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L Freysz

Publications and source records attributed to L Freysz.

At least 55 records · Page 3Linked to original sources
PubMed1986

Uptake of ethanolamine in neuronal and glial cell cultures.

The uptake of radioactive ethanolamine has been studied in exclusively neuronal and glial cell cultures from dissociated cerebral hemispheres of chick embryos. Both cell types show saturable kinetics; neurons have an apparent Km of 6.7 microM, Vmax 41.4 pmol mg prot.-1 min-1 and glial cells a Km of 119.6 microM, Vmax 3,917 pmol mg prot-1 min-1. The lower affinity of the transport and the 100 fold increase in Vmax observed in glial cells correlated with a more important accumulation of free ethanolamine found in glial cells and with a higher degree of phosphorylation of ethanolamine. The uptake appeared to be temperature and Na+ ions dependent but was not affected by CN- or ouabain. Monomethyl-, dimethylethanolamine and choline were effective in inhibiting the uptake. Little or no effect was observed with serine, methionine, carnitine, alanine or glutamate.

Animals↗
PubMed1986

Compartmentation of newly synthesized phosphatidylethanolamine in rat brain microsomes.

The compartmentation of the phosphatidylethanolamine newly synthesized in brain microsomes in vitro either by base exchange or net synthesis has been studied, using difluorodinitrobenzene as a chemical probe. The experimental results demonstrate that in rat brain microsomes the phosphatidylethanolamine molecules synthesized by base exchange and the bulk membrane lipid belong to different pools. Ca2+ bound to microsomes seems to be involved in the maintenance of the compartmentation of phosphatidylethanolamine. In the presence of Ca2+ the newly synthesized phosphatidylethanolamine molecules react with difluorodinitrobenzene as though they are organized in clusters. After biosynthesis in vivo or in vitro through the cytidine pathway, the compartmentation of the newly formed phosphatidylethanolamine appears less marked than after the synthesis through base exchange.

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PubMed1986

Effect of CDP-choline on hypocapnic neurons in culture.

Neuronal cultures from chick embryo cerebral hemispheres were protected against a hypocapnic injury by adding to their growth medium 10(-6)M CDP-choline before or after the injury. The protection obtained with CDP-choline was analyzed by a morphometric analysis and showed that pretreatment of neuronal cultures with CDP-choline maintained the number of cell aggregates and of primary neuronal processes at control values after hypocapnic shock. Various experiments showed that the intact molecule was responsible for the protective action, since pretreatment with different concentrations of various nucleosides and nucleotides (up to 10(-5) M), choline, and phosphorylcholine was without protective effect. The addition of CDP-choline after the hypocapnic injury resulted in a protection of the cultures as shown by morphological observation. Incubation of neurons with radioactive choline showed that hypocapnia increased the incorporation of the label into phospholipids whereas the presence of CDP-choline reduced it. The de novo synthesis of choline was affected by neither hypocapnia nor CDP-choline treatment. The results indicate that CDP-choline may have the capacity to protect neurons under conditions of basic pH and that cellular proliferation may be stimulated by the compound.

Animals↗
PubMed1986

Ethanolamine base-exchange reaction in rat brain microsomal subfractions.

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.

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PubMed1985

Topological biosynthesis of phosphatidylcholine in brain microsomes.

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.

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PubMed1985

Sidedness of phosphatidylcholine-synthesizing enzymes in rat brain microsomal vesicles.

The sidedness of CDP-choline:1,2-diradylglycerol choline phosphotransferase (EC 2.7.8.2) and of the choline base-exchange activity has been studied in rat brain microsomal vesicles. Proteases (trypsin and pronase) and mercury-dextran have been used as reagents for membrane surface components. All of them could inactivate both enzymes to a good extent, without affecting the morphology or the permeability to sucrose of the vesicles. It is therefore concluded that CDP-choline:1,2-diradylglycerol choline phosphotransferase and the choline base-exchange activity are localized on the outer surface of rat brain microsomal vesicles.

Animals↗
PubMed1984

Specific stimulation of phospholipase activities with phosphatidylethanolamine in transformed cells.

Phospholipase A1, A2 and C activities with phosphatidylethanolamine were enhanced in C6 cells relative to primary astrocytic cultures. Enhancement was a function of cell density. Phospholipase activities with phosphatidylcholine were unchanged as a function of cell density, while phospholipase C activity with phosphatidylinositol was reduced. All acid phospholipase activities measured were low or essentially absent in the three transformed cell lines examined. These results suggest that arachidonate release upon confluency is mainly from phosphatidylethanolamine.

Animals↗
PubMed1984

Metabolism and function of gangliosides in developing neurons.

Previous experiments have shown that the addition of a mixture of gangliosides to the growth medium induced morphological changes in primary neuronal cultures, producing especially a trophic effect and a sprouting of neurites (neuritogenesis). The study reported here examined the changes of some biochemical parameters that paralleled the morphological modifications of cultured neurons from chick brain hemispheres treated with gangliosides. Neurons cultured from 3 to 7 days in the presence of various concentrations of a purified mixture or of single-species of gangliosides (GM1, GD1a, GT1b) revealed that these glycolipids were easily incorporated into the cells as a function of their exogenous concentrations. Incubation of neurons with N-acetyl-D-[U-14C]mannosamine showed a final labeling of all endogenous cellular and exogenous incorporated gangliosides; however, the radioactivity recovered decreased as a function of the number of sialic acid units of the exogenously added gangliosides. The treatment of neuronal cells from 3 to 7 days in culture with a mixture of 10(-8) M and 10(-5) M gangliosides led to the following observations on some neurochemical parameters: no effect on the influx of choline and dopamine; no effect on the spontaneous choline efflux, whereas the K+-provoked one is abolished; decrease of the spontaneous and K+-stimulated release of dopamine; no effect on the spontaneous release of GABA for 10(-8) M gangliosides but an increase of both spontaneous and K+-provoked release for 10(-5) M gangliosides. The data suggest that the possible insertion of gangliosides into the neuronal membranes may imply structural modifications that may influence enzymatic activities, neurotransmitter transport, and finally, some nerve cell mechanisms.

Animals↗
PubMed1983

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid.

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium welchii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.

Animals↗
PubMed1983

Compartmentation of membrane phosphatidylethanolamine formed by base-exchange reaction in rat brain microsomes.

The compartmentation of membrane phosphatidylethanolamine (PE) formed by base-exchange reaction in rat brain microsomal vesicles has been investigated. After labelling membrane PE by base-exchange in vitro, microsomal vesicles were treated with trinitrobenzenesulfonic acid (TNBS). The amount of membrane PE reacting with TNBS depends on the duration and the temperature of the reaction as well as on the TNBS concentration. It was found that almost all of the labelled PE molecules, but only about 24% of membrane PE, were accessible to TNBS, under very mild reaction conditions. It is concluded that PE labelled by base-exchange is completely localized in the cytoplasmic leaflet of microsomal vesicles.

Animals↗
PubMed1982

Phospholipase activities of rat brain cytosol. Occurrence of phospholipase C activity with phosphatidylcholine.

21-day-old rat brain contains a soluble phospholipase C with the ability to hydrolyse phosphatidylcholine. This enzyme has an alkaline pH optima. The results of the DEAE-cellulose fractionation, the pH profile and the Ca2+-dependency suggest that the enzyme may be the same as that responsible for phosphatidylinositol hydrolysis. The activity of the phospholipase C is associated closely with a diacylglycerol lipase. The two enzyme activities could be separated by DEAE-cellulose fractionation, resulting in greater than 100% apparent recovery of the phospholipase C acivity. This phospholipase C activity is not the result of the back reaction of choline phosphotransferase.

Animals↗
PubMed1982

Possible role of sialocompounds in the uptake of choline into synaptosomes and nerve cell cultures.

Incubation of primary nerve cell cultures and of crude synaptosomal preparations with neuraminidase released sialic acid from both gangliosides and sialoglycoproteins. After this treatment, the pattern of ganglioside distribution was severely modified with a decrease of polysialogangliosides (GD1b, GT1b, Gt1L, GQ1) and a dramatic increase in monosialoganglioside GM1. The choline influx into neuraminidase treated cells and organelles was reduced by 30--50% but the efflux was unmodified. In particular the high affinity mechanism of choline uptake disappeared and the low affinity mechanism was modified in both cases. The disappearance of the high affinity uptake mechanism was not followed by a decreased acetylcholine synthesis as it should be if the current theories on choline uptake and acetylcholine synthesis are correct. Our present data thus confirm our previous hypothesis that choline metabolism regulates choline uptake rather than the other way round as is suggested by the theories most widely accepted at present. Choline uptake was unaffected by pretreatment of cells and organelles with tetanus toxin suggesting that the effect of neuraminidase on the choline uptake were either mediated through glycoproteins or through gangliosides other than those which bind to tetanus toxin (GD1b and GT1b). Several speculative models for explaining the effect of neuraminidase on choline uptake are proposed.

Animals↗
PubMed1982

Topographic distribution of enzymes synthesizing phosphatidylcholine and phosphatidylethanolamine in chicken brain microsomes.

The localization of phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes within the transverse plane of chicken brain microsomes was investigated by using proteases (trypsin and pronase) and neuraminidase. Treatment of intact microsomes with the proteases inactivated the phosphocholine transferase completely and the ethanolamine phosphotransferase only slightly. This latter enzyme was, however, completely inactivated when deoxycholate-treated microsomes were exposed to proteases. Treatment of intact microsomes with neuraminidase had no effect on both phosphotransferases, although 65% of the sialic acid of sialoglycoproteins and 37% of that of gangliosides were removed. With deoxycholate-disrupted microsomes nearly all sialic acid from the sialoglycoproteins and about 70% of that of gangliosides were released. In parallel, the phosphoethanolamine transferase was 90% inactivated. It is suggested that phosphocholine transferase is localized on the outer face of the microsomal vesicle, whereas the phosphoethanolamine transferase could be a sialoglycoprotein, possibly situated on the inner face of the vesicle, or perhaps a transmembrane protein.

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