Effects of Brefeldin A on Galactosphingolipid Synthesis in an Immortalized Schwann Cell Line: Evidence for Different Intracellular Locations of Galactosylceramide Sulfotransferase and Ceramide Galactosyltransferase Activities

Abstract: Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6-h labeling period, a dose-dependent inhibition of [³⁵S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 µg/ml BFA. Labeling of newly synthesized galactosphingolipids with [³H]-palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA-GalCer) to 162% of control values, whereas labeling of GalCer containing 2-hydroxy fatty acids (HFA-GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA-GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA-GalCer synthesis. Examination of [³H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2-hydroxylation or the formation of HFA ceramide. Incorporation of [³H]palmitic acid into glucosylceramide and GM3 was increased over control levels whereas labeling of GM2 was inhibited, consistent with what has been reported previously for the effect of BFA on these glycolipids in other cell types. These results suggest that there are at least two separate intracellular sites for the galactosylation of HFA and NFA ceramide, respectively, which can be distinguished by their sensitivity to BFA. Our results also indicate that the site of GalCer sulfation is not redistributed to the endoplasmic reticulum in the presence of BFA and therefore may be localized to the distal Golgi or trans-Golgi network.     

Polymorphic phases of galactocerebrosides: spectroscopic evidence of lamellar crystalline structures

Fourier transform infrared spectroscopy was applied to study the structural and thermal properties of bovine brain galactocerebroside (GalCer) containing amide linked non-hydroxylated or alpha-hydroxy fatty acids (NFA- and HFA-GalCer, respectively). Over the temperature range 0-90 degrees C, both GalCer displayed complex thermal transitions, characteristic of polymorphic phase behavior. Upon heating, aqueous dispersions of NFA- and HFA-GalCer exhibited high order-disorder transition temperatures near 80 and 72 degrees C, respectively. En route to the chain melting transition, the patterns of the amide I band of NFA-GalCer were indicative of two different lamellar crystalline phases, whereas those of HFA-GalCer were suggestive of lamellar gel and crystalline bilayers. Cooling from the liquid-crystalline phase resulted in the formation of another crystalline phase of NFA-GalCer and a gel phase of HFA-GalCer, with a phase transition near 62 and 66 degrees C, respectively. Prolonged incubation of GalCer bilayers at 38 degrees C revealed conversions among lamellar crystalline phases (NFA-GalCer) or between lamellar gel and crystalline bilayer structures (HFA-GalCer). Spectral changes indicated that the temperature and/or time induced formation of the lamellar crystalline structures of NFA- and HFA-GalCer was accompanied by partial dehydration and by rearrangements of the hydrogen bonding network and bilayer packing mode of GalCer.     

Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis

Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the "early" galactose-transferring enzymes is largely unknown. We used short-chain ceramides with either an 2-hydroxy fatty acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA- and NFA-galactosylceramide (GalCer), lactosylceramide (LacCer), and galabiosylceramide (Ga2Cer). Although the HFA-GalCer synthesizing activity colocalized with an ER marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and permeabilized cells, newly synthesized short-chain GlcCer and GalCer were accessible to serum albumin, whereas LacCer and Ga2Cer were protected. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GalCer are synthesized in the cytosolic leaflet, while LacCer and Ga2Cer are synthesized in the lumenal leaflet of the Golgi. (b) HFA-GalCer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet. (c) GlcCer, NFA-GalCer, and HFA-GalCer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.     

Sphingolipid metabolism in Bacteroideaceae.

The lipid composition of the anaerobic Bacteroides thetaiotaomikron has been analyzed. Sphingomyelin, ceramide phosphinicoethanolamine, free even-numbered and branched chain sphingosine bases and ceramide represented about 50% of the total lipid extract. The main ester phospholipid was phosphatidylethanolamine. The alkali-stable sphingophospholipids were predominantly N-acylated with 3-hydroxypalmitic acid, whereas the ester phospholipids are preferentially substituted with normal even and odd-numbered and branched-chain fatty acids. When Bacteroides was grown in a medium supplemented with labelled palmitic acid, this fatty acid was utilized for acylation reactions and to a large extent for the de novo synthesis of sphinganine. This long-chain base was incorporated into the sphingolipids and was also present in free form. The 3-hydroxypalmitic acid present in sphingolipids is not derived from palmitic acid, since labelled palmitate did not serve as a precursor. Free sphinganine added to the culture medium was also utilized efficiently for the biosynthesis of the sphingolipids by growing Bacteroides cultures. The 3H/14C ratio in sphingomyelin and ceramide phosphinicoethanolamine is the same, when [1-14C]palmitic acid and [3-3H]sphinganine serve as precursors. Sphingomyelin, which is usually only present in higher animals, is synthesized de novo in this Bacteroides strain.     

De novo synthesis and recycling pathways of sphingomyelin in rat Sertoli cells

Sertoli cells from 19-day-old rats have two molecular species of sphingomyelin (SM1 and SM2) with different kinetic characteristics and fatty acid composition. Here, we have studied the incorporation of [14C]-choline and [14C]-palmitic acid into SM in presence or absence of fumonisin B1, an inhibitor of ceramide synthesis, and beta-chloroalanine, an inhibitor of sphinganine synthesis. The contributions of de novo synthesis and recycling pathways were estimated by analysis of the inhibition caused by these drugs. SM1 was synthesized more by sphingosine recycling, and SM2 was synthesized principally by ceramide recycling than SM1. De novo synthesis seems to be important for the two SM types, but our results showed that this pathway is more extensively utilized by SM2. In conclusion, using Sertoli cell cultures, we have shown for the first time that in the same cell different molecular species of SM are synthesized by different pathways.     

Lipid composition and metabolism in megakaryocytes at different stages of maturation

The lipid composition and metabolism of isolated guinea pig megakaryocyte subgroups at various stages of maturation were investigated. Three groups were studied: 1) 67% of megakaryocytes in Group A were immature; 2) Group B was heterogeneous and contained both immature and mature subgroups of megakaryocytes; 3) 92% of megakaryocytes in Group C were mature. Lipid composition was determined by thin-layer chromatography, lipid-phosphorus, and gas-liquid chromatography. Cholesterol, ceramide, and de novo fatty acid synthesis were evaluated with [14C]acetate. [14C]Glycerol was used to assess de novo phospholipid synthesis. 14C-Labeled fatty acids were used to evaluate fatty acid uptake. The phospholipid and cholesterol content was found to be four times greater in mature megakaryocytes than that in immature megakaryocytes, which paralleled the protein content and volume of mature and immature cells. The cholesterol-phospholipid ratio was similar and there were no differences in the phospholipid species in the three groups. Phospholipid and cholesterol synthesis were established in immature megakaryocytes and persisted at about the same level in mature megakaryocytes. The uptake of arachidonic and palmitic acids also occurred primarily in immature cells, while the de novo synthesis of palmitic acid occurs predominantly in mature megakaryocytes. There was an inverse relationship between the uptake of exogenous palmitic acid and fatty acid synthesis, but the uptake of palmitic acid primarily inhibited fatty acid synthesis in mature megakaryocytes. There were differences in the acylation of phospholipid species with arachidonic acid in megakaryocytes at different stages of maturation since the acylation of phosphatidylcholine occurred primarily in immature megakaryocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.     

Glycosphingolipid fatty acid arrangement in phospholipid bilayers: cholesterol effects.

Deuterium wide line NMR spectroscopy was used to study cholesterol effects on the ceramide portions of two glycosphingolipids (GSLs) distributed as minor components in fluid membranes. The common existence of very long fatty acids on GSLs was taken into account by including one glycolipid species with fatty acid chain length matching that of the host matrix, and one longer by 6 carbons. N-stearoyl and N-lignoceroyl galactosyl ceramide with perdeuterated fatty acid (18:0[d35] GalCer and 24:0[d47] GalCer) were prepared by partial synthesis. They were dispersed in bilayer membranes having the 18-carbon-fatty-acid phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), as major component. Glycolipid fatty acid chain behavior and arrangement were analyzed using order profiles derived from their 2H-NMR spectra. Cholesterol effects on order parameter profiles for 18:0[d35] GalCer, with chain length equal to that of the host matrix, followed the pattern known for acyl chains of phospholipids. The presence of sterol led to restriction of trans/gauche isomerization along the length of the chain, with the largest absolute increase in order parameters being toward the surface, but somewhat greater relative effect just below the "plateau" region. In cholesterol-containing membranes, order parameter profiles for the long chain species, 24:0[d47] GalCer, showed a characteristic secondary "plateau" associated with carbon atoms C14 to C23, a feature also present in SOPC bilayers without cholesterol and in pure hydrated 24:0[d47] GalCer. Cholesterol-induced ordering effects on the long chain glycolipid were similar to those described for the shorter chain species, but were minimal at the methyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.     

Enhanced turnover of arachidonic acid-containing species of phosphatidylinositol and phosphatidic acid of concanavalin A-stimulated lymphocytes

The incorporation of [32P]orthophosphate into phosphatidylinositol (PI) of pig lymphocytes was markedly increased by stimulation with concanavalin A. The labeling of PI with [3H]glycerol was also enhanced significantly, indicating that both de novo synthesis and recircular system (PI response) of PI were accelerated. This rapid labeling of PI might be related to the rapid breakdown of phosphatidylinositol 4,5-bisphosphate which was observed in various stimulated tissues. Concanavalin A also accelerated the labeling of phosphatidic acid with 32P and [3H]glycerol. To determine the dependence of this phenomenon on the fatty acid composition of both phospholipids, we separated PI and phosphatidic acid into individual molecular species. The predominant molecular species in PI was tetraene (81.6%) and those in phosphatidic acid were monoene (53.0%), diene (15.8%) and tetraene (19.2%), respectively. Interestingly, the incorporation of 32P into arachidonic acid-containing species (tetraene) was most rapidly elevated. On the other hand, the increment of 32P into saturated + monoene, diene and triene was relatively smaller and resembled that of [3H]glycerol. Similarly, the incorporation of 32P into tetraene of phosphatidic acid was preferentially accelerated. This is the first report concerning the metabolism of molecular species of phosphatidic acid in stimulated cells. These results indicate that the PI recirculating system is virtually dependent on tetraenoic species and that the participation of other molecular species is small. The increased de novo synthesis mainly depends upon molecular species other than tetraene. Arachidonic acid-containing species which turn over rapidly via the PI cycle may have an important role in the mitogenic triggering.     

Glycosphingolipid acyl chain orientational order in unsaturated phosphatidylcholine bilayers.

The glycosphingolipid, galactosyl ceramide (GalCer), was studied by 2H nuclear magnetic resonance (NMR) in fluid phospholipid bilayer membranes, with regard to arrangement of its acyl chain. For this purpose, species with perdeuterated 18-carbon fatty acid (18:0[d35]GalCer) or with perdeuterated 24-carbon fatty acid (24:0[d47] GalCer) were dispersed in bilayers of the 18-carbon phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). For 18:0[d35] GalCer, smoothed profiles of the order parameter, SCD, were found to be very similar to one another over the range of glycolipid concentration, 5-40 mol%. In addition, they were very similar to orientational order parameter profiles well known from the literature on phospholipid and glycolipid acyl chains (which deals in general with membranes of homogeneous chain length in the range 14-18 carbons). Corresponding order parameter profiles for the long-chain species, 24:0[d47] GalCer, were also similar to one another for glycolipid concentrations between 5 and 40 mol%. Their shapes, however, were distinctly different from those of the shorter chain analogues. SCD profiles for the two species were quantitatively similar to a membrane depth of C15. SCD values at C16 and C17 were approximately 20 and 30%, respectively, higher for the long-chain glycosphingolipid than for its short-chain analogue in SOPC. Nitroxide spin labels attached rigidly to C16 of the long-chain glycolipid in SOPC gave electron paramagnetic resonance (EPR) order parameters that were twice as high as for a spin label at C16 on the shorter chain glycolipid. Comparison was made between spectra of 24:0[d47] GalCer in SOPC and fully hydrated bilayers of the pure 24:0[d47] GalCer, a system that is considered to be partially interdigitated in fluid and gel phases. The resultant 2H NMR order parameter profiles displayed similar features, indicating that related organizational properties exist in these fluid systems. Effective chain length of 24:0[d47] GalCer within the SOPC membrane was calculated using the method of Schindler and Seelig (1975. Biochemistry, 14:2283-2287). The result suggested that the long-chain fatty acid should protrude roughly one third of the host matrix chain length across the bilayer midplane. However, a treatment of the same order parameters making very few assumptions about chain conformation indicated a high degree of orientational flexibility for the "extra" length of the long chain fatty acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Signal transduction in EJ-H-ras-transformed cells: de novo synthesis of diacylglycerol and subversion of agonist-stimulated inositol lipid metabolism

We examined the level of 1,2-diacylglycerol and inositol phosphates in normal and EJ-H-ras-transformed BALB/3T3 fibroblasts by prelabelling the cells with [3H]glycerol, [3H]inositol, [14C]glucose, [14C]arachidonic acid, and [14C]palmitic acid. Steady-state level of inositol phosphates, however, was the same in control and transformed cells. Diacyglycerol labelling by [14C]arachidonic acid was the same in control and transformed cells. Insulin dramatically increased diacylglycerol labeling by [14C]glucose in normal cells, whereas it did not affect ras-transformed fibroblasts. Neurotransmitter-induced inositol lipid turnover was greatly enhanced in ras-transformed cells; conversely, platelet-derived growth factor and thrombin-stimulated normal cells to a greater extent than transformed fibroblasts. Taken together these results suggest that ras transformation may induce multifarious effects on signal transduction: it may cause de novo synthesis of diacylglycerol and subversion of neurotransmitter and growth factor receptor coupling to inositol lipid metabolism.     

Metabolism of fatty acids and their incorporation into phospholipids of the mitochondria and endoplasmic reticulum in isolated hepatocytes determined by isolation of fluorescence derivatives

Isolated hepatocytes were incubated in the presence of [14C]palmitic, [14C]linoleic or [14C]linolenic acid and the time-courses of incorporation of radioactivity into phosphatidylcholine and phosphatidylethanolamine of microsomes and mitochondria were followed. For this purpose a procedure was developed for HPLC separation of 9-diazomethylanthracene (ADAM) derivatives of fatty acids. When [14C]palmitic acid was used, the major product of elongation and desaturation was octadecadienoic acid, which accounted for 35-65% of the total radioactivity. Labeled palmitoleic, stearic and oleic acids could also be isolated. In fatty acids which do not participate to any large extent in deacylation-reacylation reactions, the pattern of incorporation was characteristic: a high rate of incorporation into microsomal and a low rate of incorporation into mitochondrial phospholipids during the first 40 min, followed by a decrease in the former and an increase in mitochondrial labeling. This pattern is consistent with the fact that de novo synthesis of these two phospholipids occurs in the endoplasmic reticulum in vivo. When cells were incubated in the presence of [14C]linoleic acid, 70-90% of the radioactivity recovered in phospholipids was in this same form, whereas the remaining label was mainly in arachidonic acid and, to some extent, in eicosatrienoic acid. When hepatocytes were incubated in the presence of [14C]linolenic acid, 70-85% of the radioactivity in isolated phospholipids was associated with linolenic acid. As much as 20% of the label was recovered in docosahexanoic acid and 5-10% in arachidonic acid. In the case of the two latter labeled substrates the exchange reactions seem to dominate over de novo synthesis. For phospholipids synthesized de novo the transfer from the endoplasmic reticulum to mitochondria requires about 3 h.     

Lipid remodeling leads to the introduction and exchange of defined ceramides on GPI proteins in the ER and Golgi of Saccharomyces cerevisiae.

Previous experiments with Saccharomyces cerevisiae had suggested that diacylglycerol-containing glycosylphosphatidylinositols (GPIs) are added to newly synthesized proteins in the endoplasmic reticulum (ER) and that ceramides subsequently are incorporated into GPI proteins by lipid remodeling. Here we prove this hypothesis by labeling yeast cells with [3H]dihydrosphingosine ([3H]DHS) and showing that this tracer is incorporated into many GPI proteins even when protein synthesis and, hence, anchor addition, is blocked by cycloheximide. [3H]DHS incorporation is greatly enhanced if endogenous synthesis of DHS is inhibited by myriocin. Labeled GPI anchors contain three types of ceramides which, based on previous and present results, are identified as DHS-C26:0, phytosphingosine-C26:0 and phytosphingosine-C26:0-OH, the latter being found only on proteins which have reached the Golgi. Lipid remodeling can occur both in the ER and in a later secretory compartment. In addition, ceramide is incorporated into GPI proteins a long time after their initial synthesis by a process in which one ceramide gets replaced by another ceramide. Remodeling outside the ER requires vesicular flow from the ER to the Golgi, possibly to supply the remodeling enzymes with ceramides.     

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

Short-term magnesium deficiency upregulates sphingomyelin synthase and p53 in cardiovascular tissues and cells: relevance to the de novo synthesis of ceramide

The present study tested the hypotheses that 1) short-term dietary deficiency of magnesium (21 days) in rats would result in the upregulation of sphingomyelin synthase (SMS) and p53 in cardiac and vascular (aortic) smooth muscles, 2) low levels of Mg(2+) added to drinking water would either prevent or greatly reduce the upregulation of both SMS and p53, 3) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg(2+) concentration ([Mg(2)](o)) would lead to the de novo synthesis of ceramide, 4) inhibition of either SMS or p53 in primary culture VSMCs exposed to low [Mg(2+)](o) would lead to reductions in the levels of de novo ceramide synthesis, and 5) inhibition of sphingomyelin palmitoyl-CoA transferase (SPT) or ceramide synthase (CS) in primary cultured VSMCs exposed to low [Mg(2+)](o) would lead to a reduction in the levels of de novo ceramide synthesis. The data indicated that short-term magnesium deficiency (10% normal dietary intake) resulted in the upregulation of SMS and p53 in both ventricular and aortic smooth muscles; even very low levels of water-borne Mg(2+) (e.g., 15 mg·l(-1)·day(-1)) either prevented or ameliorated the upregulation in SMS and p53. Our experiments also showed that VSMCs exposed to low [Mg(2+)](o) resulted in the de novo synthesis of ceramide; the lower the [Mg(2+)](o), the greater the synthesis of ceramide. In addition, the data indicated that inhibition of either SMS, p53, SPT, or CS in VSMCs exposed to low [Mg(2+)](o) resulted in marked reductions in the de novo synthesis of ceramide.