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

1988 ◽  
Vol 252 (3) ◽  
pp. 889-892 ◽  
Author(s):  
H Eckardt ◽  
I Filipovic ◽  
A Hasilik ◽  
E Buddecke
Keyword(s):  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Blot Hybridization ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Relative Increase ◽  
Calmodulin Antagonists ◽  
Dot Blot ◽  
Cultured Human Fibroblasts ◽  
Density Lipoprotein Receptor

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.

scholarly journals Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts.

1981 ◽  
Vol 90 (3) ◽  
pp. 595-604 ◽  
Author(s):  
L S Barak ◽  
W W Webb
Keyword(s):  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Internalization ◽  
Low Density ◽  
Receptor Complexes ◽  
Heparin Sulfate ◽  
Cultured Human Fibroblasts ◽  
Coa Reductase

The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory.

scholarly journals Cholesterol metabolism. Effect of 26-thiacholesterol and 26-aminocholesterol, analogues of 26-hydroxycholesterol, on cholesterol synthesis and low-density-lipoprotein-receptor binding

1988 ◽  
Vol 255 (3) ◽  
pp. 1049-1052 ◽  
Author(s):  
E Miao ◽  
S R Wilson ◽  
N B Javitt
Keyword(s):  
Cholesterol Synthesis ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Activity ◽  
Lipoprotein Receptor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Density Lipoprotein Receptor

1. The effects of 26-aminocholesterol and 26-thiacholesterol on cholesterol synthesis and LDL (low-density lipoprotein)-receptor activity were compared with naturally occurring 26-hydroxycholesterol utilizing both human fibroblasts and hepatoma (Hep G2) cells. 2. At equimolar concentrations (0.625 microM), down-regulation of LDL-receptor activity and cholesterol synthesis was greater with human fibroblasts than with Hep G2 cells. 3. At much higher concentrations (5-20 microM) the 26-thia analogue had little effect on either cholesterol synthesis or LDL-receptor activity.

scholarly journals Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts.

1983 ◽  
Vol 97 (5) ◽  
pp. 1635-1640 ◽  
Author(s):  
W J Schneider ◽  
C J Slaughter ◽  
J L Goldstein ◽  
R G Anderson ◽  
J D Capra ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Binding Reaction ◽  
Density Lipoprotein Receptor ◽  
Peptide Antibodies

The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti-receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.

scholarly journals Uptake and esterification of exogenous cholesterol by low-density-lipoprotein-receptor-negative human fibroblasts in culture

1984 ◽  
Vol 222 (3) ◽  
pp. 821-824 ◽  
Author(s):  
J P Slotte ◽  
S Ekman ◽  
S Björkerud
Keyword(s):  
Exchange Rate ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Low Density Lipoprotein Receptor ◽  
Exogenous Cholesterol ◽  
Density Lipoprotein Receptor

The incorporation and metabolism of both vesicle- and LDL (low-density lipoprotein)- derived [3H]cholesterol by LDL-receptor-negative fibroblasts were studied. Independent of the cholesterol source, free [3H]cholesterol was readily incorporated into the cells and was available for esterification. 7-Oxocholesterol stimulated both [3H]cholesterol incorporation, by increasing the exchange rate, and the subsequent esterification of it irrespective of the source of exogenous [3H]cholesterol. The 7-oxocholesterol-stimulated esterification of exogenously derived LDL free [3H]cholesterol was progesterone-sensitive and energy-requiring.

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