8-Nitro-cGMP (8-nitroguanosine 3′,5′-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H2O2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H2O2. Using the mitochondria-targeted chemical probe MitoSOX™ Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H2O2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.

INTRODUCTION

cGMP is a cyclic nucleotide formed from GTP by the catalytic action of the enzymes called guanylyl cyclases [1,2]. In vertebrates, two guanylyl cyclase isoforms have been identified, membrane-bound pGC (particulate-type guanylyl cyclase) and sGC (soluble-type guanylyl cyclase), that are expressed in almost all cell types [2]. These enzymes are activated in response to specific signals, such as NO for sGC and peptide ligands for pGC, to produce cGMP. Subsequently, cGMP functions as a second messenger for these signals and regulates a wide variety of cell physiological functions such as vascular smooth muscle motility, host defence, intestinal fluid and electrolyte homoeostasis, and retinal phototransduction [2]. Such biological actions of cGMP may be primarily mediated by activation of downstream effector molecules such as cGMP-dependent protein kinase, ion channels and phosphodiesterases [2].

A nitrated derivative of cGMP, 8-nitro-cGMP (8-nitroguanosine 3′,5′-cyclic monophosphate), has been identified in mammalian cells [36]. 8-Nitro-cGMP possesses unique biochemical properties, e.g. it behaves as an electrophile and reacts with protein sulfhydryls, which results in cGMP adduction to protein sulfhydryls [3,5,7,8]. This post-translational modification by 8-nitro-cGMP via cGMP adduction is named protein S-guanylation [3,5,7,8]. Furthermore, 8-nitro-cGMP can induce an antioxidant adaptive response in cells via S-guanylation of the redox sensor protein Keap1 (Kelch-like ECH-associated protein 1), which results in transcriptional activation of Nrf2 with concomitant expression of a battery of genes that encode an array of phase II detoxifying or antioxidant enzymes, as well as other cytoprotective proteins [5]. Thus 8-nitro-cGMP may function as a potent electrophilic second messenger involved in regulation of redox signalling [79].

To explore how and when 8-nitro-cGMP is involved in regulating cell physiology via its unique electrophilic properties, understanding of the molecular mechanisms regulating 8-nitro-cGMP formation in cells is essential. Nitration of the guanine moiety is a crucial step for production of nitrated nucleotides including 8-nitro-cGMP. Previous studies suggested that RNOS (reactive nitrogen oxide species), formed from the reaction of NO and ROS (reactive oxygen species), can nitrate guanine derivatives under biologically relevant conditions [10,11]. An example of RNOS include peroxynitrite (ONOO), which is a potent oxidizing and nitrating agent formed from the reaction of NO and superoxide (O2). In the present study, we investigated the role of ROS in 8-nitro-cGMP formation both in vitro and in cells. Chemical analyses revealed that ONOO was a potent agent for nitration of guanine nucleotides. In addition to ONOO, nitrite in the presence of H2O2 and MPO (myeloperoxidase) may nitrate guanine nucleotides. We used rat C6 glioma cells to study cell formation of 8-nitro-cGMP, because the cells produced a significant amount of 8-nitro-cGMP in response to stimulation with LPS (lipopolysaccharide) plus pro-inflammatory cytokines via expression of the inducible isoform of NO synthase [5]. In these C6 cells stimulated with LPS/cytokines, mitochondria-derived superoxide acted as a direct determinant of 8-nitro-cGMP formation. This demonstration is the first indicating that mitochondria-derived superoxide plays an important role in biological nitration of guanine nucleotides. Furthermore, we determined that mitochondria-derived superoxide production was regulated by H2O2 generated from Nox2 (NADPH oxidase 2), which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study thus indicate that 8-nitro-cGMP can serve as a unique secondary messenger that may be implicated in regulating ROS signalling in the presence of NO.

MATERIALS AND METHODS

Materials

cGMP, GTP, GDP, GMP and rotenone were obtained from Sigma–Aldrich. The NO-liberating agent P-NONOate (propylamine NONOate {CH3N[N(O)NO](CH2)3NH2+CH3, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene}), which has a half-life of 7.6 min in aqueous solutions at a neutral pH under the experimental conditions used in the present study, was obtained from Dojindo Laboratories. SIN-1 (3-morpholinosydnonimine), DTPA (diethylenetriamine penta-acetic acid), EDTA and tiron (1,2-dihydroxy-3,5-benzene-disulfonic acid) were obtained from Dojindo Laboratories. Tyrosine was obtained from Kyowa Hakko. MPO was purchased from Alexis Biochemicals. HRP (horseradish peroxidase) was obtained from Wako Pure Chemical Industries. Bovine SOD (Cu,Zn-superoxide dismutase) was purchased from Sigma–Aldrich. Catalase was purchased from Boehringer Mannheim. The succinimidyl derivative of PEG [poly(ethylene glycol)] propionic acid, which has an average molecular mass of 5000 Da, was obtained from NOF Corporation. NADPH oxidase p47phox siRNA (small interfering RNA) was purchased from Invitrogen. DCDHF-DA (2′,7′-dichlorodihydrofluorescein diacetate), MitoSOX™ Red and DHE (dihydroethidium) were purchased from Molecular Probes (Invitrogen). Anti-p47phox antibody (catalogue number 07-497) was purchased from Millipore. HRP-conjugated anti-mouse secondary antibody was purchased form Amersham Pharmacia Biotech. For effective delivery of SOD and catalase to the intracellular compartment, the enzymes were chemically modified by conjugation with PEG to obtain the pegylated enzymes (PEG–SOD and PEG–catalase) [12]. SOD (10 mg/ml) or catalase (10 mg/ml) was reacted with succinimidyl PEG (155 mg/ml for SOD and 120 mg/ml for catalase) in 0.5 M sodium phosphate buffer (pH 7.4) for 2 h at 4°C with stirring as described previously [13]. Authentic ONOO was synthesized from acidified nitrite and H2O2 using a quenched-flow method as described previously [14]. The concentration of ONOO was determined by means of photospectrometry with a molar absorption coefficient of ϵ302=1670 M−1·cm−1 [14]. Contaminating H2O2 was then decomposed using manganese dioxide.

Synthesis of various guanine nucleotides

Authentic 8-nitro-cGMP labelled with a stable isotope or unlabelled (8-15NO2-cGMP or 8-14NO2-cGMP respectively) was prepared according to the method we described previously [3,5]. 15N-Labelled cGMP, i.e. c[15N5]GMP ([U-15N5, 98%]GMP), was synthesized from [15N5]GTP ([U-15N5, 98%]GTP; Cambridge Isotope Laboratories) via an enzymatic reaction using purified sGC [5]. All of these stable isotope-labelled guanine nucleotides were used as internal standards in the stable isotope dilution technique with LC (liquid chromatography)-MS/MS (tandem MS) analysis, as described below. Authentic 8-nitro-GMP, 8-nitro-GDP and 8-nitro-GTP were prepared by reacting 1 mM GMP, GDP and GTP respectively, with 2 mM ONOO in 0.1 M sodium phosphate buffer (pH 7.4) containing 25 mM NaHCO3 and 0.1 mM DTPA.

Chemical analysis of guanine nucleotide nitration in vitro

Guanine nucleotides were reacted with various RNOS systems in vitro to determine the nitrating potential of each RNOS system. Formation of nitrated derivatives was analysed by means of RP (reverse-phase)-HPLC equipped with a PDA (photodiode array) or with ECD (electrochemical detection) [3]. RP-HPLC-ECD was used for analysis of cGMP nitration, whereas RP-HPLC-PDA was used for analysis of nitration of other guanine nucleotides. For RP-HPLC-PDA, HPLC plus PDA detection was performed with a UV detector using an MCM C-18 column (150 mm long, 4.6 mm inner diameter; MC Medical). Samples were eluted with 0–40% acetonitrile in 1.0% dibutylammonium acetate buffer with a 0.7 ml/min flow rate; detection was by HPLC-PDA, with a UV detector (SPD-M10A VP; Shimadzu).

Authentic ONOO system

The guanine nucleotides cGMP, GMP, GDP and GTP (each at 1 mM) were reacted under vortex-mixing with authentic ONOO (2 mM) in 0.1 M sodium phosphate buffer (pH 7.4) containing 25 mM NaHCO3 and 0.1 mM DTPA. Nitration of cGMP by ONOO was further analysed as a function of ONOO concentration and buffer pH. That is, cGMP (50 μM) was reacted under vortex-mixing with ONOO (up to 10 μM) in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM DTPA with or without 25 mM NaHCO3. The effect of pH was examined for the pH range 2.5–7.4. Buffers used included 0.1 M citric acid buffer (pH 2.5–5.0) and 0.1 M sodium phosphate buffer (pH 5.5–7.4), containing 0.1 mM DTPA with or without 25 mM NaHCO3.

SIN-1 system

SIN-1 was used to study the effect of simultaneous production of NO and superoxide [15] on guanine nucleotide nitration. cGMP (50 μM) or tyrosine (50 μM) was reacted with SIN-1 (0–100 μM) in 0.1 M sodium phosphate buffer (pH 7.4) in the presence of 0.1 mM DTPA and 25 mM NaHCO3.

Nitrite/H2O2/haem peroxidase system

Haem peroxidases catalyse oxidation of nitrite in the presence of H2O2 to form the potent nitrating agent NO2 [16,17]. In the present study, two haem peroxidases, MPO and HRP, were used as catalysts. cGMP (50 μM) or tyrosine (50 μM) was reacted at 37°C for 3 h with either MPO (10 nM) or HRP (23.8 nM) in 0.1 M sodium phosphate buffer (pH 7.4) containing 100 μM NaNO2 and 100 μM H2O2.

Aerobic NO production system

NO is oxidized to form NO2, an oxidizing and nitrating agent, under aerobic conditions [18]. To study the effect of NO and NO2 on guanine nucleotide nitration, we used the NO-releasing agent P-NONOate, which spontaneously decomposes to release NO [19]. cGMP (50 μM) and tyrosine (50 μM) were reacted with P-NONOate (0-100 μM), an NO donor in 0.1 M sodium phosphate buffer (pH 7.4), in the presence of 0.1 mM DTPA and 25 mM NaHCO3, followed by measurement of the nitrated derivatives of cGMP and tyrosine. Nitration of cGMP and tyrosine was also examined in an acidified nitrite system [18]: cGMP (50 μM) and tyrosine (50 μM) were reacted for 1 h at 37°C with NaNO2 (100 μM) in 0.1 M sodium citrate buffer (pH 2.5–4.5).

Hypochlorous acid/nitrite system

HOCl (hypochlorous acid) is a product of the MPO-catalysed oxidation of chlorine ion (Cl) in the presence of H2O2. HOCl reportedly reacts with nitrite to form NO2Cl [20]. To determine the effect of NO2Cl on guanine nucleotide nitration, cGMP (50 μM) and tyrosine (50 μM) were reacted for 4 h at 37°C with HOCl (100 μM) plus NaNO2 (100 μM) in 0.1 M sodium phosphate buffer (pH 7.4).

Effect of ROS scavengers

To determine the roles of ROS on guanine nucleotide nitration, we used three different ROS scavengers: SOD and tiron for scavenging superoxide and catalase for scavenging H2O2. For the experiments described below, PEGylated SOD and catalase were used for effective delivery of those enzymes into cells [12].

Cell treatment

Rat C6 glioma cells were cultured at 37°C in DMEM (Dulbecco's modified Eagle's medium; Wako Pure Chemical Industries) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin. Cells were plated at a density of 1.5×106 cells per 60-mm dish to prepare cell extracts for LC-ESI (electrospray ionization)-MS/MS, and at a density of 1×105 cells per chamber in BD Falcon Culture Slides (BD Biosciences) for immunocytochemistry. To study 8-nitro-cGMP formation, cells were stimulated for 36 h with a mixture of 10 μg/ml LPS (from Escherichia coli; Sigma–Aldrich, catalogue number L8274) and 200 units/ml IFN-γ (interferon-γ), 500 units/ml TNF-α (tumour necrosis factor-α) and 10 ng/ml IL-1β (interleukin-1β) (all cytokines from R&D Systems). In some experiments, to investigate the mechanism of ROS-dependent 8-nitro-cGMP production, cells were stimulated in the presence of ROS scavengers, including PEG–SOD, tiron and PEG–catalase, followed by analyses for 8-nitro-cGMP formation and ROS generation. In certain experiments, cells were treated with an LPS/cytokine mixture for 0, 3, 12, 24 and 36 h followed by detection of mitochondrial ROS by MitoSOX™ Red, as described below. In other experiments, to investigate involvement of mitochondrial ROS generation in 8-nitro-cGMP formation, cells were pretreated for 15 min with 10 μM rotenone before LPS/cytokine stimulation. To study the role of Nox2 in cellular ROS production, cells were transfected with NADPH oxidase p47phox-specific siRNA before stimulation as described below.

Immunocytochemistry

Formation of 8-nitro-cGMP in C6 cells was analysed by means of immunocytochemistry with an anti-(8-nitro-cGMP) mouse monoclonal antibody and Cy3 (indocarbocyanine)-labelled goat anti-mouse IgG antibody (10 μg/ml; Amersham Biosciences, catalogue number PA43002), as described previously [3,5]. Fluorescence intensity values from three different experiments were obtained, and the average relative fluorescence intensity (as the percentage fluorescence intensity) was determined for LPS/cytokine-stimulated cells. We confirmed that LPS/cytokines and PEG-derivatized proteins had no significant effects on quenching or augmentation of fluorescence by using non-immune antiserum (results not shown).

LC-ESI-MS/MS analysis for intracellular formation of 8-nitro-cGMP

Intracellular levels of 8-nitro-cGMP were quantified by means of LC-ESI-MS/MS as described previously [5]. Amounts of endogenous cGMP and 8-nitro-cGMP were determined via the stable isotope dilution method based on the recovery efficiency of stable isotope-labelled derivatives (c[15N5]GMP and 8-15NO2-cGMP) spiked with the cell extract as described previously [5].

Determination of cellular ROS production

Cellular ROS production was determined by means of fluorescence microspectrometry with chemical probes that become fluorescent on reaction with ROS. Specifically, we used MitoSOX™ Red [21] and DHE [22] for detection of mitochondrial superoxide production and DCDHF-DA [23] for detection of cellular oxidants. C6 cells were washed with Hank's buffer [0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4.2 mM NaHCO3 (pH 7.4)], and were then stained with 2.5 μM MitoSOX™ Red or with 100 nM DHE dissolved in Hank's buffer, or with 5 μM DCDHF-DA dissolved in PBS, for 15 min at 37°C in the dark. Cells were then washed carefully with Hank's buffer, mounted with mounting buffer, covered with coverslips and examined with a Nikon EZ-C1 confocal laser microscope. Excitation was at 420 nm for MitoSOX™ Red and 543 nm for DHE (the red photomultiplier channel of the confocal microscope was used for image acquisition); for DCDHF-DA, excitation was at 488 nm (the green photomultiplier channel of the confocal microscope was used for image acquisition). To minimize run-to-run variations, the laser intensity and photomultiplier tube voltage were kept constant, and microscopic observations were performed on all sample sets at same time. Images were captured and processed by means of Nikon EZ-C1 software. Further image processing and quantification were performed using Adobe Photoshop Elements version 2.0 (Adobe Systems). Fluorescence intensity values from three different experiments were obtained, and the average relative fluorescence intensity (as the percentage fluorescence intensity) was determined for LPS/cytokine-stimulated cells. In other experiments, the percentage relative fluorescence intensity was determined for PBS-treated cells.

Transfection of p47phox siRNA

A 25-nucleotide p47phox siRNA (manufactured by Invitrogen, catalogue number 1320003, Oligo ID, MSS206956 and MSS275934) was used for transfection using Lipofectamine™ RNAiMAX transfection reagent (Invitrogen). Briefly, C6 cells were seeded in 12-well plates at a density of 2×105 cells/well. Cells were transfected with p47phox siRNA (60 pmol/well) using Lipofectamine™ RNAiMAX transfection reagent. At 72 h after transfection, cells were harvested, but just before the harvest they were treated with LPS/cytokines for 36 h. Stealth RNAi (RNA interference) negative control (high GC; Invitrogen) was used as a negative control siRNA as described above. In other experiments, cells were treated with 10 μM rotenone for 15 min before stimulation with LPS/cytokines for 36 h.

Western blot analysis

Proteins were separated using SDS/PAGE and transferred on to a PVDF membrane. The blot was blocked with 5% non-fat skimmed milk followed by 1 h of incubation with an anti-p47phox antibody (Millipore) and a goat anti-rabbit HRP-conjugated IgG secondary antibody. The immunoreactive bands were detected using chemiluminescence reagent (Millipore) with a luminescent image analyser (LAS-1000, Fujifilm).

Statistical analysis

All cell culture results were obtained from at least three separate wells or three separate dishes, and the data are represented as means±S.E.M. Statistical analyses were performed using Student's t test.

RESULTS

Nitration of guanine nucleotides by various RNOS: in vitro chemical analyses

We investigated the effects of various RNOS on nitration of guanine nucleotides in vitro. We first examined nitration of guanine nucleotides in the reaction with ONOO, a potent nitrating and oxidizing species formed from the reaction of NO and superoxide. As shown in Figure 1(A), ONOO nitrated all guanine nucleotides. However, the efficacy of nitration varied depending on the structure of the nucleotides; GTP showed the highest production of nitrated derivative, with nitration efficiency then decreasing in the following order: GDP>GMP>cGMP.

ONOO-dependent nitration of guanine nucleotides and tyrosine

Figure 1
ONOO-dependent nitration of guanine nucleotides and tyrosine

(A) Nitration of various guanine nucleotides by ONOO. The guanine nucleotides cGMP, GMP, GDP and GTP (1 mM each) were reacted with 2 mM ONOO in 0.1 M sodium phosphate buffer (pH 7.4) in the presence of 25 mM NaHCO3. (B) Nitration of cGMP (left-hand panel) and tyrosine (right-hand panel) by ONOO as a function of ONOO concentration. (C) pH dependence of ONOO-mediated nitration of cGMP and tyrosine. In (B and C) cGMP (50 μM) or tyrosine (50 μM) was reacted with the indicated concentration of ONOO and different pH range respectively. Data are expressed as means±S.E.M. (n=3). *P<0.05 and **P<0.01, compared with the group in the absence of NaHCO3.

Figure 1
ONOO-dependent nitration of guanine nucleotides and tyrosine

(A) Nitration of various guanine nucleotides by ONOO. The guanine nucleotides cGMP, GMP, GDP and GTP (1 mM each) were reacted with 2 mM ONOO in 0.1 M sodium phosphate buffer (pH 7.4) in the presence of 25 mM NaHCO3. (B) Nitration of cGMP (left-hand panel) and tyrosine (right-hand panel) by ONOO as a function of ONOO concentration. (C) pH dependence of ONOO-mediated nitration of cGMP and tyrosine. In (B and C) cGMP (50 μM) or tyrosine (50 μM) was reacted with the indicated concentration of ONOO and different pH range respectively. Data are expressed as means±S.E.M. (n=3). *P<0.05 and **P<0.01, compared with the group in the absence of NaHCO3.

Nitration of guanine nucleotides by ONOO was further examined as a function of ONOO concentration and pH of the reaction mixtures, with cGMP used as a model substrate. As shown in Figure 1(B), formation of 8-nitro-cGMP depended on the concentration of ONOO. 8-Nitro-cGMP formation was markedly enhanced in the presence of NaHCO3. This enhanced effect of NaHCO3 on ONOO-mediated nitration was more obvious for cGMP than for tyrosine. The efficacies of ONOO-mediated nitration of both cGMP and tyrosine were maximum at neutral pH (pH 7–7.2) (Figure 1C). To exclude the possibility that nitrite that may be contaminating the ONOO solution could affect the induction of cGMP nitration, we examined cGMP nitration in a reaction with decomposed ONOO solution that would presumably contain the same amount of contaminating nitrite as an intact ONOO solution. Results indicated no 8-nitro-cGMP formation in the reaction of cGMP with decomposed ONOO (results not shown).

Table 1 summarizes the effects of various RNOS systems on nitration of cGMP and tyrosine. The NO donor P-NONOate did not cause detectable nitration of cGMP, even under aerobic conditions. An acidic nitrite system did cause tyrosine nitration, but no detectable formation of 8-nitro-cGMP. SIN-1, which simultaneously generates NO and superoxide, caused both cGMP and tyrosine nitration. This result suggests that ONOO formed from NO and superoxide is an effective nitrating agent for guanine nucleotides. In addition to SIN-1, the complete NaNO2/H2O2/MPO system led to marked nitration of cGMP. Omission of just one component from this complete system resulted in no detectable level of 8-nitro-cGMP. HOCl is a strong oxidant produced by MPO. NaNO2 in the presence of HOCl effectively nitrated tyrosine, but not cGMP. No 8-nitro-cGMP formed after replacement of MPO by HRP in the complete system, which suggests that nitration of guanine nucleotides depends on the type of peroxidase, even when both nitrite anion and H2O2 are available simultaneously. Nitration of cGMP by RNOS was inhibited by specific inhibitors and scavengers of ROS (Figure 2). SOD completely suppressed SIN-1-mediated cGMP nitration, whereas it failed to suppress nitration of cGMP mediated by authentic ONOO or NaNO2/H2O2/MPO. Catalase, however, was an effective inhibitor of only NaNO2/H2O2/MPO-mediated nitration of cGMP. Tiron is a small molecule that is a SOD mimic and has been used as a superoxide scavenger [24]. In the present study, however, tiron effectively suppressed cGMP nitration mediated by all RNOS systems examined. Similarly, tiron effectively suppressed tyrosine nitration caused by authentic ONOO (Supplementary Figure S1 at http://www.BiochemJ.org/bj/441/bj4410719add.htm). It was also found that tiron effectively suppressed tyrosine nitration by P-NONOate, suggesting that tiron can inhibit nitration by either ONOO or aerobic NO (NO2), possibly via scavenging NO2, independent of its superoxide scavenging activity.

Effect of ROS scavengers on the formation of 8-nitro-cGMP induced by various RNOS systems

Figure 2
Effect of ROS scavengers on the formation of 8-nitro-cGMP induced by various RNOS systems

cGMP (50 μM) was reacted with authentic ONOO (5 μM) (A), with SIN-1 (100 μM) (B) or with NaNO2 (100 μM)/H2O2 (100 μM)/MPO (10 nM) (C) in the absence or presence of SOD (10 and 100 units/ml), catalase (100 and 1000 units/ml) or tiron (20 and 200 μM). Data are expressed as means±S.E.M. (n=3). *P<0.05 and **P<0.01 compared with the control.

Figure 2
Effect of ROS scavengers on the formation of 8-nitro-cGMP induced by various RNOS systems

cGMP (50 μM) was reacted with authentic ONOO (5 μM) (A), with SIN-1 (100 μM) (B) or with NaNO2 (100 μM)/H2O2 (100 μM)/MPO (10 nM) (C) in the absence or presence of SOD (10 and 100 units/ml), catalase (100 and 1000 units/ml) or tiron (20 and 200 μM). Data are expressed as means±S.E.M. (n=3). *P<0.05 and **P<0.01 compared with the control.

Table 1
Nitration of cGMP (50 μM) and tyrosine (50 μM) by various RNOS systems

ND, not detected.

 Product formed (nM) 
Conditions 8-Nitro-cGMP 3-Nitrotyrosine 
NONOate (100 μM), 4h† ND 16.7±1.1* 
NaNO2 (100 μM), at pH 3.0, 1 h‡ ND 95.8±1.8* 
NaNO2 (100 μM), at pH 4.0, 1 h‡ ND 30.3±3.0* 
SIN-1 (50 μM), 2 h§ 7.7±0.3* 267.3±5.6* 
SIN-1 (100 μM), 2 h§ 14.5±0.7* 402.9±8.3* 
NaNO2 (100 μM), H2O2 (100 μM), MPO (10 nM), 4 h‖ 67.3±1.5* 9307.0±11.1* 
NaNO2 (100 μM), MPO (10 nM), 4 h‖ ND ND 
H2O2 (100 μM), MPO (10 nM), 4 h‖ ND ND 
NaNO2 (100 μM), H2O2 (100 μM), 4 h‖ ND ND 
NaNO2 (100 μM), H2O2 (100 μM), HRP (23.8 μM), 4 h‖ ND 803.5±1.6* 
HOCl (100 μM), NaNO2 (100 μM), 4 h¶ ND 48.3±1.9* 
 Product formed (nM) 
Conditions 8-Nitro-cGMP 3-Nitrotyrosine 
NONOate (100 μM), 4h† ND 16.7±1.1* 
NaNO2 (100 μM), at pH 3.0, 1 h‡ ND 95.8±1.8* 
NaNO2 (100 μM), at pH 4.0, 1 h‡ ND 30.3±3.0* 
SIN-1 (50 μM), 2 h§ 7.7±0.3* 267.3±5.6* 
SIN-1 (100 μM), 2 h§ 14.5±0.7* 402.9±8.3* 
NaNO2 (100 μM), H2O2 (100 μM), MPO (10 nM), 4 h‖ 67.3±1.5* 9307.0±11.1* 
NaNO2 (100 μM), MPO (10 nM), 4 h‖ ND ND 
H2O2 (100 μM), MPO (10 nM), 4 h‖ ND ND 
NaNO2 (100 μM), H2O2 (100 μM), 4 h‖ ND ND 
NaNO2 (100 μM), H2O2 (100 μM), HRP (23.8 μM), 4 h‖ ND 803.5±1.6* 
HOCl (100 μM), NaNO2 (100 μM), 4 h¶ ND 48.3±1.9* 
*

P<0.01, compared with the control (no RNOS treatment).

In 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 mM DTPA at 37°C.

In 0.1 M sodium citrate buffer (pH 2.5–4.5) and 100 μM NaNO2 at 37°C.

§

In 0.1 M sodium phosphate buffer (pH 7.4), 0.1 mM DTPA and 25 mM NaHCO3 at 37°C.

In 0.1 M sodium phosphate buffer (pH 7.4) at 37°C.

In 0.1 M citric acid buffer (pH 4.5) at 37°C.

Formation of 8-nitro-cGMP in rat C6 glioma cells: involvement of cellular ROS production

Our chemical analyses clearly demonstrated that NO itself is not sufficient to cause nitration of guanine nucleotides, but requires ROS, including superoxide and H2O2, for that reaction to occur. To study the roles of ROS in 8-nitro-cGMP formation in cells, we used C6 cells in culture as a model system.

Immunocytochemical analyses provided the baseline formation of 8-nitro-cGMP in non-stimulated C6 cells (Figure 3A). Formation of 8-nitro-cGMP was markedly enhanced in C6 cells when cells were stimulated with LPS/cytokines (Figure 3A). Treatment with PEG–SOD, which is reportedly a membrane-permeant SOD derivative [12], reduced the immunostaining in a manner dependent on PEG–SOD concentration (Figure 3B). This result suggests the essential role of superoxide for cell formation of 8-nitro-cGMP in stimulated C6 cells. Similar to PEG–SOD, PEG–catalase, a membrane-permeant catalase derivative, suppressed 8-nitro-cGMP formation in C6 cells stimulated with LPS/cytokines (Figure 3C). LC-ESI-MS/MS analyses verified 8-nitro-cGMP formation in C6 cells and its modulation by PEG–SOD and PEG–catalase. In agreement with immunocytochemical data, these analyses detected a certain level of 8-nitro-cGMP in non-stimulated C6 cells (Figure 4). As Figure 4 shows, stimulation by LPS/cytokines significantly promoted formation of both cGMP and 8-nitro-cGMP in C6 cells. The concentration of 8-nitro-cGMP was approximately 5.6-fold higher than that of cGMP under the experimental conditions used in the present study. PEG–SOD treatment moderately reduced the level of cGMP. A similar trend was observed with PEG–catalase, although it was not statistically significant. Formation of 8-nitro-cGMP was almost completely nullified by treatment with both PEG–SOD and PEG–catalase, a finding that agrees with results obtained by immunocytochemistry. Thus these data suggest that formation of 8-nitro-cGMP depends greatly on cell production of both superoxide and H2O2. Under these condition, PEG–SOD and PEG–catalase treatments did not affect NO production in C6 cells (Supplementary Figure S2 at http://www.BiochemJ.org/bj/441/bj4410719add.htm).

Immunocytochemical analysis of 8-nitro-cGMP formation in rat C6 glioma cells and modulation of its formation by ROS scavengers

Figure 3
Immunocytochemical analysis of 8-nitro-cGMP formation in rat C6 glioma cells and modulation of its formation by ROS scavengers

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of the indicated concentrations of PEG–SOD or PEG–catalase. Cells were then fixed with Zamboni fixative, as described in the Materials and methods section, followed by immunocytochemical detection of intracellular 8-nitro-cGMP with the use of the 1G6 monoclonal antibody against 8-nitro-cGMP. (A) Cells were treated with different concentrations of PEG–SOD (top panels) or PEG–catalase (bottom panels), 1 h before the addition of LPS/cytokines and during stimulation with LPS/cytokines for 36 h, and the immunocytochemical detection of 8-nitro-cGMP was then performed. Scale bars=50 μm. (B) Concentration-dependent decrease in relative fluorescence intensity of 8-nitro-cGMP in C6 cells after the addition of the cell-permeant superoxide scavenger PEG–SOD (1–200 units/ml) during stimulation with LPS/cytokines. (C) Concentration-dependent decrease in relative fluorescence intensity of 8-nitro-cGMP in C6 cells after the addition of the cell-permeant H2O2 scavenger PEG–catalase (1–200 units/ml) during stimulation with LPS/cytokines. Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group.

Figure 3
Immunocytochemical analysis of 8-nitro-cGMP formation in rat C6 glioma cells and modulation of its formation by ROS scavengers

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of the indicated concentrations of PEG–SOD or PEG–catalase. Cells were then fixed with Zamboni fixative, as described in the Materials and methods section, followed by immunocytochemical detection of intracellular 8-nitro-cGMP with the use of the 1G6 monoclonal antibody against 8-nitro-cGMP. (A) Cells were treated with different concentrations of PEG–SOD (top panels) or PEG–catalase (bottom panels), 1 h before the addition of LPS/cytokines and during stimulation with LPS/cytokines for 36 h, and the immunocytochemical detection of 8-nitro-cGMP was then performed. Scale bars=50 μm. (B) Concentration-dependent decrease in relative fluorescence intensity of 8-nitro-cGMP in C6 cells after the addition of the cell-permeant superoxide scavenger PEG–SOD (1–200 units/ml) during stimulation with LPS/cytokines. (C) Concentration-dependent decrease in relative fluorescence intensity of 8-nitro-cGMP in C6 cells after the addition of the cell-permeant H2O2 scavenger PEG–catalase (1–200 units/ml) during stimulation with LPS/cytokines. Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group.

LC-ESI-MS/MS analysis of 8-nitro-cGMP formation in rat C6 glioma cells and modulation of its formation by ROS scavengers

Figure 4
LC-ESI-MS/MS analysis of 8-nitro-cGMP formation in rat C6 glioma cells and modulation of its formation by ROS scavengers

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of PEG–SOD or PEG–catalase, and cell extracts were prepared as described in the Materials and methods section, followed by LC-ESI-MS/MS quantification of cGMP and 8-nitro-cGMP formed in cells. (A) LC-ESI-MS/MS chromatograms of cGMP and 8-nitro-cGMP in untreated cells and cells treated with 200 units/ml PEG–SOD or 200 units/ml PEG–catalase (from 1 h before LPS/cytokine addition), during stimulation with LPS/cytokines for 36 h. (B) Intracellular cGMP concentrations in cells after stimulation with LPS/cytokines in the presence or absence of PEG–SOD or PEG–catalase (each at 200 units/ml) determined with LC-ESI-MS/MS. (C) Intracellular 8-nitro-cGMP concentrations in cells after stimulation with LPS/cytokines in the presence or absence of PEG–SOD or PEG–catalase (each at 200 units/ml) determined with LC-ESI-MS/MS. Data are expressed as means±S.E.M. (n=3). *P<0.05 and **P<0.01 compared with the LPS/cytokine-treated group. ##P<0.01, compared with the PBS-treated group.

Figure 4
LC-ESI-MS/MS analysis of 8-nitro-cGMP formation in rat C6 glioma cells and modulation of its formation by ROS scavengers

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of PEG–SOD or PEG–catalase, and cell extracts were prepared as described in the Materials and methods section, followed by LC-ESI-MS/MS quantification of cGMP and 8-nitro-cGMP formed in cells. (A) LC-ESI-MS/MS chromatograms of cGMP and 8-nitro-cGMP in untreated cells and cells treated with 200 units/ml PEG–SOD or 200 units/ml PEG–catalase (from 1 h before LPS/cytokine addition), during stimulation with LPS/cytokines for 36 h. (B) Intracellular cGMP concentrations in cells after stimulation with LPS/cytokines in the presence or absence of PEG–SOD or PEG–catalase (each at 200 units/ml) determined with LC-ESI-MS/MS. (C) Intracellular 8-nitro-cGMP concentrations in cells after stimulation with LPS/cytokines in the presence or absence of PEG–SOD or PEG–catalase (each at 200 units/ml) determined with LC-ESI-MS/MS. Data are expressed as means±S.E.M. (n=3). *P<0.05 and **P<0.01 compared with the LPS/cytokine-treated group. ##P<0.01, compared with the PBS-treated group.

Cellular production of ROS and related oxidants was analysed by using chemical probes that become fluorescent in response to ROS and oxidants. With MitoSOX™ Red, a mitochondria-targeted superoxide-sensitive fluorigenic probe [21], we found that stimulation of C6 cells with LPS/cytokines significantly induced production of mitochondrial superoxide (Figure 5A). A time-course study showed that mitochondrial superoxide production gradually increased and reached a plateau at 24 h after LPS/cytokine stimulation (Supplementary Figure S3 at http://www.BiochemJ.org/bj/441/bj4410719add.htm). On the basis of this result, analyses of ROS production were carried out at 36 h after stimulation. We also used double staining of cells with MitoTracker® Green and MitoSOX™ Red to investigate whether the MitoSOX™ Red signal was derived from mitochondria. As shown in Supplementary Figure S4 (at http://www.BiochemJ.org/bj/441/bj4410719add.htm), MitoSOX™ Red staining co-localized well with MitoTracker® Green staining, which suggests that the superoxide detected by MitoSOX™ Red was primarily of mitochondrial origin.

Fluorescence microscopic determination of ROS production in rat C6 glioma cells stimulated with LPS/cytokines

Figure 5
Fluorescence microscopic determination of ROS production in rat C6 glioma cells stimulated with LPS/cytokines

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of PEG–SOD or PEG–catalase. Cells were then analysed for the presence of mitochondrial superoxide and cellular H2O2, as described in the Materials and methods section. (A) MitoSOX™ Red (top panels), DCDHF-DA (middle panels) and DHE (bottom panels) staining of untreated cells and cells treated with 200 units/ml PEG–SOD or 200 units/ml PEG–catalase from 1 h before the addition of LPS/cytokine and during stimulation with LPS/cytokine for 36 h, as detected by the Nikon EZ-C1 confocal laser microscope (for MitoSOX™ Red: excitation at 420 nm and red photomultiplier channel; for DCDHF-DA: excitation at 488 nm and green photomultiplier channel; and for DHE: excitation at 543 nm and red photomultiplier channel). Scale bars=50 μm. (B) Relative fluorescence intensity for MitoSOX™ Red staining (top panel), DCDHF-DA staining (middle panel) and DHE staining (bottom panel) of untreated C6 cells or cells treated with 200 units/ml PEG–SOD or 200 units/ml PEG–catalase from 1 h before the addition of LPS/cytokine and during stimulation with LPS/cytokine for 36 h. Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group.

Figure 5
Fluorescence microscopic determination of ROS production in rat C6 glioma cells stimulated with LPS/cytokines

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of PEG–SOD or PEG–catalase. Cells were then analysed for the presence of mitochondrial superoxide and cellular H2O2, as described in the Materials and methods section. (A) MitoSOX™ Red (top panels), DCDHF-DA (middle panels) and DHE (bottom panels) staining of untreated cells and cells treated with 200 units/ml PEG–SOD or 200 units/ml PEG–catalase from 1 h before the addition of LPS/cytokine and during stimulation with LPS/cytokine for 36 h, as detected by the Nikon EZ-C1 confocal laser microscope (for MitoSOX™ Red: excitation at 420 nm and red photomultiplier channel; for DCDHF-DA: excitation at 488 nm and green photomultiplier channel; and for DHE: excitation at 543 nm and red photomultiplier channel). Scale bars=50 μm. (B) Relative fluorescence intensity for MitoSOX™ Red staining (top panel), DCDHF-DA staining (middle panel) and DHE staining (bottom panel) of untreated C6 cells or cells treated with 200 units/ml PEG–SOD or 200 units/ml PEG–catalase from 1 h before the addition of LPS/cytokine and during stimulation with LPS/cytokine for 36 h. Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group.

Treatment with PEG–SOD significantly reduced the fluorescence intensity originating with MitoSOX™ Red (Figure 5B). PEG–catalase also suppressed production of superoxide, as shown by reduced MitoSOX™ Red-derived fluorescence. These results suggest that mitochondrial superoxide production may be regulated by H2O2 production. We then studied production of H2O2 using DCDHF-DA, a cell-permeant and oxidation-sensitive fluorescent probe. Microscopic observation of DCDHF-DA-derived fluorescence clearly showed oxidant production in C6 cells after stimulation with LPS/cytokines (Figure 5A). A significant inhibitory effect of PEG–catalase (Figure 5B) suggests that H2O2 is produced in stimulated C6 cells and acts as a major oxidant involved in induction of DCDHF-derived fluorescence. The specificity of superoxide detection was confirmed using DHE, and the results were consistent with those obtained by MitoSOX™ Red analysis (Figure 5).

As mentioned above, tiron can act not only as a superoxide scavenger, but also as an antioxidant to inhibit guanine nucleotide nitration caused by ONOO and NaNO2/H2O2/MPO (Figure 2). Immunocytochemical analyses revealed that tiron effectively suppressed formation of 8-nitro-cGMP in C6 cells stimulated with LPS/cytokines (Figure 6). Similarly, tiron treatment significantly reduced fluorescence derived from both MitoSOX™ Red and DCDHF-DA (Figure 6). Under these conditions, tiron did not cause any detectable cytotoxic effects (Supplementary Figure S5 at http://www.BiochemJ.org/bj/441/bj4410719add.htm).

Effect of the SOD mimic tiron on formation of 8-nitro-cGMP and ROS production in rat C6 glioma cells

Figure 6
Effect of the SOD mimic tiron on formation of 8-nitro-cGMP and ROS production in rat C6 glioma cells

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of tiron (1–100 μM), followed by immunocytochemical detection of intracellular 8-nitro-cGMP with the use of a 1G6 monoclonal antibody against 8-nitro-cGMP, or by direct staining for mitochondrial superoxide or cellular H2O2 as described in the Materials and methods section. Cells were untreated or treated with 1–100 μM tiron from 1 h before the addition of LPS/cytokine and during stimulation with LPS/cytokines for 36 h, followed by immunocytochemical detection of 8-nitro-cGMP (A, top panels); detection of MitoSOX™ Red staining (A, middle panels), via a Nikon EZ-C1 confocal laser microscope (excitation, 420 nm; red photomultiplier channel); and detection of DCDHF-DA staining (A, bottom panels), via a Nikon EZ-C1 confocal laser microscope (excitation, 488 nm; green photomultiplier channel). Scale bars=50 μm. (B) Relative fluorescence intensity of C6 cells, treated as described above, for 8-nitro-cGMP immunocytochemical staining (left-hand panel), MitoSOX™ Red staining (middle panel) and DCDHF-DA staining (right-hand panel). Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group.

Figure 6
Effect of the SOD mimic tiron on formation of 8-nitro-cGMP and ROS production in rat C6 glioma cells

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of tiron (1–100 μM), followed by immunocytochemical detection of intracellular 8-nitro-cGMP with the use of a 1G6 monoclonal antibody against 8-nitro-cGMP, or by direct staining for mitochondrial superoxide or cellular H2O2 as described in the Materials and methods section. Cells were untreated or treated with 1–100 μM tiron from 1 h before the addition of LPS/cytokine and during stimulation with LPS/cytokines for 36 h, followed by immunocytochemical detection of 8-nitro-cGMP (A, top panels); detection of MitoSOX™ Red staining (A, middle panels), via a Nikon EZ-C1 confocal laser microscope (excitation, 420 nm; red photomultiplier channel); and detection of DCDHF-DA staining (A, bottom panels), via a Nikon EZ-C1 confocal laser microscope (excitation, 488 nm; green photomultiplier channel). Scale bars=50 μm. (B) Relative fluorescence intensity of C6 cells, treated as described above, for 8-nitro-cGMP immunocytochemical staining (left-hand panel), MitoSOX™ Red staining (middle panel) and DCDHF-DA staining (right-hand panel). Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group.

mETC (mitochondrial electron transport chain) complexes, particularly complexes I and III, are the main source of ROS produced from mitochondria [25]. The mETC inhibitor rotenone reportedly accelerates or inhibits mitochondrial ROS production, depending on the cell type studied [26,27]. We thus studied whether modulation of mitochondrial ROS production with the mETC complex inhibitor rotenone affected formation of 8-nitro-cGMP in C6 cells. As shown in Figure 7, rotenone treatment increased 8-nitro-cGMP formation in C6 cells stimulated with LPS/cytokines, as shown by immunocytochemistry and LC-ESI-MS/MS results: both methods revealed a similar increase, by approximately 1.5-fold. Under the same experimental conditions, rotenone treatment significantly increased MitoSOX™ Red-derived fluorescence intensity without affecting DCDHF-DA-derived fluorescence (Figure 7B). This finding suggests that the effect of rotenone was specific to mitochondrial ROS production and hence that 8-nitro-cGMP formation in C6 cells is closely related to mitochondrial superoxide production. However, treatment of non-stimulated cells with rotenone alone did not significantly affect the production of 8-nitro-cGMP, and, in fact, an increased MitoSOX™ Red signal was observed (Figure 7). This result suggests that 8-nitro-cGMP production requires simultaneous production of both NO and ROS.

Modulation of 8-nitro-cGMP formation by rotenone in rat C6 glioma cells

Figure 7
Modulation of 8-nitro-cGMP formation by rotenone in rat C6 glioma cells

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of rotenone (10 μM). Cells were then fixed with Zamboni fixative, as described in the Materials and methods section, followed by immunocytochemical detection of intracellular 8-nitro-cGMP with the use of the 1G6 monoclonal antibody against 8-nitro-cGMP, or by direct staining for mitochondrial superoxide or cellular H2O2. Cells were treated with 10 μM rotenone for 15 min before the addition of LPS/cytokine (or were untreated), during stimulation with LPS/cytokines for 36 h, followed by immunocytochemical detection via a Nikon EZ-C1 confocal laser microscope of 8-nitro-cGMP (A, top panels); MitoSOX™ Red staining (excitation, 420 nm; red photomultiplier channel) (A, upper middle panels); DCDHF-DA staining (excitation, 488 nm; green photomultiplier channel) (A, lower middle panels) and DHE staining (excitation, 543 nm; red photomultiplier channel) (A, bottom panels). Scale bars=50 μm. (B) Relative fluorescence intensity of C6 cells, treated as described above, for 8-nitro-cGMP immunocytochemical staining (top left-hand panel); MitoSOX™ Red staining (bottom left-hand panel); DCDHF-DA staining (top right-hand panel); and DHE staining (bottom right-hand panel). (C) Intracellular 8-nitro-cGMP concentrations in cells after stimulation with LPS/cytokines with or without 10 μM rotenone pretreatment, as determined with LC-ESI-MS/MS. Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group. ##P<0.01 compared with the PBS-treated group.

Figure 7
Modulation of 8-nitro-cGMP formation by rotenone in rat C6 glioma cells

Cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h in the absence or presence of rotenone (10 μM). Cells were then fixed with Zamboni fixative, as described in the Materials and methods section, followed by immunocytochemical detection of intracellular 8-nitro-cGMP with the use of the 1G6 monoclonal antibody against 8-nitro-cGMP, or by direct staining for mitochondrial superoxide or cellular H2O2. Cells were treated with 10 μM rotenone for 15 min before the addition of LPS/cytokine (or were untreated), during stimulation with LPS/cytokines for 36 h, followed by immunocytochemical detection via a Nikon EZ-C1 confocal laser microscope of 8-nitro-cGMP (A, top panels); MitoSOX™ Red staining (excitation, 420 nm; red photomultiplier channel) (A, upper middle panels); DCDHF-DA staining (excitation, 488 nm; green photomultiplier channel) (A, lower middle panels) and DHE staining (excitation, 543 nm; red photomultiplier channel) (A, bottom panels). Scale bars=50 μm. (B) Relative fluorescence intensity of C6 cells, treated as described above, for 8-nitro-cGMP immunocytochemical staining (top left-hand panel); MitoSOX™ Red staining (bottom left-hand panel); DCDHF-DA staining (top right-hand panel); and DHE staining (bottom right-hand panel). (C) Intracellular 8-nitro-cGMP concentrations in cells after stimulation with LPS/cytokines with or without 10 μM rotenone pretreatment, as determined with LC-ESI-MS/MS. Data are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine-treated group. ##P<0.01 compared with the PBS-treated group.

Nox2 is a member of the NADPH oxidase family and is an important source of ROS, particularly in immunologically stimulated cells [28]. To clarify the implications of Nox2-dependent ROS production for 8-nitro-cGMP formation, we performed knockdown of p47phox, a critical component of Nox, by using p47phox siRNA. The efficacy of the p47phox knockdown was confirmed by Western blotting (Figure 8). In C6 cells treated with p47phox siRNA, immunocytochemistry revealed marked suppression of 8-nitro-cGMP formation (Figure 8). The p47phox knockdown also significantly suppressed production of mitochondrial superoxide and cellular oxidant as determined by fluorescence microscopy (Figure 8). As shown in Supplementary Figure S6 (at http://www.BiochemJ.org/bj/441/bj4410719add.htm), superoxide and H2O2 production were remarkably augmented by LPS/cytokine stimulation and suppressed by p47phox siRNA treatment. Levels of H2O2 were approximately 5-fold lower than those of superoxide. This may be due, at least in part, to the different methods used. Superoxide was captured by cytochrome c in situ when generated extracellularly. On the other hand, concentrations of H2O2 were determined after 10 min accumulation in culture supernatant. During a 10 min incubation, some of the H2O2 produced extracellularly would be consumed by cells so that values determined for H2O2 accumulation reflect the differences between the production and consumption.

Effects of Nox2 gene knockdown on formation of 8-nitro-cGMP and ROS production in rat C6 glioma cells

Figure 8
Effects of Nox2 gene knockdown on formation of 8-nitro-cGMP and ROS production in rat C6 glioma cells

Cells were transfected with control siRNA or p47phox-specific siRNA, as described in the Materials and methods section, followed by stimulation with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h. Immunocytochemistry with the 1G6 monoclonal antibody against 8-nitro-cGMP was used to detect intracellular 8-nitro-cGMP, or direct staining was used for mitochondrial superoxide or cellular H2O2. (A) Immunocytochemistry for 8-nitro-cGMP (1G6; top panels); fluorescent staining of mitochondrial superoxide (MitoSOX™ Red; middle panels); and fluorescent staining of intracellular H2O2 (DCDHF-DA; bottom panels). Scale bars=50 μm. (B) Relative fluorescence intensity of C6 cells, treated as described above, for 8-nitro-cGMP immunocytochemical staining (top left-hand panel), MitoSOX™ Red staining (bottom left-hand panel) and DCDHF-DA staining (bottom right-hand panel). The top right-hand panel shows the Western blot analysis for the p47phox knockdown. Results are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine plus negative control siRNA-treated group.

Figure 8
Effects of Nox2 gene knockdown on formation of 8-nitro-cGMP and ROS production in rat C6 glioma cells

Cells were transfected with control siRNA or p47phox-specific siRNA, as described in the Materials and methods section, followed by stimulation with a mixture of LPS (10 μg/ml), IFN-γ (200 units/ml), TNF-α (500 units/ml) and IL-1β (10 ng/ml) for 36 h. Immunocytochemistry with the 1G6 monoclonal antibody against 8-nitro-cGMP was used to detect intracellular 8-nitro-cGMP, or direct staining was used for mitochondrial superoxide or cellular H2O2. (A) Immunocytochemistry for 8-nitro-cGMP (1G6; top panels); fluorescent staining of mitochondrial superoxide (MitoSOX™ Red; middle panels); and fluorescent staining of intracellular H2O2 (DCDHF-DA; bottom panels). Scale bars=50 μm. (B) Relative fluorescence intensity of C6 cells, treated as described above, for 8-nitro-cGMP immunocytochemical staining (top left-hand panel), MitoSOX™ Red staining (bottom left-hand panel) and DCDHF-DA staining (bottom right-hand panel). The top right-hand panel shows the Western blot analysis for the p47phox knockdown. Results are expressed as means±S.E.M. (n=3). **P<0.01, compared with the LPS/cytokine plus negative control siRNA-treated group.

We also performed investigations to see whether mitochondria could produce ROS under conditions of p47phox knockdown. As seen in Supplementary Figure S7 (at http://www.BiochemJ.org/bj/441/bj4410719add.htm), we found that rotenone treatment along with LPS/cytokines in Nox2-knockdown cells increased mitochondrial superoxide production, independent of the Nox2-generated ROS. This finding suggests that even though the mitochondrial respiratory chain is intact in Nox2-knockdown cells, its superoxide production is impaired under the stimulation conditions used in the present study. Taken together, these results suggest that Nox2 contributes to the formation of 8-nitro-cGMP via production of H2O2, which in turn enhances mitochondrial superoxide production.

To study the role of H2O2 in formation of 8-nitro-cGMP and its association with mitochondrial superoxide production, we treated C6 cells with NO donors and H2O2. P-NONOate alone had a negligible effect on 8-nitro-cGMP formation (Figure 9A and Supplementary Figure S8A at http://www.BiochemJ.org/bj/441/bj4410719add.htm). Treatment with H2O2 slightly increased 8-nitro-cGMP formation, and PEG–SOD treatment suppressed this increase. 8-Nitro-cGMP formation significantly increased in C6 cells that were simultaneously treated with both P-NONOate and H2O2. Furthermore, PEG–SOD treatment markedly suppressed 8-nitro-cGMP formation induced by P-NONOate plus H2O2. To clarify whether extracellular H2O2 can directly accelerate mitochondrial superoxide production, we examined the effect of H2O2 treatment on MitoSOX™ Red fluorescence. In fact, we found that H2O2 treatment significantly increased mitochondrial superoxide production (Figure 9B and Supplementary Figure S8B). Taken together, in stimulated C6 cells, H2O2 derived from Nox2 may play a role in NO-dependent formation of 8-nitro-cGMP, possibly via accelerating superoxide production in mitochondria.

Increase in 8-nitro-cGMP formation and mitochondrial superoxide production in rat C6 glioma cells by H2O2 and NO treatment

Figure 9
Increase in 8-nitro-cGMP formation and mitochondrial superoxide production in rat C6 glioma cells by H2O2 and NO treatment

(A) Cells were untreated or treated with 10 or 100 μM H2O2 for 36 h, plus PEG–SOD (200 units/ml), P-NONOate (100 μM) or PEG–SOD (200 units/ml) plus P-NONOate (100 μM). Immunocytochemistry for intracellular 8-nitro-cGMP using the 1G6 monoclonal antibody against 8-nitro-cGMP, as described in the Materials and methods section, was carried out. Scale bars=50 μm. (B) In other experiments, cells were untreated or treated only with 10 or 100 μM H2O2, followed by detection of mitochondrial superoxide generation by MitoSOX™ Red staining as described in the Materials and methods section (left-hand panels). Scale bars=50 μm.

Figure 9
Increase in 8-nitro-cGMP formation and mitochondrial superoxide production in rat C6 glioma cells by H2O2 and NO treatment

(A) Cells were untreated or treated with 10 or 100 μM H2O2 for 36 h, plus PEG–SOD (200 units/ml), P-NONOate (100 μM) or PEG–SOD (200 units/ml) plus P-NONOate (100 μM). Immunocytochemistry for intracellular 8-nitro-cGMP using the 1G6 monoclonal antibody against 8-nitro-cGMP, as described in the Materials and methods section, was carried out. Scale bars=50 μm. (B) In other experiments, cells were untreated or treated only with 10 or 100 μM H2O2, followed by detection of mitochondrial superoxide generation by MitoSOX™ Red staining as described in the Materials and methods section (left-hand panels). Scale bars=50 μm.

DISCUSSION

In the present study, we have investigated the chemical and biochemical mechanisms of 8-nitro-cGMP formation, with particular focus on the roles of ROS. Our chemical analyses showed that NO itself is not sufficient to nitrate guanine nucleotides in vitro. Two reaction systems, ONOO and NaNO2/H2O2/MPO, effectively produced nitrated guanine nucleotides. At physiological pH, both ONOO and its conjugated acid ONOOH (pKa=6.8) exist, and the latter decomposes via homolysis to give the hydroxy radical (OH) and NO2 [29]. In the presence of CO2, ONOO reacts with CO2 to form nitrosoperoxycarbonate anion (ONOOCO2), which undergoes homolysis to give CO3•− and NO2 [30]. Reduction potentials have been reported for OH (E0=1.9–2.1 V) [31], NO2 (E0=1.04 V) [32], CO3•− (E0=1.5 V) [33] and guanine (E0=1.29 V) [34]. Oxidation of guanine by OH or CO3•− is thus believed to be thermodynamically favourable and would result in formation of the guanine radical cation. This cation undergoes recombination with NO2 to form 8-nitroguanine [11]. A similar mechanism may therefore operate for nitration of guanine nucleotides induced by ONOO and ONOOCO2. The NaNO2/H2O2/MPO system used in the present study was another potent mechanism for nitration of guanine nucleotides. MPO reacts with H2O2 to form MPO compound I, which can oxidize nitrite and produce NO2 [35]. This compound I may also directly oxidize guanine nucleotides because of its strong reduction potential (E0=1.35 V) [36]. In contrast, we found no production of nitrated guanine nucleotides in the reaction of guanine nucleotides with NaNO2/H2O2/HRP. HRP compound I is reportedly a much weaker one-electron oxidant compared with MPO compound I [37]. Therefore HRP compound I could not oxidize guanine nucleotides to form the corresponding cation radical.

We determined that GTP, among the guanine nucleotides examined, was the most susceptible to nitration induced by ONOO. GTP makes up nearly 25% of the total intracellular nucleotide triphosphate pool, and it acts as a versatile nucleotide as it participates in many critical physiological functions, including RNA synthesis, cell signalling through activation of GTP-binding proteins and production of the second messenger cGMP [38]. Electrophilic activity of nitrated guanine nucleotides varies depending on their structures (Md. M. Rahaman, T. Sawa and T. Akaike, unpublished work). Among the 8-nitroguanine derivatives examined, 8-nitro-cGMP showed the highest electrophilic activity at neutral pH, and the electrophilic activity decreasing in the following order: 8-nitro-cGMP>>8-nitroguanosine>8-nitro-GTP/8-nitro-GMP. The second-order rate constants for the reaction of glutathione with those molecules were determined to be 0.05 (8-nitro-cGMP), 0.018 (8-nitroguanosine), 0.01 (8-nitro-GTP), and 0.008 (8-nitro-GMP) M−1·s−1 respectively ([3] and Md. M. Rahaman, T. Sawa and T. Akaike, unpublished work). In this context, 8-nitro-GTP may stably be present in cytosol where glutathione is present abundantly. On the other hand, our previous work demonstrated that 8-nitro-GTP can act as a substrate for sGC, with 8-nitro-cGMP formed as a product [5]. These data suggest that 8-nitro-GTP may be implicated in electrophile signalling via acting as an excellent substrate for guanylyl cyclase to produce 8-nitro-cGMP, rather than inducing direct protein S-guanylation. Although ONOO has been considered as a toxic by-product of NO and ROS formation, accumulating evidence suggests a signalling function. Kang et al. [39] reported that ONOO formed under sulfur amino acid deprivation activates the Nrf2 signal via the PI3K (phosphoinositide 3-kinase) [39]. Our previous study revealed that the Nrf2 signal can be activated, at least in part, via Keap1 S-guanylation, as supported by the fact that reduction of 8-nitro-cGMP formation by sGC inhibitor appreciably suppressed Nrf2 nuclear translocation and haem oxygenase-1 gene expression, without affecting RNOS formation [5]. It is thus suggested that ONOO may contribute to activation of Nrf2-dependent responses via multiple mechanisms, including increased production of electrophilic molecules such as 8-nitro-cGMP and activation of the PI3K pathway.

The present study shows that Nox2 and mitochondria are two important sources of ROS in rat C6 glioma cells stimulated by LPS/cytokines and are critically involved in regulation of 8-nitro-cGMP formation. The levels of H2O2 from stimulated C6 cells were determined to be 1.63±0.03 nmol/min per mg of protein (or 3.0±0.2 μM in culture supernatant for 10 min incubation under the conditions used in the present study). In separate experiments, 10 μM H2O2 added exogenously to non-stimulated cells sufficiently induced mitochondrial superoxide production and 8-nitro-cGMP formation in the presence of NO donor (Supplementary Figure S8). These observations suggest that ~10 μM H2O2 is required to promote mitochondrial superoxide production in C6 cells. Recent studies have suggested that superoxide production in mitochondria is accelerated by H2O2 [40]. We therefore speculate that H2O2 derived from Nox2 contributes to the acceleration of mitochondrial superoxide generation, and hence, 8-nitro-cGMP formation as illustrated in Figure 10. In this context, a study by Zhang et al. [41] demonstrated local production of superoxide by Nox4 in sarcoplasmic reticulum, suggesting regulation of ROS signalling by different Nox enzymes in certain cell types, warranting further investigation of 8-nitro-cGMP signalling in these cells.

Schematic diagram of possible mechanisms involved in cell formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with LPS/cytokines

Figure 10
Schematic diagram of possible mechanisms involved in cell formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with LPS/cytokines

iNOS, inducible NOS; L-Arg, L-arginine.

Figure 10
Schematic diagram of possible mechanisms involved in cell formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with LPS/cytokines

iNOS, inducible NOS; L-Arg, L-arginine.

In summary, in the present study we have verified that ROS play a pivotal role in the formation of 8-nitro-cGMP in C6 cells stimulated with LPS/cytokines. Superoxide, most probably derived from mitochondria, is directly involved in the formation of 8-nitro-cGMP, whereas H2O2 generated by Nox2 also has an important role by increasing mitochondrial superoxide production. Our data thus suggest that 8-nitro-cGMP may serve as a unique second messenger for ROS signalling in the presence of NO. Greater understanding of 8-nitro-cGMP formation in relation to mitochondrial function and NADPH oxidase regulation may help us develop new diagnostic methods and treatment of diseases related to dysregulation of NO and ROS [42].

Abbreviations

     
  • DCDHF-DA

    2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • DHE

    dihydroethidium

  •  
  • DTPA

    diethylenetriamine penta-acetic acid

  •  
  • ECD

    electrochemical detection

  •  
  • ESI

    electrospray ionization

  •  
  • HRP

    horseradish peroxidase

  •  
  • IFN-γ

    interferon-γ

  •  
  • IL-1β

    interleukin-1β

  •  
  • Keap1

    Kelch-like ECH-associated protein 1

  •  
  • LC

    liquid chromatography

  •  
  • LPS

    lipopolysaccharide

  •  
  • mETC

    mitochondrial electron transport chain

  •  
  • MPO

    myeloperoxidase

  •  
  • MS/MS

    tandem MS

  •  
  • 8-nitro-cGMP

    8-nitroguanosine 3′,5′-cyclic monophosphate

  •  
  • Nox2

    NADPH oxidase 2

  •  
  • PDA

    photodiode array

  •  
  • PEG

    poly(ethylene glycol)

  •  
  • pGC

    particulate-type guanylyl cyclase

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • P-NONOate

    propylamine NONOate {CH3N[N(O)NO](CH2)3NH2+CH3, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene}

  •  
  • RNOS

    reactive nitrogen oxide species

  •  
  • ROS

    reactive oxygen species

  •  
  • sGC

    soluble-type guanylyl cyclase

  •  
  • SIN-1

    3-morpholinosydnonimine

  •  
  • siRNA

    small interfering RNA

  •  
  • SOD

    superoxide dismutase

  •  
  • TNF-α

    tumour necrosis factor-α

AUTHOR CONTRIBUTION

Khandaker Ahtesham Ahmed and Tomohiro Sawa designed and performed experiments, analysed the data and wrote the paper. Hideshi Ihara, Shingo Kasamatsu and Jun Yoshitake contributed to the acquisition of LC-MS/MS data. Tatsuya Okamoto, Md. Mizanur Rahaman and Shigemoto Fujii performed immunocytochemistry, cytotoxicity assays, superoxide and H2O2 measurements, and Western blotting experiments. Takaaki Akaike designed and supervised the project, and wrote the paper. All authors read and approved the final version of the manuscript.

We thank Judith B. Gandy for excellent editing of the paper prior to submission.

FUNDING

This work was supported, in part, by the Ministry of Education, Sciences, Sports and Technology (MEXT) Grants-in-Aid for Scientific Research [grant numbers 21390097, 21590312] and Grants-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Area) [grant numbers 20117001, 20117005] and the Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) programme [grant number 09801194].

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Supplementary data