Skip to main content
Download PDF

Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cellsin vitro

Biological Procedures Online volume 5, pages 136–142 (2003)Cite this article

Abstract

Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells invitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 µM) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikertet al.,FEBS Lett 2001, 506:131–134) on aspects of validation procedures as well as limitations and pitfalls of this method.

References

  1. Mocanda S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology.Pharmacol Rev 1991; 43:109–142.

    Google Scholar 

  2. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition.Biochem J 2001; 357:593–615.

    Article  PubMed  CAS  Google Scholar 

  3. Li H, Förstermann U. Nitric oxide in the pathogenesis of vascular disease.J Pathol 2000; 190:244–254.

    Article  PubMed  CAS  Google Scholar 

  4. Wink DA, Vodovotz Y, Laval J, Laval F, Dewhirst MW, Mitchell JB. The multifaceted roles of nitric oxide in cancer.Carcinogenesis 1998; 19:711–721.

    Article  PubMed  CAS  Google Scholar 

  5. O’Donnell VB, Freeman BA. Interactions between nitric oxide and lipid oxidation pathways. Implications for vascular disease.Circ Res 2001; 88:12–21.

    PubMed  CAS  Google Scholar 

  6. Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase.J Clin Invest 1999; 103:897–905.

    Article  PubMed  CAS  Google Scholar 

  7. Chen PY, Sanders PW. Role of nitric oxide synthesis in saltsensitive hypertension in Dahl/Rapp rats.Hypertension 1993; 22:812–818.

    PubMed  CAS  Google Scholar 

  8. Nagano T, Yoshimura T. Bioimaging of nitric oxide.Chem Rev 2002; 102(4):1235–1270

    Article  PubMed  CAS  Google Scholar 

  9. Kojima H, Sakurai K, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Akaike T, Maeda H, Nagano T. Development of a fluorescent indicator for the bioimaging of nitric oxide.Biol Pharm Bull 1997; 20:1229–1232.

    PubMed  CAS  Google Scholar 

  10. Kojima H, Hirotani M, Nakatsubo N, Kikuchi K, Urano Y, Higuchi T, Hirata Y, Nagano T. Bioimaging of nitric oxide with fluorescence indicators based on the rhodamine chromophore.Anal Chem 2001; 73:1967–1973.

    Article  PubMed  CAS  Google Scholar 

  11. Nakatsubo N, Kojima H, Sakurai K, Kikuchi K, Nagoshi H, Hirata Y, Akaike T, Maeda H, Urano Y, Higuchi T, Nagano T. Improved nitric oxide detection using 2,3- diaminonaphthalene and ist application to the evaluation of novel nitric oxide synthase inhibitors.Biol Pharm Bull 1998; 21:1247–1250.

    PubMed  CAS  Google Scholar 

  12. Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, Nagano T. Fluorescent indicators for imaging nitric oxide production.Angew Chem 1999; Int. Ed. 38:3209–3212.

    Article  CAS  Google Scholar 

  13. Kojima H, Sakurai K, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore.Chem Pharm Bull (Tokyo) 1998; 46:373–375.

    CAS  Google Scholar 

  14. Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. Detection and imaging of nitric oxide with novel fluorescence indicators: diaminofluoresceins.Anal Chem 1998; 70:2446–2453.

    Article  PubMed  CAS  Google Scholar 

  15. Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, Maeda D, Imai Y, Irimura T, Nagano T. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins.FEBS Lett 1998; 427:263–266.

    Article  PubMed  CAS  Google Scholar 

  16. Kojima H, Nakatsubo N, Kikuchi K, Urano Y, Higuchi T, Tanaka J, Kudo Y, Nagano T. Direct evidence of NO production in rat hippocampus and cortex using a new fluorescence indicator. DAF-2DA.Neuroreport 1998; 9:3345–3348.

    Article  PubMed  CAS  Google Scholar 

  17. Itoh Y, Ma FH, Hoshi H, Oka M, Noda K, Ukai Y, Kojima H, Nagano T, Toda N. Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry.Anal Biochem 2000; 287:203–209.

    Article  PubMed  CAS  Google Scholar 

  18. Nagano T, Takizawa H, Hirobe M. Reactions of nitric oxide with amines in the presence of dioxygen.Tetrahedron Letters 1995; 36:8239–8242.

    CAS  Google Scholar 

  19. Leikert JF, Räthel TR, Müller C, Vollmar AM, Dirsch VM. Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein.FEBS Lett 2001; 506:131–134.

    Article  PubMed  CAS  Google Scholar 

  20. Edgell CJ, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization.Proc Natl Acad Sci USA 1983; 80(12):3734–3737.

    Article  PubMed  CAS  Google Scholar 

  21. Nagano T, Takizawa H, Hirobe M. Reactions of nitric oxide with amines in the presence of dioxygene.Tetrahedron Letters 1995; 36:8239–8242.

    CAS  Google Scholar 

  22. Keefer LK, Nims RW, Davies KM, Wink DA. “NONOates” (1-substituted diazen-1-ium-12-diolates) as nitric oxide donors: convenient nitric oxide dosage forms.Methods Enzymol 1996; 268:281–293.

    Article  PubMed  CAS  Google Scholar 

  23. Leikert JF, Räthel TR, Wohlfahrt P, Cheynier V, Vollmar AM, Dirsch VM. Red wine polyphenols enhance endothelial nitric oxide synthase expression and subsequent nitric oxide release from endothelial cells.Circulation 2002; 106:1614–1617.

    Article  PubMed  CAS  Google Scholar 

  24. Zeng G, Quon MJ. Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells.J Clin Invest 1996; 98:894–898.

    Article  PubMed  CAS  Google Scholar 

  25. Montagnani M, Chen H, Barr VA, Quon MJ. Insulinstimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser1179.J Biol Chem 2001; 276:30392–30398.

    Article  PubMed  CAS  Google Scholar 

  26. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ. Roles for insulin receptor, PI3- kinase and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells.Circulation 2000; 101:1539–1545.

    PubMed  CAS  Google Scholar 

  27. Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC. Reciprocal phodphorylation and regulation of endothelial nitric oxide synthase in response to bradykinin stimulation.J Biol Chem 2001; 276:16587–16591.

    Article  PubMed  CAS  Google Scholar 

  28. Zhu P, Zaugg CE, Simper D, Hornstein P, Allegrini PR, Buser PT. Bradykinin improves postischaemic recovery in the rat heart: role of high energy phosphates, nitric oxide, and prostacyclin.Cardiovascular Research 1995; 29:658–663.

    PubMed  CAS  Google Scholar 

  29. Kikuchi K, Nagano T, Hayakawa H, Hirata Y, Hirobe M. Detection of nitric oxide production from a perfused organ by a luminol-H2O2 system.Anal Chem 1993; 65:1794–1799.

    Article  PubMed  CAS  Google Scholar 

  30. Faulkner KM, Liochev SI, Fridovichs I. Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo.J Biol Chem 1994; 269:23471–23476.

    PubMed  CAS  Google Scholar 

  31. Szabo C, Day BJ, Salzman AL. Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondrial respiration in immunostimulated macrophages using a manganese mesoporphyrin superoxide dismutase mimetic and peroynitrite scavenger.FEBS Letters 1996; 381:82–86.

    Article  PubMed  CAS  Google Scholar 

  32. Nagata N, Momose K, Ishida Y. Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide.J Biochem 1999; 125:658–661.

    PubMed  CAS  Google Scholar 

  33. Huang A, Vita JA, Venema RC, Keaney J. Ascorbis acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin.J Biol Chem 2000; 275:17399–17406.

    Article  PubMed  CAS  Google Scholar 

  34. Broillet M, Randin O, Chatton J. Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging.FEBS Lett 2001; 491:227–232.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacy, Center of Drug Research, University of Munich, Butenandtstr. 5-13, D-81377, Munich, Germany

    Thomas R. Räthel, Jürgen F. Leikert, Angelika M. Vollmar & Verena M. Dirsch

Authors
  1. Thomas R. Räthel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jürgen F. Leikert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Angelika M. Vollmar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Verena M. Dirsch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Verena M. Dirsch.

Additional information

Published: June 2, 2003

Rights and permissions

Reprints and permissions

About this article

Cite this article

Räthel, T.R., Leikert, J.F., Vollmar, A.M. et al. Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cellsin vitro . Biol. Proced. Online 5, 136–142 (2003). https://doi.org/10.1251/bpo55

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo55

Indexing terms

Biological Procedures Online

ISSN: 1480-9222

Contact us