Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures.

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10(-5)M and 10(-8)M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [3H] and [35S] methionine and [3H]ethanolamine as precursors showed an increased methylation of [3H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [3H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [3H]- and [35S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased 3H- and 35S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [3H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-3H] label after incubation of neurons with [3H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.

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. The Km was 405 +/- 58 microM for choline and 62 +/- 8 microM for AcCoA. Five isoelectric forms were found with pH's of 4.55, 6.05, 7.05, 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 recovered 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).(ABSTRACT TRUNCATED AT 250 WORDS)

Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex.

Using a sensitive histofluorescence staining method that allows for a quantitation of neuronal death, we compared the protective effects of gangliosides (a group of naturally occurring glycosphingolipids), phencyclidine (PCP), and MK-801 (dibenzocyclohepteneimine) on glutamate- and kainate-induced neuronal death in primary cultures of cortical and cerebellar neurons prepared from neonatal rats. PCP and MK-801 block neurotoxicity induced by glutamate doses 50 times higher than the LD50 (LD50 in Mg2+-free medium, 10 microM) but only partially block the kainate neurotoxicity (LD50 in presence of Mg2+, 100 microM). In contrast, pretreatment with gangliosides (GT1b greater than GD1b greater than GM1) results in complete and insurmountable protection against the neurotoxicity elicited by glutamate or kainate. In primary cultures of cerebellar granule cells gangliosides, unlike PCP and MK-801, fail to block glutamate-gated cationic currents and the glutamate-evoked increase of (i) inositol phospholipid hydrolysis (ii) c-fos mRNA content, and (iii) nuclear accumulation of c-fos protein. Protection of glutamate neurotoxicity by gangliosides does not require their presence in the incubation medium; however, it is proportional to the amount of glycosphingolipid accumulated in the neuronal membranes. The ganglioside concentration (30-60 microM) that blocks glutamate-elicited neuronal death also prevents glutamate- and kainate-induced protein kinase C translocation from cytosol to neuronal membranes.

Effect of exogenous gangliosides on the lipid composition of chick neurons in culture.

When exogenous gangliosides are added to the growth medium of neuronal cell cultures they are inserted into their plasma membranes and are afterwards metabolized in the cytoplasmic interior. The action of exogenous gangliosides brings important morphological and biochemical changes to neurons in culture. The present report shows that the treatment with exogenous gangliosides of a primary culture of chick neurons modified the distribution of fatty acids in phosphatidylinositol (PI), mainly that of arachidonic acid and the fatty acids of the (n - 3) series without affecting the other phospholipids. The composition of neutral lipids did not change but their content was increased up to 2-3-fold depending upon the concentration of gangliosides. The change of the growth medium from one containing fetal calf serum to a chemically defined one reduced dramatically the content of free fatty acids while the addition of gangliosides raised this content to normal levels. The increase in the amount of diacylglycerol (DG) confirmed the finding that gangliosides stimulate phosphoinositide degradation. Finally the fatty acid composition of DG suggests indirectly that this compound might be produced also by degradation of phosphatidylcholine and not only of PI.

Effect of external high potassium and pH on the uptake of choline in glial and neuronal cells in culture.

The Vmax of the uptake of choline was increased in nerve cell cultures by lowering (from 7.4 to 6.5) or increasing (from 7.4 to 8.1) the pH. In neurons no effect was observed on the value of the Km's of the uptake of either the apparent high or low affinity components. In glial cells only a low affinity component was measured at pH 6.5 and diffusion was observed at pH 8.1. An excess of K+ ions in the incubation medium reproduced the increase in Vmax observed with changes in pH suggesting a possible dependence of the uptake of choline upon the H+ and OH- gradients. Taking into account the characteristics already known of the transport of choline into nerve cells, such a dependence adds new insight in the mechanisms underlying the transport and indicates another possible regulation of choline entry, eventually directed towards the synthesis of acetylcholine.

[Effect of exogenous gangliosides on the metabolism of inositol compounds in chick neurons in primary culture]. / Effet de gangliosides exogènes sur le métabolisme des composés à inositol dans les neurones de poulet en culture primaire.

A mixture of exogenous gangliosides at 10(-8) M and 10(-5) M concentration added to the medium of chick neurons stimulates the metabolism of inositol compounds. Notably the incorporation of [3H] myo-inositol is increased in phosphatidylinositol and in inositol triphosphate. Gangliosides, hence, by increasing the production of second messengers might regulate the phosphorylation processes of neuronal proteins.

Ion dependent efflux from neuronal and glial cell cultures.

A spontaneous efflux of choline originating from the cytoplasmic free choline compartment and, partly, from metabolized form was measured from neurons and glial cells in culture. The efflux was stimulated by an excess of K(+) and by the absence of Ca(2+) ions from the incubation medium in both types of culture. The two effects did not appear to be synergistic. The stimulation produced by an excess of K(+) (100 mM) was blocked in neurons by 0.5 ?M BaCl(2) and in glia cells by 0.1 ?M BaCl(2) (in the presence of 30 mM K(+)). The stimulation produced by the absence of Ca(2+) instead was not blocked by Ba(2+) ions in either of the two types of culture. The results suggest that the stimulation induced by K(+) (high concentration and long time of incubation) might be of biochemical rather than physiological nature and that choline may be driven out of the cells in correlation with the K(+) gradient. The greater sensitivity of glial cells to K(+) ions may also suggest a supportive role of these cells with respect to neurons, as they seem capable of furnishing choline for neuronal needs during depolarization.

The effect of exogenous gangliosides on neurons in culture: A morphometric analysis.

Cultures of isolated neurons have been treated with a purified preparation of gangliosides (10(-5)M and 10(-9)M) added to the cell growth medium at the 3rd day in culture and a morphometric analysis of the cells was performed with an image analyzer after 1 and 4 days of treatment. The number of cells and the area of the cell bodies were increased following the treatment. The results indicate as well the 'sprouting' effect of the glycolipids on the number of secondary neuronal processes and an increase in the length of the primary neuntes. The present data and other biochemical evidence (Dreyfus et al., 1984, J. Neurosci. Res.) suggest that the addition of exogenous gangliosides may have a trophic effect on neurons, greatly enhances the number of cell to cell contacts, and, possibly, stimulates cell proliferation and differentiation.

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.