C. Mazière

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Abstract Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-14C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the in situ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in vitro by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.

Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo

We describe an ELISA technique able to recognize malondialdehyde-modified low-density lipoproteins (LDL). For this purpose we produced antibodies to malondialdehyde-LDL, specific for the malondialdehyde modification of LDL; these antibodies recognized essentially malondialdehyde-LDL. Coating ELISA plates with the antibodies to malondialdehyde-LDL and using peroxidase-labelled antibodies to LDL, which reveal only apolipoprotein B, we obtained an accurate method of detecting malondialdehyde-modified apolipoprotein B. Preliminary studies demonstrated that this method allows the detection of lipoproteins containing malondialdehyde-modified apolipoprotein B in the serum of patients with cardiovascular diseases.

Glucose-enriched medium enhances cell-mediated low density lipoprotein peroxidation

A 1 week preculture of endothelial or smooth muscle cells in glucose‐enriched (11.2 to 44.8 mM) media resulted in a marked enhancement of the subsequent ability of cells to oxidize low density lipoprotein, as assessed by the lipid peroxidation end product and conjugated diene content of the particle, its relative electrophoretic mobility and its degradation by macrophages. This phenomenon is correlated to a marked stimulation of superoxide anion secretion by cells. Such an effect of elevated glucose concentration on cell‐induced LDL oxidative modification could be involved in the increased occurrence of atherosclerotic lesions in diabetes mellitus.

Rapid analysis of cellular lipids without extraction

A method is described where cell suspension obtained by scraping of monolayers was directly applied on silica gel plates. Extraction and separation of different lipid classes were simultaneously obtained during chromatography. In the range of validity of the method (no more than 80 micrograms of cellular protein tested for neutral lipids and 30 micrograms for phospholipids), this technique allows rapid lipid analysis of small samples of cultured cells, bypassing all the time- and solvent-consuming extraction and evaporation steps. The method appears to be also suitable for measurement of enzymes of lipid metabolism such as acyl coenzyme A-cholesterol-acyltransferase.

PHENOTHIAZINES INHIBIT COPPER AND ENDOTHELIAL CELL-INDUCED PEROXIDATION OF LOW DENSITY LIPOPROTEIN A COMPARATIVE STUDY WITH PROBUCOL, BUTYLATED HYDROXYTOLUENE AND VITAMIN E

The effect of two phenothiazines, chlorpromazine (CPZ) and trifluoperazine (TFP) on the copper and endothelial cell-induced peroxidation of low density lipoprotein (LDL) has been studied and compared to that of drugs previously shown to protect LDL against peroxidation: probucol (PBC) and butylated hydroxytoluene (BHT). Incubation with CPZ or TFP inhibited in a dose-dependent manner LDL peroxidation induced either by copper ions or by cultured endothelial cells. Both the electrophoretic mobility and the thiobarbituric reactive substance content of LDL returned to almost normal values in the presence of 50 microM CPZ or TFP. The two studied phenothiazines also strongly inhibited the hydrolysis of LDL phosphatidylcholine which accompanies copper or endothelial cell-induced peroxidation of the particle. CPZ and TFP were as effective as PBC and BHT in inhibiting the LDL peroxidation. Whereas copper or endothelial cell-oxidized LDL were recognized and rapidly catabolized by mouse peritoneal macrophages, CPZ- or TFP-, as well as PBC- or BHT-treated LDL were not. Moreover, it was found that, in contrast to vitamin E, neither CPZ nor PBC reacted with model peroxy radicals formed by gamma irradiation of aerated ethanol. The possible mechanisms underlying this protective effect of phenothiazines against LDL oxidative modification are discussed.

Inhibition of human fibroblasts sphingomyelinase by cholesterol and 7-dehydrocholesterol.

Abstract An inhibition of human fibroblast sphingomyelinase by cholesterol and 7-dehydrocholesterol is shown. This effect is obtained for cholesterol and 7-dehydrocholesterol/sphingomyelin molar ratios above 0.1. Diffusion measurements performed on mixed liposomes demonstrated for cholesterol/sphingomyelin and 7-dehydrocholesterol/sphingomyelin molar ratios above 0.1 a sharp increase in diffusion intensity. The mechanism of the inhibition of sphingomyelinase by sterols is discussed in relation to the physical state of the substrate. A possible involvement of this phenomenon in sphingomyelin accumulation observed in aging or in atheroma is discussed.

Alterations in cholesterol metabolism in cultured fibroblasts from patients with Niemann-Pick disease type C.

SummaryCholesterol synthesis, esterification and efflux were investigated in cultured fibroblasts from patients with Niemann-Pick disease type C. Sterol synthesis from sodium acetate was markedly increased in the two Niemann-Pick disease type C strains as compared to controls, either in the presence or absence of exogenous cholesterol supply by low-density lipoproteins. By contrast, cholesterol esterification was about 2–3-fold reduced when measured by oleic acid incorporation into cholesteryl esters and 10–15-fold reduced when measured with labelled free cholesterol as precursor, although acylcoenzyme-A: cholesterol acyltransferase activity was normal when studiedin vitro on cell homogenates. Chase experiments with14C-cholesterol demonstrated that the rate of cholesterol efflux was decreased by about 3–4-fold in fibroblasts from patients with Niemann-Pick disease type C. These results provide further evidence for alterations of sterol metabolism in Niemann-Pick disease type C and support the hypothesis of a trapping of exogenous cholesterol, which cannot enter the regulatory intracellular pools.

Effect of cyclic AMP on low density lipoprotein binding and internalization by cultured human fibroblasts

Pretreatment of cultured human fibroblasts by cyclic AMP resulted in a marked decrease in the binding and internalization of the low density lipoproteins (about 55% of controls for cyclic AMP 2.10(-3) M). This effect was dose dependent and increased by theophyllin. DL propranolol, an inhibitor of adenylcyclase, had an opposite effect. Isoproterenol, which stimulates adenylcyclase, reproduced the effect of cyclic AMP. The cholesterol synthesis from [2-14C] acetate was decreased by cyclic AMP, theophyllin and isoproterenol, and increased by propranolol. The incorporation of [1-14C] oleate into cholesteryl esters was reduced by cyclic AMP, theophyllin, isoproterenol and propranolol.

Epinephrine decreases low density lipoprotein processing and lipid synthesis in cultured human fibroblasts

The effect of epinephrine on 125I-low density lipoprotein (LDL) uptake and cholesterol metabolism was investigated after a 24 hours pretreatment of cultured human fibroblasts. Epinephrine decreased LDL uptake (binding + internalization) and degradation in a dose-dependent manner. Cholesterol synthesis from 14C sodium acetate and cholesterol esterification measured by 14C oleic acid incorporation into cholesteryl esters were also decreased. These results are in agreement with the general view that epinephrine increases cyclic AMP intracellular level, as it was previously demonstrated that dibutyryl cyclic AMP or isoproterenol treatment of cultured fibroblasts had similar effect on these pathways. The decrease in LDL processing induced by epinephrine could be involved in the worsening effect of epinephrine on atherosclerosis.

Cholesterol and 7 dehydrocholesterol inhibit the in situ degradation of sphingomyelin by cultured human fibroblasts

Feeding cultured human fibroblasts with cholesterol and 7-dehydrocholesterol resulted in a strong decrease of the in situ degradation of sphingomyelin (about 20 and 40 fold reduction for cholesterol and 7-dehydrocholesterol respectively, at 50 micrograms/ml of medium, and for 24 h incubation). Measurement performed on cell homogenates showed a slight decrease of the sphingomyelinase activity (about 75% of controls), whereas the activities of other lysosomal enzymes (beta glucosaminidase, beta galactosidase) were not significantly affected.