scholarly journals The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts.

1983 ◽  
Vol 96 (3) ◽  
pp. 644-650 ◽  
Author(s):  
M Merion ◽  
W S Sly
Keyword(s):  
Plasma Membrane ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Subcellular Fractionation ◽  
Secondary Lysosome ◽  
Secondary Lysosomes ◽  
Gradient Fractionation ◽  
Intermediate Density

Recent work from several laboratories has suggested the participation of intermediate structures in the delivery of adsorbed ligands from the plasma membrane to lysosomes. This report presents subcellular fractionation studies bearing on the role of these structures in adsorptive pinocytosis of epidermal growth factor (EGF), beta-hexosaminidase, and low density lipoprotein (LDL) by human fibroblasts. Using a two-step Percoll density gradient fractionation, we identified newly internalized (5 min) EGF in two intermediate density structures that are essentially negative for plasma membrane marker, and more bouyant than secondary lysosomes. Continued incubation for 20 min resulted in transfer to (or conversion to) vesicles sedimenting with secondary lysosomes. Internalized beta-hexosaminidase and LDL behaved similarly, appearing first in structures of intermediate density, and later appearing in association with secondary lysosomes. Two drugs, NH4Cl and monensin, were found to inhibit ligand transfer to the secondary lysosome peak, although they did not inhibit entry of bound ligands into intermediate density structures. Upon removal of both inhibitors, internalized ligands were quickly transferred to the secondary lysosome peak. This "transfer process" was faster for EGF, than for the other two ligands studied. We interpret these data to indicate that the endocytosis of these three ligands, and their delivery to lysosomes in fibroblasts, proceeds through a common pathway, involving intermediate nonlysosomal structures.

scholarly journals Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Akash Das ◽  
Michael S Brown ◽  
Donald D Anderson ◽  
Joseph L Goldstein ◽  
Arun Radhakrishnan
Keyword(s):  
Plasma Membrane ◽  
Regulatory Mechanism ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Cholesterol Homeostasis ◽  
Mutant Form ◽  
Perfringolysin O ◽  
Cholesterol Levels ◽  
Plasma Membrane Cholesterol

When human fibroblasts take up plasma low density lipoprotein (LDL), its cholesterol is liberated in lysosomes and eventually reaches the endoplasmic reticulum (ER) where it inhibits cholesterol synthesis by blocking activation of SREBPs. This feedback protects against cholesterol overaccumulation in the plasma membrane (PM). But how does ER know whether PM is saturated with cholesterol? In this study, we define three pools of PM cholesterol: (1) a pool accessible to bind 125I-PFO*, a mutant form of bacterial Perfringolysin O, which binds cholesterol in membranes; (2) a sphingomyelin(SM)-sequestered pool that binds 125I-PFO* only after SM is destroyed by sphingomyelinase; and (3) a residual pool that does not bind 125I-PFO* even after sphingomyelinase treatment. When LDL-derived cholesterol leaves lysosomes, it expands PM's PFO-accessible pool and, after a short lag, it also increases the ER's PFO-accessible regulatory pool. This regulatory mechanism allows cells to ensure optimal cholesterol levels in PM while avoiding cholesterol overaccumulation.

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts

Cell ◽  
1977 ◽  
Vol 10 (3) ◽  
pp. 351-364 ◽  
Author(s):  
Richard G.W. Anderson ◽  
Michael S. Brown ◽  
Joseph L. Goldstein
Keyword(s):  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Low Density ◽  
Endocytic Vesicle

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.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

1997 ◽  
Vol 77 (04) ◽  
pp. 710-717 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
J Kar Kruijt ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Liver Uptake ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

The Role of the Endothelium in Premature Atherosclerosis: Molecular Mechanisms

2020 ◽  
Vol 27 (7) ◽  
pp. 1041-1051 ◽  
Author(s):  
Michael Spartalis ◽  
Eleftherios Spartalis ◽  
Antonios Athanasiou ◽  
Stavroula A. Paschou ◽  
Christos Kontogiannis ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Low Density Lipoprotein ◽  
Critical Role ◽  
Density Lipoprotein ◽  
Number Of Genes ◽  
Causes Of Mortality ◽  
Artery Disease ◽  
Genetic And Environmental Factors ◽  
Made In

Atherosclerotic disease is still one of the leading causes of mortality. Atherosclerosis is a complex progressive and systematic artery disease that involves the intima of the large and middle artery vessels. The inflammation has a key role in the pathophysiological process of the disease and the infiltration of the intima from monocytes, macrophages and T-lymphocytes combined with endothelial dysfunction and accumulated oxidized low-density lipoprotein (LDL) are the main findings of atherogenesis. The development of atherosclerosis involves multiple genetic and environmental factors. Although a large number of genes, genetic polymorphisms, and susceptible loci have been identified in chromosomal regions associated with atherosclerosis, it is the epigenetic process that regulates the chromosomal organization and genetic expression that plays a critical role in the pathogenesis of atherosclerosis. Despite the positive progress made in understanding the pathogenesis of atherosclerosis, the knowledge about the disease remains scarce.

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