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Histochemistry

, Volume 85, Issue 2, pp 169–175 | Cite as

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

  • A. Miki
  • P. Kugler
Article

Summary

The effect of exposure to leupeptin (25 μg/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a doublelabelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptintreated yolk sacs were labelled with Con-A Fer at 4°C and then incubated with HRP for 5, 15 or 60 min at 37°C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptintreated cells did not exhibit any labelling. These findings indicate that, after leupeptin treatment, both endocytotic activity and membrane recycling decrease, and that fusions of the apical vacuolar system with giant lysosomes are retarded or inhibited.

Keywords

Ferritin Endodermal Cell Apical Cell Membrane Membrane Recycling Vacuolar System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Aoyagi T, Umezawa H (1975) Structures and activities of proteinase inhibitors of microbial origin. In: Reich E, Ritkin D, Skaw E (eds) Proteinases and biological control. Cold Spring Harbor Symp Quant Biol, pp 429–454Google Scholar
  2. Beck F (1981) Induced cell injury and cell death as a cause of congenital malformation in rats. Histochem J 13:667–679Google Scholar
  3. Beck F, Lowy A (1982) The effect of cathepsin inhibitor on rat embryos grown in vitro. J Embryol Exp Morphol 71:1–9Google Scholar
  4. Berg T, Ose T, Tolleshaug H (1981) Intracellular degradation of 125I-labelled asialoglycoproteins: effects of leupeptin on subcellular distribution of asialofetuin. Int J Biochem 13:253–259Google Scholar
  5. Brown JC, Hunt RC (1978) Lectins. Int Rev Cytol 52:277–349Google Scholar
  6. Brown NA, Fabro S (1981) Quantitation of rat embryonic development in vitro: A morphological scoring system. Teratology 24:65–78Google Scholar
  7. Freeman SJ, Lloyd JB (1983) Inhibition of proteolysis in rat yolk sac as a cause of teratogenesis. Effects of leupeptin vitro and in vivo. J Embryol Exp Morphol 78:183–193Google Scholar
  8. Gass P, Kugler P (1984) Enzymdifferenzierung des visceralen Dottersackepithels der Ratte. Z Mikrosk Anat Forsch 98:721–745Google Scholar
  9. Hohman TC, Bowers B (1984) Hydrolase secretion is a consequence of membrane recycling. J Cell Biol 98:246–252Google Scholar
  10. Hopgood MF, Clark MG, Ballard FJ (1977) Inhibition of protein degradation in isolated rat hepatocytes. Biochem J 164:399–407Google Scholar
  11. King BF, Enders AC (1970) Protein absorption and transport by the guinea pig visceral yolk sac. Am J Anat 129:261–288Google Scholar
  12. Kirschke H, Langer J, Wiederanders B, Ansorge S, Bohley P (1977) Cathespin L. A new proteinase from rat-liver lysosomes. Eur J Biochem 74:293–301Google Scholar
  13. Knowles SE, Ballard FJ, Livesey G, Williams KE (1981) Effects of microbial proteinase inhibitors on the degradation of endogenous and internalized proteins by rat yolk sac. Biochem J 196:41–48Google Scholar
  14. Kugler P (1982) Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac. Histochemistry 75:215–218Google Scholar
  15. Kugler P (1985) Hämoproteinaufnahme und Peptidasenmuster des visceralen Dottersackepithels der Ratte während der Entwicklung. Acta Histochem (in press)Google Scholar
  16. Kugler P, Beckenbauer T (1986) Wirkung von Leupeptin auf Proteinaufnahme und-abbau des viszeralen Dottersackepithels der Ratte. Z Mikrosk Anat Forsch (in press)Google Scholar
  17. Kugler P, Miki A (1985) Study on membrane recycling in the rat visceral yolk sac endoderm using concanavalin A. Histochemistry 83:359–367Google Scholar
  18. Lambson RO (1966) An electron microscopic visualization of transport across rat visceral volk sac. Am J Anat 118:21–52Google Scholar
  19. Le Hir M, Dubach UC (1982) The cellular specificity of lectin binding in the kidney. I. A light microscopical study in the rat. Histochemistry 74:521–530Google Scholar
  20. Lewis PR, Knight DP (1977) Staining methods for sectioned material. North Holland, Amsterdam New York Oxford, pp 25–53Google Scholar
  21. Libby P, Goldberg AL (1978) Leupeptin, a protease inhibitor, decreases protein degradation in normal and disease muscles. Science 199:534–536Google Scholar
  22. Libby P, Ingwall JS, Goldberg AL (1979) Reduction of protein degradation and atrophy in cultured fetal mouse hearts by leupeptin. Am J Physiol 237:E35-E39Google Scholar
  23. Miki A, Kugler P (1984) Comparative histochemical study on the visceral yolk sac endoderm in the rat in vivo and in vitro. Histochemistry 81:409–415Google Scholar
  24. New DAT (1978) Whole-embryo culture and the study of mammahan embryos during organogenesis. Biol Rev 53:81–122Google Scholar
  25. New DAT, Coppola PT, Terry S (1973) Culture of explanted rat embryos in rotating tubes. J Reprod Fert 35:135–138Google Scholar
  26. New DAT, Coppola PT, Cockroft DL (1976) Improved development of head-fold rat embryos in culture resulting from low oxygen and modifications of the culture medium. J Reprod Fert 48:219–222Google Scholar
  27. Nicolson GL (1974) The interaction of lectins with animal cell surfaces. Int Rev Cytol 39:89–179Google Scholar
  28. Nicolson GL, Singer SJ (1974) The distribution and asymmetry of mammalian cell surface saccharides utilizing ferritin-conjugated plant agglutinins as specific sacchardie stains. J Cell Biol 60:236–248Google Scholar
  29. Seglen PO, Grinde B, Solheim AE (1979) Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem 95:215–225Google Scholar
  30. Seibel W (1974) An ultrastructural comparison of the uptake and transport of horseradish peroxidase by the rat visceral yolk-sac placenta during mid-and late gestation. Am J Anat 140:213–236Google Scholar
  31. Steele CE, New DAT (1974) Serum variants causing the formation of double hearts and other abnormalities in explanted rat embryos. J Embryol Exp Morphol 31:707–719Google Scholar
  32. Steinman RM, Cohn ZA (1972) The interaction of soluble horseradish peoxidase with mouse peritoneal macrophages in vitro. J Cell Biol 55:186–204Google Scholar
  33. Umezawa H, Aoyagi T (1977) Activities of proteinase inhibitors of microbial origin. In: Barrett A (ed) Proteinases in mammalian cells and tissues. North Holland, Amsterdam, pp 637–662Google Scholar
  34. Ward WF, Chua BL, Li JB, Morgan HB, Mortimore GE (1979) Inhibition of basal and deprivation-induced proteolysis by leupeptin and pepstatin in perfused rat liver and heart. Biochem Biophys Res Commun 87:92–98Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • A. Miki
    • 1
  • P. Kugler
    • 1
  1. 1.Anatomical Institute of the University of WürzburgWürzburgFederal Republic of Germany

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