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.     

Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.     

Expression of low density lipoprotein receptor in cultured human granulosa cells: regulation by human chorionic gonadotropin, cyclic AMP, and sterol

Steroidogenic cells utilize lipoprotein-delivered cholesterol as a primary substrate for hormone synthesis. We studied low density lipoprotein (LDL) receptors in cultured human granulosa cells to determine what factors regulate receptor expression. Granulosa cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) for 1.5 to 14 hr. The LDL receptor content of cells increased by approximately twofold within 6 hr of hCG treatment, and the content continued to increase for at least 14 hr, as determined by immunoblotting. The rate of LDL receptor synthesis was also demonstrated to increase within 2.5 to 3.5 hr of hCG treatment by immunoisolation of LDL receptor from cells metabolically labeled with a pulse of [35S]methionine. The cyclic AMP analogue, 8-bromo-cAMP, was also found to increase LDL receptor synthesis. This increased rate of synthesis was shown to be dependent on ongoing RNA synthesis, since actinomycin D abolished hCG- or 8-bromo-cAMP-stimulated LDL receptor synthesis. We also demonstrated that hCG- and 8-bromo-cAMP-mediated regulation of LDL receptor synthesis in granulosa cells supersedes the classical cholesterol-mediated regulation of the receptor described in fibroblasts. Although 25-hydroxycholesterol induced a decrease in LDL receptor content and synthesis within 6 hr, this action was overridden by simultaneous exposure to hCG. Our findings demonstrate the existence of a novel cAMP-mediated mechanism for regulation of LDL receptor synthesis in steroidogenic cells.     

Calmodulin antagonists increase the amount of mRNA for the low-density-lipoprotein receptor in skin fibroblasts.

The effects of calmodulin antagonists on the amount of LDL receptor (LDL-R) mRNA in cultured human fibroblasts was examined by hybridization with a fragment of LDL-R cDNA. In a 'Northern' blot the fragment hybridized to a 5.3-kilobase RNA, as expected for LDL-R mRNA. The concentration of this RNA was increased in preparations from cells that were treated with trifluoperazine or W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide]. The selectivity of the increase was established by using a probe for beta-actin mRNA. In dot-blot hybridization it was observed that the calmodulin antagonists cause 2-4-fold relative increase in the amount of LDL-R mRNA.     

Low density lipoprotein receptor degradation is influenced by a mediator protein(s) with a rapid turnover rate, but is unaffected by receptor up- or down-regulation

Treatment of cultured human skin fibroblasts with cycloheximide retarded the down-regulation of low density lipoprotein (LDL) receptor activity caused by 25-hydroxycholesterol. The rate of LDL receptor degradation, measured directly by means of [35S]methionine pulse-chase experiments, was also markedly inhibited by cycloheximide (or puromycin), suggesting that continuous synthesis of a short-lived mediator protein(s) was necessary for normal LDL receptor turnover. In the absence of cycloheximide, both the up- and down-regulation of LDL receptor activity took place with a half-time of approximately 12 hr. Pulse-chase measurements with [35S]methionine yielded a receptor half-life (t1/2) of 11.7 +/- 2.2 hr (n = 10) in up-regulated cells; the t1/2 in the partially down-regulated state was similar. The presence of LDL or 25-hydroxycholesterol did not alter this degradation rate. Regulation of LDL receptor activity under these various culture conditions therefore probably occurred solely as a result of changes in the rate of receptor synthesis. The cycloheximide-sensitive factor(s) that influences receptor turnover apparently did not play a regulatory role in the up- or down-regulation of the LDL receptor.     

Agents which increase cyclic AMP have diverse effects on low-density-lipoprotein-receptor function in human vascular smooth-muscle cells and skin fibroblasts.

Receptor-mediated binding and metabolism of low-density lipoproteins (LDL) in cultured human vascular smooth-muscle cells and skin fibroblasts are altered by increased cellular cyclic AMP concentrations. However, the LDL receptor does not respond to changes in cyclic AMP concentration in a simple manner. The activation of adenylate cyclase with forskolin, or the addition of membrane-permeant cyclic AMP analogues, initially decreases the expression of the LDL receptor, but is followed by a substantial increase in receptor expression after 24 h. This increase does not occur in the presence of inhibitors of RNA or protein synthesis, and is due to doubling of the Bmax. of the LDL receptor, without alteration of its affinity for LDL. By contrast, elevation of cyclic AMP concentration by inhibition of phosphodiesterases results in decreased receptor expression throughout the 24 h period. These two response patterns are reproducible phenomena, consistently observed in low-passaged cells derived from seven unrelated individuals.     

Effect of inhibiting N-glycosylation on the stability and binding activity of the low density lipoprotein receptor

Tunicamycin, a specific inhibitor of N-glycosylation, was used to study the function of asparagine-linked oligosaccharides of the low density lipoprotein (LDL) receptor in cultured human skin fibroblasts. When cells were preincubated in the presence of 0.5 micrograms/ml of the drug the incorporation of [3H]mannose into the receptor was completely prevented and that of [3H]glucosamine was reduced to approximately 41% of the control value. The [35S]methionine radioactivity detected in receptor core protein of tunicamycin-treated cells was about 52% of that measured in the receptor of control cells. The decrease in the radioactivity was similar in both the mature receptor as well as in its precursor form, and it was significantly greater than that found in total protein. The rates of receptor degradation in control- and tunicamycin-treated cells were comparable. Neither cell surface appearance of the newly synthesized LDL receptor nor its recycling were affected by tunicamycin. However, the LDL receptor produced in tunicamycin-treated cells was smaller in molecular size, and it exhibited an about 50% lower binding capacity when compared with its counterpart synthesized in control cells. This indicates that there is a relationship between N-glycosylation and the ligand binding activity of the LDL receptor. The possible role of asparagine-linked oligosaccharides in optimizing the biological activity of the LDL receptor is discussed.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.     

Regulation of synthesis and cell content of the low-density-lipoprotein receptor protein in cultured fibroblasts from normal and familial hypercholesterolaemic subjects

Incorporation of [35S]methionine into low-density-lipoprotein (LDL) receptors by normal fibroblasts and those from a homozygous familial hypercholesterolaemic (FH) subject who produced defective but immunoprecipitable receptor proteins of normal size, was compared with the ability of the cells to bind LDL and their content of LDL receptor protein determined using a double-antibody radioimmunoassay. The FH cells produced precursor protein with a longer half-life (3-4 h) than normal cells (40 min), most of which was eventually processed to a mature form of the receptor. Total receptor half-life was similar to normal (approx. 12 h) and LDL binding about 20% of normal. Incubation of normal fibroblasts with lipoprotein-deficient serum (LPDS) led to an increase in the amount of LDL receptor protein in the cells which was closely followed by the increase in their ability to bind LDL. Receptor synthesis increased rapidly at first, but then fell by more than 60% before remaining constant. The peak of synthesis coincided with the greatest rate of increase in receptor content. At equilibrium in LPDS receptor synthesis, LDL binding and receptor protein content were all approximately 3.3-fold higher than in cells maintained in 10% foetal calf serum (FCS). The FH cells also responded to LPDS with a rise and fall in the rate of receptor synthesis. They did not compensate for their inefficiency in producing active receptors with an increase in total receptor content. In LPDS, peak synthesis and maximum receptor content of the FH cells were similar to normal. In FCS receptor synthesis and content were well below maximum so that they did not fully employ even the low capacity for LDL uptake of which they were capable. With both types of cell, inhibition of mevalonic acid and cholesterol synthesis with compactin delayed, but did not prevent the secondary fall in the rate of receptor synthesis, again suggesting a regulatory role for some factor not directly related to cholesterol metabolism.     

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 G_M1 or G_D1a, 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 supression 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 α₂-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.     

Regulation of low density lipoprotein receptor synthesis in cultured luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-cyclic AMP

We investigated the regulation of synthesis of low density lipoprotein (LDL) receptor in cultured luteinized human granulosa cells using a monoclonal antibody recognizing the human LDL receptor (IgG-C7). Cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) or 8-bromo-cAMP alone or in combination with aminoglutethimide (to block conversion of cholesterol to steroid hormones) and 5-cholesten-3 beta, 25-diol (25-hydroxycholesterol, a potent suppressor of LDL receptor expression in human fibroblasts) and pulse-labeled with [35S]methionine. A labeled protein immunoisolated with IgG-C7 was identified as the mature LDL receptor in 7.5% sodium dodecyl sulfate-polyacrylamide gels on the basis of an apparent molecular mass of 160 kDa, absence of the protein from immunoisolates prepared with a monoclonal antibody against an irrelevant antigen, and an apparent decrease in molecular weight of the mature receptor upon treatment with neuraminidase or electrophoresis under nonreducing conditions. hCG and 8-bromo-cAMP consistently increased the incorporation of radioactivity into the mature LDL receptor by 2-6-fold. The effect of hCG on LDL receptor synthesis was observed with as little as 10 mIU of hCG/ml and was apparent within 2 h of addition of the hormone. A combination of 25-hydroxycholesterol and aminoglutethimide resulted in a 60% suppression of label incorporation into mature LDL receptor compared to untreated cells. This would suggest some regulation of LDL receptor synthesis by negative feedback of sterol. However, both hCG and 8-bromo-cAMP increased label incorporation into the LDL receptor in the face of these agents. We conclude that in human granulosa cells, hCG, through the intermediacy of cAMP, rapidly increases LDL receptor synthesis by a mechanism which is, at least in part, independent of alterations in cellular cholesterol balance.     

Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes.

Incubation of SV40 transformed fibroblasts with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, produced a two-fold increase in insulin receptor concentration without an effect on receptor affinity. The increase was dose-dependent, was observed after 8 hrs of treatment, and reached a maximum level by 12 to 24 hours. Upon removal of the nucleotide, receptor number decreased towards basal level.Incubation of cultured human lymphocytes (IM-9 line) with cyclic AMP derivatives or MIX also increased the number of insulin receptors without an alteration in receptor affinity. This effect was partially blocked by inhibition of protein synthesis and was independent of changes in cell cycle. The increase in insulin receptors was a specific response to cyclic AMP as the number of receptors for human growth hormone was unaltered. Incubation with 8-bromo-cyclic GMP did not alter the level of insulin binding.     

Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis

The modulation of glucocorticoid receptor activity by cyclic nucleotides was studied in cultured human skin fibroblasts. The receptors appeared to be activated in the presence of dibutyryl-cAMP and inactivated by dibutyryl-cGMP. Significantly, the cGMP content of the fibroblasts increased during cell growth, with a concomitant decrease in the glucocorticoid receptor activity, while when the cells reached early confluency the decrease in cGMP content was accompanied by an increase in cAMP and increased activity of the glucocorticoid receptors. In addition, cortisol induced (2'-5')oligoadenylate synthetase in these cells and raised the cellular (2'-5')oligoadenylate concentrations. This resulted in a decrease in both DNA and protein synthesis activity in the cells, a response which correlated with the (2'-5')oligoadenylate concentration. The combination of cortisol and dibutyryl-cAMP had a synergetic stimulatory effect on the (2'-5')oligoadenylate concentration and a synergetic inhibitory effect on protein synthesis. In conclusion, it is demonstrated here that cyclic nucleotides can modulate glucocorticoid receptor activity in cultured human skin fibroblasts, and thus these compounds may indirectly affect cellular metabolism by regulating the cellular responses to glucocorticoids.     

Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase is sensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODs used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [14C]acetate incorporation into [14C]cholesterol and by an increased [125I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.     

Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes

The effects of prostaglandin (PG) E1, PGE2, the stable prostacyclin analogue Iloprost, and PGF2 alpha on low density lipoprotein (LDL) receptor activity and cholesterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 45 hr in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of 125I-labeled LDL. Addition of PGE1 in increasing concentrations to the incubation medium inhibited cholesterol synthesis and the specific accumulation and degradation of 125I-labeled LDL; at a concentration of 10 microM, the inhibitions were 61%, 70%, and 67%, respectively, after an incubation of 20 hr. The effects of PGE2 and Iloprost were similar. The action of the prostaglandins on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. The prostaglandins yielded sigmoidal log concentration-effect curves. In contrast, PGF2 alpha had no influence on cholesterol synthesis or LDL receptor activity up to a concentration of 10 microM. PGE1, PGE2, and Iloprost, but not PGF2 alpha, led to an increase in the concentration of intracellular cyclic AMP. Dibutyryl cyclic AMP mimicked the effects of the E-prostaglandins and Iloprost on the LDL receptor activity. The results suggest that PGE1, PGE2, and prostacyclin affect LDL receptor activity and cholesterol synthesis and, therefore, may play a role in the regulation of cholesterol homeostasis and in the development of atherosclerosis.     

Interaction of lipoprotein Lp[a] with the B/E-receptor: a study using isolated bovine adrenal cortex and human fibroblast receptors

The ability of different lipoprotein Lp[a] preparations to compete with LDL-binding to the B/E-receptor was investigated by ligand blot and filter assays. Lp[a] was purified from donors with various genetic polymorphic forms by affinity chromatography using lysine-Sepharose or specific immunoadsorbers. These preparations were free of "LDL-like" material. Part of Lp[a] was reduced and freed from specific apo[a] antigen yielding "Lpa-." 125I-labeled low density lipoproteins (LDL) were incubated with B/E-receptor preparations from bovine adrenal cortex or from human skin fibroblasts, and the competition with unlabeled LDL, Lp[a], Lpa-, apo[a], and apoE-free HDL was studied by a ligand blot or filter assay technique. The following results were obtained. 1) LDL and Lpa- were equally potent in displacing 125I-labeled from B/E-receptor in the ligand blot and the filter assay. Lpa + ( = Lp[a]) also displaced LDL but to a much lesser degree: 50% displacement was observed with LDL and Lpa- at a 1-fold excess, whereas a 7.5-fold excess was required of Lpa +. 2) Apo[a], as well as apoE-free HDL, did not compete with LDL binding. 3) Competition experiments using B/E-receptors from bovine adrenal cortex or from human skin fibroblasts were comparable. 4) There was no difference in the behavior of Lp[a] isolated from the two affinity chromatography methods. 5) Lp[a] of different genetic variants behaved virtually identically. The results are discussed from the point of view of the in vivo metabolism of Lp[a].     

Uptake of 2,3,7,8-tetrachlorodibenzo-p-dioxin from plasma lipoproteins by cultured human fibroblasts

Tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to human plasma in vitro associated with the plasma lipoproteins. The effects of plasma and lipoproteins on cellular uptake of dioxin were studied using normal human skin fibroblasts and mutant fibroblasts from a patient with homozygous familial hypercholesterolemia. The latter cells lack the normal cell membrane receptor for low density lipoprotein (LDL). The time- and temperature-dependent cellular uptake of [3H]dioxin was greatest from LDL, intermediate from high density lipoprotein (HDL) and least from serum. A significantly greater uptake from LDL by the normal cells compared to the mutant cells indicated the involvement of the LDL receptor-mediated pathway. Concentration-dependent studies indicated that the cellular uptake at 37 degrees C of [3H]dioxin varied linearly with dioxin concentration at constant LDL concentration. Thin-layer chromatography (TLC) showed that conversion to more polar compounds may have occurred after 24-h incubation with cells. [3H]Dioxin could be removed from cells efficiently by incubation with 20% serum greater than HDL greater than LDL. Since the vehicle of delivery may influence subsequent location and metabolism of this compound in cells, it is concluded that the physiologic vehicles (either serum- or LDL-associated dioxin), rather than organic solvents, should be used in experiments with cultured cells or perfused organs.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).