R. Massarelli

Synthesis of choline phospholipids in neuronal and glial cell cultures by the methylation pathway.

The synthesis of choline in the nervous tissue has been a subject of debate (reviews [1,2]). Since the work of [3,4] it was believed that the stepwise methylation of ethanolamine and/or phosphatidylethanolamine in nervous tissue was non-existant or irrelevant until the suggestion that choline might be produced de novo in the rat brain [5-7] through methylation of phosphatidylethanolamine. Methyltransferase activity has been shown in rat brain synaptosomes [8,9], suggesting that nervous tissue may have the necessary machinery for the de novo synthesis of phosphatidylcholine. The possibility of obtaining cell cultures containing exclusively neurons or glial cells gave us the opportunity to check, in these isolated systems, whether neurons and/or glia can methylate phosphatidylethanolamine to phosphatidylcholine. The results obtained suggest that both cell types have this capacity and that the synthesis of choline phospholipids through the methylation pathway is much higher in glial cells than in neurons.

Molecular species of choline and ethanolamine phospholipids in rat cerebellum during development

Abstract: The molecular species composition of rat cerebellar phospholipid subclasses has been studied by HPLC after phospholipase C treatment and dinitrophenyl derivatization. During rat cerebellum development (3–90 days postpartum), cholinephosphoglycerides and ethanolamine phosphoglycerides represented ∼80% of all phospholipids, with their relative amount changing after 1 month. Among ethanolamine phosphoglycerides, the molar ratio of diacylglycerophosphoethanolamine (diacylGPE) to alkenylacylGPE decreased from ∼1.4 at 3 days to ∼0.5 after 10 days. The phospholipids investigated contained up to 12 different molecular species. The rate of accumulation of the various molecular species of diacylglycerophosphocholine (diacylGPC), diacylGPE, and alkenylacylGPE during cerebellar development allowed a classification into three main groups. The overall increase of the molecular species of the first group (6‐diacylGPC, 5‐diacylGPE, and 4‐alkenylacylGPE) was ∼ 18‐fold between 3 and 90 days, with a faster rate of accumulation between 3 and 30 days. Those of the second group (3‐diacylGPC, 5‐diacyl‐GPE, and 5‐akenylacylGPE) increased by ∼45‐fold during the same developmental period, at a slow rate before day 15 and at a faster one thereafter. The molecular species of the third group (3‐alkenylacylGPE) increased by >250‐fold between 3 and 90 days, at a very slow rate before day 21 and more quickly thereafter. The different rates of accumulation of the components of the three groups during cerebellar development suggest a preferential location of the first group in membranes of neuronal perikaryons, glial cells, and synaptosomal structures. Those of the second group appear to be located in both synaptosomal membranes and myelin sheets, and those of the third group can be considered as myelin markers.

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.

Oleoylamine and sphingosine stimulation of phosphatidylserine synthesis by LA‐N‐2 cells is protein kinase C independent

The presence of sphingosine and oleoylamine in the culture medium of LA‐N‐2 cells stimulated the incorporation of [3H]serine into its corresponding phospholipid, phosphatidylserine (PtdSer). The optimum stimulation for sphingosine and oleoylamine were 50 μM and 100 μM, respectively. Oleoylamine increased the incorporation of [3H]serine over 6‐fold while sphingosine increased the incorporation of [3H]serine over 2.5‐fold. The amount of radioactivity found in water‐soluble components and in protein was similar to that found with control LA‐N‐2 cells. The incorporation of [3H]choline and [3H]ethanolamine into their corresponding phospholipids were decreased in the presence of either oleoylamine or sphingosine. A protein kinase C (PKC) activator, DiC8, and a PKC inhibitor, H7, did not influence the enhanced phosphatidylserine formation by sphingosine and oleoylamine. In addition, there were no differences in the stimulatory effect of sphingosine and oleoylamine discernable between PKC down‐regulated cells or controls. These observations indicate that this oleoylamine and sphingosine mediated enhanced phosphatidylserine synthesis is PKC‐independent.

Stimulation of phospholipase D activity and indication of acetylcholine synthesis by oleate in rat brain synaptosomal preparations

It had been previously demonstrated that the oleate activation of synaptosomal membrane phospholipase D liberated choline which was available for acetylcholine formation. The present investigations were undertaken to determine if oleate might have an effect on choline uptake by synaptosomes. It was observed that oleate interfered with choline uptake when incubations were carried out at 37°C but uptake was stimulated at 3°C. Oleate was the most effective fatty acid of several tested. Preliminary observations suggest the presence of a membranous form of choline acetyltransferase.

Molecular Species of Choline and Ethanolamine Glycerophospholipids in Rat Brain Myelin During Development

The composition of the molecular species of various phospholipid subclasses was examined in myelin isolated from brain of 15-, 21- and 90-day-old rats. The molecular species of diacylglycerophosphocholine (PtdCho), diacylglycerophosphoethanolamine (PtdEtn) and plasmenylethanolamine (PlsEtn) were quantified by high-performance liquid chromatography (HPLC) after phospholipase C treatment and dinitrobenzoyl derivatization. In rat brain myelin, each phospholipid subclass showed a specific pattern of molecular species that changed during development. PtdCho contained large amounts of saturated/monounsaturated and disaturated species and low amounts of saturated/polyunsaturated species. During brain development, the levels of saturated/monounsaturated molecular species increased whereas those of the disaturated and saturated/polyunsaturated species decreased. PtdEtn were characterized by their low levels of disaturated species and a high content of saturated/monounsaturated and saturated/polyunsaturated species, of which those containing fatty acids of the n−3 series decreased, whereas those containing fatty acids of the n−6 series did not change during brain development. The levels of saturated/monounsaturated species increased in PtdEtn. No disaturated molecular species could be detected in PlsEtn. This alkenylacyl subclass contained large amounts of saturated/polyunsaturated, saturated/monounsaturated and dimonounsaturated molecular species. During development, the levels of saturated/polyunsaturated molecular species decreased while those of the two others increased. The data indicated that myelin sheaths undergo phospholipid changes during brain development and maturation.

Pyruvate reverses metabolic effects produced by hypoxia in glioma and hepatoma cell cultures.

The intervention of pyruvate in glucose metabolism was investigated during hypoxic stress in tumour cell cultures having respiratory capacities under normoxic conditions. Results obtained with nuclear magnetic resonance (NMR) spectroscopy showed that, under normoxic conditions, rat glioma C6 and human hepatoma Hep G2 cell cultures metabolised [(13)C(1)]glucose into lactate, alanine, glutamate and other less abundant metabolites, as already known from the literature. In the absence of pyruvate, during hypoxia or cyanide poisoning, both cell types dramatically decreased the label into glutamate and accumulated [(13)C(3)]glycerol-3-phosphate. The compound was further identified by 31P NMR spectroscopy. The accumulation of the label in glycerol-3-phosphate, however, did not occur when the cells were incubated in the presence of pyruvate. The fate of the latter, followed under normoxic conditions by incubating cells with [(13)C(3)]pyruvate and natural glucose, showed that the label was mainly found in alanine, lactate and glutamate. Anoxic conditions increased the label in lactate and reduced that of glutamate. The data show a metabolic effect of pyruvate during mitochondrial blockade due to severe lack of oxygen in tumour cell lines.

Choline acetyltransferase-like activity bound to neuronal plasma membranes.

A form of CAT-like activity was found bound present in rat brain synaptosomal membranes which could be recovered in the Triton X-114 phase. The enzyme activity was slightly activated by NaCl, had a pH maximum around 8 and showed a temperature dependence with a Q10 of 2.28. It was inhibited 100% by 10−6 M naphthyl vinyl pyridinium but not by 10−5 M diisopropyl phosphofluoridate. The kinetics of this bound form of CAT were similar to the soluble form of the enzyme. TheKm was 405±58 μM for choline and 62±8 μM for AcCoA. Five isoelectric forms were found with pHs of 4.55, 6.05, 7.06, 7.36, and 8.00 which is in contrast to the three isoelectric forms found of the soluble enzyme in rat brain. The presence of a CAT-like activity in the plasma membrane was confirmed with experiments performed using intact synaptosomes and intact cells in culture. Acetylcholine, synthesized from radioactive AcCoA by intact rat brain synaptosomes, was recovered in the incubation medium and only in the presence of exogenous choline or when the production of choline was stimulated by oleate via the activation of phospholipase D. This was also seen in experiments with intact pheochromocytoma cell cultures (PC 12) which synthesize acetylcholine that was recoverved in the incubation medium. Acetylcholine formation in the presence of choline and AcCoA was stimulated in cells that had been grown in the presence of nerve growth factor (NGF). The localization of 1% of CAT activity in a transbilayer position in the plasma membrane, could suggest a possible role of this enzymatic form in the regulation of acetylcholine synthesis.

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 ?M) or higher (150 ?M) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 ?M choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K(m)s with different affinities. The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.

Choline fluxes in primary nerve cell cultures. Correlation with the endocellular metabolism of choline.

The fluxes of choline across the plasma membrane were measured in primary nerve cell cultures from chick embryo cerebral hemispheres containing neurons and supporting cells. The incubation of cells with exogenous concentrations of choline far below the concentrations present in the growth medium (?30-50 ?M) and in the range of the high affinity uptake mechanism (about 0.5 ?M) profoundly affected the steady state of the endocellular free choline levels. The kinetics of the uptake were dependent upon the endocellular status of the choline pool since after preincubation in the absence of choline two K(m)s are observed (K(m1): 0.8 ?M; V(max)(1): 44.8 pmol/mg protein/2 min; K(m)(2): 14.3 ?M, V(max)(2): 333.3 pmol/mg protein/2 min) while only one mechanism can be found when the endocellular pool of choline was kept in steady state conditions (K(m): 14.3 ?M, V(max): 545.5 pmol/mg protein/2 min). The presence of an homoexchange phenomenon was suspected since choline efflux could be increased by increasing the concentrations of choline in the incubation medium. The results suggest that the movement of choline into nerve cells in culture appears to be mediated by a single mechanism which is regulated by the endocellular status of the choline pool.