The Critical Role of Nitric Oxide Signaling, via Protein S-Guanylation and Nitrated Cyclic GMP, in the Antioxidant Adaptive Response

Abstract

A nitrated guanine nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 μm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys434 of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response. A nitrated guanine nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 μm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys434 of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response. IntroductionNitric oxide (NO) plays diverse physiological roles in vascular regulation, neuronal transmission, inflammation, and host defense against microbial pathogens. In vascular and neuronal systems, NO performs these functions mainly through a cGMP-dependent mechanism (1Murad F. J. Clin. Invest. 1986; 78: 1-5Crossref PubMed Scopus (672) Google Scholar, 2Bredt D.S. Hwang P.M. Snyder S.H. Nature. 1990; 347: 768-770Crossref PubMed Scopus (2678) Google Scholar), but the presence and contribution of other pathways that are not directly linked to cGMP have also been suggested to operate in certain aspects of NO signaling occurring in various cells and tissues in different organisms (3Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1352) Google Scholar, 4Schopfer F.J. Baker P.R. Freeman B.A. Trends Biochem. Sci. 2003; 28: 646-654Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 5Radi R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4003-4008Crossref PubMed Scopus (1209) Google Scholar). Among these other mechanisms is chemical modification of biomolecules, including nitrosylation and nitration of amino acids, proteins, and lipids, this modification being induced by NO-derived reactive nitrogen oxide species (RNOS), 3The abbreviations used are: RNOSreactive nitrogen oxide species8-nitro-cGMP8-nitroguanosine 3′,5′-cyclic monophosphatePDEphosphodiesteraseROSreactive oxygen speciesMALDImatrix-assisted laser desorption ionizationTOFtime-of-flightMSmass spectrometryMS/MStandem mass spectrometrySNAPS-nitroso-N-acetylpenicillamineBSObuthionine sulfoximineiNOSinducible NO synthaseLPSlipopolysaccharideIFN-γinterferon-γTNFαtumor necrosis factor αIL-1βinterleukin-1βl-NMMANω-monomethyl-l-argininePBSphosphate-buffered salinesGCsoluble guanylate cyclaseLCliquid chromatographyHPLChigh performance liquid chromatographyECDelectrochemical detectionESIelectrospray ionizationDTTdithiothreitolNEMN-ethylmaleimidesiRNAsmall interfering RNAHSAhuman serum albuminSNO-HSAS-nitrosylated human serum albuminc[15N5]GMP[U-15N5, 98%]guanosine 3′,5′-cyclic monophosphate[15N5]GTP[U-15N5, 98%]guanosine 5′-triphosphate. such as peroxynitrite (ONOO−) and nitrogen dioxide (NO2) (3Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1352) Google Scholar, 4Schopfer F.J. Baker P.R. Freeman B.A. Trends Biochem. Sci. 2003; 28: 646-654Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 5Radi R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4003-4008Crossref PubMed Scopus (1209) Google Scholar).RNOS cause nitration of nucleic acids in addition to amino acids, proteins, and lipids. We previously found that nitrated guanine derivatives, including 8-nitroguanine and 8-nitroguanosine, formed in cultured cells and in tissues from murine viral pneumonia and human lung disease (6Akaike T. Okamoto S. Sawa T. Yoshitake J. Tamura F. Ichimori K. Miyazaki K. Sasamoto K. Maeda H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 685-690Crossref PubMed Scopus (138) Google Scholar, 7Yoshitake J. Akaike T. Akuta T. Tamura F. Ogura T. Esumi H. Maeda H. J. Virol. 2004; 78: 8709-8719Crossref PubMed Scopus (52) Google Scholar, 8Terasaki Y. Akuta T. Terasaki M. Sawa T. Mori T. Okamoto T. Ozaki M. Takeya M. Akaike T. Am. J. Respir. Crit. Care Med. 2006; 174: 665-673Crossref PubMed Scopus (35) Google Scholar). An important finding was that 8-nitroguanosine possessed a unique redox activity, which suggested a critical biological role of guanine nitration (9Sawa T. Akaike T. Ichimori K. Akuta T. Kaneko K. Nakayama H. Stuehr D.J. Maeda H. Biochem. Biophys. Res. Commun. 2003; 311: 300-306Crossref PubMed Scopus (53) Google Scholar). In fact, we recently discovered that a novel nitrated cyclic nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is generated after NO production (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar). 8-Nitro-cGMP had the strongest redox activity among the nitrated guanine derivatives tested, and this property was distinct from that activating cGMP-dependent protein kinases. Being an electrophile, 8-nitro-cGMP effectively reacted with sulfhydryl groups of cysteine residues and formed a protein-S-cGMP adduct, via a post-translational modification named protein S-guanylation (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar).Although we successfully determined the chemical identification of 8-nitro-cGMP in our earlier study (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar), we had not yet achieved rigorous quantification of 8-nitro-cGMP in biological systems (e.g. cells), and mechanisms of 8-nitro-cGMP action were still to be clarified. One specific question concerned what constituted a target molecule for nitration: GTP, a substrate of guanylate or Also, signaling pathways of 8-nitro-cGMP were not this we the redox protein protein as of the for the physiological significance and of S-guanylation to be (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar, M.H. Fujii S. Okamoto T. S. S. Sawa T. Akaike T. J. PubMed Scopus Google Scholar). The factor is of the cellular defense mechanisms against and H. Yamamoto M. Trends Med. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Kobayashi A. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: PubMed Scopus Google Scholar, Chem. Res. PubMed Scopus (315) Google Scholar, Y. H. K. T. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar). Nrf2 is a factor and antioxidant that in against and reactive oxygen species Nrf2 is and by Nrf2 is by an which in the Y. Kobayashi A. H. M. J. K. Yamamoto M. 2007; PubMed Scopus Google and as an of the for to or Nrf2 is and which in induction of a of have been to the reactive cysteine residues in and modification of the cysteine in that a certain of cysteine residues K. Y. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, A. A. Biochem. J. 2004; PubMed Scopus Google Scholar, A. Y. T. K. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar, S. Biol. Med. PubMed Scopus Google Scholar, A. Biochem. J. PubMed Scopus Google Scholar, M. Y. Kaneko H. Nakayama Y. M. Y. Y. Yamamoto M. Biol. PubMed Scopus Google Scholar). In fact, we cysteine residues and that are critical to activity to Nrf2 the to T. T. Kobayashi A. J. J. H. Yamamoto M. Biol. 28: PubMed Scopus Google Scholar). These were by of a with and or of T. T. Kobayashi A. J. J. H. Yamamoto M. Biol. 28: PubMed Scopus Google the present study, we to the and aspects of 8-nitro-cGMP in the cells and the potential roles of 8-nitro-cGMP in protein S-guanylation induced by We first and intracellular of 8-nitro-cGMP after NO production in C6 glioma cells. A finding was that the of 8-nitro-cGMP formation was comparable with or higher than that of cGMP formed in cells. important is the 8-nitro-cGMP generated induced S-guanylation of and increased Nrf2-dependent 8-nitro-cGMP was against effects of hydrogen through of Nrf2 target such as We via matrix-assisted laser desorption/ionization mass spectrometry critical cysteine of which was modified by 8-nitro-cGMP in cells. study substantial of the biological significance and the formation mechanism of the of 8-nitro-cGMP formed in cells. of our study is of a of by which our that are a that is the cysteine in the molecule to Nrf2 nitration of biological by NO-derived such as and been (3Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1352) Google Scholar, 4Schopfer F.J. Baker P.R. Freeman B.A. Trends Biochem. Sci. 2003; 28: 646-654Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 5Radi R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4003-4008Crossref PubMed Scopus (1209) Google Scholar). the various nitrated to 8-nitro-cGMP unique (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar, M.H. Fujii S. Okamoto T. S. S. Sawa T. Akaike T. J. PubMed Scopus Google Scholar, Y. H. Fujii S. Sawa T. Akaike T. Arimoto H. Chem. Commun. PubMed Scopus Google Scholar). study revealed that 8-nitro-cGMP Nrf2 through S-guanylation of and antioxidant and are activated in cultured C6 cells. be also that formation of a large amount of in a NO formed endogenously in the cells or induced was via our analysis a study with used C6 cells for the present express high levels of which is the for cGMP formation after with Also, C6 cells are activated by to express In fact, C6 cells to with NO and an of cGMP, as by In the of intracellular cGMP formation was in that formation increased after with NO from and declined to levels a the profile of 8-nitro-cGMP in cells, a for cGMP and 8-nitro-cGMP in that 8-nitro-cGMP formation increased h after the peak of cGMP and a at 24 h after is that the peak of 8-nitro-cGMP to be than that of In the for cGMP and 8-nitro-cGMP production in cells different from in cells, still the and much higher were for 8-nitro-cGMP with cGMP from cells activated to of the of 8-nitro-cGMP and the higher of 8-nitro-cGMP with the of cGMP, may the that cGMP is nitrated and 8-nitro-cGMP we the of a chemical nitration (e.g. that induced by mechanisms of this intracellular 8-nitro-cGMP formation may therefore in mechanisms of 8-nitro-cGMP formation are not yet NO-dependent guanine nitration through be in 8-nitro-cGMP formation cells (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar). The in of cGMP and 8-nitro-cGMP formation may be by the chemical of as this by (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar). Also, guanine nitration may be the guanine of other than cGMP (e.g. and may be for nitrated signaling occurring in which we are in our In fact, our revealed a unique to intracellular formation of which may be via from via GTP nitration by as in cGMP (e.g. the potential to and cGMP and 8-nitro-cGMP have different of production in cells, we may have an important that 8-nitro-cGMP and cGMP may have distinct roles in NO present which the biological of 8-nitro-cGMP formed in cells. the of 8-nitro-cGMP formed was and precisely such a amount was in cells may not be with the of are to and be with In 8-nitro-cGMP with to that in unique However, we are that with other biological is in of the for 8-nitro-cGMP and is of than the of T. Arimoto H. Akaike T. Chem. Google Scholar). to with the present finding that not 8-nitro-cGMP in and cells. of this 8-nitro-cGMP formation is in the of as an NO formation of an of nitration of not NO but also are to We therefore formation in of C6 cells with the of a for this is we that from as in In fact, in cultured cells was recently Am. J. 2004; PubMed Scopus Google Scholar). is therefore that formation by which led to 8-nitro-cGMP formation in the cells. formation to 8-nitro-cGMP was achieved with C6 cells activated by and which and may as study also found that NO-dependent 8-nitro-cGMP production such a through activation of the we suggested earlier that 8-nitro-cGMP may have in the (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar, M.H. Fujii S. Okamoto T. S. S. Sawa T. Akaike T. J. PubMed Scopus Google Scholar), the mechanism of activation induced by 8-nitro-cGMP In this of the important of our study is that S-guanylation of at a specific cysteine is that Cys434 is the of These suggest that 8-nitro-cGMP is in the signaling for and to and through of which in S-guanylation is a unique post-translational We recently found that protein S-guanylation is caused by 8-nitroguanosine derivatives in (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar, M.H. Fujii S. Okamoto T. S. S. Sawa T. Akaike T. J. PubMed Scopus Google Scholar, Y. H. Fujii S. Sawa T. Akaike T. Arimoto H. Chem. Commun. PubMed Scopus Google Scholar). the roles of protein S-guanylation in we the of S-guanylated We found that is the target S-guanylated by NO and S-guanylated from chemical such as sulfhydryl and that are caused by NO and were not in cysteine residues in cells, which that may a role in the NO signaling via our of S-guanylated cysteine residues in protein S-guanylation was at Cys434 of KEAP1 from cells. In KEAP1 reacted with 8-nitro-cGMP in an in of cysteine residues were that Cys434 S-guanylation was not is therefore that the Cys434 of may have a that physiological specific S-guanylation rather than S-guanylation of cysteine residues of a of have generated in in the presence of high of K. Y. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, A. A. Biochem. J. 2004; PubMed Scopus Google Scholar, A. Y. T. K. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar, S. Biol. Med. PubMed Scopus Google Scholar, A. Biochem. J. PubMed Scopus Google Scholar), to our we the first identification of cysteine modification with an signaling previously the in significance of and for functions in a in T. T. Kobayashi A. J. J. H. Yamamoto M. Biol. 28: PubMed Scopus Google Scholar). rigorous of the in for Cys434 is yet to a study that Cys434 in is of the cysteine residues to and that formation of Cys434 with causes in the of the molecule R. A.D. Chem. Res. PubMed Scopus Google Scholar). the specific S-guanylation of Cys434 by 8-nitro-cGMP may as a for is an addition to our of the cysteine in that a specific of cysteine residues in M. Y. Kaneko H. Nakayama Y. M. Y. Y. Yamamoto M. Biol. PubMed Scopus Google Scholar, T. T. Kobayashi A. J. J. H. Yamamoto M. Biol. 28: PubMed Scopus Google have determined that the is of molecule of Nrf2 and of Y. H. K. T. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar, B. Kobayashi A. Y. S. Yamamoto M. Biol. 2007; PubMed Scopus (315) Google Scholar). analysis revealed a of T. K. Y. H. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). The at the of to the and at the of the In a study that of Nrf2 and with the at the of the B. Kobayashi A. Y. S. Yamamoto M. Biol. 2007; PubMed Scopus (315) Google Scholar, B. T. Y. M. M. Kobayashi A. S. Yamamoto M. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We have a and for the induction of Nrf2 In the of and to the of Nrf2 for Y. H. K. T. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar, B. Kobayashi A. Y. S. Yamamoto M. Biol. 2007; PubMed Scopus (315) Google Scholar). that or this Nrf2 by the the and the this study, that Cys434 of is the target of S-guanylation. the of the Cys434 is at and to the of the as in B. T. Y. M. M. Kobayashi A. S. Yamamoto M. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. 2006; PubMed Scopus Google Scholar). for the mechanism of S-guanylation of Cys434 causes Nrf2 we One is that S-guanylation of Cys434 may to the and of Cys434 is to the of the other is that the Cys434 modification may the of the the of the was the of the from the of the had than the which that the of the is with the other of the the and T. K. Y. H. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). S-guanylation may the the which to of the and the of activity of the study determined that 8-nitro-cGMP the through S-guanylation of we found that of C6 cells with 8-nitro-cGMP induced by We also found that 8-nitro-cGMP increased the of Nrf2 and of of to in and M.H. Fujii S. Okamoto T. S. S. Sawa T. Akaike T. J. PubMed Scopus Google Scholar, K. Akaike T. Fujii S. S. T. S. M. Maeda H. J. PubMed Scopus Google Scholar, S. Akaike T. J. T. M. Tamura F. Y. Maeda H. J. 2003; PubMed Scopus Google Scholar). by 8-nitro-cGMP after is at in with increased the of these we that is of the activity of we NO-dependent formation of 8-nitro-cGMP and S-guanylation of by 8-nitro-cGMP in cultured C6 cells. and signaling to Nrf2 activation and of including which to be in the to in of S-guanylation of at Cys434 in that mechanisms that in of the Nrf2 An important is that S-guanylation of by 8-nitro-cGMP is a unique of the for IntroductionNitric oxide (NO) plays diverse physiological roles in vascular regulation, neuronal transmission, inflammation, and host defense against microbial pathogens. In vascular and neuronal systems, NO performs these functions mainly through a cGMP-dependent mechanism (1Murad F. J. Clin. Invest. 1986; 78: 1-5Crossref PubMed Scopus (672) Google Scholar, 2Bredt D.S. Hwang P.M. Snyder S.H. Nature. 1990; 347: 768-770Crossref PubMed Scopus (2678) Google Scholar), but the presence and contribution of other pathways that are not directly linked to cGMP have also been suggested to operate in certain aspects of NO signaling occurring in various cells and tissues in different organisms (3Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1352) Google Scholar, 4Schopfer F.J. Baker P.R. Freeman B.A. Trends Biochem. Sci. 2003; 28: 646-654Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 5Radi R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4003-4008Crossref PubMed Scopus (1209) Google Scholar). Among these other mechanisms is chemical modification of biomolecules, including nitrosylation and nitration of amino acids, proteins, and lipids, this modification being induced by NO-derived reactive nitrogen oxide species (RNOS), 3The abbreviations used are: RNOSreactive nitrogen oxide species8-nitro-cGMP8-nitroguanosine 3′,5′-cyclic monophosphatePDEphosphodiesteraseROSreactive oxygen speciesMALDImatrix-assisted laser desorption ionizationTOFtime-of-flightMSmass spectrometryMS/MStandem mass spectrometrySNAPS-nitroso-N-acetylpenicillamineBSObuthionine sulfoximineiNOSinducible NO synthaseLPSlipopolysaccharideIFN-γinterferon-γTNFαtumor necrosis factor αIL-1βinterleukin-1βl-NMMANω-monomethyl-l-argininePBSphosphate-buffered salinesGCsoluble guanylate cyclaseLCliquid chromatographyHPLChigh performance liquid chromatographyECDelectrochemical detectionESIelectrospray ionizationDTTdithiothreitolNEMN-ethylmaleimidesiRNAsmall interfering RNAHSAhuman serum albuminSNO-HSAS-nitrosylated human serum albuminc[15N5]GMP[U-15N5, 98%]guanosine 3′,5′-cyclic monophosphate[15N5]GTP[U-15N5, 98%]guanosine 5′-triphosphate. such as peroxynitrite (ONOO−) and nitrogen dioxide (NO2) (3Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1352) Google Scholar, 4Schopfer F.J. Baker P.R. Freeman B.A. Trends Biochem. Sci. 2003; 28: 646-654Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 5Radi R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4003-4008Crossref PubMed Scopus (1209) Google Scholar).RNOS cause nitration of nucleic acids in addition to amino acids, proteins, and lipids. We previously found that nitrated guanine derivatives, including 8-nitroguanine and 8-nitroguanosine, formed in cultured cells and in tissues from murine viral pneumonia and human lung disease (6Akaike T. Okamoto S. Sawa T. Yoshitake J. Tamura F. Ichimori K. Miyazaki K. Sasamoto K. Maeda H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 685-690Crossref PubMed Scopus (138) Google Scholar, 7Yoshitake J. Akaike T. Akuta T. Tamura F. Ogura T. Esumi H. Maeda H. J. Virol. 2004; 78: 8709-8719Crossref PubMed Scopus (52) Google Scholar, 8Terasaki Y. Akuta T. Terasaki M. Sawa T. Mori T. Okamoto T. Ozaki M. Takeya M. Akaike T. Am. J. Respir. Crit. Care Med. 2006; 174: 665-673Crossref PubMed Scopus (35) Google Scholar). An important finding was that 8-nitroguanosine possessed a unique redox activity, which suggested a critical biological role of guanine nitration (9Sawa T. Akaike T. Ichimori K. Akuta T. Kaneko K. Nakayama H. Stuehr D.J. Maeda H. Biochem. Biophys. Res. Commun. 2003; 311: 300-306Crossref PubMed Scopus (53) Google Scholar). In fact, we recently discovered that a novel nitrated cyclic nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is generated after NO production (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar). 8-Nitro-cGMP had the strongest redox activity among the nitrated guanine derivatives tested, and this property was distinct from that activating cGMP-dependent protein kinases. Being an electrophile, 8-nitro-cGMP effectively reacted with sulfhydryl groups of cysteine residues and formed a protein-S-cGMP adduct, via a post-translational modification named protein S-guanylation (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar).Although we successfully determined the chemical identification of 8-nitro-cGMP in our earlier study (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar), we had not yet achieved rigorous quantification of 8-nitro-cGMP in biological systems (e.g. cells), and mechanisms of 8-nitro-cGMP action were still to be clarified. One specific question concerned what constituted a target molecule for nitration: GTP, a substrate of guanylate or Also, signaling pathways of 8-nitro-cGMP were not this we the redox protein protein as of the for the physiological significance and of S-guanylation to be (10Sawa T. Zaki M.H. Okamoto T. Akuta T. Tokutomi Y. Kim-Mitsuyama S. Ihara H. Kobayashi A. Yamamoto M. Fujii S. Arimoto H. Akaike T. Nat. Chem. Biol. 2007; 3: 727-735Crossref PubMed Scopus (205) Google Scholar, M.H. Fujii S. Okamoto T. S. S. Sawa T. Akaike T. J. PubMed Scopus Google Scholar). The factor is of the cellular defense mechanisms against and H. Yamamoto M. Trends Med. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Kobayashi A. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: PubMed Scopus Google Scholar, Chem. Res. PubMed Scopus (315) Google Scholar, Y. H. K. T. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar). Nrf2 is a factor and antioxidant that in against and reactive oxygen species Nrf2 is and by Nrf2 is by an which in the Y. Kobayashi A. H. M. J. K. Yamamoto M. 2007; PubMed Scopus Google and as an of the for to or Nrf2 is and which in induction of a of have been to the reactive cysteine residues in and modification of the cysteine in that a certain of cysteine residues K. Y. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, A. A. Biochem. J. 2004; PubMed Scopus Google Scholar, A. Y. T. K. Yamamoto M. Biol. 2006; PubMed Scopus Google Scholar, S. Biol. Med. PubMed Scopus Google Scholar, A. Biochem. J. PubMed Scopus Google Scholar, M. Y. Kaneko H. Nakayama Y. M. Y. Y. Yamamoto M. Biol. PubMed Scopus Google Scholar). In fact, we cysteine residues and that are critical to activity to Nrf2 the to T. T. Kobayashi A. J. J. H. Yamamoto M. Biol. 28: PubMed Scopus Google Scholar). These were by of a with and or of T. T. Kobayashi A. J. J. H. Yamamoto M. Biol. 28: PubMed Scopus Google the present study, we to the and aspects of 8-nitro-cGMP in the cells and the potential roles of 8-nitro-cGMP in protein S-guanylation induced by We first and intracellular of 8-nitro-cGMP after NO production in C6 glioma cells. A finding was that the of 8-nitro-cGMP formation was comparable with or higher than that of cGMP formed in cells. important is the 8-nitro-cGMP generated induced S-guanylation of and increased Nrf2-dependent 8-nitro-cGMP was against effects of hydrogen through of Nrf2 target such as We via matrix-assisted laser desorption/ionization mass spectrometry critical cysteine of which was modified by 8-nitro-cGMP in cells. study substantial of the biological significance and the formation mechanism of the of 8-nitro-cGMP formed in cells. of our study is of a of by which our that are a that is the cysteine in the molecule to Nrf2