A rapidly metabolizing pool of phosphatidylglycerol as a precursor of phosphatidylethanolamine and diglyceride in Bacillus megaterium.

Pulse-chase experiments in Bacillus megaterium ATCC 14581 with [U-14C]palmitate, L-[U-14C]serine, and [U-14C]glycerol showed that a large pool of phosphatidylglycerol (PG) which exhibited rapid turnover in the phosphate moiety (PGt) underwent very rapid interconversion with the large diglyceride (DG) pool. Kinetics of DG labeling indicated that the fatty acyl and diacylated glycerol moieties of PGt were also utilized as precursors for net DG formation. The [U-14C]glycerol pulse-chase results also confirmed the presence of a second, metabolically stable pool of PG (PGs), which was deduced from [32P]phosphate studies. The other major phospholipid, phosphatidylethanolamine (PE), exhibited pronounced lags relative to PG and DG in 14C-fatty acid, [14C]glycerol, and [32P]phosphate incorporation, but not for incorporation of L-[U-14C]serine into the ethanolamine group of PE or into the serine moiety of the small phosphatidylserine (PS) pool. Furthermore, initial rates of L-[U-14C]serine incorporation into the serine and ethanolamine moieties of PS and PE were unaffected by cerulenin. The results provided compelling in vivo evidence that de novo PGt, PS, and PE synthesis in this organism proceed for the most part sequentially in the order PGt yields PS yields PE rather than via branching pathways from a common intermediate and that the phosphatidyl moiety in PS and PE is derived largely from the corresponding moiety in PGt, whereas the DG pool indirectly provides an additional source for this conversion by way of the facile PGt in equilibrium or formed from DG interconversion.     

Role of Ca2+ in phosphatidylinositol response and arachidonic acid release in formylated tripeptide- or Ca2+ ionophore A23187-stimulated guinea pig neutrophils

The role of Ca2+ in phospholipid metabolism and arachidonic acid release was studied in guinea pig neutrophils. The chemotactic peptide formylmethionyl-leucyl-phenyl-alanine (fMLP) activated [32P]Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) without any effects on the labeling of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). This activation was observed in Ca2+-free medium. Even in the neutrophils severely deprived of Ca2+ with EGTA and Ca2+ ionophore A23187, the stimulated labeling was not inhibited. When [3H]arachidonic acid-labeled neutrophils were stimulated by fMLP, a loss of [3H]arachidonic acid moiety in PI and the resultant increase in [3H]arachidonyl-diacylglycerol (DG), -PA, and free [3H]arachidonic acid was marked within 3 min. With further incubation, a loss of [3H]arachidonic acid in PC and PE became significant. These results suggest the activation of phospholipase C preceded the activation of phospholipase A2. In Ca2+-free medium, the decrease in [3H]arachidonyl-PI and the increase in [3H]arachidonyl-PA were only partially inhibited, although the release of [3H]arachidonic acid and a loss of [3H]arachidonyl-PC and -PE was completely blocked. These results show that PI-specific phospholipase C was not as sensitive to Ca2+ deprivation as arachidonic acid cleaving enzymes, phospholipase A2, and diacylglycerol lipase. Ca2+ ionophore A23187, which is known as an inducer of secretion, also stimulated [32P]Pi incorporation into PI and PA, although the incorporation into other phospholipids, such as PC and PE, was inhibited. This stimulated incorporation seemed to be caused by the activation of de novo synthesis of these lipids, because the incorporation of [3H]glycerol into PA and PI was also markedly stimulated by Ca2+ ionophore. But the chemotactic peptide did not increase the incorporation of [3H]glycerol into any glycerolipids including PI and PA. Thus, it is clear that fMLP mainly activates the pathway, PI leads to DG leads to PA, whereas Ca2+ ionophore activates the de novo synthesis of acidic phospholipids. When [3H]arachidonic acid-labeled neutrophils were treated with Ca2+ ionophore, the enhanced release of arachidonic acid and the accumulation of [3H]arachidonyl-DG, -PA with a concomitant decrease in [3H]arachidonyl-PC, -PE, and -PI were observed. Furthermore, the Ca2+ ionophore stimulated the formation of lysophospholipids, such as LPC, LPE, LPI, and LPA nonspecifically. These data suggest that Ca2+ ionophore releases arachidonic acid, unlike fMLP, directly from PC, PE, and PI, mainly by phospholipase A2. When neutrophils were stimulated by fMLP, the formation of LPC and LPE was observed by incubation for more than 3 min. Because a loss of arachidonic acid from PI occurred rapidly in response to fMLP, it seems likely the activation of PI-specific phospholipase C occurred first and was followed by the activation of phospholipase A2 when neutrophils are activated by fMLP...     

Phospholipid metabolism in human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine. Degranulation is not required for release of arachidonic acid: studies with neutrophils and neutrophil-derived cytoplasts.

Neutrophils respond to chemoattractants by aggregating, degranulating, remodelling of phospholipids and releasing arachidonic acid. To determine whether ligand-induced remodelling of phospholipids depends on redistribution of intracellular organelles (degranulation), we compared phospholipid remodelling of human neutrophils with that of neutrophil-derived cytoplasts. Cytoplasts, organelle-depleted vesicles of cytosol surrounded by plasmalemma, cannot degranulate. Without a stimulus, [3H]arachidonate was incorporated preferentially into phosphatidylinositol (PI) and phosphatidylcholine (PC). Exposure of cytoplasts and neutrophils prelabelled with [3H]arachidonate or [14C]glycerol to fMet-Leu-Phe (10(-7) M) induced rapid changes in distribution of label and mass of individual phospholipids: [3H]arachidonate in phosphatidic acid (PA) increased 500% (120 s), [14C]glycerol incorporation and mass of PA approached 200% of unstimulated values, and [3H]arachidonate in PI decreased continuously; these data are compatible with activity of a PI/PA cycle. However, the mass of PI in both preparations and [14C]glycerol label in intact neutrophils increased initially (5 s), suggesting net synthesis and mobilization of more than one pool of PI. Heterogeneity of PC pools was also observed: [3H]arachidonate was lost from PC immediately upon addition of stimulus, whereas mass and [14C]glycerol values increased. Thus, net phospholipid synthesis, redistribution of arachidonate and activation of the PI/PA cycle are immediate responses of the neutrophil to receptor occupancy by chemoattractants. Furthermore, the similarity in response to fMet-Leu-Phe of neutrophils and granule-free cytoplasts indicates that these processes are independent of degranulation.     

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.     

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

GM_3对小鼠腹腔巨噬细胞磷脂代谢转换率的影响

神经节苷脂ＧＭ_３对小鼠腹腔常驻巨噬细胞（Ｒ－Ｍ）和Ｇｅ－１３２体内激活的巨噬细胞（Ｇｅ－１３２－Ｍ）的磷脂代谢转换有显著的影响,当这两种Ｍ在体外用ＧＭ_３处理时,表现出［￣（３２）Ｐ］Ｐｉ和［￣３Ｈ］肌醇参入ＰＩ降低,参入ＰＩＰ、ＰＩＰ_２增加;但在［￣（３２）Ｐ］Ｐｉ和［￣３Ｈ］胆碱参入ＰＣ上,Ｒ－Ｍ与Ｇｅ－１３２－Ｍ不同,即ＧＭ_３促进同位素前体参入Ｒ－Ｍ的ＰＣ,抑制它们参入Ｇｅ－１３２－Ｍ的ＰＣ．以上结果表明ＧＭ３可能提高了ＰＩ或ＰＩＰ的磷酸激酶的活性,致使［￣（３２）Ｐ］ＰＩＰ和［￣（３２）Ｐ］ＰＩＰ_２增多,［￣（３２）Ｐ］ＰＩ减少．激活的Ｍ（Ｇｅ－１３２－Ｍ）本身ＰＣ代谢转换率较Ｒ－Ｍ高,当Ｇｅ－１３２－Ｍ再受ＧＭ_３刺激,ＰＣ代谢转换率降低,这提示ＧＭ_３对激活的Ｍ的ＰＣ代谢转换有调节作用．     

Differential Incorporation of Precursor Moieties into Cerebral Cortex and Cerebellum Glycerophospholipids During Aging

The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [³H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [³H]choline was inhibited. However, the incorporation of [¹⁴C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([³H]palmitic) and polyunsaturated ([³H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [³H]palmitic, [³H]oleic and [³H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [³H]oleic acid incorporation into neutral lipids (NL) and [³H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.     

Regional Distribution of Phospholipids and Polyphosphatidyl Inositides in the Rabbit's Spinal Cord

The plasticity of the membrane phospholipids in general and stimulated phosphoinositides turnover in particular are the subjects in a variety of neural paradigms studying the molecular mechanisms of neuronal changes under normal and pathological conditions. The regional modifiability of phospholipids (SM, PC, PS, PI, PA + DG, PE), polyphosphatidylinositides (PI, PIP, PIP₂) and diacylglycerol-dependent incorporation of CDP-choline into phosphatidylcholine in the gray matter, white matter, dorsal horns, intermediate zone and ventral horns of the rabbit's spinal cord was studied. We have found 1. a significant increase in the concentration of SM, PC, PS, DG + PA and PE in the white matter in comparison to the gray one, 2. the highest concentration of the outer membrane leaflet-bound phospholipids in the dorsal horns and the inner membrane phospholipids in the intermediate zone in comparison to the gray matter, 3. a substantial amount of labeled polyphosphatidylinositides (poly-PI_s) in the spinal cord white matter with descending order PIP > PI > PIP₂, 4. similar incorporation of myo-2-[³H]inositol into all poly-PI_s in ventral horns and intermediate zone, but a different, lower incorporation into PI and PIP and higher into PIP₂ in the dorsal horns, 5. higher diacylglycerol-dependent incorporation of CDP-choline into PC in the regionally undivided gray matter than in the white matter taken as a whole, 6. the high proportion of diacylglycerol-dependent incorporation of CDP-choline into PC in both the ventral and dorsal horns, whereas that in the intermediate zone remained low.     

Phospholipid Composition and Metabolism of Micrococcus denitrificans

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of ³²P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of ³²P or acetate-1-¹⁴C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.     

PHOSPHOLIPID METABOLISM IN LIGHT AND DARK ADAPTED EXCISED RETINA

Abstract— The phospholipid composition of, and the incorporation of labelled phosphorus into the different phospholipids of rat and calf retina have been studied. The influence of various conditions, such as dark and light adaptation, during the preparation of retina, lipid extraction and incubation of retina with radioactive phosphorus was investigated.
The phospholipid composition of rat retina did not differ significantly from that of calf retina and the different conditions of preparation and incubation did not modify the distributions.
The specific radioactivities of the different phospholipids of calf and rat retina, incubated in the presence of ³²P, distinguished in both species two groups of components characterized by the rate of labelling. Phosphatidic acid (PA) and inositol glycerophospholipids (PI) belonged to the first group and showed the highest uptake of labelled phosphorus; the second group, comprising choline glycerophospholipids (PC), serine glycerophospholipids (PS), sphingomyelin (SP), ethanolamine glycerophospholipids (PE) and cardiolipin (CL) showed low incorporation activities. Only SP was labelled differently in rat and calf retina. With the exception of PS, there was no evidence for the influence of light on the turnover of individual phospholipids. The finding that PS showed higher specific radioactivities when adaptation and incubation proceeded in the dark, seems to be of interest and needs further study.     

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of triacylglycerol in developing rat brain

Metabolism of triacylglycerol (TAG) in developing brain has been examined. TAG is a relatively minor fraction of brain lipid in both suckling and adult rats and cannot be accounted for as entrapped blood. When glycerol tri[1-¹⁴C]oleate and [2-³H]glycerol trioleate were simultaneously injected intracerebrally into suckling rats, both labels appeared in diacylglycerol and the major phospholipids; acyl chain label was incorporated more extensively at early time points, with choline phosphoglycerides being most actively labeled. With [1-¹⁴C]fatty acids and [2-³H] glycerol administration, the specific activity of TAG was much greater than that of the more abundant phospholipids. Although direct acyl exchange between TAG and phospholipids was not demonstrated, relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated.Abbreviations used TAG triacylglycerol - DAG diacylcerol - HPLC high performance liquid chromatography - CoA coenzyme A - BSA bovine serum albumin - TLC thin layer chromatography - DPM disintegrations per minute - ATP adenosine triphosphate - GLC gas liquid chromatography - PC choline, phosphoglyceride - PE ethanolamine phosphoglyceride - PS serine phosphoglyceride - PI inositol phosphoglyceride     

Effects of phospholipids on sphingomyelin hydrolysis induced by intestinal alkaline sphingomyelinase: an in vitro study

Digestion of dietary sphingomyelin (SM) is catalyzed by intestinal alkaline sphingomyelinase (SMase) and may have important implications in colonic tumorigenesis. Previous studies demonstrated that the digestion and absorption of dietary SM was slow and incomplete and that the colon was exposed to SM and its hydrolytic products including ceramide. In the present work, we studied the influences of glycerophospholipids and hydrolytic products of phosphatidylcholine (PC; i.e., lyso-PC, fatty acid, diacylglycerol, and phosphorylcholine) on SM hydrolysis induced by purified rat intestinal alkaline SMase in the presence of 10 mM taurocholate. It was found that various phospholipids including PC, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidic acid (PA) inhibit alkaline SMase activity in a dose-dependent manner, with the degree of inhibition being in the order PA > PS > PI > PC > PE. Similar inhibition was also seen in a buffer of pH 7.4, which is close to the physiologic pH in the middle of the small intestine. When the effects of hydrolytic products of PC were studied, lyso-PC, oleic acid, and 1,2-dioleoyl glycerol also inhibited alkaline SMase activity, whereas phosphorylcholine enhanced SMase activity. However, in the absence of bile salt, acid phospholipids including PA, PS, and PI mildly stimulated alkaline SMase activity whereas PC and PE had no effect. It is concluded that in the presence of bile salts, glycerophospholipids and their hydrolytic products inhibit intestinal alkaline SMase activity. This may contribute to the slow rate of SM digestion in the upper small intestine.     

Metabolism of [14C]arachidonic acid-labeled lipids in quiescent and OAG-stimulated ascites tumor cells.

1. A rapid uptake and esterification of [14C]arachidonic acid during the first 4 hr of cultivation of ascites cells in serum-deprived medium was observed followed by a fast turnover of the fatty acid. 2. Labeling and turnover of esterified arachidonate in individual phospholipid classes was in the order: phosphatidylcholine (PC) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol-4-phosphate (PIP) and -4,5-bisphosphate (PIP2) greater than phosphatidylethanolamine (PE) greater than PE-plasmalogens. 3. In cells stimulated with 1-oleoyl-2-acetyl-sn-glycerol a transient course of arachidonic acid incorporation into PC, PI, PIP and PIP2 was determined peaking 30 min after stimulation, indicating both esterification and release under these conditions. 4. The release of arachidonate was blocked by quinacrine which is a specific inhibitor of phospholipase A2.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.     

The effect of histone deacetylase inhibitor trichostatin A (TSA) on the incorporation of 32P (Pi) and 3H-palmitic acid into the phospholipids of Tetrahymena

Histone deacetylases (HDACs) are able to control also the acetylation of tubulin. In the present experiments the effect of trichostatin A (TSA), a HDAC inhibitor was studied on the incorporation of 3H-palmitic acid and 32P to the phospholipids (PI, PIP, PS, PC, PA, PE) of Tetrahymena pyriformis, considering earlier observations on the microtubular system's influence on signalling in this unicellular eukaryote. Treatment with 1, 5, or 10 microM TSA was studied. The incorporation of hydrophobic tail component, palmitic acid was inhibited in a concentration dependent manner into all the phospholipids, except for PA, where the incorporation was increased. 32P incorporation was also inhibited. The possible relation between the microtubular system and signalling is discussed.     

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.     

Phospholipid Biosynthesis in Synchronous Plasmodium falciparum Cultures1

The metabolism of phospholipids in synchronous Plasmodium falciparum-infected erythrocytes was studied over one cycle of 48 h by the incorporation of labeled palmitate, serine, choline, and myo-inositol into cellular lipids. The rates of incorporation of palmitate and serine into total phospholipids and of choline into phosphatidylcholine (PC) were linear with the maturation of the parasite, increasing by a factor of 2–5.6 according to the precursors. The rate of inositol incorporation into phosphatidylinositol was 9.6 times higher at the schizont stage than at the ring stage, with a marked increase in the second half of the cycle. A significant incorporation of palmitate into triglycerides also occurred during the schizont stage of the parasite. The incorporations of serine and palmitate into phosphatidylethanolamine (PE) and PC showed a net increase at approximately the twentieth hour of the cycle, while the radioactivities recovered in phosphatidylserine (PS) had already reached a maximum by this time. These findings indicate an instantaneous transformation of PS into PE and PC through a decarboxylation of PS into PE, then a methylation of PE into PC during the second half of the cycle. Although PS is a minor component of the Plasmodium parasite, our findings demonstrate the important role of this phospholipid as a precursor of PE and PC, which are major constituents of parasite phospholipids.