Calmodulin antagonist W-7 inhibits lysosomal sphingomyelinase activity in C6 glioma cells.

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) is known to be a potent calmodulin antagonist and inhibitor of calmodulin-dependent protein kinases. W-7 and 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) are inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. In C6 glioma cells, W-7 and not H-7 inhibited dose-dependently acid sphingomyelinase, a result indicating the modulation of this lysosomal enzyme by a calmodulin-dependent system. Other lysosomal enzymes, such as beta-glucosidase, alpha-galactosidase, and arylsulfatase A, were unaffected by W-7 and H-7, a finding indicating a selective effect of W-7 on sphingomyelinase.

Modifications of sphingomyelin and phosphatidylcholine metabolism by tricyclic antidepressants and phenothiazines.

Phenothiazines and tricyclic antidepressants, when added to culture medium, gave rise in several types of cells (C6 rat glioma cells and human fibroblasts), to a decrease in lysosomal sphingomyelinase activity. The effect of chlorpromazine and desipramine was dose dependent, and was observed after 3 hours of incubation with the drugs at concentrations ranging between 1 and 10 microM. In C6 glioma cell cultures, the decrease in sphingomyelinase activity was related to the clinical effectiveness of phenothiazines, tricyclic antidepressants and derivatives. Incorporation of (choline-14C) sphingomyelin showed that the metabolic pathway implying the synthesis of phosphatidylcholine from the hydrolysis of sphingomyelin and/or transfer of phosphorylcholine to phosphatidylcholine was also partially reduced.

Desipramine elicits the expression of opiate receptors and sulfogalactosylceramide synthesis in rat C6 glioma cells.

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.

Effect of tricyclic antidepressants on sphingomyelinase and other sphingolipid hydrolases in C6 cultured glioma cells.

Cationic amphiphilic drugs, which include tricyclic antidepressants, have been shown to give rise to lipidoses under experimental conditions, with a general increase of lipids especially phospholipids. We report here an early and important decrease in sphingomyelinase activity in C6 glioma cells cultured in the presence of imipramine or desipramine at final concentrations of 0.01 and 0.05 mM. The effect was both dose-dependent and time-dependent and was observed before any lipid accumulation. Cerebroside beta-glucosidase and cerebroside beta-galactosidase had normal activities under the same experimental conditions and thus there was no general effect on membrane-bound sphingolipid hydrolases. A decrease of sphingomyelinase activity has been previously reported for two amphiphilic compounds, perhexiline maleate and AY 9944. These results suggest a potential function of sphingomyelinase in the mode of action of these drugs.

[Study of thesaurismosis induced by perhexiline maleate. Confirmation of experimental data]. / Etude d'une thésaurismose induite par le maléate de perhexiline. Confirmation des données expérimentales.

Perhexiline maleate is an amphiphilic molecule. Along with many other drugs it is responsible for experimental and, in some instances, clinical lipidoses. Sphingomyelinase deficiency has been evidenced in cell cultures incubated with perhexiline maleate. We describe the occurrence of a similar defect in a patient. The disturbances in the phospholipid turnover which are responsible for the thesaurismosis may originate in the sphingomyelinase deficiency.

Tricyclic antidepressant desipramine induces stereospecific opiate binding and lipid modifications in rat glioma C6 cells.

Incubation for 48 hours of C6 glioma cell cultures with 10(-4)M tricyclic antidepressant desipramine gave rise to a quantitative increase of total lipids and to qualitative modifications of glycosphinegolipids involving detection by thin-layer chromatography of spots migrating according to cerebroside and sulfatide and presence of an abnormal ganglioside pattern. These lipid modifications were associated with the appearance of stereospecific binding of opiates (dihydromorphine) with a dissociation constant of 30-60 nM. These results favor an important role of lipids in opioid receptor function.

Tricyclic antidepressants induce sphingomyelinase deficiency in fibroblast and neuroblastoma cell cultures.

Tricyclic antidepressants (imipramine and desipramine) gave rise to an important decrease of sphingomyelinase activity in murine neuroblastoma and human fibroblast cell cultures. It occurred within 1 to 2 hours at a final concentration of 1 or 2 X 10(-5) M in cell culture medium. Other lysosomal enzymes such as acid lipase, arylsulfatases A and B and hexosaminidases were not modified. Low level of sphingomyelinase activity may be related to the amphiphilic characteristics of the drugs: iminodibenzyle which has the same tricyclic core but is devoid of the side chain necessary for amphiphilic properties had no effect. As iminodibenzyle has no therapeutic action, amphiphilic may be requisite to antidepressant properties of tricyclic drugs.

Lipid and lysosomal enzymes in human fibroblasts cultured with perhexiline maleate.

To understand the mechanism of the lysosomal lipid storage induced by perhexiline maleate, we performed simultaneous lipid analysis and lysosomal enzymes determinations. Human fibroblasts were cultured for 5 days in the presence of perhexiline maleate at a concentration of 2 micrograms/ml of culture medium. Lipid analysis showed that those non toxic levels determined the same changes as seen with higher concentrations of the drug (Hauw et al. 1980) i. e. increase of cholesterol and of all major phospholipids. Qualitative phospholipid pattern was not markedly changed. Lysosomal enzymes activities were not modified with the exception of sphingomyelinase which was reduced to 12% of its normal level.

Perhexiline maleate-induced lipidosis in cultured human fibroblasts: cell kinetics, ultrastructural and biochemical studies.

Perhexiline maleate reduced the growth of human skin fibroblasts in cell culture at a concentration range of 0.3-3 micrograms/ml. At the highest concentration, the cells survived only four days. Pleomorphic inclusions characteristic of drug-induced phospholipidoses appeared in cultured cells. Analysis of the major lipid classes was performed on cells exposed to 3 micrograms/ml at four days. Gangliosides, phospholipids and cholesterol levels four to six times above controls were found. No major qualitative abnormalities were detected in phospholipids. On the contrary, an abnormal pattern of gangliosides was seen by densitometry of silica gel thin-layer plates with increases of GD3 and of an unknown ganglioside. Drug induced lipidosis may involve other lipids than phospholipids, particularly gangliosides.

Bismuth intoxication: bismuth level in pig brain lipids and in subcellular fractions.

Experimental intoxications of pigs were performed. It seems to be confirmed that bismuth crosses the blood-brain barrier. An organic derivative, trivinyl-bismuth, is more active than the inorganic salt of bismuth. In the brain, bismuth is preferentially found in synaptosomes. Bismuth is found in brain lipids of control pigs at very low levels. In intoxicated pigs, the level of bismuth is increased mainly in cerebellum and then in thalamus. Bismuth is partly associated to lipids. There is not a correlation between the level of bismuth in blood and in brain lipids. However, in the trivinyl intoxicated pig, a high level of blood bismuth is concomittant to a high level of brain lipid bismuth. The high content of this metal in cerebellum lipid extract may be of functional significance.

[Extraction of plasma lipids preserving antigenic properties of proteins and allowing quantitation of gangliosides by neuraminic acid determination (author's transl)]. / Méthode d'extraction des lipides plasmatiques conservant les propriétés antigéniques de protéines et permettant le dosage de l'acide sialique des gangliosides.

A technique described by Cham and Knowles allows the extraction of plasma phospholipides, triglycerides, cholesterol and free fatty acids without denaturing the proteins. The method has been slightly modified by us. Under our conditions, the yield of lipid extraction is comparable to the method of Folch. It has the advantage of not extracting mineral phosphorous and blood glucose. Thus gangliosides can be also easily determined. We confirmed by immunoelectrophoresis that the antigenic properties of plasmatic proteins remain after lipid extraction.