Molecular and Cellular Biochemistry

, Volume 367, Issue 1–2, pp 175–183 | Cite as

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

  • Yanling Yan
  • Steven Haller
  • Anna Shapiro
  • Nathan Malhotra
  • Jiang Tian
  • Zijian Xie
  • Deepak Malhotra
  • Joseph I. Shapiro
  • Jiang Liu


We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular 22Na+ transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular 22Na+ transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.


Ouabain Na/K-ATPase signaling Na/K-ATPase NHE3 Redistribution 



The authors would like to thank Ms. Carol Woods her excellent help. Portions of this work were supported by National Institutes of Health Grants HL-105649 (to J.T.), HL-109015 (to Z.X. and J.I.S.) and GM-78565 (to Z.X.).


  1. 1.
    Guyton AC (1991) Blood pressure control—special role of the kidneys and body fluids. Science 252(5014):1813–1816PubMedCrossRefGoogle Scholar
  2. 2.
    Stamler J, Rose G, Elliott P, Dyer A, Marmot M, Kesteloot H et al (1991) Findings of the International Cooperative INTERSALT Study. Hypertension 17(1 Suppl):I9–I15PubMedGoogle Scholar
  3. 3.
    Meneton P, Jeunemaitre X, de Wardener HE, MacGregor GA (2005) Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. Physiol Rev 85(2):679–715PubMedCrossRefGoogle Scholar
  4. 4.
    Haddy FJ (2006) Role of dietary salt in hypertension. Life Sci 79(17):1585–1592PubMedCrossRefGoogle Scholar
  5. 5.
    Schoner W, Scheiner-Bobis G (2008) Role of endogenous cardiotonic steroids in sodium homeostasis. Nephrol Dial Transplant 23(9):2723–2729PubMedCrossRefGoogle Scholar
  6. 6.
    Bagrov AY, Shapiro JI, Fedorova OV (2009) Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets. Pharmacol Rev 61(1):9–38PubMedCrossRefGoogle Scholar
  7. 7.
    Nesher M, Dvela M, Igbokwe VU, Rosen H, Lichtstein D (2009) Physiological roles of endogenous ouabain in normal rats. Am J Physiol Heart Circ Physiol 297(6):H2026–H2034PubMedCrossRefGoogle Scholar
  8. 8.
    Manunta P, Hamilton BP, Hamlyn JM (2006) Salt intake and depletion increase circulating levels of endogenous ouabain in normal men. Am J Physiol Regul Integr Comp Physiol 290(3):R553–R559PubMedCrossRefGoogle Scholar
  9. 9.
    Fedorova OV, Doris PA, Bagrov AY (1998) Endogenous marinobufagenin-like factor in acute plasma volume expansion. Clin Exp Hypertens 20(5–6):581–591PubMedCrossRefGoogle Scholar
  10. 10.
    Lloyd MA, Sandberg SM, Edwards BS (1992) Role of renal Na+,K(+)-ATPase in the regulation of sodium excretion under normal conditions and in acute congestive heart failure. Circulation 85(5):1912–1917PubMedCrossRefGoogle Scholar
  11. 11.
    De Wardener HE (1977) Natriuretic hormone. Clin Sci Mol Med 53(1):1–8PubMedGoogle Scholar
  12. 12.
    Haddy FJ, Overbeck HW (1976) The role of humoral agents in volume expanded hypertension. Life Sci 19(7):935–947PubMedCrossRefGoogle Scholar
  13. 13.
    Blaustein MP (1977) Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and a hypothesis. Am J Physiol 232(5):C165–C173PubMedGoogle Scholar
  14. 14.
    Blaustein MP, Zhang J, Chen L, Song H, Raina H, Kinsey SP et al (2009) The pump, the exchanger, and endogenous ouabain: signaling mechanisms that link salt retention to hypertension. Hypertension 53(2):291–298PubMedCrossRefGoogle Scholar
  15. 15.
    Dostanic-Larson I, Van Huysse JW, Lorenz JN, Lingrel JB (2005) The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation. Proc Natl Acad Sci USA 102(44):15845–15850PubMedCrossRefGoogle Scholar
  16. 16.
    Loreaux EL, Kaul B, Lorenz JN, Lingrel JB (2008) Ouabain-sensitive alpha1 Na,K-ATPase enhances natriuretic response to saline load. J Am Soc Nephrol 19(10):1947–1954PubMedCrossRefGoogle Scholar
  17. 17.
    Liu J, Yan Y, Liu L, Xie Z, Malhotra D, Joe B et al (2011) Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension. J Biol Chem 286(26):22806–22813PubMedCrossRefGoogle Scholar
  18. 18.
    Liu J, Kesiry R, Periyasamy SM, Malhotra D, Xie Z, Shapiro JI (2004) Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism. Kidney Int 66(1):227–241PubMedCrossRefGoogle Scholar
  19. 19.
    Cai H, Wu L, Qu W, Malhotra D, Xie Z, Shapiro JI, Liu J (2008) Regulation of apical NHE3 trafficking by ouabain-induced activation of basolateral Na/K-ATPase receptor complex. Am J Physiol Cell Physiol 294(2):C555–C563PubMedCrossRefGoogle Scholar
  20. 20.
    Liu J, Xie ZJ (2010) The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter trafficking. Biochim Biophys Acta 1802(12):1237–1245PubMedGoogle Scholar
  21. 21.
    Oweis S, Wu L, Kiela PR, Zhao H, Malhotra D, Ghishan FK et al (2006) Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK1 cells. Am J Physiol Renal Physiol 290(5):F997–F1008PubMedCrossRefGoogle Scholar
  22. 22.
    Liang M, Cai T, Tian J, Qu W, Xie ZJ (2006) Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay. J Biol Chem 281(28):19709–19719PubMedCrossRefGoogle Scholar
  23. 23.
    Haber RS, Pressley TA, Loeb JN, Ismail-Beigi F (1987) Ionic dependence of active Na–K transport: “clamping” of cellular Na+ with monensin. Am J Physiol 253(1 Pt 2):F26–F33PubMedGoogle Scholar
  24. 24.
    Haggerty JG, Agarwal N, Reilly RF, Adelberg EA, Slayman CW (1988) Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1). Proc Natl Acad Sci USA 85(18):6797–6801PubMedCrossRefGoogle Scholar
  25. 25.
    Soleimani M, Watts BA 3rd, Singh G, Good DW (1998) Effect of long-term hyperosmolality on the Na+/H+ exchanger isoform NHE-3 in LLC-PK1 cells. Kidney Int 53(2):423–431PubMedCrossRefGoogle Scholar
  26. 26.
    Klisic J, Zhang J, Nief V, Reyes L, Moe OW, Ambuhl PM (2003) Albumin regulates the Na+/H+ exchanger 3 in OKP cells. J Am Soc Nephrol 14(12):3008–3016PubMedCrossRefGoogle Scholar
  27. 27.
    Maxfield FR, McGraw TE (2004) Endocytic recycling. Nat Rev Mol Cell Biol 5(2):121–132PubMedCrossRefGoogle Scholar
  28. 28.
    Algharably N, Owler D, Lamb JF (1986) The rate of uptake of cardiac glycosides into human cultured cells and the effects of chloroquine on it. Biochem Pharmacol 35(20):3571–3581PubMedCrossRefGoogle Scholar
  29. 29.
    McDonough AA, Biemesderfer D (2003) Does membrane trafficking play a role in regulating the sodium/hydrogen exchanger isoform 3 in the proximal tubule? Curr Opin Nephrol Hypertens 12(5):533–541PubMedCrossRefGoogle Scholar
  30. 30.
    Yang LE, Maunsbach AB, Leong PK, McDonough AA (2004) Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes. Am J Physiol Renal Physiol 287(5):F896–F906PubMedCrossRefGoogle Scholar
  31. 31.
    Aperia A (2001) Regulation of sodium/potassium ATPase activity: impact on salt balance and vascular contractility. Curr Hypertens Rep 3(2):165–171PubMedCrossRefGoogle Scholar
  32. 32.
    Liu J, Liang M, Liu L, Malhotra D, Xie Z, Shapiro JI (2005) Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1. Kidney Int 67(5):1844–1854PubMedCrossRefGoogle Scholar
  33. 33.
    Lingrel JB, Kuntzweiler T (1994) Na+,K(+)-ATPase. J Biol Chem 269(31):19659–19662PubMedGoogle Scholar
  34. 34.
    Blanco G, Mercer RW (1998) Isozymes of the Na–K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol 275(5 Pt 2):F633–F650PubMedGoogle Scholar
  35. 35.
    Sweadner KJ (1989) Isozymes of the Na+/K+-ATPase. Biochim Biophys Acta 988(2):185–220PubMedGoogle Scholar
  36. 36.
    Aizman O, Uhlen P, Lal M, Brismar H, Aperia A (2001) Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc Natl Acad Sci USA 98(23):13420–13424PubMedCrossRefGoogle Scholar
  37. 37.
    Liu J, Tian J, Haas M, Shapiro JI, Askari A, Xie Z (2000) Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations. J Biol Chem 275(36):27838–27844PubMedGoogle Scholar
  38. 38.
    Aydemir-Koksoy A, Abramowitz J, Allen JC (2001) Ouabain-induced signaling and vascular smooth muscle cell proliferation. J Biol Chem 276(49):46605–46611PubMedCrossRefGoogle Scholar
  39. 39.
    Haas M, Wang H, Tian J, Xie Z (2002) Src-mediated inter-receptor cross-talk between the Na+/K+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases. J Biol Chem 277(21):18694–18702PubMedCrossRefGoogle Scholar
  40. 40.
    Lingrel JB (2010) The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na,K-ATPase. Ann Rev Physiol 72:395–412CrossRefGoogle Scholar
  41. 41.
    Yates NA, McDougall JG (1993) Effects of direct renal arterial infusion of bufalin and ouabain in conscious sheep. Br J Pharmacol 108(3):627–630PubMedGoogle Scholar
  42. 42.
    Kelly MP, Quinn PA, Davies JE, Ng LL (1997) Activity and expression of Na+–H+ exchanger isoforms 1 and 3 in kidney proximal tubules of hypertensive rats. Circ Res 80(6):853–860PubMedCrossRefGoogle Scholar
  43. 43.
    Harris RC, Brenner BM, Seifter JL (1986) Sodium-hydrogen exchange and glucose transport in renal microvillus membrane vesicles from rats with diabetes mellitus. J Clin Invest 77(3):724–733PubMedCrossRefGoogle Scholar
  44. 44.
    Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR et al (1998) Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. Nat Genet 19(3):282–285PubMedCrossRefGoogle Scholar
  45. 45.
    Lorenz JN, Schultheis PJ, Traynor T, Shull GE, Schnermann J (1999) Micropuncture analysis of single-nephron function in NHE3-deficient mice. Am J Physiol 277(3 Pt 2):F447–F453PubMedGoogle Scholar
  46. 46.
    McDonough AA (2010) Mechanisms of proximal tubule sodium transport regulation that link extracellular fluid volume and blood pressure. Am J Physiol Regul Integr Comp Physiol 298(4):R851–R861PubMedCrossRefGoogle Scholar
  47. 47.
    Liu J, Shapiro JI (2007) Regulation of sodium pump endocytosis by cardiotonic steroids: molecular mechanisms and physiological implications. Pathophysiology 14(3–4):171–181PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Yanling Yan
    • 1
    • 3
  • Steven Haller
    • 1
  • Anna Shapiro
    • 1
  • Nathan Malhotra
    • 1
  • Jiang Tian
    • 1
  • Zijian Xie
    • 1
    • 2
  • Deepak Malhotra
    • 1
  • Joseph I. Shapiro
    • 1
    • 2
  • Jiang Liu
    • 1
  1. 1.Department of MedicineUniversity of Toledo College of MedicineToledoUSA
  2. 2.Department of Physiology and PharmacologyUniversity of Toledo College of MedicineToledoUSA
  3. 3.Institute of Biomedical EngineeringYanshan UniversityQinhuangdaoChina

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