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M Record

Publications and source records attributed to M Record.

At least 37 records · Page 2Linked to original sources
PubMed1990

PAF-acether transfer activity in HL-60 cells is induced during differentiation.

Platelet-activating factor is a proinflammatory lipid active at subnanomolar concentrations. The intermembrane transfer of a biologically active PAF analog has been previously demonstrated in macrophages. Here we demonstrate that the specific activity of this transfer activity increases when HL-60 cells are induced to differentiate by treatment with dimethyl sulfoxide, dibutyryl cAMP or phorbol diester. In undifferentiated HL-60 cells, methylcarbamyl-PAF transfer activity was only 0.56 U.min-1.mg-1. This basal value was increased 2.6 and 6.7 times upon granulocytic and macrophagic differentiation, respectively. On the other hand, the transfer of 2-O-methyl-PAF, a cytotoxic analog with no PAF biological activity, remained very low and did not vary during differentiation.

Bucladesine↗
PubMed1990

Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator.

A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites. Very little (less than 10%) of the [3H]PAF produced from [3H]alkylacetyl-G was released from intact cells under a variety of incubation conditions but 50% of the de novo-derived mediator was recovered in the medium of cells that were permeabilized with saponin. Our results indicate that PAF is rapidly translocated from its intracellular site of enzymatic synthesis to the plasma membrane where it is apparently sequestered in a pool that is not accessible to extracellular acceptors in contact with intact cells.

Cell Line↗
PubMed1989

O-alkyl-O-methylglycerophosphocholine, an antineoplastic lipid, undergoes spontaneous redistribution between biological membranes prepared from HL-60 cells.

Cytotoxic actions of the unnatural phospholipid 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholine (alkylmethyl-GPC) appear to be targeted to the plasma membrane of sensitive cells. We analyzed the distribution of [3H]alkylmethoxy-GPC in subcellular membranes isolated from human promyelocytic leukemia (HL-60) cells. [3H]Alkylmethyl-GPC added to intact cells selectively labels plasma membrane-enriched fractions of the postnuclear supernatant, but the labeling profile is independent of the temperature and duration of the incubation, and concentration of the molecule. Also, an identical distribution pattern is obtained when [3H]alkylmethyl-GPC is directly added to postnuclear supernatants. Moreover, [3H]alkylmethyl-GPC translocates between subcellular membranes in a manner that does not depend on membrane-adsorbed or cytosolic transfer proteins. These results indicate that the subcellular localization studies reported for alkylmethyl-GPC and structurally-related molecules must be interpreted with caution.

Antineoplastic Agents↗
PubMed1989

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil.

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30-45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PEt [corrected] in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.

Cell Membrane↗
PubMed1989

Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism.

The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs-II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso-PAF to the acetyltransferase to form PAF-acether.

Acetyltransferases↗
PubMed1988

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum.

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.

5'-Nucleotidase↗
PubMed1985

Evidence that biosynthesis of platelet-activating factor (paf-acether) by human neutrophils occurs in an intracellular membrane.

Human polymorphonuclear leukocytes (PMN) were incubated in the absence or presence of the calcium ionophore A23187 (6 microM) for 10 min at 37 degrees C. They were then lysed by nitrogen cavitation and fractionated using Percoll gradients. Three major fractions of increasing density corresponding to plasma membrane, intracellular membranes and secretory granules were detected using [3H]concanavalin A, NADH-dehydrogenase and beta-D-glucuronidase as respective markers. In both cases, the acetyltransferase activity responsible for biosynthesis of paf-acether (platelet-activating factor of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was detected in the intermediary fraction, the enzyme activity being increased 3-4-fold in stimulated cells. From the comparison with the distribution of various markers, it is concluded that in human PMN the final step of paf-acether assembly occurs in an intracellular membrane, possibly the endoplasmic reticulum.

Acetyltransferases↗
PubMed1985

A rapid isolation procedure of plasma membranes from human neutrophils using self-generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes.

In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self-generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils.

5'-Nucleotidase↗
PubMed1985

Ether-phospholipid composition in neutrophils and platelets.

It is well documented that ether-lipids, especially the 1-0-alkyl-2-0-acyl-sn-glycero-3-phosphocholine, can serve as precursors in paf-acether (platelet-activating factor) biosynthesis. This study was undertaken to determine the amount of these compounds in two cell types which are good producers of paf-acether: human neutrophils and rabbit platelets. The method of phospholipid analysis was based on selective destruction of diacyl molecules by lipase from guinea pig pancreas and of plasmalogens by acidolysis; phospholipids were then separated by bidimensional thin-layer chromatography. In platelets as in neutrophils, the major phospholipids were choline (37%) and ethanolamine (30 and 32%, respectively) phosphoglycerides and sphingomyelin (18 and 16%, respectively). In rabbit platelets the 1-alkyl molecules represented 15% of the choline class (0.7 nmol/10(7) cells). Neutrophils differed strikingly from platelets by the high level of these molecules which constituted 50% of the choline class (16.3 nmol/10(7) cells). It therefore appears that in the two cell types, the amount of 1-0-alkyl-2-0-acyl-sn-glycero-3-phosphocholine largely exceeded that would be necessary for paf-acether biosynthesis.

Animals↗
PubMed1985

Turnover of phosphocholine and phosphoethanolamine in ether-phospholipids of Krebs II ascite cells.

Krebs II ascite cells suspended in Eagle medium were incubated at 37 C for up to 6 hr in the presence of [3H] glycerol or [32P] orthophosphate. After extraction, their lipids were treated with guinea pig phospholipase A1 under conditions where all diacyl-phospholipids (diacyl-PL) became hydrolyzed with 55% recovery of lyso-PL. Using a bidimensional thin layer chromatography (TLC) involving exposure to HCl fumes between the two runs, it then became possible to determine at once the specific radioactivity of the three subclasses (diacyl-, alkylacyl- and alkenylacyl-) present in choline glycerophospholipids (CGP) and ethanolamineglycerophospholipids (EGP). Compared to diacyl-PL, a lower de novo synthesis of ether subclasses was evidenced in both CGP and EGP by [3H] glycerol incorporation. Although the same profile was obtained for CGP with [32P] orthophosphate, the three EGP subclasses displayed in this case the same specific radioactivity. These data indicate a higher turnover rate of the polar head group of ether-EGP compared to either-CGP. The simple methodology used in the present study might thus prove helpful in developing enzymatic studies dealing with the mechanism of this accelerated renewal.

Animals↗
PubMed1984

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells.

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A2 and Staphylococcus aureus sphingomyelinase C and on phospholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A1 and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).

Animals↗
PubMed1984

Different susceptibility of alkylacyl--versus diacyl--and alkenylacyl--phosphatidylcholine subclasses to stimulation of biosynthesis by phospholipase C.

Krebs II ascites cells were incubated with [3H] or [14C] choline in the presence or in the absence of Clostridium welchii phospholipase C (PLC). At enzyme concentrations where cell lysis remained limited, PLC specifically enhanced phosphatidylcholine (PC) biosynthesis, as shown by comparison with [14C] ethanolamine. Further analysis revealed that the stimulating effect of PLC remained limited to 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and 1-alkenyl-2-acyl-GPC, whereas the biosynthesis of 1-alkyl-2-acyl-GPC, the putative precursor of platelet activating factor (PAF-acether) remained unchanged. These differences reflect different localizations of the three PC subclasses in the plasma membrane and are discussed in relation to the regulation mechanism of PC biosynthesis.

Animals↗
PubMed1984

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells.

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.

Animals↗
PubMed1984

Studies on ether phospholipids. II. Comparative composition of various tissues from human, rat and guinea pig.

The ether phospholipid composition of various tissues (brain, heart, lung, liver, kidney, testis, erythrocytes and plasma) has been investigated in human, rat and guinea pig, using a new method of determination (El Tamer, A., Record, M., Fauvel, J., Chap, H. and Douste-Blazy, L. (1984) Biochim. Biophys. Acta 793, 213-220). This is based on the selective removal of diacyl phospholipid species by phospholipase A1 degradation followed by acidolysis of the plasmalogens. Our results fit rather well with other literature data available for human and rat tissues, illustrating the good reliability of the method. Among various differences noted between the three mammalian species, guinea pig is characterized by a relatively higher content of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and of ethanolamine plasmalogens in blood plasma. Alkylacyl-GPC, a putative precursor of platelet activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC), is also more abundant in guinea pig lung and in human kidney. This study also revealed a striking parallelism between the tissue content of alkylacyl-GPC and alkylacyl-GPE (1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine). This new observation is discussed in relation to a possible metabolic link between these two phospholipids.

Animals↗
PubMed1982

Isolation and characterization of plasma membranes from krebs II ascite cells using Percoll gradient.

1. Plasma membranes were isolated from Krebs II ascite cells grown in the mouse. Cells were disrupted by nitrogen cavitation in an isotonic alkaline buffer containing magnesium and ATP. Isolation was performed in an alkaline-buffered self-generating gradient of Percoll with an angular rotor. At each step of the preparation, the pH appeared as the critical aspect of our procedure. 2. External membrane markers were concanavalin A and 5'-nucleotidase (EC 3.1.3.5). They reached a relative specific activity of 10, whereas this value was only of 0.7 for the endoplasmic reticulum marker, NADH dehydrogenase (EC 1.6.99.3). 3. Plasma membrane from 4 ml packed cells were isolated within 1 h after homogenization with good yield: 50% and 67% of total [3H]concanavalin A and 5'-nucleotidase, respectively, were recovered in the two plasma membrane fractions. 4. Electron microscopy examination showed the presence of vesicles of different sizes devoid of other structural contaminants. 5. Using the specific binding of concanavalin A to the external cell membrane, it was calculated that about 50% of the total cell phospholipid and 10% protein are located in the plasma membrane. Their sphingomyelin content is much higher than in the whole cell, in contrast to phosphatidylinositol, known as a more specific endoplasmic reticulum phospholipid.

Animals↗
PubMed1980

Utilization of membranous lipid substrates by membranous enzymes. Hydrolysis of sphingomyelin in erythrocyte 'ghosts' and liposomes by the membranous sphingomyelinase of chicken erythrocyte 'ghosts'.

Incubation at 37 degrees C of haemolysed chicken erythrocytes ('chicken erythrocyte ghosts') resulted in hydrolysis of the membrane sphingomyelin, suggesting an activation of a latent sphingomyelinase during the haemolysis procedure. When this incubation was continued for several hours, the entire sphingomyelin of the erythrocyte 'ghosts' was hydrolysed and membranes were obtained that were devoid of sphingomyelin, but had an active sphingomyelinase. Mixing the latter membranes with human erythrocyte 'ghosts' or positively charged liposomes led to hydrolysis of the sphingomyelin in these two membranes. This suggested that, after haemolysis, the activated sphingomyelinase in the membrane of the chicken erythrocyte 'ghosts' could hydrolyse sphingomyelin in its own membrane ('intramembrane utilization') or adjacent membranes ('intermembrane utilization').

Animals↗