Degradation Of Low Density Lipoprotein Research Articles

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13- cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2.

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of uptake and degradation of low density lipoproteins by arteries and veins of rabbits

The uptake and degradation of low density lipoproteins were compared in arteries and veins of rabbits in vivo. Rabbits were injected with low density lipoproteins labelled singly with 125I ( 125I-LDL) or doubly with 131I and with [ 125I]tyramine cellobiose (TC-LDL) which remains trapped intracellularly. After increasing times up to 24 h the largest vessels were perfused to remove blood, fixed in situ and excised. When the uptake of 125I-LDL by arteries and veins was compared on the basis of luminal surface area, there were no significant differences up to 3.5 h. When degradation rates of double labelled LDL were compared after 24 h on the same basis, the arteries contained significantly more TC label than the veins, but when the comparison was based on dry weights there were no significant differences. These results show that veins, like arteries, take up LDL from the circulating blood; and they suggest that the preferential accumulation of LDL in arteries susceptible to atherosclerosis may be accounted for, at least in part, by more degradation of LDL in arteries than in veins.

Enhanced degradation of low density lipoprotein in human monocyte-derived macrophages associated with an increase in its free fatty acid content

Low density lipoprotein (LDL) rich in oleic acid (designated FFA-rich LDL) was produced by the reconstitution technique. FFA-rich LDL, like acetyl LDL, moved faster than native LDL in agarose gel electrophoresis. While FFA-rich LDL was observed to degrade far less than natural LDL in lymphocytes, its degradation in monocyte-derived macrophages was three times higher than that of natural LDL or LDL reconstituted without the addition of oleic acid. A competitive study showed that the catabolism of FFA-rich LDL in macrophages may be influenced by systems other than the acetyl LDL receptor.

A possible antiatherogenic role for phosphocitrate through modulation of low density lipoprotein uptake and degradation in aortic smooth muscle cells

The present study reports the influence of a phosphorylated polycarboxylic acid, phosphocitrate, on low density lipoprotein (LDL) metabolism in cultured rabbit aortic smooth muscle cells. Phosphocitrate profoundly influenced both LDL binding and degradation. At the maximal effective concentration (2 mM), phosphocitrate released approximately 90% of the receptor-bound [ 125I]LDL whilst the total amount of [ 125I]LDL degraded was reduced by 60%. Measurement of total cholesterol accumulation revealed that even in the presence of high concentrations of added LDL, phosphocitrate (2 mM) diminished cholesterol levels close to the basal levels seen in incubations in lipoprotein-deficient serum. Further, this inhibitory effect of phosphocitrate was demonstrable after 24 h at 37°C. Phosphocitrate, a recognized anticalcifying agent, possesses a strong negative charge to size ratio at physiological pH. It is postulated that the observed effects probably arise from charge interference and/or its ability to modulate cellular calcium concentration.

Non-saturable degradation of LDL by monocyte-derived macrophages leads to a reduction in HMG-CoA reductase activity with little synthesis of cholesteryl esters

In monocyte-derived macrophages from both normal and familial hypercholesterolaemic (1711) subjects, degradation of low density lipoprotein (LDL) through non-saturable pathways produced the same fall in 3-hydroxy-3-methylglutaryl-CoA reductase activity as receptor-mediated degradation of acetylated LDL, yet did not lead to as great an increase in incorporation of [ 14C]oleate into cholesteryl esters. Studies using FH cells showed that the simultaneous addition of LDL did not reduce oleate incorporation resulting from degradation of acetylated LDL, and that there was a similar relationship for both lipoproteins between the increase in net oleate incorporation and the increase in the cholesteryl ester content of the cells. FH cells maintained in serum-free medium accumulated more free cholesterol than cells in lipoprotein-deficient serum when incubated with LDL but not when incubated with acetylated LDL. The results suggest that the cholesterol released from non-saturable degradation of LDL is more easily removed from the cells by acceptors in the medium than cholesterol released from receptor-mediated uptake of acetylated LDL, and is not readily available for esterification.

Low density lipoprotein degradation by secretory granules of rat mast cells. Sequential degradation of apolipoprotein B by granule chymase and carboxypeptidase A.

The secretory granules of rat serosal mast cells are able efficiently to degrade the apolipoprotein B component of low density lipoproteins (LDL) Kokkonen, J. O., and Kovanen, P. T. (1985) J. Biol. Chem. 260, 14756-14763). The granules are known to contain two neutral proteases with complementary specificities: a chymotrypsin-like endopeptidase called chymase, and an exopeptidase, the granule carboxypeptidase A. The role of this enzyme pair in the proteolytic degradation of LDL was studied with the aid of specific enzyme inhibitors. Incubation of LDL with intact granules (both enzymes active) led to the formation of numerous low molecular weight peptides and the liberation of free amino acids, most of which (95%) were aromatic (Phe, Tyr, Trp) or branched-chain aliphatic (Leu, Ile, Val). Selective inhibition of granule carboxypeptidase A (leaving chymase active) blocked the liberation of free amino acids, but left the formation of peptides uninhibited. On the other hand, selective inhibition of granule chymase (leaving carboxypeptidase A active) totally abolished the proteolytic degradation of LDL. The results are consistent with a model according to which the proteolytic degradation of LDL by mast cell granules results from coordinated action of the two granule-bound enzymes, whereby the chymase first cleaves peptides from the apolipoprotein B of LDL, and thereafter the carboxypeptidase A cleaves amino acids from the peptides formed.

Regulation of low density lipoprotein receptor activity in primary cultures of human hepatocytes by serum lipoproteins.

The low density lipoprotein receptor activity was measured in primary cultures of human hepatocytes. The receptor-mediated association and degradation of low density lipoprotein increased gradually up to 140 and 190%, respectively, upon incubation of the cells with increasing amounts of whole serum (up to 100%). Preincubation of the cells with low density lipoprotein resulted in a weak downregulation of the receptor-mediated association of low density lipoprotein (only 35% reduction at 100 micrograms low density lipoprotein per ml). However, preincubation with high density lipoproteins with density between 1.16 and 1.20 gm per ml (heavy high density lipoprotein) resulted in a more than 2-fold stimulation of the receptor-mediated association of low density lipoprotein. This heavy high density lipoprotein-mediated stimulation could not be antagonized by a simultaneous addition of low density lipoprotein during that preincubation. We conclude that, in primary cultures of human hepatocytes, the downregulation of the low density lipoprotein receptor activity by low density lipoprotein is weak and completely overruled by heavy high density lipoprotein. If these results for human hepatocytes in vitro hold true for hepatocytes in vivo, our results might explain why in vivo liver cells still display low density lipoprotein receptor activity notwithstanding the exposure of these cells to physiological concentrations of low density lipoprotein.

Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor.

Incubation of low density lipoprotein (LDL) with endothelial cells or smooth muscle cells overnight has resulted in an oxidative modification of LDL that results in its recognition by macrophages by way of the acetyl LDL receptor. In the present study, we examined whether macrophages themselves can oxidize and modify LDL in a manner similar to that of endothelial cells. Incubation of 125I-labeled LDL with resident or thioglycollate-elicited macrophages for 24 hours in Ham's F-10 medium resulted in the appearance of thiobarbituric acid (TBA) reactive materials and trichloroacetic acid (TCA) soluble radioactivity in the medium. The LDL harvested from these incubations showed increased electrophoretic mobility and was degraded rapidly when added to fresh macrophages as compared to LDL previously incubated in the absence of cells. These macrophage-induced modifications could be prevented if the first incubation was carried out in the presence of the antioxidant butylated hydroxytoluene (BHT) or in Dulbecco's modified Eagle's medium (DMEM). The degradation of 125I-labeled macrophage-modified LDL by macrophages was competitively inhibited by unlabeled acetyl LDL or unlabeled endothelial cell-modified LDL but not by native LDL, indicating that the degradation was mediated by the acetyl LDL receptor.

Internalization of low-density-lipoprotein-specific receptors in leukemic guinea pig lymphocytes.

Leukemic guinea pig lymphocytes (L2C) have ten times as many low-density lipoprotein (LDL) receptors as healthy lymphocytes, but LDL accounts for only 38% of the cholesterol in L2C cells, compared to more than 95% in normal cells. Our data show that LDL fails to regulate cholesterol biosynthesis and that there is a defect in LDL internalization and receptor turnover in L2C cells. We also demonstrate that the degradation of LDL is not a limiting process. By discriminating between binding and internalization, we show that internalization in L2C is much slower than in normal cells and that the decrease in metabolism is related to the slow turnover of the LDL receptors.

Effect of proteolysis of low-density serum lipoproteins on their interaction with macrophages

125I-labelled human serum low density lipoproteins (LDL) were incubated with cultured mouse peritoneal macrophages at 37 degrees C, with the following study of cellular uptake and 125I-LDL degradation by measuring the content of TCA-soluble products of LDL hydrolysis in the cultural medium. It was shown that limited pepsin proteolysis of LDL (10%) led to a more effective LDL uptake and degradation by macrophages. The data suggest that enzyme-induced modification of LDL may increase their atherogenicity.

Impaired hepatocyte binding, uptake and degradation of glucosylated low-density lipoproteins

The catabolism of low-density lipoproteins (LDL), the major cholesterol-carrying lipoproteins in plasma, is mediated in part via a high-affinity uptake pathway in the liver. Non-enzymatic glucosylation of lysine residues of apolipoprotein B, the major protein of LDL, blocks receptor-mediated uptake of LDL by fibroblasts and endothelial cells. We investigated the effect of the degree of glucosylation on the binding, uptake and degradation of radioiodinated LDL by the human hepatoma cell line Hep G2. Human LDL was glucosylated with 250 mM glucose and 30 mM cyanoborohydride at 37 °C. Incubations ranging from 3 to 48 h in duration resulted in the formation of 6–27% of glucitol-lysine adducts as demonstrated by coincubation with [ 14C]glucose. The degree of glucose incorporation corresponded to the extent of inhibition of binding, uptake and degradation of LDL (10–90%). The data are consistent with the view that glucosylation of LDL markedly impairs their catabolism. This phenomenon may be related to the pathophysiology of the premature atherosclerosis observed in diabetes mellitus.

Insulin regulates low density lipoprotein metabolism by swine granulosa cells.

Insulin synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells. The mechanisms subserving this facilitative interaction included the following: 1) insulin's synergism with LDL was profoundly attenuated by covalent modification of arginine residues in LDL by 1,2-cyclohexanedione treatment; 2) insulin increased by 2- to 6-fold the number of specific high affinity LDL receptors on granulosa cells, with no change in apparent binding affinity; 3) insulin augmented rates of [125I]iodo-LDL internalization and degradation without enhancing nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrollidone); 4) insulin increased by 2.5- to 3-fold granulosa cell content of free and esterified cholesterol (measured by fluorometry) in response to treatment with unlabeled LDL; 5) insulin stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester, and amplified [3H]progesterone secretion by granulosa cells exposed to [3H]cholesteryl linoleate-labeled LDL; and 6) insulin action was specific in that it was not mimicked by desoctapeptide insulin, epidermal growth factor, fibroblast growth factor, or relaxin. We conclude that insulin and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via specific mechanisms that depend upon 1,2,-cyclohexanedione-sensitive residues within LDL apoprotein. Insulin action results in significantly augmented binding, internalization, and degradation of LDL, which is accompanied by increased effectual delivery of cholesterol substrate into cellular sterol pools that participate in enhanced steroidogenesis.

Role of receptor-independent low density lipoprotein transport in the maintenance of tissue cholesterol balance in the normal and WHHL rabbit.

These studies were undertaken to determine the role of receptor-independent low density lipoprotein (LDL) transport in cholesterol balance across individual tissues and the whole animal. Homologous LDL, which measures total LDL transport, and methylated heterologous LDL, which measures receptor-independent LDL uptake, were cleared from the plasma at very different rates in the NZ control rabbit (3,900 and 1,010 microliter/hr per kg, respectively) whereas in the WHHL rabbit both preparations were cleared at essentially the same rate (approximately 1,070 microliter/hr per kg). Receptor-independent LDL clearance was detected in all tissues of the NZ control rabbit and these varied from 32 (spleen) to less than 0.5 (skeletal muscle) microliter/hr per g. In contrast, receptor-dependent LDL uptake was found in only about half of these same organs. In the WHHL rabbit, the rates of receptor-independent LDL transport were the same as in the NZ control rabbit, but no receptor-dependent uptake was detected. Using these clearance values it was calculated that in the control rabbit nearly 70% of LDL-cholesterol was removed from the plasma by the liver and 89% of this was receptor-mediated. With loss of receptor activity, however, the burden of LDL degradation was shifted away from the liver so that approximately 70% of LDL-cholesterol uptake took place in the extra-hepatic tissues of the WHHL rabbit. Thus, in the normal animal, the primary function of receptor-dependent LDL transport is to promote the rapid uptake and disposal of plasma LDL by the liver. In the absence of such receptor activity, cholesterol balance across most individual organs and the whole animal remains essentially normal and is mediated by the receptor-independent process. Because of the much lower absolute clearance rates manifested by this transport mechanism, however, substantial and predictable elevations in the circulating plasma LDL-cholesterol levels are required to maintain this balance.

Low apparent affinity for low-density lipoprotein of receptors expressed by human macrophages maintained with whole serum.

Normal human monocyte-derived macrophages maintained in medium containing whole serum exhibited saturable degradation of low-density lipoprotein (LDL) that was mediated by LDL receptors. This degradation required a higher concentration of LDL to achieve one-half saturation than that in cells preincubated with lipoprotein-deficient serum (LPDS). Studies of short-term uptake and of heparin-releasable binding of LDL showed that binding to the surface receptors was the limiting factor for degradation under both conditions and that the LDL receptors expressed by cells in whole serum had a significantly lower affinity for LDL than those in cells pre-incubated in LPDS. LDL receptors in monocyte-macrophages could mediate the uptake and degradation of complexes between apolipoprotein E (apoE) and phospholipid. The receptors in cells pre-incubated in LPDS bound the complexes and LDL with apparently the same affinity and in approximately the same molar ratio. Receptors in cells maintained with whole serum did not have a lower affinity for the complexes than cells pre-incubated in LPDS, although the molar ratio of maximum degradation of LDL to that of complexes was greater.

Conservation of the low density lipoprotein receptor-binding domain of apoprotein B. Demonstration by a new monoclonal antibody, MB47.

The fact that low density lipoprotein (LDL) from multiple animal species binds to the human LDL receptor suggested that the LDL-receptor binding domain of apoprotein (apo)B must be evolutionarily conserved. To determine if a common receptor domain epitope existed on apo B, we generated a monoclonal antibody that was specific for the LDL-receptor domain of apo B. This was accomplished by using a screening procedure that selected for a hybridoma supernatant that could block specific cellular uptake and degradation of LDL. Western blots showed that this antibody, termed MB47, was specific for apo B-100. Fluid phase assays indicated a high binding affinity (Ka = 4 X 10(9) M-1) and demonstrated that all human LDL particles expressed the MB47 epitope. Scatchard analysis indicated that a maximum of one MB47 molecule bound to each LDL particle. In solid phase assays, antibody MB47 bound to plasma or LDL of multiple mammalian species, including guinea pig, rabbit, pig, dog, cat, seal, whale, bear, and lion, but it did not bind to mouse or rat LDL. In contrast, a rabbit antiserum to LDL and two other anti-apo B monoclonal antibodies, MB3 and MB19, which do not bind to the receptor domain, were specific only for human LDL. LDL from multiple species, including mouse LDL, competed effectively with 125I-human LDL for binding to human fibroblasts. MB47 effectively inhibited uptake and degradation of labeled human, guinea pig, and rabbit LDL by both human and guinea pig fibroblasts. We conclude that antibody MB47 binds to a single receptor domain on LDL and identifies a vital region conserved through mammalian evolution.

Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor.

Studies of low density lipoprotein (LDL) metabolism in nonhuman model systems have indicated that the mammalian liver has dual mechanisms for the uptake and regulation of the concentration of plasma LDL. Heretofore, direct evaluation of lipoprotein uptake mechanisms in human hepatocytes has not been possible. In order to compare hepatocyte LDL uptake with fibroblast LDL metabolism, human hepatocytes were isolated and cultured from small biopsy specimens obtained from normolipidemic and homozygous familial hypercholesterolemic patients. Cells cultured in serum-free culture medium retained the morphological and biochemical characteristics of hepatocytes for at least 7 days. The uptake and degradation of LDL by hepatocytes was compared to that of the cultured human fibroblasts. Like fibroblasts, hepatocytes bound, internalized, and degraded LDL. In both cell types, uptake approached saturation at a concentration of 50 micrograms of LDL protein/ml. Competition for LDL binding by LDL, high density lipoprotein, and modified LD revealed that the hepatocyte binding was specific for LDL. Cellular cholesterol loading by incubation in LDL-enriched culture medium resulted in diminished LDL uptake in both cell types. Chemical modification of LDL by acetoacetylation, acetylation, and reductive methylation abolished LDL uptake and degradation by fibroblasts. However, hepatocytes bound and degraded the modified LDL at 30-50% the level of native LDL. Homozygous familial hypercholesterolemic hepatocytes were devoid of the LDL receptor pathway but metabolized native LDL to the extent observed with modified LDL uptake by normal hepatocytes. In contrast to the classic LDL receptor pathway, the second or alternate pathway does not lead to regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity. These findings indicate the presence of two separate pathways of LDL uptake in human hepatocytes which have different effects on hepatocytic cholesterol metabolism.

Low density lipoprotein receptor-dependent prostaglandin synthesis in Swiss 3T3 cells stimulated by platelet-derived growth factor.

We studied the effects of human plasma lipoproteins on the synthesis of prostaglandin (PG) E2 in Swiss 3T3 mouse fibroblasts. Quiescent cells, maintained in medium deficient in both platelet-derived growth factor (PDGF) and lipoproteins, synthesized less than 8 ng of PGE2 per 10(6) cells per 22 hr, and this rate did not change in response to the addition of lipoproteins. In contrast, PDGF-stimulated cells, incubated in medium deficient in lipoproteins, synthesized 45-110 ng of PGE2 per 10(6) cells during the same period of time, and this rate increased 2- to 5-fold in the presence of added low density lipoproteins (LDL). This stimulatory effect of LDL seemed to depend on LDL receptor-mediated binding, uptake, and degradation of the lipoproteins because: both LDL and very low density lipoproteins were active, whereas high density lipoproteins were not; low concentrations of LDL were effective; the effect of native LDL was blocked by acetylation of the LDL; PDGF increased both the expression of LDL receptors and the cellular uptake of LDL; chloroquine blocked the effect of LDL but not that of exogenous arachidonic acid. These results provide evidence that the LDL pathway is critically linked to PG synthesis in PDGF-stimulated cells.

Dexamethasone modulates lipoprotein metabolism in cultured human monocyte-derived macrophages. Stimulation of scavenger receptor activity.

Human monocyte-derived macrophages (HMM) play a key role in the formation of atherosclerotic plaques by accumulating cholesteryl ester (CE) to become foam cells. HMM have receptors for native low density lipoprotein (LDL) and acetylated-LDL (ALDL), and uptake of ALDL can promote substantial cellular CE accumulation. Furthermore, macrophages specifically and saturably bind glucocorticoids, which in turn modulate numerous macrophage functions. Preincubating HMM in dexamethasone-inhibited LDL degradation (230 +/- 12 vs. 515 +/- 21 ng/mg cell protein X 18 h, P less than 0.001) but stimulated ALDL degradation (5.3 +/- 0.5 vs. 2.5 +/- 0.3 micrograms/mg X 18 h, P less than 0.01). These effects were time- and dose-dependent, occurring maximally by 24 h and with 2.5 X 10(-8) M dexamethasone. Dexamethasone increased the maximum velocity for ALDL degradation (16.2 vs. 12.0 micrograms/mg X 18 h, P less than 0.01) without changing the apparent Michaelis constant. Progesterone, 11 alpha-epicortisol, and 17 alpha-OH progesterone (a competitive antagonist of the glucocorticoid receptor) had no effect on HMM ALDL degradation, but 17 alpha-OH progesterone abolished the stimulatory action of dexamethasone. In he presence of ALDL, incorporation of [14C]oleic acid into CE was enhanced over fourfold by dexamethasone (4015 +/- 586 vs. 943 +/- 91 cpm/mg X 2 h, P less than 0.01), and HMM incubated with ALDL and dexamethasone accumulated more free cholesterol (34.6 +/- 1.9 vs. 26.2 +/- 0.8 micrograms/mg, P less than 0.02) and CE (32.8 +/- 2.3 vs. 14.8 +/- 0.8 micrograms/mg, P less than 0.002) than did macrophages without dexamethasone. In cultured human umbilical vein endothelial cells, dexamethasone did not change ALDL degradation, but reduced LDL degradation by 30% (P less than 0.001). In summary, dexamethasone inhibits LDL receptor activity by both macrophages and endothelial cells, but stimulates ALDL receptor activity only in macrophages. These observations provide evidence for the regulation of macrophage endocytic receptors by glucocorticoid hormones.

家族性高コレステロール血症患者のLDLレセプター活性とそれに対する薬物療法の影響

We examined low density lipoprotein (LDL) receptor activities of 13 heterozygous patients with familial hypercholesterolemia (FH) using peripheral lymphocytes.The receptor activities were measured as the degradation of 125I-labeled LDL in lymphocytes which had been cultured for three days in 10% lipoprotein-deficient serum. After incubation, 10μg/ml of 125I-LDL were added to the medium in the presence or absence of 250μg/ml of nonlabeled LDL and then the lymphocytes were incubated for five hours further. The radioactivity of the trichloroacetic acid-soluble fraction of the medium was measured.As a result, LDL receptor activities of FH heterozygotes were revealed to correlate significantly and negatively with serum LDL-cholesterol concentration. If we combined them with those of two FH homozygotes and 18 hyperlipidemic patients other than FH and normolipidemic persons, the latter of which we reported previously (J. Jpn. Atheroscler. Soc. 12: 303-308, 1984), not only the grade of the negative correlation between LDL receptor activities and LDL-cholesterol became greater, but LDL receptor activities also correlated negatively with total cholesterol (TC) (p<0.01). Extrapolation from these data suggested that LDL receptor activity would disappear when TC concentration was higher than 718mg/dl. The value estimated agreed well with the clinical findings of FH.Then, probucol (750mg a day) was administered to eight heterozygous patients with FH for four to six months, by which TC concentration decreased significantly from 319.6±36.7 (mg/dl) to 282.3±17.4 (mg/dl). The result suggests that probucol is effective in the treatment of FH. The levels of LDL receptor activities in the pretreatment state was not correlated to the effectiveness of probucol treatment in each case.LDL receptor activities were also measured after probucol administration and were compared with those of the pretreatment state. There were a great variations of change of LDL receptor activity among the eight patients, and no definite trend of the activity could be found. But, it was suggested that the percentage change of TC concentration was correlated negatively with the difference in LDL receptor activity before and after probucol administration, that is, the more the TC concentration decreased, the more the LDL receptor activity increased.These results may suggest that the main action of probucol is not on LDL receptor and probucol indirectly influences LDL receptor activity by the change in serum cholesterol concentration.