Original contribution
Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide

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Abstract

4,5 Diaminofluorescein (DAF-2) is increasingly utilized as a fluorescent detector for nitric oxide (radical dotNO) in cells and tissues. In oxygenated solutions, reactive nitrogen species derived from radical dotNO autoxidation nitrosate DAF-2 to yield the highly fluorescent DAF-2 triazole. In the present study, we investigated the nitrosation of DAF-2 at a neutral pH by absorption and fluorescence spectroscopy using NONOates as chemical sources of radical dotNO. We found that both chemically synthesized peroxynitrite and horseradish peroxidase in the presence of hydrogen peroxide (H2O2) oxidized DAF-2 to a relatively stable nonfluorescent intermediate (t1/2 ∼ 90 s). Oxidation of DAF-2 prior to the addition of the radical dotNO donor DEA/NO resulted in an increase in fluorescence that was approximately 7-fold higher than treatment with DEA/NO alone. The increase in DAF-2 triazole formation upon oxidation of DAF-2 was confirmed by high performance liquid chromatography. Peroxynitrite generated in situ from the equimolar production of radical dotNO and superoxide (O2•−) also increased the yields of DAF-2 triazole formation, which was completely inhibited when O2•− was in excess of radical dotNO. We propose that DAF-2 is oxidized to a free radical intermediate that directly reacts with radical dotNO, thereby bypassing the requirement for radical dotNO autoxidation for the formation of DAF-2 triazole. Our findings indicate that DAF-2 fluorometric assays are quantitatively difficult to interpret in cells and in solution when oxidants and radical dotNO are co-generated.

Introduction

The diatomic free radical nitric oxide (radical dotNO) plays multiple regulatory functions in the nervous, immune, and cardiovascular systems. The biological reactivity of radical dotNO 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 radical dotNO with O2•− [2] and dinitrogen trioxide (N2O3) for the reaction of radical dotNO 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 radical dotNO 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 radical dotNO [7].

While the measurement of radical dotNO is a necessary adjunct to the understanding of the mechanisms by which radical dotNO functions, the instability and rapid diffusion of radical dotNO have rendered difficult its detection in cells and tissues. A new approach has been described by Kojima and co-workers to visualize intracellular radical dotNO 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 radical dotNO with molecular oxygen (O2) rather than radical dotNO itself nitrosates DAF-2 [9], [11]. The radical dotNO/O2 reaction is second order with respect to radical dotNO, indicating that this reaction is slow at physiological concentration of radical dotNO [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 radical dotNO/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 radical dotNO, 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 radical dotNO. The results described herein show that both horseradish peroxidase and peroxynitrite oxidize DAF-2 to yield a nonfluorescent intermediate, which directly reacts with radical dotNO 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 radical dotNO-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 radical dotNO 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 radical dotNO 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 radical dotNO to bypass the radical dotNO/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.

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