Glucose-induced phospholipid hydrolysis in isolated pancreatic islets: quantitative effects on the phospholipid content of arachidonate and other fatty acids

Our recent findings indicate that glucose-induced insulin secretion from isolated pancreatic islets is temporally associated with accumulation of substantial amounts of free arachidonic acid and that arachidonate may serve as a second messenger for intracellular calcium mobilization in islets. In an effort to determine the source of this released arachidonate, the endogenous fatty acid composition of phospholipids from islets has been determined by thin-layer chromatographic separation of the phospholipids, methanolysis to the fatty acid methyl esters, and quantitative gas chromatographic analyses. The relative abundance of phospholipids in islets as judged by their fatty acid content was phosphatidylcholine (PC), 0.63; phosphatidylethanolamine (PE), 0.23; phosphatidylinositol (PI), 0.067; phosphatidylserine (PS), 0.049. Arachidonate constituted 17% of the total islet fatty acid content, and PC contained 43% of total islet arachidonate. Islets incubated with [3H]arachidonate in the presence of 28 mM D-glucose incorporated radiolabel into PC with a considerably higher specific activity than that of PE, PS or PI. The total fatty acid content of PC from islets incubated with 28 mM glucose for 30 min was significantly lower than that of islets incubated with 3 mM glucose, and smaller effects were observed with PE, PS and PI. The molar decrement in PC arachidonate was 3.2 pmol/islet under these conditions, which is sufficient to account for the previously observed accumulation of free arachidonate (2 pmol/islet). A sensitive method involving negative ion-chemical ionization-mass spectrometric analyses of the pentafluorobenzyl esters of fatty acids derived from trace amounts of lysophosphatidylcholine (lyso-PC) was developed, and glucose-stimulation was found to reduce islet lyso-PC content by about 10-fold. These findings indicate that the insulin secretagogue D-glucose induces phospholipid hydrolysis in islets and suggest that PC may be the major source of free arachidonate which accumulates in glucose-stimulated islets.     

Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation.

The objective of the present study was to better understand the remodeling of arachidonic acid (AA) in phospholipids of the mouse bone marrow-derived mast cell (BMMC) during Ag and ionophore A23187 activation. Initial studies were designed to understand the movement of AA in phospholipid classes under resting conditions. BMMC pulse labeled with AA incorporated greater than 95% of the label into the major phospholipid classes. Phosphatidylcholine (PC) subclasses, 1-acyl-2-arachidonoyl-(sn-glycero-3-phosphocholine (GPC)) in particular, initially accounted for most of the label incorporated into the cells with phosphatidylinositol/phosphatidylserine (PI/PS) and phosphatidylethanolamine (PE) subclasses containing much smaller quantities. Prolonged incubation of labeled BMMC resulted in a decrease in the radioactivity in PC with a concomitant increase in PE such that 1-alk-1-enyl-2-arachidonoyl-(sn-glycero-3-phosphoethanolamine (GPE)) became the single largest labeled AA pool by 24 h. Further experiments indicated that 24 h was the time required to reach isotopic equilibrium among AA-containing phospholipids of the BMMC. In the next series of experiments, BMMC phospholipids were labeled to different specific activities by either labeling the cells for 0.5 h or for 24 h followed by stimulation. Under isotopic equilibrium conditions (24 h), stimulation resulted in AA release from PE greater than PC much greater than PI/PS with 1-alk-1-enyl-2-arachidonoyl-GPE providing the bulk of AA released from the BMMC. By contrast, cells labeled for 0.5 h released AA from PC much greater than PI/PS, with 1-acyl-2-arachidonoyl-GPC accounting for most of the AA released from BMMC phospholipids. Label associated with PE subclasses under nonequilibrium conditions remained unchanged or slightly increased throughout a 10-min stimulation period. Finally, BMMC were double labeled with [14C]-AA for 24 h and then with [3H]-AA for 0.5 h. Cell stimulation resulted in a decrease in the [3H]/[14C] ratio in PC and PI and an increase in the ratio in PE. The decrease in [3H]/[14C] ratio in PC was mainly in 1-acyl-2-arachidonoyl-GPC, whereas the increase in PE subclasses was primarily in 1-alk-1-enyl-2-arachidonoyl-GPE. The [3H]/[14C] ratio in cellular neutral lipids and in supernatant fluid products were at values between PC and PE subclasses. Taken together, these data suggest that during Ag activation, the release of free arachidonic acid is from predominantly PE subclasses. Concomitant with the release of AA, there is a rapid remodeling of AA from PC subclasses into PE subclasses (1-alk-1-enyl-2-acyl-GPE).     

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.     

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.     

Selective utilization of omega 6 and omega 3 polyunsaturated fatty acids by human skin fibroblasts

Incorporation of exogenous [14C] arachidonate by human skin fibroblasts was found to be significantly greater than that of either [14C]linoleate or alpha-[14C] linolenate. Arachidonate was preferentially esterified in the PI + PS and PE classes of phospholipids. Over 40% of the incorporated [14C] arachidonate was chain elongated in 24 hours. Cells were also grown in lipid-free medium to enhance PUFA desaturation and elongation and the utilization of various omega 6 and omega 3 metabolites examined. Whereas [14C] linoleate partitioned approximately 50:50 between PL and TAG, eicosatrienoate (20:3 omega 6) was selectively sequestered in TAG. Arachidonate and docosatetraenoate (22:4 omega 6) were preferentially incorporated into phospholipids; the PI + PS fraction was most highly enriched with arachidonate. Modification of alpha-[14C] linolenate was more extensive than that of [14C] linoleate. Docosapentaenoate (22:5 omega 3) was the major omega 3 [14C] PUFA of PI + PS and PE. Eicosapentaeonate was not selectively incorporated into phospholipids; within phospholipids the 20:5 omega 3 was primarily in PC. These results indicate that human skin fibroblasts exhibit acyl specificity in the esterification of polyunsaturated fatty acids, including preferential utilization of arachidonate rather than other prostaglandin precursors in the PI + PS fraction.     

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...     

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.     

Phospholipid biosynthesis in mature human erythrocytes

Mature human erythrocytes were tested for their ability to synthetize membrane phospholipids from simple precursors: [32P]-orthophosphate (32Pi), [U-14C] glycerol, [U-14C] glucose, [U-14C] serine, and [U-14C] choline. The incorporation of these labels into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (lyso-PC), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) was measured. All the phospholipids tested incorporated 32Pi, glycerol, and glucose in a time dependent manner. According to the rate of 32Pi incorporation, three groups of phospholipids could be distinguished: 1) PA, PIP2, PIP, lyso-PC; 2) PI and PS; 3) PC and PE, which incorporated 5 x 10(3), 40, and 6 nmol 32Pi/mmol phospholipid per 1 h, respectively. Moreover, [U-14C] serine and [U14C] choline were found to incorporate into phospholipids, and PS-decarboxylase activity could be measured. The possibility that the observed incorporation was due to contamination with bacteria or other blood cells could be ruled out. Our results bring evidence for de novo phospholipid synthesis of human red blood cells.     

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1-14C]oleate to individual phospholipids of platelets

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-¹⁴C]oleate resulting in esterification of [1-¹⁴C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-¹⁴C]oleate and esterification of [1-¹⁴C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 μM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 μM 2′,5′ dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM chloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.     

Differential effect of progesterone on the labeling of phosphatidylinositol with [3H]inositol and [32P]phosphate in the uterus of the estrogen-treated ovariectomized rat

In rat uterine mince incubated in vitro [3H]inositol was found to be incorporated into phosphatidylinositol (PI) predominantly via a pathway which could be markedly and dose dependently activated with Mn2+ (0.1-10 mM) and inhibited by Ca2+ (1-10 mM). These ions had no effect on the incorporation of [32P]phosphate (32P) into PI indicating a distinct inositol-exchange mechanism for the labeling of PI with [3H]inositol. Treatment of ovariectomized rats for 5 days with 2 micrograms estradiol dipropionate (EDP) increased about 3-fold (when measured in the presence of 1 mM Mn2+) and 4-5-fold (when measured in the presence of 1 mM Ca2+) the inositol-exchange activity in the rat uterus, and these effects were suppressed by 40 and 30% respectively by the concomitant administration of 2 mg progesterone (P). EDP alone or in combination with P increased to the same extent (by a factor of 2-3) the rate of labeling with 32P of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and plasmenylethanolamine (PmE). The labeling rate of PI was increased 1.5-1.7-fold by treatment with EDP and this increase was selectively augmented further to about 2.5-fold by the simultaneous administration of P. Treatment with P alone had no significant effect on the incorporation of either labeled precursor. Steroid hormone treatments had no effect on the amount of these phospholipids in 100 mg uterine tissue, but they increased about 1.7-fold the rate of labeling of ATP with 32P. We conclude that P, when administered together with estradiol, regulates differentially the turnover of the inositol and phosphate moieties of PI with possible physiological consequences.     

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.     

Effect of estradiol and progesterone on the rate of incorporation of radiolabeled arachidonate into phospholipids and triglycerides of the rat uterus

To obtain information on the effect of estradiol (E) and progesterone (P) on the overall rate of the acylation and deacylation reactions with [3H]arachidonate ([3H]AA) in the rat uterus, we investigated the effect of chronic treatment of ovariectomized rats with 2-200 micrograms/day E-dipropionate (EPP) and with 2 mg P alone or with the combination of these steroids on the extent and the time course of the in vitro incorporation of [3H]AA into various phospholipids (PLs) and the triacylglycerol fraction (TG). The results demonstrate that physiologic doses of E leads to a rapid equilibrium of the deacylation-acylation cycle only in the case of phosphatidylinositol (PI) whereas at high (200 micrograms/day) dose level it exerts the same effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as on TG. P alone has no remarkable effects in the ovariectomized rat, but it decreases markedly the incorporation of [3H]AA into PI and TG in intact animals. Furthermore, P decreases the incorporation of [3H]AA into PI in the ovariectomized rat treated with 2 micrograms/day EPP as well as attenuating the enhanced labeling of PC, PE and PI caused by higher doses. Time-course studies provide evidence that all of these effects of progesterone can be accounted for by its ability to decrease the rate of deacylation and, therefore, to prolong the time needed to attain equilibrium in the rates of acylation and deacylation reactions of the various lipids with arachidonate. These data offer a novel outlook on the regulatory role of progesterone and estrogens in uterine function.     

Remodeling of arachidonate-containing phosphoglycerides within the human neutrophil

Initial incorporation and subsequent remodeling of 16 phosphoglyceride molecular species containing arachidonate in the human neutrophil have been studied. Neutrophils were pulse-labeled with [3H]arachidonic acid (AA) for 5 min, then phospholipids were analyzed either at this time point or after a subsequent 120-min incubation. [3H]AA was found to be incorporated into phosphoglycerides phosphatidylinositol (PI) greater than phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) by 5 min. Incorporation of [3H]AA was not related to pool size, but reflected an increase in phosphoglyceride turnover. Following the 120-min incubation, only PE gained a significant amount of labeled arachidonate. Specific activity analysis revealed that PI contained the highest labeled/unlabeled ratio at both 5 min and 120 min. After the initial 5-min pulse, the majority of [3H]arachidonate was incorporated into 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-PC, -PE, and -PI showing no preference for fatty acyl chains at the sn-1 position. However, [3H]AA was remodeled into 1-alkyl-acyl-and 1-alk-1-enyl-acyl-sn-glycero-3-PC and -PE molecular species in those neutrophils incubated for the additional 120 min. Specific activities of [3H]AA within all diacyl molecular species were initially higher relative to those alkyl-acyl and alk-1-enyl-acyl molecular species, but for PC and PE became more uniform as label shifted into ether and plasmalogen pools during the additional 120-min incubation. In contrast, the specific activity of 1-stearoyl-2-arachidonoyl-sn-glycero-3-PI remained constant throughout the 120-min incubation.     

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.     

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.     

Hexadecylphosphocholine inhibits phosphatidylcholine synthesis via both the methylation of phosphatidylethanolamine and CDP-choline pathways in HepG2 cells

We reported in a recent publication that hexadecylphosphocholine (HePC), a lysophospholipid analogue, reduces cell proliferation in HepG2 cells and at the same time inhibits the biosynthesis of phosphatidylcholine (PC) via CDP-choline by acting upon CTP:phosphocholine cytidylyltransferase (CT). We describe here the results of our study into the influence of HePC on other biosynthetic pathways of glycerolipids. HePC clearly decreased the incorporation of the exogenous precursor [1,2,3-3H]glycerol into PC and phosphatidylserine (PS) whilst increasing that of the neutral lipids diacylglycerol (DAG) and triacylglycerol (TAG). Interestingly, the uptake of L-[3-3H]serine into PS and other phospholipids remained unchanged by HePC and neither was the activity of either PS synthase or PS decarboxylase altered, demonstrating that the biosynthesis of PS is unaffected by HePC. We also analyzed the water-soluble intermediates and final product of the CDP-ethanolamine pathway and found that HePC caused an increase in the incorporation of [1,2-14C]ethanolamine into CDP-ethanolamine and phosphatidylethanolamine (PE) and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine cytidylyltransferase activity. Since PE can be methylated to give PC, we studied this process further and observed that HePC decreased the synthesis of PC from PE by inhibiting the PE N-methyltransferase activity. These results constitute the first experimental evidence that the inhibition of the synthesis of PC via CDP-choline by HePC is not counterbalanced by any increase in its formation via methylation. On the contrary, in the presence of HePC both pathways seem to contribute jointly to a decrease in the overall synthesis of PC in HepG2 cells.     

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.     

Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (adseverin) by some phospholipids

A 74-kDa protein (adseverin) derived from adrenal medulla severs actin filaments and nucleates actin polymerization in a Ca2(+)-dependent manner but does not form an EGTA-resistant complex with actin monomers, which is different from the gelsolin-actin interaction. The dissociation of gelsolin-actin complexes by phosphatidylinositol 4,5-bisphosphate (PIP2) and the inhibitory effect on actin filament severing by gelsolin was recently reported. This study shows that the activity of adseverin is inhibited not only by PIP2 but also by some common phospholipids including phosphatidylinositol (PI) and phosphatidylserine (PS). Other phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no effect. The addition of PC or PE to PI diminished the inhibitory effect of PI. Triton X-100 and neomycin were also found effective in suppressing the effect of PI, suggesting that the arrangement of polar head groups is important in exerting the inhibitory effect. Ca2(+)-dependent binding of adseverin to PS liposomes but not to PC or PE liposomes was observed by a centrifugation assay.     

Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.     

Phorbol ester binding to a human lymphoblastoid B-cell line, LA350, stimulates 32P incorporation into selected phospholipids and immunoglobulin secretion

Anti-mu antibody binds to surface IgM on LA350, a transformed human B-cell line, and causes the immediate (5 min) hydrolysis of phosphatidylinositol (PI) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol followed by a subsequent (48-72 hr) increase in immunoglobulin M (IgM) production. Phorbol myristate acetate (PMA) in a dose-dependent fashion inhibited completely the anti-mu-stimulated hydrolysis of PI and its resynthesis (PI cycle) from phosphatidic acid (PA) (P less than 0.001). Phorbol dibutyrate (PD), but not the inactive methyl ester derivative of PMA (PMA-ME), inhibited the anti-mu stimulation of the PI cycle (P less than 0.001). Conversely, PMA and PD, but not PMA-ME, stimulated in a dose-dependent fashion the metabolic events consistent with an activation of a putative phosphatidylcholine (PC) cycle. For example, at 10(-8) M PMA there was a 300% increase in the acute (1 hr) incorporation of [3H]choline into PC (P less than 0.001), a 680% increase in the acute (1 hr) incorporation of 32P into PC (P less than 0.001), but no net synthesis of PC as measured by the lack of PMA-stimulated incorporation of 32P into PC in LA350 prelabeled for 24 hr. Also in cells labeled to equilibrium with [3H]choline and in pulse-chase experiments we established that PMA produces a rapid incorporation of choline phosphate into PC and a rapid breakdown of PC, yielding choline metabolites released as choline itself into external medium surrounding the cell. Binding studies with [3H]PD demonstrated a dissociation constant of 20 mM and 5.3 x 10(5) total binding sites per cell. PMA was as effective as cold PD in inhibiting [3H]PD binding (P less than 0.001), but PMA-ME was ineffective. PMA and PD, but not PMA-ME, produced a similar dose-dependent (maximal at 10(-8) M) increase (300%) in immunoglobulin production as measured by either an ELISA assay or a reverse hemolytic plaque assay (P less than 0.001). Thus, activation of either the PI or the PC cycle results in significant enhancement in immunoglobulin production in LA350. Although PMA turns off the PI cycle, it turns on the PC cycle. A common mechanism to explain these findings might be the activation of protein kinase C, indirect via diacylglycerol release in the PI cycle stimulation by anti-mu and direct in the PC cycle stimulation by PMA by virtue of direct binding to protein kinase C.