Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process

Free Radical Biology & Medicine 38 (2005) 1361 – 1371 www.elsevier.com/locate/freeradbiomed

Original Contribution

Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process Lisa A. Ridnoura,b,T, Julia E. Sima, Jinah Choic, Dale A. Dickinsond, Henry J. Formanc, Iman M. Ahmade, Mitchell C. Colemane, Clayton R. Hunta, Prahbat C. Goswamia,e, Douglas R. Spitza,e,T a

Division of Radiation and Cancer Biology, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park Boulevard, Room 411, St. Louis, MO 63108, USA b Radiation Biology Branch, National Cancer Institute, Building 10 B3B69, 9000 Rockville Pike, Bethesda, MD 20892, USA c School of Natural Science, University of California at Merced, P.O. Box 2039, Merced, CA 95344, USA d Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 1665 University Boulevard, Ryals 31, Birmingham, AL 35294-0022, USA e Free Radical and Radiation Biology Program, Department of Radiation Oncology, Holden Comprehensive Cancer Center, B180 Medical Labs, University of Iowa, Iowa City, IA 52242, USA Received 18 August 2004; revised 24 January 2005; accepted 28 January 2005 Available online 23 February 2005

Abstract Nitric oxide (SNO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an SNO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after SNO exposure. Altered thiol metabolism was observed immediately after SNO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, g-glutamylcysteine, and cysteine), which then reaccumulated during the SNO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to SNO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in SNO-exposed cells increased approximately twofold 24 h after H2O2 challenge. These results demonstrate that SNO exposure is capable of inducing an adaptive response to H2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity. D 2005 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Stress response; Hydrogen peroxide; Glutamate cysteine ligase; Glutathione; Thiols; Free radicals

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Abbreviations: NO, nitric oxide; GGC, g-glutamylcysteine; CYS, cysteine; GCL, glutamate cysteine ligase; RNS, reactive nitrogen species; ROS, reactive oxygen species; ONOO
, peroxynitrite; SNAP, S-nitroso-Nacetyl-l-penicillamine; O2
, superoxide; BSO, buthionine-l-sulfoximine; CHX, cycloheximide; DMF, dose-modifying factor; NEDD, N-(1-naphthyl) ethylenediamine dihydrochloride. T Corresponding authors. Fax: (301) 480 2238. E-mail address: [email protected] (L.A. Ridnour).

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0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.01.023

Nitric oxide ( NO) is a free radical generated by the oxidation of l-arginine to l-citrulline via the nitric oxide S synthase enzyme [1]. Physiologically NO can be either beneficial or deleterious depending upon the concentration at which it is formed and the length of time of exposure. In S endothelial cells and neurons, NO is generated in relatively low concentrations over shorter periods of time and functions as an effector molecule regulating vascular tone and neuronal excitation processes, respectively [2–4].

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Conversely, cytotoxicity caused by exposure to increased S concentrations of NO spanning a longer duration has been observed in a number of other systems, most notably cytokine-stimulated macrophages [5]. Nitric oxide and S reactive nitrogen species (RNS) derived from NO, such
as ONOO and NO2, have also been implicated as mediators of lung injury caused by exposure to cigarette smoke and air pollutants [6–8]. In addition, RNS have been suggested to play a role in tumor cell killing as well as cytotoxic processes that are known to occur during diabetogenesis, reperfusion injury, neurotoxicity, autoimmune disease, and inflammation [5,9–12]. The complexity of S these diverse affects may be explained by the fact that NO is known to rapidly interact with molecular oxygen and ROS, such as superoxide, to form a number of RNS, including ONOO
, NO2, N2O3, and other NOx species [13,14]. In addition, RNS can act as oxidizing agents as well as nitrosating, nitrating, and nitrosylating agents and are capable of eliciting a range of biological affects that relate to cellular stress [15–17]. Response to cellular stress is defined as the ability of a cell to modulate protective biochemical pathways after exposure to a slightly toxic insult (i.e., heat shock, radiation, and oxidative stress) that results in the preconditioning of the system and its ability to adapt to and survive a subsequent more toxic challenge. Toward this end, the ability of both prokaryotic and eukaryotic systems to induce S adaptive stress responses after exposure to H2O2, O2
, oxygen toxicity, and drugs capable of acting as oxidizing agents has been well documented [18–22]. However, S considerably less is known of the NO-induced stress S response or the ability of NO to induce resistance to subsequent, more toxic oxidant challenge. Nunoshiba et al. S have demonstrated the ability of NO to activate the SoxRS regulon, which is associated with the superoxide-induced stress response in Escherichia coli[23]. Kim et al. have S shown that treatment of primary hepatocytes with the NOreleasing drug SNAP is capable of inducing resistance to further treatment with H2O2, but this response to H2O2mediated cytotoxicity has not been well characterized [24]. Another study has shown that exposure of IMR-90 human S embryonic lung fibroblasts to sublethal fluxes of pure NO resulted in the induction of at least 12 proteins, one being S heme oxygenase (HO-1) [25]. Moreover, the NO-mediated induction of HO-1 in NSC34 mouse motor neurons induced S resistance to NO- as well as H2O2-mediated cell killing [26]. S To date, the mechanisms associated with NO and stress response induction are just beginning to be S unraveled. Because NO is produced in the presence of S ROS in vivo, this report investigates the ability of NO to induce resistance to oxidative stress mediated by H2O2. This study demonstrates that exposure of HA1 cells to S 1.4–1.7 mM NO-saturated medium under aerobic conditions is capable of inducing resistance to H2O2-mediated oxidative stress. The induced resistance occurs as early as

8 h and persists through at least 48 h after exposure to Furthermore, our results demonstrate (1) the immeS diate consumption of intracellular thiol pools, (2) NOmediated decreases in GCL activity, and (3) the requirement of subsequent increases in GCL activity and S glutathione synthesis for the induction of NO-mediated resistance to H2O2 challenge. This study suggests that S S exposure to NO or RNS derived from NO results in alterations in thiol metabolism that may represent important stimulators of biologically significant oxidative stress responses in mammalian cells.

SNO.

Experimental procedures Cell culturing techniques Chinese hamster fibroblasts (HA1) were cultured in EagleTs MEM supplemented with 10% fetal bovine serum and 0.1% penicillin–streptomycin (complete medium) and maintained at 378C in a humidified atmosphere of 5% CO2 and air. For experimental purposes the cells were seeded at 1  105 cells per 60-mm tissue culture dish, yielding S approximately 1  106 cells at the time of NO exposure [27]. The exposure medium employed in this work was serum-free EagleTs MEM without phenol red or glutamine (Washington University Tissue Culture Facility). Nitric oxide-treated cells were allowed to recover at 378C at times ranging from 0 to 48 h and then challenged with increasing doses of H2O2. Challenged cells were trypsinized and plated for clonogenic cell survival [28] or harvested for the measurement of GCL activity (described below). For the assessment of cell survival, colony formation was allowed to progress for 7–9 days, then the colonies were fixed, stained, and counted and the normalized surviving fraction was calculated as previously described [29]. To determine the kinetics associated with the development of resistance, survival curves of N2- or SNO-pretreated and H2O2-challenged cells were generated. Dose-modifying factors (DMFs) at isosurvival levels (either 20 or 50%) were determined for SNO- vs N2-treated cells and plotted vs recovery time. Dose-modifying factors were calculated according to the equation DM F ¼ ðd in S NOQpretreated cellsÞ =ðd in N2 Qpretreated cellsÞ; where d is the dose of H2O2 necessary to reach the specified percentage of survival.

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Preparation of NO-containing medium Nitric oxide gas (CP grade 99%) was obtained from Scott Specialty Gases (Philadelphia, PA, USA). Nitrogen gas (oxygen-free grade) was obtained from Genex (St. Louis, MO, USA). One-hundred-milliliter volumes of pretreatment

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medium were aliquoted into sterile 200-ml glass bottles and then sealed with rubber stoppers. Initially, the medium was bubbled with N2 to remove all O2. This was accomplished by connecting an N2 gas line (5 psi) to a gas flow regulator adjusted to a flow rate of 10 ml/min. Briefly, the N2 gas was then bubbled through the medium for a time corresponding to 1 min per 2-ml volume. The N2-saturated medium was S S then saturated with NO gas for 1 h, resulting in NO concentrations ranging from 1.4 to 1.7 mM. During this S process, NO gas was passed through a 1 M NaOH liquid S solution for the removal of species other than NO. Nitric oxide concentrations were directly measured using a modification of the neutral Griess reaction described in the S method of Nims et al. [30,31]. Briefly, NO-saturated medium was withdrawn from the bottles using a gas-tight 100-Al syringe (Osage) and then added to 100 mM potassium phosphate buffer, pH 7.4, containing 17 mM sulfanilic acid and 0.4 mM NEDD to a final volume of 1 ml. The solution was immediately mixed by inversion and incubated at room temperature for 5 min, and then absorbance at 496 nm was determined on a Beckman DUS 65 spectrophotometer. The NO concentration of the solution was calculated according to BeerTs law using a chemiluminescence-derived extinction coefficient of 6600 cm
1 M
1. Nitrite levels were determined in the same cuvette by the addition of 10 Al 85% phosphoric acid to yield an acidic Griess reaction. The solution was immediately mixed by inversion and incubated at room temperature for 15 min, and then absorbance at 540 nm was determined. S Nitrite levels of the NO-saturated medium were undetectable before exposure of medium to room air.

SNO exposure and H2O2 challenge S

To determine the ability of NO to induce crossresistance to a subsequent challenge by oxidants, 4 ml SNO-saturated medium was added to exponentially growS ing HA1 cells. This NO treatment resulted in surviving fractions of 60–70%. The cells were then incubated for 30 min at 378C in 5% CO2 in air. At the end of the incubation period, the treatment medium was replaced with fresh complete medium and the cells were allowed to recover for 0–48 h at 378C in 5% CO2 in air. At the end of the recovery period, the cells were challenged with increasing doses of H2O2 calculated as mol/cell  10
13 for 1 h at 378C in 5% CO2 in air. After challenge, the cells were trypsinized and plated for clonogenic survival. In a separate experiment, 5 mM BSO and 5 Ag/ml CHX were employed during the first 8 h of recovery after SNO exposure to inhibit glutathione synthesis and de novo protein synthesis, respectively. Analyses of glutathione and thiol pools Total and oxidized glutathione were measured in cell homogenates spectrophotometrically according to the

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method of Anderson [32]. Reduced glutathione, cysteine (CYS), and g-glutamylcysteine (GGC) pools were analyzed using high-performance liquid chromatography (HPLC) as described by Ridnour et al. [33]. Glutathione and thiol content were normalized per milligram protein as determined from the method of Lowry et al. [34]. HPLC GCL activity assay GCL activities were determined using an adaptation of the monobromobimane method described by Nardi to the NPM-conjugation method of Ridnour et al. [35,33]. Briefly, 50 Al of sample homogenate was added to 250 Al of 100 mM Tris buffer, pH 8.2, containing 0.75 mM l-cysteine, 0.75 mM l-glutamic acid, 6 mM ATP, 50 mM KCl, 6 mM DTT, and 20 mM MgCl2 and then incubated at 378C. Samples were vortexed and 20-Al aliquots were withdrawn from the reaction every 2.5–5.0 min for 15–20 min and added to a solution containing 230 Al H2O followed by derivatization with 750 Al of 1 mM N-(1-pyrenyl)maleimide (NPM) or 750 Al of 0.50 mM Thioglo 3 (Covalent Associates, Inc.). Sample derivatization was allowed to occur for 5 min, followed by acidification (pH 2.5) with 1 Al 1:6 HCl, and then the mixture was filtered. Thiols were separated on a C18 non-end-capped ReliaSil column (Column Engineering, Ontario, CA, USA) and quantified using a fluorescence detector. Sample GGC levels were determined against a standard curve, normalized per milligram protein, and plotted vs time. GCL activity was reported as nmol GGC mg
1 min
1. Immunoblotting The antibodies against rat GCL used in this work specifically recognized the catalytic (70 kDa) and regulatory (30 kDa) subunits of the GCL enzyme [36]. Twenty micrograms of heat denatured protein was loaded per lane and electrophoresed on 12.5% SDS–polyacrylamide gels and then transferred onto nitrocellulose membranes as described [37,38]. Transferred protein was incubated with polyclonal antibody recognizing the catalytic or regulatory subunits for 4 h at room temperature. The blots were then washed and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive protein was visualized using chemiluminescence reagents from Amersham. Immunoreactive protein using actin antibody was used as a loading control for densitometric quantification of GCL protein. Statistical analysis The data were statistically analyzed using ANOVA and DunnettTs test to determine differences between three or more groups and a Student t test for determining the differences between two groups.

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Results

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To investigate the ability of NO to induce resistance to oxidative stress mediated by H2O2, clonogenic cell survival was measured in exponentially grown HA1 cells exposed to S N2- or NO-saturated medium for 30 min at 378C. Cells exposed to H2O2 (2.5 mol/cell  10
13 or 0.25 pmol/cell) at 378C for 1 h served as a positive control. In addition, a nonexposed group of cells was challenged and served as a negative control. After pretreatment, the cells were allowed to recover in fresh complete medium for 20 h at 378C. At the end of recovery, the cells were challenged with increasing doses of H2O2 for 1 h and then trypsinized and plated for survival. Fig. 1 illustrates the surviving fractions (normalized to respective unchallenged plating efficiencies) after exposure to increasing doses of H2O2. Control and N2pretreated cells (negative controls) exhibit similar levels of sensitivity to H2O2-mediated cytotoxicity, whereas the H2O2-positive control demonstrated greater resistance to subsequent H2O2 challenge (DMF20% isosurvival ~ 2.4). S Similar to the H2O2-positive control, NO-pretreated cells show a higher degree of resistance to H2O2 challenge, as defined by a DMF20% isosurvival of 1.8. These data S demonstrate that NO exposure induced resistance to clonogenic cell killing mediated by H2O2 challenge in a pattern similar to the resistance induced by the H2O2pretreated positive control.

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Fig. 1. Exposure of HA1 cells to 1.7 mM NO-saturated medium induces resistance to H2O2-mediated cytotoxicity. Exponentially growing cells were exposed to NO and then allowed to recover at 378C for 20 h as described under Experimental procedures. After recovery the cells were challenged for 1 h with the indicated concentrations of H2O2 and then plated for clonogenic cell survival. Cells pretreated with 2.5  10
13 mol/cell H2O2 were included as a positive control, whereas cells exposed to N2-saturated medium, as well as a nonpretreated group, served as negative controls. Dose-modifying factors were calculated at 20% isosurvival.

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A time-course study was performed to investigate the S kinetics associated with the development of NO-induced resistance to H2O2-mediated cell killing. Log-phase cells were exposed as described above except that the recovery time ranged from 0 to 48 h before H2O2 challenge. Dosemodifying factors (relative to N2-exposed controls) at 50% isosurvival were determined from survival curves at each time point and plotted vs recovery time. Results presented in Fig. 2 demonstrate that the development of SNO-induced resistance to H2O2 occurs as early as 8 h and persists through at least 48 h. Because glutathione levels are a primary determinant of S sensitivity to cell killing by NO, O2, and H2O2 [27,39–41], we examined the effects of inhibition of glutathione synthesis on the development of resistance. Cells were incubated with 5 mM BSO, a specific inhibitor of GCL, during the first 8 h of recovery after SNO exposure [27]. The 8-h recovery time was chosen as this was the earliest time of measurable resistance to H2O2-mediated cytotoxicity as shown in Fig. 2. Five millimolar BSO inhibited the resynthesis of glutathione by 80 to 90% compared to untreated controls (Fig. 3, inset). Also, 5 Ag/ml CHX, a concentration known to inhibit up to 85% of de novo protein synthesis [42], was added to a S separate group of N2- or NO-exposed cells to investigate the involvement of de novo protein synthesis in the SNOinduced stress response. After recovery, the cells were challenged with 4  10
13 mol/cell H2O2 and plated for clonogenic survival. Fig. 3 demonstrates that the inhibition of either glutathione synthesis or de novo protein synthesis, during the first 8 h of recovery, equally sensitized N2pretreated controls to H2O2-mediated cytotoxicity by approximately fourfold compared to the uninhibited control. This result suggests that in unadapted cells, both glutathione synthesis and de novo protein synthesis provide protection against H2O2-mediated cell killing. The inhibition of de novo S protein synthesis in NO-exposed cells only slightly S inhibited (~ 20%) the NO-induced stress response to H2O2-mediated cytotoxicity compared to the respective, uninhibited control. In contrast, the inhibition of glutathione synthesis via inhibition of the GCL enzyme not only abolished the SNO-induced stress response, but also S sensitized NO-exposed cells to H2O2-mediated cell killing. These results indicate that, whereas both glutathione synthesis and de novo protein synthesis protect against H2O2S mediated cell killing in the unadapted N2 controls, the NOinduced stress response is more dependent upon GCL activity and the synthesis of glutathione for the development of resistance against H2O2-mediated cell killing. Consistent with this interpretation Richman and Meister have shown that acute depletion of glutathione leads to a transient increase in the activity of preexisting GCL enzyme, which allows the cell to maintain intracellular glutathione content without de novo synthesis of GCL subunits [43]. This observation may at least partially account for the relatively S modest inhibition of H2O2 resistance seen in NO-pretreated cells incubated in the presence of CHX for 8 h.

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Fig. 2. The kinetics of NO-induced resistance to H2O2 cell killing as determined by DMF at 50% isosurvival vs time. Log-phase cells were exposed to N2- or NO-saturated medium and then allowed to recover at 378C for 0 to 48 h. After recovery, the cells were challenged with H2O2 and plated for clonogenic cell survival.

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To further evaluate a role of glutathione metabolism S during the development of NO-induced resistance, a timecourse study was conducted to determine the levels of intracellular thiol pools including GSH, GGC, and CYS S after pretreatment with NO. Fig. 4A shows an immediate S reduction (75%) in glutathione levels after the 30 min NO exposure, which persisted through the first 8 h of recovery. Interestingly, NO-pretreated cells demonstrated a growth delay during the same time interval (data not shown), which supports the hypothesis of an involvement of glutathione in cell proliferation as described by Shaw and Chou [44]. S Twenty-four hours after NO exposure, glutathione levels had increased nearly 2-fold above the levels found in the respective N2-pretreated control. In addition, Fig. 4A shows a reduction in glutathione levels between 8 and 24 h in the N2-pretreated controls. Decreases in the levels of glutathione were associated with an increase in cell density and contact inhibition that occurred over the time-course study in the N2 control cells and is consistent with other published reports demonstrating an inverse correlation between glutathione levels and cell density [44,45]. Fig. 4B demonstrates a slight increase in GSSG levels between 1 and 8 h after SNO pretreatment; however, this increase accounted for only about 5% of the reduction in glutathione levels. Consistent with the consumption followed by reaccumulation in glutathione levels, GGC and CYS levels decreased 100 and 65%, respectively, after pretreatment S with NO, and by 24 h of recovery, GGC and CYS pools had increased to 2- and 1.5-fold above the levels measured in the N2 controls (Figs. 4C and 4D). Because GGC and

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CYS are precursors in the synthesis of glutathione, and because GGC and CYS pools are also consumed immediS ately after exposure to NO, these data suggest an inability to resynthesize glutathione during the early phase of S recovery, after exposure to NO. In addition, these results demonstrate a dramatic modulation in glutathione metaboS lism during the development of NO-mediated resistance. The cellular ability to resynthesize glutathione during the development of the SNO-induced stress response was further evaluated by the assessment of GCL immunoreactive S protein levels after exposure to NO. Because treatment S with NO resulted in the immediate consumption of GSH S and GGC (Fig. 4), the effects of NO on GCL immunoreactive protein levels were evaluated over the 24-h recovery S period after exposure to NO. The results in Fig. 5 demonstrate an immediate reduction in immunoreactive protein corresponding to both the catalytic (HS) and the regulatory (LS) subunits of the GCL protein at 0 h after S exposure to NO. By 24 h of recovery, immunoreactive protein for the catalytic subunit reaccumulated to control levels or slightly greater. Similarly, immunoreactive protein levels of the LS subunit were reduced to undetectable levels S immediately after exposure to NO. The reaccumulation of this subunit was apparent by 8 h of recovery, and then by

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Fig. 3. Inhibition of glutathione synthesis abolishes NO-induced resistance to H2O2 cell killing. Exponentially growing cells were treated with N2- or NO-saturated medium as described. After NO exposure 5 mM BSO (GCL inhibitor) or 5 Ag/ml CHX (de novo protein synthesis inhibitor) was added to triplicate dishes, and the cells were allowed to recover for 8 h. The cells were then challenged with 4  10
13 mol/cell H2O2 for 1 h at 378C and then trypsinized and plated for clonogenic cell survival. Surviving fractions were normalized to the appropriate non-H2O2challenged plating efficiencies after the 8-h recovery period. *p b 0.05 for comparison to the respective N2 control; **p b 0.05 for comparison of N2 + drug to N2 control; ***p b 0.05 for comparison of NO + drug to NO control. The inset shows the total glutathione in cell homogenates taken from each treatment group, relative to the N2-treated control.

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Fig. 4. NO exposure of HA1 cells results in the consumption, followed by reaccumulation, of intracellular thiol pools. Exponentially growing cells were treated with N2- or NO-saturated medium and a time-course recovery experiment was conducted for 0–24 h of recovery. The cells were then scrape harvested and immediately prepared for HPLC evaluation of intracellular thiol pools including (A) GSH, (B) GSSG, (C) CYS, and (D) GGC, as described under Experimental procedures using NPM. The data are single-point measurements and are representative of two experiments each.

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24 h the regulatory subunit had increased to a level that was slightly greater than the N2 control. The modulatory pattern of GCL-immunoreactive protein levels was consistent with those observed in the thiol pools (Figs. 4 and 5), suggesting the involvement of decreased levels of GCL protein and the inability to replenish glutathione levels during the early S period of recovery after exposure to NO. S An association between NO-mediated cytoprotection and stimulation of the ubiquitin proteasomal pathway has

Fig. 5. Immediate loss followed by the reaccumulation of immunoreactive protein levels of the glutathione-metabolizing enzyme GCL after NO exposure. Exponentially growing cells were treated with N2- or NOsaturated medium for 30 min and then allowed to recover for 0–24 h. Cells were scrape harvested and protein homogenates were prepared. Twenty micrograms of protein was loaded per lane and electrophoresed on a 12.5% SDS–polyacrylamide gel. The protein was transferred onto nitrocellulose membrane and incubated for 4 h at room temperature with antibodies recognizing the GCL catalytic (HS) and regulatory (LS) protein subunits. Immunoreactive protein was visualized by chemiluminescence and is representative of three blots.

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recently been demonstrated by the NO-mediated inhibition of H2O2-induced transferrin receptor-dependent apoptosis in bovine aortic endothelial (BAEC) cells [46]. In this study, Kalyanaraman and co-workers have shown enhanced levels of high-molecular-weight ubiquitinated protein in BAECs pretreated with lactacystin (an inhibitor S of the 26S proteasome) and the NO donor DETA/NO, compared to cells treated with DETA/NO alone, suggesting S that NO up-regulates the proteasomal pathway. To begin to address the possible involvement of alterations in S proteolysis in the NO-mediated loss of immunoreactive GCL protein shown in Fig. 5, the presence of highmolecular-weight ubiquitinated protein was evaluated in whole-cell homogenates of N2 control and SNO-exposed cells pretreated with lactacystin. Fig. 6A shows increased levels of high-molecular-weight ubiquitinated protein in S both N2 control and NO-exposed cells pretreated with lactacystin, indicating that the 26S proteasome had been inhibited. The Coomassie blue-stained gel demonstrated equal loading (data not shown). Although there were minimal differences in the levels of ubiquitinated protein in SNO-exposed cells compared to N2 controls, GCL activity S in the NO-treated cells, both in the presence and in the absence of lactacystin, was dramatically reduced compared to the respective N2 control (Fig. 6). These data suggest that S the NO-mediated inhibitory effect on GCL activity does not seem to involve proteasomal degradation but may involve S direct modification(s) of the protein. Moreover, the NOmediated inhibitory effects of GCL activity shown in Fig. 6

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Fig. 6. NO-mediated inhibition of GCL activity and the effects of a proteasome inhibitor (lactacystin). Exponentially growing cells were pretreated with 15 AM lactacystin for 2 h and then exposed to NO- or N2-saturated medium for 30 min. The cells were scrape harvested and whole-cell homogenates prepared. Twenty micrograms of protein was electrophoresed on a 4–20% SDS–polyacrylamide gel and then transferred onto nitrocellulose membrane and incubated for 1 h at room temperature with primary antibody recognizing ubiquitinated protein. Immunoreactive protein was visualized by chemifluorescence detection. (A) Increases in high-molecular-weight ubiquitinated protein (monoclonal Ub antibody; Santa Cruz) in the cells pretreated with lactacystin, an inhibitor of the 26S proteasome complex, are shown. (B) NO-mediated inhibition of GCL activity in the presence and absence of lactacystin, compared to the N2 control, is demonstrated. GCL activity was determined by the HPLC assay as described under Experimental procedures (using Thioglo 3), normalized per milligram protein, and plotted as a function of time.

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may be of further significance in explaining the enhanced H2O2-mediated toxicity exhibited in the NO-pretreated cells that were further exposed to BSO (Fig. 3), when considering the observations of Richman and Meister regarding transient increases in preexisting GCL enzyme activity after acute depletion of glutathione, which occurred in the absence of de novo synthesis of GCL subunits [43]. Taken together these results allow for the speculation that compared to both CHXand BSO-treated N2 controls, the enhanced H2O2 toxicity exhibited by the BSO-treated cells after SNO exposure may involve inhibition of preexisting GCL activity. S Now that NO-induced alterations in thiol metabolism had been established, glutathione synthetic capability was S examined in cells 24 h after exposure to NO both before and after H2O2 challenge. In this experiment, log-phase cells S were exposed to N2- and NO-saturated medium and then allowed to recover for 24 h. Unchallenged cells were harvested immediately after the 24-h recovery period. H2O2challenged cells were exposed to 5  10
13 mol/cell H2O2 (a dose selected from the midrange of the survival curve shown in Fig. 1), for 1 h, and then the challenge medium was replaced with fresh complete medium and the cells were incubated at 378C for 0, 8, and 24 h. The cells were then harvested at these time points and evaluated for the effects S of NO vs N2 pretreatment on GCL activity and glutathione levels in response to H2O2 challenge. The results in Fig. 7 demonstrate 2-fold increases in GCL activity 24 h after S H2O2 challenge in NO-pretreated cells, compared to N2 controls (Fig. 7A). Concomitantly, an approximate 1.5-fold S increase in reduced glutathione was observed in NO exposed cells 24 h after H2O2 challenge (Fig. 7B). Compared to N2 controls, CYS levels were slightly lower S in NO-pretreated cells, whereas GGC levels were slightly elevated after H2O2 challenge (Figs. 7C and 7D). Interest-

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ingly, the immediate effects of NO exposure on thiol consumption as demonstrated in Fig. 4 are in contrast to the S elevated thiol levels in response to H2O2 challenge in NOpretreated cells shown in Fig. 7. Moreover, compared to N2 control, H2O2 challenge leads to increases in the immunoreactive protein levels of both the heavy and the light S subunits of GCL in NO-pretreated cells (Fig. 8). These S results support the hypothesis that prior exposure to NO induces resistance to H2O2-mediated oxidative stress, which is mediated at least in part by increased GCL activity and elevated glutathione levels in response to the H2O2 challenge.

Discussion

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In this study the ability of NO to induce a cellular stress response to a subsequent, more toxic challenge by H2O2 was investigated. Previously it has been shown that two S mechanisms by which NO may elicit a stress response involve the induction of HO-1 or Fe-containing proteins [25,26,47]. Alternatively, thiol metabolism could provide an additional mechanism by which the cell can induce S protection after exposure to NO [48,49]. Glutathione is a nonprotein thiol that exists in millimolar concentrations in most cell types and has a wide range of intracellular functions [50,51]. The current study demonstrates the ability S of NO-adapted cells to induce a stress response to H2O2mediated cytotoxicity. In our model, the development of resistance involves thiol metabolism as defined by the initial consumption followed by reaccumulation in the levels of intracellular thiol pools, which include GSH, GGC, and CYS. Possible mechanisms that may account for the loss of glutathione demonstrated in this study and elsewhere [27]

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Fig. 7. NO-pretreated cells demonstrate increases in the levels of GCL activity in response to H2O2 challenge. Exponentially growing cells were pretreated with N2- or NO-saturated medium for 30 min and allowed to recover for 24 h, and cells from triplicate dishes were harvested. The remaining cells were challenged with 5  10
13 mol/cell H2O2 for 1 h at 378C. After 1 h incubation, the challenge medium was replaced with fresh complete medium and the cells were incubated at 378C. H2O2-challenged cells were then harvested in triplicate at 0, 8, and 24 h postchallenge. (A) GCL activity levels were determined by the HPLC assay as described under Experimental procedures (using NPM) and normalized per mg protein min
1. (B) GSH, (C) CYS, and (D) GGC levels were determined at the indicated times after H2O2 treatment and normalized per milligram of protein. The data are single-point measurements and are representative of two experiments each.

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may involve the formation of S-nitrosoglutathione (GSNO), which functions as a signaling molecule and has a half-life of approximately 90 min [52]. Once formed, GSNO can S decompose to form NO and thiyl radical, which further reacts to form protein mixed disulfides [50]. In addition to the immediate reduction in thiol pools (Fig. 4) and the loss of GCL-immunoreactive protein and GCL activity, the first and rate-limiting enzyme in the glutathione synthetic pathS way was also observed after NO exposure (Figs. 5 and 6).

Fig. 8. H2O2 challenge results in increased levels of GCL immunoreactive protein in NO-pretreated cells. Exponentially growing cells were pretreated with N2- or NO-saturated medium for 30 min and then allowed to recover for 24 h and then challenged with H2O2 as described for Fig. 6. Samples were harvested at the indicated times after H2O2 challenge. Twenty micrograms of protein was loaded per lane and electrophoresed on a 12.5% SDS–polyacrylamide gel. The protein was transferred onto nitrocellulose membrane and incubated for 4 h at room temperature with antibodies recognizing the GCL catalytic (HS) and regulatory (LS) protein subunits. Immunoreactive protein was visualized by chemiluminescence and is representative of two blots.

S

S

Because glutathione contributes significantly to the maintenance of intracellular redox equilibrium, the sustained decrease in GCL protein and activity during the first 4 h S after NO suggests an inability of the cell to resynthesize glutathione for maintenance of intracellular redox equilibrium. Whereas other studies have shown that acute depletion of glutathione leads to a transient increase in the activity of preexisting GCL enzyme, allowing the cell to maintain intracellular glutathione content [43], our results S demonstrate an immediate NO-induced inhibitory effect on both GCL protein and enzymatic activity followed by recovery of GCL levels and thiol pools during development of resistance to H2O2-mediated oxidative stress. The involvement of GCL in mediating the induction of cellular steady-state levels of glutathione under conditions of stress has been documented [53–56]. In our model, recovery of GCL activity and the resynthesis of glutathione seem to be S important factors in the NO-induced stress response to oxidative stress and are supported by two observations. First, S exposure of NO-pretreated cells to BSO, a specific inhibitor of GCL, during the first 8 h of recovery resulted in inhibition of GSH synthesis by 80–90%, which not only inhibited SNO-induced resistance to H2O2-mediated cell killing, but S actually sensitized the NO-pretreated cells to H2O2-induced cytotoxicity (Fig. 3). In addition, compared to N2 controls, S H2O2 challenge of NO-adapted cells resulted in up to a twofold increase in GCL activity levels 24 h after H2O2

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challenge (Fig. 7A). Therefore, this work demonstrates that S cells exposed to NO-saturated medium are primed for a dramatic induction of GCL activity and GSH synthesis in response to a subsequent H2O2-mediated oxidant challenge. Moreover, GCL activity and glutathione synthesis seem to be S required for the NO-induced resistance to H2O2-mediated oxidative stress. S The involvement of NO in thiol metabolism and glutathione synthesis has been shown in previous reports and has been reviewed by Dickinson et al. [56]. Kuo et al. S have demonstrated that endogenous NO production regulates hepatocyte glutathione levels through a mechanism that is dependent upon the transcriptional regulation of GCL, as the inhibition of iNOS dramatically decreased the levels of intracellular glutathione and GCL activity and the rate of transcription of GCL in IL-1-stimulated hepatocytes [57]. In addition, the decrease in the levels of transcription and activity of GCL, as well as intracellular glutathione, S were restored to control levels by the addition of NO donors to iNOS-inhibited cells, supporting the hypothesis S for the involvement of NO in the regulation of GCL and intracellular glutathione levels [57]. Also, Moellering et al. S and White et al. have demonstrated an NO-mediated induction of glutathione synthesis through a mechanism involving GCL [48,49,58]. Moreover, in a comparison of the effects of sustained elevated glutathione levels vs the induction of glutathione synthesis via increased GCL, Woods et al. have demonstrated a requirement for the synthesis of glutathione in the attenuation of free radical formation and associated oxidative stress in cells treated with pro-oxidants [59]. S In summary, this work has shown that NO exposure is capable of inducing resistance to oxidative stress mediated S by H2O2. The NO-induced stress response seems to involve altered thiol metabolism as characterized by the immediate consumption, followed by reaccumulation, of cellular thiols including glutathione, GGC, and CYS, as well as GCL immunoreactive protein. Compared to the N2 S control, the advantage of NO-adapted cells seems to involve the ability to increase the activity levels of the GCL enzyme (approximately twofold) in response to H2O2 challenge. This hypothesis is strengthened by the effects of the inhibition of GCL enzyme activity during the first 8 h of S recovery after exposure to NO, in which the development of resistance was inhibited (Fig. 3). To our knowledge, these S data also provide the first evidence identifying an NOinduced adaptive response that seems to afford cells the enhanced ability to induce GCL activity and GSH synthesis, which is required for the development of resistance to oxidative stress.

Acknowledgments This work was supported by NIH Grants 2RO1HL51469 (D.R.S.), F32ES05781 (L.A.R.), R29CAG9593 (P.C.G.),

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ES05511 (H.J.F.), and DOE DE-FG02-02ER63447 (M.C.C., D.R.S.).

References [1] Ortega, M.; Amaya, A. Nitric oxide reactivity and mechanisms involved in its biological effects. Pharmacol. Res. 42:421 – 427; 2000. [2] Ignarro, L. J. Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling. Pharmacol. Toxicol. 67:1 – 7; 1990. [3] Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43:109 – 142; 1991. [4] OTDell, T. J.; Hawkins, R. D.; Kandel, E. R.; Arancio, O. Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger. Proc. Natl. Acad. Sci. USA 88:11285 – 11289; 1991. [5] Stuehr, D. J.; Nathan, C. F. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169:1543 – 1555; 1989. [6] Nakajima, T.; Oda, H.; Kusumoto, F.; Nogami, H. Nitric oxides and their effects on health. 1980:121 – 142. [7] Barnes, P. J.; Belvisi, M. G. Nitric oxide and lung disease. Thorax 48:1034 – 1043; 1993. [8] Pryor, W. A.; Stone, K. Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrite, and peroxynitrite. Ann. N. Y. Acad. Sci. 686:12 – 28; 1993. [9] Kwon, N. S.; Lee, S. H.; Choi, C. S.; Kho, T.; Lee, H. S. Nitric oxide generation from streptozotocin. FASEB J. 8:529 – 533; 1994. [10] Forman, L. J.; Liu, P.; Nagele, R. G.; Yin, K.; Wong, P. Y. Augmentation of nitric oxide, superoxide, and peroxynitrite production during cerebral ischemia and reperfusion in the rat. Neurochem. Res. 23:141 – 148; 1998. [11] Dawson, V. L.; Dawson, T. M.; London, E. D.; Bredt, D. S.; Snyder, S. H. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA 88:6368 – 6371; 1991. [12] Kolb, H.; Kolb-Bachofen, V. Nitric oxide: a pathogenetic factor in autoimmunity. Immunol. Today 13:157 – 160; 1992. [13] Wink, D. A.; Darbyshire, J. F.; Nims, R. W.; Saavedra, J. E.; Ford, P. C. Reaction of the bioregulatory agent nitric oxide in oxygenated aqueous media: determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. 6:23 – 27; 1993. [14] Lipton, S. A.; Choi, Y. B.; Pan, Z. H.; Lei, S. Z.; Chen, H. S. V.; Sucher, N. J.; Loscalzo, J.; Singel, D. J.; Stamler, J. S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364:626 – 632; 1993. [15] Luperchio, S.; Tamir, S.; Tannenbaum, S. R. NO-induced oxidative stress and glutathione metabolism in rodent and human cells. Free Radic. Biol. Med. 21:513 – 519; 1996. [16] Ischiropoulos, H.; Zhu, L.; Beckman, J. S. Peroxynitrite formation from macrophage-derived nitric oxide. Arch. Biochem. Biophys. 298:446 – 451; 1992. [17] Stamler, J. S. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78:931 – 936; 1994. [18] Demple, B.; Amabile-Cuevas, C. F. Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon. Cell 67:837 – 839; 1991. [19] Spitz, D. R.; Adams, D. T.; Sherman, C. M.; Roberts, R. J. Mechanisms of cellular resistance to hydrogen peroxide, hyperoxia, and 4-hydroxy-2-nonenal toxicity: the significance of increased catalase activity in H2O2-resistant fibroblasts. Arch. Biochem. Biophys. 292:221 – 227; 1992.

1370

L.A. Ridnour et al. / Free Radical Biology & Medicine 38 (2005) 1361–1371

[20] Walkup, L.; Kogoma, T. Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical. J. Bacteriol. 171:1476 – 1484; 1989. [21] Sullivan, S. J.; Oberley, T. D.; Roberts, R. J.; Spitz, D. R. A stable O2-resistant cell line: role of lipid peroxidation byproducts in O2mediated injury. Am. J. Physiol. (Lung Cell. Mol. Physiol) 262 (6 Pt 1):L748 – L756; 1992. [22] Greenberg, J. T.; Monach, P. A.; Chou, J. H.; Josephy, P. D.; Demple, B. Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc. Natl. Acad. Sci. USA 87:6181 – 6185; 1990. [23] Nunoshiba, T.; DeRojas-Walker, T.; Wishnok, J. S.; Tannenbaum, S. R.; Demple, B. Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. Proc. Natl. Acad. Sci. USA 90:9993 – 9997; 1993. [24] Kim, Y. M.; Bergonia, H.; Lancaster, J. R. Nitrogen oxide-induced autoprotection in isolated rat hepatocytes. FEBS Lett. 374:228 – 232; 1995. [25] Marquis, J. C.; Demple, B. Complex genetic response of human cells to sublethal levels of pure nitric oxide. Cancer Res. 58:3435 – 3440; 1998. [26] Bishop, A.; Marquis, J. C.; Cashman, N. R.; Demple, B. Adaptive resistance to nitric oxide in motor neurons. Free Radic. Biol. Med. 26:978 – 988; 1999. [27] Walker, M. W.; Kinter, M. T.; Roberts, R. J.; Spitz, D. R. Nitric oxide-induced cytotoxicity: involvement of cellular resistance to oxidative stress and the role of glutathione in protection. Pediatr. Res. 37:41 – 49; 1995. [28] Spitz, D. R.; Dewey, W. C.; Li, G. C. Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts. J. Cell. Physiol. 131:364 – 373; 1987. [29] Spitz, D. R.; Li, G. C. Heat-induced cytotoxicity in H2O2resistant Chinese hamster fibroblasts. J. Cell. Physiol. 142:255 – 260; 1990. [30] Nims, R. W.; Darbyshire, J. F.; Saavedra, J. E.; Christodoulou, D.; Hanbauer, I.; Cox, G. W.; Grisham, M. B.; Laval, F.; Cook, J. A.; Krishna, M. C.; Wink, D. A. Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods: Companion Methods Enzymol. 7:48 – 54; 1995. [31] Ridnour, L. A.; Sim, J. E.; Wink, D. A.; Martin, S.; Buettner, G. R.; Spitz, D. R. A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media. Anal. Biochem. 281:223 – 229; 2000. [32] Anderson, M. E. In: Greenwald, R. A. (Ed.), Handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press; 1985: 317 – 323. [33] Ridnour, L. A.; Winters, R. A.; Ercal, N.; Spitz, D. R. Measurement of glutathione, glutathione disulfide, and other thiols in mammalian cell and tissue homogenates using high-performance liquid chromatography separation of N-(1-pyrenyl)maleimide derivatives. Methods Enzymol. 299:258 – 267; 1998. [34] Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem 193: 265 – 275; 1951. [35] Nardi, G.; Cipollaro, M.; Loguercio, C. Assay of gamma-glutamylcysteine synthetase and glutathione synthase in erythrocytes by high-performance liquid chromatography with fluorimetric detection. J. Chromatogr. 530:122 – 128; 1990. [36] Liu, R. M.; Gao, L.; Choi, J.; Forman, H. J. Gamma-glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal. Am. J. Physiol. 275 (5 Pt 1):L861 – L869; 1998. [37] Oberley, L. W.; McCormick, M. L.; Sierra, E.; Kasemeset-St. Clair, D. Manganese superoxide dismutase in normal and transformed human embryonic lung fibroblasts. Free Radic. Biol. Med. 6: 379 – 384; 1989.

[38] Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350 – 4354; 1979. [39] Biaglow, J. E.; Varnes, M. E.; Clark, E. P.; Epp, E. R. The role of thiols in cellular response to radiation and drugs. Radiat. Res. 95:437 – 455; 1983. [40] Mitchell, J. B.; Russo, A. Thiols, thiol depletion, and thermosensitivity. Radiat. Res. 95:471 – 485; 1983. [41] Spitz, D. R.; Kinter, M. T.; Roberts, R. J. Contribution of increased glutathione content to mechanisms of oxidative stress resistance in hydrogen peroxide resistant hamster fibroblasts. J. Cell. Physiol. 165:600 – 609; 1995. [42] Lee, Y. J.; Dewey, W. C. Protection of Chinese hamster ovary cells from hyperthermic killing by cycloheximide or puromycin. Radiat. Res. 106:98 – 110; 1986. [43] Richman, P. G.; Meister, A. Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J. Biol. Chem. 250:1422 – 1426; 1975. [44] Shaw, J. P.; Chou, I. N. Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts. J. Cell. Physiol. 129:193 – 198; 1986. [45] Lu, S. C.; Ge, J. L. Loss of suppression of GSH synthesis at low cell density in primary cultures of rat hepatocytes. Am. J. Physiol. 263 (6 Pt 1):C1181 – C1189; 1992. [46] Kotamraju, S.; Tampo, Y.; Keszler, A.; Chitambar, C. R.; Joseph, J.; Haas, A. L.; Kalyanaraman, B. Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: role of ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 100: 10653 – 10658; 2003. [47] Domachowske, J. B. The role of nitric oxide in the regulation of cellular iron metabolism. Biochem. Mol. Med. 60:1 – 7; 1997. [48] Moellering, D.; McAndrew, J.; Patel, R.; Cornwell, T.; Lincoln, T.; Cao, X.; Messing, J.; Forman, H. J.; Jo, H.; Darley-Usmar, V. M. Nitric oxide-dependent induction of glutathione synthesis through increased expression of gamma-glutamylcysteine synthetase. Arch. Biochem. Biophys. 358:74 – 82; 1998. [49] Moellering, D.; McAndrew, J.; Patel, R. P.; Forman, H. J.; Mulcahy, R. T.; Jo, H.; Darley-Usmar, V. M. The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. FEBS Lett. 448:292 – 296; 1999. [50] Anderson, M. E. Glutathione and glutathione delivery compounds. Adv. Pharmacol. 38:65 – 78; 1997. [51] Arrigo, A. P. Gene expression and the thiol redox state. Free Radic. Biol. Med. 27:936 – 944; 1999. [52] Mohr, S.; Hallak, H.; de Boitte, A.; Lapetina, E. G.; Brune, B. Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde3-phosphate dehydrogenase. J. Biol. Chem. 274:9427 – 9430; 1999. [53] Shi, M. M.; Kugelman, A.; Iwamoto, T.; Tian, L.; Forman, H. J. Quinone-induced oxidative stress elevates glutathione and induces gamma-glutamylcysteine synthetase activity in rat lung epithelial L2 cells. J. Biol. Chem. 269:26512 – 26517; 1994. [54] Lui, R. M.; Hu, H. P.; Robison, T. W.; Forman, H. J. Differential enhancement of gamma-glutamyl transpeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells. Am. J. Respir. Cell Mol. Biol. 14:186 – 191; 1996. [55] Lui, R. M.; Hu, H. P.; Robison, T. W.; Forman, H. J. Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity. Am. J. Respir. Cell Mol. Biol. 14:192 – 197; 1996. [56] Dickinson, D. A.; Moellering, D. R.; Iles, K. E.; Patel, R. P.; Levonen, A. L.; Wigley, A.; Darley-Usmar, V. M.; Forman, H. J. Cytoprotection against oxidative stress and the regulation of glutathione synthesis. Biol. Chem. 384:527 – 537; 2003. [57] Kuo, P. C.; Abe, K. Y.; Schroeder, R. A. Interleukin-1-induced nitric oxide production modulates glutathione synthesis in cultured rat

L.A. Ridnour et al. / Free Radical Biology & Medicine 38 (2005) 1361–1371 hepatocytes. Am. J. Physiol. (Cell Physiol.) 271 (3 Pt 1):C851 – C862; 1996. [58] White, A. C.; Maloney, E. K.; Boustani, M. R.; Hassoun, P. M.; Fanburg, B. L. Nitric oxide increases cellular glutathione levels in rat lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 13:442 – 448; 1995.

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[59] Woods, J. S.; Kavanagh, T. J.; Corral, J.; Reese, A. W.; Diaz, D.; Ellis, M. E. The role of glutathione in chronic adaptation to oxidative stress: studies in a normal rat kidney epithelial (NRK52E) cell model of sustained upregulation of glutathione biosynthesis. Toxicol. Appl. Pharmacol. 160:207 – 216; 1999.