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Journal of Fluorescence

, Volume 14, Issue 1, pp 17–23 | Cite as

Fluorescence Measurements of Steady State Peroxynitrite Production Upon SIN-1 Decomposition: NADH Versus Dihydrodichlorofluorescein and Dihydrorhodamine 123

  • Francisco Javier Martin-Romero
  • Yolanda Gutiérrez-Martin
  • Fernando Henao
  • Carlos Gutiérrez-MerinoEmail author
Article

Abstract

The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity ≤ 0.1 μM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Locke's buffer or Dulbecco's modified Eagle's medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14–18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Locke's buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 μM SIN-1 amounted to 1.4 ± 0.2 μM in Locke's buffer, 3.2–3.6 μM in DMEM-F12 (plus and minus phenol red) and 1.8 μM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2′,7′-dichlorodihydrofluorescein diacetate to oxidation by H2O2, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (±5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.

Peroxynitrite 3-morpholinosydnonimine NADH fluorescence 

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REFERENCES

  1. 1.
    J. S. Beckman, M. Carson, C. D. Smith, and W. H. Koppenol (1993). ALS, SOD and peroxynitrite. Nature 364(6438), 584.PubMedGoogle Scholar
  2. 2.
    B. Halliwell (1997). What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? FEBS Lett. 411(2/3), 157-160.PubMedGoogle Scholar
  3. 3.
    M. P. Murphy, M. A. Packer, J. L. Scarlett, and S. W. Martin (1998). Peroxynitrite: A biologically significant oxidant. Gen. Pharmacol. 31(2), 179-186.PubMedGoogle Scholar
  4. 4.
    R. M. Uppu and W. A. Pryor (1996). Synthesis of peroxynitrite in a two-phase system using isoamyl nitrite and hydrogen peroxide. Anal. Biochem. 236(2), 242-249.Google Scholar
  5. 5.
    S. Pfeiffer, A. C. Gorren, K. Schmidt, E. R. Werner, B. Hansert, D. S. Bohle, and B. Mayer (1997). Metabolic fate of peroxynitrite in aqueous solution. Reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry. J. Biol. Chem. 272(6), 3465-3470.PubMedGoogle Scholar
  6. 6.
    W. H. Koppenol, J. J. Moreno, W. A. Pryor, H. Ischiropoulos, and J. S. Beckman (1992). Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5(6), 834-842.PubMedGoogle Scholar
  7. 7.
    G. E. Arteel, K. Briviba, and H. Sies (1999). Protection against peroxynitrite FEBS Lett. 445(2/3), 226-230.PubMedGoogle Scholar
  8. 8.
    R. Radi (1998). Peroxynitrite reactions and diffusion in biology. Chem. Res. Toxicol. 11(7), 720-721.PubMedGoogle Scholar
  9. 9.
    J. S. Beckman, J. Chen, H. Ischiropoulos, and J. P. Crow (1994). Oxidative chemistry of peroxynitrite. Methods Enzymol. 233, 229-240.PubMedGoogle Scholar
  10. 10.
    Y. Gutierrez-Martin, F. J. Martin-Romero, F. Henao, and C. Gutierrez-Merino (2002). Synaptosomal plasma membrane Ca2+ pump activity inhibition by repetitive micromolar ONOO- pulses. Free Radical. Biol. Med. 32(1), 46-55.Google Scholar
  11. 11.
    M. Kirsch and H. de Groot (1999). Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide. J. Biol. Chem. 274(35), 24664-24670.PubMedGoogle Scholar
  12. 12.
    S. A. Lipton, Y. B. Choi, Z. H. Pan, S. Z. Lei, H. S. Chen, N. J. Sucher, J. Loscalzo, D. J. Singel, and J. S. Stamler (1993). A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364(6438), 626-632.PubMedGoogle Scholar
  13. 13.
    L. Brunelli, J. P. Crow, and J. S. Beckman (1995). The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch. Biochem. Biophys. 316(1), 327-334.PubMedGoogle Scholar
  14. 14.
    A. J. Gow, Q. Chen, M. Gole, M. Themistocleous, V. M. Lee, and H. Ischiropoulos (2000). Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency. Am. J. Physiol. Cell. Physiol. 278(6), C1099-C1107.PubMedGoogle Scholar
  15. 15.
    J. L. Trackey, T. F. Uliasz, and S. J. Hewett (2001). SIN-1-induced cytotoxicity in mixed cortical cell culture: Peroxynitrite-dependent and-independent induction of excitotoxic cell death. J. Neurochem. 79(2), 445-455.PubMedGoogle Scholar
  16. 16.
    A. Schrammel, S. Pfeiffer, K. Schmidt, D. Koesling, and B. Mayer (1998). Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione. Mol. Pharmacol. 54(1), 207-212.PubMedGoogle Scholar
  17. 17.
    J. P. Crow (1997). Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1(2), 145-157.PubMedGoogle Scholar
  18. 18.
    J. A. Royall and H. Ischiropoulos (1993). Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch. Biochem. Biophys. 302(2), 348-355.PubMedGoogle Scholar
  19. 19.
    D. Jourd'heuil, L. Mills, A. M. Miles, and M. B. Grisham (1998). Effect of nitric oxide on hemoprotein-catalyzed oxidative reactions. Nitric Oxide 2(1), 37-44.PubMedGoogle Scholar
  20. 20.
    S. L. Hempel, G. R. Buettner, Y. Q. O'Malley, D. A. Wessels, and D. M. Flaherty (1999). Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radical. Biol. Med. 27(1/2), 146-159.Google Scholar
  21. 21.
    T. Ohashi, A. Mizutani, A. Murakami, S. Kojo, T. Ishii, and S. Taketani (2002). Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: Formation of the fluorescein is independent of the generation of reactive oxygen species. FEBS Lett. 511 (1-;3), 21-27.PubMedGoogle Scholar
  22. 22.
    R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman (1991). Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266(7), 4244-4250.PubMedGoogle Scholar
  23. 23.
    A. Claiborne (1985). In R. A. Greenwald (Ed.), Handbook of Methods for Oxygen Radical Research, CRC Press, Boca Raton, FL, pp. 283-284.Google Scholar
  24. 24.
    H. Bohn and K. Schonafinger (1989). Oxygen and oxidation promote the release of nitric oxide from sydnonimines. J. Cardiovasc. Pharmacol. 14 Suppl. 11, S6-S12.Google Scholar
  25. 25.
    M. Feelisch, J. Ostrowski, and E. Noack (1989). On the mechanism of NO release from sydnonimines. J. Cardiovasc. Pharmacol. 14(Suppl 11), S13-S22.Google Scholar
  26. 26.
    F. J. Martin-Romero, E. Garcia-Martin, and C. Gutierrez-Merino (2002). Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low-potassium-induced apoptosis of cerebellar granule cells. J. Neurochem. 82(3), 705-715.PubMedGoogle Scholar
  27. 27.
    F. J. Martin-Romero, B. Santiago-Josefat, J. Correa-Bordes, C. Gutierrez-Merino, and P. Fernandez-Salguero (2000). Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition. J. Mol. Neurosci. 15(3), 155-165.PubMedGoogle Scholar
  28. 28.
    B. Halliwell (2003). Oxidation stress in cell culture: An under-appreciated problem? FEBS Lett. 540(1-;3), 3-6.PubMedGoogle Scholar
  29. 29.
    L. Hua Long and B. Halliwell (2001). Oxidation and generation of hydrogen peroxide by thiol compounds in commonly used cell culture media. Biochem. Biophys. Res. Commun. 286(5), 991-994.PubMedGoogle Scholar
  30. 30.
    M. Kirsch, E. E. Lomonosova, H. G. Korth, R. Sustmann, and H. de Groot (1998). Hydrogen peroxide formation by reaction of peroxynitrite with Hepes and related tertiary amines. J. Biol. Chem. 273(21), 12716-12724.PubMedGoogle Scholar
  31. 31.
    M. Kirsch and H. de Groot (2000). Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions. Evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite. J. Biol. Chem. 275(22), 16702-16708.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 2004

Authors and Affiliations

  • Francisco Javier Martin-Romero
    • 1
  • Yolanda Gutiérrez-Martin
    • 1
  • Fernando Henao
    • 1
  • Carlos Gutiérrez-Merino
    • 1
    Email author
  1. 1.Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias and Escuela de Ingenierías AgrariasUniversidad de ExtremaduraBadajozSpain

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