Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Aug 1;111(2):379–389. doi: 10.1083/jcb.111.2.379

Endocytic vesicles from renal papilla which retrieve the vasopressin- sensitive water channel do not contain a functional H+ ATPase

PMCID: PMC2116183  PMID: 1696262

Abstract

The water permeability of the kidney collecting duct epithelium is regulated by vasopressin (VP)-induced recycling of water channels between an intracellular vesicular compartment and the plasma membrane of principal cells. To test whether the water channels pass through an acidic endosomal compartment during the endocytic portion of this pathway, we measured ATP-dependent acidification of FITC-dextran- labeled endosomes in isolated microsomal fractions from different regions of Brattleboro rat kidneys. Both VP-deficient controls and rat treated with exogenous VP were examined. ATP-dependent acidification was not detectable in endosomes containing water channels from distal papilla (osmotic water permeability Pf = 0.038 +/- 0.004 cm/s). In contrast, the addition of ATP resulted in a strong acidification of renal cortical endosomes (pHmin = 5.8, initial rate = 0.18-0.25 pH U/s). Acidification of cortical endosomes was reversed with nigericin and strongly inhibited by N-ethyl-maleimide. Passive proton permeability was similar and low in both cortical and papillary endosomes from rats treated or not treated with VP. The fraction of labeled endosomes present in microsomal preparations was determined by fluorescence imaging microscopy of microsomes nonspecifically bound to poly-l-lysine-coated coverslips and was 25% in cortical preparations compared to 14% (+VP) and 9% (-VP) in papillary preparations. The fraction of cortical endosomes was enriched 1.5-fold by immunoabsorption to coverslips coated with mAbs against the bovine vacuolar proton pump. In contrast, the fraction of papillary endosomes was depleted more than twofold by immunoabsorption to identical coverslips. Finally, sections of distal papilla stained with antibodies against the lysosomal glycoprotein LGP120 showed that most of the entrapped FITC-dextran did not colocalize with this lysosomal protein. These results demonstrate that vesicles which internalize water channels in kidney collecting duct principal cells lack functional proton pumps, and do not deliver the bulk of their FITC-dextran content to lysosomes. The data suggest that the principal cell contains a specialized nonacidic apical endocytic compartment which functions primarily to recycle membrane components, including water channels, to the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson R. G., Falck J. R., Goldstein J. L., Brown M. S. Visualization of acidic organelles in intact cells by electron microscopy. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4838–4842. doi: 10.1073/pnas.81.15.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bomsel M., Prydz K., Parton R. G., Gruenberg J., Simons K. Endocytosis in filter-grown Madin-Darby canine kidney cells. J Cell Biol. 1989 Dec;109(6 Pt 2):3243–3258. doi: 10.1083/jcb.109.6.3243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Breitfeld P. P., Harris J. M., Mostov K. E. Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells. J Cell Biol. 1989 Aug;109(2):475–486. doi: 10.1083/jcb.109.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown D., Gluck S., Hartwig J. Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H+ATPase. J Cell Biol. 1987 Oct;105(4):1637–1648. doi: 10.1083/jcb.105.4.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown D., Hirsch S., Gluck S. An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations. Nature. 1988 Feb 18;331(6157):622–624. doi: 10.1038/331622a0. [DOI] [PubMed] [Google Scholar]
  6. Brown D., Hirsch S., Gluck S. Localization of a proton-pumping ATPase in rat kidney. J Clin Invest. 1988 Dec;82(6):2114–2126. doi: 10.1172/JCI113833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown D. Membrane recycling and epithelial cell function. Am J Physiol. 1989 Jan;256(1 Pt 2):F1–12. doi: 10.1152/ajprenal.1989.256.1.F1. [DOI] [PubMed] [Google Scholar]
  8. Brown D., Orci L. Vasopressin stimulates formation of coated pits in rat kidney collecting ducts. Nature. 1983 Mar 17;302(5905):253–255. doi: 10.1038/302253a0. [DOI] [PubMed] [Google Scholar]
  9. Brown D., Weyer P., Orci L. Vasopressin stimulates endocytosis in kidney collecting duct principal cells. Eur J Cell Biol. 1988 Jun;46(2):336–341. [PubMed] [Google Scholar]
  10. Chen P. Y., Pearce D., Verkman A. S. Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore. Biochemistry. 1988 Jul 26;27(15):5713–5718. doi: 10.1021/bi00415a048. [DOI] [PubMed] [Google Scholar]
  11. Clapp W. L., Madsen K. M., Verlander J. W., Tisher C. C. Intercalated cells of the rat inner medullary collecting duct. Kidney Int. 1987 May;31(5):1080–1087. doi: 10.1038/ki.1987.111. [DOI] [PubMed] [Google Scholar]
  12. Forgac M. Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev. 1989 Jul;69(3):765–796. doi: 10.1152/physrev.1989.69.3.765. [DOI] [PubMed] [Google Scholar]
  13. Fuller S. D., Simons K. Transferrin receptor polarity and recycling accuracy in "tight" and "leaky" strains of Madin-Darby canine kidney cells. J Cell Biol. 1986 Nov;103(5):1767–1779. doi: 10.1083/jcb.103.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giloh H., Sedat J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982 Sep 24;217(4566):1252–1255. doi: 10.1126/science.7112126. [DOI] [PubMed] [Google Scholar]
  16. Gluck S., Al-Awqati Q. Vasopressin increases water permeability in inducing pores. Nature. 1980 Apr 17;284(5757):631–632. doi: 10.1038/284631a0. [DOI] [PubMed] [Google Scholar]
  17. Gluck S., Caldwell J. Immunoaffinity purification and characterization of vacuolar H+ATPase from bovine kidney. J Biol Chem. 1987 Nov 15;262(32):15780–15789. [PubMed] [Google Scholar]
  18. Griffiths G., Hoflack B., Simons K., Mellman I., Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. doi: 10.1016/s0092-8674(88)80026-6. [DOI] [PubMed] [Google Scholar]
  19. Handler J. S. Antidiuretic hormone moves membranes. Am J Physiol. 1988 Sep;255(3 Pt 2):F375–F382. doi: 10.1152/ajprenal.1988.255.3.F375. [DOI] [PubMed] [Google Scholar]
  20. Lencer W. I., Weyer P., Verkman A. S., Ausiello D. A., Brown D. FITC-dextran as a probe for endosome function and localization in kidney. Am J Physiol. 1990 Feb;258(2 Pt 1):C309–C317. doi: 10.1152/ajpcell.1990.258.2.C309. [DOI] [PubMed] [Google Scholar]
  21. Lewis V., Green S. A., Marsh M., Vihko P., Helenius A., Mellman I. Glycoproteins of the lysosomal membrane. J Cell Biol. 1985 Jun;100(6):1839–1847. doi: 10.1083/jcb.100.6.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Macey R. I. Transport of water and urea in red blood cells. Am J Physiol. 1984 Mar;246(3 Pt 1):C195–C203. doi: 10.1152/ajpcell.1984.246.3.C195. [DOI] [PubMed] [Google Scholar]
  23. Madsen K. M., Tisher C. C. Structural-functional relationships along the distal nephron. Am J Physiol. 1986 Jan;250(1 Pt 2):F1–15. doi: 10.1152/ajprenal.1986.250.1.F1. [DOI] [PubMed] [Google Scholar]
  24. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  25. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  26. Muller J., Kachadorian W. A., DiScala V. A. Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells. J Cell Biol. 1980 Apr;85(1):83–95. doi: 10.1083/jcb.85.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ohkuma S., Moriyama Y., Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982 May;79(9):2758–2762. doi: 10.1073/pnas.79.9.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schmid S. L., Fuchs R., Male P., Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell. 1988 Jan 15;52(1):73–83. doi: 10.1016/0092-8674(88)90532-6. [DOI] [PubMed] [Google Scholar]
  29. Schwartz G. J., Al-Awqati Q. Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules. J Clin Invest. 1985 May;75(5):1638–1644. doi: 10.1172/JCI111871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schwartz G. J., Barasch J., Al-Awqati Q. Plasticity of functional epithelial polarity. 1985 Nov 28-Dec 4Nature. 318(6044):368–371. doi: 10.1038/318368a0. [DOI] [PubMed] [Google Scholar]
  31. Shi L. B., Verkman A. S. Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder. J Gen Physiol. 1989 Dec;94(6):1101–1115. doi: 10.1085/jgp.94.6.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sklar L. A., Oades Z. G., Jesaitis A. J., Painter R. G., Cochrane C. G. Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7540–7544. doi: 10.1073/pnas.78.12.7540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stone D. K., Xie X. S. Proton translocating ATPases: issues in structure and function. Kidney Int. 1988 Apr;33(4):767–774. doi: 10.1038/ki.1988.65. [DOI] [PubMed] [Google Scholar]
  34. Strange K., Willingham M. C., Handler J. S., Harris H. W., Jr Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated, perfused rabbit cortical collecting tubule. J Membr Biol. 1988 Jul;103(1):17–28. doi: 10.1007/BF01871929. [DOI] [PubMed] [Google Scholar]
  35. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  36. Valtin H. The discovery of the Brattleboro rat, recommended nomenclature, and the question of proper controls. Ann N Y Acad Sci. 1982;394:1–9. doi: 10.1111/j.1749-6632.1982.tb37405.x. [DOI] [PubMed] [Google Scholar]
  37. Verkman A. S., Lencer W. I., Brown D., Ausiello D. A. Endosomes from kidney collecting tubule cells contain the vasopressin-sensitive water channel. Nature. 1988 May 19;333(6170):268–269. doi: 10.1038/333268a0. [DOI] [PubMed] [Google Scholar]
  38. Verkman A. S. Mechanisms and regulation of water permeability in renal epithelia. Am J Physiol. 1989 Nov;257(5 Pt 1):C837–C850. doi: 10.1152/ajpcell.1989.257.5.C837. [DOI] [PubMed] [Google Scholar]
  39. Verkman A. S. Passive H+/OH- permeability in epithelial brush border membranes. J Bioenerg Biomembr. 1987 Oct;19(5):481–493. doi: 10.1007/BF00770031. [DOI] [PubMed] [Google Scholar]
  40. Verkman A. S., Weyer P., Brown D., Ausiello D. A. Functional water channels are present in clathrin-coated vesicles from bovine kidney but not from brain. J Biol Chem. 1989 Dec 5;264(34):20608–20613. [PubMed] [Google Scholar]
  41. Wade J. B., Stetson D. L., Lewis S. A. ADH action: evidence for a membrane shuttle mechanism. Ann N Y Acad Sci. 1981;372:106–117. doi: 10.1111/j.1749-6632.1981.tb15464.x. [DOI] [PubMed] [Google Scholar]
  42. Ye R. G., Shi L. B., Lencer W. I., Verkman A. S. Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule. J Gen Physiol. 1989 May;93(5):885–902. doi: 10.1085/jgp.93.5.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yurko M. A., Gluck S. Production and characterization of a monoclonal antibody to vacuolar H+ATPase of renal epithelia. J Biol Chem. 1987 Nov 15;262(32):15770–15779. [PubMed] [Google Scholar]
  44. van Deurs B., Röpke C., Thorball N. Kinetics of pinocytosis studied by flow cytometry. Eur J Cell Biol. 1984 May;34(1):96–102. [PubMed] [Google Scholar]
  45. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES