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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
H2S2014 in Kyoto: The 3rd International Conference on H2S in Biology and Medicine Hideo Kimura * National Institute of Neuroscience, National Center for Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
A R T I C L E
I N F O
Article history: Received 2 September 2014 Revised 6 October 2014 Available online
A B S T R A C T
About 20 years ago, a pungent gas was found to be the physiological mediator of cognitive function and vascular tone. Since then, studies on hydrogen sulfide (H2S) have uncovered its numerous physiological roles such as protecting various tissues/organs from ischemia and regulating inflammation, cell growth, oxygen sensing, and senescence. These effects of H2S were extensively studied, and some of the corresponding mechanisms were also studied in detail. Previous studies on the synergistic interaction between H2S and nitric oxide (NO) have led to the discovery of several potential signaling molecules. Polysulfides are considerably potent and are one of the most active forms of H2S. H2S has a significant therapeutic potential, which is evident from the large number of novel H2S-donating compounds and substances developed for manipulating endogenous levels of H2S. The Third International Conference on H2S was held in Kyoto in June 2014. One hundred and sixty participants from 21 countries convened in Kyoto to report new advances, discuss conflicting findings, and make plans for future research. This article summarizes each oral presentation presented at the conference. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Nitric oxide (NO) was discovered as a gaseous signaling molecule in 1986 [1,2]; carbon monoxide (CO) was then discovered in 1993 [3–5]. The H2S-producing enzymes were extensively studied between the 1950s and the 1970s [6–8]. However, H2S was considered a mere by-product of the various metabolic pathways or just a marker for enzyme activities. In 1989, endogenous sulfide was discovered in mammalian brains [9,10]. Unfortunately, the H2S measured in these studies was found to be contaminated with other forms of sulfur that also release this gas. Although the re-evaluated levels were much lower than those initially reported, these studies confirmed the existence of H2S inside the tissues [11–13]. The role of H2S as a neuromodulator that acts in association with cystathionine β-synthase (CBS) as an H2S-producing enzyme in the brain was demonstrated in 1996 [14]. Its other role as a smooth-muscle relaxant in conjugation with cystathionine γ–lyase (CSE), which generates H2S in the smooth muscles, was discovered in the following year [15]. The involvement of ATP-dependent K+ channels in vascular relaxation was subsequently demonstrated [16]. Because H2S is a relatively toxic gas, its cytoprotective effects had been completely overlooked. It was found to protect neurons from oxidative stress by increasing the levels of glutathione, a major en-
* National Institute of Neuroscience, National Center for Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. Fax: +81 42 346 1755. E-mail address: [email protected]
dogenous antioxidant and scavenger of reactive oxygen species (ROS) [17,18]. This finding led to the identification of the protective effect of H2S on various other organs/tissues. H2S protects organs/tissues from ischemia-reperfusion injury and oxidative insults [19,20]. It induces angiogenesis, which is also mediated by the vascular endothelial growth factor (VEGF) [21,22]. H 2 S is oxidized in the mitochondria for the generation of adenosine triphosphate (ATP) [23,24], and it presumably functions as an oxygen sensor [25,26]. The activity of CBS is enhanced by S-adenosyl methionine [14], and the activity of CSE is increased by Ca2+ [27]. However, the latter result proved to be controversial, when it was reported that CSE was suppressed by Ca2+ [28]. Cysteine aminotransferase (CAT) is identical to aspartate aminotransferase, and produces 3-mercaptopyruvate, a substrate for 3-mercaptopyruvate sulfurtransferase (3MST). The enzyme 3MST generates H2S [29]. The activity of CAT is also suppressed by Ca2+ [30]. In contrast to CBS and CSE, 3MST requires an accompanying reducing agent to produce H2S. Thioredoxin was identified to be the reductant that assisted 3MST in producing H2S [31,32]. An H2S-generating pathway involving D-cysteine was identified recently [33]. One of the proposed modes through which H2S transmits subcellular signals involves sulfhydration or sulfuration [34]. It involves the addition of sulfur to the cysteine residues of the target proteins to induce a conformational change that modifies their activities. Previous studies show that Keap1, NF-kB, transient receptor potential ankyrin 1 (TRPA1), and perkin are activated by sulfhydration or sulfuration [35–40]. Recently, polysulfides (H2Sn, n = 3–7; n = 2 for the persulfide) discovered in the mammalian brain were found to
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activate the TRPA1 channels [39], thereby facilitating the translocation of Nuclear-factor-E2-related factor 2 (Nrf2) to the nucleus after being released from the sulfhydrated Keap1 [36]. The polysulfides were also found to modulate the activity of phosphatase and tensin homolog (PTEN) [41] more efficiently than H2S. After carefully studying their conversion efficiency and oxidation states, it was proposed that polysulfides, rather than H2S, sulfhydrate (or sulfurate) their target proteins [42]. H2S exerts regulatory effects on inflammatory processes and promotes their resolution. Owing to these beneficial effects of H2S, several H2S-releasing anti-inflammatory drugs and H2S-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) exhibiting no significant side effects on the gastrointestinal tract have been developed [43]. Many of these drugs have shown considerable promise in the relevant animal models, and are presently undergoing clinical trials. H2S also inhibits cancer development at various stages, and H2S donors are cytotoxic to human cancer cells [44]. They also induce cell cycle arrest in the G2/M phase and promote apoptosis. However, they do not affect the survival of normal human cells. Several recent studies have shown that H2S-releasing NSAIDs exhibit enhanced chemopreventive effects in cancer cells, whereas parental NSAIDs alone do not confer any such beneficial effects to cancer cells [45]. Contrary to these observations, CBS is upregulated in the colorectal and ovarian cancer cells, where H2S is involved in promoting cellular bioenergetics, proliferation, and migration [46]. In these cancer cells, the suppression of CBS activity can be used as a potential therapy. Because the metabolism, oxidation, evaporation, and absorption of sulfane-sulfur occur rapidly, it is difficult to accurately measure the concentrations of H2S, or to track its movements inside the cells. Recently, several fluorescent probes have been developed for the specific detection of H2S and sulfane-sulfur [47–50]. These probes consist of two functional sites, a site sensitive to H2S and a site harboring a fluorophore. The first conference on H2S was successfully held in 2009 in Shanghai, China, and the second conference was held in 2012 in Atlanta, Georgia, USA. During the same period, the first European conference on H2S was held in 2012 in Blatislava, Slovakia, and the second in 2013 in Exeter, England. Two weeks before this Kyoto conference, the international symposium on H2S and NO was held in Naples, Italy. Research on H2S is witnessing enormous growth at present.
2. Session 1, biology and chemistry of H2S In his presentation entitled “H2S and beyond,” Hideo Kimura from National Institute of Neuroscience discussed his group’s recent findings on the production of H2S from D-cysteine and the potential roles of polysulfides as signaling molecules. D-cysteine is metabolized to H2S by a specific pathway, and it efficiently prevents ischemia-reperfusion injury. The study group has also shown that polysulfides activate ions channels and a transcription factor much more potently than H2S by sulfhydration or sulfuration of these targets. Rui Wang from Lakehead University discussed the importance of the interaction among the gasotransmitters such as H2S, NO, and carbon monoxide (CO), for the functioning of the cellular signaling networks. In his presentation entitled “The next wave of gasotransmitter research”, he also mentioned that ammonia, another gasotransmitter, is an endogenously produced gas with specific substrates and enzymes. It functions at physiological concentrations through the activation of specific molecular targets. Gasotransmitters share common target molecules, but exert their activities using different mechanisms. In some cases, they produce the same outcome through the activation of entirely different pathways.
The cellular regulation of H2S levels and the enzyme activities necessary for H2S production and metabolism are not well understood. In her presentation entitled “Enzymology of H2S turnover”, Ruma Banerjee from University of Michigan Medical School discussed the structures of CBS and 3MST at the reactive intermediates in the active sites, as well as the relative contribution of CBS, CSE and 3MST to H2S production. H2S clearance plays an important role during the cessation of H2S signaling. The study group has recently shown that the activity of CBS is increased by its glutathionilation under oxidative stress, thereby increasing the levels of glutathione and H2S. This is a plausible mechanism for cellular survival during oxidative stress. 3. Session 2, H2S in cardiovascular diseases David Lefer from LSU Cardiovascular Center of Excellence discussed the protective effect of an H2S-donor and a major garlic extract (diallyl trisulfide; DATS) on a pressure-overloaded heart. H2S therapy increases the expression of the proangiogenic factor vascular endothelial cell growth factor, but decreases the expression of the angiogenesis inhibitory factor, angiostatin. In his presentation entitled “Hydrogen sulfide in heart failure,” he also mentioned that the phosphorylation of endothelial NO synthase (eNOS) at serine 1177 is increased by H2S treatment, which causes an increase in the bioavailable NO, a peripheral factor that protects the heart from cardiac arrest. The involvement of H2S in cleaving the cysteine disulfide bonds for activating the target proteins such as VEGFR2, EGFR, the insulin receptor, and the potassium channels was discussed by Yi-Chun Zhu from Fudan University in his presentation entitled “Identification of hydrogen sulfide “receptors” in the receptor tyrosine kinase family and ion channels”. H2S activates Na+/K+-ATPase through EGFR, leading to an increase in Na+ excretion and a decrease in blood pressure in chronic salt-loaded rats. The activation of potassium channels in cardiomyocytes improves fatal arrhythmia during acute myocardial infarction. In his presentation entitled “BRG1-dependent epigenetic control of vascular smooth muscle cell proliferation by hydrogen sulfide”, Junbao Du from Peking University First Hospital discussed the role of H2S in suppressing the proliferation of vascular smooth muscle cells by downregulating Brg1, the central catalytic subunit of the ATP-dependent chromatin remodeling complex, and by reducing the recruitment of Brg1 to the promoter regions of proliferationrelated genes such as the proliferating cell nuclear antigen, neurotrophin3, and platelet-derived growth factor subunit A. John Calvert from Emory University School of Medicine discussed the role of Nrf2 in mediating the cardioprotective effects of H2S therapy in a mouse model of ischemia-induced heart failure. In his presentation entitled “Nrf2 signaling is essential for hydrogen sulfide mediated cardioprotection in the setting of ischemiainduced heart failure” he also mentioned that the effect of H2S was confirmed in Nrf2 KO mice, which display exacerbated dysfunction and hypertrophy as compared with the wild-type mice. 3. Session 3, Inflammation Matt Whiteman from University of Exeter Medical School discussed the development of novel compounds that release H2S in a physiologically relevant manner, and examined the precise role of H2S in inflammation and therapeutic application. To circumvent the problems involving the immediate release of H2S (in solution) via NaHS, and the presence of residual solvent contaminants in GYY4137, novel compounds with a slow and well-controlled release have been developed. In his presentation entitled “H2S in inflammation: Time for resolution?” he mentioned that his group has also developed compounds targeted to the mitochondria.
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In his presentation entitled “Roles of the hydrogen sulfideCAV3.2 T-type calcium channel pathway in neuronal and neuroendocrine differentiation,” Atsufumi Kawabata from Kinki University, showed that H2S enhances the activity of the Cav3.2 T-type calcium channels that are involved in pain signaling and neuronal differentiation. The upregulated expression of CSE and Cav3.2 channels enhances the secretory responses in differentiationinduced human prostate cancer cells. This pathway may serve as a target for prostate cancer therapy. Fumito Ichinose from Harvard Medical School discussed the endotoxemia-induced decrease in the plasma sulfide levels. In his presentation entitled “H2S in sepsis and shock – A story of mice and men,” he also mentioned that breathing H 2 S attenuates the endotoxin-induced systemic inflammation, and restores the sulfide levels. A marked increase in the levels of thiosulfate is also observed. The application of thiosulfate improves survival rates. Recent data from his group shows intriguing results: the deficiency and suppression of CSE increases (instead of decreasing) the levels of thiosulfate in the liver and prevents acute liver failure. In her presentation entitled “H2S targets transient receptor potential receptors to relieve from inflammation and pain,” Nathalie Vegnolle from INSERM, mentioned that NaHS (200–1000 μM) decreases the calcium responses of TRPV4 and TRPA1 to their corresponding agonists in the intestinal epithelial cells and dorsal ganglion cells. H2S efficiently reduces visceral inflammation and pain by inhibiting the pro-inflammatory and pro-nociceptive effects of TRPV4 and TRPA1.
4. Session 4, analysis of H2S Makoto Suematsu, Keio University mentioned that the regulation of CBS by CO determines the balance between NADPH and glutathione, as well as the directional glucose utilization in cancer cells that protects them from oxidative stress. In his presentation entitled “CO-CBS system for regulating tenuous balance between NADPH and persulfides in cancer”, he also mentioned stressinduced CO suppresses CBS, which leads to a decrease in the methylation of PFKFB3, thereby shifting the glucose utilization from glycolysis to the pentose phosphate pathway, and stimulating cancer cell proliferation during the process. In contrast, the activation of CBS increases NADPH and glutathione, thereby protecting the cells from oxidative stress. In his presentation entitled “Development of an H2S fluorescent probe and its application to inhibitor screening for H 2 S producing enzyme,” Kenjiro Hanaoka from Tokyo University, discussed the development of a novel fluorescence probe for H2S, HSip1, which specifically reacts with 10 μM H2S, even in the presence of 10 mM glutathione. By using this probe, they successfully developed selective inhibitors for 3MST and CSE. The development of slow H2S-releasing donors and specific inhibitors of H2S-producing enzymes was discussed by Andreas Papapetropoulos from University of Athens in his presentation entitled “Novel pharmacological tools for the H2S pathway”. In contrast to inorganic compounds such as NaHS and Na 2 S, which immediately release H2S, the organic donors slowly release H2S in a physiologically relevant manner. Hidehiko Nakagawa from Nagoya City University discussed the development of a photochemical method for H2S generation in his presentation entitled “Photo-inducible hydrogen sulfide releaser using ketoprofenate photocages”. A ketoprofenate photocage releases H2S in an appropriate location, timing, and dosage proportional to the timing and intensity of the incident electromagnetic radiation. In his presentation entitled “New chemistry and chemical tools for hydrogen sulfide research,” Ming Xian from Washington State
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University discussed the development of controllable H2S donors and fluorescent probes for H2S and sulfane-sulfur species. 5. Session 5, therapeutics and development of H2S-based drugs John Wallace from University of Calgary discussed the development of H2S-releasing NSAIDs in his presentation entitled “H2Sbased therapeutics for inflammatory diseases”. NSAIDs are effective at reducing pain and inflammation, but they show serious adverse effects especially on the gastrointestinal (GI) tract. The H 2 Sreleasing NSAIDs show a dramatically better GI safety and antiinflammatory efficacy than the parental NSAIDs. Preclinical studies have confirmed the safety of these compounds and have led to clinical trials in humans. In his presentation entitled “Hydrogen sulfide accounts for the peripheral vascular effects of sulfhydrylated ACE inhibitors beyond ACE inhibition: A proof of concept of the role of H2S in cardiovascular?,” Giuseppe Cirino, University Degli Studi Di Napoli, mentioned that the sulfhydrylated ACE inhibitor, zofenopril, relaxes vascular smooth muscles and improves vascular function by potentiating the effect of H2S in a spontaneous hypertension model. Jinsong Bian from National University of Singapore mentioned that H 2 S reduces the polymerization of tau, a major component of the neurofibrillary tangle by sulfhydrating its cysteine residues, and inhibits amyloid β–formation, a major component of plaque, in the animal models of Alzheimer’s disease in his presentation entitled “Can hydrogen sulfide be used to treat neurodegenerative diseases?”. H2S also protects the dopaminergic neurons in the animal models of Parkinson’s disease. In his presentation entitled “Phase 1, heart failure clinical trial to evaluate the safety of SG1002 – A novel hydrogen sulfide donor,” Tony Giordano from Sulfagenix, discussed the safety of SG1002 for normal individuals. A double-blind, placebo-controlled Phase 1 study of SG1002 showed that it is well tolerated in normal individuals, and causes limited side effects. His group is repeating this study on a heart failure subject. Jerzy Beltowski from Medical University of Lublin mentioned that nucleoside monophosphorothioates, which are nucleotide analogs with one oxygen atom replaced by sulfur, release H2S in perivascular adipose tissue and induce vasorelaxation. In his presentation entitled “Nucleoside mono-phosphorothioates as the H2S donors in perivascular adipose tissue” he also mentioned that these substances could be effectively used as vasodilators especially in patients suffering from the metabolic syndrome. 6. Session 6, H2S signaling In his presentation entitled “Hydrogen sulfide as an oxygen sensor,” Ken Olson from Indiana University School of Medicine, mentioned that the O2-dependent metabolism of H2S could be an effective O2-sensing mechanism. Tissue H2S concentrations are inversely related to the O 2 concentrations, and this reciprocal relationship between H2S and O2 is responsible for the similarity between the effect of hypoxia and the effect of H2S. The determination of this mechanism at sub-cellular level will strongly support the above-mentioned hypothesis. Frederic Bouillaud, INSERM, mentioned in his presentation entitled “Mitochondrial oxidation of H2S in mammals” that H2S is oxidized in the mammalian mitochondria. It is metabolized by sulfide quinone reductase and dioxygenase, and is involved in the production of ATP in the liver. In his presentation entitled “H2S signal interacts with hypoxia signal in a mitochondria-dependent manner,” Kiichi Hirota, Kansai Medical University, mentioned that H2S modulates the intracellular O2 homeostasis, reduces the accumulation of hypoxia-inducible
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factor 1 (HIF-1), and also reduces the subsequent gene expression induced by hypoxia. H2S decreases cellular O2 consumption and inhibits the HIF-1 accumulation induced by hypoxia by increasing its metabolism without affecting its production. Koji Takeuchi from Kyoto Pharmaceutical University discussed that H2S modulates the acid-induced HCO3- secretion by enhancing the production of mucosal PGE2 and NO, and it is involved in mucosal protection in the duodenum in his presentation entitled “Stimulation of duodenal HCO3− secretion by H2S in rats: Regulatory mechanism and importance in mucosal defense”. The duodenal damage caused by the acid is healed by H2S produced by CSE. Mark Hellmich from University of Texas Medical Branch mentioned that the H2S produced by CBS plays an important role in promoting cellular bioenergetics, proliferation, and migration in colorectal and ovarian cancer. He also mentioned in his presentation entitled “Role of cystathionine-beta synthase and its product, hydrogen sulfide in cancer biology” that targeting CBS to suppress H2S production in cancer cells may provide novel anticancer therapies.
7. Session 7, H2S and NO Christopher Kevil from LSU Health Science Center mentioned that ischemia increases CSE expression and the levels of H2S that restores blood flow in the ischemic tissues in his presentation entitled “Vascular sulfide metabolism during ischemia”. Once blood starts flowing, the levels of CSE and H2S return to the control level. This blood flow restoration is blunted in CSE-deficient animals and augmented in CSE-transgene animals. An increase in the bioavailability of H2S is an important physiological adaptive response under ischemic conditions. In his presentation entitled “H2S induced decomposition of nitroso-compounds and consequent biological effect,” Karol Ondrias from Institute of Molecular Physiology and Genetics and INPP SAS mentioned that H2S acts on S-nitrosoglutathione to produce polysulfides and SSNO−, which subsequently releases NO, thereby relaxing the aorta smooth muscle more effectively than S-nitrosoglutathione. The interaction between H 2 S and S-nitrosoglutathione may have an important physiological role. The identification of the endogenous products is a challenge for future research. Martin Feelisch from University of Southampton mentioned that the interaction between H2S and NO produces multiple reaction products including reactive nitrogen and sulfur species, which induce the expression of downstream signaling molecules such as cGMP and Keap1/Nrf2. In his presentation entitled “The NO/ sulfide system and its significance for biological signaling” he also discussed the production of SSNO−, nitroxyl, and polysulfides. Yi-Zhun Zhu from Fudan University mentioned that S-propargyl-cysteine, ZYZ-802 exerts cardiovascular- and neuroprotective effects by modulating the CSE activity in his presentation entitled “Novel effect of endogenous H2S donor, ZYZ802 on heart and brain”. This compound promotes cell proliferation, adhesion, migration, tube formation of vascular endothelial cells, and angiogenesis. It also suppresses the formation of the amyloid beta protein, and ZYZ 802 shows therapeutic benefits for cardiovascular diseases and neurodegenerative disorders. In his presentation entitled “The mechanism(s) of protein persulfide formation: Detection of protein S-sulfhydration by a tagswitch assay,” Milos Filipovic from Friedrich-Alexander University of Erlangen-Nurenberg discussed a new method to measure the levels of sulfhydrated molecules. In this tag-switch method, sulfhydrated residues are detected by forming stable thioether conjugates. He also proposed a possible mechanism for the formation
of persulfide, including the reaction with protein sulfenic acids, and the oxidation of H2S with metal centers. Konstantin Shatalin from New York University School of Medicine mentioned that Bacillus anthracis-derived NO is critical for infection, and that CBS/CSE/NOS mutations change the global gene transcription profile, as well as spore formation, and diminish virulence. In his presentation entitled “Role of H2S and NO in Bacillus anthracis spore formation and virulence” he suggested that the interaction of H2S and NO plays an important role in bacterial virulence. Their proposed mechanism could be used in antimicrobial therapy. 8. Session 8, diabetes and renal diseases Csaba Szabo from University of Texas Medical Branch mentioned that hyperglycemia- or streptozotocin-induced diabetes causes endothelial- and mitochondrial-dysfunction, and that lipoic acid, a substance used for the 3MST-mediated production of H2S, restores this dysfunction. In his presentation entitled “Pathophysiological changes in hydrogen sulfide homeostasis during diabetic complications” he suggested that diabetes is associated with the impairment of the 3MST/H2S pathway, and that this impairment could lead to the pathogenesis of diabetic complications. In his presentation entitled “The role of hydrogen sulfide in diabetic nephropathy (DN),” Yukio Yuzawa from Fujita Health University School of Medicine mentioned that, in the model of diabetic nephropathy, the expression of CSE is markedly reduced, and it is similarly to that of eNOS. The subsequent vasoconstriction and loss of blood flow in the peritubular capillary is restored by H2S. His research group suggests that H 2 S and NO regulate the tubulointerstitial microcirculation, and that the H2S system could be used as a target for treating DN. Alp Sener from London Health Sciences Center mentioned that supplemental H2S treatment during prolonged warm as well as cold ischemia associated with transplantation, dramatically improves the renal vascular flow patterns as well as the renal graft survival and function. It also decreases renal injury and inflammation as compared with grafts preserved in standard preservative solutions. In his presentation entitled “Hydrogen sulphide: The magic bullet in organ transplantation?” he also presented their preliminary data showing that D-cysteine treatment efficiently protects renal grafts from ischemia. In his presentation entitled “Hydrogen sulphide in renal disease and transplantation,” Harry van Goor from University Medical Center Groningen mentioned that thiosulfate protects kidneys from experimental renal diseases. Based on the result that the effectiveness of such a protective mechanism is different in mice and rats, they suggested that H2S treatment holds great promise for use in the treatment of these diseases. However, it has been a challenge to successfully apply this technique in humans. Utpal Sen from University of Louisville discussed that matrix metalloproteinase-9 (MMP-9)-mediated H2S production triggers reno-vascular remodeling in diabetes, and that H2S modulates autophagy to mitigate adverse remodeling in his presentation entitled “Diabetic renal remodeling: Role of hydrogen sulfide”. Toshihide Kimura from Oita University mentioned that glucose increases the production of H2S by inducing the expression of CSE, which leads to the protection of islet cells from apoptotic cell death. In his presentation entitled “Protective roles of hydrogen sulfide in pancreatic beta-cells” he also suggested that H2S produced by CSE prevents the pancreatic beta cells from releasing insulin, thereby reducing the cellular stress caused by glucose. 9. Session 9, H2S, persulfide, and polysulfide Maria Wrobel from Jagiellonian University mentioned that 3MST transfers sulfur to cyanide for detoxification or to proteins for
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modification of their activity in her presentation entitled “Sulfurtransferases and their role in hydrogen sulfide generation”. Suppression of the 3MST-mediated mitochondrial production of H2S by oxidative stress may cause various pathophysiological conditions. According to her, the identification of sulfane-sulfur donors and acceptors as well as the determination of their regulatory roles in cellular metabolism would be the target for future studies. In his presentation entitled “Mercaptopyruvate sulfurtransferase and hydrogen sulfide,” Noriyuki Nagahara from Nippon Medical School discussed the three-step mechanism of H2S production by 3MST, and the physiological roles of 3MST. In addition to the production of H2S, 3MST has three other activities: carrying out the degradation of cysteine to pyruvate via the mercaptopyruvate pathway, serving as an antioxidant, and producing sulfur oxides. Peter Nagy from National Institute of Oncology mentioned in his presentation entitled “Mechanistic considerations of sulfide versus polysulfide mediated signaling events from a chemist’s perspective” that the interaction of sulfides with the protein metal center plays an important role in sulfide-mediated signaling, in addition to the sulfhydration of protein cysteine residues, and interactions with the molecules involved in NO signaling. In his presentation entitled “Nociceptive action of hydrogen sulfide through the activation of transient receptor potential ankyrin 1 (TRPA1),” Toshio Ohta, Tottori University, mentioned that the activation of TRPA1 channels by polysulfides and H2S is involved in neurogenic inflammation and hyperalgesia. Polysulfides activate the TRPA1 channels more efficiently than does H2S, and the injection of these compounds into the mouse hind paw causes pain-related behavior. These sulfur compounds and TRPA1 are attractive therapeutic targets for the treatment of hyperalgesia. Yuki Ogasawara from Meiji Pharmaceutical University mentioned that polysulfide adds bound sulfane-sulfur to Keap1 during sulfhydration to release Nrf2 to the nucleus, where it upregulates the transcription of glutathione synthetase and heme-oxygenase 1 genes. This leads to the protection of neuronal cells from oxidative damage. In his presentation entitled “Bound sulfur exerts protective effect against cytotoxicity in neuronal cells” he also demonstrated the acceleration of Akt phosphorylation by polysulfides. Dennis Searcy from University of Massachussetts mentioned in his presentation entitled “Elemental sulfur reduction by eukaryotic cytoplasm consistent with an ancient sulfur symbiosis” that H2S is consumed during aerobic conditions, and produced through cysteine metabolism during anoxic conditions. S0 is metabolized to produce H2S through a pathway that does not involve cysteine metabolism. During the sulfur cycle, S0 is reduced in the cytoplasm, and H2S is oxidized inside the mitochondria. In his presentation entitled “Isolation, characterization, and reactivity of biomimetic hydrodisulfide (RSSH) complexes” Michael Pluth from University of Oregon showed that small molecule hydrodisulfides react with biologically relevant reactive sulfur, nitrogen, and oxygen species including thiols, NO x , and H 2 O 2 . Hydrodisulfides could be important in H2S storage and signaling.
10. Future perspective The endogenous concentrations of H2S have recently been reevaluated and confirmed its existence under the steady-state conditions. However, the physiological stimuli which induce changes in H2S concentrations and the range of such changes have not been determined. One of the approaches to address these problems is to understand the metabolic turnover of H2S: a balance between H2S production and degradation. CBS activity is regulated by S-adenosylmethionine, NO, CO and glutathionylation. CSE and CAT are regulated by Ca2+ [14,27,28,30]. The regulation of H2S degrading enzymes in mitochondrial has not well been understood.
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Polysulfides have recently emerged as candidate signaling molecules. Polysulfides activate TRPA1 channels, facilitates the translocation of transcription factor Nrf2, and a cancer suppressor PTEN by sulfurating (sulfhydrating) the cysteine residues of these target proteins to modify their activity [36,39,41,42]. It is necessary to understand both enzymatic- and non-enzymatic-production of polysulfides and the mechanism of their effects. The cross-talk between H2S and NO has been extensively studied. A synergistic effect between both substances was initially reported, subsequently the regulation of H2S production and its transcriptional regulation by NO has been identified [15,16]. Regulation of NO production by H2S through the modification of NOS activity was recently demonstrated [51]. Novel candidates for signaling molecules such as HSNO, HNO, and HSSNO have been proposed to be generated by the chemical interaction between H2S and NO [52,53]. It is necessary to clarify whether H2S and NO are present in cells at the same time, and if enzymes producing these molecules are activated under the same conditions. The biochemical nature of H2S and related molecules, as well as their mechanisms of action and regulation, will be clarified, and the stimulating discussion will be expected in the 4th International Conference on H2S in Biology and Medicine held in Naples, Italy in 2016. Acknowledgments This work was supported by a grant from the National Institute of Neuroscience, KAKENHI (23659089) Grant-in-Aid for Challenging Exploratory Research, and KAKENHI (26460115) Grantin-Aid for Scientific Research to HK. The third International Conference on H2S in Biology and Medicine was supported by Commemorative organization for the Japan World Exposition ’70, Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Naito Foundation, The Research Foundation for Pharmaceutical Sciences, Terumo Life Science Foundation, The Tokyo Biochemical Research Foundation, The Uehara Memorial Foundation, Antibe Therepeutics Inc., Sulfagenix, Inc., Fondazione Internazionale Menarini, Asahi Kasei Pharma Corp., Kobayashi Pharmaceutical Co., Ltd., Noevir Holdings Co., Ltd., and The Federation of Pharmaceutical Manufactures’ Associations of Japan. Finally, I thank A & E Planning Co. Ltd or H2S 2014 secretariat for organizing the conference. References [1] R.F. Furchgott, Studies on relaxation of rabbit aorta by sodium nitrate: basis for the proposal that the acid-activatable component of the inhibitory factor from retractor penis is inorganic nitrate and the endothelium-derived relaxing factor is nitric oxide, in: P.M. Vanhoutte (Ed.), Mechanisms of Vasodilatation, Raven, New York, 1988, pp. 401–414. [2] L.J. Ignarro, R.E. Byrns, K.S. Wood, Biochemical and pharmacological properties of endothelium-derived relaxing factor and its similarity to nitric oxide radical, in: P.M. Vanhoutte (Ed.), Mechanisms of Vasodilatation, Raven, New York, 1988, pp. 427–435. [3] A. Verma, D.J. Hirsch, E.E. Glatt, G.V. Ronnett, S.H. Snyder, Carbon monoxide: a putative neural messenger, Science 259 (1993) 381–384. [4] C.F. Stevens, Y. Wang, Reversal of long-term potentiation by inhibitors of haem oxygenase, Nature 364 (1993) 147–149. [5] M. Zhuo, S.A. Small, E.R. Kandel, R.D. Hawkins, Nitric oxide and carbon monoxide produce activity-dependent long-term synaptic enhancement in hippocampus, Science 260 (1993) 1946–1950. [6] A. Meister, P.E. Fraser, S.V. Tice, Enzymatic desulfuration of mercaptopyruvate to pyruvate, J. Biol. Chem. 206 (1954) 561–575. [7] D. Cavallini, B. Mondovi, C. De Marco, A. Scioscia-Santoro, The mechanism of desulphhydration of cysteine, Enzymologia 24 (1962) 253–266. [8] A.E. Braunstein, E.V. Goryachenkowa, E.A. Tolosa, I.H. Willhardt, L.L. Yefremova, Specificity and some other properties of liver serine sulphhydrase: evidence for its identity with cystathionine β–synthase, Biochim. Biophys. Acta 242 (1971) 247–260. [9] M.W. Warenycia, L.R. Goodwin, C.G. Benishin, R.J. Reiffenstein, D.M. Grancom, J.D. Taylor, et al., Acute hydrogen sulfide poisoning. Demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels, Biochem. Pharmacol. 38 (1989) 973–981.
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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
H2S2014 in Kyoto: The 3rd International Conference on H2S in Biology and Medicine Hideo Kimura * National Institute of Neuroscience, National Center for Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
A R T I C L E
I N F O
Article history: Received 2 September 2014 Revised 6 October 2014 Available online
A B S T R A C T
About 20 years ago, a pungent gas was found to be the physiological mediator of cognitive function and vascular tone. Since then, studies on hydrogen sulfide (H2S) have uncovered its numerous physiological roles such as protecting various tissues/organs from ischemia and regulating inflammation, cell growth, oxygen sensing, and senescence. These effects of H2S were extensively studied, and some of the corresponding mechanisms were also studied in detail. Previous studies on the synergistic interaction between H2S and nitric oxide (NO) have led to the discovery of several potential signaling molecules. Polysulfides are considerably potent and are one of the most active forms of H2S. H2S has a significant therapeutic potential, which is evident from the large number of novel H2S-donating compounds and substances developed for manipulating endogenous levels of H2S. The Third International Conference on H2S was held in Kyoto in June 2014. One hundred and sixty participants from 21 countries convened in Kyoto to report new advances, discuss conflicting findings, and make plans for future research. This article summarizes each oral presentation presented at the conference. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Nitric oxide (NO) was discovered as a gaseous signaling molecule in 1986 [1,2]; carbon monoxide (CO) was then discovered in 1993 [3–5]. The H2S-producing enzymes were extensively studied between the 1950s and the 1970s [6–8]. However, H2S was considered a mere by-product of the various metabolic pathways or just a marker for enzyme activities. In 1989, endogenous sulfide was discovered in mammalian brains [9,10]. Unfortunately, the H2S measured in these studies was found to be contaminated with other forms of sulfur that also release this gas. Although the re-evaluated levels were much lower than those initially reported, these studies confirmed the existence of H2S inside the tissues [11–13]. The role of H2S as a neuromodulator that acts in association with cystathionine β-synthase (CBS) as an H2S-producing enzyme in the brain was demonstrated in 1996 [14]. Its other role as a smooth-muscle relaxant in conjugation with cystathionine γ–lyase (CSE), which generates H2S in the smooth muscles, was discovered in the following year [15]. The involvement of ATP-dependent K+ channels in vascular relaxation was subsequently demonstrated [16]. Because H2S is a relatively toxic gas, its cytoprotective effects had been completely overlooked. It was found to protect neurons from oxidative stress by increasing the levels of glutathione, a major en-
* National Institute of Neuroscience, National Center for Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. Fax: +81 42 346 1755. E-mail address: [email protected]
dogenous antioxidant and scavenger of reactive oxygen species (ROS) [17,18]. This finding led to the identification of the protective effect of H2S on various other organs/tissues. H2S protects organs/tissues from ischemia-reperfusion injury and oxidative insults [19,20]. It induces angiogenesis, which is also mediated by the vascular endothelial growth factor (VEGF) [21,22]. H 2 S is oxidized in the mitochondria for the generation of adenosine triphosphate (ATP) [23,24], and it presumably functions as an oxygen sensor [25,26]. The activity of CBS is enhanced by S-adenosyl methionine [14], and the activity of CSE is increased by Ca2+ [27]. However, the latter result proved to be controversial, when it was reported that CSE was suppressed by Ca2+ [28]. Cysteine aminotransferase (CAT) is identical to aspartate aminotransferase, and produces 3-mercaptopyruvate, a substrate for 3-mercaptopyruvate sulfurtransferase (3MST). The enzyme 3MST generates H2S [29]. The activity of CAT is also suppressed by Ca2+ [30]. In contrast to CBS and CSE, 3MST requires an accompanying reducing agent to produce H2S. Thioredoxin was identified to be the reductant that assisted 3MST in producing H2S [31,32]. An H2S-generating pathway involving D-cysteine was identified recently [33]. One of the proposed modes through which H2S transmits subcellular signals involves sulfhydration or sulfuration [34]. It involves the addition of sulfur to the cysteine residues of the target proteins to induce a conformational change that modifies their activities. Previous studies show that Keap1, NF-kB, transient receptor potential ankyrin 1 (TRPA1), and perkin are activated by sulfhydration or sulfuration [35–40]. Recently, polysulfides (H2Sn, n = 3–7; n = 2 for the persulfide) discovered in the mammalian brain were found to
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activate the TRPA1 channels [39], thereby facilitating the translocation of Nuclear-factor-E2-related factor 2 (Nrf2) to the nucleus after being released from the sulfhydrated Keap1 [36]. The polysulfides were also found to modulate the activity of phosphatase and tensin homolog (PTEN) [41] more efficiently than H2S. After carefully studying their conversion efficiency and oxidation states, it was proposed that polysulfides, rather than H2S, sulfhydrate (or sulfurate) their target proteins [42]. H2S exerts regulatory effects on inflammatory processes and promotes their resolution. Owing to these beneficial effects of H2S, several H2S-releasing anti-inflammatory drugs and H2S-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) exhibiting no significant side effects on the gastrointestinal tract have been developed [43]. Many of these drugs have shown considerable promise in the relevant animal models, and are presently undergoing clinical trials. H2S also inhibits cancer development at various stages, and H2S donors are cytotoxic to human cancer cells [44]. They also induce cell cycle arrest in the G2/M phase and promote apoptosis. However, they do not affect the survival of normal human cells. Several recent studies have shown that H2S-releasing NSAIDs exhibit enhanced chemopreventive effects in cancer cells, whereas parental NSAIDs alone do not confer any such beneficial effects to cancer cells [45]. Contrary to these observations, CBS is upregulated in the colorectal and ovarian cancer cells, where H2S is involved in promoting cellular bioenergetics, proliferation, and migration [46]. In these cancer cells, the suppression of CBS activity can be used as a potential therapy. Because the metabolism, oxidation, evaporation, and absorption of sulfane-sulfur occur rapidly, it is difficult to accurately measure the concentrations of H2S, or to track its movements inside the cells. Recently, several fluorescent probes have been developed for the specific detection of H2S and sulfane-sulfur [47–50]. These probes consist of two functional sites, a site sensitive to H2S and a site harboring a fluorophore. The first conference on H2S was successfully held in 2009 in Shanghai, China, and the second conference was held in 2012 in Atlanta, Georgia, USA. During the same period, the first European conference on H2S was held in 2012 in Blatislava, Slovakia, and the second in 2013 in Exeter, England. Two weeks before this Kyoto conference, the international symposium on H2S and NO was held in Naples, Italy. Research on H2S is witnessing enormous growth at present.
2. Session 1, biology and chemistry of H2S In his presentation entitled “H2S and beyond,” Hideo Kimura from National Institute of Neuroscience discussed his group’s recent findings on the production of H2S from D-cysteine and the potential roles of polysulfides as signaling molecules. D-cysteine is metabolized to H2S by a specific pathway, and it efficiently prevents ischemia-reperfusion injury. The study group has also shown that polysulfides activate ions channels and a transcription factor much more potently than H2S by sulfhydration or sulfuration of these targets. Rui Wang from Lakehead University discussed the importance of the interaction among the gasotransmitters such as H2S, NO, and carbon monoxide (CO), for the functioning of the cellular signaling networks. In his presentation entitled “The next wave of gasotransmitter research”, he also mentioned that ammonia, another gasotransmitter, is an endogenously produced gas with specific substrates and enzymes. It functions at physiological concentrations through the activation of specific molecular targets. Gasotransmitters share common target molecules, but exert their activities using different mechanisms. In some cases, they produce the same outcome through the activation of entirely different pathways.
The cellular regulation of H2S levels and the enzyme activities necessary for H2S production and metabolism are not well understood. In her presentation entitled “Enzymology of H2S turnover”, Ruma Banerjee from University of Michigan Medical School discussed the structures of CBS and 3MST at the reactive intermediates in the active sites, as well as the relative contribution of CBS, CSE and 3MST to H2S production. H2S clearance plays an important role during the cessation of H2S signaling. The study group has recently shown that the activity of CBS is increased by its glutathionilation under oxidative stress, thereby increasing the levels of glutathione and H2S. This is a plausible mechanism for cellular survival during oxidative stress. 3. Session 2, H2S in cardiovascular diseases David Lefer from LSU Cardiovascular Center of Excellence discussed the protective effect of an H2S-donor and a major garlic extract (diallyl trisulfide; DATS) on a pressure-overloaded heart. H2S therapy increases the expression of the proangiogenic factor vascular endothelial cell growth factor, but decreases the expression of the angiogenesis inhibitory factor, angiostatin. In his presentation entitled “Hydrogen sulfide in heart failure,” he also mentioned that the phosphorylation of endothelial NO synthase (eNOS) at serine 1177 is increased by H2S treatment, which causes an increase in the bioavailable NO, a peripheral factor that protects the heart from cardiac arrest. The involvement of H2S in cleaving the cysteine disulfide bonds for activating the target proteins such as VEGFR2, EGFR, the insulin receptor, and the potassium channels was discussed by Yi-Chun Zhu from Fudan University in his presentation entitled “Identification of hydrogen sulfide “receptors” in the receptor tyrosine kinase family and ion channels”. H2S activates Na+/K+-ATPase through EGFR, leading to an increase in Na+ excretion and a decrease in blood pressure in chronic salt-loaded rats. The activation of potassium channels in cardiomyocytes improves fatal arrhythmia during acute myocardial infarction. In his presentation entitled “BRG1-dependent epigenetic control of vascular smooth muscle cell proliferation by hydrogen sulfide”, Junbao Du from Peking University First Hospital discussed the role of H2S in suppressing the proliferation of vascular smooth muscle cells by downregulating Brg1, the central catalytic subunit of the ATP-dependent chromatin remodeling complex, and by reducing the recruitment of Brg1 to the promoter regions of proliferationrelated genes such as the proliferating cell nuclear antigen, neurotrophin3, and platelet-derived growth factor subunit A. John Calvert from Emory University School of Medicine discussed the role of Nrf2 in mediating the cardioprotective effects of H2S therapy in a mouse model of ischemia-induced heart failure. In his presentation entitled “Nrf2 signaling is essential for hydrogen sulfide mediated cardioprotection in the setting of ischemiainduced heart failure” he also mentioned that the effect of H2S was confirmed in Nrf2 KO mice, which display exacerbated dysfunction and hypertrophy as compared with the wild-type mice. 3. Session 3, Inflammation Matt Whiteman from University of Exeter Medical School discussed the development of novel compounds that release H2S in a physiologically relevant manner, and examined the precise role of H2S in inflammation and therapeutic application. To circumvent the problems involving the immediate release of H2S (in solution) via NaHS, and the presence of residual solvent contaminants in GYY4137, novel compounds with a slow and well-controlled release have been developed. In his presentation entitled “H2S in inflammation: Time for resolution?” he mentioned that his group has also developed compounds targeted to the mitochondria.
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In his presentation entitled “Roles of the hydrogen sulfideCAV3.2 T-type calcium channel pathway in neuronal and neuroendocrine differentiation,” Atsufumi Kawabata from Kinki University, showed that H2S enhances the activity of the Cav3.2 T-type calcium channels that are involved in pain signaling and neuronal differentiation. The upregulated expression of CSE and Cav3.2 channels enhances the secretory responses in differentiationinduced human prostate cancer cells. This pathway may serve as a target for prostate cancer therapy. Fumito Ichinose from Harvard Medical School discussed the endotoxemia-induced decrease in the plasma sulfide levels. In his presentation entitled “H2S in sepsis and shock – A story of mice and men,” he also mentioned that breathing H 2 S attenuates the endotoxin-induced systemic inflammation, and restores the sulfide levels. A marked increase in the levels of thiosulfate is also observed. The application of thiosulfate improves survival rates. Recent data from his group shows intriguing results: the deficiency and suppression of CSE increases (instead of decreasing) the levels of thiosulfate in the liver and prevents acute liver failure. In her presentation entitled “H2S targets transient receptor potential receptors to relieve from inflammation and pain,” Nathalie Vegnolle from INSERM, mentioned that NaHS (200–1000 μM) decreases the calcium responses of TRPV4 and TRPA1 to their corresponding agonists in the intestinal epithelial cells and dorsal ganglion cells. H2S efficiently reduces visceral inflammation and pain by inhibiting the pro-inflammatory and pro-nociceptive effects of TRPV4 and TRPA1.
4. Session 4, analysis of H2S Makoto Suematsu, Keio University mentioned that the regulation of CBS by CO determines the balance between NADPH and glutathione, as well as the directional glucose utilization in cancer cells that protects them from oxidative stress. In his presentation entitled “CO-CBS system for regulating tenuous balance between NADPH and persulfides in cancer”, he also mentioned stressinduced CO suppresses CBS, which leads to a decrease in the methylation of PFKFB3, thereby shifting the glucose utilization from glycolysis to the pentose phosphate pathway, and stimulating cancer cell proliferation during the process. In contrast, the activation of CBS increases NADPH and glutathione, thereby protecting the cells from oxidative stress. In his presentation entitled “Development of an H2S fluorescent probe and its application to inhibitor screening for H 2 S producing enzyme,” Kenjiro Hanaoka from Tokyo University, discussed the development of a novel fluorescence probe for H2S, HSip1, which specifically reacts with 10 μM H2S, even in the presence of 10 mM glutathione. By using this probe, they successfully developed selective inhibitors for 3MST and CSE. The development of slow H2S-releasing donors and specific inhibitors of H2S-producing enzymes was discussed by Andreas Papapetropoulos from University of Athens in his presentation entitled “Novel pharmacological tools for the H2S pathway”. In contrast to inorganic compounds such as NaHS and Na 2 S, which immediately release H2S, the organic donors slowly release H2S in a physiologically relevant manner. Hidehiko Nakagawa from Nagoya City University discussed the development of a photochemical method for H2S generation in his presentation entitled “Photo-inducible hydrogen sulfide releaser using ketoprofenate photocages”. A ketoprofenate photocage releases H2S in an appropriate location, timing, and dosage proportional to the timing and intensity of the incident electromagnetic radiation. In his presentation entitled “New chemistry and chemical tools for hydrogen sulfide research,” Ming Xian from Washington State
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University discussed the development of controllable H2S donors and fluorescent probes for H2S and sulfane-sulfur species. 5. Session 5, therapeutics and development of H2S-based drugs John Wallace from University of Calgary discussed the development of H2S-releasing NSAIDs in his presentation entitled “H2Sbased therapeutics for inflammatory diseases”. NSAIDs are effective at reducing pain and inflammation, but they show serious adverse effects especially on the gastrointestinal (GI) tract. The H 2 Sreleasing NSAIDs show a dramatically better GI safety and antiinflammatory efficacy than the parental NSAIDs. Preclinical studies have confirmed the safety of these compounds and have led to clinical trials in humans. In his presentation entitled “Hydrogen sulfide accounts for the peripheral vascular effects of sulfhydrylated ACE inhibitors beyond ACE inhibition: A proof of concept of the role of H2S in cardiovascular?,” Giuseppe Cirino, University Degli Studi Di Napoli, mentioned that the sulfhydrylated ACE inhibitor, zofenopril, relaxes vascular smooth muscles and improves vascular function by potentiating the effect of H2S in a spontaneous hypertension model. Jinsong Bian from National University of Singapore mentioned that H 2 S reduces the polymerization of tau, a major component of the neurofibrillary tangle by sulfhydrating its cysteine residues, and inhibits amyloid β–formation, a major component of plaque, in the animal models of Alzheimer’s disease in his presentation entitled “Can hydrogen sulfide be used to treat neurodegenerative diseases?”. H2S also protects the dopaminergic neurons in the animal models of Parkinson’s disease. In his presentation entitled “Phase 1, heart failure clinical trial to evaluate the safety of SG1002 – A novel hydrogen sulfide donor,” Tony Giordano from Sulfagenix, discussed the safety of SG1002 for normal individuals. A double-blind, placebo-controlled Phase 1 study of SG1002 showed that it is well tolerated in normal individuals, and causes limited side effects. His group is repeating this study on a heart failure subject. Jerzy Beltowski from Medical University of Lublin mentioned that nucleoside monophosphorothioates, which are nucleotide analogs with one oxygen atom replaced by sulfur, release H2S in perivascular adipose tissue and induce vasorelaxation. In his presentation entitled “Nucleoside mono-phosphorothioates as the H2S donors in perivascular adipose tissue” he also mentioned that these substances could be effectively used as vasodilators especially in patients suffering from the metabolic syndrome. 6. Session 6, H2S signaling In his presentation entitled “Hydrogen sulfide as an oxygen sensor,” Ken Olson from Indiana University School of Medicine, mentioned that the O2-dependent metabolism of H2S could be an effective O2-sensing mechanism. Tissue H2S concentrations are inversely related to the O 2 concentrations, and this reciprocal relationship between H2S and O2 is responsible for the similarity between the effect of hypoxia and the effect of H2S. The determination of this mechanism at sub-cellular level will strongly support the above-mentioned hypothesis. Frederic Bouillaud, INSERM, mentioned in his presentation entitled “Mitochondrial oxidation of H2S in mammals” that H2S is oxidized in the mammalian mitochondria. It is metabolized by sulfide quinone reductase and dioxygenase, and is involved in the production of ATP in the liver. In his presentation entitled “H2S signal interacts with hypoxia signal in a mitochondria-dependent manner,” Kiichi Hirota, Kansai Medical University, mentioned that H2S modulates the intracellular O2 homeostasis, reduces the accumulation of hypoxia-inducible
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factor 1 (HIF-1), and also reduces the subsequent gene expression induced by hypoxia. H2S decreases cellular O2 consumption and inhibits the HIF-1 accumulation induced by hypoxia by increasing its metabolism without affecting its production. Koji Takeuchi from Kyoto Pharmaceutical University discussed that H2S modulates the acid-induced HCO3- secretion by enhancing the production of mucosal PGE2 and NO, and it is involved in mucosal protection in the duodenum in his presentation entitled “Stimulation of duodenal HCO3− secretion by H2S in rats: Regulatory mechanism and importance in mucosal defense”. The duodenal damage caused by the acid is healed by H2S produced by CSE. Mark Hellmich from University of Texas Medical Branch mentioned that the H2S produced by CBS plays an important role in promoting cellular bioenergetics, proliferation, and migration in colorectal and ovarian cancer. He also mentioned in his presentation entitled “Role of cystathionine-beta synthase and its product, hydrogen sulfide in cancer biology” that targeting CBS to suppress H2S production in cancer cells may provide novel anticancer therapies.
7. Session 7, H2S and NO Christopher Kevil from LSU Health Science Center mentioned that ischemia increases CSE expression and the levels of H2S that restores blood flow in the ischemic tissues in his presentation entitled “Vascular sulfide metabolism during ischemia”. Once blood starts flowing, the levels of CSE and H2S return to the control level. This blood flow restoration is blunted in CSE-deficient animals and augmented in CSE-transgene animals. An increase in the bioavailability of H2S is an important physiological adaptive response under ischemic conditions. In his presentation entitled “H2S induced decomposition of nitroso-compounds and consequent biological effect,” Karol Ondrias from Institute of Molecular Physiology and Genetics and INPP SAS mentioned that H2S acts on S-nitrosoglutathione to produce polysulfides and SSNO−, which subsequently releases NO, thereby relaxing the aorta smooth muscle more effectively than S-nitrosoglutathione. The interaction between H 2 S and S-nitrosoglutathione may have an important physiological role. The identification of the endogenous products is a challenge for future research. Martin Feelisch from University of Southampton mentioned that the interaction between H2S and NO produces multiple reaction products including reactive nitrogen and sulfur species, which induce the expression of downstream signaling molecules such as cGMP and Keap1/Nrf2. In his presentation entitled “The NO/ sulfide system and its significance for biological signaling” he also discussed the production of SSNO−, nitroxyl, and polysulfides. Yi-Zhun Zhu from Fudan University mentioned that S-propargyl-cysteine, ZYZ-802 exerts cardiovascular- and neuroprotective effects by modulating the CSE activity in his presentation entitled “Novel effect of endogenous H2S donor, ZYZ802 on heart and brain”. This compound promotes cell proliferation, adhesion, migration, tube formation of vascular endothelial cells, and angiogenesis. It also suppresses the formation of the amyloid beta protein, and ZYZ 802 shows therapeutic benefits for cardiovascular diseases and neurodegenerative disorders. In his presentation entitled “The mechanism(s) of protein persulfide formation: Detection of protein S-sulfhydration by a tagswitch assay,” Milos Filipovic from Friedrich-Alexander University of Erlangen-Nurenberg discussed a new method to measure the levels of sulfhydrated molecules. In this tag-switch method, sulfhydrated residues are detected by forming stable thioether conjugates. He also proposed a possible mechanism for the formation
of persulfide, including the reaction with protein sulfenic acids, and the oxidation of H2S with metal centers. Konstantin Shatalin from New York University School of Medicine mentioned that Bacillus anthracis-derived NO is critical for infection, and that CBS/CSE/NOS mutations change the global gene transcription profile, as well as spore formation, and diminish virulence. In his presentation entitled “Role of H2S and NO in Bacillus anthracis spore formation and virulence” he suggested that the interaction of H2S and NO plays an important role in bacterial virulence. Their proposed mechanism could be used in antimicrobial therapy. 8. Session 8, diabetes and renal diseases Csaba Szabo from University of Texas Medical Branch mentioned that hyperglycemia- or streptozotocin-induced diabetes causes endothelial- and mitochondrial-dysfunction, and that lipoic acid, a substance used for the 3MST-mediated production of H2S, restores this dysfunction. In his presentation entitled “Pathophysiological changes in hydrogen sulfide homeostasis during diabetic complications” he suggested that diabetes is associated with the impairment of the 3MST/H2S pathway, and that this impairment could lead to the pathogenesis of diabetic complications. In his presentation entitled “The role of hydrogen sulfide in diabetic nephropathy (DN),” Yukio Yuzawa from Fujita Health University School of Medicine mentioned that, in the model of diabetic nephropathy, the expression of CSE is markedly reduced, and it is similarly to that of eNOS. The subsequent vasoconstriction and loss of blood flow in the peritubular capillary is restored by H2S. His research group suggests that H 2 S and NO regulate the tubulointerstitial microcirculation, and that the H2S system could be used as a target for treating DN. Alp Sener from London Health Sciences Center mentioned that supplemental H2S treatment during prolonged warm as well as cold ischemia associated with transplantation, dramatically improves the renal vascular flow patterns as well as the renal graft survival and function. It also decreases renal injury and inflammation as compared with grafts preserved in standard preservative solutions. In his presentation entitled “Hydrogen sulphide: The magic bullet in organ transplantation?” he also presented their preliminary data showing that D-cysteine treatment efficiently protects renal grafts from ischemia. In his presentation entitled “Hydrogen sulphide in renal disease and transplantation,” Harry van Goor from University Medical Center Groningen mentioned that thiosulfate protects kidneys from experimental renal diseases. Based on the result that the effectiveness of such a protective mechanism is different in mice and rats, they suggested that H2S treatment holds great promise for use in the treatment of these diseases. However, it has been a challenge to successfully apply this technique in humans. Utpal Sen from University of Louisville discussed that matrix metalloproteinase-9 (MMP-9)-mediated H2S production triggers reno-vascular remodeling in diabetes, and that H2S modulates autophagy to mitigate adverse remodeling in his presentation entitled “Diabetic renal remodeling: Role of hydrogen sulfide”. Toshihide Kimura from Oita University mentioned that glucose increases the production of H2S by inducing the expression of CSE, which leads to the protection of islet cells from apoptotic cell death. In his presentation entitled “Protective roles of hydrogen sulfide in pancreatic beta-cells” he also suggested that H2S produced by CSE prevents the pancreatic beta cells from releasing insulin, thereby reducing the cellular stress caused by glucose. 9. Session 9, H2S, persulfide, and polysulfide Maria Wrobel from Jagiellonian University mentioned that 3MST transfers sulfur to cyanide for detoxification or to proteins for
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modification of their activity in her presentation entitled “Sulfurtransferases and their role in hydrogen sulfide generation”. Suppression of the 3MST-mediated mitochondrial production of H2S by oxidative stress may cause various pathophysiological conditions. According to her, the identification of sulfane-sulfur donors and acceptors as well as the determination of their regulatory roles in cellular metabolism would be the target for future studies. In his presentation entitled “Mercaptopyruvate sulfurtransferase and hydrogen sulfide,” Noriyuki Nagahara from Nippon Medical School discussed the three-step mechanism of H2S production by 3MST, and the physiological roles of 3MST. In addition to the production of H2S, 3MST has three other activities: carrying out the degradation of cysteine to pyruvate via the mercaptopyruvate pathway, serving as an antioxidant, and producing sulfur oxides. Peter Nagy from National Institute of Oncology mentioned in his presentation entitled “Mechanistic considerations of sulfide versus polysulfide mediated signaling events from a chemist’s perspective” that the interaction of sulfides with the protein metal center plays an important role in sulfide-mediated signaling, in addition to the sulfhydration of protein cysteine residues, and interactions with the molecules involved in NO signaling. In his presentation entitled “Nociceptive action of hydrogen sulfide through the activation of transient receptor potential ankyrin 1 (TRPA1),” Toshio Ohta, Tottori University, mentioned that the activation of TRPA1 channels by polysulfides and H2S is involved in neurogenic inflammation and hyperalgesia. Polysulfides activate the TRPA1 channels more efficiently than does H2S, and the injection of these compounds into the mouse hind paw causes pain-related behavior. These sulfur compounds and TRPA1 are attractive therapeutic targets for the treatment of hyperalgesia. Yuki Ogasawara from Meiji Pharmaceutical University mentioned that polysulfide adds bound sulfane-sulfur to Keap1 during sulfhydration to release Nrf2 to the nucleus, where it upregulates the transcription of glutathione synthetase and heme-oxygenase 1 genes. This leads to the protection of neuronal cells from oxidative damage. In his presentation entitled “Bound sulfur exerts protective effect against cytotoxicity in neuronal cells” he also demonstrated the acceleration of Akt phosphorylation by polysulfides. Dennis Searcy from University of Massachussetts mentioned in his presentation entitled “Elemental sulfur reduction by eukaryotic cytoplasm consistent with an ancient sulfur symbiosis” that H2S is consumed during aerobic conditions, and produced through cysteine metabolism during anoxic conditions. S0 is metabolized to produce H2S through a pathway that does not involve cysteine metabolism. During the sulfur cycle, S0 is reduced in the cytoplasm, and H2S is oxidized inside the mitochondria. In his presentation entitled “Isolation, characterization, and reactivity of biomimetic hydrodisulfide (RSSH) complexes” Michael Pluth from University of Oregon showed that small molecule hydrodisulfides react with biologically relevant reactive sulfur, nitrogen, and oxygen species including thiols, NO x , and H 2 O 2 . Hydrodisulfides could be important in H2S storage and signaling.
10. Future perspective The endogenous concentrations of H2S have recently been reevaluated and confirmed its existence under the steady-state conditions. However, the physiological stimuli which induce changes in H2S concentrations and the range of such changes have not been determined. One of the approaches to address these problems is to understand the metabolic turnover of H2S: a balance between H2S production and degradation. CBS activity is regulated by S-adenosylmethionine, NO, CO and glutathionylation. CSE and CAT are regulated by Ca2+ [14,27,28,30]. The regulation of H2S degrading enzymes in mitochondrial has not well been understood.
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Polysulfides have recently emerged as candidate signaling molecules. Polysulfides activate TRPA1 channels, facilitates the translocation of transcription factor Nrf2, and a cancer suppressor PTEN by sulfurating (sulfhydrating) the cysteine residues of these target proteins to modify their activity [36,39,41,42]. It is necessary to understand both enzymatic- and non-enzymatic-production of polysulfides and the mechanism of their effects. The cross-talk between H2S and NO has been extensively studied. A synergistic effect between both substances was initially reported, subsequently the regulation of H2S production and its transcriptional regulation by NO has been identified [15,16]. Regulation of NO production by H2S through the modification of NOS activity was recently demonstrated [51]. Novel candidates for signaling molecules such as HSNO, HNO, and HSSNO have been proposed to be generated by the chemical interaction between H2S and NO [52,53]. It is necessary to clarify whether H2S and NO are present in cells at the same time, and if enzymes producing these molecules are activated under the same conditions. The biochemical nature of H2S and related molecules, as well as their mechanisms of action and regulation, will be clarified, and the stimulating discussion will be expected in the 4th International Conference on H2S in Biology and Medicine held in Naples, Italy in 2016. Acknowledgments This work was supported by a grant from the National Institute of Neuroscience, KAKENHI (23659089) Grant-in-Aid for Challenging Exploratory Research, and KAKENHI (26460115) Grantin-Aid for Scientific Research to HK. The third International Conference on H2S in Biology and Medicine was supported by Commemorative organization for the Japan World Exposition ’70, Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Naito Foundation, The Research Foundation for Pharmaceutical Sciences, Terumo Life Science Foundation, The Tokyo Biochemical Research Foundation, The Uehara Memorial Foundation, Antibe Therepeutics Inc., Sulfagenix, Inc., Fondazione Internazionale Menarini, Asahi Kasei Pharma Corp., Kobayashi Pharmaceutical Co., Ltd., Noevir Holdings Co., Ltd., and The Federation of Pharmaceutical Manufactures’ Associations of Japan. Finally, I thank A & E Planning Co. Ltd or H2S 2014 secretariat for organizing the conference. References [1] R.F. Furchgott, Studies on relaxation of rabbit aorta by sodium nitrate: basis for the proposal that the acid-activatable component of the inhibitory factor from retractor penis is inorganic nitrate and the endothelium-derived relaxing factor is nitric oxide, in: P.M. Vanhoutte (Ed.), Mechanisms of Vasodilatation, Raven, New York, 1988, pp. 401–414. [2] L.J. Ignarro, R.E. Byrns, K.S. Wood, Biochemical and pharmacological properties of endothelium-derived relaxing factor and its similarity to nitric oxide radical, in: P.M. Vanhoutte (Ed.), Mechanisms of Vasodilatation, Raven, New York, 1988, pp. 427–435. [3] A. Verma, D.J. Hirsch, E.E. Glatt, G.V. Ronnett, S.H. Snyder, Carbon monoxide: a putative neural messenger, Science 259 (1993) 381–384. [4] C.F. Stevens, Y. Wang, Reversal of long-term potentiation by inhibitors of haem oxygenase, Nature 364 (1993) 147–149. [5] M. Zhuo, S.A. Small, E.R. Kandel, R.D. Hawkins, Nitric oxide and carbon monoxide produce activity-dependent long-term synaptic enhancement in hippocampus, Science 260 (1993) 1946–1950. [6] A. Meister, P.E. Fraser, S.V. Tice, Enzymatic desulfuration of mercaptopyruvate to pyruvate, J. Biol. Chem. 206 (1954) 561–575. [7] D. Cavallini, B. Mondovi, C. De Marco, A. Scioscia-Santoro, The mechanism of desulphhydration of cysteine, Enzymologia 24 (1962) 253–266. [8] A.E. Braunstein, E.V. Goryachenkowa, E.A. Tolosa, I.H. Willhardt, L.L. Yefremova, Specificity and some other properties of liver serine sulphhydrase: evidence for its identity with cystathionine β–synthase, Biochim. Biophys. Acta 242 (1971) 247–260. [9] M.W. Warenycia, L.R. Goodwin, C.G. Benishin, R.J. Reiffenstein, D.M. Grancom, J.D. Taylor, et al., Acute hydrogen sulfide poisoning. Demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels, Biochem. Pharmacol. 38 (1989) 973–981.
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Please cite this article in press as: Hideo Kimura, H2S2014 in Kyoto: The 3rd International Conference on H2S in Biology and Medicine, Nitric Oxide (2014), doi: 10.1016/ j.niox.2014.10.001
