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Oxidation of Adrenaline and Its Derivatives by S-Nitrosoglutathione
NITRIC OXIDE: Biology and Chemistry Vol. 5, No. 1, pp. 39 – 46 (2001) doi:10.1006/niox.2000.0323, available online at http://www.idealibrary.com on
Oxidation of Adrenaline and Its Derivatives by S-Nitrosoglutathione Maria Pia Rigobello, Guido Scutari, Rita Boscolo, and Alberto Bindoli 1 Centro di Studio delle Biomembrane (CNR) and Dipartimento di Chimica Biologica, Universita` di Padova, Padova, Italy
Received April 19, 2000, and in revised form October 17, 2000; published online January 16, 2001
An oxidizing effect of S-nitrosoglutathione toward adrenaline and its cyclic derivatives (adrenochrome and adrenolutin) is reported. The oxidation was monitored either spectrophotometrically or as oxygen uptake. Adrenaline was first oxidized to adrenochrome that, after isomerization to adrenolutin, was further oxidized to products monitored as fluorescence decrease. To occur to a significant extent, this oxidation requires copper ions that, in addition to a direct effect on the oxidation of the ortho-diphenol moiety, are also able to decompose nitrosothiols, giving rise to nitric oxide. The latter, after interaction with oxygen and superoxide, produces nitrogen oxides and peroxynitrite, respectively, that are important contributors to the oxidative process. In this context, catecholamines might act as regulatory factors toward nitric oxide and its derivatives. © 2001 Academic Press Key Words: adrenaline; adrenochrome; adrenolutin; copper ions; nitric oxide; nitrogen oxides; peroxynitrite; S-nitrosoglutathione.
Nitrogen monoxide (currently called “nitric oxide”) is formed enzymatically in biological systems where it plays a fundamental physiological role as a messenger after binding to guanilyl cyclase (1). However, if formed in relatively high concentration, it might also secondarily act as a cytotoxic agent (2). 1
To whom correspondence and reprint requests should be addressed. Fax: 39 (49) 807.3310. E-mail: labbind@ civ.bio.unipd.it. 1089-8603/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
In fact, due to its free radical nature, it forms, after interaction with oxygen and superoxide, several reactive derivatives. These derivatives, collectively called nitrogen oxides (NOx) are responsible for nitrosative stress (3) and exhibit a high reactivity with GSH. 2 Because of the large abundance of the latter in biological systems, formation of GSNO can easily take place. Therefore, glutathione acts as a scavenger of the nitrogen oxides, playing a critical role in the prevention of nitrosative stress (2). Once formed, S-nitrosoglutathione can have different roles acting as a carrier of NO or in transnitrosation reactions. In addition, it exhibits antioxidant properties (4, 5). S-Nitrosothiols were reported to occur naturally in tissues, plasma, and other body fluids as GSNO (6, 7) and S-nitrosoalbumin (8). S-Nitrosothiols can deliver NO and the corresponding disulfide by photochemical and thermal pathways (9). The presence in solution of transition metal ions and, in particular, of copper ions (10 –12) or copper-containing enzymes (13), has a marked effect on the rate of GSNO decomposition. Copper acts in a catalytic way and its cuprous form is the active one that cleaves the S-N bond (12). However, the cellular fate of GSNO and, in particular, the molecules interacting with it are not completely defined. In the present paper we have examined the interactions between adrenaline and GSNO. Previous research has shown that nitric oxide and its derivatives cause the oxidation and 2
Abbreviations used: GSNO, S-nitrosoglutathione; GSH, reduced glutathione; GSSG, oxidized glutathione. 39
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nitration of catecholamines (14 –16). The catabolism of catecholamines proceeds through two major pathways involving the enzymes monoamine oxidase and catechol-ortho-methyl transferase. However, the catechol moiety can easily undergo oxidation and, consequently, a nonenzymatic oxidative pathway should be taken into account (17). The oxidation of catecholamines is a rather complex process where quinone, hydroquinone, and semiquinone species are involved. Catecholamines, after conversion to semiquinone, disproportionate to form the corresponding ortho-quinone; the latter undergo an irreversible 1,4-intramolecular cyclization leading to the formation of an unstable leucoaminochrome which is rapidly oxidized to an aminochrome (17– 20). Therefore, the major products formed from the autoxidation of adrenaline are: adrenochrome, its rearrangement product adrenolutin, and dihydroxyindole formed after dehydration of leucoadrenochrome. However, these products are relatively unstable and undergo further transformation giving rise to dimers and oligomers (21). In previous papers (17, 22), we have demonstrated that the oxidation products of catecholamines such as adrenochrome, particularly in the presence of reducing agents or systems, give rise to a large production of oxygenreduced species, such as superoxide anion and hydrogen peroxide. From the reported results it is apparent that GSNO, in the presence of copper ions, strongly stimulates the formation of the oxidation products of adrenaline. MATERIALS AND METHODS
S-Nitrosoglutathione was prepared according to Hart (23). The visible and ultraviolet spectra of the product obtained are consistent with the data reported in the literature (24). The concentration of GSNO solutions was assessed using ⑀ M ⫽ 15 M ⫺1 cm ⫺1 at 544 nm (24). Oxygen consumption was measured with an oxygraph apparatus using a Clarktype oxygen electrode (25) connected to a personal computer (26). Spectrophotometric and spectrofluorometric measurements are described in the legend of the relevant figures. The data from different experiments generated via the oxygraph or the spectrophotometer/ spectrofluorometer software were stored and con-
verted to ASCII format. The data pairs format was used to transfer the data to numerical analysis and graphics software. Therefore, the data were utilized for averaging the various curves that are the mean of four or five experiments obtained with 1 (oxygraph) and 5 (spectrophotometer/spectrofluorometer) sampling points/min. RESULTS
At physiological pH the uncatalyzed oxidation of adrenaline is very low (27). In the presence of copper ions there is a moderate stimulation of oxygen uptake (Fig. 1). The effect of Cu 2⫹ has been observed and, in particular, it was reported that the rate of autoxidation of adrenaline is linearly dependent on the concentration of copper, which appears to form a Cu 2⫹–adrenaline complex (28). As apparent in Fig. 1, the addition of GSNO is scarcely effective on the oxidation of adrenaline; however, if copper ions are also present, a significant increase of oxygen uptake occurs. The oxidation of adrenaline in the presence of GSNO ⫹ Cu 2⫹ was followed at 480 nm, where adrenochrome, an intermediate of the oxidative process of catecholamines, can be observed. As apparent from Fig. 2, a transient formation of adrenochrome occurs since there is an initial rapid increase of the optical density at 480 nm in the first 10 min followed by a decrease (Inset A of Fig. 2). On the contrary, the absorption observable at 400 nm and indicative of adrenolutin formation constantly increases in the time interval measured (Inset B of Fig. 2). The effect of copper ion alone, in the same time interval, gives rise to a comparatively lower increase of optical density both at 400 and 480 nm (not shown), indicating again that the combination GSNO ⫹ Cu 2⫹ is of fundamental importance in stimulating the oxidation of adrenaline. Other metal ions such as Zn 2⫹, Mn 2⫹, and Fe 2⫹, in our conditions, are almost totally ineffective in stimulating oxygen consumption (not shown). Adrenochrome alone determines, in our conditions, a slow, but significant, oxygen uptake that is increased by the presence of copper ions while the addition of GSNO slightly stimulates oxygen consumption (Figs. 3b–3d). However, when Cu 2⫹ and GSNO are added together, a large consumption of
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ADRENALINE OXIDATION BY S-NITROSOGLUTATHIONE
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FIG. 1. Stimulation of oxygen consumption by adrenaline in the presence of CuSO 4 and GSNO. Reactions were performed in 1.2 ml of 0.5 M Na, K-phosphate buffer (pH 7.5) constantly stirred; the various additions were made after about 1 min of equilibration with air at 25°C. The final concentrations of the reagents were: 5 mM adrenaline, 40 M CuSO 4, and 1 mM GSNO. Adrenaline (a); adrenaline ⫹ GSNO (b); adrenaline ⫹ CuSO 4 (c); adrenaline ⫹ CuSO 4 ⫹ GSNO (d). Catalase (40 g) was added at the arrows. 100% oxygen corresponds to 0.237 mM O 2.
oxygen is apparent (Fig. 3e). Inset A of Fig. 3 reports the oxygen uptake occurring in the presence of GSNO/Cu 2⫹ and increasing concentrations of adrenochrome. From the comparison of Figs. 1 and 3, it is apparent that oxidation of adrenochrome occurs to an extent larger than that of adrenaline. In order to investigate the further transformation of adrenochrome, the fluorescence of adrenolutin was followed. The addition of adrenochrome to a phosphate medium (pH 7.4) in aerobic conditions gives rise to a large increase of fluorescence that reaches a maximum and, afterwards, starts decreasing at a rather slow rate (Fig. 4a). However, the addition of ascorbate before the fluorescence decrease is complete, brings again the fluorescence to the maximum level, indicating that the fading of fluorescence is a reversible autoxidation of adrenolutin, possibly to the corresponding quinone (not shown). The latter, however does not appear to be stable and, together with the parent compounds,
brings to the formation of dimers and oligomers identified by Palumbo et al. (21). The addition of GSNO gives rise to a slow decrease of fluorescence; on the contrary, copper is able to significantly stimulate the oxidation of adrenolutin. The effect of copper is further stimulated by the addition of GSNO but, after a few minutes, fluorescence starts increasing again and, after reaching a maximum, slowly fades again. This effect does not occur with copper or GSNO alone, indicating that the combination GSNO/Cu 2⫹ possibly brings the formation of products different from those derived from the spontaneous oxidation of adrenolutin. DISCUSSION
At variance with lipid peroxidation where nitric oxide acts essentially as an inhibitor (5), in the case of thiols (2, 29, 30) and phenolic compounds (31–34) nitric oxide and its derivatives act as oxidants bringing
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FIG. 2. Oxidation of adrenaline by nitrosoglutathione and copper. Adrenaline (5 mM, final concentration) was dissolved in 0.5 M Na, K-phosphate buffer (pH 7.5). After recording the first spectrum, 1 mM GSNO and 40 M CuSO 4 were added and the subsequent scan intervals were of 3 min. Inset A: time course of absorbance at 480 nm (adrenochrome). Inset B: time course of absorbance at 400 nm (adrenolutin).
to the formation of disulfides and quinones, respectively. This behavior is viewed essentially as an antioxidant action exerted by thiols and reduced quinones and devoted to the interception of nitrogen oxides, such as peroxynitrite and NO2. Nitric oxide, in anaerobic conditions, is able to oxidize ubiquinol to ubiquinone and is reduced to NO⫺ (31). In aerobic conditions, the reaction of nitric oxide with ubiquinol is associated with oxygen consumption. The superoxide anion formed is rapidly removed by NO yielding peroxynitrite subsequently involved in the propagation of ubiquinol oxidation (31). It was also reported that 5,6dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid are oxidized by nitric oxide under aerobic conditions (32). These precursors of melanin are targets for
the reactive nitrogen oxides that are converted to nitrite ions and are far more reactive with NO-derived species than ␣-tocopherol, suggesting a sparing action toward this antioxidant (32). In this context, it was previously observed that the exposure of microsomes to nitric oxide or sydnonimine causes a loss of ␣-tocopherol paralleled by the formation of ␣-tocopherol quinone (33). Polyhydroxyaromatic compounds such as catechol, 1,4-hydroquinone, pyrogallol, and chlorogenic acid in combination with NOreleasing compounds are able to induce a DNA strand break (34) due to peroxynitrite formed from NO and the large amount of superoxide anion derived from the ready autoxidation of these compounds (34). In the present paper an oxidizing effect of GSNO
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ADRENALINE OXIDATION BY S-NITROSOGLUTATHIONE
FIG. 3. Stimulation of oxygen consumption by adrenochrome in the presence of CuSO 4 and GSNO. Reactions were performed in 1.2 ml of 0.5 M Na, K-phosphate buffer (pH 7.5) constantly stirred; the various additions were made after about 1 min of equilibration with air at 25°C. The final concentrations of the reagents in the mixture were 0.5 mM adrenochrome, 40 M CuSO 4, and 1 mM GSNO. CuSO 4 ⫹ GSNO (a); adrenochrome (b); adrenochrome ⫹ GSNO (c); adrenochrome ⫹ CuSO 4 (d); adrenochrome ⫹ CuSO 4 ⫹ GSNO (e). Catalase (40 g) was added at the arrows. Inset A reports oxygen uptake in the presence of 40 M CuSO 4, 1 mM GSNO and increasing concentrations of adrenochrome: 0.1 mM (a⬘), 0.5 mM (b⬘), and 1 mM (c⬘). 100% oxygen corresponds to 0.237 mM O 2.
toward adrenaline and its derivatives is reported. A scheme of the redox reactions that might occur during the oxidation of adrenaline by GSNO in the presence of CuSO 4 is reported (Scheme 1). The oxidation process requires copper ions in order to occur to a significant extent. Copper ions appear to play an essential role either in the oxidation of catecholamines (28) and in the decomposition of nitrosothiols (10 –12). In the latter case the reduction of Cu 2⫹ to its cuprous form is necessary (10 –12). The splitting of GSNO elicits the production of NO that, in the presence of oxygen, generates an oxidizing environment constituted of nitrogen oxides collectively indicated as NOx and mostly consisting of NO 2 and N 2O 3, which are better oxidizing species as compared to NO (35). Therefore, they appear as the
RH2 ⫹ O23 RH • ⫹ O 2•⫺ ⫹ H ⫹
[1] ⫹
RH2 ⫹ Cu 3 Cu ⫹ RH ⫹ H 2GSNO—Cu 1⫹ 3 GSSG ⫹ 2 •NO RH2 ⫹ •NO 3 RH • ⫹ NO ⫺ ⫹ H ⫹ 2 •NO ⫹ O2 3 2 •NO2 RH2 ⫹ •NO2 3 RH • ⫹ HNO2
[2] [3] [4] [5] [6]
O 2•⫺ ⫹ O 2•⫺ ⫹ 2H ⫹ 3 H2O2 ⫹ O2
[7]
2⫹
•
1⫹
•
NO ⫹ O 2•⫺ 3 ONOO ⫺—H ⫹ 3 ONOOH
2RH2 ⫹ ONOOH 3 2 RH ⫹ HNO2 ⫹ H2O 6RH • 3 3RH2 ⫹ 3R •
[8] [9] [10]
SCHEME 1. Oxidation mechanism of adrenaline and its cyclic derivatives by the system GSNO/CuSO 4 in aerobic conditions. RH 2, RH • , and R indicate the ortho-diphenol, semiquinone, and ortho-quinone forms of adrenaline and its cyclic derivatives (leucoadrenochrome and adrenolutin), respectively.
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RIGOBELLO ET AL.
FIG. 4. Effect of CuSO 4 and GSNO on adrenolutin decomposition. Adrenolutin formation from adrenochrome (0.12 mM) was followed fluorometrically (excitation: 400 nm, emission: 520 nm) at 25°C in 0.5 M Na, K-phosphate buffer (pH 7.5). After spontaneous formation of adrenolutin (about 10 min), 2 mM GSNO (b), 12 M CuSO 4 (c), and 2 mM GSNO ⫹ 12 M CuSO 4 (d) were added (arrow). Control, no additions (a).
major responsible for the stimulation of the oxidation of adrenaline (Scheme 1, reaction 6). In addition, since the oxidation products of adrenaline give rise to the production of superoxide (17, 22) also the formation of peroxynitrite, which acts as a potent oxidizing agent (35), should be taken into account (Scheme 1, reaction 8). Addition of catalase after the oxidation has occurred is a simple way to indicate, by the oxygen production, the presence of hydrogen peroxide derived from the dismutation of the superoxide anion generated during oxidation of catecholamines. As observed in Figs. 1 and 3, the addition of catalase shows the formation of oxygen in the presence of adrenaline ⫹ Cu 2⫹, adrenochrome alone, adrenochrome ⫹ Cu 2⫹. In the presence of adrenochrome ⫹ GSNO there is a very slight stimulation of oxygen production, while with the complete system formed by GSNO ⫹ Cu 2⫹, both in the presence of adrenaline and adrenochrome, no stimulation, after the addition of catalase, is apparent even though a large consumption of oxygen has occurred (Figs. 1 and 3). The lack of effect of catalase is a good evidence of peroxynitrite formation since superoxide anion formed
by autoxidation of ortho-diphenol forms of adrenaline and its derivatives (leucoadrenochrome and adrenolutin), preferentially interacts with NO instead of dismutating to hydrogen peroxide. In addition, the presence of superoxide dismutase in the system adrenochrome/Cu2⫹/GSNO is without effect on oxygen uptake (not reported). However, this lack of effect of catalase might also depend on its reversible inhibition by nitric oxide (36) since only the addition of a large amount of hydrogen peroxide elicits a significant formation of oxygen after a marked lag time (not shown). It should also be noted that part of the oxygen taken up in the overall oxidative process is directed to the formation of NO 2 after interaction with nitric oxide derived from GSNO, without forming hydrogen peroxide (Scheme 1). The ortho-diphenol moiety of adrenaline and its transformation products appear to play a primary role in the mechanism of GSNO splitting catalyzed by copper. In fact, the ortho-diphenol can bind cupric copper and reduce it to the cuprous form (28), which actively decomposes GSNO giving rise to NO (10 – 13) as reported in Scheme 1 (reactions 2 and 3). In
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ADRENALINE OXIDATION BY S-NITROSOGLUTATHIONE
the same scheme, reaction 1 indicates the autoxidation of adrenaline and its cyclic derivatives. This autoxidation is, in our conditions, extremely limited for adrenaline (Fig. 1) but occurs more consistently with the nonisolatable intermediate leucoadrenochrome (39) and with adrenochrome (Fig. 3). In the latter case the formation of superoxide anion is indicated by the splitting of hydrogen peroxide by catalase (Fig. 3). Nitric oxide can act as a direct oxidant of catecholamines (reaction 4). A similar process was shown for hydroquinones in anaerobic conditions. Reaction 9 is the overall reaction of oxidation of the catecholamines by peroxynitrite. In fact, peroxynitrite is able to oxidize several reducing substrates, including antioxidants, with a rather complex reaction mechanism involving one and twoelectron processes (40 – 42). However, other reactions not apparent in Scheme 1 might occur; for instance, a factor that adds complexity to the system when using nitric oxide donors is represented by the contribution of the moiety remaining after the release of nitric oxide (37). In the case of GSNO, this moiety is represented by the thiyl radical of glutathione (GS •). Copper is an essential trace element participating in the catalytic reactions of several enzymes. However, when its concentration exceeds the needs, copper is toxic and causes deleterious effects to tissues and organs; consequently, its transport regulation is particularly important (43). Copper exist almost entirely complexed with small molecules or proteins (43); therefore, in living cells, a direct interaction between free copper ions and GSNO appears improbable. However, not only copper, but also coppercontaining enzymes such as Cu-Zn-superoxidedismutase (13) and a copper-dependent GSNO lyase (44) found in fibroblasts and platelets actively decomposes GSNO. The controlled production of nitrogen oxide exerts a fundamental role in the vascular, nervous and immune system. However, its excessive of inappropriate production causes cellular damage collectively indicated as nitrosative stress (3, 38). Considering catecholamines, or, more generally, orthodiphenol structures, nitric oxide appears to act essentially as a prooxidant factor by itself or after interaction with oxygen or superoxide anion. Therefore, it appears of interest to evaluate the potential
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Oxidation of Adrenaline and Its Derivatives by S-Nitrosoglutathione Maria Pia Rigobello, Guido Scutari, Rita Boscolo, and Alberto Bindoli 1 Centro di Studio delle Biomembrane (CNR) and Dipartimento di Chimica Biologica, Universita` di Padova, Padova, Italy
Received April 19, 2000, and in revised form October 17, 2000; published online January 16, 2001
An oxidizing effect of S-nitrosoglutathione toward adrenaline and its cyclic derivatives (adrenochrome and adrenolutin) is reported. The oxidation was monitored either spectrophotometrically or as oxygen uptake. Adrenaline was first oxidized to adrenochrome that, after isomerization to adrenolutin, was further oxidized to products monitored as fluorescence decrease. To occur to a significant extent, this oxidation requires copper ions that, in addition to a direct effect on the oxidation of the ortho-diphenol moiety, are also able to decompose nitrosothiols, giving rise to nitric oxide. The latter, after interaction with oxygen and superoxide, produces nitrogen oxides and peroxynitrite, respectively, that are important contributors to the oxidative process. In this context, catecholamines might act as regulatory factors toward nitric oxide and its derivatives. © 2001 Academic Press Key Words: adrenaline; adrenochrome; adrenolutin; copper ions; nitric oxide; nitrogen oxides; peroxynitrite; S-nitrosoglutathione.
Nitrogen monoxide (currently called “nitric oxide”) is formed enzymatically in biological systems where it plays a fundamental physiological role as a messenger after binding to guanilyl cyclase (1). However, if formed in relatively high concentration, it might also secondarily act as a cytotoxic agent (2). 1
To whom correspondence and reprint requests should be addressed. Fax: 39 (49) 807.3310. E-mail: labbind@ civ.bio.unipd.it. 1089-8603/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
In fact, due to its free radical nature, it forms, after interaction with oxygen and superoxide, several reactive derivatives. These derivatives, collectively called nitrogen oxides (NOx) are responsible for nitrosative stress (3) and exhibit a high reactivity with GSH. 2 Because of the large abundance of the latter in biological systems, formation of GSNO can easily take place. Therefore, glutathione acts as a scavenger of the nitrogen oxides, playing a critical role in the prevention of nitrosative stress (2). Once formed, S-nitrosoglutathione can have different roles acting as a carrier of NO or in transnitrosation reactions. In addition, it exhibits antioxidant properties (4, 5). S-Nitrosothiols were reported to occur naturally in tissues, plasma, and other body fluids as GSNO (6, 7) and S-nitrosoalbumin (8). S-Nitrosothiols can deliver NO and the corresponding disulfide by photochemical and thermal pathways (9). The presence in solution of transition metal ions and, in particular, of copper ions (10 –12) or copper-containing enzymes (13), has a marked effect on the rate of GSNO decomposition. Copper acts in a catalytic way and its cuprous form is the active one that cleaves the S-N bond (12). However, the cellular fate of GSNO and, in particular, the molecules interacting with it are not completely defined. In the present paper we have examined the interactions between adrenaline and GSNO. Previous research has shown that nitric oxide and its derivatives cause the oxidation and 2
Abbreviations used: GSNO, S-nitrosoglutathione; GSH, reduced glutathione; GSSG, oxidized glutathione. 39
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nitration of catecholamines (14 –16). The catabolism of catecholamines proceeds through two major pathways involving the enzymes monoamine oxidase and catechol-ortho-methyl transferase. However, the catechol moiety can easily undergo oxidation and, consequently, a nonenzymatic oxidative pathway should be taken into account (17). The oxidation of catecholamines is a rather complex process where quinone, hydroquinone, and semiquinone species are involved. Catecholamines, after conversion to semiquinone, disproportionate to form the corresponding ortho-quinone; the latter undergo an irreversible 1,4-intramolecular cyclization leading to the formation of an unstable leucoaminochrome which is rapidly oxidized to an aminochrome (17– 20). Therefore, the major products formed from the autoxidation of adrenaline are: adrenochrome, its rearrangement product adrenolutin, and dihydroxyindole formed after dehydration of leucoadrenochrome. However, these products are relatively unstable and undergo further transformation giving rise to dimers and oligomers (21). In previous papers (17, 22), we have demonstrated that the oxidation products of catecholamines such as adrenochrome, particularly in the presence of reducing agents or systems, give rise to a large production of oxygenreduced species, such as superoxide anion and hydrogen peroxide. From the reported results it is apparent that GSNO, in the presence of copper ions, strongly stimulates the formation of the oxidation products of adrenaline. MATERIALS AND METHODS
S-Nitrosoglutathione was prepared according to Hart (23). The visible and ultraviolet spectra of the product obtained are consistent with the data reported in the literature (24). The concentration of GSNO solutions was assessed using ⑀ M ⫽ 15 M ⫺1 cm ⫺1 at 544 nm (24). Oxygen consumption was measured with an oxygraph apparatus using a Clarktype oxygen electrode (25) connected to a personal computer (26). Spectrophotometric and spectrofluorometric measurements are described in the legend of the relevant figures. The data from different experiments generated via the oxygraph or the spectrophotometer/ spectrofluorometer software were stored and con-
verted to ASCII format. The data pairs format was used to transfer the data to numerical analysis and graphics software. Therefore, the data were utilized for averaging the various curves that are the mean of four or five experiments obtained with 1 (oxygraph) and 5 (spectrophotometer/spectrofluorometer) sampling points/min. RESULTS
At physiological pH the uncatalyzed oxidation of adrenaline is very low (27). In the presence of copper ions there is a moderate stimulation of oxygen uptake (Fig. 1). The effect of Cu 2⫹ has been observed and, in particular, it was reported that the rate of autoxidation of adrenaline is linearly dependent on the concentration of copper, which appears to form a Cu 2⫹–adrenaline complex (28). As apparent in Fig. 1, the addition of GSNO is scarcely effective on the oxidation of adrenaline; however, if copper ions are also present, a significant increase of oxygen uptake occurs. The oxidation of adrenaline in the presence of GSNO ⫹ Cu 2⫹ was followed at 480 nm, where adrenochrome, an intermediate of the oxidative process of catecholamines, can be observed. As apparent from Fig. 2, a transient formation of adrenochrome occurs since there is an initial rapid increase of the optical density at 480 nm in the first 10 min followed by a decrease (Inset A of Fig. 2). On the contrary, the absorption observable at 400 nm and indicative of adrenolutin formation constantly increases in the time interval measured (Inset B of Fig. 2). The effect of copper ion alone, in the same time interval, gives rise to a comparatively lower increase of optical density both at 400 and 480 nm (not shown), indicating again that the combination GSNO ⫹ Cu 2⫹ is of fundamental importance in stimulating the oxidation of adrenaline. Other metal ions such as Zn 2⫹, Mn 2⫹, and Fe 2⫹, in our conditions, are almost totally ineffective in stimulating oxygen consumption (not shown). Adrenochrome alone determines, in our conditions, a slow, but significant, oxygen uptake that is increased by the presence of copper ions while the addition of GSNO slightly stimulates oxygen consumption (Figs. 3b–3d). However, when Cu 2⫹ and GSNO are added together, a large consumption of
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ADRENALINE OXIDATION BY S-NITROSOGLUTATHIONE
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FIG. 1. Stimulation of oxygen consumption by adrenaline in the presence of CuSO 4 and GSNO. Reactions were performed in 1.2 ml of 0.5 M Na, K-phosphate buffer (pH 7.5) constantly stirred; the various additions were made after about 1 min of equilibration with air at 25°C. The final concentrations of the reagents were: 5 mM adrenaline, 40 M CuSO 4, and 1 mM GSNO. Adrenaline (a); adrenaline ⫹ GSNO (b); adrenaline ⫹ CuSO 4 (c); adrenaline ⫹ CuSO 4 ⫹ GSNO (d). Catalase (40 g) was added at the arrows. 100% oxygen corresponds to 0.237 mM O 2.
oxygen is apparent (Fig. 3e). Inset A of Fig. 3 reports the oxygen uptake occurring in the presence of GSNO/Cu 2⫹ and increasing concentrations of adrenochrome. From the comparison of Figs. 1 and 3, it is apparent that oxidation of adrenochrome occurs to an extent larger than that of adrenaline. In order to investigate the further transformation of adrenochrome, the fluorescence of adrenolutin was followed. The addition of adrenochrome to a phosphate medium (pH 7.4) in aerobic conditions gives rise to a large increase of fluorescence that reaches a maximum and, afterwards, starts decreasing at a rather slow rate (Fig. 4a). However, the addition of ascorbate before the fluorescence decrease is complete, brings again the fluorescence to the maximum level, indicating that the fading of fluorescence is a reversible autoxidation of adrenolutin, possibly to the corresponding quinone (not shown). The latter, however does not appear to be stable and, together with the parent compounds,
brings to the formation of dimers and oligomers identified by Palumbo et al. (21). The addition of GSNO gives rise to a slow decrease of fluorescence; on the contrary, copper is able to significantly stimulate the oxidation of adrenolutin. The effect of copper is further stimulated by the addition of GSNO but, after a few minutes, fluorescence starts increasing again and, after reaching a maximum, slowly fades again. This effect does not occur with copper or GSNO alone, indicating that the combination GSNO/Cu 2⫹ possibly brings the formation of products different from those derived from the spontaneous oxidation of adrenolutin. DISCUSSION
At variance with lipid peroxidation where nitric oxide acts essentially as an inhibitor (5), in the case of thiols (2, 29, 30) and phenolic compounds (31–34) nitric oxide and its derivatives act as oxidants bringing
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FIG. 2. Oxidation of adrenaline by nitrosoglutathione and copper. Adrenaline (5 mM, final concentration) was dissolved in 0.5 M Na, K-phosphate buffer (pH 7.5). After recording the first spectrum, 1 mM GSNO and 40 M CuSO 4 were added and the subsequent scan intervals were of 3 min. Inset A: time course of absorbance at 480 nm (adrenochrome). Inset B: time course of absorbance at 400 nm (adrenolutin).
to the formation of disulfides and quinones, respectively. This behavior is viewed essentially as an antioxidant action exerted by thiols and reduced quinones and devoted to the interception of nitrogen oxides, such as peroxynitrite and NO2. Nitric oxide, in anaerobic conditions, is able to oxidize ubiquinol to ubiquinone and is reduced to NO⫺ (31). In aerobic conditions, the reaction of nitric oxide with ubiquinol is associated with oxygen consumption. The superoxide anion formed is rapidly removed by NO yielding peroxynitrite subsequently involved in the propagation of ubiquinol oxidation (31). It was also reported that 5,6dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid are oxidized by nitric oxide under aerobic conditions (32). These precursors of melanin are targets for
the reactive nitrogen oxides that are converted to nitrite ions and are far more reactive with NO-derived species than ␣-tocopherol, suggesting a sparing action toward this antioxidant (32). In this context, it was previously observed that the exposure of microsomes to nitric oxide or sydnonimine causes a loss of ␣-tocopherol paralleled by the formation of ␣-tocopherol quinone (33). Polyhydroxyaromatic compounds such as catechol, 1,4-hydroquinone, pyrogallol, and chlorogenic acid in combination with NOreleasing compounds are able to induce a DNA strand break (34) due to peroxynitrite formed from NO and the large amount of superoxide anion derived from the ready autoxidation of these compounds (34). In the present paper an oxidizing effect of GSNO
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ADRENALINE OXIDATION BY S-NITROSOGLUTATHIONE
FIG. 3. Stimulation of oxygen consumption by adrenochrome in the presence of CuSO 4 and GSNO. Reactions were performed in 1.2 ml of 0.5 M Na, K-phosphate buffer (pH 7.5) constantly stirred; the various additions were made after about 1 min of equilibration with air at 25°C. The final concentrations of the reagents in the mixture were 0.5 mM adrenochrome, 40 M CuSO 4, and 1 mM GSNO. CuSO 4 ⫹ GSNO (a); adrenochrome (b); adrenochrome ⫹ GSNO (c); adrenochrome ⫹ CuSO 4 (d); adrenochrome ⫹ CuSO 4 ⫹ GSNO (e). Catalase (40 g) was added at the arrows. Inset A reports oxygen uptake in the presence of 40 M CuSO 4, 1 mM GSNO and increasing concentrations of adrenochrome: 0.1 mM (a⬘), 0.5 mM (b⬘), and 1 mM (c⬘). 100% oxygen corresponds to 0.237 mM O 2.
toward adrenaline and its derivatives is reported. A scheme of the redox reactions that might occur during the oxidation of adrenaline by GSNO in the presence of CuSO 4 is reported (Scheme 1). The oxidation process requires copper ions in order to occur to a significant extent. Copper ions appear to play an essential role either in the oxidation of catecholamines (28) and in the decomposition of nitrosothiols (10 –12). In the latter case the reduction of Cu 2⫹ to its cuprous form is necessary (10 –12). The splitting of GSNO elicits the production of NO that, in the presence of oxygen, generates an oxidizing environment constituted of nitrogen oxides collectively indicated as NOx and mostly consisting of NO 2 and N 2O 3, which are better oxidizing species as compared to NO (35). Therefore, they appear as the
RH2 ⫹ O23 RH • ⫹ O 2•⫺ ⫹ H ⫹
[1] ⫹
RH2 ⫹ Cu 3 Cu ⫹ RH ⫹ H 2GSNO—Cu 1⫹ 3 GSSG ⫹ 2 •NO RH2 ⫹ •NO 3 RH • ⫹ NO ⫺ ⫹ H ⫹ 2 •NO ⫹ O2 3 2 •NO2 RH2 ⫹ •NO2 3 RH • ⫹ HNO2
[2] [3] [4] [5] [6]
O 2•⫺ ⫹ O 2•⫺ ⫹ 2H ⫹ 3 H2O2 ⫹ O2
[7]
2⫹
•
1⫹
•
NO ⫹ O 2•⫺ 3 ONOO ⫺—H ⫹ 3 ONOOH
2RH2 ⫹ ONOOH 3 2 RH ⫹ HNO2 ⫹ H2O 6RH • 3 3RH2 ⫹ 3R •
[8] [9] [10]
SCHEME 1. Oxidation mechanism of adrenaline and its cyclic derivatives by the system GSNO/CuSO 4 in aerobic conditions. RH 2, RH • , and R indicate the ortho-diphenol, semiquinone, and ortho-quinone forms of adrenaline and its cyclic derivatives (leucoadrenochrome and adrenolutin), respectively.
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RIGOBELLO ET AL.
FIG. 4. Effect of CuSO 4 and GSNO on adrenolutin decomposition. Adrenolutin formation from adrenochrome (0.12 mM) was followed fluorometrically (excitation: 400 nm, emission: 520 nm) at 25°C in 0.5 M Na, K-phosphate buffer (pH 7.5). After spontaneous formation of adrenolutin (about 10 min), 2 mM GSNO (b), 12 M CuSO 4 (c), and 2 mM GSNO ⫹ 12 M CuSO 4 (d) were added (arrow). Control, no additions (a).
major responsible for the stimulation of the oxidation of adrenaline (Scheme 1, reaction 6). In addition, since the oxidation products of adrenaline give rise to the production of superoxide (17, 22) also the formation of peroxynitrite, which acts as a potent oxidizing agent (35), should be taken into account (Scheme 1, reaction 8). Addition of catalase after the oxidation has occurred is a simple way to indicate, by the oxygen production, the presence of hydrogen peroxide derived from the dismutation of the superoxide anion generated during oxidation of catecholamines. As observed in Figs. 1 and 3, the addition of catalase shows the formation of oxygen in the presence of adrenaline ⫹ Cu 2⫹, adrenochrome alone, adrenochrome ⫹ Cu 2⫹. In the presence of adrenochrome ⫹ GSNO there is a very slight stimulation of oxygen production, while with the complete system formed by GSNO ⫹ Cu 2⫹, both in the presence of adrenaline and adrenochrome, no stimulation, after the addition of catalase, is apparent even though a large consumption of oxygen has occurred (Figs. 1 and 3). The lack of effect of catalase is a good evidence of peroxynitrite formation since superoxide anion formed
by autoxidation of ortho-diphenol forms of adrenaline and its derivatives (leucoadrenochrome and adrenolutin), preferentially interacts with NO instead of dismutating to hydrogen peroxide. In addition, the presence of superoxide dismutase in the system adrenochrome/Cu2⫹/GSNO is without effect on oxygen uptake (not reported). However, this lack of effect of catalase might also depend on its reversible inhibition by nitric oxide (36) since only the addition of a large amount of hydrogen peroxide elicits a significant formation of oxygen after a marked lag time (not shown). It should also be noted that part of the oxygen taken up in the overall oxidative process is directed to the formation of NO 2 after interaction with nitric oxide derived from GSNO, without forming hydrogen peroxide (Scheme 1). The ortho-diphenol moiety of adrenaline and its transformation products appear to play a primary role in the mechanism of GSNO splitting catalyzed by copper. In fact, the ortho-diphenol can bind cupric copper and reduce it to the cuprous form (28), which actively decomposes GSNO giving rise to NO (10 – 13) as reported in Scheme 1 (reactions 2 and 3). In
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ADRENALINE OXIDATION BY S-NITROSOGLUTATHIONE
the same scheme, reaction 1 indicates the autoxidation of adrenaline and its cyclic derivatives. This autoxidation is, in our conditions, extremely limited for adrenaline (Fig. 1) but occurs more consistently with the nonisolatable intermediate leucoadrenochrome (39) and with adrenochrome (Fig. 3). In the latter case the formation of superoxide anion is indicated by the splitting of hydrogen peroxide by catalase (Fig. 3). Nitric oxide can act as a direct oxidant of catecholamines (reaction 4). A similar process was shown for hydroquinones in anaerobic conditions. Reaction 9 is the overall reaction of oxidation of the catecholamines by peroxynitrite. In fact, peroxynitrite is able to oxidize several reducing substrates, including antioxidants, with a rather complex reaction mechanism involving one and twoelectron processes (40 – 42). However, other reactions not apparent in Scheme 1 might occur; for instance, a factor that adds complexity to the system when using nitric oxide donors is represented by the contribution of the moiety remaining after the release of nitric oxide (37). In the case of GSNO, this moiety is represented by the thiyl radical of glutathione (GS •). Copper is an essential trace element participating in the catalytic reactions of several enzymes. However, when its concentration exceeds the needs, copper is toxic and causes deleterious effects to tissues and organs; consequently, its transport regulation is particularly important (43). Copper exist almost entirely complexed with small molecules or proteins (43); therefore, in living cells, a direct interaction between free copper ions and GSNO appears improbable. However, not only copper, but also coppercontaining enzymes such as Cu-Zn-superoxidedismutase (13) and a copper-dependent GSNO lyase (44) found in fibroblasts and platelets actively decomposes GSNO. The controlled production of nitrogen oxide exerts a fundamental role in the vascular, nervous and immune system. However, its excessive of inappropriate production causes cellular damage collectively indicated as nitrosative stress (3, 38). Considering catecholamines, or, more generally, orthodiphenol structures, nitric oxide appears to act essentially as a prooxidant factor by itself or after interaction with oxygen or superoxide anion. Therefore, it appears of interest to evaluate the potential
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