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At least 19 recordsLinked to original sources
PubMed1992

Stimulation of sphingomyelin biosynthesis by brefeldin A and sphingomyelin breakdown by okadaic acid treatment of rat hepatocytes.

Studies on sphingomyelin metabolism in rat hepatocytes were facilitated by the use of choline-deficient cells which allowed for the rapid labeling of phosphatidylcholine and as a result sphingomyelin. Pulse and pulse-chase studies with [methyl-3H]choline and [methyl-3H]methionine demonstrated that both compounds were effectively used for sphingomyelin biosynthesis and that newly made and pre-existing phosphatidylcholine could be used for sphingomyelin biosynthesis. When hepatocytes were incubated with brefeldin A, there was a 2.4-fold stimulation of the conversion of phosphatidylcholine into sphingomyelin. Since brefeldin A causes collapse of the cis/medial Golgi into the endoplasmic reticulum the stimulation of sphingomyelin biosynthesis could be due to more rapid access of the labeled phosphatidylcholine in the endoplasmic reticulum to sphingomyelin synthase in the collapsed Golgi. Forskolin inhibited the brefeldin A-induced stimulation of sphingomyelin biosynthesis. To investigate whether or not phosphorylation reactions regulate sphingomyelin metabolism, hepatocytes were incubated with okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A. Rather than stimulating sphingomyelin biosynthesis, okadaic acid enhanced the catabolism of sphingomyelin. In contrast, a cyclic AMP analogue and forskolin had no effect on sphingomyelin biosynthesis or catabolism. Surprisingly, other pulse-chase studies demonstrated that okadaic acid stimulated the catabolism of only newly made sphingomyelin. The brefeldin A and okadaic acid effects were independent of lysosomal involvement. Subcellular fractionation studies revealed that brefeldin A and okadaic acid effects were generalized in all sphingomyelin containing membranes. The brefeldin A studies suggest that the rate of transfer of phosphatidylcholine from the endoplasmic reticulum to the Golgi might be limiting for sphingomyelin biosynthesis. The okadaic acid studies indicate that the catabolism of sphingomyelin by a sphingomyelinase is regulated by an unidentified protein kinase and by either protein phosphatase 1 and/or 2A activity in hepatocytes.

Animals↗
PubMed2002

A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.

Animals↗
PubMed1991

Interaction of cholesterol with sphingomyelin in bilayer membranes: evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles.

Cholesterol undergoes exchange between membranes containing sphingomyelin at a much slower rate than between membranes lacking sphingomyelin. To investigate the role of the hydroxy group at the 3-position of sphingomyelin in the interaction between sphingomyelin and cholesterol, we have measured the rates of [4-14C]cholesterol exchange between unilamellar vesicles prepared with N-stearoylsphingomyelin or with synthetic analogues in which the hydroxy group is replaced with an O-alkyl group or with hydrogen. Vesicles prepared from 3-deoxy- and 3-O-methyl-N-stearoylsphingomyelin had the same rate of [14C]-cholesterol desorption. The half-times for exchange from vesicles prepared with 3-O-methyl- and 3-deoxy-N-stearoylsphingomyelins and 10 mol % of cholesterol were only slightly faster (a factor of only 1.5) than that found from vesicles prepared from N-stearoylsphingomyelin and 10 mol % cholesterol. The rate of cholesterol desorption from vesicles could be accelerated by preparing vesicles from bulky 3-O-alkyl analogues of sphingomyelin. Vesicles containing 3-O-ethyl-N-stearoylsphingomyelin and 3-O-tetrahydropyranyl egg sphingomyelin gave rate enhancements of approximately 14 and 35, compared with the rates observed in vesicles made from N-stearoyl- and egg sphingomyelin, respectively. These data indicate that insertion of sterically bulky groups at the 3-position of sphingomyelin (such as ethoxy and tetrahydropyranyloxy) in place of hydroxy interferes markedly with the molecular packing of cholesterol and sphingomyelin in bilayer membranes; however, the hydroxy group of sphingomyelin is not critical for the strong interaction of cholesterol with sphingomyelin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions.

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S possess a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind to sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters (Km, Vmax). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5-10-times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.

Acetylcholinesterase↗
PubMed1997

Effect of sphingomyelin composition on the phase structure of phosphatidylcholine-sphingomyelin bilayers.

In this study, we examine the phase behavior as well as lateral diffusion and percolation in the region of coexisting gel and fluid phases in binary mixtures composed of dimyristoylphosphatidylcholine and one of two totally synthetic D-erythro-sphingomyelins (having either C16 or C24 acyl chains, both having similar gel to liquid-crystalline phase transition temperatures). This study stems from the uniqueness of sphingomyelins having gel to liquid-crystalline transition temperatures in the range of physiological interest, and the fact that more than 50% of the naturally occurring sphingomyelin species have a chain mismatch. The presence of sphingomyelin in biological membranes can thus be expected to give rise to a complex phase structure. Fluorescence recovery after photobleaching, differential scanning calorimetry, and electron microscopy are used to show that, despite similarity in the temperature range of the gel to liquid-cystalline phase transition of the two sphingomyelins, the two differ in their phase structure. Also they differ to a large extent in their mixing with dimyristoylphosphatidylcholine. Dimyristoylphosphatidylcholine and C16 sphingomyelin mix nearly ideally, with the percolation threshold locus lying close to the liquidus on the phase diagram. In contrast, the C24 sphingomyelin and dimyristoylphosphatidylcholine mix nonideally, with the percolation threshold locus lying close to the solidus. In addition, mixtures containing C24 sphingomyelin have a complex thermotropic behavior which may be related to the observation that these dispersions contain several types of particles, some of which are not multilamellar vesicles. These studies suggest that the degree of sphingomyelin chain mismatch is an important factor in determining lateral organization in the membrane.

Calorimetry, Differential Scanning↗
PubMed1983

Pathways of sphingomyelin metabolism in cultured fibroblasts from normal and sphingomyelin lipidosis subjects.

The metabolism of endogenous sphingomyelin labeled with 32P or [methyl-3H]choline and of exogenous [choline-methyl-3H], [32P]-, or [N-acyl-1-14C]sphingomyelin was studied in normal and Niemann-Pick Type A (NP-A) cultured fibroblasts. Despite a greater than 96% decrease in lysosomal sphingomyelinase activity in the NP-A cells, they were able to degrade endogenously produced [32P]- or [methyl-3H]sphingomyelin at normal or near normal rates. Exogenous [methyl-3H]-, [methyl-3H, 32P]-, and [methyl-3H, N-acyl-1-14C] sphingomyelin was taken up intact by normal and NP-A cells, with NP-A cells accumulating 4-8 times more lipid. By 20 h, 50% of the control cell-associated 3H and 32P was recovered in lecithin, and the ratio of activities (3H/32P) indicated most of the phosphorylcholine derived from sphingomyelin had been transferred intact. By comparison in NP-A cells, after a 40-h incubation only 20% of the labeled phosphorylcholine derived from sphingomyelin was recovered in lecithin. With both cell lines, 20 to 50 times more sphingomyelin was hydrolyzed than was taken up by the cells; the reaction products in the medium were ceramide and a mixture of water-soluble compounds such as phosphorylcholine and choline. These results indicate that there are at least two metabolic pathways for sphingomyelin modification in cultured fibroblasts in addition to degradation by the lysosomal acid sphingomyelinase. One route is hydrolysis by a cellular sphingomyelinase. The second is the hydrolysis and/or transfer of phosphorylcholine from sphingomyelin and results in the synthesis of lecithin.

Carbon Radioisotopes↗
PubMed1996

Characterization of the sphingomyelin content of isolated pancreatic islets. Evaluation of the role of sphingomyelin hydrolysis in the action of interleukin-1 to induce islet overproduction of nitric oxide.

Inflammatory cytokines may participate in the destruction of pancreatic islets during the pathogenesis of insulin-dependent diabetes mellitus, and the cytokine interleukin-1 (IL-1) strongly inhibits insulin secretion from rat pancreatic islets by a process which involves induction of expression of the inducible isoform of nitric oxide synthase and the overproduction of nitric oxide. The signaling events between IL-1 receptor occupancy and induction of nitric oxide synthase in rat islets involve activation of the transcriptional activator NFkappa B. Because sphingomyelin hydrolysis has been implicated as a signaling process both in NFkappa B activation and in IL-1 action in some cells, we have examined the potential involvement of sphingomyelin hydrolysis in the induction of islet nitric oxide overproduction by IL-1. Rat islet sphingomyelin pools were radiolabeled with [3H]choline, and sphingomyelin was then isolated by normal phase HPLC. Electrospray ionization-mass spectrometric analysis revealed islet sphingomyelin consists of at least 4 distinct molecular species, and the most abundant of them contained sphingosine as the long chain base and a residue of palmitic acid as the fatty acid substituent. Molecular species containing residues of stearic acid and arachidic acid were also observed. Neither interleukin-1 nor tumor necrosis factor-alpha was found to induce hydrolysis of islet sphingomyelin species, and neither an exogenous, cell-permeant ceramide species (N-acetyl-D-sphingosine) nor exogenous sphingomyelinase mimicked or potentiated the effect of IL-1 to increase rat islet nitric oxide generation, as reflected by nitrite production. Similar findings were obtained with RINm5F insulinoma cells and with mouse pancreatic islets. These findings provide the first information on the molecular species of sphingomyelin in pancreatic islets and suggest that sphingomyelin hydrolysis is not involved in the signaling pathway whereby IL-1 induces the overproduction of nitric oxide by pancreatic islets.

Animals↗
PubMed2004

Keratinocytes rapidly readjust ceramide-sphingomyelin homeostasis and contain a phosphatidylcholine-sphingomyelin transacylase.

Ceramide as central second messenger of the apoptosis-related sphingomyelin signaling pathway is a potential target for the control of cancer. A complex metabolizing network defines cell type and stage-specific final ceramide concentrations. Successful therapeutic control of ceramide levels requires a knowledge of multiple related turnover rates. The metabolism of ceramide and sphingomyelin was studied in keratinocytes under the condition of an unstimulated sphingomyelin signaling pathway. Preparations enriched in plasma membranes contain a neutral Mg(2+)-dependent sphingomyelinase and a Mg(2+)-independent sphingomyelin synthase that vigorously preserve balanced ceramide and sphingomyelin levels. Ceramide regulates neutral sphingomyelinase. Inhibition of sphingomyelin synthase by D609 treatment results in temporary loss of intercelluar contacts and in cellular shrinking. It is ineffective for sustained elevation of ceramide levels. Ceramide phosphorylating and deacylating activities are insignificant. Recently, fatty-acid remodeling in sphingomyelin was reported as likely to counteract the membrane-rigidifying effects of cholesterol. Keratinocytes transfer fluorescence labeled acyl-chains between phosphatidylcholine and sphingomyelin. A transferase of that kind would allow rapid adjustment of local lipid composition in response to acutely changed conditions. In addition, this transferase might have a function in the formation of the epidermal permeability barrier.

Bridged-Ring Compounds↗
PubMed1997

Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity.

Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative pathway to the cell surface exists for sphingomyelin, brefeldin A and mitotic cells were used to block vesicular traffic between the Golgi complex and the plasma membrane. Exogenous sphingomyelinase was applied in the cold to assay for the presence of sphingomyelin on the surface of CHO cells. Newly synthesized radiolabeled sphingomyelin was found to equilibrate with cell surface sphingomyelin within 1.5 hours at 37 degrees C. Brefeldin A and mitosis inhibited this transport but, surprisingly, not the surface appearance of the short-chain sphingomyelin analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl(C6-NBD)-sphingo myelin as assayed by depletion of this lipid in the medium by the scavenger albumin. Transport of C6-NBD-sphingomyelin in the presence of brefeldin A was blocked by cyclosporin A and PSC 833, inhibitors of the multidrug resistance P-glycoprotein. The same was observed in HepG2 and HeLa cells, and for short-chain glucosylceramide, which demonstrates the general nature of the transporter-dependent sphingolipid translocation across the plasma membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Monensin inhibits synthesis of plasma membrane sphingomyelin by blocking transport of ceramide through the Golgi: evidence for two sites of sphingomyelin synthesis in BHK cells.

The monovalent cationophore monensin, which is known to interfere with vesicular transport through the Golgi apparatus, inhibits synthesis of sphingomyelin in BHK cells by up to 40%. The monensin-sensitive component of sphingomyelin synthesis appears to be the pool which normally reaches the cell surface since treatment of cells with exogenous sphingomyelinase causes an almost identical loss of sphingomyelin. Monensin causes increases in ceramide and glucosylceramide labelling which together are equivalent to the decrease in sphingomyelin labelling. Monensin also increases synthesis of cholesterol ester, probably due to the decreased delivery of sphingomyelin to the plasma membrane. However, monensin has no effect on resynthesis of plasma membrane sphingomyelin which has been degraded by extracellular sphingomyelinase. The results support the idea that synthesis of sphingomyelin destined for the plasma membrane does not occur in the cis- or medial-Golgi but depends on vesicular transport of ceramide to a second synthesis site which is distal to the medial-Golgi.

Animals↗
PubMed1993

Microsomal sphingomyelin accumulation in thioacetamide-injured regenerating rat liver: involvement of sphingomyelin synthase activity.

The purpose of this work was to determine whether alterations in the lipid composition of rat liver microsomal membranes existed during thioacetamide-induced injury prior to the development of hepatic cancer and biochemical mechanisms involved. Rats were injected intraperitoneally with (50 mg/kg body wt per day) thioacetamide or diluent for 8 days. Liver homogenates and microsomal membranes from liver homogenates were obtained. Incorporation of [32P]orthophosphate into whole liver lipids and hepatic microsomal lipids was evaluated 75 min after isotope administration. These determinations were made after two separate periods of treatment (3 and 8 days). Activity of sphingomyelin synthase was assayed in rat liver homogenates as well as in the purified microsomal fractions. Results demonstrated a maintenance of liver and hepatic microsomal contents of phosphatidylcholine during thioacetamide-induced injury even when the biosynthesis of this glycerophospholipid in both liver and their microsomal fractions appeared decreased. Also observed was a considerable increase of microsomal sphingomyelin, as well as an increased hepatic biosynthesis of sphingomyelin caused by thioacetamide treatment. The microsomal sphingomyelin/phosphatidylcholine radioactivity ratio significantly increased. Sphingomyelin synthase activity in liver homogenate appeared stimulated. In conclusion, our data are consistent with a thioacetamide-induced increase in microsomal sphingomyelin by a stimulation of sphingomyelin synthase. Based on this and previous studies, accumulation of sphingomyelin in the microsomal purified fraction is associated with the number of thioacetamide doses and is an early event clearly detected prior to tumoral characteristics of hepatocytes.

Animals↗
PubMed2001

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase.

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.

Ceramides↗
PubMed1975

The optical activity of D-erythro-sphingomyelin and its contribution to the circular dichroism of sphingomyelin-containing systems.

Circular dichroism studies on bovine brain sphingomyelin show the presence of a strong negative cotton effect below 200 nm, the position and magnitude of which depend on the physical state of the lipid. This cotton effect is thought to arise from the pi-pi transition of the amide group in the sphingomyelin backbone. The sphingomyelin contribution to the observed ellipticity of membranes and lipoprotein complexes depends on the mol fraction of amide groups present as sphingomyelin: this contribution is calculated to be less than 2% in the case of serum high density lipoprotein and the order of 20% below 200 nm in the case of the erythrocyte ghost membrane. Due to the similarity of the CD spectrum of sphingomyelin to that of a random coil polypeptide, use of uncorrected ellipticity data is expected to lead to an overestimate of the random coil content of proteins in systems containing a high sphingomyelin content.

Animals↗
PubMed1978

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus.

The hydrolysis of D-erythro beef brain sphingomyelin and D,L-erythro-N-palmitoylsphingomyelin dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30 degrees C for beef brain sphingomyelin and 41 degrees C for N-palmitoylsphingosine-phosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase-transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.

Liposomes↗
PubMed1990

The role of sphingomyelin in phosphatidylcholine metabolism in cultured human fibroblasts from control and sphingomyelin lipidosis patients and in Chinese hamster ovary cells.

Human fibroblasts in culture take up exogenous [choline-Me-3H,32P]sphingomyelin (SM) from the medium and incorporate it into cellular SM and phosphatidylcholine [Spence, Clarke & Cook (1983) J. Biol. Chem. 258, 8595-8600]. The ratio of [3H]choline/[32P]Pi is similar in SM and phosphatidylcholine, indicating that the phosphocholine (P-Cho) moiety is transferred intact. Similar results are obtained with Niemann-Pick (NP) cells which are deficient in lysosomal sphingomyelinase activity, suggesting that the P-Cho transfer may not be mediated by the lysosomal sphingomyelinase and that alternative pathways of sphingomyelin catabolism are present in cultured cells. In this study we have shown that: (1) the P-Cho pool in control and NP cells incubated with exogenous labelled SM has a specific radioactivity intermediate between that of SM and PtdCho; (2) expansion of the intracellular P-Cho pool by incubation with exogenous choline reduces the incorporation of [3H]choline from SM into PtdCho; and (3) incorporation of P-Cho from SM into PtdCho is decreased at the non-permissive temperature in Chinese hamster ovary cells with a temperature-sensitive mutation in the cytidylyltransferase reaction. These results suggest that incorporation of P-Cho from SM into PtdCho involves a reaction sequence in which P-Cho is hydrolysed from SM by a sphingomyelinase, followed by incorporation of P-Cho into PtdCho via the cytidine pathway of biosynthesis (SM----P-Cho----CDP-Cho----PtdCho). The appreciable incorporation of P-Cho from SM into PtdCho in sphingomyelinase-deficient NP cells suggests a more substantial or effective lysosomal sphingomyelinase activity in intact cells than is measured in vitro, and/or a significant contribution by other sphingomyelinase activities in these cells.

Animals↗
PubMed1998

Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?

Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite directions, the levels of ceramide and diacylglycerol. In this study SMS activity was investigated in normal and SV40-transformed human lung fibroblasts (WI38). The addition of [3H]C2-ceramide to cells resulted in a time-dependent formation of [3H]C2-SM. At 24 h after treatment, normal WI38 cells cleared 17% of [3H]C2-ceramide producing [3H]C2-SM, which accounted for 13% of total radioactivity. On the other hand, SV40-transformed cells cleared 45% of [3H]C2-ceramide and produced C2-SM, which accounted for 24% of total radioactivity. This enhanced production of C2-SM was also supported by an increase in the total SMS activity of cells (measured in vitro), such that SV40-transformed cells had SMS activity of 222 pmol/mg of protein/h, whereas wild type cells had 78 pmol/mg of protein/h of activity. Additional studies aimed at examining the SMS activity directed at ceramide produced in the plasma membrane. Treatment of cells with exogenous bacterial sphingomyelinase (SMase) for 25 min resulted in cleavage of 90-95% of total SM and the concomitant generation of ceramide. After bacterial SMase treatment, wild type WI38 cells cleared ceramide very slowly (19.2 pmol of ceramide/nmol of phosholipid Pi after 6 h of incubation) and hardly regenerated any SM. On the other hand, SV40-transformed cells cleared ceramide much faster (41.1 pmol/nmol of Pi after 6 h of incubation) and regenerated approximately 80% of the original SM. These results show that the enhanced SMS activity of transformed cells is particularly pronounced when ceramide is produced in the plasma membrane. Finally, several observations led us to consider the relationship of SMS to the "putative" phosphatidylcholine-specific phospholipase C (PC-PLC). We, therefore, tested the effects of D609, a purported PC-PLC-specific inhibitor on the activity of SMS. D609 inhibited SMS activity in vitro. In addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC (10-50 microg/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attributable to SMS.

Bridged-Ring Compounds↗
PubMed1993

Cholesterol and sphingomyelin syntheses are regulated independently in cultured human intestinal cells, CaCo-2: role of membrane cholesterol and sphingomyelin content.

There is a presumed association between cellular cholesterol and sphingomyelin metabolism. To study this relationship in the intestine, the activity of the rate controlling enzyme of sphingolipid synthesis, serine palmitoyltransferase (SPT), and the biosynthesis of long-chain bases were characterized in cultured human intestinal cells, CaCo-2. Cells were then incubated with substances known to alter cholesterol biosynthesis, and the effect of these mediators on SPT activity and long-chain base synthesis was determined and compared with their effects on HMG-CoA reductase activity and cholesterol synthesis. The polar sterol, 25-hydroxycholesterol, the squalene epoxide inhibitor, U18666A, and the inhibitor of HMG-CoA reductase, lovastatin, all significantly inhibited the synthesis of cholesterol without altering either SPT activity or long-chain base synthesis. Mevalonate, which increased cholesterol production 3-fold, also had no affect on SPT activity or sphingoid base synthesis. Serine, which significantly increased the synthesis of long-chain bases, did not alter cholesterol biosynthesis. Moreover, the suicide inhibitors of SPT, beta-chloroalanine and cycloserine, did not alter cholesterol synthesis while markedly decreasing long chain base synthesis. Cells were incubated with palmitic, oleic, linoleic, and eicosapentaenoic acids. Only palmitic acid, the preferred substrate for SPT, increased the production of long-chain bases. Both palmitic and oleic acids, however, increased the synthesis of cholesterol. Cells enriched in sphingomyelin had higher rates of synthesis of both cholesterol and long-chain bases compared to their controls. In contrast, cholesterol and long-chain base syntheses were significantly decreased in cells enriched in cholesterol. Control cells incubated with phospholipid liposomes alone had higher rates of synthesis of both lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗
PubMed2000

The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.

We have demonstrated previously that there is an abnormal expression of sphingomyelin (SM) deacylase in the epidermis of patients with atopic dermatitis (ADe). In the present study, we have prepared N-[palmitic acid-1-(14)C]SM and N-[palmitic acid-1-(14)C]glucosylceramide (GCer) to use as substrates and have quantified SM deacylase activity by detecting the release of [(14)C]palmitic acid in extracts of the stratum corneum or the epidermis of ADe patients. In studies using [palmitic acid-1-(14)C]SM as a substrate, a pH dependency of catalytic activity with a peak at pH 5.0 was found. Preparative SDS/PAGE using an extract of ADe epidermis revealed that the molecular mass of SM deacylase is 40000 Da, which is consistent with its apparent molecular mass of 42000 Da estimated by gel-filtration analysis of stratum corneum extracts. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, beta-glucocerebrosidase (GlcCDase), sphingomyelinase (SMase) and acid ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. In enzymic analysis using pI-4.2 SM deacylase partially purified by IEF, which had no detectable contamination with acid ceramidase, GlcCDase or SMase, radio-TLC analysis revealed that radiolabelled sphingosylphosphocholine or [1-(14)C]palmitic acid was enzymically liberated from [choline-methyl-(14)C]SM or N-[palmitoyl-1-(14)C]GCer, respectively, used as substrates. Further the pI-4.2 protein purified from extracts of the stratum corneum of ADe patients was able to hydrolyse N-[palmitoyl-1-(14)C]SM and GCer, but not N-[palmitoyl-1-(14)C]ceramide. These results indicate that a hitherto undiscovered epidermal enzyme, termed here glucosylceramide sphingomyelin deacylase, is expressed in the skin of ADe patients, which plays an important role in ceramide deficiency (including acylceramides) in the stratum corneum.

Carboxylic Ester Hydrolases↗