Gérard Rebel

pH drift of "physiological buffers" and culture media used for cell incubation during in vitro studies.

In pharmacological or toxicological studies performed at room atmosphere comparison of various media used for cell incubation revealed discrepancies among results due to pH instability when these media contain bicarbonate. With the classically used protocols, a relatively fast and notable rise of the pH of such media has been observed, and values higher than 8.5 could be reached after 1 h of incubation. A less important rise in pH was also observed for media containing low amounts of sodium bicarbonate, e.g., Hanks formula-derived media. Because Hepes-buffered media or media with abnormal osmolarity cannot always be used for such studies, our choice of media is limited.

Activation of protein kinase C down-regulates glial but not neuronal taurine uptake.

Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator significantly decreased in a time- and dose-dependent manner taurine uptake by rat astroglial but not neuronal cells. The PMA-induced inhibition of taurine uptake by rat astrocytes was prevented by chelerythrine, a potent and selective inhibitor of PKC. The differential effect of PMA on rat neuronal and astroglial taurine transport was also obtained with the protein phosphatase inhibitor okadaic acid. This was not only the feature of rat cells since the same differential effects were obtained with human glioma GL15 and human neuroblastoma IMR32 cell lines. The results suggest that the neuronal and astroglial taurine transporter may be structurally different.

Lipid composition in liver and brain of genetically obese (ob/ob), heterozygote (ob/+) and normal (+/+) mice

Lipid composition was studied in liver and brain of normal (+/+), heterozygote (ob/+) and obese (ob/ob) mice. It was found that this genetic defect is expressed differently in the lipid composition of these organs. Cholesterol is increased in liver but strongly decreased in brain of obese animals. Phosphatide fatty acid composition is modified in liver and not in brain. In contrast, phospholipids and total ganglioside sialic acid are affected similarly in both organs. Although clinically normal, heterozygote (ob/+) mice already show an abnormal lipid composition in liver and brain. The potential importance of these results is presented.

Reversible intramitochondrial release of protein related to unsaturated fatty acids of membranes

Abstract The influence of temperature on intramitochondrial protein and enzyme release was studied in control and “lipid-deficient” rat liver mitochondria and in synaptosomal and “cell body” mitochondria of rat brain. (i) The fatty acid composition of the phospolipid fraction was shown to be different in control and lipid-deficient preparations. (ii) Arrhenius curves for temperature-dependent release of protein showed breaks. (iii) When comparing control to lipid-free rat liver mitochondria and cell body to synaptosomal rat brain mitochondria, shifts in the breaks in the Arrhenius plots were observed for release of aspartate aminotransferase, protein and malate dehydrogenase. (iv) Intramitochondrial temperature-dependent, succinate-induced protein release was also studied in rat liver mitochondria which had previously undergone a succinate-induced release and rebinding cycle. These mitochondria showed a temperature-dependent protein release identical to that of untreated mitochondria.

Taurine analog modulation of taurine uptake by two different mechanisms in cultured glial cells

Previous data have shown that HEPES, a taurine structural analog, inhibits the uptake of taurine by cultured cells differently, depending on its addition either to the culture medium or to the Krebs-Ringer buffer used for cell incubation during taurine uptake measurements (Lleu and Rebel, J Neurosci Res 23: 78-86, 1989). An extensive study of the effect of numerous other taurine structural analogs on taurine uptake by cultured glial cells was carried out. Our results show that taurine uptake modulation by structural analogs follows two different mechanisms. For the first mechanism, observable after the simultaneous presence of taurine and of its analog during the incubation time of the uptake experiment (10 min), the amine function on the molecule is essential. The sulfonate group could be replaced either by a sulfinic group or by a carboxylic group. beta-Alanine, hypotaurine, acetyltaurine, guanidinoethanesulfonate and guanidinopropionate are the most potent inhibitors in this first mechanism. For the second mechanism, which requires the presence of the analog in the culture medium during the 48 hr preceding the taurine uptake measurement, the simultaneous presence of an amine and of a sulfonate group or of an amine and a sulfinate group is required. Carboxylates are ineffective in modulating taurine uptake in this mechanism. The sulfonate buffers synthesized by Good et al. (Biochemistry 5: 467-477, 1966) also affect taurine uptake in both mechanisms.

Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.

Glycolipids and Cell Differentiation

Differentiation of a cultured cell refers generally to a change in the expression of some peculiar functions specific to this cell. Generally differentiation involves only a modification of some biochemical properties of the cell such as synthesis of melanin by melanocytes (KREIDER et al., 1975) or synthesis of collagen by bone fibroblasts (MANNER and KULEBA, 1974) or tendon cells (SCHWARTZ et al., 1976). However, differentiation is sometimes characterized by a net change in the cell morphology which accompanies the biochemical changes as for example with myoblasts.

Membrane lipids in bromodeoxyuridine-differentiated astroglial cells in culture.

Embryonic hamster astroblasts (NN strain) grown in continuous line were cultivated in the presence of bromodeoxyuridine (BrdU). A decrease in the growth rate of the cells and striking changes in their morphology were observed, the morphology of the cells resembling that of mature astrocytes. Membrane lipids of BrdU-differentiated and standard cells were compared. No modification of the lipid/protein ratio was observed. Phospholipids and cholesterol were increased in the same proprotions in the cells, and no modification of the phospholipid distribution was observed. Ganglioside sialic acid remained at the same level, but the ganglioside distribution was highly modified. Complex gangliosides appeared (GM1 and GD1a), while the proportion of simple gangliosides (GM3 and GD3) decreased. However, neither GT1 nor GQ1 were detected in differentiated cells. The distribution of phosphoglyceride acyl groups was highly modified, the proportion of arachidonic and docosapentaenoic acids being 2 to 3 times higher in BrdU-treated cells than in proliferating ones. These results were compared to those obtained with another clonal line of glial cells (C6) which exhibited no morphological differentiation in the presence of BrdU; the lipids of these cells were not modified by such a treatment.

Polar head groups manipulation of phospholipids in cultured neuroblastoma cells

Abstract The phospholipid polar head groups composition of neuroblastoma cells growing in surface cultures was altered as a result of ethanolamine and N-methyl ethanolamine base analogues addition. After 2 days incubation a significant increase of about 30% of the cellular phospholipids and the cholesterol content was evident. These changes were accompanied by a decrease in the cellular adhesive properties and the total cell number. The fatty acid profiles of the phosphoglycerides were practically identical irrespective of the base supplement.

Seasonal variation of the composition of membrane lipids in liver mitochondria of the hibernator cricetus cricetus relation to intramitochondrial intermembranal protein movement

Abstract 1. 1. European hamster ( Cricetus cricetus ) raised under constant temperature conditions show a seasonal variation in the profiles of the structural fatty acids of the liver mitochondrial membrane. In these animals there are but small differences between sleeping and aroused animals in winter. 2. 2. The most important variation in lipid composition occurs in the prehibernating phase—at the end of summer and the beginning of autumn. 3. 3. However, there is no apparent coordinated relation between the seasonal variation of the composition of the fatty acids and the membrane “fluidity” as expressed by the break in the Arrhenius curve for protein release in intermembranal space of the liver mitochondria. 4. 4. This break occurred at a higher temperature in active animals in winter (arousedl than in summer. 5. 5. No general correlation could be found between the breaks in the Arrhenius curves and the variations of the different fatty acid species during the seasonal cycle, except for the most polyunsaturated fatty acid (docosahexanoic acid) where an excellent inverse correlation was observed. 6. 6. Our results suggest that the more fluid parts of the lipidic leaflet of the mitochondrial membrane are those more specifically involved in such phenomena as succinate induced intramitochondrial protein movement and that the changes in composition of the mitochondrial lipids are a possible adaptative advantages for the hibernator.