Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. II. Uptake and cytotoxicity of ultraviolet-treated LDL on lymphoid cell lines.

The 'cytotoxicity' of ultraviolet-treated low-density lipoproteins (LDL) has been investigated using cultured lymphoid cell lines from normal subjects and from a patient with receptor-negative familial hypercholesterolemia. The ultraviolet-treated LDL were taken up by control lymphoblasts through the classical apo B/E-receptor pathway, while they were slowly taken up by receptor-negative lymphoblasts by non-specific endocytosis. These LDL were found highly 'cytotoxic' on normal lymphoblasts as demonstrated by Trypan blue dye uptake, [3H]thymidine incorporation, lactate dehydrogenase release and by electron microscopy.

Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cell. I. Chemical modifications of ultraviolet-treated low-density lipoproteins.

A new experimental model system constituted by ultraviolet-treated low-density lipoproteins (LDL) has been designed in order to investigate the biological effects of lipid peroxides entering the cell through the endocytotic pathway. This paper reports the chemical modifications of the lipid components and apolipoproteins of the ultraviolet-treated LDL. Human LDL were submitted to short ultraviolet radiations (254 nm, 0.

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols.

Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins.

Human HDL subfractions (HDL2, HDL3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [14C]cholesterol and [3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A2 from Crotalus adamanteus in presence of albumin leading to a 72-82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes.

Acid lability of the mutated glucosylceramide-beta-glucosidase in a lymphoid cell line from type 2 Gaucher disease.

Lymphoid cell lines from patients with infantile (type-2) and juvenile (type 3) Gaucher disease have been established by Epstein-Barr virus transformation and investigated and compared with the adult phenotype (type 1) with the view to enzymology. The enzymatic defect in glucosylceramide(GlcCer)-beta-glucosidase activity was more severe in type 2 and 3 than in type 1 cells. The mutant GlcCer-beta-glucosidase from our studied type 2 lymphoid cells was profoundly labile at pH 4.

[The cytotoxicity of oxidized lipoproteins].

The peroxidation of low-density lipoproteins (LDL) and their subsequent cytotoxicity is believed to be involved in the atherogenesis. The aim of this work was to determine the possible involvement of lipid peroxides in the cytotoxicity of lipoproteins. We report a new experimental model system constituted by lipoproteins treated by short-UV radiations which result in a major lipid peroxidation without functional alteration of apoproteins.

Metabolism of pyrenedecanoic acid in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and from a patient with multisystemic lipid storage myopathy.

A lymphoid cell line has been established from a patient with multisystemic lipid storage myopathy and showed a major triacylglycerol storage, whereas the content of other neutral lipids and phospholipids was in the normal range. The metabolism of the triacylglycerols has been investigated in this lymphoid cell line from multisystemic lipid storage myopathy as well as in control cells through pulse-chase experiments using 10-(1-pyrene)decanoic acid (P10), a fluorescent fatty acid derivative, as precursor. After 1 h incubation, the uptake of P10 was not significantly different in multisystemic lipid storage myopathy and control lymphoid cells.

Stimulation of 12-HETE production in human platelets by an immunomodulator, LF 1695. Evidence for activation of arachidonate liberation coupled to cyclo-oxygenase inhibition.

Upon incubation with human platelets previously labelled with [14C]arachidonic acid, a new immunomodulator, LF 1695, induced the accumulation of [14C]-12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Although the time course of [14C]HETE accumulation was identical with 60 microM LF 1695 and calcium ionophore A23187, the latter compound also promoted the formation of 14C-labelled thromboxane B2 and 12-(S)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), whereas 12-HETE was the only arachidonic acid metabolite generated under the action of LF 1695, suggesting that the drug inhibited cyclo-oxygenase. This was further confirmed by the fact that LF 1695 inhibited the second wave of platelet aggregation induced by ADP as well as arachidonic acid effects.

In vitro detergent activation of lysosomal acid beta-glucosidase in the spleen of normal and type 1 Gaucher patients is not accompanied by change in aggregation state.

The genetic defect in Gaucher disease consists in a deficiency of a membrane-bound lysosomal acid beta-glucosidase. Using the radiation inactivation method, we have previously reported a subunit coupling of the mutated acid beta-glucosidase from Gaucher type 1 spleen in contrast to the normal one (Maret, A., Potier, M.

Independence of triacylglycerol-containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy.

The functional relationship between the two subcellular compartments involved in catabolism of triglycerides, i.e. lysosomes and lipid-containing cytoplasmic vacuoles, has been investigated using cultured fibroblasts from patients affected with two different genetic lipid (triacylglycerol) storage disorders: Wolman disease and multisystemic lipid storage myopathy.

Phosphatidylcholine and triacylglycerol hydrolysis in HDL as induced by hepatic lipase: modulation of the phospholipase activity by changes in the particle surface or in the lipid core.

(1) Human HDL2 (d 1.063-1.125) and HDL3 (d 1.

Modified lipid-protein interactions in Tangier beta lipoprotein (LDL2) demonstrated by fluorescence quenching.

Fluorescence quenching by iodide ions has been found to be higher in isolated Tangier low density lipoprotein (LDL2) than in isolated normal LDL2. Apolipoprotein (apo) B-100 is the main protein component of these lipoproteins and its tryptophanyl residues (Trp) are known to be the most hydrophobic and to be responsible for protein fluorescence. Trp exposure can thus be calculated; it was 0.

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice.

Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.

[Effects of pentoxifylline and propentofylline on the turnover of polyphosphoinositides of the erythrocyte membrane and on the metabolism of arachidonic acid in platelets].

[gamma-32p] ATP incorporation into polyphosphoinositides of the erythrocyte membrane has been studied in the presence of increasing concentrations of pentoxifylline and propentofylline. Our results show an inhibitory effect on the labelling of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, higher with propentofylline. We have then demonstrated that these two drugs block the metabolism of arachidonic acid in platelets stimulated by thrombin.

Effects of two methylxanthines, pentoxifylline and propentofylline, on arachidonic acid metabolism in platelets stimulated by thrombin.

[3H]Pentoxifylline and [3H]propentofylline were taken up by human platelets in a dose-dependent manner probably involving a passive diffusion through the plasma membrane. In vitro, the two drugs were able to inhibit platelet activation induced by thrombin. serotonin secretion was reduced from 57% to 38% and 28% in the presence of 1 mM pentoxifylline and 1 mM propentofylline, respectively.

Accumulation of large VLDL in cyclophosphamide treated rabbits. Relationship with lipoprotein lipase deficiency.

Rabbit very low density lipoproteins (VLDL) have been fractionated by heparin sepharose chromatography into two subpopulations: an unretained fraction (UR) and a retained fraction (R). The separation profiles of VLDL from cyclophosphamide treated rabbits differed from those obtained in normal animals: UR fraction was far more important in treated rabbits than in control animals. Comparative studies of the two VLDL subfractions isolated from treated rabbits have been performed.

Effect of PCR 4099 on ADP-induced calcium movements and phosphatidic acid production in rat platelets.

Antiplatelet activity of PCR 4099, an analogue of ticlopidine, resides in its specific effect against exogenous as well as released ADP. This study investigated in rat platelets the effects of the drug on ADP-induced shape change, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Shape change and influx of Ca2+ ions across the plasma membrane were not modified after PCR 4099 administration using aspirin-treated platelets.

Relative fluorescence of normal and acid lipase-deficient cultured fibroblasts following administration of pyrene decanoic acid.

Skin fibroblasts, derived from normal individuals or patients with Wolman's disease (an autosomal recessive disorder due to acid lysosomal lipase deficiency) were incubated with the fluorescent fatty acid, pyrene-decanoic acid (P10). Measurements of the fluorescence intensities of the total lipid extracts indicated that equal quantities of P10 were incorporated into both cell types. The fluorescence emitted by the intact cells was subsequently recorded in a fluorescence microscope equipped with a microdetector unit, which permitted determination of the fluorescence emitted by the intact cell or by specific regions thereof.

Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase.

A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein.

Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors.

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers.

Effect of a stimulant of guanylate cyclase, sin 1, on calcium movements and phospholipase C activation in thrombin-stimulated human platelets.

The effects of sin 1, a metabolite of an antianginal agent, molsidomine, were investigated on human platelet activation induced by thrombin. This drug promoted a slight inhibition of serotonin release in a medium containing 1 mM Ca2+ or 1 mM EGTA (from 63% to 46% and from 57% to 41% of total serotonin secretion, respectively, with the highest dose used). Under these conditions, Ca2+ movements, monitored by quin 2 fluorescence, were markedly impaired.