Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits.

Huettinger, Manfred; Schneider, Wolfgang J.; Ho, Y. K.; Goldstein, Joseph L.; Brown, Michael S.

doi:10.1172/jci111469

Cited by 54 publications

(25 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Individual strips were incubated in 1 ml of incubation buffer (Tris-buffered saline (TBS) 50 mM Tris, 150 mM NaCl, pH 7.4, 2 mM CaC12, 0.01% Triton X-100, Pierce Rockford, IL) after blocking with 5 mg non-fat dry milk per ml (2 h each). Immunodetection was done essentially as described [31] using HRP-conjugated second antibodies (Bio-Rad, Vienna) and the ECL reagent (Amersham, Vienna) according to the specifications of the manufacturer for visualisation. For quantification of bound radioactive ligands densitometric scanning of autoradiographs was performed with a scanner system (IMAGE-MASTER, Pharmacia, Sweden).…”

Section: Electrophoresis and Blottingmentioning

confidence: 99%

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α₂‐macroglobulin receptor and transport to endosomes

et al. 1995

View full text Add to dashboard Cite

LDL receptor related protein (LRP) is a ubiquitously expressed cell surface receptor that binds, at least in vitro, a plethora of ligands among them c~2-macroglobulin and lactoferrin (Lf). The function of LRP in internalisation and distribution of ligands within cellular metabolism is still unclear. We here investigated by combined ligand-and immunoblotting the participation of LRP/a2MR and its associated protein (RAP) in receptor mediated endocytosis of Lf into rat liver. We found LRP highly enriched in sucrose density gradient fractions around density I.I0 g/ml, previously characterised as endosomal fractions. RAP was concentrated in distinct fractions around density 1.14 g/ml. This separation of RAP from LRPIa2MR is physiologically meaningful as RAP avidly binds to LRPIa2MR and by that shuts off aH ligand binding function. In endosomal fractions we found one single binding protein for ~2Sl-labelled Lf. With a specific anti LRPla2MR antibody and ligand blotting with 125I-labelled RAP this endosomal Lf binding site was verified to be LRP/azMR. Endosomes did not bind labelled Lf when prepared from rats that received an intravenous injection of Lf (20 mg per animal) 20 rain prior to preparation. Surprisingly we immunodetected Lf in these endosomes at a position around 600 kDa, comigrating with LRP/ c~2MR. We determined Lf binding to be optimal at pH 5.8, what led us to suggest the existence of a very stable LF-LRP/c~2MR complex in endosomes. These data support the idea of effective binding of Lf at pH as found in inflamed tissue environment where Lf is reported to be involved in leukocyte mediated inflammation regulation.

show abstract

Section: Electrophoresis and Blottingmentioning

confidence: 99%

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α₂‐macroglobulin receptor and transport to endosomes

et al. 1995

View full text Add to dashboard Cite

show abstract

“…After binding to the LDL receptor, these antibodies are carried into the cell and delivered to lysosomes, as is LDL (23). When such a monoclonal antibody is labeled with 1251 and injected into rabbits, most of the radioactivity is taken up rapidly in the liver in a process that depends on the antibody binding to the LDL receptor (24).…”

Section: Methodsmentioning

confidence: 99%

Imaging of hepatic low density lipoprotein receptors by radionuclide scintiscanning in vivo.

Huettinger¹,

Corbett²,

Schneider³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The low density lipoprotein (LDL) receptor mediates the cellular uptake of plasma lipoproteins that are derived from very low density lipoproteins (VLDL). Most of the functional LDL receptors in the body are located in the liver. Here, we describe a radionuclide scintiscanning technique that permits the measurement of LDL receptors in the livers of intact rabbits. '231-labeled VLDL were administered intravenously, and scintigraphic images of the liver and heart were obtained at intervals thereafter. In seven normal rabbits, radioactivity in the liver increased progressively between 1 and 20 min after injection, while radioactivity in the heart (reflecting that in plasma) decreased concomitantly. In Watanabeheritable hyperlipidemic rabbits, which lack LDL receptors on a genetic basis, there was little uptake of 1231-labeled VLDL into the liver and little decrease in cardiac radioactivity during this interval. These findings demonstrate that the LDL receptor is necessary for the hepatic uptake of VLDL-derived lipoproteins in the rabbit. Two conditions that diminish hepatic LDL receptor activity, cholesterol-feeding and prolonged fasting, also reduced the uptake of 123I-labeled VLDL in the liver as measured by scintiscanning. The In experimental animals (2-5) and in man (6-8), the total number of LDL receptors in the body has been estimated by measuring the rate of disappearance of 125I-labeled LDL from the circulation. In animals, the number of receptors in various tissues can be estimated by removal of organs after intravenous administration of radiolabeled lipoproteins and measurement of the radioactive lipoprotein that has been taken up by those organs. Such studies have revealed that the vast bulk of LDL receptors in animals are in the liver (4, 9, 10) and that these hepatic receptors are subject to regulation (2,5,(10)(11)(12)(13). In rabbits, LDL receptor activity is suppressed by high cholesterol diets (11), by diets composed of wheat starch and casein (12), and by prolonged fasting (13). Conversely, LDL receptor activity is stimulated by agents that lower the cholesterol content of liver, such as bile acidbinding resins and inhibitors of cholesterol synthesis (2, 8).A method for the noninvasive measurement of LDL receptor activity in liver would be desirable, initially for experimental studies in animals, and eventually for clinical studies in humans. The possibility of such measurement emerges from recent advances in scintiscanning and computer techniques and in the availability of low-energy isotopes (14). Theoretically, these methods should allow quantitative estimates in vivo of the receptor-mediated uptake of radiolabeled lipoproteins in the liver.To this end, we administered 123I-labeled VLDL intravenously to rabbits and measured the uptake of the lipoprotein in livers of these animals by radionuclide scintiscanning. We have chosen VLDL as a ligand because this lipoprotein is converted in the bloodstream to IDL, which enters the liver rapidly, due to its content of apoprotein E (15,16). In ...

show abstract

“…8 For the in vivo determination of LDL turnover, 40xl0 6 cpm labeled LDL was injected into the marginal ear vein, and the disappearance of radioactivity was determined as described. 8 4 cpm/nmol).…”

Section: Iodination Of Lipoproteins and Assay Of Internalization And mentioning

confidence: 99%

Hypolipidemic activity of HOE-402 is mediated by stimulation of the LDL receptor pathway.

Huettinger

Hermann

Goldenberg

et al. 1993

Arterioscler Thromb

Self Cite

View full text Add to dashboard Cite

HOE-402 (4 -amlno-2 -[4,4 -dimethyl-2 -oxo-1 -imldazolldinyl] -pyrimldlne-5 -A' -[trifluoromethylphenyl] -carboxamide-monohydrochloride) has been shown to exhibit hypolipidemic action in heterozygous Watanabe heritable hyperlipidemic rabbits. In all animals, elevated cholesterol levels were reduced to normal (from 3.0 to 1.5 mmol/L) after 3 weeks of HOE-402 treatment This was due entirely to reduction of low density lipoprotein (LDL) cholesterol and was paralleled by accelerated removal of plasma '"I-LDL. This reduction of LDL levels was not found in homozygous LDL receptor-defective animals, emphasizing the necessity of a functional LDL receptor system for the hypolipidemic action. The effect of HOE-402 on LDL receptor activity in the cultured hepatoma cell line HepG2 was also determined. When cells were incubated with plasma from treated animals (containing cholesterol 1.5 mmol/L and HOE-402 80 ng/mL), high-affinity cell-surface binding sites for LDL were induced more than threefold, as shown by Scatchard analysis of cell-surface binding data. Induction of the LDL receptor was detectable after 6 hours and was 300% after 18 hours. This induction was specific for LDL, as '"I-transferrin and ["Feltransferrin were internalized normally in HOE-402-treated cells. The increase of LDL receptor protein was related to induced LDL receptor mRNA levels (400%), as shown by quantification of Northern blotting experiments. These findings suggest that HOE-402 mediated its hypolipidemic action mainly via the LDL receptor pathway. It enhanced mRNA levels for LDL receptor, hence increasing its synthesis, which subsequently resulted in reduced plasma LDL levels. Apart from having pharmacological implications, this compound could be very useful in elucidating the components that regulate the gene expression of the LDL receptor. through a concerted regulation of two pathways J. A-that supply them with exogenous and endogenous cholesterol. When cells need cholesterol, they start the buildup of low density lipoprotein receptor (LDLr) protein to enhance the uptake of exogenous cholesterol. In addition, they increase the amount of enzymes responsible for de novo cholesterol synthesis. The balance of these pathways is particularly delicate, as it controls not only the intracellular level of cholesterol but also the extracellular blood cholesterol level. As a therapeutic method of keeping blood cholesterol levels down, this balance can be shifted toward enhanced uptake of cholesterol into the cells. By blocking de novo synthesis of cholesterol with certain drugs, cells rely on exogenous uptake and mediate this demand by increasing the synthesis of mRNA coding for the LDLr protein. 13 Besides blocking intracellular de novo synthesis, treatment with ethinyl estradiol also leads to dramatically enhanced LDLr activity. The enhanced LDL uptake from plasma into the liver leads to reduced plasma cholesterol levels. In both systems, the series of events that are triggered follow the most powerful pathway to reduce plasma cholesterol: synthesis o...

show abstract

Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits.

Cited by 54 publications

References 30 publications

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α₂‐macroglobulin receptor and transport to endosomes

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α₂‐macroglobulin receptor and transport to endosomes

Imaging of hepatic low density lipoprotein receptors by radionuclide scintiscanning in vivo.

Hypolipidemic activity of HOE-402 is mediated by stimulation of the LDL receptor pathway.

Contact Info

Product

Resources

About

Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits.

Cited by 54 publications

References 30 publications

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α2‐macroglobulin receptor and transport to endosomes

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α2‐macroglobulin receptor and transport to endosomes

Imaging of hepatic low density lipoprotein receptors by radionuclide scintiscanning in vivo.

Hypolipidemic activity of HOE-402 is mediated by stimulation of the LDL receptor pathway.

Contact Info

Product

Resources

About

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α₂‐macroglobulin receptor and transport to endosomes

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor‐related protein/α₂‐macroglobulin receptor and transport to endosomes