosis. Adhesion of the cells to a sulfatide-coated surface resulted in a dose-dependent enhancement of phagocytosis mediated via FcγR or CR3, or both receptors. Galactocerebroside, but not glucocerebroside, also enhanced phagocytosis by neutrophils; therefore, galactose residue is thought to be required on ceramide molecules for the activation. Chymotrypsin-treated neutrophils, from which most L-selectin had been removed, reacted with sulfatide and galactocerebroside to enhance phagocytosis. These results suggest that an unidentified receptor for these cerebrosides exists on neutrophils and participates in the enhancement of phagocytosis.

Different Sphingolipids Show Differential Partitioning into Sphingolipid/Cholesterol-Rich Domains in Lipid Bilayers

Two fluorescence-based approaches have been applied to examine the differential partitioning of fluorescent phospho- and sphingolipid molecules into sphingolipid-enriched domains modeling membrane “lipid rafts.” Fluorescence-quenching measurements reveal that N-(diphenylhexatrienyl)propionyl- (DPH3:0-)-labeled gluco- and galactocerebroside partition into sphingolipid-enriched domains in sphingolipid/phosphatidylcholine/cholesterol bilayers with substantially higher affinity than do analogous sphingomyelin, ceramide, or phosphatidylcholine molecules. By contrast, the affinity of sphingomyelin and ceramide for such domains is only marginally greater than that of a phosphatidylcholine with similar hydrocarbon chains. By using direct measurements of molecular partitioning between vesicles of different compositions, we show that the relative affinities of different C 6-NBD- and C 5-Bodipy-labeled sphingolipids for sphingolipid-enriched domains are quantitatively, and in most circumstances even qualitatively, quite different from those found for species whose N-acyl chains more closely resemble the long saturated chains of cellular sphingolipids. These findings lend support in principle to previous suggestions that differential partitioning of different sphingolipids into “raft” domains could contribute to the differential trafficking of these species in eukaryotic cells. However, our findings also indicate that short-chain sphingolipid probes previously used to examine this phenomenon are in general ill-suited for such applications.

Sphingomyelinase and ceramide analogs induce contraction and rises in [Ca(2+)](i) in canine cerebral vascular muscle.

Studies were designed to investigate effects of neutral sphingomyelinase (N-SMase) and ceramide analogs as well as phosphorylcholine on vascular tone and Ca(2+) mobilization in isolated canine cerebral arterial smooth muscle. N-SMase (0.001-0.1 U/ml) provoked a gradual but sustained vasoconstriction of arterial rings in a concentration-related manner that was endothelium independent. Incubation of denuded arterial rings in Ca(2+)-free medium or pretreatment with verapamil in extracellular Ca(2+) resulted in a reduction of the N-SMase-evoked constriction. Exposure of arterial rings to 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA)-AM did not, however, result in a reduction of N-SMase-induced constriction. Both staurosporine and bisindolymaleimide I attenuated N-SMase-induced contractions to 66% and 72% of control, respectively. N-SMase caused gradual and sustained rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) in primary cultured cerebral vascular smooth muscle cells. Pretreatment of these cultured cells with nimodipine and verapamil caused a steady decline in N-SMase-induced rises in [Ca(2+)](i). Exposure of the cells to Ca(2+)-free solution reversed the [Ca(2+)](i)-induced rise triggered by N-SMase to the resting baseline. Both C(8) and C(16) ceramide (10(-9)-10(-6) M), but not phosphorylcholine, constricted denuded canine arterial rings in a concentration-related manner and elevated [Ca(2+)](i). Our results suggest that the sphingomyelin-signaling pathway, via a probable release of ceramide molecules, may play an important role in regulation of cerebral arterial wall tone.

Biophysical Studies of Model Stratum Corneum Lipid Monolayers by Infrared Reflection−Absorption Spectroscopy and Brewster Angle Microscopy

One, two, and three component lipid models of the stratum corneum (SC) consisting of non hydroxy fatty acid (NFA) ceramide, cholesterol, and perdeuterated palmitic acid have been investigated as monolayers at the air/water interface. Infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy (BAM), and π−A isotherms have provided a molecular level understanding and a macromolecular picture of SC lipid organization and phase behavior. BAM studies of pure ceramide monolayers reveal discrete domains of highly ordered molecules, which upon addition of fatty acid and cholesterol become more loosely packed fluid-like phases. IRRAS measurements of the multicomponent films reveal that the ceramide molecules remain conformationally ordered in these apparently more fluid films. However, both the chain packing and the headgroup hydrogen bonding are very different in monolayers than in the bulk phase, multilamellar system. Chain packing is hexagonal in the monolayer, whereas it is orthorhombic for multilayers. Hydrogen bonding patterns in the headgroup region of NFA ceramide molecules in monolayers is significantly different from multilayers. The splitting of headgroup amide modes, previously observed in multilamellar samples, is not observed in monolayers. This lack of amide mode splitting in monolayers is consistent with the hypothesis (Moore et al. J. Phys. Chem. 1997, 101, 8933−8940) that amide mode splitting in NFA ceramide multilamellar samples is a transverse interaction between opposite headgroups in adjacent bilayers going down through the bilayers along the z-axis. The consequences of the observed lipid phase behavior, and headgroup bonding interactions, for skin barrier lipid cohesion and organization are discussed. The implications of this structure on skin barrier function are then considered.

Specific processing of poly(ADP-ribose) polymerase, accompanied by activation of caspase-3 and elevation/reduction of ceramide/hydrogen peroxide levels, during induction of apoptosis in host HL-60 cells infected by the human granulocytic ehrlichiosis (HGE) agent.

We studied the mechanism by which the human granulocytic ehrlichiosis (HGE) agent induces programmed cell death (apoptosis) in human promyelocytic HL-60 leukemia cells. Using several New York HGE isolates, we show that the HGE agent-elicited apoptosis is accompanied by increased processing of nuclear enzyme poly(ADP-ribose) polymerase (PARP), concurrent with a noticeable increase in caspase 3 activities. A marked increase in the amounts of the signaling molecule ceramide but not of diacylglycerol was also observed in HGE agent-infected HL-60 cells, compared with the amounts in uninfected controls. Simultaneous or prior treatment of infected HL-60 cells with the ceramide synthase inhibitor fumonisin B1 did not affect the magnitude of infection by the intracellular pathogen, as determined by both the presence of morulae and the expression of its outer surface membrane protein, p44. These results suggest that the observed changes in ceramide are generated through the sphingomyelinase pathway and not by way of de novo synthesis of ceramide. We also assayed for changes in intracellular hydrogen peroxide and show that the HGE agent causes a decrease in its concentrations in infected cells.

Caspases mediate C 2-ceramide-induced apoptosis of the human oligodendroglial cell line, MO3.13

The signalling molecule ceramide participates in the sphingomyelin pathway and accumulates intracellularly in response to inflammatory mediators. Here we show that membrane permeable C 2-ceramide is apoptogenic in the immortalised human oligodendroglial cell line MO3.13. Apoptosis (defined by cell shrinkage and chromatin condensation) is accompanied by caspase enzyme activation. Immunoblotting analysis of extracts from differentiated MO3.13 cells revealed the presence of caspase-3 proenzyme, activation by cleavage of pro-caspase-3 in cells treated with C 2-ceramide and cleavage of the caspase substrates fodrin and rabaptin. Lysates also showed cleavage of a fluorogenic peptide substrate. Addition of the general caspase inhibitor BAF markedly attenuated apoptosis of MO3.13 oligodendroglia. A role for caspase-3-like enzymes in ceramide-induced apoptosis of oligodendroglia may have important implications for approaches to treatment of demyelinating diseases.

Enantioselective Synthesis of a Novel Trans Double Bond Ceramide Analogue via Catalytic Asymmetric Dihydroxylation of an Enyne. The Role of the Trans Double Bond of Ceramide in the Fusion of Semliki Forest Virus with Target Membranes

Intensive interest is currently focused on the role of ceramide (1), a key lipid molecule that functions as a second messenger. The first asymmetric synthesis of a D-erythro-ceramide analogue that contains a C(5)-C(6) trans double bond (3), together with its biological evaluation in a viral-liposome fusion system, is described. Sharpless asymmetric dihydroxylation of 4'-methoxyphenyl trans-5-octadecyn-2-enyl ether (enyne 8a), prepared by the coupling reaction of 1-[(E)-(4'-bromo-2'-butenyl)oxy]-4-methoxybenzene (7) with Lithium tetradecyne, generated yne-diol 9 in 96% yield with the desired stereochemistry at the C(2)and (3) positions and high enantiomeric purity (95% enantiomeric excess). Birch reduction (Li/EtNH2) of yne-diol 9 furnished (2R,3R,5E)-octadecene-1,2,3-triol (10) stereospecifically. The latter was converted to 2 azido derivative 13 in three steps (via a 2-O-triflate-1,3-O-benzylidene intermediate) and 55% overall yield. Reduction of azide 13 and in situ N-acylation with p-nitrophenyl cis-hexadec-4-enoate provided D-erythro-Delta(5)-trans-ceramide (3) in 91% yield. The role of the trans double bond of ceramide in mediating fusion of an alphavirus (Semliki Forest virus) was assessed in a liposomal model system, using target phospholipid/cholesterol vesicles containing either D-erythro-ceramide 1 or 3. The kinetics of virus fusion, as monitored by a change in pyrene excimer fluorescence over a period of 60 s, showed that Delta(5)-trans-ceramide 3 was completely inactive, indicating that there is an absolute requirement for the trans double bond to be located between C(4) and C(5). These data indicate that the molecular determinants on the viral envelope glycoprotein are highly specific for recognition of the unsaturated site in the ceramide molecule.

Nitric Oxide Stimulates Chronic Ceramide Formation in Glomerular Endothelial Cells

Exposure of glomerular endothelial cells for 24 h to compounds releasing NO, including spermine-NO, MAHMA-NO, andS-nitroso-glutathione, results in a dose-dependent and delayed (after 24 h) increase in the lipid signaling molecule ceramide. This NO-induced stimulation occurs in a cGMP-independent fashion since the membrane-permeant cGMP analogue dibutyryl cGMP has no effect on chronic ceramide production. Short-term incubation of endothelial cells for 20 min reveals that NO and dibutyryl cGMP fail to stimulate an acute ceramide increase, whereas TNF-α, a well-known activator of sphingomyelinases, is able to acutely increase ceramide formation. Interestingly,N-oleoylethanolamine, an acidic ceramidase inhibitor, potentiates NO-induced chronic ceramide production, indicating that ceramide generation rather than ceramide metabolism is modulated by NO. Furthermore, NO-induced delayed ceramide formation is partially inhibited by the thiol-specific inhibitor iodoacetamide and the radical scavenger α-tocopherol, suggesting a regulatory role of thiol-containing enzymes and the involvement of a redox-sensitive mechanism. In addition, NO causes an increased DNA fragmentation in glomerular endothelial cells which is further enhanced byN-oleoylethanolamine and can be mimicked by exogenous ceramide. In summary, these results imply that ceramide represents an important mediator of NO-triggered chronic cell responses like apoptosis. Inhibition of ceramide synthesis may provide a new therapeutic approach to the treatment of pathological conditions involving increased NO formation.

Nitric Oxide Donors Induce Stress Signaling via Ceramide Formation in Rat Renal Mesangial Cells

Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.

Identification of ceramide targets in interleukin-1 and tumor necrosis factor-α signaling in mesangial cells

An increasing number of cell-surface receptors have been shown to trigger sphingomyelin turnover and generation of the lipid signaling molecule ceramide. Ceramide plays a role in mediating cellular responses as diverse as inflammation, differentiation, gene expression, growth suppression, and apoptosis. A radioiodinated, photoaffinity-labeling analog of ceramide ([125I]TID-ceramide) was used to identify downstream signaling targets of ceramide. Ceramide was found to bind specifically to and activate protein kinase c-Raf, leading to subsequent activation of the extracellular signal-regulated kinase (ERK) module in mesangial cells. We found also that ceramide binds to and differentially modulates the activity of distinct protein kinase C isoenzymes. These data are discussed in the context of interleukin 1beta-induced inflammatory gene expression in mesangial cells.

Role of an acidic compartment in tumor-necrosis-factor-alpha-induced production of ceramide, activation of caspase-3 and apoptosis.

Tumor necrosis factor-alpha (TNF-alpha) apoptosis by recruiting a complex of cytosolic proteins at its plasma membrane receptor. Among them is caspase-8, an interleukin-1beta-converting enzyme (ICE)-like protease that initiates an amplified protease cascade to activate the cell-death machinery. The latter comprises at least caspase-3 and caspase-7, which execute cell death by cleaving numerous protein substrates, including poly(ADP-ribose) polymerase. In addition, TNF-alpha stimulates the production of ceramide, which also activates the death machinery. Whether the signaling pathways elicited by caspase-8 and ceramide proceed independently or intersect at a specific subcellular site is unknown. Using the lysosomotropic agent NH4Cl and the vesicularization inhibitor brefeldin A, we show here the convergence of TNF-alpha-induced death signaling on an acidic, subcellular compartment reminiscent of lysosomes. This compartment generates at least two signaling pathways that account for the caspase-3 activation and apoptosis induced by TNF-alpha, one involving ceramide and caspase-unrelated adapter molecules and another involving yet unknown lysosomal mediators. The apoptosis inhibitor Bcl-2 specifically acts on the ceramide-activated pathway to block caspase-3 activation and apoptosis. The latter result explains why Bcl-2 only partially blocks TNF-alpha-induced apoptosis.

Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH2-terminal kinase. Correlation with its inhibitory effect on tumor necrosis factor-alpha production.

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-alpha (TNFalpha) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNFalpha release, (ii) the suppressor effect on TNFalpha production, which originates from the lack of TNFalpha mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNFalpha production by inhibiting ERK1/2, p38, and JNK kinase activities.

C 2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C 2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O 2 −) production. The preincubation of PMN for 15 min with C 2-ceramide increased by up to almost 3-fold the amounts of O 2 − generated in response to 0.1 and 1 μM fMLP. Priming was detected at C 2-ceramide concentrations of 2 μM to 4 μM per million PMN. Though less potent than C 2-ceramide, C 6-ceramide ( N-hexanoylsphingosine) could prime for O 2 − generated in response to 0.1 μM fMLP, with maximal effects obtained at 10–20 μM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O 2 − generation. As expected, TNF-α (1000 U/ml), also primed for fMLP-induced O 2 − production; however, the combination of TNF-α and C 2-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C 2-ceramide and TNF-α. C 2-ceramide at 2 μM did not enhance the production of O 2 − induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B 4 (LTB 4), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C 2-ceramide (2 μM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A 2 or D (PLC, PLA 2, PLD) were involved in the priming effect by C 2-ceramide. However, C 2-ceramide inhibited in a dose-related manner the production of O 2 − induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C 2-ceramide. The priming O 2 − production by C 2-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-α have different mechanisms than C 2-ceramide.

Lipids Are Covalently Attached to Rigid Corneocyte Protein Envelopes Existing Predominantly as β-Sheets: A Solid-State Nuclear Magnetic Resonance Study

C solid-state nuclear magnetic resonance at natural abundance was used to study isolated corneocyte envelopes from porcine stratum corneum. The presence of lipids covalently attached to the protein envelopes was detected by chemical shifts of methylene and methyl groups of the bound lipids. The corneocyte protein envelopes are rigid, as suggested by efficient 1H to 13C cross polarization and 13C spin-lattice relaxation studies. The chemical shift of the carbonyl carbons of the protein envelopes supports the prediction that the chemically bound lipid envelope is attached to proteins arranged predominantly in the beta-sheet conformation, allowing a dense palisade of ceramide molecules to form a water-impermeable external sheath.

Ceramide mediates tumor necrosis factor effects on P450-aromatase activity in cultured granulosa cells

In [3H]serine-labelled granulosa cells treatment with TNF alpha (10 ng/ml) resulted in a transient decrease in cellular [3H]sphingomyelin and generation of [3H]ceramide that remained elevated 60 min later. In cells labelled with [methyl-14C]choline, TNF alpha induced a similar reduction in [14C]sphingomyelin content that was accompanied by a sustained elevation in [14C]phosphorylcholine levels. In FSH-primed cells, TNF alpha inhibited P450-AROM activity in a dose-dependent manner, an effect that was also observed in cells treated with bacterial sphingomyelinase (SMase 0.003-0.3 U/ml) or increasing concentrations (0.1-10 microM) of N-acetylsphingosine (C2-cer) a membrane-permeable analogue of ceramide. These results support the notion that sphingomyelin degradation to a bioeffector molecule ceramide, may be an early event involved in TNF alpha-induced signal transduction in granulosa cells.

Ceramide mediates tumor necrosis factor effects on P450-aromatase activity in cultured granulosa cells.

In [3H]serine-labelled granulosa cells treatment with TNF alpha (10 ng/ml) resulted in a transient decrease in cellular [3H]sphingomyelin and generation of [3H]ceramide that remained elevated 60 min later. In cells labelled with [methyl-14C]choline, TNF alpha induced a similar reduction in [14C]sphingomyelin content that was accompanied by a sustained elevation in [14C]phosphorylcholine levels. In FSH-primed cells, TNF alpha inhibited P450-AROM activity in a dose-dependent manner, an effect that was also observed in cells treated with bacterial sphingomyelinase (SMase 0.003-0.3 U/ml) or increasing concentrations (0.1-10 microM) of N-acetylsphingosine (C2-cer) a membrane-permeable analogue of ceramide. These results support the notion that sphingomyelin degradation to a bioeffector molecule ceramide, may be an early event involved in TNF alpha-induced signal transduction in granulosa cells.

Thermotropic behavior of phosphatidylcholine-glucosyl ceramide mixtures: Effects of phospholipid acyl chain composition and interaction with water

The thermotropic behavior of multilamellar vesicles composed of mixtures of dimyristoyl phosphatidylcholine-glucosyl ceramide and of egg phosphatidylcholine-glucosyl ceramide was investigated using differential scanning calorimetry. Macroscopic demixing of the lipid components occurred when multilamellar vesicles were prepared from mixtures of glucosyl ceramide and egg phosphatidylcholine by conventional methods. This problem was overcome by a technique based on spray drying of the lipid mixture. The results obtained for the two systems are compared with data available for dipalmitoyl phosphatidylcholine-glucosyl ceramide mixtures ( Biochemistry 22 (1983) 3497–3501). All three phosphatidylcholines perturb the complex thermotropic behavior of glucosyl ceramide. The data suggest that the interference with intermolecular interactions among glycosyl ceramide molecules by phospholipid molecules is related to the molecular miscibility of the two components. This is strongly dependent on the acyl chain composition of the phosphatidylcholine and the water activity of the ambient aqueous phase.