Skip to main content
SpringerLink
Log in

Cytoskeletal disruption in A6 kidney cells: Impact on endo/exocytosis and NaCl transport regulation by antidiuretic hormone

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

Antidiuretic hormone (ADH; 2.5 × 10−8 m vasotocin) produces a stimulation of apical fluid phase endocytosis, protein secretion and NaCl reabsorption in Xenopus laevis A6 distal nephron cell epithelia pretreated with aldosterone (10−6 m). The increase of NaCl transport is mediated by a sequential opening of apical Cl and Na conductances. The aim of this study was to characterize the actin and tubulin cytoskeleton of A6 cells and to assess the impact of its disruption on baseline and ADH-induced apical vesicular membrane movements and ion transport to test for possible functional links. The microfilament (MF) and microtubule (MT) networks and their disruption were visualized by confocal laser microscopy. Conditions of depolimerization were selected, by cytochalasin D or cold and nocodazole, respectively. MF disruption produced an increase in baseline apical protein secretion (exocytic movements) (plus 18%) and a decrease of its induction by ADH (minus 35%). MF disruption also increased baseline horseradish peroxidase uptake (endocytic movements) (plus 21%), however, without affecting its ADH-induced increase. In the case of MT disruption, the ADH-induced stimulation of both protein secretion and fluid phase endocytosis was decreased by 70 and 44%, respectively. At the ion transport level, MF and MT disruption only insignificantly affected the ADH-induced Cl conductance, while they decreased the ADH-induced stimulation of Na transport (amiloride-sensitive short-circuit current and conductance) by a factor of 2 to 4. In conclusion, both MT and MF disruption decrease ADH-induced apical protein secretion and Na conductance, while the ADH-induced apical Cl conductance is not significantly affected. Taken together the data support the hypothesis that the modulation of Na channel expression by apical vesicular membrane movements plays a role in Na transport expression and its regulation by ADH.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Acher, R. 1974. Chemistry of the neurohypophyseal hormones: An example of molecular evolution. In: Handbook of Physiology, Endocrinology. E. Knobil and W.H. Sawyer, editors. Vol 4. pp. 119–130. Am. Physiol. Soc., Washington DC

    Google Scholar 

  2. Beron, J., Verrey, F. 1994. Aldosterone induces early activation and late accumulation of Na-K-ATPase at surface of A6 cells. Am. J. Physiol. 266:C1278-C1290

    Google Scholar 

  3. Boll, W., Partin, J.S., Katz, A.I., Caplan, M.J., Jamieson. J.D. 1991. Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 88:8592–8596.

    Google Scholar 

  4. Breitfeld, P.P., McKinnon, W.C., Mostov, K.E. 1990. Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells. J. Cell Biol. 111:2365–2373

    Google Scholar 

  5. Brown, D., Sabolic, I. 1993. Endosomal pathways for water channel and proton pump recycling in kidney epithelial cells. J. Cell Sci. Suppl. 17:49–59

    Google Scholar 

  6. Canessa, C.M., Schild, L., Buell, B., Thorens, B., Gautschi, I., Horisberger, J.D., Rossier, B.C. 1994. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467

    Google Scholar 

  7. Cantiello, H.F., Prat, A.G., Bonventre, J.V., Cunningham, C.C., Hartwig, J.H., Ausiello, D.A. 1993. Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells. J. Biol. Chem. 268:4596–4599

    Google Scholar 

  8. Chalfant, M.L., Coupaye-Gerard, B., Kleyman, T.R. 1993. Distinct regulation of Na+ reabsorption and Cl− secretion by arginine vasopressin in the amphibian cell line A6. Am. J. Physiol. 264:C1480-C1488

    Google Scholar 

  9. DeAlmeida, J.B., Stow, J.L. 1991. Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells. Am. J. Physiol. 261:C691-C700

    Google Scholar 

  10. Driscoll, M. 1992. Molecular genetics of cell death in the nematode caenorhabditis elegans. J. Neurobiol. 23:1327–1351

    Google Scholar 

  11. Duchatelle, P., Ohara, A., Ling, B.N., Kemendy, A.E., Kokko, K.E., Matsumoto, P.S., Eaton, D.C. 1992. Regulation of renal epithelial sodium channels. Mol. Cell. Biochem. 114:27–34

    Google Scholar 

  12. Eilers, U., Klumperman, J., Hauri, H.P. 1989. Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2). J. Cell Biol. 108:13–22

    Google Scholar 

  13. Els, W.J., Chou, K.Y. 1993. Sodium-dependent regulation of epithelial sodium channel densities in frog skin—a role for the cytoskeleton. J. Physiol. 462:447–464

    Google Scholar 

  14. Els, W.J., Helman, S.I. 1991. Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action. J. Gen. Physiol. 98:1197–1220

    Google Scholar 

  15. Fath, K.R., Mamajiwalla, S.N., Burgess, D.R. 1993. The cytoskeleton in development of epithelial cell polarity. J. Cell Sci. Suppl. 17:65–73

    Google Scholar 

  16. Franki, N., Ding, G., Gao, Y., Hays, R.M. 1992. Effect of cytochalasin D on the actin cytoskeleton of the toad bladder epithelial cell. Am. J. Physiol. 263:C995-C1000

    Google Scholar 

  17. Fuller, C.M., Bridges, R.J., Benos, DJ. 1994. Forskolin- but not ionomycin-evoked Cl− secretion in colonic epithelia depends on intact microtubules. Am. J. Physiol. 266:C661-C668

    Google Scholar 

  18. Garty, H. 1994. Molecular properties of epithelial, amilorideblockable Na+ channels. FASEB J. 8:522–528

    Google Scholar 

  19. Garty, H., Benos, D.J. 1988. Characteristics and regulatory mechanisms of the amiloride-blockable Na+ channel. Physiol. Rev. 68:309–373

    Google Scholar 

  20. Gilbert, T., Le Bivic, A., Quaroni, A., Rodriguez-Boulan, E. 1991. Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 cells. J. Cell Biol. 113:275–288

    Google Scholar 

  21. Gottlieb, T.A., Ivanov, I.E., Adesnik, M., Sabatini, D.D. 1993. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells. J. Cell Biol. 120:695–710

    Google Scholar 

  22. Hansch, E., Forgo, J., Murer, H., Biber, J. 1993. Role of microtubules in the adaptive response to low phosphate of Na/Pi cotransport in opossum kidney cells. Pfluegers Arch. 422:516–522

    Google Scholar 

  23. Kemendy, A.E., Kleyman, T.R., Eaton, D.C. 1992. Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. Am. J. Physiol. 263:C825-C837

    Google Scholar 

  24. Kleyman, T.R., Ernst, S.A., Coupaye-Gerard, B. 1994. Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells. Am. J. Physiol. 266:F506-F511

    Google Scholar 

  25. Kriz, W., Kaissling, B. 1992. Structural organization of the mammalian kidney. In: The Kidney: Physiology and Pathophysiology (2nd ed.). D.W. Seldin and G. Giebisch, editors. pp. 707–777. Raven, New York

    Google Scholar 

  26. Lieuvin, A., Labbé, J., Dorée, M., Job, D. 1994. Intrinsic microtubule stability in interphase cells. J. Cell. Biol. 124:985–996

    Google Scholar 

  27. Marunaka, Y., Eaton, D.C. 1990. Chloride channels in the apical membrane of a distal nephron A6 cell line. Am. J. Physiol. 258:C352-C368

    Google Scholar 

  28. Marunaka, Y., Eaton, D.C. 1991. Effects of vasopressin and cAMP on single amiloride-blockable Na channels. Am. J. Physiol. 260:C1071-C1084

    Google Scholar 

  29. Marunaka, Y., Tohda, H. 1993. Effects of vasopressin on single Cl− channels in the apical membrane of distal nephron cells (A6). Biochim. Biophys. Acta 1153:105–110

    Google Scholar 

  30. Masson, D., Kreis, T.E. 1993. Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells. J. Cell Biol. 123:357–371

    Google Scholar 

  31. Matthews, J.B., Smith, J.A., Tally, K.J., Awtrey, C.S., Nguyen, H.V., Rich, J., Madara, J.L. 1994. Na-K-2Cl cotransport in intestinal epithelial cells—influence of chloride efflux and F-actin on regulation of cotransporter activity and bumetanide binding. J. Biol. Chem. 269:15703–15709

    Google Scholar 

  32. Oh, Y.S., Smith, P.R., Bradford, A.L., Keeton, D., Benos, D.J. 1993. Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers. Am. J. Physiol. 265:C85-C91

    Google Scholar 

  33. Ojakian, G.K., Schwimmer, R. 1992. Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells. J. Cell Sci. 103:677–687

    Google Scholar 

  34. Paccolat, M.P., Geering, K., Gaeggeler, H.P., Rossier, B.C. 1987. Aldosterone regulation of Na+ transport and Na+-K+-ATPase in A6 cells: role of growth conditions. Am. J. Physiol. 252:C468-C476

    Google Scholar 

  35. Palmer, L.G. 1992. Epithelial Na channels—Function and diversity. Annu. Rev. Physiol. 54:51–66

    Google Scholar 

  36. Parczyk, K., Haase, W., Kondor-Koch, C. 1989. Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby Canine Kidney. J. Biol. Chem. 264:16837–16846

    Google Scholar 

  37. Perkins, F.M., Handler, J.S. 1981. Transport properties of toad kidney epithelia in culture. Am. J. Physiol. 241:C154-C159

    Google Scholar 

  38. Prat, A.G., Bertorello, A.M., Ausiello, D.A., Cantiello, H.F. 1993. Activation of epithelial Na+ channels by protein kinase-A requires actin filaments. Am. J. Physiol. 265:C224-C233

    Google Scholar 

  39. Puoti, A., Canessa, C., Rossier, B.C. 1994. Cloning and functional analysis of the amiloride-sensitive epithelial sodium channel of X. laevis. Experientia 50:A41 (Abstr.)

    Google Scholar 

  40. Rossier, B.C., Canessa, C.M., Schild, L., Horisberger, J.D. 1994. Epithelial sodium channels. Curr. Opinion Nephrol. Hypertension 3:487–496

    Google Scholar 

  41. Rothe, C.F., Quay, J.F., Armstrong, W.M. 1969. Measurement of epithelial electrical characteristics with an automatic voltage clamp device with compensation for solution resistance. IEEE Trans. Bio-Medical Engineering 16:160–164

    Google Scholar 

  42. Schafer, J.A., Hawk, C.T. 1992. Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids. Kidney Int. 41:255–268

    Google Scholar 

  43. Schwiebert, E.M., Mills, J.W., Stanton, B.A. 1994. Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line. J. Biol. Chem. 269:7081–7089.

    Google Scholar 

  44. Tousson, A., Alley, C.D., Sorcher, E.J., Brinkley, B.R., Benos, D.J. 1989. Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia. J. Cell Science 93:349–362

    Google Scholar 

  45. Verrey, F. 1994. Antidiuretic hormone action in A6 cells: effect on apical Cl and Na conductances and synergism with aldosterone for NaCl reabsorption. J. Membrane Biol. 138:65–76

    Google Scholar 

  46. Verrey, F., Digicaylioglu, M., Bolliger, U. 1993. Polarized membrane movements in A6 kidney cells are regulated by aldosterone and vasopressin/vasotocin. J. Membrane Biol. 133:213–226

    Google Scholar 

  47. Verrey, F., Schaerer, E., Zoerkler, P., Paccolat, M.P., Geering, K., Kraehenbuhl, J.P., Rossier, B.C. 1987. Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. J. Cell Biol. 104:1231–1237

    Google Scholar 

  48. Watson, A.J., Levine, M.S., Donowitz, M., Montrose, M.H. 1992. Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin. J. Biol. Chem. 267:956–962

    Google Scholar 

  49. Yanase, M., Handler, J.S. 1986. Adenosine 3′,5′-cyclic monophosphate stimulates chloride secretion in A6 epithelia. Am. J. Physiol. 251:C810–814

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland

    F. Verrey & U. Bolliger

  2. Institute of Anatomy, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland

    P. Groscurth

Authors
  1. F. Verrey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Groscurth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. U. Bolliger
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

The authors wish to thank Jörg Beron, Jean-Daniel Horisberger and Ian Forster for reading the manuscript, Walter Scherle for the scanning electron microscopy and Christian Gasser for the artwork. This work was supported by Grant 31.30132.90 and 31.39449.93 from the Swiss National Science Foundation.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Verrey, F., Groscurth, P. & Bolliger, U. Cytoskeletal disruption in A6 kidney cells: Impact on endo/exocytosis and NaCl transport regulation by antidiuretic hormone. J. Membarin Biol. 145, 193–204 (1995). https://doi.org/10.1007/BF00237377

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00237377

Key words

Search

Navigation