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S9-6 Elemental sulfur reduction by eukaryotic cytoplasm consistent with an ancient sulfur symbiosis
Abstracts / Nitric Oxide 39 (2014) S1–S14
References [1] P. Nagy et al., Chemical aspects of hydrogen sulfide measurements in physiological samples BBA Gen, Subj. Curr. Meth. Stud. ROS Spec. Issue 1840 (2014) 876– 891. [2] P. Nagy, C.C. Winterbourn, Rapid reaction of hydrogen sulfide with the neutrophil oxidant hypochlorous acid to generate polysulfides Chem, Res. Toxicol. 23 (2010) 1541–1543. [3] R. Greiner et al., Polysulfides link H2S to protein thiol oxidation Antioxid, Redox Signal. 19 (15) (2013) 1749–1765. [4] M.M. Cortese-Krott et al., Nitrosopersulfide (ONSS ) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide, Redox Biol. 2 (2014) 234–244. [5] Pálinkás Z et al. Interactions of hydrogen sulfide with human myeloperoxidase in revision. http://dx.doi.org/10.1016/j.niox.2014.03.045
S9-4 Nociceptive action of hydrogen sulfide through the activation of transient receptor potential ankyrin 1 (TRPA1) Hitomi Ogawa a, Yukari Hatakeyama a, Kenji Takahashi a, Makoto Tominaga b, Hideo Kimura c, Toshio Ohta a a Department of Veterinary Pharmacology, Faculty of Agriculture, Tottori University, Tottori, Japan b Division of Cell Signaling, National Institutes of Natural Sciences, Okazaki, Japan c Department of Molecular Pharmacology, National Institute of Neuroscience, Tokyo, Japan Hydrogen sulfide (H2S), an endogenous gasotransmitter, modulates various biological functions. It is known that H2S causes neurogenic inflammation and elicits hyperalgesia. Here we show that H2S selectively stimulates TRPA1 channels, which are involved in nociception in mice. In wild-type mouse sensory neurons, H2S increased the intracellular Ca2+ concentration ([Ca2+]i), which was inhibited by ruthenium red, a nonselective TRP channel blocker and HC-030031, a TRPA1 blocker, but not BCTC, a TRPV1 blocker. H2S-responding neurons highly corresponded to a TRPA1 agonist (AITC)-sensitive ones. [Ca2+]i responses to H2S were observed in neurons from TRPV1( / ) mice but not from TRPA1( / ) mice. Heterologously expressed mouse TRPA1 was activated by H2S. Analyses of the TRPA1 mutant channel revealed that two cysteine residues located in the N-terminal internal domain were responsible for the activation by H2S. Polysulfides as possible H2S-derived bioactive molecules also stimulated TRPA1 with more efficiently than H2S. Intraplantar injection of H2S or polysulfides into the mouse hind paw caused pain-related behaviors, which were significantly less in TRPA1( / ) mice. The [Ca2+]i responses to H2S in sensory neurons and in heterologously expressed channels, and pain-related behavior induced by H2S were enhanced under acidic conditions. These results suggest that H2S and polysulfides function as nociceptive messengers through the activation of TRPA1 channels. TRPA1 may be a therapeutic target for sulfidesrelated algesic action, especially under inflammatory conditions. http://dx.doi.org/10.1016/j.niox.2014.03.046
S9-5 Bound sulfur exerts protective effect against cytotoxicity in neuronal cells Yuki Ogasawara a, Shin Koike a, Norihiro Shibuya b, Kazuyuki Ishii a, Hideo Kimura b a Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
S13
b Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
Over 20 years ago, we developed a sensitive method for the determination of H2S liberated by reduction and applied this method to mammalian tissue [1]. As a result of these experiments, we termed ‘‘bound sulfur’’ to describe the preferred state of endogenous H2S. Bound sulfur is defined as sulfur that is rapidly liberated as sulfide by reduction with dithiothreitol. Polysulfide is a bound sulfur species derived from endogenous H2S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells.
References [1] Y. Ogasawara et al., Anal. Biochem. 215 (1993) 73–81.
http://dx.doi.org/10.1016/j.niox.2014.03.047
S9-6 Elemental sulfur reduction by eukaryotic cytoplasm consistent with an ancient sulfur symbiosis Dennis G. Searcy Biology Department, University of Massachussetts, Amherst, USA An accepted theory for the origin of eukaryotic cells postulates a methanogenic ancestor, although no evidence of methanogenesis exists in modern eukaryotic cells. Instead, sulfur reactions are ubiquitous. The seminal event in eukaryotic evolution might have been a sulfur-based symbiosis between a S°-reducing host and a sulfide-oxidizing bacterium. A vestige of those activities might remain in modern cells. To test this, Tetrahymena thermophila was examined as a representative eukaryotic cell. Aerobic cells consumed H2S at a rate of 140 nmol (g protein) 1 s 1. Anoxic cells produced H2S at 1.1 nmol g 1 s 1, but H2S production was suppressed by AOAA, an inhibitor of cysteine catabolism. When aerobic cells were fed H2S slowly so that all the H2S was oxidized, and then made anoxic, H2S was produced at up to 3.6 nmol g 1 s 1, and not inhibited by AOAA. Evidently, H2S is oxidized to a compound that is available for H2S production. When aerobic incubation was continued after H2S addition had stopped, subsequent H2S production decreased with t1/2 = 11 min. When S° was added to 7 mg cells at 85 pmol s 1, H2S production was 4.4 nmol g 1 s 1 and not inhibited by AOAA. When cells were lysed and fractionated, there was NADH-stimulated S° reductase activity in the membrane fraction, distinguishing it from NADPH disulfide oxidoreductases and linking S° reduction to glycolysis. Thus, there is evidence of a sulfur cycle in which S° is reduced in the cytoplasm and H2S is oxidized in mitochondria. http://dx.doi.org/10.1016/j.niox.2014.03.048
References [1] P. Nagy et al., Chemical aspects of hydrogen sulfide measurements in physiological samples BBA Gen, Subj. Curr. Meth. Stud. ROS Spec. Issue 1840 (2014) 876– 891. [2] P. Nagy, C.C. Winterbourn, Rapid reaction of hydrogen sulfide with the neutrophil oxidant hypochlorous acid to generate polysulfides Chem, Res. Toxicol. 23 (2010) 1541–1543. [3] R. Greiner et al., Polysulfides link H2S to protein thiol oxidation Antioxid, Redox Signal. 19 (15) (2013) 1749–1765. [4] M.M. Cortese-Krott et al., Nitrosopersulfide (ONSS ) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide, Redox Biol. 2 (2014) 234–244. [5] Pálinkás Z et al. Interactions of hydrogen sulfide with human myeloperoxidase in revision. http://dx.doi.org/10.1016/j.niox.2014.03.045
S9-4 Nociceptive action of hydrogen sulfide through the activation of transient receptor potential ankyrin 1 (TRPA1) Hitomi Ogawa a, Yukari Hatakeyama a, Kenji Takahashi a, Makoto Tominaga b, Hideo Kimura c, Toshio Ohta a a Department of Veterinary Pharmacology, Faculty of Agriculture, Tottori University, Tottori, Japan b Division of Cell Signaling, National Institutes of Natural Sciences, Okazaki, Japan c Department of Molecular Pharmacology, National Institute of Neuroscience, Tokyo, Japan Hydrogen sulfide (H2S), an endogenous gasotransmitter, modulates various biological functions. It is known that H2S causes neurogenic inflammation and elicits hyperalgesia. Here we show that H2S selectively stimulates TRPA1 channels, which are involved in nociception in mice. In wild-type mouse sensory neurons, H2S increased the intracellular Ca2+ concentration ([Ca2+]i), which was inhibited by ruthenium red, a nonselective TRP channel blocker and HC-030031, a TRPA1 blocker, but not BCTC, a TRPV1 blocker. H2S-responding neurons highly corresponded to a TRPA1 agonist (AITC)-sensitive ones. [Ca2+]i responses to H2S were observed in neurons from TRPV1( / ) mice but not from TRPA1( / ) mice. Heterologously expressed mouse TRPA1 was activated by H2S. Analyses of the TRPA1 mutant channel revealed that two cysteine residues located in the N-terminal internal domain were responsible for the activation by H2S. Polysulfides as possible H2S-derived bioactive molecules also stimulated TRPA1 with more efficiently than H2S. Intraplantar injection of H2S or polysulfides into the mouse hind paw caused pain-related behaviors, which were significantly less in TRPA1( / ) mice. The [Ca2+]i responses to H2S in sensory neurons and in heterologously expressed channels, and pain-related behavior induced by H2S were enhanced under acidic conditions. These results suggest that H2S and polysulfides function as nociceptive messengers through the activation of TRPA1 channels. TRPA1 may be a therapeutic target for sulfidesrelated algesic action, especially under inflammatory conditions. http://dx.doi.org/10.1016/j.niox.2014.03.046
S9-5 Bound sulfur exerts protective effect against cytotoxicity in neuronal cells Yuki Ogasawara a, Shin Koike a, Norihiro Shibuya b, Kazuyuki Ishii a, Hideo Kimura b a Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
S13
b Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
Over 20 years ago, we developed a sensitive method for the determination of H2S liberated by reduction and applied this method to mammalian tissue [1]. As a result of these experiments, we termed ‘‘bound sulfur’’ to describe the preferred state of endogenous H2S. Bound sulfur is defined as sulfur that is rapidly liberated as sulfide by reduction with dithiothreitol. Polysulfide is a bound sulfur species derived from endogenous H2S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells.
References [1] Y. Ogasawara et al., Anal. Biochem. 215 (1993) 73–81.
http://dx.doi.org/10.1016/j.niox.2014.03.047
S9-6 Elemental sulfur reduction by eukaryotic cytoplasm consistent with an ancient sulfur symbiosis Dennis G. Searcy Biology Department, University of Massachussetts, Amherst, USA An accepted theory for the origin of eukaryotic cells postulates a methanogenic ancestor, although no evidence of methanogenesis exists in modern eukaryotic cells. Instead, sulfur reactions are ubiquitous. The seminal event in eukaryotic evolution might have been a sulfur-based symbiosis between a S°-reducing host and a sulfide-oxidizing bacterium. A vestige of those activities might remain in modern cells. To test this, Tetrahymena thermophila was examined as a representative eukaryotic cell. Aerobic cells consumed H2S at a rate of 140 nmol (g protein) 1 s 1. Anoxic cells produced H2S at 1.1 nmol g 1 s 1, but H2S production was suppressed by AOAA, an inhibitor of cysteine catabolism. When aerobic cells were fed H2S slowly so that all the H2S was oxidized, and then made anoxic, H2S was produced at up to 3.6 nmol g 1 s 1, and not inhibited by AOAA. Evidently, H2S is oxidized to a compound that is available for H2S production. When aerobic incubation was continued after H2S addition had stopped, subsequent H2S production decreased with t1/2 = 11 min. When S° was added to 7 mg cells at 85 pmol s 1, H2S production was 4.4 nmol g 1 s 1 and not inhibited by AOAA. When cells were lysed and fractionated, there was NADH-stimulated S° reductase activity in the membrane fraction, distinguishing it from NADPH disulfide oxidoreductases and linking S° reduction to glycolysis. Thus, there is evidence of a sulfur cycle in which S° is reduced in the cytoplasm and H2S is oxidized in mitochondria. http://dx.doi.org/10.1016/j.niox.2014.03.048