Original contributionIncreased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide
Introduction
The diatomic free radical nitric oxide (NO) plays multiple regulatory functions in the nervous, immune, and cardiovascular systems. The biological reactivity of NO is dictated primarily by its reaction with transition metals, superoxide (O2•−), and molecular oxygen (O2) [1]. The latter two reactions yield a variety of reactive nitrogen oxide species that include peroxynitrite (ONOO−/ONOOH) for the reaction of NO with O2•− [2] and dinitrogen trioxide (N2O3) for the reaction of NO with O2 [3]. These species represent potentially important modulators of cell and tissue functions by virtue of their ability to oxidize, nitrate (addition of NO2), and nitrosate (addition of NO+) biologically relevant molecules [4]. Over recent years, nitrosation reactions have received increasing attention as a potential mechanism by which cellular homeostasis may be altered under physiological and pathophysiological conditions [5]. The nitrosation of specific thiols on proteins may serve as a signaling process in cells and as a mechanism by which NO bioactivity may be stored and transported in tissues [6]. Little is known regarding the role of N-nitroso adducts as posttranslational modifications, but it is recognized that the nitrosation of primary amines on DNA bases and the formation of N-nitrosamines from secondary amines may contribute to the increased incidence of carcinogenesis associated with the overproduction of NO [7].
While the measurement of NO is a necessary adjunct to the understanding of the mechanisms by which NO functions, the instability and rapid diffusion of NO have rendered difficult its detection in cells and tissues. A new approach has been described by Kojima and co-workers to visualize intracellular NO based on the utilization of the cell permeable 4,5 diaminofluorescein diacetate (DAF-2DA) [8], [9], [10]. DAF-2DA is hydrolyzed by intracellular esterases to liberate the cell impermeable 4,5 diaminofluorescein (DAF-2), which upon nitrosation yields a highly fluorescent derivative DAF-2 triazole. Mechanistically, previous reports suggest that dinitrogen trioxide (N2O3), the product of the reaction of NO with molecular oxygen (O2) rather than NO itself nitrosates DAF-2 [9], [11]. The NO/O2 reaction is second order with respect to NO, indicating that this reaction is slow at physiological concentration of NO [4]. These kinetic considerations impose an intrinsic limitation to the amount of N2O3 produced in cells and it is not known whether these are high enough to account for nitrosation reactions, especially if competing reactions such as N2O3 hydrolysis and trapping by ascorbate and thiols are also taken into account. This is in sharp contrast with the detection limit of the DAF-2 assay in cells estimated at 5 nM [9]. The NO/O2 reaction is accelerated approximately 300-fold in the hydrophobic milieu of biological membranes but the fate of the reactive nitrogen species generated in this environment is unknown at this time [12].
Despite an increasing interest for the utilization of DAF-2 as a “dosimeter” of intracellular NO, little is known regarding its selectivity. A recent report indicates that fluorescein derivatives such as 2′, 7′ dichlorofluorescein (DCF) react with horseradish peroxidase-compound I and II to form a phenoxyl free radical [13]. The reaction yields superoxide (O2•−) via the intermediate oxidation of reducing agents such as glutathione and NADPH. These observations led us to entertain the possibility that DAF-2 might be oxidized by horseradish peroxidase in a fashion similar to DCF and that this may affect its reaction with NO. The results described herein show that both horseradish peroxidase and peroxynitrite oxidize DAF-2 to yield a nonfluorescent intermediate, which directly reacts with NO to form the fluorescent triazole derivative. Our study shows that results from DAF-2 fluorometric assays are difficult to interpret in cells and tissues undergoing oxidative and nitrosative stress.
Section snippets
Materials
Xanthine oxidase was purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). 4,5-Diaminofluorescein (DAF-2) was purchased from Calbiochem (San Diego, CA, USA). DEA/NO and Sp/NO was obtained from Cayman Chemicals (Ann Arbor, MI, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Generation of superoxide and/or nitric oxide
Superoxide was generated at 37°C using 250 μM hypoxanthine and various concentration of xanthine oxidase (0 to 10 mU/ml) in phosphate-buffered saline (PBS; pH 7.4)
Effect of horseradish peroxidase on the nitrosation of DAF-2 by DEA/NO
The NO-dependent conversion of DAF-2 to its triazole derivative restores the fluorescent properties of the fluorescein moiety, via reduction of the electron-donating properties of the aromatic vicinal amines [9], [14]. In agreement with previous studies [9], [11], we confirmed that the addition of the NO donor DEA/NO (0–10 μM) caused a concentration-dependent increase in fluorescence at 517 nm (excitation = 490 nm) that was inhibited by the removal of oxygen from the solution (data not shown).
Discussion
Products derived from the autoxidation of NO are essential for the nitrosation of DAF-2 to form the fluorescent DAF-2 triazole in oxygenated solutions [11]. However, in the presence of either a peroxidase or peroxynitrite, DAF-2 is oxidized to an intermediate that directly combines with NO to bypass the NO/O2 reaction. This is most evident if considering the results obtained with bolus addition of peroxynitrite where the formation of an intermediate with a half-life of approximately 90 s was a
Acknowledgements
This work was supported by a grant from the National Institute of Health (CA89366). I am very grateful to Frances L. Jourd’heuil for her expert technical assistance.
References (20)
- et al.
Nitrosylationthe prototypic redox-based signaling mechanism
Cell
(2001) - et al.
Orthogonality of calcium concentration and ability of 4,5-diaminofluorescein to detect NO
J. Biol. Chem.
(2002) - et al.
Distinction between nitrosating mechanisms within human cells and aqueous solution
J. Biol. Chem.
(2001) - et al.
Phenoxyl free radical formation during the oxidation of the fluorescent dye 2′, 7′-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements
J. Biol. Chem.
(1999) - et al.
Bicarbonate inhibits N-nitrosation in oxygenated nitric oxide solutions
J. Biol. Chem.
(1996) - et al.
The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate
Arch. Biochem. Biophys.
(1999) - et al.
Reaction of superoxide and nitric oxide with peroxynitrite
J. Biol. Chem.
(2001) - et al.
Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2)implications for cellular NO imaging
FEBS Lett.
(2001) - et al.
Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolitesimplications in inflammation
Am. J. Physiol.
(1999) - et al.
Apparent hydroxyl radical production by peroxynitriteimplications for endothelial injury from nitric oxide and superoxide
Proc. Natl. Acad. Sci. USA
(1990)