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Iron Released by Sodium Nitroprusside Contributes to Heme Oxygenase-1 Induction via the cAMP-Protein Kinase A-Mitogen-Activated Protein Kinase Pathway in RAW 264.7 Cells

Department of Pharmacology, College of Medicine and Institute of Health Sciences, Gyeongsang National University, Jinju, South Korea

Received October 31, 2005; accepted January 26, 2006

	Abstract

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Nitric oxide (NO) is a potent inducer of heme oxygenase (HO)-1, and NO-induced HO-1 expression is dependent on the cGMP-signaling pathway. Sodium nitroprusside (SNP) produces NO and iron. However, it is unclear whether NO is exclusively responsible for induction of HO-1 by SNP in RAW 264.7 cells. We tested our hypothesis that iron may contribute more to the SNP induction of HO-1 than does NO by comparing the HO-1 protein level and the production of NO in RAW 264.7 cells treated with SNP and S-nitroso-N-acetyl-DL-penicillamine (SNAP). Although SNP induced less NO production than SNAP, SNP induced the production of more HO-1 protein than did SNAP. Deferoxamine (DFO) decreased SNP- but not SNAP-induced HO-1 expression but did not decrease the production of NO. SNP-induced HO-1 was significantly inhibited by specific protein kinase A (PKA) inhibitors or an antagonist of cAMP but not by guanylyl cyclase inhibitors. Exogenous iron (ferric ammonium citrate or ferricyanide) and forskolin increased the level of HO-1, which was inhibited by PKA inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). These results indicate that iron and cAMP, but not cGMP, play crucial roles in the induction of HO-1 in RAW 264.7 cells. Moreover, DFO and inhibitors of extracellular signal-related kinases 1/2 or c-Jun NH₂-terminal kinase inhibited HO-1 production induced by SNP. This study illustrates that iron rather than NO from SNP contributes to HO-1 induction. Therefore, studies on the effects of SNP should consider the role of iron in some biological functions. We concluded that iron released by SNP contributes to HO-1 induction via the cAMP-PKA-mitogen-activated protein kinase pathway.

NO is a free radical involved in the regulation of many physiological functions, including endothelium-dependent vasodilation, neurotransmission, and the cell-mediated immune response (Feldman et al., 1993; Moncada and Higgs, 1993; MacMicking et al., 1997). Alterations in NO synthesis are implicated in the pathophysiology of inflammation, septic shock, atherosclerosis, and glomerulonephritis (Laskin et al., 1994; Vane et al., 1994; Nathan, 1997; Furusu et al., 1998). NO is regarded as a pharmacologically active molecule of SNP; therefore, many of the biological actions of SNP are known to be mediated through the activation of guanylate cyclase and cGMP production (Kim et al., 1995; Polte et al., 2000). Because NO is known as a potent inducer of HO-1, one may think with no doubt that the induction of HO-1 by SNP is dependent on the cGMP-signaling pathway. However, it is quite conceivable that SNP may regulate HO-1 induction, a gene known to be sensitive to oxidative stress, via a change in the redox state by releasing free iron from SNP rather than only by the action of NO.

We investigated the possibility that a factor or factors other than NO, especially iron, are involved in the regulation of SNP-mediated HO-1 induction. We compared HO-1 induction in response to SNP, a NO⁺ generator, with that induced by SNAP, a NO· generator without any iron in its structure. We also investigated the possible signaling pathway involved in SNP-induced HO-1 expression, particularly the roles of cAMP and MAPK.

	Materials and Methods

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Materials. Dulbecco's modified Eagle's medium, fetal bovine serum, and antibiotics (penicillin/streptomycin) were obtained from Gibco BRL (Rockville, MD). Anti-HO-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-p-ERK 1/2, anti-p-JNK, and anti-p-p38 antibodies were obtained from Cell Signaling Technology (Beverly, MA). LY83583, H89, carboxy-PTIO, SB203580, and PD98059 were obtained from Calbiochem (San Diego, CA). All other chemicals, including SNP, SNAP, deferoxamine (DFO), KT5720, KT5823, Rp-cAMPS, hydroxocobalamin, ferric ammonium citrate (FAC), and potassium ferricyanide [K₃Fe(CN)₆] were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell Culture. RAW 264.7 murine macrophages were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in a humidified atmosphere of 95% air and 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Cell Treatment. Cells were pretreated with H89 or KT5720 [a specific protein kinase A (PKA) inhibitor], Rp-cAMPS (an antagonist of cAMP), LY83583 or ODQ (an inhibitor of soluble guanylyl cyclase), KT5823 (a specific PKG inhibitor), PD98059 (a selective ERK inhibitor), SB203580 (a p38 inhibitor), SP600125 (a selective JNK inhibitor), or DFO (a free iron chelator) for 1 h in serum-free medium, after which SNP or SNAP was added to the cells.

Assay for Nitrite Production. NO was measured as its stable oxidative metabolite, nitrite, as described previously (Green and Schaefer, 1981). After 18-h incubation, 500 µl of the culture medium was mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). The absorbance at 550 nm was measured, and the nitrite concentration was determined using a curve calibrated with sodium nitrite standards.

Western Blot Analysis. The cells were harvested and lysed with buffer containing 0.5% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5, and protease inhibitors. The protein concentration of each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL). To detect HO-1, 20 µg of the total protein was electrophoresed on a 10% polyacrylamide gel, and to detect phospho-MAPKs, 30 µg of the total protein was electrophoresed on a 12% polyacrylamide gel. The gels were transferred to polyvinylidene difluoride membranes by a semidry electrophoretic transfer at 15 V for 60 to 75 min. The polyvinylidene difluoride membranes were blocked overnight at 4°C in 5% BSA. The cells were incubated with the primary antibodies, diluted 1:500 in Tris-buffered saline/Tween 20 containing 5% BSA for 2 h, and then incubated with the secondary antibody at room temperature for 1 h. Anti-goat IgG specific for HO-1 or anti-rabbit IgG specific for p-ERK 1/2, p-JNK, or p-p38 was used as the secondary antibody (1:5000 dilution in Tris-buffered saline/Tween 20 containing 1% BSA). The signals were detected by enhanced chemoluminescence (Amersham, Piscataway, NJ).

Determination of cAMP Content. The cells were treated with SNP and test substances for 4 h, and then medium was aspirated from plate. After adding 1 ml of 0.1 M HCl to the cells, the cells were incubated for 20 min at room temperature. The cells were scraped and centrifuged at 1000g for 10 min. The cAMP in the supernatant was analyzed with the cAMP EIA Kit (Cayman Chemical, Ann Arbor, MI) as described by the manufacturer.

Data Analysis. Scanning densitometry was performed using an Image Master VDS (Pharmacia Biotech Inc., San Francisco, CA). Treatment groups were compared using one-way analysis of variance, and the Newman-Keuls test was used to locate any significant differences identified in the analysis of variance. P < 0.05 or <0.01 was accepted as significant.

	Results

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The NO Donors SNP and SNAP Have Different Effects on the Production of Nitrite and HO-1 Level in RAW 264.7 Cells. NO is a potent inducer of HO-1, and NO-induced HO-1 induction is dependent on the GMP-signaling pathway (Maines, 1997; Immenschuh et al., 1998a). We compared the ability of SNP and SNAP to induce nitrite production in RAW 264.7 cells and investigated whether SNP- or SNAP-induced NO production coincides with HO-1 induction. The amount of nitrite produced after 24 h in the medium was 26.6 µM, with 500 µM SNP and 151.1 µM with SNAP, indicating that efficiency of generation of nitrite by SNAP was 5.7 times greater than that of SNP (Fig. 1A). SNAP also had a greater capability for nitrite production than did SNP in terms of concentration dependence (Fig. 1B). For example, at 24 h of treatment, SNAP-induced nitrite production (340.7 µM) was 7.7 times that induced by SNP (44.4 µM). In contrast, SNP was more effective at increasing the HO-1 protein level than SNAP, and SNAP showed only a weak ability to induce HO-1 in a time- and concentration-dependent manner (Fig. 2, A and B). Treatment with 500 µM SNP caused HO-1 production to reach a maximum level at 8 h, and this level was sustained until 24 h. These results suggest that the amount of NO is not proportional to HO-1 production in RAW 264.7 cells. Furthermore, it indicates that factor(s) other than NO possibly contribute more to SNP induction of HO-1.

View larger version (24K): [in this window] [in a new window]   Fig. 1. The NO donors SNP and SNAP have different effects on nitrite production in the RAW 264.7 murine macrophage cell line. SNAP caused the production of more nitrite than SNP in RAW 264.7 cells. The kinetics of nitrite production by 500 µM SNP and 500 µM SNAP (A) and concentration-dependent nitrite production by SNP and SNAP after 24 h of treatment were measured in the culture medium using the Griess reagent (B). Data are mean ± S.E.M. values from three or more independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 2. SNP and SNAP regulate HO-1 induction differently and in a concentration- and time-dependent manner. RAW 264.7 cells were treated with SNP or SNAP at doses of 1, 10, 100, 250, or 500 µM (A) for 8 h and at 500 µM for 1, 2, 4, 8, 16, or 24 h (B). HO-1 protein levels were measured in SNP- or SNAP-treated cells by Western blot analysis as described under Materials and Methods. Thirty micrograms of protein extract from SNP- and SNAP-treated cells was loaded in same SDS-polyacrylamide gel to compare the level of HO-1 protein induced by SNP and SNAP. The band intensities were assessed by scanning densitometry. The data are presented as the mean ± S.E.M. of three independent experiments. One-way analysis of variance was used to compare the multiple group means followed by Newman-Keuls test (significance compared with the control, **, P < 0.01; control level = 1).

View larger version (38K): [in this window] [in a new window]   Fig. 3. The role of iron in the regulation of HO-1 and NO production by SNP and SNAP. Cells were pretreated with the iron chelator DFO (100, 300, or 500 µM) for 1 h before treatment with SNP or SNAP. After 8-h induction with SNP (A and B) or SNAP (C and D), the HO-1 protein level was measured in the cell extract using Western blot analysis, and nitrite production in the medium was measured using the Griess reagent. The band intensities were assessed by scanning densitometry. The data are presented as the mean ± S.E.M. of three independent experiments. One-way analysis of variance was used to compare the multiple group means followed by Newman-Keuls test (significance compared with the control, **, P < 0.01; significance compared with SNP: , P < 0.05 or , P < 0.01; control level = 1).

View larger version (24K): [in this window] [in a new window]   Fig. 4. The involvement of the PKA pathway via cAMP on SNP-induced HO-1 expression. A, cells were exposed to the specific PKA inhibitor (H89 or KT5720, 10 µM) and an antagonist of cAMP (Rp-cAMPS, 100 µM) or to an inhibitor of soluble guanylyl cyclase (ODQ or LY83583, 10 µM) and a specific PKG inhibitor (KT5823, 10 µM), for 1 h and then treated with 500 µM SNP for 8 h. B, to confirm the involvement of the PKA pathway via cAMP on SNP-induced HO-1 production, RAW 264.7 cells were treated with the cAMP generator forskolin. The data are presented as the mean ± S.E.M. of three independent experiments. One-way analysis of variance was used to compare the multiple group means followed by Newman-Keuls test (significance compared with the control, *, P < 0.05 or **, P < 0.01; significance compared with SNP or forskolin, , P < 0.05 or , P < 0.01; control level = 1).

Induction of HO-1 by SNP through the ERK and JNK Pathways. HO-1 is induced by many stimuli that also enhance the activity of MAPKs. We hypothesized that activation of MAPKs might be involved in the signaling pathways that induce HO-1 gene expression. We investigated the signal-transduction pathway mediating the SNP-induced increase in HO-1 expression. After SNP treatment, the HO-1 protein level decreased significantly after the addition of PD98059 (40 µM), a specific ERK inhibitor, and SP600125 (50 µM), a specific JNK inhibitor. In contrast, pretreatment with SB203580 (5 µM), a p38 inhibitor, did not alter SNP-induced HO-1 expression (Fig. 5A). To confirm the specificity of the inhibitors PD98059 and SP600125, we performed Western blot analysis using different doses of inhibitors and found that PD98059 and SP600125 inhibited specifically SNP-induced HO-1 expression (Fig. 5B). These results indicate that MAPKs differentially regulate SNP-induced HO-1 expression, which means that ERK and JNK, but not p38, are involved in the SNP-mediated induction of HO-1 protein.

View larger version (28K): [in this window] [in a new window]   Fig. 5. SNP induction of HO-1 protein through the ERK and JNK pathways. A, the HO-1 protein level was measured in the cells treated with SNP (500 µM) for 8 h after pretreatment with PD98059 (40 µM), SB203580 (5 µM), or SP600125 (50 µM). B, PD98059 (10, 20, or 40 µM) and SP600125 (10, 25, or 50 µM) were added to specifically inhibit the ERK or JNK pathway in a concentration-dependent manner. The data are presented as the mean ± S.E.M. of three independent experiments. One-way analysis of variance was used to compare the multiple group means followed by Newman-Keuls test (significance compared with the control, **, P < 0.01; significance compared with SNP, , P < 0.01; control level = 1).

Phosphorylation of ERK and JNK by SNP through the cAMP-Dependent PKA Pathway. Treatment with MAPK inhibitors showed that up-regulation of HO-1 by SNP occurs through the ERK and JNK pathways. We next examined whether SNP facilitates phosphorylation of ERK or JNK. Phosphorylation of ERK1/2 was first detected 10 min after SNP treatment, was sustained until 8 h after treatment, and then decreased thereafter (Fig. 6A). The phosphorylation level of JNK (p54/p46) was relatively weak and peaked 8 h after SNP treatment (Fig. 6A). These results suggest that the ERK and JNK pathways are involved in HO-1 induction by SNP. Because the cAMP-dependent PKA pathway is involved in SNP-induced HO-1 expression, we investigated the relationship between PKA and MAPK. Pre-incubation of cells with H89 significantly inhibited the SNP-induced phosphorylation of ERK1/2 and JNK, indicating that SNP induces HO-1 via PKA-MAPK (Fig. 6B). Moreover, we confirmed that cAMP directly activates ERK1/2 and JNK phosphorylation by showing that a cAMP analog, 8-Br-cAMP (100 µM), activated ERK1/2 or JNK phosphorylation, which was inhibited by pretreatment of Rp-cAMPS, an antagonist of cAMP (Fig. 6C).

View larger version (28K): [in this window] [in a new window]   Fig. 6. SNP-induced phosphorylation of ERK and JNK through the PKA pathway. A, the cell lysate was extracted from the cells treated with SNP (500 µM) at the indicated times, and Western blot analysis was performed using anti-p-ERK1/2 and anti-ERK1/2 or anti-p-SAPK/JNK (p54/p46) and anti-SAPK/JNK antibodies (Thr183/Tyr185). B, the activation of the SNP-induced ERK or JNK pathway was prevented by pretreatment with the specific PKA inhibitor H89 or the free iron chelator DFO. C, TO confirm the direct activation of ERK1/2 or JNK pathway by a cAMP analog, cells were treated with 8-Br-cAMP (100 µM) for 30 min with or without Rp-cAMPS. Each lane was loaded with 60 µg of the cell lysates. The data were confirmed in two experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 7. The involvement of iron in HO-1 induction through cAMP. A, RAW 264.7 cells were treated with FAC (1 mM) or potassium ferricyanide (1 mM) with or without H89, a specific PKA inhibitor. After 8 h of treatment, the HO-1 protein level was determined by Western blot analysis. The data are presented as the mean ± S.E.M. of three independent experiments. One-way analysis of variance was used to compare the multiple group means followed by Newman-Keuls test (significance compared with the control, **, P < 0.01; significance compared with FAC or ferricyanide, , P < 0.01; control level = 1). B, cell lysates were obtained from RAW 264.7 cells treated with FAC (1 mM) or potassium ferricyanide (1 mM) for 4 h, and the cAMP analysis was performed as described under Materials and Methods. C and D, to investigate the role of NO or cyanide in the SNP-induced HO-1 expression, cells were pretreated with the NO scavenger carboxy-PTIO (250 or 500 µM) or HCB (100 or 300 µM) and the CN- inhibitor, composed of 8 U/ml rhodanese with 5 mM Na2S2O3, for 30 min and then treated with SNP for 8 h. The data were confirmed by repeated experiments.

	Discussion

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In general, it is well recognized that NO and cGMP are signal-coupling molecules that are responsible for many pharmacological actions manifested by SNP. Thus, NO is regarded as an exclusive molecule responsible for SNP-induced relaxation of vascular smooth muscle. However, apart from NO, SNP has been described as an NO⁺ donor (Stamler et al., 1992). Thus, we wanted to address whether there is a possibility that SNP induces HO-1 in RAW 264.7 cells not by NO. The major finding of the present study was that SNP induced HO-1 expression in RAW 264.7 cells is via cAMP-MAPK pathways, which is mediated by iron, not by NO. By comparing nitrite production and HO-1 expression based on concentrations, we found that SNP induced a much higher HO-1 protein level than SNAP, although the amount of nitrite donated from SNP in the medium was much lesser than SNAP. Furthermore, we found that HO-1 expression is dependent on cellular free iron. The iron chelator DFO inhibited the induction of HO-1 in response to SNP without reducing the nitrite production; rather, it increased SNP-mediated nitrite production to some degree. This suggests that iron rather than NO contributed to SNP induction of HO-1. The reason for the increase of nitrite by DFO in SNP-treated cells is not clear. However, it can be speculated that reduced cellular iron concentrations by DFO significantly influenced inducible nitric-oxide synthase expression (Weiss et al., 1994). Thus, iron deprivation caused the enhanced inducible nitric-oxide synthase expression, which results in an increase of NO formation. However, it will require further investigation. On the other hand, DFO has been also suggested to directly scavenge reactive oxygen species (ROS), including hydroxyl radical and superoxide anion (Halliwell, 1989), which may increase the efficiency of NO, such as prolongation of half-life without affecting the change of concentration, so that it enhanced NO action in the case of SNAP, which caused an increase of HO-1 induction. Therefore, SNAP-induced HO-1 expression is NO-dependent. We believe that the inhibitory effects of DFO shown in SNP-induced HO-1 expression are related with chelatable iron released from SNP. Therefore, different effects of DFO on SNP- and SNAP-exposed cells suggest that induction of HO-1 by these chemicals is under different regulatory mechanisms. Indeed, free iron is known to have effects on the gene expression of several proteins involved in iron metabolism, transferrin receptor, and ferritin (Suematsu et al., 1994; Raju and Maines, 1996; Brenneisen et al., 1998), and on enzymes potentially involved in oxidant or inflammatory conditions—inducible nitric-oxide synthase (Tenhunen et al., 1969), the aconitases (Tenhunen et al., 1970), and HO-1 (Ryter et al., 2000). Furthermore, evidence from the results of carboxy-PTIO or HCB, a known scavenger of NO, strongly indicates that iron is more critical than NO in SNP induction of HO-1 in RAW 264.7 cells. We found that any concentrations of carboxy-PTIO (250500 µM) or 100 to 300 µM HCB failed to inhibit SNP-induced HO-1 protein level; even though 500 µM HCB showed weak inhibition of SNP induction of HO-1. Cyanide does not seem to induce HO-1 in vitro (Motterlini et al., 1996) and is less likely to have contributed to the stimulation of HO activity by SNP, because the specific cyanide chelator rhodanese in Na₂S₂O₃ did not inhibit HO-1 induction by SNP. Thus, the present study emphasizes that the metabolites of SNP other than NO are functionally important in some biological system, such as HO-1 induction. In fact, SNP generates ROS during the redox cycling of nitroprusside (Bates et al., 1991; Ramakrishna Rao and Cederbaum, 1996), and it is metabolized to a number of products, such as NO, iron, cyanide, or oxygen-free radicals (Ramakrishna Rao and Cederbaum, 1996). It is well known that SNP forms a coordination complex of a ferrous ion (Fe²⁺) with five CN^- anions and a nitrosonium ion (NO⁺). We propose here that iron released by SNP plays a critical role for the induction of HO-1 in RAW 264.7 cells. However, contribution of S-nitrosothiol, which may have been produced by an interaction of the NO⁺ from SNP with thiol groups in HO-1 induction, cannot be excluded. Because thiols, by virtue of their ability to be reversibly oxidized, are recognized as key components involved in the maintenance of redox balance. Furthermore, increasing evidence suggests that thiol groups located on various molecules act as redox-sensitive switches, thereby providing a common trigger for a variety of ROS and reactive nitrogen species-mediated signaling events. However, it is unlikely that SNAP can react directly with thiol groups to form S-nitrosothiol because NO reacts with metals to form NO⁺ (Stamler et al., 1992), which then reacts with thiol groups to form S-nitrosothiols. We believe that the differences in iron homeostasis, and, to a lesser extent, the formation of S-nitrosothiol, may differentially affect the redox state of the cell. This might explain the observed differences in HO-1 induction by SNP and SNAP.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Possible mechanism by which SNP induces HO-1 expression. SNP-generated iron may activate the cAMP-PKA pathway, which is linked to MAPK pathway, resulting in HO-1 expression. SNP-donated NO maybe involve in the HO-1 expression partly through cGMP-mediated inhibition of cAMP breakdown (Polte and Schroder, 1998).

However, the inhibition of SNP-induced HO-1 expression by DFO was relatively modest compared with the inhibition by the inhibitor of cAMP or PKA pathway, which suggests that some other pathway is involved in the SNP-activated PKA pathway. Actually, the inhibitor of soluble guanylyl cyclase ODQ or LY83583 little inhibited the HO-1 induction by SNP, suggesting that SNP-generated NO possibly activated the cGMP pathway, which is linked to the cAMP-PKA pathway. This possibility had been suggested by the report that cAMP mediates antioxidant protection by NO donors in endothelial cells as a likely consequence of cGMP-dependent inhibition of cAMP breakdown (e.g., through the blockade of phosphodiesterase III) (Polte and Schroder, 1998). The reports that HO-1 is also responsive to gene activation by cAMP (Nakagawa et al., 1988; Pizurki and Polla, 1994; Durante et al., 1997a) and that HO-1 is induced by cAMP and cAMP response element activation (Immenschuh et al., 1998a) support firmly our results that SNP induces HO-1 by activation of iron-cAMP-PKA-MAPK.

We conclude that SNP changes cellular iron homeostasis by generating iron and NO⁺. The increased cellular free iron level may activate the cAMP-PKA pathway, which is linked to the MAPK pathway, especially ERK1/2 or JNK, which regulates HO-1 expression via the cAMP response element/activator protein-1 site of the HO-1 gene (Alam et al., 1994). To our knowledge, ours is the first study to report that the free iron-cAMP-ERK1/2 or JNK signaling pathways are involved in SNP-mediated HO-1 induction. It should be noted, therefore, that care is needed when interpreting studies measuring the effects of SNP, because the role of iron molecules sometimes exceeds that of NO in some conditions such as HO-1 induction. After this article was accepted for publication, Wang et al. (2006) reported data supporting our conclusion that iron, not NO, is responsible for degradation of the iron regulation protein IRP2 by SNP. Therefore, SNP can no longer be regarded as an "NO-donor".

	Acknowledgements

 
We thank Young Shin Ko for excellent technical assistance.

	Footnotes

 
This work was supported by the Korea Research Foundation (R03-2004-000-10033-0).

ABBREVIATIONS: HO, heme oxygenase; BSA, bovine serum albumin; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DFO, deferoxamine; ERK, extracellular signal-regulated kinase; FAC, ferric ammonium citrate; HCB, hydroxocobalamin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKG, protein kinase G; SNAP, S-nitroso-N-acetyl-DL-penicillamine; SNP, sodium nitroprusside; ODQ, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; ROS, reactive oxygen species; p-, phospho-; Rp-cAMPS, adenosine-3',5'-cyclic monophosphorothioate, Rp isomer; LY83583, 6-anilino-5,8-quinolinedione; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PD98059, 2'-amino-3'-methoxyflavone; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone; KT-5823, (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester; KT-5720, (9S,10S,12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester.

Address correspondence to: Dr. Ki Churl Chang, Department of Pharmacology, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, South Korea. E-mail: kcchang{at}gsnu.ac.kr

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