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Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases
BBRC Biochemical and Biophysical Research Communications 295 (2002) 1125–1134 www.academicpress.com
Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases Teh-Min Hu,a William L. Hayton,a Mark A. Morse,b and Susan R. Malleryc,* a Division of Pharmaceutics, College of Pharmacy, Columbus, OH 43210-1241, USA Department of Environmental Health Science, School of Public Health, Columbus, OH 43210-1241, USA Department of Oral and Maxillofacial Surgery and Pathology, College of Dentistry, The Ohio State University, Columbus, OH 43210-1241, USA b
c
Received 24 June 2002
Abstract It has been shown that superoxide dismutase (SOD) can both potentiate and attenuate NO-mediated toxicity. This present study investigated the role of SOD and GSH in a sustained nitrosative and oxidative environment simulated by the nitric oxide (NO) and superoxide ðO 2 Þ donor, 3-morpholinosydnonimine (SIN-1). We describe, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results show that SOD’s effects on nitrosation are biphasic in nature i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD predominantly exhibits a pronitrosative effect, with a complete loss of antinitrosative effects noted at higher levels of GSH. Our findings likely reflect the complex and dynamic nature of SOD interactions with oxidative and nitrosative species. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Superoxide dismutase; Nitric oxide; SIN-1; Nitrosation; Kinetics; Biphasic; Dynamics
The free radicals superoxide anion ðO 2 Þ and nitric oxide (NO) play important roles in numerous physiological and pathological processes. Because of their free radical characteristics, both O 2 and NO can be converted to various other reactive species, which can be further subcategorized into reactive oxygen species (ROS) and reactive nitrogen species (RNS) [1]. Under normal physiological conditions, NO, O 2 and their reactive species derivatives are maintained at the state of ‘‘redox homeostasis’’ [1], at which reactive species production is balanced by various types of scavengers such as superoxide dismutase (SOD) and reduced glutathione (GSH) [1]. Under such circumstances, ROS and RNS function as regulatory mediators of a broad spectrum of physiological responses [1]. However, in the scenarios where the production of ROS and/or RNS is persistently high (e.g., sites of sustained inflammation) [2] or where mutations of SOD (e.g., in neurodegenerative diseases
*
Corresponding author. Fax: 614-292-9384. E-mail address: [email protected] (S.R. Mallery).
[1,3,4]) occur, oxidative or nitrosative damage could be inflicted by excessive amounts of either ROS or RNS upon DNA, proteins, lipids, and other cellular components [5–8]. Previous studies have demonstrated that SOD’s concentration is critical in determining whether SOD is antioxidative or prooxidative [9]. When exogenous SOD was used to alleviate the oxidative stress in the ischemia-reperfusion models, bell-shaped dose–response relationships were observed [10–12]. While low concentrations (<5 lg=ml) of SOD reduced lipid peroxidation and were cardioprotective, high concentrations (>50 lg=ml) increased lipid peroxidation and can exacerbate acute cardiac injury [12]. Therefore, the results of previous investigations suggested that optimal concentrations existed for SOD when protecting biological systems from oxidative damages [9]. Several mechanisms have been proposed to account for the adverse effect of high [SOD], which include overproduction of hydrogen peroxide resulting from the SOD-catalyzed dismutation of O 2 [13,14], SOD’s peroxidase activity [15], and dual roles played by
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 8 2 0 - 3
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superoxide in both initiating and terminating radical chain reactions [12]. Recently, a study by Offer et al. [16] showed that low concentrations (<20 U/ml) of Cu,ZnSOD inhibited O 2 -induced ferrocyanide oxidation, while its antioxidative effect was lost at high [Cu,ZnSOD]. It was concluded that, in the presence of ultralow steady-state O 2 levels, the reaction kinetics of Cu,ZnSOD is altered; i.e., the oxidized form of Cu,Zn-SOD not only reacts with O 2 but also oxidizes the target molecule that it was supposed to protect [16]. Later, Liochev and Fridovich [17] demonstrated that Cu,ZnSOD functions both as a superoxide reductase and a superoxide oxidase. The findings of Offer et al. [16] were consequently attributed to the increase in SOR activity that accompanies increased concentrations of Cu,Zn-SOD. Cumulatively, these previous investigations suggested that, in addition to its superoxide dismutation activity, SOD also exhibits other enzymatic functions. However, all additional activities of SOD are somewhat related to the redox properties of SOD and have direct or indirect involvement of O 2 . High levels of O2 and NO are concurrently generated in many pathophysiological processes [2]. Further, the pathways of the generation and elimination of both ROS and RNS are closely related [5]. Therefore, it is of biological and pharmacological relevance to investigate how SOD’s disparate activities modulate RNS-mediated nitrosative and oxidative chemistry in the context of sustained O 2 and NO production. It has been shown that SOD can both potentiate [18–20] and attenuate [21–23] NO-mediated cytotoxicity. The opposing findings again reflect the complexity of SOD’s biological actions. From the kinetics standpoint, SOD could impinge on the chemical balance between ROS- and RNS-associated reaction pathways [5]. While previous studies by Wink et al. [24] importantly demonstrated the chemical aspects of SOD/RNS/ROS interactions at a static time point, little is known about the dynamics of SOD’s overall effect on the RNS-mediated nitrosative and oxidative chemistry. The objective of our study was to investigate the effect of SOD over time on RNS-mediated nitrosation kinetics. Nitrosation, i.e., the addition of NOþ to a nucleophile [25], is one of the major NO-mediated reactions in biological systems. Nitrosation products of albumin, hemoglobin, and glutathione (GSH) could act as reservoirs for NO storage or transportation in plasma or in cells [26]. Recently, protein nitrosation has been proposed to be a prototypic redox-based signaling mechanism [27,28]. Moreover, nitrosation may cause damage to DNA bases [29] and enzymes [30–33]. S-nitrosation was shown to inactivate enzymes that contain catalytically essential sulfhydryl groups, such as glutathione peroxidase [30], glutathione reductase [31], and glyceraldehyde3-phosphate dehydrogenase [32,33]. Consequently,
SOD’s ability to suppress or enhance nitrosation is of physiological and pathological significance. This present study investigated the role of SOD in a sustained nitrosative and oxidative environment simulated by an NO and O 2 donor. Our paper describes, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results showed that SOD’s effects on nitrosation are biphasic in nature, i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation.
Materials and methods Materials. SIN-1 was purchased from Calbiochem (La Jolla, CA). Cu,Zn-SOD, Mn-SOD, Fe-SOD, catalase, diaminonaphthalene (DAN), dihydrorhodamine 123 (DHR), glutathione (GSH), diethyltriaminepentaacetic acid (DTPA), and all other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). The chemical structures of SIN-1, DAN, and DHR are shown in Fig. 1. Kinetics of DAN nitrosation. The nitrosation kinetics of DAN was studied using a fluorescence spectrometer (Perkin–Elmer LS 50B, Norwalk, CT). Reactions were performed in a 96-well microtiter plate format at 25 1 °C in the sample chamber. The fluorescence intensity of nitrosated DAN was measured at excitation and emission wavelengths of 375 and 430 nm, respectively. A stock solution of DAN (30 mM) was freshly prepared in dimethyl sulfoxide (DMSO), protected from light exposure, and stored at 4 °C. The dynamic range of DAN in the nitrosation kinetics was studied by varying the concentration of DAN (0.78–300 lM) in 200 ll of phosphate-buffered saline (pH 7.4) that contained 0.1 mM DTPA and 120 U/ml catalase to eliminate hydrogen peroxide. Reactions were initiated by adding an aliquot of 4 ll SIN-1 stock solution (5 mM in DMSO) into 200 ll DAN-containing reaction buffers. The fluorescence intensity after the addition of SIN-1 was measured at 10-min intervals. The nitrosation of DAN in the presence of SOD (0–1000 U/ml) and SIN-1 ð100 lMÞ was
Fig. 1. The chemical structures of 3-morpholinosydnonimine (SIN-1), 2,3-diaminonaphthalene (DAN), and dihydrorhodamine 123 (DHR).
T.-M. Hu et al. / Biochemical and Biophysical Research Communications 295 (2002) 1125–1134 studied in the same pH 7.4 reaction buffer at 25 1 °C by measuring the increase in fluorescence every 10 min up to 8 h. Oxidation of DHR by SIN-1. The oxidation of DHR was determined at 25 1 °C in the above-mentioned reaction buffer except that DAN was replaced by DHR (10 mM stock in dimethylformamide stored at 20 °C and protected from light). The fluorescence of rhodamine 123 generated from the oxidation reaction was measured at excitation and emission wavelengths of 500 and 530 nm, respectively. The effect of SOD (0–1000 U/ml) on SIN-1-mediated DHR oxidation was studied in 200-ll DHR-containing reaction buffer, where the DHR concentration ( 0:39 lM in the final reaction mixture) was chosen based on a pilot experiment in which the dynamic range of DHR was determined. The oxidation kinetics of DHR was typically monitored every 5-min up to 4 h. HPLC determination of SIN-1 degradation kinetics. An HPLC method [34] was modified to study the effect of SOD on the degradation kinetics of SIN-1. The method consisted of a reversed phase column (Waters Nova-Pak C18 3:9 300 mm), a mobile phase (10 mM sodium acetate buffer (pH 3.1)/acetonitrile/methanol ¼ 92/6.4/1.6, flow rate ¼ 1 ml/min), and UV detection at 254 nm. SIN-1 was separated from its two degradation products, SIN-1A and SIN-1C, with retention times of 4.0, 9.0, and 9.8 min for SIN-1, SIN-1A, and SIN-1C, respectively. The kinetics of the SIN-1 degradation at 25 °C was studied by serial sampling (0, 30, 60, 90, 120, 180, 240, 300, 360, and 480 min), with an initial [SIN-1] of 100 lM, with and without 1000 U/ ml Cu,Zn-SOD in the same reaction buffer as described in the nitrosation kinetics section. The dose–response relationship of Cu,Zn-SOD for the degradation of SIN-1 was further investigated by determining the relative quantity (expressed as percentage control) of SIN-1 and its degradation products after 4 h incubation in reaction buffer containing 100 lM SIN-1 and various concentrations of Cu,Zn-SOD (1000, 250, 62.5, 15.6, 3.13, and 0.78 U/ml). Method validation. The concentrations of DAN and DHR used in the study were determined in pilot studies to exclude the possibility of fluorescence quenching. In addition, nitrogen oxide species such as NO 2 , NO3 , and ONOO , which were likely generated during the reaction, were evaluated and shown not to nitrosate DAN. SOD was also shown not to affect the fluorescence of DAN and DHR. Nevertheless, suitable controls (e.g., SOD/DAN for SIN-1-mediated nitrosation kinetics, SOD/DHR for oxidation kinetics) were included in each experimental run and the fluorescence reported in this paper was after subtracting out the background fluorescence of the controls.
Results Kinetics of DAN nitrosation The kinetic profile of SIN-1-mediated DAN nitrosation, Fig. 2, was characterized by the formation of fluorescence at 430 nm. The nitrosation for the control (curve 1) peaked at 180 min after the addition of SIN-1 ð100 lMÞ. After the maximum, the fluorescence intensity tended to decline gradually. With increasing concentrations of Cu,Zn-SOD up to 15.6 U/ml, the rate and extent of DAN nitrosation increased disproportionately, curves 2–6 of Fig. 2A. In contrast, [Cu,Zn-SOD] above 15.6 U/ml increasingly attenuated DAN nitrosation, curves 7–11 of Fig. 2B. The kinetics of fluorescence formation was dramatically affected by the presence of the highest [Cu,Zn-SOD], at which the fluorescence intensity accumulated in a slow and sustained fashion (Fig. 2B, curve 11).
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Fig. 2. Nitrosation kinetics of DAN in the absence and presence of different levels of Cu,Zn-SOD.The reactions were performed in a 96well microtiter plate format. SIN-1 ð100 lMÞ was added to PBS-based reaction buffer containing 3:13 lM DAN, 0.1 mM DTPA, 120 U/ml catalase, and Cu,Zn-SOD (0–1000 U/ml), pH 7.4, at 25 1 °C. The fluorescence intensity after the addition of SIN-1 was measured at 10min intervals by fluorescence spectroscopy. Data represent the means of three measurements. Each curve represents a different [Cu,ZnSOD]: 1, control; 2, 0.98 U/ml; 3, 1.95 U/ml; 4, 3.9 U/ml; 5, 7.8 U/ml; 6, 15.6 U/ml; 7, 31.2 U/ml; 8, 62. 5 U/ml; 9, 125 U/ml; 10, 500 U/ml; 11, 1000 U/ml.
SOD modulated SIN-1-mediated DAN nitrosation in a biphasic fashion To further characterize the interaction between SOD and DAN nitrosation, a dose–response relationship was constructed in accordance with the relative degree of nitrosation compared with the control versus [Cu,ZnSOD] at different measurement times. Remarkably, multiple biphasic dose–response curves characterized by a dynamic transition among those curves emerged (Fig. 3A). These data enabled a 3-D representation of the dynamic dose–response relationship (Fig. 3B). The maximum stimulatory effect of SOD occurred initially at ½Cu; Zn-SOD < 10 U/ml, which corresponded to a
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bly, high [Cu,Zn-SOD] reduced N-nitrosation to the extent that DAN nitrosation levels were generally below control levels. Heat inactivated (>90 °C, 2 h) Cu,Zn-SOD lost both its stimulatory and inhibitory effects on N-nitrosation (data not shown). Free metal ions such as Cuþ , Cuþþ , and Znþþ also had no effect on N-nitrosation. Bovine serum albumin (BSA) was used to test whether a nonspecific protein effect might have been involved in the reaction. While BSA had no effect on nitrosation at low concentrations, it inhibited the reaction to some extent at concentrations >1 lM (data not shown). DAN concentrations affected SOD modulation of nitrosation kinetics
Fig. 3. Time- and concentration-dependent, biphasic, dose–response relationship for the effect of Cu,Zn-SOD on DAN nitrosation. (A) The fluorescence intensities at the same time point in Fig. 2 were expressed as percentage control and plotted against [Cu,Zn-SOD]. Each curve (n ¼ 3) represents the dose–response relationship at 20-min intervals. The arrows indicate the direction of time progression starting at 45 min and ending at 485 min. (B) Saddle-like dose–response surface was generated by plotting the relative fluorescence intensities against [Cu,Zn-SOD] and the reaction time.
hump in this dose region at early incubation time. The size of the hump in this region diminished and then disappeared at 180 min (Fig. 3B). After this mark, the dose–response curve in the low Cu,Zn-SOD concentration region changed insignificantly along time and became the ascending part of a new emerging hump whose size grew continuously and the concentration of Cu,ZnSOD corresponding to the hump also tended to increase. In contrast with the low concentration region, the dose– response relationship in the high concentration region (30–1000 U/ml) appeared to be steady in the early period while changing substantially in the later period. Nota-
To investigate whether the substrate concentration affected the observed dose–response phenomena, the concentration of DAN was halved or doubled while keeping other experimental conditions unchanged. Fig. 4 depicts the sharp contrast between two experiments that used fourfold differences of [DAN]. While the time-dependent, bell-shaped, dose–response relationship remained in both cases, there was a fundamental difference in the way in which N-nitrosation was affected by the presence of Cu,Zn-SOD. At ½DAN ¼ 1:56 lM, Fig. 4A, the hump in the higher SOD region dominated that in the lower region (600% at [Cu,ZnSOD] 30 U/ml at 480 min versus 450% at [Cu,ZnSOD] 3 U/ml at 45 min). In addition, the dynamic transition between the two regions in Fig. 4A occurred much earlier than that in Fig. 3. However, the result was somewhat reversed when [DAN] was increased by fourfold (Fig. 4B). The effect of Cu,Zn-SOD peaked at the initial period and then dropped more slowly until it achieved a second phase in which the maximum effect was shifted to the right (Fig. 4B). Moreover, the dose– response relationship at [Cu,Zn-SOD] >100 U/ml was less perturbed during the reaction period (Fig. 4B). To account for this observation, the kinetic profiles for the three controls with different levels of DAN were compared (Fig. 4B). The time to attain maximum nitrosation appeared to be increased from 2 h for the control with the lowest [DAN] to 3 h and 5 h for the control with two- and fourfold higher [DAN], respectively. The extent of nitrosation increased almost linearly. All SOD isoforms demonstrated biphasic reaction kinetics Also examined was whether Mn- and Fe-SOD elicited a biphasic effect similar to that of Cu,Zn-SOD. A similar dose–response relationship occurred for these SOD isoforms as well (Fig. 5), although a smaller inhibitory effect was observed for both high [Mn-SOD] and [Fe-SOD]; the maximal effective concentration at t ¼ 480 min was increased from 31.3 U/ml for Cu,Zn-
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Fig. 5. Time- and concentration-dependent, biphasic, dose–response relationships for the effects of Mn- and Fe-SOD. Procedures were described in Figs. 2 and 3, except (A) Mn-SOD and (B) Fe-SOD were studied. Reactions were initiated by adding SIN-1 ð100 lMÞ to PBSbased reaction buffer containing 3:13 lM DAN, 0.1 mM DTPA, 120 U/ml catalase, and various activities of SOD (0–1000 U/ml). Values represent the means of three measurements.
Fig. 4. Effect of DAN concentration on SOD modulation of nitrosation kinetics. Procedures were as described in Fig. 2, except (A) 1=2 ½DAN or (B) 2 ½DAN was used. Mean dose–response curves (n ¼ 4) were obtained by plotting the relative fluorescence intensities (percentage control) against [Cu,Zn-SOD]. The time interval for each curve is 20 min. The arrows indicate the direction of time progression starting at 45 min and ending at 485 min. (C) The kinetic profiles for the three controls with different levels of DAN.
SOD (Fig. 3A) to 62.5 U/ml for both Mn- and Fe-SOD (Figs. 5A and B). Glutathione modulated the biphasic effect of Cu,Zn-SOD in a concentration-dependent fashion Glutathione (GSH), a reduced thiol and an antioxidant, is an NO sink that modulates cellular redox re-
actions. It was therefore postulated that GSH would modify the effect of SOD on the N-nitrosation reaction mediated by SIN-1-derived reactive nitrogen species. GSH at or above 0.5 mM inhibited SIN-1-mediated Nnitrosation by >90% (Fig. 6A). Accordingly, the effect of Cu,Zn-SOD was examined in the presence of 0.2, 1, and 5 mM GSH, which represented low, intermediate, and high physiologically relevant levels. While the biphasic effect of Cu,Zn-SOD was retained at 0.2 mM GSH, it disappeared with higher [GSH] (Fig. 6B). When the data were expressed as percentage control and compared with the dose–response curve obtained from the experiment in which GSH was absent, it was remarkable that the effect of Cu,Zn-SOD was enhanced by the presence of increasing [GSH] (up to 1 mM), which was reflected in the upward shift of the entire dose–response curve (Fig. 6C). However, the whole dose–response curve of Cu,Zn-SOD fell dramatically at 5 mM GSH (Fig. 6C). This result may be explained by the fact that the maximum inhibitory effect of GSH on DAN nitrosation was achieved at ½GSH 1 mM (Fig. 6A). Above this level, the detection limit might be approached; therefore, no further inhibition was
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Fig. 7. Effect of Cu,Zn-SOD on SIN-1-mediated oxidation of DHR. SIN-1 ð100 lMÞ was added to pH 7.4 PBS buffer containing 0:39 lM DHR, 0.1 mM DTPA, 120 U/ml catalase, and Cu,Zn-SOD (0–1000 U/ ml), at 25 1 °C. The fluorescence intensity after the addition of SIN-1 was measured at a 5-min interval; fluorescence was expressed as percentage control ðn ¼ 4 SDÞ and plotted at a 20-min interval against [Cu,Zn-SOD]. The arrows indicate the direction of time progression starting at 25 min and ending at 245 min.
and O 2 , SIN-1-mediated oxidation of the fluorescence dye DHR 123 was studied in the presence of various levels of Cu,Zn-SOD. Cu,Zn-SOD inhibited the SIN-1mediated oxidation of DHR and the IC50 increased over time (Fig. 7). Unlike N-nitrosation, Cu,Zn-SOD did not stimulate SIN-1-mediated oxidation of DHR. Cu,Zn-SOD inhibited the release of NO from SIN-1
observable for the baseline. In this case, normalization may have resulted in an underestimation of the actual effect of GSH on the Cu,Zn-SOD-mediated response.
An HPLC method was used to follow SIN-1 as well as its degradation intermediate, SIN-1A, and its stable end product, SIN-1C, whose formation was indicative of NO release from SIN-1. Inclusion of 1000 U/ml Cu,ZnSOD significantly inhibited the formation of SIN-1C and thereby the release of NO (Fig. 8A). This inhibitory effect required functional Cu,Zn-SOD, as indicated from the heat-inactivation result (Fig. 8B). To construct a dose–response curve over the same concentration range of Cu,Zn-SOD studied in the previous experiments, the peak intensities of SIN-1, SIN-1A, and SIN-1C were measured after 4 h incubation of SIN-1. High [Cu,ZnSOD] preserved SIN-1 and inhibited the formation of SIN-1C (Fig. 8C). However, Cu,Zn-SOD had no effect on the degradation of SIN-1 and the formation of its degradation products at the low concentration range, during which Cu,Zn-SOD stimulated the N-nitrosation reaction.
The effect of Cu,Zn-SOD on SIN-1-mediated oxidation of DHR was monophasic
Discussion
To investigate whether the biphasic effect of SOD might be observed in other reactions mediated by NO
During chronic inflammation, activated macrophages release high levels of both NO and O 2 [2]. This present
Fig. 6. Effect of glutathione (GSH) on Cu,Zn-SOD modulation of DAN nitrosation. (A) SIN-1 ð100 lMÞ was added to PBS-based reaction buffer pH 7.4 containing 3:13 lM DAN, 0.1 mM DTPA, 120 U/ ml catalase, and GSH (0–10 mM), at 25 1 °C. The fluorescence intensity after the addition of SIN-1 was measured at 4 h and was plotted against [GSH]. (B) At three fixed GSH levels (0.2 mM, 1 mM, and 5 mM), DAN nitrosation was studied in the absence and the presence of 10, 33.3, 100, 333, and 1000 U/ml Cu,Zn-SOD. Fluorescence intensity at 4 h for each group was plotted against [Cu,Zn-SOD]. Values represent the means SD (n ¼ 4). (C) The data in B were expressed as percentage control. The dose–response curve at t ¼ 4 h for Cu,Zn-SOD in the absence of GSH was included for the purpose of comparison.
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Fig. 8. Effect of Cu,Zn-SOD on NO release kinetics of SIN-1 measured by HPLC. SIN-1 ð100 lMÞ was added to a pH 7.4 PBS buffer containing 0.1 mM DTPA and 120 U/ml catalase, at 25 1 °C. SIN-1 and its degradation products (SIN-1A and SIN-1C) were determined by HPLC. (A) SIN-1C formation in the absence and the presence of 1000 U/ml Cu,Zn-SOD. (B) SIN-1C formation at 4 h in the absence or presence of either intact Cu,Zn-SOD (1000 U/ml) or heat-inactivated (HI) Cu,Zn-SOD. (C) Cu,Zn-SOD (1–1000 U/ml) was included; the solutions were incubated for 4 h and the peak intensity of SIN-1 and its degradation products were determined. Mean data were presented as percentage control, n ¼ 3 SD.
study simulated such a scenario using the well-characterized NO and O 2 donor, SIN-1 [34,35]. Our results showed that all three isoforms of SOD (CuZn, Mn, Fe) modulate nitrosative chemistry in a dynamic fashion that is both dose- and time-dependent. Specifically, we
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determined that SOD’s effects on nitrosation are biphasic in nature, i.e., while lower concentrations of SOD were pronitrosative, higher SOD concentrations inhibited nitrosation. However, even those initially inhibitory, higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD’s pronitrosative effect became dominant, with complete loss of antinitrosative effects noted at higher levels of GSH. Previous studies have focused on SOD’s roles in ROS-mediated oxidative chemistry [10–17]. The results indicated that SOD elicits a bell-shaped dose–response relationship, in which high concentrations of SOD elicit prooxidative activities [10–12]. In contrast, SOD’s role in modulating nitrosative chemistry remains less characterized. While some studies have shown that SOD may directly interact with NO or NO-derived RNS [36– 38], most studies have centered on SOD’s O 2 -scavenging activity that results in an indirect modulation of nitrosative reactions [24,39]. A previous investigation has shown that SOD can enhance RNS-mediated nitrosation reactions [24]. The dose–response relationship followed a monotonous function line: increased [SOD], which resulted in a higher NO=O 2 ratio, consequently increased ½N2 O3 and enhanced nitrosation [24]. However, our results demonstrated a biphasic dose–response relationship for SOD modulation of N-nitrosation. While the current study employed SIN-1 to simultaneously generate NO and O 2 , the previous study used DEA/NO and xanthine/xanthine oxidase system to generate NO and O 2 , respectively. The generation of NO and O from SIN-1 was sustained for at least 8 h 2 (Fig. 8A), which allowed us to investigate the dynamics of SOD’s action on RNS-mediated N-nitrosation reaction. In contrast, the generation of NO from DEA/NO was near completion within 30 min, given that the rate constant is 0.3 min1 [24]. Since the previous study measured SOD’s effect on RNS-mediated nitrosation at a single time point where the generation of RNS has gone to completion, it represented a static measurement of SOD’s overall effect on the extent, but not the rate of RNS-mediated nitrosation. We should emphasize that the present study investigated SOD’s role in the modulation of nitrosation kinetics over an extended period, during which RNS and ROS are continuously generated and eliminated. Low levels of SOD enhanced both the rate and extent of nitrosation, which epitomized the pronitrosative characteristics of SOD. However, high levels of SOD dramatically reduced the nitrosation rate and completely distorted the curvature of the kinetic profiles. Although high [SOD] (P500 U/ml) initially either showed no effect (Mn- and Fe-SOD, Fig. 5) on nitrosation or exhibited reduced nitrosation (Cu,Zn-SOD, Fig. 3), the pronitrosative characteristics of SOD were revealed only after extended reaction time. Therefore, our data suggest that
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SOD is both pro- and antinitrosative depending on its concentrations and the duration of reaction. These findings are of particular biologic and therapeutic relevance. For example: (i) Sustained overexpression of SOD in tissues where NO and O 2 are also generated at high concentrations may result in pronitrosative conditions, which along with ROS-mediated oxidative stress may contribute to the pathogenesis of many diseases. (ii) Dynamic interactions among SOD, ROS, and RNS and other antioxidants at SOD’s critical sites of action may determine the overall therapeutic efficacy of targeted, long-lived SOD derivatives [40]. SIN-1 generates 1:1 ratio of NO and O 2 , which based on the previous results by Wink et al. [24] would result in maximal oxidation and minimal nitrosation. SOD can increase the NO=O 2 ratio to a value greater than 1. Under such a circumstance, an excess of NO can block ONOO -mediated oxidation action [24,41], which is attributable to the reaction between NO and ONOO [24,41] or between NO and the free radical intermediates ð NO2 ; OHÞ from the decomposition of ONOO [42]. Further, NO reacts with the free radical intermediate NO2 , which gives rise to the nitrosating agent N2 O3 [24,41,42]. Accordingly, the previous studies suggested that SOD is fundamentally pronitrosative. Our findings, which suggested that high concentrations of SOD are antinitrosative, therefore, provide a novel insight into SOD’s actions in the NO=O 2 system. We propose a schematic model (Fig. 9) to explain SOD’s pronitrosative properties. First, SOD can function as a RNS sink. Since NO can interact with copper ions in Cu,Zn-SOD [36], this interaction may limit the formation of the nitrosating species N2 O3 . Moreover, SOD itself is likely a target for N2 O3 -mediated nitrosation, given that protein
Fig. 9. Proposed mechanisms for biphasic SOD modulation of DAN nitrosation.
nitrosation can be autocatalyzed within the hydrophobic cores of protein [43]. As SOD concentrations increased, SOD may compete with DAN for reaction with N2 O3 , thereby resulting in a reduction of DAN nitrosation. High levels of GSH would mask this competition, since GSH has high affinity towards N2 O3 [5]. Our data, which showed that GSH effectively quenched DAN nitrosation and intensified SOD’s pronitrosative activities, support this premise. Another mechanism for SOD to limit the formation of N2 O3 is the reaction between SOD and ONOO [38,44], which may outcompete the NO=ONOO reactions at high concentrations of SOD. Second, SOD’s ability to inhibit NO release could further reduce the generation of nitrosating species. However, our data suggested that this pathway has limited contribution to the antinitrosative activities of SOD. Overall, the net effect of SOD on nitrosation is determined by the counterbalance of SOD’s abilities to both promote and quench reactive species. Many human diseases are attributable to the deleterious consequences of oxidative and nitrosative stress, e.g., ischemia/reperfusion injury and rheumatoid arthritis [1,2]. Consequently, reactive species scavengers and antioxidants have been advocated as therapeutic agents for these conditions. Intuitively, high doses of antioxidant enzymes such as SOD may initially appear beneficial in the management of diseases associated with reactive species stress. However, our results indicate that similar to many other therapeutic agents, SOD’s protective effects lie within a restricted therapeutic range. The results of our study demonstrate the importance of considering both dose and duration of treatment when contemplating use of SOD in a therapeutic setting. By its abilities to directly interact with both ROS and RNS, SOD functions as a key regulatory molecule in reactive species chemistry. Furthermore, SOD’s effects extend beyond cytoprotection to include modulation of intracellular signaling, and antimicrobial and antitumorigenic functions. In conclusion, our study demonstrates that all three SOD isoforms (Cu,Zn-, Mn-, and Fe-SOD) exhibit a biphasic effect on modulation of N-nitrosation. We describe, for the first time, the dynamics of SOD’s actions in a sustained NO=O 2 system. Our data show that, while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations may become pronitrosative over time. In the presence of physiologically relevant concentrations of GSH, SOD predominantly exhibits a pronitrosative effect. Our findings likely reflect the complex and dynamic nature of SOD interactions with ROS and RNS. This study provided insights into the unsettled nature of NO and O 2 in the cellular milieu, where the balance of various reactants and scavengers may affect NO-mediated nitrosation in a nonlinear fashion.
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Acknowledgments This work was supported by NIH/NIDCR P01 DE 12704, P30CA16058. Teh-Min Hu was supported by a scholarship from National Defense Medical Center, Taipei, Taiwan.
[20]
[21]
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Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases Teh-Min Hu,a William L. Hayton,a Mark A. Morse,b and Susan R. Malleryc,* a Division of Pharmaceutics, College of Pharmacy, Columbus, OH 43210-1241, USA Department of Environmental Health Science, School of Public Health, Columbus, OH 43210-1241, USA Department of Oral and Maxillofacial Surgery and Pathology, College of Dentistry, The Ohio State University, Columbus, OH 43210-1241, USA b
c
Received 24 June 2002
Abstract It has been shown that superoxide dismutase (SOD) can both potentiate and attenuate NO-mediated toxicity. This present study investigated the role of SOD and GSH in a sustained nitrosative and oxidative environment simulated by the nitric oxide (NO) and superoxide ðO 2 Þ donor, 3-morpholinosydnonimine (SIN-1). We describe, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results show that SOD’s effects on nitrosation are biphasic in nature i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD predominantly exhibits a pronitrosative effect, with a complete loss of antinitrosative effects noted at higher levels of GSH. Our findings likely reflect the complex and dynamic nature of SOD interactions with oxidative and nitrosative species. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Superoxide dismutase; Nitric oxide; SIN-1; Nitrosation; Kinetics; Biphasic; Dynamics
The free radicals superoxide anion ðO 2 Þ and nitric oxide (NO) play important roles in numerous physiological and pathological processes. Because of their free radical characteristics, both O 2 and NO can be converted to various other reactive species, which can be further subcategorized into reactive oxygen species (ROS) and reactive nitrogen species (RNS) [1]. Under normal physiological conditions, NO, O 2 and their reactive species derivatives are maintained at the state of ‘‘redox homeostasis’’ [1], at which reactive species production is balanced by various types of scavengers such as superoxide dismutase (SOD) and reduced glutathione (GSH) [1]. Under such circumstances, ROS and RNS function as regulatory mediators of a broad spectrum of physiological responses [1]. However, in the scenarios where the production of ROS and/or RNS is persistently high (e.g., sites of sustained inflammation) [2] or where mutations of SOD (e.g., in neurodegenerative diseases
*
Corresponding author. Fax: 614-292-9384. E-mail address: [email protected] (S.R. Mallery).
[1,3,4]) occur, oxidative or nitrosative damage could be inflicted by excessive amounts of either ROS or RNS upon DNA, proteins, lipids, and other cellular components [5–8]. Previous studies have demonstrated that SOD’s concentration is critical in determining whether SOD is antioxidative or prooxidative [9]. When exogenous SOD was used to alleviate the oxidative stress in the ischemia-reperfusion models, bell-shaped dose–response relationships were observed [10–12]. While low concentrations (<5 lg=ml) of SOD reduced lipid peroxidation and were cardioprotective, high concentrations (>50 lg=ml) increased lipid peroxidation and can exacerbate acute cardiac injury [12]. Therefore, the results of previous investigations suggested that optimal concentrations existed for SOD when protecting biological systems from oxidative damages [9]. Several mechanisms have been proposed to account for the adverse effect of high [SOD], which include overproduction of hydrogen peroxide resulting from the SOD-catalyzed dismutation of O 2 [13,14], SOD’s peroxidase activity [15], and dual roles played by
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 8 2 0 - 3
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superoxide in both initiating and terminating radical chain reactions [12]. Recently, a study by Offer et al. [16] showed that low concentrations (<20 U/ml) of Cu,ZnSOD inhibited O 2 -induced ferrocyanide oxidation, while its antioxidative effect was lost at high [Cu,ZnSOD]. It was concluded that, in the presence of ultralow steady-state O 2 levels, the reaction kinetics of Cu,ZnSOD is altered; i.e., the oxidized form of Cu,Zn-SOD not only reacts with O 2 but also oxidizes the target molecule that it was supposed to protect [16]. Later, Liochev and Fridovich [17] demonstrated that Cu,ZnSOD functions both as a superoxide reductase and a superoxide oxidase. The findings of Offer et al. [16] were consequently attributed to the increase in SOR activity that accompanies increased concentrations of Cu,Zn-SOD. Cumulatively, these previous investigations suggested that, in addition to its superoxide dismutation activity, SOD also exhibits other enzymatic functions. However, all additional activities of SOD are somewhat related to the redox properties of SOD and have direct or indirect involvement of O 2 . High levels of O2 and NO are concurrently generated in many pathophysiological processes [2]. Further, the pathways of the generation and elimination of both ROS and RNS are closely related [5]. Therefore, it is of biological and pharmacological relevance to investigate how SOD’s disparate activities modulate RNS-mediated nitrosative and oxidative chemistry in the context of sustained O 2 and NO production. It has been shown that SOD can both potentiate [18–20] and attenuate [21–23] NO-mediated cytotoxicity. The opposing findings again reflect the complexity of SOD’s biological actions. From the kinetics standpoint, SOD could impinge on the chemical balance between ROS- and RNS-associated reaction pathways [5]. While previous studies by Wink et al. [24] importantly demonstrated the chemical aspects of SOD/RNS/ROS interactions at a static time point, little is known about the dynamics of SOD’s overall effect on the RNS-mediated nitrosative and oxidative chemistry. The objective of our study was to investigate the effect of SOD over time on RNS-mediated nitrosation kinetics. Nitrosation, i.e., the addition of NOþ to a nucleophile [25], is one of the major NO-mediated reactions in biological systems. Nitrosation products of albumin, hemoglobin, and glutathione (GSH) could act as reservoirs for NO storage or transportation in plasma or in cells [26]. Recently, protein nitrosation has been proposed to be a prototypic redox-based signaling mechanism [27,28]. Moreover, nitrosation may cause damage to DNA bases [29] and enzymes [30–33]. S-nitrosation was shown to inactivate enzymes that contain catalytically essential sulfhydryl groups, such as glutathione peroxidase [30], glutathione reductase [31], and glyceraldehyde3-phosphate dehydrogenase [32,33]. Consequently,
SOD’s ability to suppress or enhance nitrosation is of physiological and pathological significance. This present study investigated the role of SOD in a sustained nitrosative and oxidative environment simulated by an NO and O 2 donor. Our paper describes, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results showed that SOD’s effects on nitrosation are biphasic in nature, i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation.
Materials and methods Materials. SIN-1 was purchased from Calbiochem (La Jolla, CA). Cu,Zn-SOD, Mn-SOD, Fe-SOD, catalase, diaminonaphthalene (DAN), dihydrorhodamine 123 (DHR), glutathione (GSH), diethyltriaminepentaacetic acid (DTPA), and all other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). The chemical structures of SIN-1, DAN, and DHR are shown in Fig. 1. Kinetics of DAN nitrosation. The nitrosation kinetics of DAN was studied using a fluorescence spectrometer (Perkin–Elmer LS 50B, Norwalk, CT). Reactions were performed in a 96-well microtiter plate format at 25 1 °C in the sample chamber. The fluorescence intensity of nitrosated DAN was measured at excitation and emission wavelengths of 375 and 430 nm, respectively. A stock solution of DAN (30 mM) was freshly prepared in dimethyl sulfoxide (DMSO), protected from light exposure, and stored at 4 °C. The dynamic range of DAN in the nitrosation kinetics was studied by varying the concentration of DAN (0.78–300 lM) in 200 ll of phosphate-buffered saline (pH 7.4) that contained 0.1 mM DTPA and 120 U/ml catalase to eliminate hydrogen peroxide. Reactions were initiated by adding an aliquot of 4 ll SIN-1 stock solution (5 mM in DMSO) into 200 ll DAN-containing reaction buffers. The fluorescence intensity after the addition of SIN-1 was measured at 10-min intervals. The nitrosation of DAN in the presence of SOD (0–1000 U/ml) and SIN-1 ð100 lMÞ was
Fig. 1. The chemical structures of 3-morpholinosydnonimine (SIN-1), 2,3-diaminonaphthalene (DAN), and dihydrorhodamine 123 (DHR).
T.-M. Hu et al. / Biochemical and Biophysical Research Communications 295 (2002) 1125–1134 studied in the same pH 7.4 reaction buffer at 25 1 °C by measuring the increase in fluorescence every 10 min up to 8 h. Oxidation of DHR by SIN-1. The oxidation of DHR was determined at 25 1 °C in the above-mentioned reaction buffer except that DAN was replaced by DHR (10 mM stock in dimethylformamide stored at 20 °C and protected from light). The fluorescence of rhodamine 123 generated from the oxidation reaction was measured at excitation and emission wavelengths of 500 and 530 nm, respectively. The effect of SOD (0–1000 U/ml) on SIN-1-mediated DHR oxidation was studied in 200-ll DHR-containing reaction buffer, where the DHR concentration ( 0:39 lM in the final reaction mixture) was chosen based on a pilot experiment in which the dynamic range of DHR was determined. The oxidation kinetics of DHR was typically monitored every 5-min up to 4 h. HPLC determination of SIN-1 degradation kinetics. An HPLC method [34] was modified to study the effect of SOD on the degradation kinetics of SIN-1. The method consisted of a reversed phase column (Waters Nova-Pak C18 3:9 300 mm), a mobile phase (10 mM sodium acetate buffer (pH 3.1)/acetonitrile/methanol ¼ 92/6.4/1.6, flow rate ¼ 1 ml/min), and UV detection at 254 nm. SIN-1 was separated from its two degradation products, SIN-1A and SIN-1C, with retention times of 4.0, 9.0, and 9.8 min for SIN-1, SIN-1A, and SIN-1C, respectively. The kinetics of the SIN-1 degradation at 25 °C was studied by serial sampling (0, 30, 60, 90, 120, 180, 240, 300, 360, and 480 min), with an initial [SIN-1] of 100 lM, with and without 1000 U/ ml Cu,Zn-SOD in the same reaction buffer as described in the nitrosation kinetics section. The dose–response relationship of Cu,Zn-SOD for the degradation of SIN-1 was further investigated by determining the relative quantity (expressed as percentage control) of SIN-1 and its degradation products after 4 h incubation in reaction buffer containing 100 lM SIN-1 and various concentrations of Cu,Zn-SOD (1000, 250, 62.5, 15.6, 3.13, and 0.78 U/ml). Method validation. The concentrations of DAN and DHR used in the study were determined in pilot studies to exclude the possibility of fluorescence quenching. In addition, nitrogen oxide species such as NO 2 , NO3 , and ONOO , which were likely generated during the reaction, were evaluated and shown not to nitrosate DAN. SOD was also shown not to affect the fluorescence of DAN and DHR. Nevertheless, suitable controls (e.g., SOD/DAN for SIN-1-mediated nitrosation kinetics, SOD/DHR for oxidation kinetics) were included in each experimental run and the fluorescence reported in this paper was after subtracting out the background fluorescence of the controls.
Results Kinetics of DAN nitrosation The kinetic profile of SIN-1-mediated DAN nitrosation, Fig. 2, was characterized by the formation of fluorescence at 430 nm. The nitrosation for the control (curve 1) peaked at 180 min after the addition of SIN-1 ð100 lMÞ. After the maximum, the fluorescence intensity tended to decline gradually. With increasing concentrations of Cu,Zn-SOD up to 15.6 U/ml, the rate and extent of DAN nitrosation increased disproportionately, curves 2–6 of Fig. 2A. In contrast, [Cu,Zn-SOD] above 15.6 U/ml increasingly attenuated DAN nitrosation, curves 7–11 of Fig. 2B. The kinetics of fluorescence formation was dramatically affected by the presence of the highest [Cu,Zn-SOD], at which the fluorescence intensity accumulated in a slow and sustained fashion (Fig. 2B, curve 11).
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Fig. 2. Nitrosation kinetics of DAN in the absence and presence of different levels of Cu,Zn-SOD.The reactions were performed in a 96well microtiter plate format. SIN-1 ð100 lMÞ was added to PBS-based reaction buffer containing 3:13 lM DAN, 0.1 mM DTPA, 120 U/ml catalase, and Cu,Zn-SOD (0–1000 U/ml), pH 7.4, at 25 1 °C. The fluorescence intensity after the addition of SIN-1 was measured at 10min intervals by fluorescence spectroscopy. Data represent the means of three measurements. Each curve represents a different [Cu,ZnSOD]: 1, control; 2, 0.98 U/ml; 3, 1.95 U/ml; 4, 3.9 U/ml; 5, 7.8 U/ml; 6, 15.6 U/ml; 7, 31.2 U/ml; 8, 62. 5 U/ml; 9, 125 U/ml; 10, 500 U/ml; 11, 1000 U/ml.
SOD modulated SIN-1-mediated DAN nitrosation in a biphasic fashion To further characterize the interaction between SOD and DAN nitrosation, a dose–response relationship was constructed in accordance with the relative degree of nitrosation compared with the control versus [Cu,ZnSOD] at different measurement times. Remarkably, multiple biphasic dose–response curves characterized by a dynamic transition among those curves emerged (Fig. 3A). These data enabled a 3-D representation of the dynamic dose–response relationship (Fig. 3B). The maximum stimulatory effect of SOD occurred initially at ½Cu; Zn-SOD < 10 U/ml, which corresponded to a
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bly, high [Cu,Zn-SOD] reduced N-nitrosation to the extent that DAN nitrosation levels were generally below control levels. Heat inactivated (>90 °C, 2 h) Cu,Zn-SOD lost both its stimulatory and inhibitory effects on N-nitrosation (data not shown). Free metal ions such as Cuþ , Cuþþ , and Znþþ also had no effect on N-nitrosation. Bovine serum albumin (BSA) was used to test whether a nonspecific protein effect might have been involved in the reaction. While BSA had no effect on nitrosation at low concentrations, it inhibited the reaction to some extent at concentrations >1 lM (data not shown). DAN concentrations affected SOD modulation of nitrosation kinetics
Fig. 3. Time- and concentration-dependent, biphasic, dose–response relationship for the effect of Cu,Zn-SOD on DAN nitrosation. (A) The fluorescence intensities at the same time point in Fig. 2 were expressed as percentage control and plotted against [Cu,Zn-SOD]. Each curve (n ¼ 3) represents the dose–response relationship at 20-min intervals. The arrows indicate the direction of time progression starting at 45 min and ending at 485 min. (B) Saddle-like dose–response surface was generated by plotting the relative fluorescence intensities against [Cu,Zn-SOD] and the reaction time.
hump in this dose region at early incubation time. The size of the hump in this region diminished and then disappeared at 180 min (Fig. 3B). After this mark, the dose–response curve in the low Cu,Zn-SOD concentration region changed insignificantly along time and became the ascending part of a new emerging hump whose size grew continuously and the concentration of Cu,ZnSOD corresponding to the hump also tended to increase. In contrast with the low concentration region, the dose– response relationship in the high concentration region (30–1000 U/ml) appeared to be steady in the early period while changing substantially in the later period. Nota-
To investigate whether the substrate concentration affected the observed dose–response phenomena, the concentration of DAN was halved or doubled while keeping other experimental conditions unchanged. Fig. 4 depicts the sharp contrast between two experiments that used fourfold differences of [DAN]. While the time-dependent, bell-shaped, dose–response relationship remained in both cases, there was a fundamental difference in the way in which N-nitrosation was affected by the presence of Cu,Zn-SOD. At ½DAN ¼ 1:56 lM, Fig. 4A, the hump in the higher SOD region dominated that in the lower region (600% at [Cu,ZnSOD] 30 U/ml at 480 min versus 450% at [Cu,ZnSOD] 3 U/ml at 45 min). In addition, the dynamic transition between the two regions in Fig. 4A occurred much earlier than that in Fig. 3. However, the result was somewhat reversed when [DAN] was increased by fourfold (Fig. 4B). The effect of Cu,Zn-SOD peaked at the initial period and then dropped more slowly until it achieved a second phase in which the maximum effect was shifted to the right (Fig. 4B). Moreover, the dose– response relationship at [Cu,Zn-SOD] >100 U/ml was less perturbed during the reaction period (Fig. 4B). To account for this observation, the kinetic profiles for the three controls with different levels of DAN were compared (Fig. 4B). The time to attain maximum nitrosation appeared to be increased from 2 h for the control with the lowest [DAN] to 3 h and 5 h for the control with two- and fourfold higher [DAN], respectively. The extent of nitrosation increased almost linearly. All SOD isoforms demonstrated biphasic reaction kinetics Also examined was whether Mn- and Fe-SOD elicited a biphasic effect similar to that of Cu,Zn-SOD. A similar dose–response relationship occurred for these SOD isoforms as well (Fig. 5), although a smaller inhibitory effect was observed for both high [Mn-SOD] and [Fe-SOD]; the maximal effective concentration at t ¼ 480 min was increased from 31.3 U/ml for Cu,Zn-
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Fig. 5. Time- and concentration-dependent, biphasic, dose–response relationships for the effects of Mn- and Fe-SOD. Procedures were described in Figs. 2 and 3, except (A) Mn-SOD and (B) Fe-SOD were studied. Reactions were initiated by adding SIN-1 ð100 lMÞ to PBSbased reaction buffer containing 3:13 lM DAN, 0.1 mM DTPA, 120 U/ml catalase, and various activities of SOD (0–1000 U/ml). Values represent the means of three measurements.
Fig. 4. Effect of DAN concentration on SOD modulation of nitrosation kinetics. Procedures were as described in Fig. 2, except (A) 1=2 ½DAN or (B) 2 ½DAN was used. Mean dose–response curves (n ¼ 4) were obtained by plotting the relative fluorescence intensities (percentage control) against [Cu,Zn-SOD]. The time interval for each curve is 20 min. The arrows indicate the direction of time progression starting at 45 min and ending at 485 min. (C) The kinetic profiles for the three controls with different levels of DAN.
SOD (Fig. 3A) to 62.5 U/ml for both Mn- and Fe-SOD (Figs. 5A and B). Glutathione modulated the biphasic effect of Cu,Zn-SOD in a concentration-dependent fashion Glutathione (GSH), a reduced thiol and an antioxidant, is an NO sink that modulates cellular redox re-
actions. It was therefore postulated that GSH would modify the effect of SOD on the N-nitrosation reaction mediated by SIN-1-derived reactive nitrogen species. GSH at or above 0.5 mM inhibited SIN-1-mediated Nnitrosation by >90% (Fig. 6A). Accordingly, the effect of Cu,Zn-SOD was examined in the presence of 0.2, 1, and 5 mM GSH, which represented low, intermediate, and high physiologically relevant levels. While the biphasic effect of Cu,Zn-SOD was retained at 0.2 mM GSH, it disappeared with higher [GSH] (Fig. 6B). When the data were expressed as percentage control and compared with the dose–response curve obtained from the experiment in which GSH was absent, it was remarkable that the effect of Cu,Zn-SOD was enhanced by the presence of increasing [GSH] (up to 1 mM), which was reflected in the upward shift of the entire dose–response curve (Fig. 6C). However, the whole dose–response curve of Cu,Zn-SOD fell dramatically at 5 mM GSH (Fig. 6C). This result may be explained by the fact that the maximum inhibitory effect of GSH on DAN nitrosation was achieved at ½GSH 1 mM (Fig. 6A). Above this level, the detection limit might be approached; therefore, no further inhibition was
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Fig. 7. Effect of Cu,Zn-SOD on SIN-1-mediated oxidation of DHR. SIN-1 ð100 lMÞ was added to pH 7.4 PBS buffer containing 0:39 lM DHR, 0.1 mM DTPA, 120 U/ml catalase, and Cu,Zn-SOD (0–1000 U/ ml), at 25 1 °C. The fluorescence intensity after the addition of SIN-1 was measured at a 5-min interval; fluorescence was expressed as percentage control ðn ¼ 4 SDÞ and plotted at a 20-min interval against [Cu,Zn-SOD]. The arrows indicate the direction of time progression starting at 25 min and ending at 245 min.
and O 2 , SIN-1-mediated oxidation of the fluorescence dye DHR 123 was studied in the presence of various levels of Cu,Zn-SOD. Cu,Zn-SOD inhibited the SIN-1mediated oxidation of DHR and the IC50 increased over time (Fig. 7). Unlike N-nitrosation, Cu,Zn-SOD did not stimulate SIN-1-mediated oxidation of DHR. Cu,Zn-SOD inhibited the release of NO from SIN-1
observable for the baseline. In this case, normalization may have resulted in an underestimation of the actual effect of GSH on the Cu,Zn-SOD-mediated response.
An HPLC method was used to follow SIN-1 as well as its degradation intermediate, SIN-1A, and its stable end product, SIN-1C, whose formation was indicative of NO release from SIN-1. Inclusion of 1000 U/ml Cu,ZnSOD significantly inhibited the formation of SIN-1C and thereby the release of NO (Fig. 8A). This inhibitory effect required functional Cu,Zn-SOD, as indicated from the heat-inactivation result (Fig. 8B). To construct a dose–response curve over the same concentration range of Cu,Zn-SOD studied in the previous experiments, the peak intensities of SIN-1, SIN-1A, and SIN-1C were measured after 4 h incubation of SIN-1. High [Cu,ZnSOD] preserved SIN-1 and inhibited the formation of SIN-1C (Fig. 8C). However, Cu,Zn-SOD had no effect on the degradation of SIN-1 and the formation of its degradation products at the low concentration range, during which Cu,Zn-SOD stimulated the N-nitrosation reaction.
The effect of Cu,Zn-SOD on SIN-1-mediated oxidation of DHR was monophasic
Discussion
To investigate whether the biphasic effect of SOD might be observed in other reactions mediated by NO
During chronic inflammation, activated macrophages release high levels of both NO and O 2 [2]. This present
Fig. 6. Effect of glutathione (GSH) on Cu,Zn-SOD modulation of DAN nitrosation. (A) SIN-1 ð100 lMÞ was added to PBS-based reaction buffer pH 7.4 containing 3:13 lM DAN, 0.1 mM DTPA, 120 U/ ml catalase, and GSH (0–10 mM), at 25 1 °C. The fluorescence intensity after the addition of SIN-1 was measured at 4 h and was plotted against [GSH]. (B) At three fixed GSH levels (0.2 mM, 1 mM, and 5 mM), DAN nitrosation was studied in the absence and the presence of 10, 33.3, 100, 333, and 1000 U/ml Cu,Zn-SOD. Fluorescence intensity at 4 h for each group was plotted against [Cu,Zn-SOD]. Values represent the means SD (n ¼ 4). (C) The data in B were expressed as percentage control. The dose–response curve at t ¼ 4 h for Cu,Zn-SOD in the absence of GSH was included for the purpose of comparison.
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Fig. 8. Effect of Cu,Zn-SOD on NO release kinetics of SIN-1 measured by HPLC. SIN-1 ð100 lMÞ was added to a pH 7.4 PBS buffer containing 0.1 mM DTPA and 120 U/ml catalase, at 25 1 °C. SIN-1 and its degradation products (SIN-1A and SIN-1C) were determined by HPLC. (A) SIN-1C formation in the absence and the presence of 1000 U/ml Cu,Zn-SOD. (B) SIN-1C formation at 4 h in the absence or presence of either intact Cu,Zn-SOD (1000 U/ml) or heat-inactivated (HI) Cu,Zn-SOD. (C) Cu,Zn-SOD (1–1000 U/ml) was included; the solutions were incubated for 4 h and the peak intensity of SIN-1 and its degradation products were determined. Mean data were presented as percentage control, n ¼ 3 SD.
study simulated such a scenario using the well-characterized NO and O 2 donor, SIN-1 [34,35]. Our results showed that all three isoforms of SOD (CuZn, Mn, Fe) modulate nitrosative chemistry in a dynamic fashion that is both dose- and time-dependent. Specifically, we
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determined that SOD’s effects on nitrosation are biphasic in nature, i.e., while lower concentrations of SOD were pronitrosative, higher SOD concentrations inhibited nitrosation. However, even those initially inhibitory, higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD’s pronitrosative effect became dominant, with complete loss of antinitrosative effects noted at higher levels of GSH. Previous studies have focused on SOD’s roles in ROS-mediated oxidative chemistry [10–17]. The results indicated that SOD elicits a bell-shaped dose–response relationship, in which high concentrations of SOD elicit prooxidative activities [10–12]. In contrast, SOD’s role in modulating nitrosative chemistry remains less characterized. While some studies have shown that SOD may directly interact with NO or NO-derived RNS [36– 38], most studies have centered on SOD’s O 2 -scavenging activity that results in an indirect modulation of nitrosative reactions [24,39]. A previous investigation has shown that SOD can enhance RNS-mediated nitrosation reactions [24]. The dose–response relationship followed a monotonous function line: increased [SOD], which resulted in a higher NO=O 2 ratio, consequently increased ½N2 O3 and enhanced nitrosation [24]. However, our results demonstrated a biphasic dose–response relationship for SOD modulation of N-nitrosation. While the current study employed SIN-1 to simultaneously generate NO and O 2 , the previous study used DEA/NO and xanthine/xanthine oxidase system to generate NO and O 2 , respectively. The generation of NO and O from SIN-1 was sustained for at least 8 h 2 (Fig. 8A), which allowed us to investigate the dynamics of SOD’s action on RNS-mediated N-nitrosation reaction. In contrast, the generation of NO from DEA/NO was near completion within 30 min, given that the rate constant is 0.3 min1 [24]. Since the previous study measured SOD’s effect on RNS-mediated nitrosation at a single time point where the generation of RNS has gone to completion, it represented a static measurement of SOD’s overall effect on the extent, but not the rate of RNS-mediated nitrosation. We should emphasize that the present study investigated SOD’s role in the modulation of nitrosation kinetics over an extended period, during which RNS and ROS are continuously generated and eliminated. Low levels of SOD enhanced both the rate and extent of nitrosation, which epitomized the pronitrosative characteristics of SOD. However, high levels of SOD dramatically reduced the nitrosation rate and completely distorted the curvature of the kinetic profiles. Although high [SOD] (P500 U/ml) initially either showed no effect (Mn- and Fe-SOD, Fig. 5) on nitrosation or exhibited reduced nitrosation (Cu,Zn-SOD, Fig. 3), the pronitrosative characteristics of SOD were revealed only after extended reaction time. Therefore, our data suggest that
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SOD is both pro- and antinitrosative depending on its concentrations and the duration of reaction. These findings are of particular biologic and therapeutic relevance. For example: (i) Sustained overexpression of SOD in tissues where NO and O 2 are also generated at high concentrations may result in pronitrosative conditions, which along with ROS-mediated oxidative stress may contribute to the pathogenesis of many diseases. (ii) Dynamic interactions among SOD, ROS, and RNS and other antioxidants at SOD’s critical sites of action may determine the overall therapeutic efficacy of targeted, long-lived SOD derivatives [40]. SIN-1 generates 1:1 ratio of NO and O 2 , which based on the previous results by Wink et al. [24] would result in maximal oxidation and minimal nitrosation. SOD can increase the NO=O 2 ratio to a value greater than 1. Under such a circumstance, an excess of NO can block ONOO -mediated oxidation action [24,41], which is attributable to the reaction between NO and ONOO [24,41] or between NO and the free radical intermediates ð NO2 ; OHÞ from the decomposition of ONOO [42]. Further, NO reacts with the free radical intermediate NO2 , which gives rise to the nitrosating agent N2 O3 [24,41,42]. Accordingly, the previous studies suggested that SOD is fundamentally pronitrosative. Our findings, which suggested that high concentrations of SOD are antinitrosative, therefore, provide a novel insight into SOD’s actions in the NO=O 2 system. We propose a schematic model (Fig. 9) to explain SOD’s pronitrosative properties. First, SOD can function as a RNS sink. Since NO can interact with copper ions in Cu,Zn-SOD [36], this interaction may limit the formation of the nitrosating species N2 O3 . Moreover, SOD itself is likely a target for N2 O3 -mediated nitrosation, given that protein
Fig. 9. Proposed mechanisms for biphasic SOD modulation of DAN nitrosation.
nitrosation can be autocatalyzed within the hydrophobic cores of protein [43]. As SOD concentrations increased, SOD may compete with DAN for reaction with N2 O3 , thereby resulting in a reduction of DAN nitrosation. High levels of GSH would mask this competition, since GSH has high affinity towards N2 O3 [5]. Our data, which showed that GSH effectively quenched DAN nitrosation and intensified SOD’s pronitrosative activities, support this premise. Another mechanism for SOD to limit the formation of N2 O3 is the reaction between SOD and ONOO [38,44], which may outcompete the NO=ONOO reactions at high concentrations of SOD. Second, SOD’s ability to inhibit NO release could further reduce the generation of nitrosating species. However, our data suggested that this pathway has limited contribution to the antinitrosative activities of SOD. Overall, the net effect of SOD on nitrosation is determined by the counterbalance of SOD’s abilities to both promote and quench reactive species. Many human diseases are attributable to the deleterious consequences of oxidative and nitrosative stress, e.g., ischemia/reperfusion injury and rheumatoid arthritis [1,2]. Consequently, reactive species scavengers and antioxidants have been advocated as therapeutic agents for these conditions. Intuitively, high doses of antioxidant enzymes such as SOD may initially appear beneficial in the management of diseases associated with reactive species stress. However, our results indicate that similar to many other therapeutic agents, SOD’s protective effects lie within a restricted therapeutic range. The results of our study demonstrate the importance of considering both dose and duration of treatment when contemplating use of SOD in a therapeutic setting. By its abilities to directly interact with both ROS and RNS, SOD functions as a key regulatory molecule in reactive species chemistry. Furthermore, SOD’s effects extend beyond cytoprotection to include modulation of intracellular signaling, and antimicrobial and antitumorigenic functions. In conclusion, our study demonstrates that all three SOD isoforms (Cu,Zn-, Mn-, and Fe-SOD) exhibit a biphasic effect on modulation of N-nitrosation. We describe, for the first time, the dynamics of SOD’s actions in a sustained NO=O 2 system. Our data show that, while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations may become pronitrosative over time. In the presence of physiologically relevant concentrations of GSH, SOD predominantly exhibits a pronitrosative effect. Our findings likely reflect the complex and dynamic nature of SOD interactions with ROS and RNS. This study provided insights into the unsettled nature of NO and O 2 in the cellular milieu, where the balance of various reactants and scavengers may affect NO-mediated nitrosation in a nonlinear fashion.
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Acknowledgments This work was supported by NIH/NIDCR P01 DE 12704, P30CA16058. Teh-Min Hu was supported by a scholarship from National Defense Medical Center, Taipei, Taiwan.
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