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  • 1
    ISSN: 1573-904X
    Keywords: epidermal growth factor ; receptor-mediated endocytosis ; transcytosis ; Madin-Darby Canine Kidney (MDCK) epithelial cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. The aim of this study is to clarify the intracellular fate and a rate limiting step in transcytosis of epidermal growth factor (EGF) in Madin-Darby Canine Kidney (MDCK) epithelial cells. Methods. The kinetics of transcytosis of 125I-EGF was investigated. To examine the fate of EGF molecules bound to its receptor on the cell surface, 125I-EGF was prebound to the basal surface at 4°C, followed by extensive washing and subsequent incubation at 37°C in EGF-free medium. Results. Saturable transport of 125I-EGF through the cell monolayer could only be observed from the basal to apical side. Most (~90%) of the EGF molecules bound to the surface receptor are internalized with a half-life of 1−3 min, followed by intracellular degradation with a half-life of 20−50 min. The exocytosis of internalized EGF into the apical medium is much slower with a half-life of 130−250 min. Even when 125I-EGF was incubated with MDCK cells at 37°C and washed with acid to remove cell-surface 125I-EGF, intact 125I-EGF appeared in the basal medium with a half life of 160−170 min. Conclusions. The exocytosis of internalized EGF into the apical medium is a rate limiting step in EGF transcytosis. At least a small amount of internalized EGF is recycled.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-904X
    Keywords: hepatocyte growth factor ; receptor-mediated endocytosis ; pharmacokinetics ; liver
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. The distribution of 125I-hepatocyte growth factor (HGF) to either liver parenchymal cells (PC) or non-parenchymal cells (NPC) was investigated in rats. Methods. After injection of a trace amount of 125I-HGF, the distribution of radioactivity determined by microautoradiography closely resembled that of 125I-epidermal growth factor which distributes mainly to PC. Results. The uptake clearance of 125I-HGF estimated by determining the radioactivity of isolated liver cells was three times higher for PC than for NPC. This suggests that HGF distributes mainly to PC at relatively low doses. On the other hand, the uptake clearance by PC fell on coadministering an excess (80 µg/kg) of unlabeled HGF, while no change was observed for NPC, indicating that a saturable process for the hepatic handling of HGF exists only in PC where the HGF receptor is expressed. Conclusions. At such a dose the uptake clearance was comparable for both PC and NPC showing that HGF distributes to both cell types although NPC have few HGF receptors. Since the distribution to NPC was relatively non-specific and heparin-sensitive, it may be that heparin-like substances, which are believed to exist on PC and/ or the extracellular matrix, also exist on NPC.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Pharmaceutical research 6 (1989), S. 192-202 
    ISSN: 1573-904X
    Keywords: receptor-mediated endocytosis ; peptide hormones ; epidermal growth factor ; down-regulation ; silent receptor ; internalization ; liver perfusion method ; multiple indicator dilution method ; physiological pharmacokinetic model
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A remarkable feature of the pharmacokinetics of polypeptide hormones is the contribution of specific binding sites (receptors) to the polypeptide hormone distribution and clearance in the body. The concept of “transport receptor” is now well established, and receptor-mediated endocytosis (RME) is recognized as a general mechanism in the uptake of biologically important peptide hormones. This article focuses on the kinetic analysis of the RME of polypeptides, based mainly upon the observations of the kinetics of epidermal growth factor in the liver. The following points are emphasized: (1) How can we determine the existence and the kinetic constants of polypeptide RME in vivo and in the perfused liver system? A liver perfusion method, the single-pass multiple-indicator dilution technique, has been shown to be suitable for analyzing the dynamics of interaction of peptide hormones with their cell surface receptors. (2) What is the importance of down-regulation of transport receptors to the overall kinetics of polypeptides in vivo? Time profiles of polypeptide plasma concentrations and their surface receptors in the liver after iv administration of epidermal growth factor were simulated with a physiologic pharmacokinetic model that includes kinetic constants representing the interaction of polypeptides and their receptors.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-904X
    Keywords: receptor-mediated endocytosis ; acid wash ; internalization ; filtering kidney ; epidermal growth factor (EGF) ; nonsaturable uptake of EGF from the luminal side of the kidney, luminal uptake of EGF in the proximal convoluted tubules
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract We investigated the renal distribution of 125I-EGF in the filtering perfused rat kidney using an acid washing technique. Trichloroacetic acid-precipitable 125I-EGF radioactivity was eluted from both the renal vein and the urinary cannulae, the former regarded as representing the antiluminal, and the latter the luminal, cell surface bound 125I-radioactivity. The addition of excess unlabeled EGF (20 nM) to the perfusate completely inhibited the binding of 125I-EGF to the antiluminal membrane but did not inhibit that of 125I-EGF to the luminal membrane. On the other hand, the order of relative density of 125I-EGF binding sites in the in vivo kidney determined by auto-radiography was cortex 〉 inner medulla 〉 outer medulla. After the i.v. administration of excess unlabeled EGF together with 125I-EGF, the renal uptake of 125I-EGF was inhibited completely in the inner medulla, but only by 50% in the cortex and outer medulla, suggesting the presence of nonsaturable luminal uptake of EGF in the cortex and outer medulla. After i.v. administration of 125I-EGF, a change in position of silver grains from the luminal cell surface membrane to the intracellular space was observed in the proximal convoluted tubules. In conclusion, in addition to the previously identified uptake mechanism of circulating EGF through high-affinity binding sites on the antiluminal cell surface membrane, the reabsorption mechanism of filtered EGF through low-affinity binding sites on the luminal cell surface membrane was demonstrated. In vivo autoradiography showed the gradual internalization of EGF from the luminal cell surface membrane to the intracellular space of the proximal convoluted tubule.
    Type of Medium: Electronic Resource
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