Advertisement

Pflügers Archiv - European Journal of Physiology

, Volume 460, Issue 5, pp 915–923 | Cite as

Nitric oxide release follows endothelial nanomechanics and not vice versa

  • Johannes FelsEmail author
  • Chiara Callies
  • Kristina Kusche-Vihrog
  • Hans Oberleithner
Signaling and Cell Physiology

Abstract

In the vascular endothelium, mechanical cell stiffness (К) and nitric oxide (NO) release are tightly coupled. “Soft” cells release more NO compared to “stiff” cells. Currently, however, it is not known whether NO itself is the primary factor that softens the cells or whether NO release is the result of cell softening. To address this question, a hybrid fluorescence/atomic force microscope was used in order to measure changes in К and NO release simultaneously in living vascular endothelial cells. Aldosterone was applied to soften the cells transiently and to trigger NO release. NO synthesis was then either blocked or stimulated and, simultaneously, К was measured. Cell indentation experiments were performed to evaluate К, while NO release was measured either by an intracellular NO-dependent fluorescence indicator (DAF-FM/DA) or by NO-selective electrodes located close to the cell surface. After the application of aldosterone, К decreases, within 10 min, to 80.5 ± 1.7% of control (100%). DAF-FM fluorescence intensity increases simultaneously to 132.9 ± 2.2%, which indicates a significant increase in the activity of endothelial NO synthase (eNOS). Inhibition of eNOS (by N ω-nitro-l-arginine methyl ester) blocks the NO release, but does not affect the aldosterone-induced changes in К. Application of an eNOS-independent NO donor (NONOate/AM) raises intracellular NO concentration, but, again, does not affect К. Data analysis indicates that a decrease of К by about 10% is sufficient to induce a significant increase of eNOS activity. In conclusion, these nanomechanic properties of endothelial cells in vascular endothelium determine NO release, and not vice versa.

Keywords

Endothelium Mechanical properties Endothelium-derived relaxing factor (EDRF) Nitric oxide synthase Aldosterone 

Notes

Acknowledgment

Work was supported by the graduate program “Cell Dynamics and Disease,” University of Münster (international PhD scholarship to first author) and by grants from the Deutsche Forschungsgemeinschaft (OB 63/17-1 and Koselleck Grant OB 63/18). We thank Prof. Hugh E. de Wardener, Imperial College, London, for critically reading the manuscript.

Ethical standards

This study was conducted complying current laws and ethical standards.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Martin W, White DG, Henderson AH (1988) Endothelium-derived relaxing factor and atriopeptin II elevate cyclic GMP levels in pig aortic endothelial cells. Br J Pharmacol 93:229–239PubMedGoogle Scholar
  2. 2.
    Fleming I, Busse R (2003) Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 284:R1–R12PubMedGoogle Scholar
  3. 3.
    Sessa WC (2005) Regulation of endothelial derived nitric oxide in health and disease. Mem Inst Oswaldo Cruz 100(Suppl 1):15–18PubMedGoogle Scholar
  4. 4.
    Balligand JL, Feron O, Dessy C (2009) eNOS activation by physical forces: from short-term regulation of contraction to chronic remodeling of cardiovascular tissues. Physiol Rev 89:481–534CrossRefPubMedGoogle Scholar
  5. 5.
    Rubanyi GM, Romero JC, Vanhoutte PM (1986) Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 250:H1145–H1149PubMedGoogle Scholar
  6. 6.
    Kuchan MJ, Frangos JA (1994) Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am J Physiol 266:C628–C636PubMedGoogle Scholar
  7. 7.
    Nilius B, Droogmans G (2001) Ion channels and their functional role in vascular endothelium. Physiol Rev 81:1415–1459PubMedGoogle Scholar
  8. 8.
    Oberleithner H, Callies C, Kusche-Vihrog K, Schillers H, Shahin V, Riethmuller C, MacGregor GA, de Wardener HE (2009) Potassium softens vascular endothelium and increases nitric oxide release. Proc Natl Acad Sci USA 106:2829–2834CrossRefPubMedGoogle Scholar
  9. 9.
    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 USA 104:16281–16286CrossRefPubMedGoogle Scholar
  10. 10.
    Oberleithner H, Schneider SW, Albermann L, Hillebrand U, Ludwig T, Riethmuller C, Shahin V, Schafer C, Schillers H (2003) Endothelial cell swelling by aldosterone. J Membr Biol 196:163–172CrossRefPubMedGoogle Scholar
  11. 11.
    Kidoaki S, Matsuda T (2007) Shape-engineered vascular endothelial cells: nitric oxide production, cell elasticity, and actin cytoskeletal features. J Biomed Mater Res A 81:728–735PubMedGoogle Scholar
  12. 12.
    Kondrikov D, Han HR, Block ER, Su Y (2006) Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 290:L41–L50CrossRefPubMedGoogle Scholar
  13. 13.
    Oberleithner H, Kusche-Vihrog K, Schillers H (2010) Endothelial cells as vascular salt sensors. Kidney Int 77:490–494CrossRefPubMedGoogle Scholar
  14. 14.
    Callies C, Schon P, Liashkovich I, Stock C, Kusche-Vihrog K, Fels J, Strater AS, Oberleithner H (2009) Simultaneous mechanical stiffness and electrical potential measurements of living vascular endothelial cells using combined atomic force and epifluorescence microscopy. Nanotechnology 20:175104CrossRefPubMedGoogle Scholar
  15. 15.
    Oberleithner H (2007) Is the vascular endothelium under the control of aldosterone? Facts and hypothesis. Pflugers Arch 454:187–193CrossRefPubMedGoogle Scholar
  16. 16.
    Heylen E, Huang A, Sun D, Kaley G (2009) Nitric oxide-mediated dilation of arterioles to intraluminal administration of aldosterone. J Cardiovasc Pharmacol 54:535–542CrossRefPubMedGoogle Scholar
  17. 17.
    Liu SL, Schmuck S, Chorazcyzewski JZ, Gros R, Feldman RD (2003) Aldosterone regulates vascular reactivity: short-term effects mediated by phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation. Circulation 108:2400–2406CrossRefPubMedGoogle Scholar
  18. 18.
    Schmidt BM, Sammer U, Fleischmann I, Schlaich M, Delles C, Schmieder RE (2006) Rapid nongenomic effects of aldosterone on the renal vasculature in humans. Hypertension 47:650–655CrossRefPubMedGoogle Scholar
  19. 19.
    Fels J, Oberleithner H, Kusche-Vihrog K (2010) Ménage à trois: aldosterone, sodium and nitric oxide in vascular endothelium. Biochim Biophys Acta. doi: 10.1016/j.bbadis.2010.03.006
  20. 20.
    Mutoh A, Isshiki M, Fujita T (2008) Aldosterone enhances ligand-stimulated nitric oxide production in endothelial cells. Hypertens Res 31:1811–1820CrossRefPubMedGoogle Scholar
  21. 21.
    Asai M, Takeuchi K, Saotome M, Urushida T, Katoh H, Satoh H, Hayashi H, Watanabe H (2009) Extracellular acidosis suppresses endothelial function by inhibiting store-operated Ca2+ entry via non-selective cation channels. Cardiovasc Res 83:97–105CrossRefPubMedGoogle Scholar
  22. 22.
    Itoh Y, Ma FH, Hoshi H, Oka M, Noda K, Ukai Y, Kojima H, Nagano T, Toda N (2000) Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal Biochem 287:203–209CrossRefPubMedGoogle Scholar
  23. 23.
    Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, Nagano T (1999) Fluorescent indicators for imaging nitric oxide production. Angew Chem Int Ed Engl 38:3209–3212CrossRefPubMedGoogle Scholar
  24. 24.
    Pittner J, Wolgast M, Persson AE (2003) Perfusate composition influences nitric oxide homeostasis in rat juxtamedullary afferent arterioles. Acta Physiol Scand 179:85–91CrossRefPubMedGoogle Scholar
  25. 25.
    Fujita S, Roerig DL, Bosnjak ZJ, Stowe DF (1998) Effects of vasodilators and perfusion pressure on coronary flow and simultaneous release of nitric oxide from guinea pig isolated hearts. Cardiovasc Res 38:655–667CrossRefPubMedGoogle Scholar
  26. 26.
    Levine DZ, Iacovitti M, Burns KD, Zhang X (2001) Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo. Am J Physiol Renal Physiol 281:F189–F194PubMedGoogle Scholar
  27. 27.
    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
  28. 28.
    Kasas S, Dietler G (2008) Probing nanomechanical properties from biomolecules to living cells. Pflugers Arch 456:13–27CrossRefPubMedGoogle Scholar
  29. 29.
    Grinspan JB, Mueller SN, Levine EM (1983) Bovine endothelial cells transformed in vitro by benzo(a)pyrene. J Cell Physiol 114:328–338CrossRefPubMedGoogle Scholar
  30. 30.
    Kojima H, Hirata M, Kudo Y, Kikuchi K, Nagano T (2001) Visualization of oxygen-concentration-dependent production of nitric oxide in rat hippocampal slices during aglycemia. J Neurochem 76:1404–1410CrossRefPubMedGoogle Scholar
  31. 31.
    Park JM, Higuchi T, Kikuchi K, Urano Y, Hori H, Nishino T, Aoki J, Inoue K, Nagano T (2001) Selective inhibition of human inducible nitric oxide synthase by S-alkyl-l-isothiocitrulline-containing dipeptides. Br J Pharmacol 132:1876–1882CrossRefPubMedGoogle Scholar
  32. 32.
    Heinz WF, Hoh JH (1999) Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope. Trends Biotechnol 17:143–150CrossRefPubMedGoogle Scholar
  33. 33.
    Mathur AB, Collinsworth AM, Reichert WM, Kraus WE, Truskey GA (2001) Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy. J Biomech 34:1545–1553CrossRefPubMedGoogle Scholar
  34. 34.
    Iyer S, Gaikwad RM, Subba-Rao V, Woodworth CD, Sokolov I (2009) Atomic force microscopy detects differences in the surface brush of normal and cancerous cells. Nat Nanotechnol 4:389–393CrossRefPubMedGoogle Scholar
  35. 35.
    Kasas S, Wang X, Hirling H, Marsault R, Huni B, Yersin A, Regazzi R, Grenningloh G, Riederer B, Forro L, Dietler G, Catsicas S (2005) Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly. Cell Motil Cytoskeleton 62:124–132CrossRefPubMedGoogle Scholar
  36. 36.
    Schmidt BM, Oehmer S, Delles C, Bratke R, Schneider MP, Klingbeil A, Fleischmann EH, Schmieder RE (2003) Rapid nongenomic effects of aldosterone on human forearm vasculature. Hypertension 42:156–160CrossRefPubMedGoogle Scholar
  37. 37.
    Uhrenholt TR, Schjerning J, Hansen PB, Norregaard R, Jensen BL, Sorensen GL, Skott O (2003) Rapid inhibition of vasoconstriction in renal afferent arterioles by aldosterone. Circ Res 93:1258–1266CrossRefPubMedGoogle Scholar
  38. 38.
    Cucina A, Sterpetti AV, Pupelis G, Fragale A, Lepidi S, Cavallaro A, Giustiniani Q, Santoro DL (1995) Shear stress induces changes in the morphology and cytoskeleton organisation of arterial endothelial cells. Eur J Vasc Endovasc Surg 9:86–92CrossRefPubMedGoogle Scholar
  39. 39.
    Schnittler HJ, Schneider SW, Raifer H, Luo F, Dieterich P, Just I, Aktories K (2001) Role of actin filaments in endothelial cell–cell adhesion and membrane stability under fluid shear stress. Pflugers Arch 442:675–687CrossRefPubMedGoogle Scholar
  40. 40.
    Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, Haussinger D (1998) Functional significance of cell volume regulatory mechanisms. Physiol Rev 78:247–306PubMedGoogle Scholar
  41. 41.
    Cornet M, Lambert IH, Hoffmann EK (1993) Relation between cytoskeleton, hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells. J Membr Biol 131:55–66CrossRefPubMedGoogle Scholar
  42. 42.
    Pedersen SF, Mills JW, Hoffmann EK (1999) Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells. Exp Cell Res 252:63–74CrossRefPubMedGoogle Scholar
  43. 43.
    Knudsen HL, Frangos JA (1997) Role of cytoskeleton in shear stress-induced endothelial nitric oxide production. Am J Physiol 273:H347–H355PubMedGoogle Scholar
  44. 44.
    Su Y, Edwards-Bennett S, Bubb MR, Block ER (2003) Regulation of endothelial nitric oxide synthase by the actin cytoskeleton. Am J Physiol Cell Physiol 284:C1542–C1549PubMedGoogle Scholar
  45. 45.
    Schneider M, Ulsenheimer A, Christ M, Wehling M (1997) Nongenomic effects of aldosterone on intracellular calcium in porcine endothelial cells. Am J Physiol 272:E616–E620PubMedGoogle Scholar
  46. 46.
    Trepat X, Deng L, An SS, Navajas D, Tschumperlin DJ, Gerthoffer WT, Butler JP, Fredberg JJ (2007) Universal physical responses to stretch in the living cell. Nature 447:592–595CrossRefPubMedGoogle Scholar
  47. 47.
    Takeda Y (2004) Vascular synthesis of aldosterone: role in hypertension. Mol Cell Endocrinol 217:75–79CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Johannes Fels
    • 1
    Email author
  • Chiara Callies
    • 1
  • Kristina Kusche-Vihrog
    • 1
  • Hans Oberleithner
    • 1
  1. 1.Institute of Physiology IIUniversity of MuensterMuensterGermany

Personalised recommendations