In vivo catabolism of alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine

The clearances of 125I-labeled alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine (CH3NH2) were compared in our previously described mouse model. alpha 1-Proteinase inhibitor-trypsin cleared with a t 1/2 of 20 min, antithrombin III-thrombin of 7 min and 125I-labeled alpha 2-macroglobulin-methylamine of 2 min. Competition studies were performed to determine whether one or several pathways clear these three ligands. The clearance of 125I-labeled alpha 1-proteinase inhibitor-trypsin and 125I-labeled antithrombin III-thrombin was blocked by large molar excesses of either ligand, but not by alpha 2-macroglobulin-methylamine. The clearance of 125I-labeled alpha 2-macroglobulin-methylamine can be blocked by a large molar excesses of unlabeled alpha 2-macroglobulin-methylamine but not by alpha 1-proteinase inhibitor-trypsin. These studies demonstrate that the clearance of alpha 1-proteinase inhibitor-trypsin complexes is independent of alpha 2-macroglobulin-methylamine and utilizes the same pathway which is involved in the clearance of antithrombin III-thrombin complexes.     

Reciprocal expressions of alpha 1- and beta-adrenergic receptors, but constant expression of glucagon receptor by rat hepatocytes during development and primary culture

The stimulations of cyclic AMP formation and adenylate cyclase activity by glucagon and isoproterenol were both found to be highest in neonatal rat hepatocytes and to decrease during development. Adult hepatocytes still showed a considerable response to glucagon, but a negligible response to isoproterenol. The decrease in cyclic AMP formation during development can be explained in the case of the response to beta-adrenergic agonist as due to decrease of its receptor number, judging from binding of [125I]iodocyanopindolol to purified plasma membranes. But in the case of the glucagon response, the decrease in the response may be due to change of post-receptor components of the adenylate cyclase system, because the receptor number tended to increase during development, as shown by binding of [125I]iodoglucagon. Similarly, alpha 1-adrenergic receptors increased in number during development, but their IC50 value did not change, as measured by binding of [3H]prazosin to plasma membranes. Previous studies on primary cultures of adult rat hepatocytes showed that the beta-adrenergic response and its receptor number increased markedly during short-term culture (Nakamura, T., Tomomura, A., Noda, C., Shimoji, M., & Ichihara, A. (1983) J. Biol. Chem. 258, 9283-9289). However, in this work the amount of alpha 1-adrenergic receptor of adult rat hepatocytes was found to decrease by one third during 1-2 days culture. Therefore, changes of alpha 1- and beta-adrenergic receptors during development of rat liver and during primary culture of adult rat hepatocytes were reciprocal, although the directions of change in the two conditions were opposite. The additions of various hormones to primary cultures of adult rat hepatocytes did not affect the reciprocal changes of adrenergic receptors, suggesting that these hormones did not regulate the changes of the receptors.     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

Identification and change in the receptor for hepatocyte growth factor in rat liver after partial hepatectomy or induced hepatitis

Specific binding of 125I-labeled human recombinant HGF to the primary cultured rat hepatocytes or liver plasma membranes was observed to be temperature- and time-dependent. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 24-32 pM, a value in good accord with half maximum dose for HGF activity and a receptor density of about 500-600 sites/cell. Affinity cross-linking of the receptor with 125I-HGF revealed the HGF receptor in rat liver membranes to be a polypeptide of Mr approximately 220,000. After partial hepatectomy, specific binding of 125I-HGF to the membranes of residual livers decreased by 60-70% between 3 and 6 h, and was scanty at 12 h after hepatectomy. After one week, the binding was recovered to the 1.7 fold level in the untreated rat liver. This rapid down-regulation of HGF receptors was also observed in plasma membranes of rat livers in the presence of hepatitis induced by CCl4. We propose that HGF which can be immediately supplied to the liver after hepatic injury will function as a trigger for regeneration of this organ.     

Characterization of a membrane-associated receptor from rat sinusoidal liver cells that binds formaldehyde-treated serum albumin

When treated with formaldehyde, serum albumin is known to be taken up and degraded by sinusoidal liver cells via adsorptive endocytosis. The present study aimed at characterization and identification of the membrane-associated receptor on rat sinusoidal liver cells. Kinetic studies of binding of 125I-labeled formaldehyde-treated serum albumin (125I-f-Alb) with the membranes of sinusoidal liver cells demonstrated the presence of specific, high-affinity, saturable membrane-bound receptors with an apparent Kd = 8 micrograms of f-Alb/ml and the optimal pH at around 8.0. The 125I-f-Alb binding to the membranes was not inhibited by either native albumin, asialofetuin, methylamine-treated alpha 2-macroglobulin, mannan, or immune complexes. The binding process exhibited independence of calcium and susceptibility both to heat treatment and to destruction by proteases. The binding was inhibited by concanavalin A and the inhibition was effectively reversed by the presence of alpha-methyl-D-glucoside, a haptenic inhibitor for this lectin, indicating the glycoprotein nature of the receptor. The binding protein was extracted from the membrane preparations with octyl beta-D-glucopyranoside and immunoprecipitated by anti-ligand antibody as a complex with the ligand. Sodium dodecyl sulfate-gel electrophoresis of the immunoprecipitate revealed two polypeptide chains with molecular weights of approximately 53,000 and 30,000, respectively.     

Distinct properties of the recognition sites for beta-very low density lipoprotein (remnant receptor) and alpha 2-macroglobulin (low density lipoprotein receptor-related protein) on rat parenchymal cells.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

α1-Adrenergic Receptors in Neuronal Cultures from Rat Brain: Increased Expression in the Spontaneously Hypertensive Rat

Neuronal cells in primary culture from 1-day-old brains of normotensive, Wistar-Kyoto strain (WKY) and spontaneously hypertensive (SH) rats have been utilized to study the expression of alpha 1-adrenergic receptors. Binding of a selective alpha 1 antagonist, [125I]2-[beta-(4-hydroxy-3-iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) to neuronal membranes prepared from primary brain cultures of WKY and SH rats was 75-80% specific, rapid, and time-dependent although the binding was 1.5-2 times higher in neuronal membranes from SH rat brain cultures. Kinetic analysis of the association and dissociation data demonstrated no significant differences between rat strains. Competition-inhibition experiments provided IC50 values for various antagonists and agonists in the following order: prazosin less than phentolamine less than yohimbine less than phenylephrine less than norepinephrine less than propranolol, suggesting that [125I]HEAT bound selectively to alpha 1-adrenergic receptors. Scatchard analysis of the binding data provided straight lines for both strains of rats, indicating the presence of a homogeneous population of binding sites. It also showed that the increase in the binding in neuronal cells from SH rat brains over those from normotensive WKY controls was a result of an increase in the number of alpha 1-adrenergic receptors. Incubation of neuronal cultures from both strains of rats with phenylephrine, an alpha 1-adrenergic agonist, caused a time- and dose-dependent decrease in the binding of [125I]HEAT. This decrease was due to a decrease in the number of alpha 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein.

gp330, a large glycoprotein located in renal proximal tubules, has sequence similarities with the low-density lipoprotein receptor and the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. The 40 KD human alpha 2-macroglobulin receptor-associated protein is a newly discovered heparin binding protein homologous to a major rat Heymann nephritis factor and exhibiting high affinity binding to the alpha 2-macroglobulin receptor. The present study shows by ligand blotting, light and electron microscopic autoradiography, and cytochemistry that gp330 located in coated apical membrane regions of the rat proximal tubule strongly binds the 40 KD protein. Furthermore, 45Ca2+ blotting experiments disclosed gp330 as a quantitatively important Ca2+ binding protein in renal cortex. Binding of 125I-labeled 40 KD protein to electroblotted gp330 and to coated apical membrane regions in sections of renal proximal tubules was abolished by excess unlabeled 40 KD protein, heparin, and EDTA. The endocytic properties of gp330 were investigated by in vivo microperfusion of rat proximal tubules. After 6 min, 125I-labeled 40 KD protein was mainly found in endocytic vacuoles and later accumulated in lysosomes. These data demonstrate that gp330 is a Ca2+ binding receptor for endocytosis of protein and is functionally related to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Furthermore, our results demonstrate the usefulness of semi-thin and ultra-thin cryosections in studies of ligand binding and subcellular localization of receptors with autoradiographic techniques.     

Purification of the rat hepatic alpha 2-macroglobulin receptor as an approximately 440-kDa single chain protein

alpha 2-Macroglobulin-trypsin complex (alpha 2M.T) and alpha 2M-methylamine bind in a Ca2+-dependent way to a 400- to 500-kDa receptor in rat and human liver membranes (Gliemann, J., Davidsen, O., and Moestrup, S. K. (1989) Biochim. Biophys. Acta 980, 326-332). Here we report the preparation of alpha 2M receptors from rat liver membranes solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonic acid (CHAPS) dihydrate and incubated with Sepharose-immobilized alpha 2M-methylamine. The receptor preparation eluted with EDTA (pH 6.0) contained a protein larger than the 360-kDa alpha 2M (nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and some minor contaminants. The reduced large protein was about 440 kDa using reduced laminin (heavy chain: 400 kDa) as a standard. About 10 micrograms of receptor protein was obtained from 100 mg of liver membranes. The receptor preparation immobilized on nitrocellulose sheets bound 125I-alpha 2M.T, and the binding activity co-eluted with the 440-kDa protein. 125I-Labeled rat alpha 1-inhibitor-3 (alpha 1I3), a 200-kDa analogue of the alpha 2M subunit which binds to the alpha 2M receptors, was cross-linked to the 440-kDa protein. The receptor preparation was iodinated, and the 125I-labeled 440-kDa protein was isolated. It showed Ca2+-dependent saturable binding to alpha 2M-methylamine. In conclusion, we have purified the major hepatic alpha 2M receptor as an approximately 440-kDa single chain protein.     

Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes

Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors.     

Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.

In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

Hepatocyte receptors for antithrombin III-proteinase complexes

The in vivo clearance of antithrombin III-proteinase complexes occurs via a specific and saturable pathway located on hepatocytes. We now report studies of the catabolism of antithrombin III-proteinase complexes in vitro using rat hepatocytes in primary culture. Antithrombin III-thrombin and trypsin complexes were prepared and purified to homogeneity. Ligand uptake by hepatocytes was concentration, temperature, and time dependent. Initial rate studies were performed to characterize the maximum rate of uptake, V, and apparent Michaelis constant Kapp. These studies yielded a V of 12.8 fmol/mg cell protein/min and a Kapp of 144 nM for antithrombin-trypsin complexes. Competition experiments with antithrombin III, antithrombin III-proteinase complexes, alpha 2-macroglobulin-methylamine, asialoorosomucoid and the neoglycoproteins, fucosyl-bovine serum albumin (BSA), N-acetylglucosaminyl-BSA, and mannosyl-BSA indicated that only antithrombin III-proteinase complexes were recognized by the hepatocyte receptor. Uptake studies were performed at 37 degrees C with 125I-antithrombin III-trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with autoradiography. These studies demonstrate time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

Species variation in the binding of hGH to hepatic membranes

The binding of 125I-labeled human growth hormone (hGH) to liver membranes from several different species was studied to determine the lactogenic or somatotropic hormone nature of the receptors. Liver membranes from several species of the class of Mammalia bound significant quantities of 125I-hGH. Goat, sheep, rat, mouse, and rabbit liver membranes exhibited the highest binding with cow, pig, human, and hamster liver membranes exhibiting severalfold less binding. The binding of the dog and cat liver membranes exhibited relatively high nonspecific binding. Fish and chicken liver membranes did not bind appreciable quantities of 125I-hGH. In all species except for dog and cat in which 125I-hGH bound to the membranes, hGH was the most effective competitor for binding. The mean ID50 for hGH and all membranes was 2.4 X 10(-9) M. Human liver membranes exhibited the smallest ID50, 4.9 X 10(-10) M. In sheep liver membranes, bovine growth hormone (bGH) was equipotent to hGH in competing for 125I-hGH binding. bGH also demonstrated significant competition for 125I-hGH binding in pig and cow membranes. Ovine prolactin (oPrl) exhibited significant competition for 125I-hGH only in rodent membranes. The ID50 for oPrl was 3- to 10-fold greater than for hGH in the rat, hamster, and mouse liver membranes. The ID50 for oPrl in the sheep liver membranes was 13-fold greater than that of hGH. We conclude the following: (1) There appears to be a species specificity of hGH binding that may be phylogenetically significant and may result from variations in the structure of the hormone or the receptor. (2) The competitive binding properties of hGH are fairly consistent within phylogenetic orders. (3) The simple designation of lactogenic or somatotropic for hormones and receptors is insufficient to characterize the binding properties of this group of hormones.     

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.     

Normalization of epidermal growth factor receptor and transforming growth factor production in drug resistant variants derived from adenovirus transformed cells

Variants (G2, G5) resistant to the cancer chemotherapeutic drug methylglyoxal bis (guanylhydrazone) (MGBG) were isolated from adenovirus type 2 transformed rat brain cells (F4; Sircar et al., 1987). Although at least one of these variants continued to express the adenovirus Ela and Elb transforming proteins, they both exhibited a detransformed phenotype as witnessed by flat morphology, loss of anchorage independent growth, and tumor forming capacity. Reverse transformation suggested the possibility of changes in growth factor receptors and the production of transforming growth factors. To test this possibility, we investigated the status of epidermal growth factor receptors (EGF-r) and transforming growth factor alpha (TGF-alpha) production in F4, G2 and G5 cells. The level of 125I-labeled EGF binding to intact drug resistant cells increased by 2- to 3-fold compared to the transformed parental cell. Scatchard analysis suggests that increased binding was the result of increased receptor levels rather than altered affinity of receptor for ligand. The production of growth factors which compete with 125I-labeled EGF binding declined in the detransformed G2 and G5 cells to a level intermediate between transformed (F4) and normal cells (FR3T3). EGF-receptor increase and the complementary decrease in growth factor production in the drug resistant variants may be associated with detransformation.     

Purification of the human placental alpha 2-macroglobulin receptor

The alpha 2-macroglobulin receptor was solubilized from human placental membranes, purified and characterized. Affinity cross-linking of labelled ligand to intact membranes showed a receptor size compatible with 400-500 kDa. The membranes were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and affinity chromatography was performed using Sepharose-immobilized alpha 2-macroglobulin-methylamine with elution in buffer containing 2 mM EDTA, pH 6.0. SDS-PAGE of the resulting receptor preparation showed a predominant approx. 440 kDa band (reducing conditions) and some minor accompanying proteins of 70-90 kDa and 40 kDa. The yield was 400-800 micrograms receptor preparation per placenta. The receptor preparation immobilized on nitrocellulose bound the alpha 2-macroglobulin-trypsin complex with a dissociation constant of about 400 pM. 125I-iodinated receptor preparation bound almost quantitatively to Sepharose-immobilized alpha 2-macroglobulin-methylamine in the presence of CHAPS alone, and bound 70-80% in the presence of 0.2% SDS. The labelled proteins were separated in the presence of 0.2% SDS by gel filtration or SDS-PAGE (unboiled samples). The 440 kDa protein accounted for the major part of the binding, although some approx. 80 kDa proteins, perhaps proteolytic degradation products, also showed binding activity.