Abstract
We compared methods for determining the distribution of epidermal growth factor (EGF) receptors between the cell surface and the cell interior in the rat liver. Incubation of isolated hepatocytes with 100 nM EGF for 20 min at 37°C remarkably decreased the cell-surface EGF receptor density (internalization of receptors). The detergent Brij 35 was previously reported to permit assay of the intra-cellular latent EGF receptors in liver homogenates, but in the present investigation, Brij 35 lowered the affinity of EGF for the receptor depending on the detergent concentration, and the appearance of latent receptors was not observed. In contrast, permeabilization of the cells with digitonin, followed by an acid-washing procedure, increased the EGF binding capacity to close to the control level. Hence, the EGF receptors, internalized together with EGF molecules, were not degraded for at least 20 min, and the digitonin method is suitable for quantifying the intracellular EGF receptors. The binding capacities of the digitonin-treated and untreated control cells showed no difference upon digitonin treatment, suggesting that the bulk of EGF receptors exists on the cell surface. Further, cell-surface EGF receptor density was determined after the i.v. administration of EGF (300 µg/kg) to rats. Isolated hepatocytes prepared 30 min after the administration of EGF showed little binding for EGF on the cell surface, while the cell-surface EGF receptor density recovered to close to control values in cells prepared after 3 hr.
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REFEREMCES
S. Yanai, Y. Sugiyama, D. C. Kim, T. Iga, T. Fuwa, and M. Hanano. Kinetic analysis of receptor mediated endocytosisi of epidermal growth factor by isolated rat hepatocytes. Am. J. Physiol. 260 (Cell Physiol. 29) (1991).
E. Maratos-Flier, C. Y. Y. Kao, E. M. Verdin, and G. L. King. Receptor-mediated vectorial transport of epidermal growth factor by Madin-Darby canine kidney cells. J. Cell Biol. 105:1595–1601 (1987).
H. Sato, Y. Sugiyama, Y. Sawada, T. Iga, S. Sakamoto, T. Fuwa, and M. Hanano. Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell surface receptors in the perfused rat liver by multiple-indicator dilution method. Proc. Natl. Acad. Sci. USA 85:8355–8359 (1988).
H. Sato, Y. Sugiyama, Y. Sawada, T. Iga, T. Fuwa, and M. Hanano. Internalization of EGF in perfused rat liver is independent of the degree of receptor occupancy. Am. J. Physiol. 258:G682–G689 (1990).
D. C. Kim, Y. Sugiyama, H. Sato, T. Fuwa, T. Iga, and M. Hanano. Kinetic analysis of in vivo receptor dependent binding of human epidermal growth factor by rat tissues. J. Pharm. Sci. 77:200–207 (1988).
S. Yanai, Y. Sugiyama, T. Iga, T. Fuwa, and M. Hanano. Kinetic analysis of the down-regulation of epidermal growth factor receptors in rats in vivo. Am. J. Physiol. 258:C593–C598 (1990).
Y. Sugiyama, D. C. Kim, H. Sato, S. Yanai, H. Sato, T. Iga, and M. Hanano. Receptor-mediated disposition of polypeptides: Kinetic analysis of the transport of epidermal growth factor as a model peptide using in vitro isolated perfused organs and in vivo system. J. Control. Rel. 13:157–174 (1990).
W. A. Dunn, T. P. Connolly, and A. L. Hubbard. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: Receptor pathway J. Cell Biol. 102:24–36 (1986).
W. A. Dunn and A. L. Hubbard. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: Ligand and receptor dynamics. J. CellBiol. 98:2148–2159 (1984).
D. A. McClain and J. M. Olefsky. Evidence for two independent pathways of insulin-receptor internalization in hepatocytes and hepatoma cells. Diabetes 37:806–815 (1988).
P. H. Weigel, D. A. Ray, and J. A. Oka. Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells. Anal. Biochem. 133:437–449 (1983).
P. H. Weigel and J. A. Oka. Recycling of the hepatic asialoglyoprotein receptor in isolated rat hepatocytes. J. Biol. Chem. 259:1150–1154 (1984).
Y. Sugiyama, H. Sato, S. Yanai, D. C. Kim, S. Miyauchi, Y. Sawada, T. Iga, and M. Hanano. Receptor-mediated clearance of peptide hormones. In D. D. Breimer, D. J. A. Crommelin, and K. K. Midha (eds.), Topics in Pharmaceutical Sciences 1989, Amsterdam Medical Press, Amsterdam, 1989, pp. 429–443.
T. Oka, S. Sakamoto, K. Miyosi, T. Fuwa, K. Yoda, M. Yamasaki, G. Tamura, and T. Miyake. Synthesis and secretion of human epidermal growth factor by Esherichia coli. Proc. Natl. Acad. Sci. USA 82:7212–7216 (1985).
I. Vlodavsky, K. D. Brown, and D. Gospodarowicz. A comparison of the binding of epidermal growth factor to cultured granulosa and luteal cells. J. Biol. Chem. 253:3744–3750 (1978).
H. Baur, S. Kasperek, and E. Pfaff. Criteria of viability of isolated liver cells. Hoppe-Seyler Z. Physiol. Chem. 356:827–838 (1975).
S. Yanai, Y. Sugiyama, D. C. Kim, H. Sato, T. Fuwa, T. Iga, and M. Hanano. Binding of human epidermal growth factor to tissue homogenates of the rat. Chem. Pharm. Bull. 35:4891–4897 (1987).
C. Hertel and J. P. Perkins. Sequential appearance of epidermal growth factor in plasma membrane associated and intracellular vesicles during endocytosis. J. Biol. Chem. 262:11407–11409 (1987).
H. T. Haigler, F. R. Maxfield, M. C. Willingham, and I. Pastan. Dansylcadaverine inhibits internalization of 125Iepidermal growth factor in BALB 3T3 cells. J. Biol. Chem. 255:1239–1241 (1980).
B. S. Andersson and D. P. Jones. Use of digitonin fractionation to determine mitochondrial transmembrane ion distribution in cells during anoxia. Anal. Biochem. 146:164–172 (1985).
K. Yamaoka, Y. Tanigawara, T. Nakagawa, and T. Uno. A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobio-dyn. 4:879–889 (1981).
B. A. L. Jones, R. H. Renston, and S. J. Burwen. Uptake and intracellular disposition of plasma-derived proteins and apoproteins by hepatocytes. In H. Popper and F. Schaffner (eds.), Progress in Liver Diseases, Grune &; Stratton, New York, 1982, Vol. VII, pp. 51–69.
Y. Sugiyama and M. Hanano. Receptor-mediated transport of peptide hormones and its importance in the overall hormone disposition in the body. Pharm. Res. 6:194–204 (1989).
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Yanai, S., Sugiyama, Y., Iga, T. et al. Comparison of the Methods for Determining Cell-Surface and Intracellular Receptors for Epidermal Growth Factor in the Rat Liver. Pharm Res 8, 557–562 (1991). https://doi.org/10.1023/A:1015836219502
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DOI: https://doi.org/10.1023/A:1015836219502