L. Freysz

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‐‐6M 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‐‐5M), 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.

A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine

It had been previously speculated upon but never proved that the methylation of phosphorylethanolamine could contribute to the production of choline containing compounds. However, experimental evidence obtained with neuronal cultures was interpreted as showing that the stepwise methylation of phosphobases may be an important route for this biosynthesis. We demonstrate that cytosolic fraction from rat brain possesses a N-methyltransferase activity capable of methylating phosphorylethanolamine and its mono- and dimethyl-derivatives into phosphorylcholine. The level of activity detectable in rat liver cytosol is only 18% of that found in the brain cytosol.

Ethanolamine base-exchange reaction in rat brain microsomal subfractions.

Abstract: Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subtractions 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.

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.

Effect of Monomethylethanolamine, Dimethylethanolamine, Gangliosides, Isoproterenol, and 2‐Hydroxyethylhydrazine on the Conversion of Ethanolamine to Methylated Products by Cultured Chick Brain Neurons

Abstract: The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline‐containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water‐soluble ethanolamine‐containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10−8M and 10−5M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2‐hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10−6M and 10−5M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 × 10−5M propranolol.

Asymmetric synthesis of ethanolamine phospholipids in chicken brain microsomes, through the cytidine pathway.

Phosphatidylcholine and phosphatidylethanolamine are the major phospholipids in the membranes of nervous tissue. Studies on their intramembrane localization showed that these phospholipids have an asymmetric distribution in microsomes [l] and in synaptosomal plasma membranes [2-31. These observations raise the question on the mechanisms by which this asymmetry is produced. The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in brain is largely ensured by Kennedy’s pathway [4]. The last step of their synthesis is catalyzed by phosphocholine transferase (EC 2.7.8.1) and phosphoethanolamine transferase (EC 2.7.8.2), respectively. Recent studies on the localization of these enzymes suggest that they are differentially embedded in the microsomal membrane [S]. Therefore, it seems likely that both enzymes may participate in the asymmetric assembly of phospholipids in the membranes. The synthesis of phosphatidylethanolamine and the corresponding plasmalogens by the cytidine pathway was therefore investigated, and the site of incorporation of the labelled precursors was determined. The results are the subject of this report.

Synthesis De Novo of Choline, Production of Choline from Phospholipids, and Effects of CDP-Choline on Nerve Cell Survival

The repair of nerve cell membranes may overcome or, at least, alleviate neurological damage and diseases. The mechanisms responsible for such a repair are not known but they appear to be mediated by some components present in the plasma membrane of nerve cells. Much experimental data suggest that glycolipids, especially of the ganglio series present in large amount in neuronal membranes, may mediate one of the steps implicated in the repair and regeneration of nerve cells (De Felice and Ellenberg, 1984). Among other components of plasma membranes the fundamental “bricks” of all animal cell membranes are the phospholipids. Their function has been considered for a long time to be purely structural and this concept has often assumed the idea of passivity. It was customary, until recently, to think of phospholipids simply as a wall of constraint for the cytoplasmic content of the cells and as a useful support for proteins floating in a hydrophobic bilayer.

Nuclear Triiodothyronine Receptors and Mechanisms of Triiodothyronine and Insulin Action on the Synthesis of Cerebroside Sulfotransferase by Cultures of Cells Dissociated from Brains of Embryonic Mice

Many studies have shown that the CNS is markedly dependent on thyroid hormones for its overall growth and its biochemical and morphological development (1–6). Indeed, thyroid hormone deficiency at birth, if not recognized and corrected at an early stage, results in irreversible brain damage such as: impaired growth nerve cell processes resulting in a decrease in the number of neuronal contacts, increased cell death, reduced myelination and severe mental retardation (6–16). Significantly, these defects are amenable to hormone therapy only during an early critical age period (6). In contrast, in the hyperthyroid state, myelin synthesis commences and terminates earlier (17).

Topological Biosynthesis of Phosphatidylcholine in Brain Microsomes

Abstract: 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 Kennedys 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 Kennedys 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.

In vitro synthesis and transbilayer movement of phosphatidylethanolamine molecules labelled with different fatty acids in chick brain microsomes.

The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocation of newly synthesized phosphatidylethanolamine molecules labelled with different fatty acids has been investigated in chick brain microsomes using trinitrobenzensulfonic acid. The determination of the fatty acid composition of diacylglycerophosphoethanolamine in both the outer and the inner leaflet of the microsomal vesicles revealed a similar distribution indicating that both leaflets share the same molecular species. The in vitro incorporation of radioactive fatty acids (16:0, 18:1 and 20:4(n-6] into ethanolamine phospholipids, known to be catalyzed by the lyosphosphatidylethanolamine acyl transferase, showed that the radioactive diacylglycerophosphoethanolamine molecules appeared first in the outer leaflet and were thereafter transferred to the inner leaflet. The apparent rate of translocation of the newly synthesized ethanolamine phospholipid molecules was the highest for those labelled with 16:0 and the lowest for those labelled with 20:4(n-6). The results indicate that the active site of the acyl-CoA:lysophosphatidylethanolamine acyltransferases is located on the outer leaflet of the microsomal vesicles and that the different newly synthesized molecular species of diacylglycerophosphoethanolamine may be translocated from the outer to the inner leaflet at different rates.