Labeling Of Phospholipids Research Articles

Trimetazidine increases phospholipid turnover in ventricular myocyte.

Trimetazidine (TMZ) is an anti-ischemic compound devoid of hemodynamic effects. It was recently suggested to induce cardiomyocyte protection by a mechanism involving lipid metabolism. The effects of TMZ were evaluated in rats on cardiac lipid composition, and in cultured rat cardiomyocytes on phospholipid metabolism. Rats were treated with TMZ for 4 weeks, and the fatty acid compositions were determined. Treatment with TMZ induced a significant decrease in phospholipid linoleic acid, balanced by a small increase in oleic and stearic acids. These changes were not correlated to alterations in plasma fatty acid composition. Cultured ventricular myocytes were treated with TMZ, 16 and 1 before experimentation. The time-dependent incorporation of radio labelled precursors of membrane phospholipids (3-inositol, 14C-ethanolamine, 14C-choline, 14C-arachidonic acid, 10 mumol/L) was investigated. The cells were harvested 30, 60, 105 or 150 min after precursor addition. In TMZ-cells, arachidonic acid (AA) incorporation was increased in the phospholipids, but not in other lipid fractions. This increase elicited a net increase in the total AA uptake. The incorporation of 3-inositol in the phospholipids was strongly stimulated by TMZ, although the uptake of inositol was not altered. The difference was significant within 30 min, and after 150 min the phospholipid labelling in TMZ cells was higher by 70%. A similar result was obtained with ethanolamine as precursor, which turnover increased by 50% in TMZ-treated cells. Conversely, the incorporation of choline was not significantly affected by the presence of TMZ. In conclusion TMZ appears to interfere with the metabolism of phospholipids in cardiac myocytes in a manner which could indicate an increase of membrane phospholipid turnover.

Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling.

We propose the use of membrane splitting by freeze-fracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, freeze-fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unfractured membranes and to release cytoplasm or contents. While the detergent dissolves unfractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested freeze-fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca(2+)-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.

Acylation of endogenous acyl acceptors by mouse sciatic nerve microsomes

Phospholipid (chiefly phosphatidylcholine) labeling from radioactive acyl-CoAs by mouse sciatic nerve microsomes is observed in the absence of added acyl acceptors. The maximal acylation ( ca 10% of administered) for 10 μg microsomal proteins is observed at relatively low amounts of oleoyl-CoA (0.2–0.3 nmol) and decreases as the acyl-CoA amount increases. Labeled lysophosphatidylcholine (almost exclusively esterified at position 2) is also observed, particularly when the [1- 14C]oleoyl-CoA concentration is higher than 0.2–0.3 nmol/50 μl. The labeled acyl group is mainly inserted in position 2 of the glyceroposphorylcholine. With 0.15 nmol labeled oleoyl-CoA, phosphatidylcholine acylation increases as a function of the protein amount and reaches 25% of the added label at 40 μg proteins. It is evaluated that, in the presence of 10 μg proteins, 2% of the microsomal phosphatidylcholine molecules are acylated from 0.1 nmol acyl-CoA. The acylation mechanism seems to involve an acyl exchange between acyl-CoA and phosphatidylcholine.

Immediate early gene c-fos regulates the synthesis of phospholipids but not of gangliosides.

Retinal ganglion cells isolated from chicks that in vivo were exposed to light have a higher phospholipid labeling capacity than those obtained from animals in the dark. Actinomycin D or a mixture of protein synthesis inhibitors or of antisense oligonucleotides to c-fos plus c-jun injected intraocularly 1 hr prior to the stimulation period, abolished the light-dark differences for phospholipids but not for gangliosides. Light stimulation induced the formation (and/or stabilization) of c-fos mRNA and of the protein c-Fos, indicating that immediate early gene induction, and consequently the synthesis of the protein(s) encoded, is essential to increase the synthesis of phospholipids but not of gangliosides. These results suggest a novel mechanism by which immediate early genes engram neural cells, modifying specifically the metabolism of cell constituents producing long-lasting changes in the cells.

Evidence for substrate-cycling of 3-, 3,4-, 4-, and 4,5-phosphorylated phosphatidylinositols in plants.

Short-term 32P labelling and enzymic dissection of inositol phospholipids was used to study the turnover of 3-, 3,4-, 4-, and 4,5-phosphorylated phosphatidylinositols in the plant Spirodela polyrhiza L. Analysis of label in the whole headgroup reveals that phosphatidylinositol 3- and 4-monophosphates (PtdIns3P and PtdIns4P) and phosphatidylinositol 3,4- and 4,5-bisphosphates [PtdIns(3,4)P2 and PtdIns(4,5)P2] all turn over with a half-life of approximately 2-5 h. Analysis of the labelling of individual phosphomonoesters and phosphodiesters of these lipids indicates a rapid equilibration of label between the 4- and 5-monoester phosphates of PtdIns(4,5)P2 within 5 h and largely independent of changes of labelling in the diester. We observed substantially slower equilibration of label (within approximately 27 h) between the monoester and diester of PtdIns4P. These studies therefore indicate that PtdIns4P and PtdIns(4,5)P2 participate in substrate-cycling reactions, evidence for which has been described experimentally only in erythrocytes, and give confirmation in vivo of the previous detection of inositol phospholipid phosphomonoesterase activity. Similar analyses of label in PtdIns3P and PtdIns(3,4)P2 reveal the likely participation of these molecules in substrate cycles and hence for the first time the presence of PtdIns3P 3-phosphatase and PtdIns(3,4)P2 4-phosphatase activities in plants. PtdIns3P and PtdIns(3,4)P2 undergo turnover at rates similar to those of PtdIns4P and PtdIns(4,5)P2. Estimates are made of the relative sizes of the pools of phospholipid participating in the turnover process.

Acute meal-induced changes in hepatic glycerolipid metabolism are unimpaired in severely diabetic rats: implications for the role of insulin

The effect of food intake on the partitioning of diacylglycerol between phospholipid and triacylglycerol synthesis, and on the fractional rate of secretion of triacylglycerol was studied in starved-refed diabetic rats by using the technique of selective labelling of hepatic fatty acids in vivo. Acute and phasic responses in these parameters similar to those observed previously in normal animals were obtained, in spite of the absence of any insulin response to refeeding. Labelling of the major phospholipids (phosphatidylcholine and phosphatidylethanolamine) increased markedly at the expense of triacylglycerol labelling. In addition, the fractional rate of secretion of newly-labelled triacylglycerol was decreased. The data suggest that insulin is not obligatorily involved in any decreases in hepatic triacylglycerol secretion in the prandial period, but that it may act synergistically with other meal-induced signals to mediate this effect in normal animals.

Effects of Ca++ and glycine on lipid breakdown and death of ATP-depleted MDCK cells

The relationships between cytosolic free calcium (Caf), cell associated glycine, phospholipid hydrolysis and cell death were investigated in Madin-Darby canine kidney (MDCK) cells injured by depletion of adenosine triphosphate (ATP). Glucose free incubation for three hours with a mitochondrial uncoupler resulted in progressive loss of glycine from cells. However, they were not lethally injured unless a perturbation of Ca++ homeostasis was also induced. Exposure to a Ca++ ionophore and uncoupler in 1.25 mM Ca++ medium (+Ca) resulted in accelerated cell death. ATP depleted cells with ionophore in 100 nM Ca++ medium (-Ca) were also lethally injured, but after a significant delay. Depletion of glycine preceded death in both groups of cells. Exogenous glycine (5 mM) protected +Ca cells against lethal membrane damage, but the beneficial effects were lost over a period of time. In contrast, -Ca cells were completely protected throughout. Phospholipid mass and radioactive label in lipid fractions of cells prelabeled with 3H-oleic acid were measured. Accelerated death of +Ca cells was accompanied by large decreases of phospholipid mass, loss of phospholipid label, and accumulation of unesterified labeled fatty acid. These changes were greatly decreased by incubation in -Ca medium. On the other hand, protection by glycine could not be attributed to modifications of either the massive breakdown of phospholipids that occurred in +Ca cells, or the modest changes seen in -Ca cells. In +Ca cells, the deleterious effects of increased Caf and phospholipid breakdown ultimately prevailed over protection by the amino acid. Thus, separate pathways of cell death associated with increased Caf and decreased glycine were defined in ATP depleted, Ca(+)- permeabilized MDCK cells. Calcium excess and massive phospholipid loss are features of a damage process that occurs independently of whether cells are protected by glycine or not. Conversely, the glycine sensitive component of injury is expressed regardless of whether intracellular Ca++ is increased, or large phospholipid losses occur. ATP depletion in -Ca medium provides a system to study mechanisms of glycine cytoprotection uncomplicated by Ca++ toxicity.

Quantification in vivo of the effects of different types of dietary fat on the loci of control involved in hepatic triacylglycerol secretion

Polyunsaturated fatty acids (PUFA) have been suggested to exert their hypotriglyceridaemic effect through several possible mechanisms that would be expected to decrease the rate of hepatic very-low-density-lipoprotein-triacylglycerol secretion. We have quantified the role played in vivo by changes in the pattern of partitioning of (i) acyl-CoA between oxidation and esterification, (ii) diacylglycerol between synthesis of triacylglycerol and of the major phospholipids, and (iii) triacylglycerol between secretion and storage within the liver, in response to two dietary levels of n-6 and n-3 PUFA. In order to achieve this we used the technique of selective labelling of hepatic fatty acids in vivo. Compared with a predominantly saturated fatty acid diet, both n-6 and n-3 PUFA intake resulted in a decrease in the proportion of acyl moieties that were secreted by the liver, through an increased diversion of acyl-CoA towards oxidation and a lower fractional rate of secretion of newly synthesized triacylglycerol. In addition, a diet rich in n-3 fatty acids resulted not only in a greater magnitude of these effects but also in a doubling of the partitioning of diacylglycerol towards phospholipid labelling. It is shown that the overall 50% reduction achieved by fish oil feeding in the proportion of acyl groups that were secreted by the liver was distributed over all three branch points. The contribution of each of these adaptations was quantified. The application of such an approach, i.e. the localization and in vivo quantification of the importance of loci of control, in studies on dietary and pharmacological agents that affect lipaemia, is discussed.

Immunogold localization of actin in the testis and exocrine pancreas: spatial relationship with tight junctional strands.

The fracture-label technique was used in conjunction with a monoclonal antibody to actin and the phospholipase A2-colloidal gold (PLA2-CG) method to examine the spatial distribution of actin filaments in relation to the three-dimensional arrangement of tight junctional strands in rat testes and exocrine pancreatic acinar cells. The intimate association of actin filaments with tight junctional strands in the pancreas and testis was also illustrated by a double-labeling experiment in which freeze-fractured pancreas or testis was labeled with monoclonal antibody-protein A-gold (30 nm gold size) followed by incubation with a PLA2-CG complex (11 nm gold size). Freeze-fracture-exposed tight junctional strands in both testicular and exocrine pancreatic cells labeled by PLA2-CG complex, indicated the presence of phospholipids in these cylindrical membranous structures. Immunolabeling of freeze-fractured testes with a monoclonal antibody to actin revealed a narrow band of gold particles juxtaposed to the cytoplasmic aspect of the protoplasmic membrane halves decorated with parallel linear arrays of cylindrical tight junctional strands. Many of the gold particles representing actin antigenic sites were in direct contact with the cross-fractured tight junctional strands. Fracture-label preparations of exocrine pancreas labeled with the monoclonal anti-actin antibody also exhibited a similar labeling pattern at the apex of acinars cells where the tight junction complex is located. Double-labeling experiments revealed the simultaneous labeling of actin and phospholipids in the same fracture-label preparations. Digestion of testicular and pancreatic tissue samples in a free PLA2 solution prior to labeling with the monoclonal antibody or PLA2-CG complex removed not only the gold labeling previously seen over the tight junctional strands but also reduced drastically the immunolabeling for actin that was previously seen associated with the tight junction complex. Taken together, results of the present study showed that actin filaments are structural components of the tight junction strands and are connected to the cytoplasmic aspect of the latter structures. The interaction between this particular cytoskeletal element and the tight junction may be through the binding of a special domain of the actin filament to the phospholipids that partially make up the tight junctional complex.

Alteration of epithelial cell lipid synthesis by n-nitrosonornicotine

Changes in the lipid composition of a cell membrane due to the binding of one cell modulator may affect binding of a second modulator, whether that binding is receptor-mediated (specific) or non-receptor-mediated (nonspecific). Such altered binding interactions have been demonstrated in oral epithelial cells, wherein N-nitrosonornicotine (NNN), a nonspecific ligand, enhances phorbol ester binding. To characterize membrane changes that may be responsible for such an effect, the current study examined lipid changes in hamster oral epithelial (HCP) cells associated with NNN binding. HCP cultures at two cell densities, 5 x 10(6) cells/100 mm plate (subconfluent cultures) or 10 x 10(6) cells/100 mm plate (confluent cultures) were incubated in Keratinocyte-Serum-Free Medium and exposed to 10 microM NNN or DMSO (solvent control) for 48 h. Lipids were labeled with 14C-acetate, then extracted, separated by thin layer chromatography, and the 14C-lipids located by autoradiography and counted. Exposure of subconfluent cultures to NNN for 48 h, with 14C-acetate present during the final 24 h, resulted in altered phospholipid and fatty acid labeling. Phospholipid labeling increased slightly in the presence of NNN compared to controls, while fatty acid labeling showed a modest but significant decrease in the presence of NNN. Similar changes occurred in the confluent cultures. Prelabeling of lipids in subconfluent cultures, followed by exposure to NNN in the absence of radiolabel, resulted in significantly (P < 0.05) greater phospholipid labeling in the presence of NNN compared to control cultures. At the same time, fatty acid labeling decreased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Transbilayer distribution of phosphatidylcholine and phosphatidyl-ethanolam in the vacuolar membrane of Acer pseudoplatanus cells

The distribution of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) among the outer and inner monolayers of the vacuolar membrane of Acer pseudoplatanus was investigated using isolated vacuoles, chemical labelling agents (trinitrobenzenesulfonate and fluorescamine), phospholipase A 2 from bee venom, phospholipase C and phospholipase D. Treatments were performed with intact or sonicated vacuoles. Analysis of the transbilayer distribution of PC and PE in the vacuolar membrane of Acer was limited by phospholipid fractions which were inaccessible to the probes. Lipid-protein interactions and modification of the surface charge and surface pressure in the membrane layers during treatments may obviously exert a strong influence on labelling or hydrolysis of membrane phospholipids. However, simultaneous treatments carried out with phospholipase A 2 and phospholipase C show that PE is approximately 20% more abundant in the outer monolayer than in the inner monolayer and PC is equally distributed between both leaflets of tonoplast. Compared to the phospholipids asymmetrical distribution observed in plasma membrane of erythrocyte, the vacuolar membrane of Acer is not characterized by a marked asymmetrical distribution of its major phospholipids.

A New Fluorescence Method for the Continuous Determination of Surface Lipid Oxidation in Lipoproteins and Plasma

We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu2+ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu2+ us very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation.

Effects of insulin treatment of diabetic rats on hepatic partitioning of fatty acids between oxidation and esterification, phospholipid and acylglycerol synthesis, and on the fractional rate of secretion of triacylglycerol in vivo

1. The hypothesis that insulin treatment of streptozotocin-diabetic rats does not alter acutely the ability of acylcarnitine synthesis to compete successfully for cytosolic long-chain acyl-CoA [Grantham and Zammit (1988) Biochem. J. 249, 409-414], was tested in vivo by using the technique of selective labelling of hepatic fatty acids in awake unrestrained rats. In the same animals, the partitioning of hepatic fatty acids between acylglycerol and phospholipid synthesis, and of newly labelled triacylglycerol between secretion into the plasma and retention in the liver, was also studied. 2. In untreated diabetic animals, the ratio of fatty acid oxidation to esterification was double that found in normal fed animals, whereas there were no differences in the values of the above-mentioned parameters of glycerolipid metabolism. Thus the insulin status of the rats only has chronic effects on specific aspects of fatty acid metabolism in the liver. 3. Treatment of diabetic rats with insulin resulted in no change in the oxidation/esterification ratio for the first 5 h after the start of insulin administration. Thereafter, there were reciprocal changes in the 14CO2 expired (an index of oxidation) and 14C label recovered in hepatic and plasma glycerolipids. However, the pattern of partitioning observed in normal fed rats was still not re-established after 8 h of insulin treatment. 4. There was a small and transient decrease in the fractional rate of triacylglycerol secretion by the liver at the start of insulin treatment and an increase in the proportion of labelled fatty acid that was utilized for phospholipid synthesis such that phospholipid labelling as a proportion of that of total glycerolipids was doubled after 8 h of insulin treatment. 5. The data are discussed in relation to the roles of insulin in mediating acute changes in hepatic fatty acid metabolism and very-low-density-lipoprotein triacylglycerol secretion by the liver.

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene.

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-14C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2-24 h) and concentration (0-120 microM). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6-8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6 > 20:3n-6 > 18:2n-6 = 18:3n-6. Throughout the incubation (2-24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases.

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.

Clofibrate and other peroxisomal proliferating agents relatively specifically inhibit synthesis of ethanolamine phosphoglycerides in cultured human fibroblasts

Effects of several classes of peroxisomal proliferators on peroxisomal functions, hepatomegaly, hepatocarcinogenesis and lipid metabolism have been extensively investigated in rodents. Less is known about influences of these agents, some used as hypolipidemic drugs, on various metabolic parameters in humans. We examined effects of clofibrate, di(2-ethyl-hexyl)phthalate (DEHP) and pirinixic acid (WY-14,643) on phospholipid metabolism in human fibroblasts in culture. Clofibrate inhibited incorporation of [1- 14C]chexadecanol and clinolenic acid into ethanolamine phosphoglycerides in a time- and concentration-dependent manner; labeling of plasmalogens and non-plasmalogen ethanolamine phosphoglycerides was reduced by 40–80% compared to a generalized 10–30% inhibition of labeling of other phospholipids, including phosphatidylcholine. In pulse and pulse-chase experiments, selective inhibition of incorporation of [1,2- 14C]ethanolamine, compared to [methyl- 3H]choline, confirmed relative specificity of inhibition of ethanolamine phosphoglycerides. Similar concentration dependence and specificity for inhibition of phospholipid turnover was observed for DEHP and WY-14,643, in both control and mutant (Zeilweger and adrenoleukodystrophy) fibroblasts, in the absence of major effects on peroxisomal markers. These observations that peroxisomal proliferators specifically inhibit ethanolamine phosphoglyceride turnover in human fibroblasts should be considered when assessing the efficacy and safety of such agents as hypolipidemic drugs or when evaluating mechanisms of proliferator action at the cellular level.

Enhancement of gastric mucus phospholipid secretion by an antiulcer agent, ebrotidine

1. Rat gastric mucosal cells, subjected to phospholipid labeling by incubating the cell suspension in DMEM with [ 3H]choline, were exposed to different concentrations (0-150 μM) of H 2-receptor antagonists, ebrotidine and ranitidine, and the phospholipid secretory responses were evaluated. 2. In the absence of the drugs, the secretion of choline-containing phospholipids over a 1 hr period averaged 3.97% of the total cellular labeled phospholipids. Ebrotidine caused a dose-dependent increase in the rate of phospholipid secretion which was most pronounced at 1 hr and persisted for at least 2 hr. The maximal effect was attained at 120 μM ebrotidine giving a 36% increase in phospholipid secretion. 3. The phospholipid secretory response to ebrotidine was accompanied by an increase in gastric mucosal cell cAMP level which reached a maximum value of 2.1-fold over that of controls at 1 hr. Ranitidine, in contrast, neither evoked increase in cAMP level nor caused any stimulation in phospholipid secretion. 4. The results indicate that the gastroprotective properties of ebrotidine are associated with the ability of the drug to elicit a rapid stimulation in gastric mucus phospholipid secretion, and that ranitidine does not possess such property.

Effects of sodium butyrate on the transfer of arachidonic acid to phosphatidylcholine in a clonal oligodendrocyte cell line (CB-II).

The effect of sodium butyrate on membrane phospholipid metabolism in a neonate rat cerebellum derived clonal oligodendrocyte cell line (CB-II) was investigated. Sodium butyrate is an agent known to induce cell differentiation and morphological transformations. A comparison of the in vivo phospholipid labeling patterns obtained by incubating CB-II cells with [3H]choline, [14C]myristic acid or [3H]arachidonic acid indicated that butyrate altered the route of acylation-deacylation in phosphatidylcholine (PC) biosynthesis. Using an in vitro incubation system containing homogenates of CB-II cells, the largest proportion of radioactivity was found in PC, and addition of sodium butyrate resulted in a further increase in the transfer of arachidonic acid to PC, but not to phosphatidylinositol. Similar results were obtained when this in vitro acylation activity was tested using homogenates from sodium butyrate pretreated cells. The butyrate effect was observed regardless of whether or not exogenous lysophosphatidylcholine (LPC) was added to the incubation system. Addition of butyrate did not result in a change in the activity of LPC:acyl-CoA (coenzyme A) acyltransferase (EC 2.3.1.23) in CB-II cells upon incubating cell homogenates with [1-14C]arachidonoyl-CoA and LPC. However, when cell homogenates were incubated with [3H]arachidonic acid in the presence of 2.5-10 mM sodium butyrate, arachidonoyl-CoA synthesis was stimulated. A time course study demonstrated that significant stimulation occurred after three minutes. Taken together, the results suggest that in CB-II cells, sodium butyrate stimulates the transfer of arachidonic acid into PC and that this effect is at least partially due to a stimulation of arachidonoyl-CoA ligase (EC 6.2.1.3).

Synthesis of Trilaurin by Developing Pisa Seeds (Actinodaphne hookeri)

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C16:0, C18:0, C18:2, and C18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% FL. At this stage the TAG contained exclusively C12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C12:0. Experiments on [1-14C]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in FL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into FL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into FL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering, Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C12:0 and 61% of C14:0, but not C16:0, C18:2, and C18:3, into TAG. In contrast the LCFA were incorporated preferentially into FL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering.

ChemInform Abstract: Coupling and Labeling of Phospholipids

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