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Nitric oxide release follows endothelial nanomechanics and not vice versa

  • Signaling and Cell Physiology
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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.

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References

  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 93:229–239

    CAS  PubMed  Google Scholar 

  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–R12

    CAS  PubMed  Google Scholar 

  3. Sessa WC (2005) Regulation of endothelial derived nitric oxide in health and disease. Mem Inst Oswaldo Cruz 100(Suppl 1):15–18

    CAS  PubMed  Google Scholar 

  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 89:481–534

    Article  CAS  PubMed  Google Scholar 

  5. Rubanyi GM, Romero JC, Vanhoutte PM (1986) Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 250:H1145–H1149

    CAS  PubMed  Google Scholar 

  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–C636

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  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 106:2829–2834

    Article  CAS  PubMed  Google Scholar 

  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 104:16281–16286

    Article  CAS  PubMed  Google Scholar 

  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 196:163–172

    Article  CAS  PubMed  Google Scholar 

  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 81:728–735

    PubMed  Google Scholar 

  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–L50

    Article  CAS  PubMed  Google Scholar 

  13. Oberleithner H, Kusche-Vihrog K, Schillers H (2010) Endothelial cells as vascular salt sensors. Kidney Int 77:490–494

    Article  CAS  PubMed  Google Scholar 

  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 20:175104

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. Heylen E, Huang A, Sun D, Kaley G (2009) Nitric oxide-mediated dilation of arterioles to intraluminal administration of aldosterone. J Cardiovasc Pharmacol 54:535–542

    Article  CAS  PubMed  Google Scholar 

  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 108:2400–2406

    Article  CAS  PubMed  Google Scholar 

  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 47:650–655

    Article  CAS  PubMed  Google Scholar 

  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. Mutoh A, Isshiki M, Fujita T (2008) Aldosterone enhances ligand-stimulated nitric oxide production in endothelial cells. Hypertens Res 31:1811–1820

    Article  CAS  PubMed  Google Scholar 

  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 83:97–105

    Article  CAS  PubMed  Google Scholar 

  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 287:203–209

    Article  CAS  PubMed  Google Scholar 

  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 38:3209–3212

    Article  CAS  PubMed  Google Scholar 

  24. Pittner J, Wolgast M, Persson AE (2003) Perfusate composition influences nitric oxide homeostasis in rat juxtamedullary afferent arterioles. Acta Physiol Scand 179:85–91

    Article  CAS  PubMed  Google Scholar 

  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 38:655–667

    Article  CAS  PubMed  Google Scholar 

  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–F194

    CAS  PubMed  Google Scholar 

  27. Carl P, Schillers H (2008) Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing. Pflugers Arch 457:551–559

    Article  CAS  PubMed  Google Scholar 

  28. Kasas S, Dietler G (2008) Probing nanomechanical properties from biomolecules to living cells. Pflugers Arch 456:13–27

    Article  CAS  PubMed  Google Scholar 

  29. Grinspan JB, Mueller SN, Levine EM (1983) Bovine endothelial cells transformed in vitro by benzo(a)pyrene. J Cell Physiol 114:328–338

    Article  CAS  PubMed  Google Scholar 

  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 76:1404–1410

    Article  CAS  PubMed  Google Scholar 

  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 132:1876–1882

    Article  CAS  PubMed  Google Scholar 

  32. Heinz WF, Hoh JH (1999) Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope. Trends Biotechnol 17:143–150

    Article  CAS  PubMed  Google Scholar 

  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 34:1545–1553

    Article  CAS  PubMed  Google Scholar 

  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 4:389–393

    Article  CAS  PubMed  Google Scholar 

  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 62:124–132

    Article  CAS  PubMed  Google Scholar 

  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 42:156–160

    Article  CAS  PubMed  Google Scholar 

  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 93:1258–1266

    Article  CAS  PubMed  Google Scholar 

  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 9:86–92

    Article  CAS  PubMed  Google Scholar 

  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 442:675–687

    Article  CAS  PubMed  Google Scholar 

  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 78:247–306

    CAS  PubMed  Google Scholar 

  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 131:55–66

    Article  CAS  PubMed  Google Scholar 

  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 252:63–74

    Article  CAS  PubMed  Google Scholar 

  43. Knudsen HL, Frangos JA (1997) Role of cytoskeleton in shear stress-induced endothelial nitric oxide production. Am J Physiol 273:H347–H355

    CAS  PubMed  Google Scholar 

  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–C1549

    CAS  PubMed  Google Scholar 

  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–E620

    CAS  PubMed  Google Scholar 

  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 447:592–595

    Article  CAS  PubMed  Google Scholar 

  47. Takeda Y (2004) Vascular synthesis of aldosterone: role in hypertension. Mol Cell Endocrinol 217:75–79

    Article  CAS  PubMed  Google Scholar 

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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.

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This study was conducted complying current laws and ethical standards.

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The authors declare that they have no conflict of interest.

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  1. Institute of Physiology II, University of Muenster, Robert-Koch-Str. 27b, 48149, Muenster, Germany

    Johannes Fels, Chiara Callies, Kristina Kusche-Vihrog & Hans Oberleithner

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  1. Johannes Fels
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  2. Chiara Callies
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Correspondence to Johannes Fels.

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Fels, J., Callies, C., Kusche-Vihrog, K. et al. Nitric oxide release follows endothelial nanomechanics and not vice versa. Pflugers Arch - Eur J Physiol 460, 915–923 (2010). https://doi.org/10.1007/s00424-010-0871-8

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  • DOI: https://doi.org/10.1007/s00424-010-0871-8

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