Choline Phosphate Research Articles

Lipid Composition and Structural Studies on Lipids from the Land Snail Eobania vermiculata

The total lipids of the commercial land snail Eobania vermiculata (Gastropoda, Pulm onata, Stylom m atophora) are found to constitute a small percentage (0.8% ) of the wet tissue, which is comparable to that reported for other gastropods. Polar lipid components comprise 61.4% of the total lipids. The individual lipid classes obtained by column chromatographic fractionation were purified by preparative TLC or by column chromatography and their structure was confirmed by a combination of chromatographic and analytical determinations before and after mild alkaline hydrolysis and/or (dry) acid methanolysis and by IR analysis. Neutral lipids represent 36.4% of total lipids, containing cholesterol, cholesterol esters and triglycerides as their major components (26.2% , 29.1% and 25.5% respectively). They contain also a significant amount (14%) of free glyceryl ethers, which are found in a mollusc for the first time. The overall composition of the polar lipids (mol/100 mol lipid-P) was found as follows: Cardiolipin, 2.9; phosphatidylethanolamine, 24.9 (of which 19.8% plasmalogen analog); phosphatidylcholine, 49.2 (of which 45.6% glycerylether analog); ceramide aminoethylphosphonate, 7.5 plus 0.01 (another three minor species); diglyceride-am noethylphosphonate, 6.3; Sphingoethanolamine 1.65 (for the first time found and structurally studied in a land gastropod); and phosphatidic acid 1.1. Unsaturated fatty acyl groups represent about 72.6 and 44.1 respectively in phosphatidylethanolamine and phosphatidylcholine. A significant amount (70.5% ) of unsaturated fatty acids is concentrated in neutral lipids. The C16:0 alk-1-enyl chain was found to predominate (55.6% ) in the side chains of ethanolamine plasmalogen. Batyl alcohol was found as the main glycerylether bound to choline phosphate (97.5% ). Saturated fatty acyl groups with 16 carbon atoms were main components (54%) of the major ceramide aminoethylphosphonate species.

Stimulation of phosphorylcholine turnover and diacylglycerol production in human polymorphonuclear leukocytes. Novel assay for phosphorylcholine.

Receptor-bypassing stimulants of human polymorphonuclear leukocytes (PMNLs), such as ionomycin or phorbol 12-myristate 13-acetate (PMA), generate an increase in diacylglycerol (DAG) which is independent of a phospholipase C specific for phosphatidylinositol 4,5,-bisphosphate (PIP2). Activation of a phospholipase C specific for phosphatidylcholine (PC) has been implicated as a source of DAG in other cells by measuring the release of radiolabelled phosphorylcholine. However, since PMNLs could not be labelled sufficiently with [3H]choline, we developed an h.p.l.c. assay to quantify mass levels of phosphorylcholine after enzymic conversion to [32P]CDP-choline with CTP-phosphorylcholine (choline phosphate) cytidylyltransferase (EC 2.7.7.15). This assay was linear to at least 20 nmol, and was sensitive to 10 pmol of phosphorylcholine. Baseline phosphorylcholine levels in unstimulated PMNLs were 2300 +/- 510 pmol/10(7) cells and were decreased by pretreatment with PMA (166 nM) or ionomycin (1 microM) for 10 min by 360 +/- 130 and 600 +/- 290 pmol/10(7) cells respectively (P less than 0.05). In contrast, baseline DAG levels were 147.6 +/- 11.7 pmol/10(7) cells in unstimulated PMNLs, and were increased by PMA or ionomycin by 1320 +/- 222 and 1891 +/- 264 pmol/10(7) cells respectively (P less than 0.05). Similarly, the chemoattractant fMet-Leu-Phe raised DAG levels by 731 +/- 111 pmol/10(7) cells and decreased phosphorylcholine levels by 180 +/- 60 pmol/10(7) cells. Activation of PMNLs by PMA, ionophore or fMet-Leu-Phe thus leads to the sustained production of DAG accompanied by the disappearance of phosphorylcholine. This suggests that these stimulants enhance PC turnover via a hydrolytic mechanism which is independent of phospholipase C, with activation of a PC-specific phospholipase D being a plausible mechanism.

Activation of Phospholipase D by the Fucose-Sulfate Glycoconjugate That Induces An Acrosome Reaction in Spermatozoa

We report for the first time that phospholipase D activity in sea urchin spermatozoa can be regulated by a component of egg jelly known to induce an acrosome reaction. The fucose-sulfate glycoconjugate (FSG) of egg jelly that induces an acrosome reaction in spermatozoa caused Ca2+-dependent increases in 1,2-diacylglycerol and phosphatidic acid. Diacylglycerol concentrations were increased 2-fold, and phosphatidic acid concentrations were elevated up to 10-fold 2 min after the addition of FSG to spermatozoa. FSG also caused increases in choline, but not in choline phosphate concentrations. Neither phosphorylation of diacylglycerol nor de novo synthesis from glycerol were significant routes of synthesis of phosphatidic acid during the acrosome reaction. When spermatozoa were incubated with FSG in the presence of ethanol, phosphatidylethanol was produced. As ethanol concentrations in the extracellular medium were increased from 0.1 to 2.5%, the amount of phosphatidylethanol increased, whereas phosphatidic acid concentrations decreased, suggesting a competitive transphosphatidylation reaction catalyzed by phospholipase D. Furthermore, when a phosphatidylcholine pool in spermatozoa was radiolabeled using [3H]1-O-alkyl-2-lyso-glycerol-3-phosphorylcholine, the subsequent addition of FSG caused a 4-fold accumulation of [3H]phosphatidic acid. FSG-induced elevations in [3H]phosphatidic acid were positively correlated with the percent of cells that had undergone an acrosome reaction.

DISTRIBUTION AND MOBILITY OF PHOSPHORUS IN GRASSLAND AND FOREST SOILS OF SASKATCHEWAN

Soil phosphorus transformations and losses were measured in three profiles representative of an environmental gradient covering a grassland to forest transition: a Calcareous Brown, an Orthic Black and an Orthic Gray Luvisol. A sequential extraction procedure was used to measure the different forms of both inorganic (Pi) and organic (Po) phosphorus present in soil horizons. The mobilities of phosphate anions and of some selected (Po) compounds (glucose 6-phosphate, choline phosphate and adenosine triphosphate) were estimated through isotopic dilution kinetic experiments, and indicated that all the tested Po compounds had a higher mobility than phosphate anions in B horizons; also, Po was predominant over Pi in the water extracts from these soils. A mass-balance approach (Pedogenic Index) which incorporates depth-bulk density parameters in measuring the composition of soil horizons was used to calculate losses of Pt and apatite-P (HCl-Pi). Both grassland soils behaved similarly; they lost 500–550 kg P ha−1 (20%) of their original Pt and 30% of the initial HCl-Pi fraction was either transformed to various other forms of P or lost from the solum. Losses of Pt (3020 kg P ha−1: 41 %)and of HCl-Pi (70%) were much higher in the Gray Luvisol. Since losses of Pt could not be due to leaching of phosphate anions through the profile, the importance of Po in P leaching should be considered along with surface runoff in determining the fate of P in these soils. Key words: Pedogenic index, P mass balance, P fractionation, organic P leaching, P mobility

Ｃ‐反応性蛋白（ＣＲＰ）のリガンド結合性のクロマトグラフィー的検討

Human C-reactive protein (CRP), a typical acute-phase protein, increases about hundreds-fold in concentration in response to infection, inflammation or tissue injury.CRP was first described by Tillet and Francis in 1930 as a serum protein capable of precipitating pneumococcal C-polysaccharide where phosphorylcholine (PC) was identified later to be a ligand. Clinical and biological aspects, especially immunological ones, of human CRP have been studied. And some of them have been ascribed to its capacity of ligand-binding with PC calcium-dependently. While type 4 pneumococcal polysaccharide which lacks phosphate and choline has been pointed out to be able to bind human CRP in calcium-dependent manner, ligands other than PC should be considered besides PC.We find specific affinity chromatography bearing new ligand of negatively-charged aminocaproic acid which binds human CRP calcium-dependently (buffer ; 0.15 M NaCl, 0.02 M Tris, 0.01 M CaCl2, 0.01 M NaN3, pH 7.8, room temp.), being almost comparable to PC-affinity chromatography. In addition, another specific ligand of positively-charged aminohexyl-agarose is found to bind human CRP calcium-independently (buffer; 0.09 M NaCl, 0.01 M Tris, pH 7.4, room temp.) . These new ligands may provide simple model to understand human CRP.1) Tillet, W.S., Francis, T. Jr.: Serological reactions in pneumonia with a non-protein somatic fraction of pneumococcus. J. Exp. Med. 52: 561-571, 1930.2) Volanakis, J.E., Kaplan, M.H.: Specificity of C-reactive protein for choline phosphate residues of pneumococcal C-polysaccharide. Proc. Soc. Exp. Biol. Med. 136: 612-614, 1971.3) Kaplan, M.H., Volanakis, J.E.: Interaction of C-reactive protein complexes with the complement system. I. Consumption of human complement associated with the reaction of C-reactive protein with pneumococcal C-polysaccharide and with the choline phosphatides, lecithin and sphingomyelin. J. Immunol. 112: 2135-2147, 1974.4) Heidelberger, M., Gotschlich, E. C., Higginbotham, J.D.: Inhibition experiments with pneumococcal C and depyruvylated type-IV polysaccharides. Carbohyd. Res. 22: 1-4, 1972.5) Richards, R.L., Gewurz, H., Osmand, A.P., Alving, C.R.: Interactions of C-reactive protein and complement with liposomes. Proc. Natl. Acad. Sci. USA 74: 5672-5676, 1977.6) Volanakis, J.E., Wirtz, K.W.A.: Interaction of C-reactive protein with artificial phosphatidylcholine bilayers. Nature 281: 155-157, 1979.7) Narkates, A.J., Volanakis, J.E.: C-reactive protein binding specificities : Artificial and natural phospholipid bilayers. Ann. N.Y. Acad. Sci. 389: 172-182, 1982.8) Kushner, I., Kaplan, M.H.: Studies of acute phase protein. I. An immunohistochemical method for the localization of Cx-reactive protein in rabbits. Association with necrosis in local inflammatory lesions. J. Exp. Med. 114: 961-973, 1961.9) Kushner, I., Rakita, L., Kaplan, M.H.: Studies of acute-phase protein. II. Localization of Cx-reactive protein in heart in induced myocardial infarction in rabbits. J. Clin. Invest. 42: 286-292, 1963.10) Shephard, E.G., Van Helden, PD., Strauss, M., Bohm, L., De Beer, F.C.: Functional effects of CRP binding to nuclei. Immunol. 58: 489-494, 1986.11) Whisler, R.L., Newhouse, Y.G., Mortensen, R.F.: C-reactive protein reduces the promotion of human B-cell colony formation by autoreactive T4 cells and T-cell proliferation during the autologous mixed-lymphocyte reaction. Cell Immunol. 102: 287-298, 1986.12) Kilpatrick, J.M., Virella, G.: Inhibition of platelet-activating factor by rabbit C-reactive protein. Clin. Immunol. Immunophathol. 37: 276-281, 1985.13) Tatsumi, N., Hashimoto, K., Okuda, K., Kyougoku, T.: Neutrophil chemiluminescence induced by platelet activating factor and suppressed by C-reactive protein. Clin. Chim. Acta 172: 85-92, 1988.

Regional and Developmental Estimations of Glycerophosphorylcholine Phosphodiesterase Activities in Rat Brain

The activity of glycerophosphorylcholine phosphodiesterases was determined in the mesencephalon, diencephalon, cerebral hemispheres, cerebellum and olfactory bulb during postnatal development from P5 to P70 of rat brain. These activities are low and gradually increase to near adult levels by the end of the first postnatal month similar to that for CNPase activity. This is in accord with glycerophosphorylcholine choline phosphate phosphodiesterase being present in the myelin membrane.

Phosphatidylcholine biosynthesis in castor bean endosperm. Mechanisms of regulation during the immediate postgermination period

Changes in phosphatidylcholine content, lipid precursor incorporation and lipid biosynthetic enzyme activities were observed in castor bean endosperms during a six day postgermination period. The metabolism of phosphatidylcholine was regulated independently of other phospholipids, as evidenced by the ratio of phosphatidylcholine to total phospholipid not being constant during development. The main pathway for phosphatidylcholine biosynthesis was the nucleotide pathway, although studies with labelled glycerol-3-phosphate suggested that the diacylglycerol for this pathway was not synthesized de novo, but probably obtained by degradation of pre-existing lipids. The control of the nucleotide pathway appears to reside in choline phosphate cytidylyltransferase and choline phosphotransferase, the activities of which were coordinated, but not in choline kinase. The cytidylyltransferase is regulated in part by translocation of the soluble form of the enzyme to the endoplasmic reticulum, but this regulation could not account for all of the observed changes in phosphatidylcholine synthesis.

Kinetic and biochemical properties of CTP:choline-phosphate cytidylyltransferase from the rat brain.

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15), the rate-limiting enzyme of the two-step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis. CTP:choline-phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7. Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.

Fatty acids stimulate phosphatidylcholine synthesis and CTP:choline-phosphate cytidylyltransferase in type II pneumocytes isolated from adult rat lung.

The regulation by cyclic AMP and fatty acids of phosphatidylcholine (PC) synthesis in rat alveolar type II cells was studied. In contrast with results with hepatocytes, cyclic AMP and its potent chlorophenylthio analogue had no inhibitory effect on [Me-14C]choline incorporation into PC in pulse-chase studies with alveolar type II cells. The inclusion of the fatty acids palmitate, oleate or linoleate in the chase incubation medium stimulated the incorporation of [Me-14C]choline into PC by type II cells. The effect of palmitate, which was more pronounced than that of the other fatty acids, appeared to be concentration-dependent. Increased [Me-14C]choline incorporation into PC was paralleled by an accelerated disappearance of the radiolabel from choline phosphate, which is consistent with an activation of CTP:choline-phosphate cytidylyltransferase. This enzyme is considered to be rate-limiting in the synthesis of PC de novo by type II cells. As fatty acids are also substrate for PC synthesis, their effect could also be due to compensation for a fatty acid deficiency. To test this possibility, fatty acid synthesis in the type II cells was stimulated by addition of lactate. Even then, an additional stimulation of PC synthesis by palmitate was observed, which supports the regulatory influence of exogenous fatty acids. Incubation of type II cells in the presence of 0.2 mM-palmitate resulted in a 45% increase in the membrane-bound CTP:choline-phosphate cytidylyltransferase activity, whereas the soluble activity remained unchanged. Choline kinase activity in the soluble fraction increased by 48%. However, the increase in choline kinase is unlikely to be responsible for the increased metabolic flux through the choline phosphate pathway, because there is a relatively large pool of choline phosphate in type II cells. Therefore it is suggested that the microsomal CTP:choline-phosphate cytidylyltransferase is the form of this enzyme which is active in surfactant PC synthesis, and possibly has a regulatory role in this pathway.

Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism.

A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

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

Anti-μ 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 (IP 3) 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-μ-stimulated hydrolysis of PI and its resynthesis (PI cycle) from phosphatidic acid (PA) ( P < 0.001). Phorbol dibutyrate (PD), but not the inactive methyl ester derivative of PMA (PMA-ME), inhibited the anti-μ stimulation of the PI cycle ( P < 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 < 0.001), a 680% increase in the acute (1 hr) incorporation of 32P into PC ( P < 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 pulsechase 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 m M and 5.3 × 10 5 total binding sites per cell. PMA was as effective as cold PD in inhibiting [ 3H]PD binding ( P < 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 < 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-μ and direct in the PC cycle stimulation by PMA by virtue of direct binding to protein kinase C.

Choline-phosphate cytidyltransferase activity and phosphatidylcholine synthesis in rat granular pneumocytes are increased with exogenous fatty acids

We investigated the effect of exogenous fatty acids on phosphatidylcholine (PC) and disaturated phosphati-dylcholine (DSPC) synthesis by rat granular pneumocytes in primary culture. Synthesis of PC and DSPC from [ 3- methyl]choline, as evaluated by increasing specific activity (pmol choline incorporated /μ g phosphorus), was linear for 3 h. Exogenous palmitic, oleic, linoleic, or linolenic acid (100 μM each) increased the synthesis of PC by approx. 50% during incubation for 3 h. In contrast, synthesis of DSPC was increased only by palmitic acid. The increase in DSPC synthesis was approx. 150% after 3 h. Conversion of choline phosphate to PC was increased in the presence of palmitic or oleic acid as indicated by pulse-chase studies with [ 3H- methylcholine in the intact cells. Cells incubated for 3 h with either oleic or palmitic acid showed increased choline-phosphate cytidyltransferase activity in the cells and the microsomal fraction. In addition, oleic acid increased the activity of this enzyme in the cytosolic fraction. The distribution of this enzyme in cytosolic and microsomal fraction was 24 and 76% in the cells incubated with palmitic acid and 32 and 68% in control cells. These results suggest that exogenous fatty acids stimulate the de novo pathway of PC synthesis in granular pneumocytes by increasing the microsomal choline-phosphate cytidyltransferase activity.

Synthesis of phosphatidylcholines in ozone-exposed alveolar type II cells isolated from adult rat lung: is glycerolphosphate acyltransferase a rate-limiting enzyme?

Type II cells were exposed to ozone by gas diffusion through the thin Teflon bottom of culture dishes. The rate of phosphatidylcholine synthesis by type II cells, monitored by the incorporation of [Me-14C]choline, was impaired by ozone at concentrations that did not affect other cellular parameters. The enzymes choline kinase and cholinephosphate cytidylyltransferase were not susceptible to inactivation by ozone at concentrations at which the activity of glycerolphosphate acyltransferase was decreased. The enzyme activity of lactate dehydrogenase increased after ozone exposure. The specific activity of choline kinase in the cytosolic fraction of type II cells was fivefold that in whole lung. The metabolism of [Me-14C]choline was studied as a function of the choline concentration. Maximal rates of phosphatidylcholine synthesis were already attained at a concentration of 20 microM choline. Exposure of type II cells to ozone did not affect the recovery of label from [Me-14C]choline in choline phosphate and CDP choline. However, the maximal rate of phosphatidylcholine synthesis decreased after ozone exposure, which indicates that the decreased apparent activity of glycerolphosphate acyltransferase limits the supply of diacylglycerols and thereby the rate of phosphatidylcholine synthesis. If the flux through the diacylglycerol pathway was stimulated by the addition of palmitic acid, a higher maximal rate of phosphatidylcholine synthesis was observed. The uptake of [Me-14C]choline and the recovery of label in CDPcholine were not altered by the addition of different concentrations of palmitate. It is concluded that type II cells take up choline very efficiently, probably due to the high specific activity of choline kinase. At low choline concentrations the rate of phosphatidylcholine synthesis is determined by the supply of CDPcholine. At concentrations of choline in the upper physiological range, the rate of phosphatidylcholine synthesis is determined by the availability of diacylglycerols, which in turn is limited by the apparent activity of glycerolphosphate acyltransferase.

Phosphatidylcholine synthesis in castor bean endosperm: Characteristics and reversibility of the choline kinase reaction

Choline kinase (EC 2.7.1.32) was measured in concentrated 100,000 g av supernatants from castor bean endosperm ( Ricinus communis L. var. Hale). Initial velocity analysis, along with competitive inhibitor (hemicholinium-3) and product inhibition (ADPMg 2+) studies suggested that the forward reaction followed a sequentially ordered mechanism with ATPMg 2+ binding to the enzyme first, followed by choline and then activation of the ternary complex by free Mg 2+. The kinetic constants of the forward reaction are reported. A reverse reaction was measured which had a pH optimum of 6.5 and produced 1 mol of ATP for every mole of choline phosphate. The estimated maximum possible K eq at 7.25 was 5 × 10 −3 which suggested that this reaction is highly reversible in this tissue. The possible physiological significance of this is discussed.

Uptake and degradation of natural surfactant by isolated rat granular pneumocytes.

It has been previously shown that isolated granular pneumocytes internalize and degrade dipalmitoylphosphatidylcholine (DPPC) in synthetic lipid vesicles and reutilize degradation products for phosphatidylcholine (PC) synthesis. In this study, we evaluated the uptake and degradation of radiolabeled natural surfactant (NS) isolated from lung lavage after perfusing isolated rat lungs with [3H]choline. Uptake of NS by isolated granular pneumocytes was increased approximately fourfold compared with synthetic liposomes, suggesting that physical form or a component (e.g., a protein) of NS plays a role in phospholipid uptake by these cells. Uptake was significantly decreased by metabolic inhibitors, indicating an energy requirement for this process. After 2-h incubation, the pattern of radioactivity in cells compared with NS showed a significant decrease in PC and DSPC and increase in free choline, choline phosphate, and CDP-choline. This pattern of metabolism indicates degradation of PC and metabolic reutilization of products. These studies support the hypothesis that alveolar phospholipids are accumulated and reutilized by granular pneumocytes for surfactant synthesis.

Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates.

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 degrees C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP X G1P and G6P were synthesized from glucose and inorganic phosphate at 70 degrees C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.

Developmental aspects of renal beta-amino acid transport. VI. The role of membrane fluidity and phospholipid composition in the renal adaptive response in nursing animals.

Accumulation of the beta-amino acid taurine is higher in adult rat renal brush border membrane vesicles than in nursing animals, which relates to a higher initial rate Vmax. A low sulfur amino acid diet increases and a 3% taurine diet reduces the Vmax of Na+-taurine cotransport in brush border membrane vesicles at all ages after 7 days as compared to values on a normal diet. To determine if changes in membrane fluidity account for these developmental and adaptive events, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was used to measure fluorescence anisotropy. A two-component, single break curve fit the data over the range 4 to 56 degrees C. Values for the break temperature centered around 23 degrees C, not different than a break temperature determined in adult membranes. The values for membrane polarization range from 0.311 to 0.329 in membranes from 7-, 14-, and 21-day-old pups exposed to each of the three diets, significantly lower than the value in adult membranes (p less than 0.02). The slopes of each component, equivalent to the apparent energy of activation, did not differ in relation to diet. The ontogenic changes in taurine uptake by brush border membrane vesicles related to the exposure to different dietary sulfur amino acid levels are not related to changes in membrane fluidity using 1,6-diphenyl-1,3,5-hexatriene as a probe. However, there is a decrease in fluidity with age. Total phospholipid content falls postweaning, and the percent of total content of phosphatidyl choline and glycerol phosphate fall, and phosphatidyl serine and ethanolamine rise as the rat ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the phorbolester TPA and of the ionophore A 23187 on phospholipase A2 and C activities in the mouse epidermal cell line HEL-30

The 12-O-tetradecanoylphorbol-13-acetate (TPA)- or ionophore A 23187-induced release of 14C-arachidonic acid from prelabeled murine HEL-30 keratinocytes was studied in vitro. Starting 8 min after drug treatment, a linear increase in the arachidonic acid content in the extracellular medium was observed with a concomitant loss of label in cellular phosphatidylcholine and phosphatidylinositol, but not in phosphatidylethanolamine and phosphatidylserine. No increase in intracellular diacylglycerol and phosphatidic acid was observed. The TPA-induced arachidonic acid release was inhibited by fluocinolone acetonide. The results indicate a direct activation of phosphatidylcholine- and phosphatidylinositol-specific phospholipases A2 by TPA and A23187. Cells prelabeled with 3H-choline released choline, choline phosphate and CDP-choline upon TPA but not upon A 23187 treatment. This could indicate activation of a phospholipase C-type enzyme by the phorbol ester. However, concomitant generation of diacylglycerol and phosphatidic acid was not detected.

Inhibition of cytolysin activity in large granular lymphocyte granules by lipids: Evidence for a membrane insertion mechanism of lysis

The ability of various compounds to inhibit the lytic activity of purified cytoplasmic granules from LGL tumors was tested. The lytic activity of granule cytolysin was modestly inhibited by inorganic phosphate, and various monophosphoesters, with I 50 values in the range of 8–20 m M. Among monophosphoesters, choline phosphate was exceptionally potent, with an I 50 of 1.4m M. In contrast to the inhibition by phosphate esters, the parent compounds such as neutral sugars, glycerol, and choline, showed no detectable inhibition at 50 m M. A lysolipid bearing a short aliphatic chain and some detergents were inhibitory with I 50 values in the range of 1 m M. Lysolipids with longer aliphatic chains, phospholipids as liposomes, and related lipid compounds were found to display potent inhibition of the hemolytic activity of LGL granule cytolysin, with I 50 values in the range of 0.2–30 μ M. Soluble globular proteins inhibited LGL granule cytolysin hemolytic activity with I 50 of 0.07–0.4 mg/ml. However, lipoproteins were 2–3 orders of magnitude more potent inhibitors, with L 50 values of less than 1 μg/ml. The observed potent inhibition by lipid compounds lends support to a model of the lytic action of granule cytolysin in which soluble cytolysin molecules insert into the membrane lipid bilayer during the course of the lytic event.

Degradation and reutilization of alveolar phosphatidylcholine by rat lungs.

We have shown previously that phospholipids instilled through the trachea are removed from the air spaces in isolated rat lungs by a process that is stimulated by beta-adrenergic agonists. In this study, we evaluated the fate of radiolabeled lipid vesicles [50% [3H]dipalmitoyl phosphatidylcholine (DPPC), 25% phosphatidylcholine (PC), 15% cholesterol, and 10% phosphatidylglycerol (PG)]. Vesicles were instilled through the trachea of anesthetized rats, and the lungs removed for perfusion. The percent of instilled 3H that could not be removed from lungs by extensive lung lavage increased progressively; at 3 h this fraction was 25.8 +/- 0.63% (mean +/- SE; n = 8). The percent of dpm in the lung homogenate accounted for by PC decreased progressively while dpm in lyso-PC, unsaturated PC, and aqueous soluble metabolites [choline, choline phosphate, glycerophosphorycholine, and cytidine 5'-diphosphate (CDP) choline (CDP-choline) increased. The dpm in microsomal and lamellar body fractions isolated from lung homogenates also increased progressively with time of perfusion. The presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) significantly stimulated both uptake of DPPC and the appearance of radioactivity in metabolites and subcellular organelles. This effect of 8-BrcAMP was not due to stimulation of phospholipase A activity. These results indicate that exogenous phospholipids instilled into the air spaces of rat lungs are internalized and degraded by a process that is stimulated by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)