Uptake and aminoacylation of exogenous Escherichia coli tRNA by mouse fibroblasts.

Mouse fibroblasts (L-cells) in suspension culture take up exogenous $\text{Escherichia coli}$ tRNA in the presence of DEAE-Dextran. Tritium-labeled formylmethionine tRNA and valine tRNA are both taken up at very low levels. Tritium activity is associated solely with 4S material as judged by chromatography of cell extracts on Sephadex G-100. Further analysis of this material on a dihydroxyboryl-substituted cellulose indicates that a small portion of the tRNA taken up is acylated by the L-cells.     

Effects of different types of polyunsaturated fatty acids on cholesterol esterification in human fibroblasts.

We have enriched human fibroblasts with oleic acid, with linoleic acid and with eicosapentaenoic acid. The accumulation of cholesteryl esters in the cells and the rate of esterification of cholesterol by microsomal acyl-CoA:cholesterol acyltransferase (ACAT) were measured in these cells. Cholesteryl ester levels were lower in cells enriched with eicosapentaenoic acid compared with cells enriched with oleate or linoleate. We also observed significantly lower ACAT activities in the microsomes from fibroblasts enriched with the n-3 polyunsaturated fatty acids relative to cells enriched with oleic acid or linoleic acid. We suggest that the presence of n-3 polyunsaturated fatty acids might suppress cholesteryl ester accumulation and inhibit atherogenesis.     

Inhibition of cellular cholesterol esterification can decrease low density lipoprotein receptor number in human fibroblasts

In fibroblasts deprived of exogenous cholesterol to induce low density lipoprotein receptors there is a continuing flux of cholesterol esterification. The structurally unrelated inhibitors of acyl-CoA; cholesterol acyl-transferase, progesterone, trimethylcyclohexanyl mandelate and 3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl] propanamide, (58035), could all inhibit this basal rate of esterification within 1h of addition. Exposure of cholesterol-deprived fibroblasts for 17h to progesterone or trimethylcyclohexanyl mandelate caused decreased specific binding and metabolism of low density lipoprotein. The effect was not a direct inhibition of lipoprotein binding; it was time dependent and followed from the reversible inhibition of cholesterol esterification by these two compounds. The irreversible inhibition of esterification by 58035 left the receptor number unaffected. The results indicate that down regulation of low density lipoprotein receptors is initiated by accumulation of cholesterol in a specific intracellular pool. Inhibition of cholesterol esterification by progesterone and trimethylcyclohexanyl mandelate causes accumulation of cholesterol in this pool but 58035 does not.     

Niemann-Pick type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis.

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (greater than 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [3H]oleic acid into cholesterol-[3H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimulated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquine abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

Changes in the biosynthesis, esterification and efflux of cholesterol in fibroblasts in culture from patients with Niemann-Pick disease type C

Cholesterol synthesis, esterification and efflux have been comparatively studied in control fibroblasts and in fibroblasts from patients with Niemann-Pick disease type C (NPC). Sterol synthesis was markedly increased in NPC cells as compared to controls, either in whole medium or in medium devoid of lipoproteins. 14C-oleic acid incorporation into cholesteryl-esters was 2 to 3 fold reduced in NPC cells, and esterification of 14C-exogenous cholesterol was dramatically (15-30 fold) decreased. ACAT activity, measured in vitro, was not significantly altered in NPC cells. Finally, cholesterol efflux appeared to be decreased in NPC fibroblasts as compared to controls. The hypothesis of a defect in exogenous cholesterol access to intracellular regulatory pools is proposed.     

Uptake of HDL unesterified and esterified cholesterol by human endothelial cells. Modulation by HDL phospholipolysis and cell cholesterol content

Human HDL (1.070-1.210), doubly labelled with ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol were incubated for 1–5 h with monolayer cultures of human endothelial cells. HDL were preincubated for 60–120 min the presence of albumin and with/without purified phospholipase A₂ (control HDL, phospholipase A₂ HDL) before dilution in the cell culture medium. Average phosphatidyl-choline (PC) degradation was 62.10% ± 2.57% (range 45–80%). A purified lipase /phospholipase A₁ from guinea pig pancreas was used in some experiments (range of PC hydrolysis: 16–70%). (1) ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol appeared in cells during 0–5 h incubations. Trypsin treatment allowed a simple adsorption of HDL onto the cell surface to be avoided, and most of the ³H-labelled esterified cholesterol transferred to cells was hydrolysed. Cell uptake of radioactive cholesterol increased as a function of HDL concentration but no saturation was achieved at the highest lipoprotein concentration used (200 μg cholesterol/ml). Flux of ³H/¹⁴C-labelled unesterified cholesterol was related to the cell cholesterol content, suggesting that it might partly represent an exchange process. The cell cholesterol content was slightly increased after 5 h incubation with HDL (+16%). (2) Pretreatment of HDL with purified phospholipase A₂ doubled on average the amount of cell recovered ³H-labelled esterified cholesterol, while the flux of ³H/¹⁴C-labelled unesterified cholesterol was enhanced by 15–25%. Both transfer and cell hydrolysis of ³H-labelled esterified cholesterol were increased. A stimulation was also observed using purified lipase/phospholipase A₁, provided that a threshold phospholipid degradation was achieved (between 27 and 45%). (3) Endothelial cells were conditioned in different media so as to modulate their charge in cholesterol. The uptake of ³H-labelled esterified cholesterol was found to be significantly higher in cholesterol-enriched cells compared to the sterol-depleted state. Finally, movements of ³H-labelled esterified cholesterol from HDL to endothelial cells were essentially unaffected by cell density or by the presence of partially purified cholesterol ester transfer protein. The possible roles of the transfer of HDL esterified cholesterol to endothelial cells and its modulation by phospholipases are discussed.     

Inhibition of human fibroblasts sphingomyelinase by cholesterol and 7-dehydrocholesterol

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.     

Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to shingomyelin hydrolysis

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (> 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [³H]oleic acid into cholesterol-[³H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimuated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquinone abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

Regulation of cholesterol esterification by micellar cholesterol in CaCo-2 cells

The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.     

Disposition of intracellular cholesterol in human fibroblasts

We have examined the intracellular distribution of unesterified cholesterol in cultured human fibroblasts. Intact cells were treated with cholesterol oxidase to selectively transform cell surface cholesterol to cholestenone. Isopycnic centrifugation of homogenates showed that the cholestenone had a peak buoyant density of 1.13 g/cm3. The approximately 10% of total cholesterol which remained unoxidized was distributed in two peaks of roughly equal size: a sharp peak at approximately 1.09 g/cm3 and a broad peak centered at 1.18 g/cm3. When intact cells were incubated with exogenous [3H]cholesterol, the radiolabel entered the nonoxidizable pool in a temperature-dependent fashion with a half time of 3 h at 37 degrees C. This label initially was associated with the dense but not the buoyant peak of nonoxidized cholesterol. After 40 h, the buoyant peak also became labeled; both peaks then had a specific activity slightly less than the surface cholestenone. The buoyant density of the unoxidized cholesterol did not coincide with markers for the Golgi apparatus, endoplasmic reticulum, or lysosomes. However, two ingested markers of pinocytosis, calcein and horseradish peroxidase, comigrated with the dense peak of unoxidized cholesterol. That the size of the unoxidized cholesterol pool was greater in cells deprived of serum lipoproteins than in fed cells suggested that none of the intracellular cholesterol need be ascribed to ingested sterols. The mass of unoxidizable cholesterol was not diminished when cholesterol biosynthesis was inhibited by lovastatin in lipoprotein-deprived cells. Furthermore, the newly synthesized radiolabeled cholesterol resistant to cholesterol oxidase did not migrate with intracellular cholesterol mass on sucrose density gradients. The newly synthesized cholesterol amounted to about 10% of the total unoxidized sterol. These data indicate that most of the intracellular cholesterol was not newly synthesized. We conclude that a) approximately 90% of fibroblast cholesterol is associated with the cell surface; b) the bulk of intracellular cholesterol, approximately 10% of total, is derived from internalized (endocytic) plasma membrane; and c) the most recently synthesized cholesterol, approximately 1% of the total, is in a discrete organelle.     

Modulation of sphingomyelinase-induced cholesterol esterification in fibroblasts, CaCo2 cells, macrophages and smooth muscle cells.

The present study has focused on three questions concerning the effect of sphingomyelinase on release of free cholesterol from the plasma membrane and its intracellular translocation: (i) Can one change the direction of the flow of cholesterol? (ii) Can one modulate the flow? (iii) May such a mechanism be relevant in atherogenesis? (i) The results obtained show that even in the presence of potent nonlipoprotein cholesterol acceptors in the medium, the intracellular flow of cholesterol is not reduced as measured by cholesterol esterification. Moreover, in sphingomyelinase-treated cells, cholesterol efflux in presence of nonlipoprotein acceptors was not enhanced even when intracellular esterification was inhibited. (ii) Modulation of the sphingomyelinase induced cholesterol flow can be obtained by 100 microM verapamil which reduces it. In human skin fibroblast, interference with the delivery of free cholesterol to its site of esterification was found in the presence of brefeldin A. (iii) Aortic smooth muscle cells in culture are sensitive to low concentrations of sphingomyelinase and the increase in esterified cholesterol is evident also after exposure to the enzyme for 24 h. The present results suggest that in the plasma membrane, free cholesterol bound to sphingomyelin may be in a compartment which renders it more available for transport to the cell interior than for efflux. In view of the sensitivity of aortic smooth muscle cells to sphingomyelinase, this mechanism for enhanced esterification of cholesterol could be relevant to the transformation of arterial smooth muscle cells into foam cells in the process of atherogenesis.     

Factors affecting the esterification of lipoprotein cholesterol by lecithin: cholesterol acyl transferase.

Lecithin: Cholesterol Acyltransferase (LCAT) esterified relatively small amounts of cholesterol from very low density lipoproteins (VLDL), low density lipoproteins (LDL) or high density lipoproteins (HDL) in the presence of 5% human serum albumin (HSA). On the other hand, in the presence of very high density (>1.225 g/ml) plasma fraction (F-4), the enzyme esterified cholesterol from VLDL at considerably higher rates than from LDL or HDL. VLDL together with some component present in the very high density plasma fraction (F-4) may thus provide a highly efficient complex resulting in a favorable configuration of substrate lipids for the enzyme.     

Lipoprotein affinity of human apolipoprotein A-IV during cholesterol esterification

We have studied the lipoprotein distribution of human apo A-IV during cholesterol esterification by the action of endogenous lecithin-cholesterol acyltransferase. Using immunologic and radiotracer techniques at 4 degrees C, apo A-IV was found in two discrete monomeric and dimeric populations, unassociated with plasma lipoproteins. With incubation at 37 degrees C, apo A-IV initially associated with the high density lipoprotein-3 fraction, but thereafter dissociated from its surface, and reappeared as unbound protein and in association with a complex in the low density lipoprotein size range. Inclusion of LCAT inhibitors in the incubations abolished these changes. We conclude that the changes in lipoprotein distribution of human apo A-IV closely parallel the formation and exchange of plasma cholesteryl esters.