Ivan Filipovic

Calcium channel blockers stimulate LDL receptor synthesis in human skin fibroblasts

Human skin fibroblasts incubated in lipoprotein-deficient medium in the presence of 50-100 microM of the calcium channel blockers verapamil or diltiazem incorporated up to 2.5 times more [35S]methionine into immunoprecipitable LDL receptor protein than did control cells. Verapamil was found to be more potent in this regard than diltiazem. The calcium channel blockers did not influence the overall synthesis of cellular proteins or the half-life of the LDL receptor, and they were not able to prevent the suppression of LDL receptor synthesis caused by exogenous LDL or 25-hydroxycholesterol. The calcium channel blocker-induced stimulation of LDL receptor synthesis was accompanied by a corresponding increase in binding and internalization of [125I]LDL, but the degradation of internalized lipoprotein was slightly decreased. The results suggest that intracellular Ca2+ levels modulate LDL receptor metabolism in human skin fibroblasts.

Role of net charge of low density lipoproteins in high affinity binding and uptake by cultured cells.

Abstract Selective modification of arginine residues of LDL by cyclohexanedione or acetylation of lysine residues of LDL deminishes their high affinity binding and internalisation by human skin fibroblast up to 50% as compared with native LDL. The enhanced negative charge of the modified LDL particles results in an accelerated electrophoretic mobility towards the anode. Neuraminidase treatment of cyclohexanedione-modified LDL and acetyllysine-LDL normalizes not only their electrophoretic mobility, but also restores more than 80% of the original binding and uptake capacity, the specificity of this effect being indicated by using fibroblasts deficient in LDL receptor and by competitive binding and internalization experiments.

Comparative studies on fatty acid synthesis in atherosclerotic and hypoxic human aorta.

The oxygen and glucose uptake, lactate formation, ATP/ADP and NADH/NAD ratios and incorporation of [14C]acetate and [14C]linolenic acid into lipids of early fatty streaks and more advanced complicated atherosclerotic lesions of human aorta were determined during aerobic and hypoxic incubation. Compared with grossly normal appearing sections of the aorta in intima and media preparations of early fatty streaks the oxygen uptake was increased while that in further developed atheroma was slightly diminished. Under aerobic incubation conditions the metabolic state of fatty streaks and atheroma was characterized by increased lactate formation, NADH/NAD ratio and incorporation of [14C]acetate and [14C]linolenic acid into the lipids, but by a lowered ATP/ADP ratio. More pronounced changes in these metabolic parameters were observed when the aortic tissue segments were incubated under hypoxic conditions. The analysis by argentation TLC of fatty acid methylesters derived from total lipids of aerobically incubated fatty streaks revealed an increased incorporation of [14C]acetate into the highly unsaturated long-chain fatty acids. In developed atherosclerotic lesions and in hypoxia the incorporation of radioacetate into the polyunsaturated fatty acids and the formation of 20:4 fatty acid from [14C]linolenic acid were, in contrast to the above finding, decreased while the synthesis of eicosatrienoic acid was increased. This finding suggests a block in the desaturation step of linoleic into 20:4 fatty acid in further developed atheroma and in hypoxia. In aerobically incubated atherosclerotic lesions and in hypoxia the palmitic acid was synthesized mainly by chain elongation while in grossly normal areas of the aorta at least part of this acid was synthesized de novo.

Calmodulin antagonists stimulate LDL receptor synthesis in human skin fibroblasts

The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.

Calmodulin antagonists suppress cholesterol synthesis by inhibiting sterol Δ24 reductasereductase

Preincubation of hepatoma cells and human skin fibroblasts in the presence of the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide resulted in a dose-dependent suppression of [14C] mevalonolactone incorporation into cholesterol. At a calmodulin antagonist concentration of 25 μmol, the incorporation of [14C] mevalonolactone into cellular cholesterol was suppressed to about 30% (hepatoma cells) and 10% (human skin fibroblasts) of control values. When the total nonsaponifiable [14C] lipids were separated and analyzed by two-dimensional thin layer chromatography, an accumulation of [14C] desmosterol was observed along with reduced formation of [14C] cholesterol. However, when cells were preincubated in the presence of [14C] dihydrolanosterol, [14C] cholesterol formation was not inhibited by the calmodulin antagonists. About 25% of the cell-associated dihydrolanosterol radioactivity was converted to cholesterol in both control and calmodulin antagonist-pretreated cells. The data suggest that calmodulin antagonists prevent the conversion of desmosterol into cholesterol by inhibiting sterol Δ24 reductase and that the enzymes catalyzing sterol ring modifications are not affected by the inhibitors.

SPHINGOLIPID-INDUCED ENHANCEMENT OF RECEPTOR-MEDIATED UPTAKE OF LOW DENSITY LIPOPROTEINS IN NORMAL AND RECEPTOR-DEFICIENT HUMAN SKIN FIBROBLASTS

(1) The receptor mediated endocytosis of homologous LDL by human skin fibroblasts can be significantly enhanced by prior incubation of the cells with sphingolipids. Gangliosides GM1 or GD1a, their desialylated derivatives and sphingosine stimulate binding and uptake to LDL by up to 40% of normal values. The effect is observed in normal fibroblasts, LDL receptor deficient fibroblasts or in tunicamycin-treated cells with a reduced number of functional receptors but is dependent on the time of preincubation of the cells and the concentration of the sphingolipid in the medium. (2) Detailed studies on the ganglioside effect revealed, that cell bound gangliosides intensify the LDL-induced suppression of [14C] acetate incorporation into cholesterol. (3) The receptor dependence and relative receptor specificity of the sphingolipid effect is evident from the fact that (a) after complete suppression of receptor synthesis gangliosides fail to stimulate uptake of LDL, that (b) fatty acids or lipids not containing sphingosine are without effect and that (c) the receptor specific internalisation of alpha 2-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.

Comparative Studies on the Chemistry and the Metabolism of Arterial and Venous Tissue

Comparative studies of the chemistry and metabolism of human and bovine arterial and venous tissue had the following results (values for bovine tissue in parenthesis): 1. In arterial tissue the contents of collagen were 21% (37%) and of elastin 20% (31%) referred to the dry weight. In veins collagen amounted to 47% (30%) and elastin to 7% (18%). The mucopolysaccharide concentration was higher in arterial (1.2 and 1.5 resp.) than in venous tissue (0.4 and 0.2% resp.). Hyaluronate, chondroitin 6-sulphate (chondroitin 4-sulphate), dermatan sulphate and heparan sulphate were identified. In arterial tissue chondroitin sulphate was predominant with 59 % and 49 % resp. In human venous tissue the main component was dermatan sulphate (65%), in bovine veins it was hyaluronate (75%). No dermatan sulphate was found in bovine venous tissue. The number of cells was higher in arteries (4.5 × 107 nuclei/g w. wt.) than in veins (2.8 × 107). 2. On in vitro incubation bovine arterial tissue metabolized 73 %, venous tissue 32% of the added [U-I4C] glucose within 12 h. In this period 2.8 and 2.4 mg lactate resp./g wet tissue were formed by arteries and veins. The specific radioactivity of lactate synthesized in arterial tissue reached that of the added [U-I4C] glucose whereas lactate formed by veins had only 36 % of the specific activity of the [U-I4C] glucose. The specific radioactivity of the acid mucopolysaccharides was 5–6 times higher in venous tissue than in arterial tissue. After labelling with 35S-sulphate a 3-fold higher specific radioactivity of venous sulphated mucopolysaccharides was observed. 3. After incubation of bovine venous tissue in the presence of I4C-acetate the specific radioactivity of phospholipids was 45 % higher than that of arterial tissue in contrast to a 50 % lower labelling of cholesterol in venous tissue.

p-Chlorophenoxyisobutyrate enhanced retention of homologous lipoproteins by human aortic smooth muscle cells.

Human aortic smooth muscle cells (SMC) specifically bind and take up indiscriminately both the lipid and protein moietics of homologous25I-very low density lipoproteins (VLDL) and125I-low density lipoproteins LDL). Sixty-five to 80% of absorbed lipids are incorporated into the cell lipids, preferentially into the phospholipid fraction. Twenty to 35% of the lipid bound and the protein moiety are eliminated from the cells. Half of the eliminated protein label is recovered as TCA soluble products. Five mM of p-chlorophenoxyisobutyrate (CPIB) raise the level of intracellular radioactivity derived from the lipid moieties of VLDL and LDL by about 40% via a reduced elimination. The processing of the protein moiety and lipoprotein binding to the cell surface are not affected by 5.0 mM of CPIB. CPIB lowers the incorporation of14C-acetate,14C-pyruvate, and32phosphate radioactivity into fatty acids and phospholipids of aortic SMC. Five mM of CPIB reduce the overall palmitic acid synthesis by shifting from de novo synthesis to the mechanism of chain elongation, although the further elongation to saturated C18–C24 fatty acids is also depressed. The CPIB-enhanced retention of the lipid-derived lipoprotein radio-activity is interpreted as a compensatory mechanism providing cellular fatty acids which are deficient as a result of the CPIB inhibited synthetic processes.

Glucose and Lipid Metabolism in the Human Arteriosclerotic Aorta

The chemical composition and the origin of human arteriosclerotic lesions have been extensively studied, but information on the metabolism of human arterial tissue and its changes in arteriosclerosis is limited. Though the ability of mammalian arterial tissue to synthesize fatty acids has been the subject of several reports (Whereat et al., 1967; Howard Jr., 1968; Filipovic and Buddecke, 1971; Filipovic et al., 1973), the lipid accumulation in human arteriosclerosis is thought to be the result of infiltration of plasma lipids into the arterial wall. In this study it will be shown that 14C-fatty acid and triglyceride synthesis, respectively, in arteriosclerotic parts is much greater than that in nonarteriosclerotic parts of the same aorta.

In vitro glucose metabolism and lipid biosynthesis in bovine ventricular valves

Abstract In vitro oxygen uptake, pathways of [U- 14 C] glucose degradation and lipogenesis in the mitral and tricuspid valve of normal bovine heart were compared. In the mitral valve the oxygen consumption, ATP ADP ratio, lipid content and the amounts of [ 14 C]-glucose and [ 14 C]-acetate incorporated into total lipids per mg DNA were higher, while lactate production and NADH NAD ratio were lower, than those found in tricuspid valve. Triglycerides and phospholipids represented the largest lipid fractions in the mitral and tricuspid valve, respectively, and contained about 60% of the radioactivity incorporated into total lipids, but the highest relative rate of synthesis in the mitral and tricuspid valve was calculated to be for phospholipids and triglycerides, respectively. The bulk (94 to 98%) of [ 14 C]-glucose incorprated into phospholipids and triglycerides by ventricular valves was found in the glycerol moiety. Incorporation of [ 14 C]-glucose into fatty acids was markedly lower in the tricuspid than in the mitral valve. In the mitral valve the synthesis of 16 : 0 fatty acid from [I- 14 C] acetate proceeded de novo while in the tricuspid valve by a combination of de novo synthesis and chain elongation.