Phosphatidylserine Synthesis Research Articles

Alzheimer's Abeta1-40 peptide modulates lipid synthesis in neuronal cultures and intact rat fetal brain under normoxic and oxidative stress conditions.

The effect of amyloid beta (Abeta), the major constituent of the Alzheimer's (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [14C]acetate in the absence or presence of Abeta1-40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by Abeta1-40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5-6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing Abeta1-40 concentration up to 0.1 microg/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. Abeta1-40 peptide (5 microg) was administered intraperitoneally into rat fetuses (18 days gestation) along with [14C]acetate (2 microCi/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. Abeta1-40 peptide increased synthesis of different classes of lipids up to 20-40% in brain tissue compared to controls. Labeling of liver lipids was decreased by Abeta1-40 by 20-30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that Abeta1-40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that Abeta1-40 modulation of lipid metabolism may have Alzheimer's pathological relevance, particularly at high peptide concentrations.

One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity

In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane.

Regulation of Phosphatidylserine Synthesis in Jurkat T Cell Clones: Caffeine Bypasses CD3/TCR-Induced Protein Tyrosine Kinases and Calcium Signals

Phosphatidylserine synthesis as measured by the incorporation of [3H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca2+ from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca2+ content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56lck and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca2+-ATPase of the ER, known for its Ca2+ releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca2+ release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.

Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact.

Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem. 270, 29836-29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.

Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate modulate phosphatidylserine homeostasis in glioma C6 cells.

The effect of sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate on L-[U-14C]serine incorporation into phosphatidylserine and phosphatidylserine-derived phosphatidylethanolamine was investigated in intact glioma C6 cells. Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate are potent signalling molecules which, due to their physicochemical features, may function as amphiphilic compounds. It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) significantly increase [14C]phosphatidylserine synthesis and decrease the amount of 14C-labeled phosphatidylethanolamine. Sphingosine 1-phosphate (an amphiphilic anion) was without effect on phosphatidylserine synthesis but, similarly as sphingosine and sphingosylphosphorylcholine, reduced the conversion of phosphatidylserine to phosphatidylethanolamine. These results strongly suggest that sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate can modulate cellular phospholipid homeostasis by stimulation of phosphatidylserine synthesis and an interference with phosphatidylserine decarboxylase.

Cloning and Expression of a Wheat (Triticum aestivumL.) Phosphatidylserine Synthase cDNA: OVEREXPRESSION IN PLANTS ALTERS THE COMPOSITION OF PHOSPHOLIPIDS

We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices. Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.

Incorporation of extracellular phospholipids and their effect on the growth and lipid metabolism of the Saccharomyces cerevisiae cho1/pss mutant

The cho1/pss mutant of Saccharomyces cerevisiae, which is auxotrophic for choline or ethanolamine because of the deficiency in phosphatidylserine synthesis, grew in the presence of 0.05 mM phosphatidylcholine (PC) with octanoic acids (diC8PC) or decanoic acids (diC10PC), but not in the presence of PC with longer acyl residues. It did not grow in the presence of the soluble hydrolytic products of PC, phosphorylcholine or glycerophosphorylcholine, at comparable concentrations. Addition of 10 mM hemicholinium-3, a choline transport inhibitor, or disruption of the CTR gene, which encodes a choline transporter, inhibited the growth of the cho1/pss mutant in the presence of choline, but not in the presence of 0.1 mM diC8PC. Under diC8PC-supported growth conditions, octanoic acid was barely detectable in the cellular phospholipid fraction, but was recovered in the culture medium as the free acid, and the phosphatidylethanolamine (PE) content was low in comparison to the choline-supported conditions. These results suggest that PCs with short acyl residues were taken up by the cho1/pss mutant and remodeled as they were used, and that PCs with short acyl residues do not inhibit conversion of PE to PC. The current results provide a new direction in the analysis of intracellular phospholipid movement and metabolism in yeast.

CD95 (Fas/APO-1) induces an increased phosphatidylserine synthesis that precedes its externalization during programmed cell death

CD95 (Fas, APO-1)-induced programmed cell death (apoptosis) in T cell lines is accompanied by a rapid flip-flop of phosphatidylserine (PtdSer). Externalization of this phospholipid has been previously recognized as one of the early detectable events of cells undergoing apoptosis. We show here that CD95 induces a rapid (detectable at time <15 min), strong (2.5-fold) but transitory neosynthesis of PtdSer in the Jurkat cell line that precedes its externalization. PtdSer decarboxylation, a mitochondrial specific process, was strongly inhibited by CD95 suggesting that changes in mitochondrial activity take place in the early events of Fas-induced apoptosis and participate in the increased PtdSer synthesis observed. In cells undergoing apoptosis, newly synthesized PtdSer first exposed at the cell surface was in part shed with CD95-induced plasma membrane vesicles, a process that likely explains the transitory effect observed.

Formation of phospholipid nitrogenous bases in euryhaline fish and crustaceans. I. Effects of salinity and temperature on synthesis of phosphatidylserine and its decarboxylation

Contribution of base-exchange of serine with other phospholipids for the synthesis of phosphatidylserine (PS), followed by PS decarboxylation to phosphatidylethanolamine (PE) and N-methylation of PE was studied, in vivo and in vitro, in euryhaline fish and crustaceans. Incorporation of [ 3H]serine and [ 3H]ethanolamine into PS, PE and PC was determined to assess metabolic dependence of these three phospholipids in different conditions of salinity and temperature. Salinity has the stronger effect on the PS decarboxylation and later on the methylation of PE species provided by this pathway in tissues of euryhaline fish and crustacea. The specific changes observed in the metabolism of the molecular species synthesized through these pathways might be one mechanism allowing the rapid adaptation of euryhaline fish and crustaceans to different salinity and/or temperature.

Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.

A Genetic Screen for Aminophospholipid Transport Mutants Identifies the Phosphatidylinositol 4-Kinase, Stt4p, as an Essential Component in Phosphatidylserine Metabolism

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells.

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an approximately 2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

Effect of docosahexaenoic acid on the synthesis of phosphatidylserine in rat brain in microsomes and C6 glioma cells.

Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 +/- 0.1%) when compared with control animals (15.0 +/- 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [3H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10-40 microM) showed a significant increase in the synthesis of [3H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.

Serine Base Exchange Enzyme Activity Is Modulated by Sphingosine and Other Amphiphilic Compounds: Possible Role of Positive Charge in Increasing the Synthesis of Phosphatidylserine

It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) have a stimulatory, and cholesterol 3-sulfate (an amphiphilic anion), an inhibitory, effect on [14C]serine incorporation into phosphatidylserine in glioma C6 and rat liver microsomes. In glioma intact cells sphingosine stimulates phosphatidylserine synthesis in a process independent of protein kinase C, but suppressed by thapsigargin. We suggest that the stimulation of the enzyme occurs by the interaction of amphiphilic cations with the membrane cosubstrate phospholipids, leading to a charge redistribution on their phosphate groups, and hence facilitating the enzyme action. A new hypothesis concerning the mechanism of the serine base exchange reaction is discussed.

An antagonist of ATP-regulated potassium channels, the guanidine derivative U-37883A, stimulates the synthesis of phosphatidylserine in rat liver endoplasmic reticulum membranes

The guanidine derivative U-37883A has been found to stimulate in vitro synthesis of phosphatidylserine in endoplasmic reticulum membranes, catalyzed exclusively by a serine-specific base exchange enzyme. The stimulation of the enzyme activity by the drug was concentration-dependent, with EC 50 of 54 μM, while the biologically inactive analog of U-37883A, U-42069, was without effect. The stimulation caused by U-37883A was enhanced under the conditions when active transport of Ca 2+ into the lumen of microsomal vesicles was induced, whereas it was inhibited by a calcium ionophore, A23187, and by a specific inhibitor of Ca 2+-ATPase, thapsigargin. On the other hand, a potassium ionophore, valinomycin, had no effect on phosphatidylserine synthesis. U-37883A did not affect the K m of the base exchange enzyme for serine, but greatly reduced the EC 50 value of the enzyme for calcium. Furthermore, Ca 2+ uptake by endoplasmic reticulum vesicles has been found to increase in the presence of U-37883A. These observations suggest that U-37883A enhances phosphatidylserine synthesis indirectly by acting on calcium transport, thus affecting calcium concentration within the lumen of endoplasmic reticulum membranes. Alternatively, the effect of the drug could be propagated via the mechanism by which phospholipid flip-flop movement, known to regulate the serine-specific base exchange reaction, is modulated.

Regulation of phospholipid biosynthesis by Ca 2+ -calmodulin-dependent protein kinase inhibitors

Inhibitors of Ca 2+-calmodulin (CaM)-dependent protein kinases strongly modify phospholipid metabolism. Two compounds, KN62 and KT5926 recognized as blockers of Ca 2+ -CaM-dependent protein kinase II, induced a specific increase in phosphatidylserine (PtdSer) synthesis without noticeable changes in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) biosynthesis. The increase of PtdSer synthesis was dependent on the presence of Ca 2+ in the incubation medium and was impaired in cells whose Ca 2+ stores were depleted by pretreatment with CD3 mAb, thapsigargin or EGTA. The mechanism of the stimulation of PtdSer synthesis by these two compounds seems to involve an accumulation of Ca 2+ into the endoplasmic reticulum, possibly due to an increased activity of the endoplasmic reticulum Ca 2+ -ATPase. By contrast, ML-7 and ML-9, two inhibitors of the myosin light chain kinase (MLCK), another Ca 2+-CaM-dependent kinase, were both capable of increasing PtdSer synthesis and decreasing PtdCho and PtdEtn synthesis, reproducing the effect previously described with CaM-antagonists. The increase of PtdSer caused by ML-7 and ML-9 was Ca 2+-dependent while the inhibition of PtdCho and PtdEtn synthesis was not. The use of these four protein kinase inhibitors thus suggests the possible existence of two CaM-dependent pathways that differentially regulates phospholipid metabolism in T cells.

The protein tyrosine kinase p56lck regulates the serine-base exchange activity in Jurkat T cells

Different classes of protein kinase inhibitors for protein kinase C, cAMP-dependent protein kinase or protein tyrosine kinases have been studied for their effect on phospholipid metabolism. The results show that among the compounds studied, only 4′-aminohydroxyflavone (AHF), previously described as a specific inhibitor of the protein tyrosine kinase p56lck, markedly increased phosphatidylserine synthesis in Jurkat T cells. The biosyntheses of phosphatidylcholine and phosphatidylethanolamine were not affected. Also, the synthesis of phospholipids from tritium-labeled fatty acid as precursor was left unchanged by the p56lck inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56lck could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56lck in the regulation of the serine-base exchange enzyme system was obtained by using p56lck-deficient Jurkat cells (J.CaM 1.6) in which the basal base exchange activity was markedly increased and on the other hand AHF had no effect. In addition, transfection of J.Cam 1.6 cells with p56lck-cDNA allowed recovery of the AHF activity.© 1997 Federation of European Biochemical Societies.

Different mechanisms regulate phosphatidylserine synthesis in rat cerebral cortex.

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-gamma-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-gamma-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-gamma-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis in the homogenate. The preincubation of plasma membranes in the buffer without any other addition at 37 degrees C for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-gamma-S but was insensitive to 5 mM AlF-4, a concentration that inhibited by 60% the enzyme assayed without preincubation. These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF-4 was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.

Protein kinase C inhibitors and PAF stimulate phosphatidylserine synthesis in human leucocytes

To study the regulation and turnover of phosphatidylserine (PtdSer) in human leucocytes, we investigated the effect of 12- O-tetradecanoylphorbol 13-acetate (TPA), 1- O-octadecyl-2-O-methyl- rac-glycero-3-phosphocholine (ET-18-OCH3 or edelfosine), staurosporine and platelet activating factor (PAF) on [ 14C]serine incorporation into phospholipids. More than 80% of lipid radioactivity was in PtdSer. ET-18-OCH3 stimulated incorporation into PtdSer 5-fold, without increasing incorporation into other lipids. PAF stimulated PtdSer synthesis 3-fold after 1 h, while staurosporine stimulated the synthesis 2-fold after 3 h. TPA inhibited PtdSer synthesis. It abolished the ET-18-OCH3 stimulation, and reduced the staurosporine stimulation. ET-18-OCH3 and TPA did not significantly alter the incorporation of [ 14C]arachidonic acid into PtdSer, and did not increase PtdSer turnover judged from chase and stability experiments. The results demonstrate that PKC inhibitors and PAF induce increased incorporation of [ 14C]serine into PtdSer, while TPA inhibits stimulated PtdSer synthesis. This suggests that modulation of PtdSer synthesis may regulate PKC activity in PMN cells.

Phosphatidylserine Synthesis in Glioma C6 Cells Is Inhibited by Ca2+Depletion from the Endoplasmic Reticulum: Effects of 2,5-Di-tert-butylhydroquinone and Thimerosal

The effects of 2,5-di-tertbutylhydroquinone (DBHQ) and thimerosal on phosphatidylserine synthesis by the base exchange reaction and on calcium mobilization in intact glioma C6 cells were compared with that of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+-ATPase. It has been found that all these agents inhibit phosphatidylserine synthesis by 70%, but their effectiveness are different. The data show that this inhibition is caused by Ca2+depletion of the endoplasmic reticulum, indicating that phosphatidylserine synthesis requires high concentration of Ca2+within this structure. On this basis and on literature data, a new model for the localization of the serine base exchange enzyme in the endoplasmic reticulum membrane is proposed.