Skip to main content
SpringerLink
Log in

Aldosterone and amiloride alter ENaC abundance in vascular endothelium

  • Cell and Molecular Physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC) is usually found in the apical membrane of epithelial cells but has also recently been described in vascular endothelium. Because little is known about the regulation and cell surface density of ENaC, we studied the influence of aldosterone, spironolactone, and amiloride on its abundance in the plasma membrane of human endothelial cells. Three different methods were applied, single ENaC molecule detection in the plasma membrane, quantification by Western blotting, and cell surface imaging using atomic force microscopy. We found that aldosterone increases the surface expression of ENaC molecules by 36% and the total cellular amount by 91%. The aldosterone receptor antagonist spironolactone prevents these effects completely. Acute application of amiloride to aldosterone-pretreated cells led to a decline of intracellular ENaC by 84%. We conclude that, in vascular endothelium, aldosterone induces ENaC expression and insertion into the plasma membrane. Upon functional blocking with amiloride, the channel disappears from the cell surface and from intracellular pools, indicating either rapid degradation and/or membrane pinch-off. This opens new perspectives in the regulation of ENaC expressed in the vascular endothelium.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11:36–42

    Google Scholar 

  2. Agarwal MK, Mirshahi M (1999) General overview of mineralocorticoid hormone action. Pharmacol Ther 84:273–326

    Article  PubMed  CAS  Google Scholar 

  3. Alvarez de la Rosa D, Canessa CM, Fyfe GK, Zhang P (2000) Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol 62:573–594

    Article  PubMed  CAS  Google Scholar 

  4. Alvarez de la Rosa D, Li H, Canessa CM (2002) Effects of aldosterone on biosynthesis, traffic, and functional expression of the epithelial sodium channel in A6 cells. J Gen Physiol 119:427–442

    Article  PubMed  CAS  Google Scholar 

  5. Apodaca G (2002) Modulation of membrane traffic by mechanical stimuli. Am J Physiol Renal Physiol 282:F179–F190

    PubMed  Google Scholar 

  6. Awayda MS, Tousson A, Benos DJ (1997) Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits. Am J Physiol 273:C1889–C1899

    PubMed  CAS  Google Scholar 

  7. Canessa CM, Merillat A-M, Rossier BC (1994) Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol 267:C1682–C1690

    PubMed  CAS  Google Scholar 

  8. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D, Rossier BC (1994) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467

    Article  PubMed  CAS  Google Scholar 

  9. Chen W, Valamanesh F, Mirshahi T, Soria J, Tang R, Agarwal MK, Mirshahi M (2004) Aldosterone signaling modifies capillary formation by human bone marrow endothelial cells. Vasc Pharmacol 40(6):269–277

    Article  CAS  Google Scholar 

  10. Connell JM, Davies E (2005) The new biology of aldosterone. J Endocrinol 186:1–20

    Article  PubMed  CAS  Google Scholar 

  11. Dagenais A, Frechette R, Yamagata Y, Yamagata T, Carmel JF, Clermont ME, Brochiero E, Masse C, Berthiaume Y (2004) Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 286:L301–L311

    Article  PubMed  CAS  Google Scholar 

  12. Fakitsas P, Adam G, Daidie D, van Bemmelen MX, Fouladkou F, Patrignani A, Wagner U, Warth R, Camargo SM, Staub O, Verrey F (2007) Early aldosterone-induced gene product regulates the epithelial sodium channel by deubiquitylation. J Am Soc Nephrol 18:1084–1092

    Article  PubMed  CAS  Google Scholar 

  13. Falkenstein E, Christ M, Feuring M, Wehling M (2000) Specific nongenomic actions of aldosterone. Kidney Int 57:1390–1394

    Article  PubMed  CAS  Google Scholar 

  14. Fuller PJ, Young MJ (2005) Mechanism of mineralocorticoid action. Hypertension 46:1227–1235

    Article  PubMed  CAS  Google Scholar 

  15. Garty H, Palmer LG (1997) Epithelial sodium channels: function, structure, and regulation. Phys Rev 77:359–396

    CAS  Google Scholar 

  16. Golestaneh N, Klein C, Valamanesh F, Suarez G, Agarwal MK, Mirshahi M (2001) Mineralocorticoid receptor-mediated signaling regulates the ion gated sodium channel in vascular endothelial cells and requires an intact cytoskeleton. Biochem Biophys Res Com 280(5):1300–1306

    Article  PubMed  CAS  Google Scholar 

  17. Henderson RM, Schneider S, Li Q, Hornby D, White SJ, Oberleithner H (1996) Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy. Proc Natl Acad Sci U S A 93:8756–8760

    Article  PubMed  CAS  Google Scholar 

  18. Hillebrand U, Hausberg M, Stock C, Shahin V, Nikova D, Riethmuller C, Kliche K, Ludwig T, Schillers H, Schneider SW, Oberleithner H (2006) 17beta-estradiol increases volume, apical surface and elasticity of human endothelium mediated by Na+/H+ exchange. Cardiovasc Res 69:916–924

    Article  PubMed  CAS  Google Scholar 

  19. Hillebrand U, Schillers H, Riethmüller C, Stock C, Wilhelmi M, Oberleithner H, Hausberg M (2007) Dose-dependent endothelial cell growth and stiffening by aldosterone: endothelial protection by eplerenone. J Hypertens 25:639–647

    Article  PubMed  CAS  Google Scholar 

  20. Hughey RP, Mueller GM, Bruns JB, Kinlough CL, Poland PA, Harkleroad KL, Carattino MD, Kleyman TR (2003) Maturation of the epithelial Na+ channel involves proteolytic processing of the α- and γ-subunits. J Biol Chem 278:37073–37082

    Article  PubMed  CAS  Google Scholar 

  21. Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, Kleyman TR (2004) Epithelial sodium channels are activated by furin-dependent proteolysis. J Biol Chem 279:18111–18114

    Article  PubMed  CAS  Google Scholar 

  22. Ismailov II, Berdiev BK, Bradford AL, Awayda MS, Fuller CM, Benos DJ (1996) Associated proteins and renal epithelial Na+ channel function. J Membr Biol 149:123–132

    Article  PubMed  CAS  Google Scholar 

  23. Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived frim umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756

    Article  PubMed  CAS  Google Scholar 

  24. Ji H-L, Fuller CM, Benos DJ (1998) Osmotic pressure regulates abg-rENaC expressed in Xenopus oocytes. Am J Physiol 275:C1182–C1190

    PubMed  CAS  Google Scholar 

  25. Kellenberger S, Gautschi I, Schild L (2003) Mutations in the epithelial Na+ channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate. Mol Pharmacol 64:848–856

    Article  PubMed  CAS  Google Scholar 

  26. Lewis SA, de Moura JL (1982) Incorporation of cytoplasmic vesicles into apical membrane of mammalian urinary bladder epithelium. Nature 297:685–688

    Article  PubMed  CAS  Google Scholar 

  27. Morris CE, Homann U (2001) Cell surface area regulation and membrane tension. J Membr Biol 179:79–102

    PubMed  CAS  Google Scholar 

  28. Mullins LJ, Bailey MA, Mullins JJ (2006) Hypertension, kidney, and transgenics: a fresh perspective. Physiol Rev 86:709–746

    Article  PubMed  CAS  Google Scholar 

  29. Nechyporuk-Zloy V, Stock C, Schillers H, Oberleithner H, Schwab A (2006) Single plasma membrane potassium channel detection by using dual-color quantum dot labeling. Am J Physiol Cell Physiol 291:266–269

    Article  CAS  Google Scholar 

  30. Nilius B, Droogmans G (2001) Ion channels and their functional role in vascular endothelium. Physiol Rev 81:1415–1459

    PubMed  CAS  Google Scholar 

  31. Oberleithner H (2007) Is the vascular endothelium under the control of aldosterone? Facts and hypothesis. Pflugers Arch 454:187–193

    Article  PubMed  CAS  Google Scholar 

  32. Oberleithner H, Ludwig T, Riethmüller C, Hillebrand U, Albermann L, Schäfer C, Shahin V, Schillers H (2004) Human endothelium: Target for aldosterone. Hypertension 43:1–5

    Article  CAS  Google Scholar 

  33. Oberleithner H, Riethmüller C, Ludwig T, Shahin V, Stock C, Schwab A, Hausberg M, Kusche K, Schillers H (2006) Differential action of steroid hormones on human endothelium. J Cell Sci 119:1926–1932

    Article  PubMed  CAS  Google Scholar 

  34. Oberleithner H, Schneider SW, Albermann L, Hillebrand U, Ludwig T, Riethmüller C, Shahin V, Schäfer C, Schillers H (2003) Endothelial cell swelling by aldosterone. J Membr Biol 196:163–172

    Article  PubMed  CAS  Google Scholar 

  35. Palmer LG, Frindt G (2000) Aldosterone and potassium secretion by the cortical collecting duct. Kidney Int 57:1324–1328

    Article  PubMed  CAS  Google Scholar 

  36. Schneider SW, Matzke R, Radmacher M, Oberleithner H (2004) Shape and volume of living aldosterone-sensitive cells imaged with the atomic force microscope. Methods Mol Biol 242:255–279

    PubMed  Google Scholar 

  37. Segal A, Awayda MS, Eggermont J, Van Driessche W, Weber W-M (2002) Influence of voltage and extracellular Na+ concentration on amiloride block and transport kinetics of rat ENaC expressed in Xenopus oocytes. Pflugers Arch 443:882–891

    Article  PubMed  CAS  Google Scholar 

  38. Staub O, Abriel H, Plant P, Ishikawa T, Kanelis V, Saleki R, Horisberger J-D, Schild L, Rotin D (2000) Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. Kidney Int 57:809–815

    Article  PubMed  CAS  Google Scholar 

  39. Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild D, Rotin D (1997) Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J 16:6325–6336

    Article  PubMed  CAS  Google Scholar 

  40. Teiwes J, Toto RD (2007) Epithelial sodium channel inhibition in cardiovascular disease. A potential role for amiloride. Am J Hypertens 20:109–117

    Article  PubMed  CAS  Google Scholar 

  41. Traub LM, Lukacs GL (2007) Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs. J Cell Sci 120:543–553

    Article  PubMed  CAS  Google Scholar 

  42. Vallon V, Wulff P, Huang DY, Loffing J, Volkl H, Kuhl D, Lang F (2005) Role of Sgk1 in salt and potassium homeostasis. Am J Physiol Regul Integr Comp Physiol 288:R4–R10

    PubMed  CAS  Google Scholar 

  43. Verrey F, Loffing J, Zecevic M, Heitzmann D, Staub O (2003) SGK1: aldosterone-induced relay of Na+ transport regulation in distal kidney nephron cells. Cell Physiol Biochem 13:21–28

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

We are grateful to Prof. Dr. W.-M. Weber (Institute of Animal Physiology, University of Muenster, Germany) for support and discussion and to Dr. Peter Hanley for critically reading the manuscript. The project was supported by grants of the Deutsche Forschungsgemeinschaft (Re1284/2-1 and Ob 63/16-1) and EU grant Tips4cells.

Author information

Authors and Affiliations

  1. Institute of Animal Physiology, University of Muenster, Hindenburgplatz 55, 48143, Muenster, Germany

    Kristina Kusche-Vihrog, Katja Sobczak & Nadine Bangel

  2. Institute of Physiology II, University of Muenster, Muenster, Germany

    Marianne Wilhelmi, Volodymyr Nechyporuk-Zloy, Albrecht Schwab, Hermann Schillers & Hans Oberleithner

Authors
  1. Kristina Kusche-Vihrog
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katja Sobczak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nadine Bangel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marianne Wilhelmi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Volodymyr Nechyporuk-Zloy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Albrecht Schwab
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hermann Schillers
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hans Oberleithner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kristina Kusche-Vihrog.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kusche-Vihrog, K., Sobczak, K., Bangel, N. et al. Aldosterone and amiloride alter ENaC abundance in vascular endothelium. Pflugers Arch - Eur J Physiol 455, 849–857 (2008). https://doi.org/10.1007/s00424-007-0341-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-007-0341-0

Keywords

Search

Navigation