Pflügers Archiv - European Journal of Physiology

, Volume 469, Issue 10, pp 1401–1412 | Cite as

Salt-induced Na+/K+-ATPase-α/β expression involves soluble adenylyl cyclase in endothelial cells

  • Mirja Mewes
  • Johanna Nedele
  • Katrin Schelleckes
  • Olga Bondareva
  • Malte Lenders
  • Kristina Kusche-Vihrog
  • Hans-Joachim Schnittler
  • Stefan-Martin Brand
  • Boris Schmitz
  • Eva BrandEmail author
Signaling and cell physiology
Part of the following topical collections:
  1. Signaling and cell physiology


High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca2+- and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na+/K+-ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na+)-induced Na+/K+-ATPase-α and Na+/K+-ATPase-β protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na+/K+-ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na+/K+-ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na+/K+-ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na+/K+-ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na+/K+-ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.


Sodium CREB Endothelial stiffness cAMP Aging 



EA.hy926 cells were a kind gift of Cora-Jean S. Edgell, University of NC, USA. pADneo2-C6-BGL was kindly provided by Elwyn Isaac, University of Leeds, UK. We thank Samira Schiwek and Birgit Orlowski for excellent technical assistance.

Compliance with ethical standards

Role of the funding source

Parts of the study were funded by a grant from the Else Kröner-Fresenius-Foundation (project. no. 2010_A116) to E. Brand and K. Kusche-Vihrog.

Conflict of interest

The authors declare that they have no competing of interest.

Supplementary material

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  1. 1.
    Acin-Perez R, Salazar E, Kamenetsky M, Buck J, Levin LR, Manfredi G (2009) Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation. Cell Metab 9:265–276CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Aperia A (2001) Regulation of sodium/potassium ATPase activity: impact on salt balance and vascular contractility. Curr Hypertens Rep 3:165–171CrossRefPubMedGoogle Scholar
  3. 3.
    Blaustein MP, Leenen FH, Chen L, Golovina VA, Hamlyn JM, Pallone TL, Van Huysse JW, Zhang J, Wier WG (2012) How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension. Am J Physiol Heart Circ Physiol 302:H1031–H1049CrossRefPubMedGoogle Scholar
  4. 4.
    Boegehold MA (2013) The effect of high salt intake on endothelial function: reduced vascular nitric oxide in the absence of hypertension. J Vasc Res 50:458–467CrossRefPubMedGoogle Scholar
  5. 5.
    Boisen L, Drasbek KR, Pedersen AS, Kristensen P (2010) Evaluation of endothelial cell culture as a model system of vascular ageing. Exp Gerontol 45:779–787CrossRefPubMedGoogle Scholar
  6. 6.
    Brandes RP, Fleming I, Busse R (2005) Endothelial aging. Cardiovasc Res 66:286–294CrossRefPubMedGoogle Scholar
  7. 7.
    Buschmann MH, Dieterich P, Adams NA, Schnittler HJ (2005) Analysis of flow in a cone-and-plate apparatus with respect to spatial and temporal effects on endothelial cells. Biotechnol Bioeng 89:493–502CrossRefPubMedGoogle Scholar
  8. 8.
    Buyck JF, Blacher J, Kesse-Guyot E, Castetbon K, Galan P, Safar M, Hercberg S, Czernichow S (2009) Differential associations of dietary sodium and potassium intake with blood pressure: a focus on pulse pressure. J Hypertens 27:1158–1164CrossRefPubMedGoogle Scholar
  9. 9.
    Cantiello HF (1995) Actin filaments stimulate the Na(+)-K(+)-ATPase. Am J Phys 269:F637–F643Google Scholar
  10. 10.
    Cantiello HF (1997) Changes in actin filament organization regulate Na1, K1-ATPase activity. Role of actin phosphorylation. Ann N Y Acad Sci 834:559–561CrossRefPubMedGoogle Scholar
  11. 11.
    Carl P, Schillers H (2008) Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing. Pflugers Arch 457:551–559CrossRefPubMedGoogle Scholar
  12. 12.
    Cook NR, Cutler JA, Obarzanek E, Buring JE, Rexrode KM, Kumanyika SK, Appel LJ, Whelton PK (2007) Long term effects of dietary sodium reduction on cardiovascular disease outcomes: observational follow-up of the trials of hypertension prevention (TOHP). BMJ 334:885–888CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Devarajan P, Stabach PR, De Matteis MA, Morrow JS (1997) Na, K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells. Proc Natl Acad Sci U S A 94:10711–10716CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Edgell CJ, McDonald CC, Graham JB (1983) Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S A 80:3734–3737CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Garty H, Palmer LG (1997) Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77:359–396PubMedGoogle Scholar
  16. 16.
    Gloor SM (1997) Relevance of Na, K-ATPase to local extracellular potassium homeostasis and modulation of synaptic transmission. FEBS Lett 412:1–4CrossRefPubMedGoogle Scholar
  17. 17.
    Gumz ML, Cheng KY, Lynch IJ, Stow LR, Greenlee MM, Cain BD, Wingo CS (2010) Regulation of αENaC expression by the circadian clock protein Period 1 in mpkCCD(c14) cells. Biochim Biophys Acta 1799:622–629CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hald PM (1947) The flame photometer for the measurement of sodium and potassium in biological materials. J Biol Chem 167:499–510PubMedGoogle Scholar
  19. 19.
    Hess KC, Jones BH, Marquez B, Chen Y, Ord TS, Kamenetsky M, Miyamoto C, Zippin JH, Kopf GS, Suarez SS, Levin LR, Williams CJ, Buck J, Moss SB (2005) The “soluble” adenylyl cyclase in sperm mediates multiple signaling events required for fertilization. Dev Cell 9:249–259CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hu H, Juvekar A, Lyssiotis CA, Lien EC, Albeck JG, Oh D, Varma G, Hung YP, Ullas S, Lauring J, Seth P, Lundquist MR, Tolan DR, Grant AK, Needleman DJ, Asara JM, Cantley LC, Wulf GM (2016) Phosphoinositide 3-kinase regulates glycolysis through mobilization of aldolase from the actin cytoskeleton. Cell 164:433–446CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Huveneers S, Daemen MJ, Hordijk PL (2015) Between rho (k) and a hard place: the relation between vessel wall stiffness, endothelial contractility, and cardiovascular disease. Circ Res 116:895–908CrossRefPubMedGoogle Scholar
  22. 22.
    Isaac RE, Johnson EC, Audsley N, Shirras AD (2007) Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila. J Exp Biol 210:4465–4470CrossRefPubMedGoogle Scholar
  23. 23.
    Jeggle P, Callies C, Tarjus A, Fassot C, Fels J, Oberleithner H, Jaisser F, Kusche-Vihrog K (2013) Epithelial sodium channel stiffens the vascular endothelium in vitro and in Liddle mice. Hypertension 61:1053–1059CrossRefPubMedGoogle Scholar
  24. 24.
    Kaeffer B, Pardini L (2005) Clock genes of mammalian cells: practical implications in tissue culture. In Vitro Cell Dev Biol Anim 41:311–320CrossRefPubMedGoogle Scholar
  25. 25.
    Keebaugh ES, Schlenke TA (2012) Adaptive evolution of a novel Drosophila lectin induced by parasitic wasp attack. Mol Biol Evol 29:565–577CrossRefPubMedGoogle Scholar
  26. 26.
    Kliche K, Jeggle P, Pavenstädt H, Oberleithner H (2011) Role of cellular mechanics in the function and life span of vascular endothelium. Pflugers Arch 462:209–217CrossRefPubMedGoogle Scholar
  27. 27.
    Kobayashi M, Kawakami K (1997) Synergism of the ATF/CRE site and GC box in the housekeeping Na, K-ATPase alpha1 subunit gene is essential for constitutive expression. Biochem Biophys Res Commun 241:169–174CrossRefPubMedGoogle Scholar
  28. 28.
    Korte S, Strater AS, Druppel V, Oberleithner H, Jeggle P, Grossmann C, Fobker M, Nofer JR, Brand E, Kusche-Vihrog K (2014) Feedforward activation of endothelial ENaC by high sodium. FASEB J 28:4015–4025CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Kusche-Vihrog K, Schmitz B, Brand E (2015) Salt controls endothelial and vascular phenotype. Pflugers Arch 467:499–512CrossRefPubMedGoogle Scholar
  30. 30.
    Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, Guize L, Ducimetiere P, Benetos A (2001) Aortic stiffness is an independent predictor of all-cause and cardiovascular mortality in hypertensive patients. Hypertension 37:1236–1241CrossRefPubMedGoogle Scholar
  31. 31.
    Lescale-Matys L, Putnam DS, McDonough AA (1993) Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates. Am J Phys 264:C583–C590Google Scholar
  32. 32.
    Li J, White J, Guo L, Zhao X, Wang J, Smart EJ, Li XA (2009) Salt inactivates endothelial nitric oxide synthase in endothelial cells. J Nutr 139:447–451CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Maurya PK, Prakash S (2013) Decreased activity of Ca(++)-ATPase and Na(+)/K(+)-ATPase during aging in humans. Appl Biochem Biotechnol 170:131–137CrossRefPubMedGoogle Scholar
  34. 34.
    McDonough AA, Magyar CE, Komatsu Y (1994) Expression of Na(+)-K(+)-ATPase alpha- and beta-subunits along rat nephron: isoform specificity and response to hypokalemia. Am J Phys 267:C901–C908Google Scholar
  35. 35.
    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 85:679–715CrossRefPubMedGoogle Scholar
  36. 36.
    Montrose MH (1991) Measurement of intracellular sodium and potassium in cultured epithelial cells. J Tissue Cult Methods 13:211CrossRefGoogle Scholar
  37. 37.
    Mozaffarian D, Fahimi S, Singh GM, Micha R, Khatibzadeh S, Engell RE, Lim S, Danaei G, Ezzati M, Powles J (2014) Global sodium consumption and death from cardiovascular causes. N Engl J Med 371:624–634CrossRefPubMedGoogle Scholar
  38. 38.
    Oberleithner H, Riethmuller C, Schillers H, MacGregor GA, de Wardener HE, Hausberg M (2007) Plasma sodium stiffens vascular endothelium and reduces nitric oxide release. Proc Natl Acad Sci U S A 104:16281–16286CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Oda T, Makino K, Yamashita I, Namba K, Maeda Y (2001) Distinct structural changes detected by X-ray fiber diffraction in stabilization of F-actin by lowering pH and increasing ionic strength. Biophys J 80:841–851CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Paar M, Pavenstädt H, Kusche-Vihrog K, Drüppel V, Oberleithner H, Kliche K (2014) Endothelial sodium channels trigger endothelial salt sensitivity with aging. Hypertension 64:391–396CrossRefPubMedGoogle Scholar
  41. 41.
    Paterna S, Gaspare P, Fasullo S, Sarullo FM, Di Pasquale P (2008) Normal-sodium diet compared with low-sodium diet in compensated congestive heart failure: is sodium an old enemy or a new friend? Clin Sci (Lond) 114:221–230CrossRefGoogle Scholar
  42. 42.
    Rieu I, Powers SJ (2009) Real-time quantitative RT-PCR: design, calculations, and statistics. Plant Cell 21:1031–1033CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Safar ME, Benetos A (2003) Factors influencing arterial stiffness in systolic hypertension in the elderly: role of sodium and the reninangiotensin system. Am J Hypertens 16:249–258CrossRefPubMedGoogle Scholar
  44. 44.
    Safar ME, Temmar M, Kakou A, Lacolley P, Thornton SN (2009) Sodium intake and vascular stiffness in hypertension. Hypertension 54:203–209CrossRefPubMedGoogle Scholar
  45. 45.
    Schmitz B, Nedele J, Guske K, Maase M, Lenders M, Schelleckes M, Kusche-Vihrog K, Brand SM, Brand E (2014) Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/β, and mineralocorticoid receptor. Hypertension 63:753–761CrossRefPubMedGoogle Scholar
  46. 46.
    Schmitz B, Salomon A, Rötrige A, Ritter M, Ringelstein EB, Fischer JW, Paul M, Brand E, Brand SM (2013) Interindividual transcriptional regulation of the human biglycan gene involves three common molecular haplotypes. Arterioscler Thromb Vasc Biol 33:871–880CrossRefPubMedGoogle Scholar
  47. 47.
    Schnittler HJ, Franke RP, Akbay U, Mrowietz C, Drenckhahn D (1993) Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells. Am J Phys 265:C289–C298Google Scholar
  48. 48.
    Schnoor M, Lai FP, Zarbock A, Kläver R, Polaschegg C, Schulte D, Weich HA, Oelkers JM, Rottner K, Vestweber D (2011) Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo. J Exp Med 208:1721–1735CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Soltoff SP, Mandel LJ (1984) Active ion transport in the renal proximal tubule. II Ionic dependance of the Na pump. J Gen Physiol 84:623–642CrossRefPubMedGoogle Scholar
  50. 50.
    Soták M, Polidarová L, Musílková J, Hock M, Sumová A, Pácha J (2011) Circadian regulation of electrolyte absorption in the rat colon. Am J Physiol Gastrointest Liver Physiol 301:G1066–G1074CrossRefPubMedGoogle Scholar
  51. 51.
    Stessin AM, Zippin JH, Kamenetsky M, Hess KC, Buck J, Levin LR (2006) Soluble adenylyl cyclase mediates nerve growth factor-induced activation of Rap1. J Biol Chem 281:17253–17258CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Therien AG, Blostein R (2000) Mechanisms of sodium pump regulation. Am J Phys Cell Phys 279:C541–C566Google Scholar
  53. 53.
    Tuomilehto J, Jousilahti P, Rastenyte D, Moltchanov V, Tanskanen A, Pietinen P, Nissinen A (2001) Urinary sodium excretion and cardiovascular mortality in Finland: a prospective study. Lancet 357:848–851CrossRefPubMedGoogle Scholar
  54. 54.
    Vallon V, Rieg T (2011) Regulation of renal NaCl and water transport by the ATP/UTP/P2Y2 receptor system. Am J Physiol Ren Physiol 301:F463–F475CrossRefGoogle Scholar
  55. 55.
    van der Loo B, Fenton MJ, Erusalimsky JD (1998) Cytochemical detection of a senescence-associated beta-galactosidase in endothelial and smooth muscle cells from human and rabbit blood vessels. Exp Cell Res 241:309–315CrossRefPubMedGoogle Scholar
  56. 56.
    Vinciguerra M, Deschênes G, Hasler U, Mordasini D, Rousselot M, Doucet A, Vandewalle A, Martin PY, Féraille E (2003) Intracellular Na+ controls cell surface expression of Na, K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells. Mol Biol Cell 14:2677–2688CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Warnock DG (2013) The amiloride-sensitive endothelial sodium channel and vascular tone. Hypertension 61:952–954CrossRefPubMedGoogle Scholar
  58. 58.
    Weinberger MH (1996) Salt sensitivity of blood pressure in humans. Hypertension 27:481–490CrossRefPubMedGoogle Scholar
  59. 59.
    Weinberger MH, Miller JZ, Luft FC, Grim CE, Fineberg NS (1986) Definitions and characteristics of sodium sensitivity and blood pressure resistance. Hypertension 8:II127–II134CrossRefPubMedGoogle Scholar
  60. 60.
    Zippin JH, Chen Y, Nahirney P, Kamenetsky M, Wuttke MS, Fischman DA, Levin LR, Buck J (2003) Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains. FASEB J 17:82–84PubMedGoogle Scholar
  61. 61.
    Zippin JH, Chen Y, Straub SG, Hess KC, Diaz A, Lee D, Tso P, Holz GG, Sharp GW, Levin LR, Buck J (2013) CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor. J Biol Chem 288:33283–33291CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Mirja Mewes
    • 1
  • Johanna Nedele
    • 1
  • Katrin Schelleckes
    • 1
  • Olga Bondareva
    • 2
  • Malte Lenders
    • 1
  • Kristina Kusche-Vihrog
    • 3
  • Hans-Joachim Schnittler
    • 2
  • Stefan-Martin Brand
    • 4
  • Boris Schmitz
    • 4
  • Eva Brand
    • 1
    Email author
  1. 1.Internal Medicine D, Department of Nephrology, Hypertension and RheumatologyUniversity Hospital MuensterMuensterGermany
  2. 2.Institute of Anatomy and Vascular BiologyUniversity of MuensterMuensterGermany
  3. 3.Institute of Physiology IIUniversity of MuensterMuensterGermany
  4. 4.Institute of Sports Medicine, Molecular Genetics of Cardiovascular DiseaseUniversity Hospital MuensterMuensterGermany

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