Fluid-phase endocytosis of horseradish peroxidase by cerebral endothelial cells in primary culture: Characterization and kinetic analysis

1994 ◽  
Vol 38 (6) ◽  
pp. 654-663 ◽  
Author(s):  
L. J. Noble ◽  
J. E. Kalinyak ◽  
L. H. Pitts ◽  
J. J. Hall
Keyword(s):  
Endothelial Cells ◽  
Horseradish Peroxidase ◽  
Kinetic Analysis ◽  
Primary Culture ◽  
Fluid Phase ◽  
Cerebral Endothelial Cells ◽  
Fluid Phase Endocytosis

Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins

1993 ◽  
Vol 264 (5) ◽  
pp. G880-G885 ◽  
Author(s):  
Y. Yamaguchi ◽  
E. Dalle-Molle ◽  
W. G. Hardison
Keyword(s):  
Horseradish Peroxidase ◽  
Parenchymal Cell ◽  
Fluid Phase ◽  
Mannose Receptor ◽  
Isolated Hepatocytes ◽  
Cell Counting ◽  
Cell Fractionation ◽  
Morphological Studies ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of colchicine on endocytosis of horseradish peroxidase by rat peritoneal macrophages

1980 ◽  
Vol 45 (1) ◽  
pp. 59-71 ◽  
Author(s):  
A. Piasek ◽  
J. Thyberg
Keyword(s):  
Horseradish Peroxidase ◽  
Digestive Enzymes ◽  
Cytochalasin B ◽  
Fluid Phase ◽  
Peritoneal Macrophages ◽  
The Other ◽  
Fluid Phase Endocytosis ◽  
Cytoplasmic Microtubules ◽  
Endocytic Vesicles ◽  
Cytoplasmic Complex

Horseradish peroxidase (HRP) was used as an exogenous marker to study the effects of microtubule-disruptive drugs on endocytosis in cultures of thioglycollate-elicited rat peritoneal macrophages. Colchicine and vinblastine, but not lumicolchicine or cytochalasin B, reduced HRP uptake by about 30–40%. However, as determined by stereological measurements, the size of the HRP-containing compartment within the cells remained unaltered. In both control cells and cells treated with colchicine or vinblastine the HRP-reactive vesicles were preferentially located close to the dictyosomes (stacks of cisternae) despite the fact that the Golgi complex was disorganized in the treated cells. These results suggest that intact cytoplasmic complex was disorganized in the treated cells. These results suggest that intact cytoplasmic microtubules are required to maintain a normal rate of fluid phase endocytosis in macrophages. On the other hand, it seems as if microtubules are not necessary for the translocation of newly formed endocytic vesicles/lysosomes to the dictyosomes, from which they probably are supplied with digestive enzymes.

ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 650-658 ◽  
Author(s):  
Silvia Muro ◽  
Christine Gajewski ◽  
Michael Koval ◽  
Vladimir R. Muzykantov
Keyword(s):  
Drug Delivery ◽  
Endothelial Cells ◽  
Fluid Phase ◽  
Therapeutic Agents ◽  
Intercellular Adhesion Molecule ◽  
Major Fraction ◽  
Intercellular Adhesion Molecule 1 ◽  
Lysosomal Trafficking ◽  
Fluid Phase Endocytosis

AbstractIntercellular adhesion molecule-1 (ICAM-1) is a target for drug delivery to endothelial cells (ECs), which internalize multivalent anti-ICAM nanocarriers (anti-ICAM/NCs) within 15 to 30 minutes. The concomitant ICAM-1 disappearance from the EC surface transiently inhibited subsequent binding and uptake of anti-ICAM/NCs. Within 1 hour, internalized ICAM-1 diverged from anti-ICAM/NCs into prelysosomal vesicles, resurfaced, and enabled uptake of a subsequent anti-ICAM/NC dose. Thus, internalized ICAM-1 was able to recycle back to the plasma membrane. In vivo pulmonary targeting of a second anti-ICAM/NC dose injected 15 minutes after the first dose was decreased by 50% but recovered between 30 minutes and 2.5 hours, comparable to cultured ECs. Anti-ICAM/NCs affected neither EC viability nor fluid-phase endocytosis and traffic to lysosomes. However, lysosomal trafficking of the second dose of anti-ICAM/NCs was decelerated at least 2-fold versus the first dose; hence the major fraction of anti-ICAM/NCs resided in prelysosomal vesicles for at least 5 hours without degradation. Two successive doses of anti-ICAM/NC/catalase protected ECs against H2O2 for at least 8 hours versus 2 hours afforded by a single dose, suggesting that recurrent targeting to ICAM-1 affords longer effects. ICAM-1 recycling and inhibited lysosomal traffic/degradation of subsequent doses may help to prolong activity of therapeutic agents delivered into ECs by anti-ICAM/NCs.

scholarly journals Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells

2008 ◽  
Vol 19 (12) ◽  
pp. 5490-5505 ◽  
Author(s):  
Nancy A. Goebl ◽  
Clifford M. Babbey ◽  
Amita Datta-Mannan ◽  
Derrick R. Witcher ◽  
Victor J. Wroblewski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fluid Phase ◽  
Surface Expression ◽  
Fc Receptor ◽  
Expression Level ◽  
Surface Binding ◽  
Neonatal Fc Receptor ◽  
Binding Forms ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.

scholarly journals Angiotensin Peptide Regulation of Fluid-Phase Endocytosis in Brain Microvessel Endothelial Cell Monolayers

1990 ◽  
Vol 10 (6) ◽  
pp. 827-834 ◽  
Author(s):  
François L. Guillot ◽  
Kenneth L. Audus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Lucifer Yellow ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Ang Ii ◽  
Microvessel Endothelial Cell ◽  
Fluid Phase Endocytosis ◽  
Microvessel Endothelial Cells ◽  
Brain Microvessel

An in vitro model comprised of primary cultures of brain microvessel endothelial cells was used to investigate angiotensin II (Ang II) effects on blood–brain barrier fluid-phase endocytosis. The effects of Ang II, saralasin, sarathrin, bradykinin (BK), and phorbol myristate acetate (PMA) on brain microvessel endothelial cell fluid-phase endocytosis were determined using the fluorescent marker, Lucifer yellow. Nanomolar concentrations of saralasin (a partial Ang II agonist) stimulated brain microvessel endothelial cell endocytosis by 30% whereas Ang II treatment enhanced Lucifer yellow uptake by 20%. Sarathrin (an Ang II antagonist) had no effect on Lucifer yellow uptake. Nanomolar concentrations of BK and PMA also stimulated Lucifer yellow uptake by the brain microvessel endothelial cell by 40 and 95%, respectively. Stimulatory effects of Ang II and saralasin on Lucifer yellow uptake by brain microvessel endothelial cells could be completely blocked by pretreatment with either sarathrin or indomethacin (an inhibitor of prostaglandin synthesis). In contrast, the effects of neither BK nor PMA on brain microvessel endothelial cell uptake of Lucifer yellow were altered by indomethacin pretreatment. Results indicated that Ang II, saralasin, BK, and PMA produce similar stimulatory effects on brain microvessel endothelial cell fluid-phase endocytosis with only Ang II and saralasin, producing increases in brain microvessel endothelial cell fluid-phase endocytosis that appeared to be mediated by prostaglandins.

scholarly journals Okadaic acid induces Golgi apparatus fragmentation and arrest of intracellular transport

1991 ◽  
Vol 100 (4) ◽  
pp. 753-759 ◽  
Author(s):  
J. Lucocq ◽  
G. Warren ◽  
J. Pryde
Keyword(s):  
Golgi Apparatus ◽  
Horseradish Peroxidase ◽  
Vesicular Stomatitis Virus ◽  
Okadaic Acid ◽  
Intracellular Transport ◽  
Fluid Phase ◽  
Fluid Phase Endocytosis ◽  
Vesicular Stomatitis ◽  
Dose Dependent ◽  
Mitotic Cells

The specific phosphatase inhibitor okadaic acid (OA) induced fragmentation of the Golgi apparatus in interphase HeLa cells. Immunoelectron microscopy for galactosyltransferase identified a major Golgi fragment composed of a cluster of vesicles and tubules that was morphologically indistinguishable from the ‘Golgi cluster’ previously described in mitotic cells. The presence of homogeneous immunofluorescence staining for galactosyltransferase in OA-treated cells also suggested that isolated Golgi vesicles, previously found in mitotic cells, existed along with the clusters. After removal of OA, both clusters and vesicles appeared to participate in a reassembly pathway that strongly resembled that occurring during telophase. OA also induced inhibition of intracellular transport, another feature of mitotic cells. OA treatment prevented newly synthesised G protein of vesicular stomatitis virus (VSV) from acquiring resistance to endoglycosidase H and from arriving at the cell surface. In addition, fluid phase endocytosis of horseradish peroxidase (HRP) was reduced to less than 10% of control values. All these effects were dose-dependent and reversible. OA should be a useful tool to study the Golgi division and membrane traffic.

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