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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.

Hydrogen sulfide (H2S) has been considered to be a physiological mediator since the identification of endogenous sulfides in the mammalian brain. H2S is produced from L-cysteine by enzymes such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3MST), and cysteine aminotransferase (CAT). CSE and CAT are regulated by Ca(2+). At steady-state low intracellular concentrations of Ca(2+), CSE and the 3MST/CAT pathway produce H2S. However, after intracellular concentrations of Ca(2+) increase in stimulated cells, the production of H2S by these enzymes decreases. We recently identified a fourth pathway, by which H2S is produced from D-cysteine by the enzymes D-amino acid oxidase (DAO) and 3MST. This pathway is mainly localized in the cerebellum and the kidney. The production of H2S from D-cysteine is 80 times more efficient than that from L-cysteine in the kidney, and the administration of D-cysteine to mice ameliorates renal ischemia-reperfusion injury more effectively than L-cysteine. These results suggest that D-cysteine might be used to treat renal diseases or even increase the success of kidney transplantation. We found that H2S-derived polysulfides exist in the brain and activate transient receptor potential ankyrin-1 (TRPA1) channels 300 times more potently than H2S. Although TRPA1 channels mediate sensory transduction and respond to a variety of stimuli, including cold temperature, pungent compounds and environmental irritants, their endogenous ligand(s) has not been identified. The sulfane sulfur of polysulfides is a reactive electrophile that is readily transferred to a nucleophilic protein thiolate to generate the protein persulfide or bound sulfane sulfur by sulfhydration (as referred to as sulfuration). The bound sulfane sulfur-producing activity of polysulfides is much greater than that of H2S. This review focuses on the physiological roles of H2S and H2S-derived polysulfides as signaling molecules.

Hydrogen sulfide (H2S) is a familiar toxic gas that smells of rotten eggs. After the identification of endogenous H2S in the mammalian brain two decades ago, studies of this molecule uncovered physiological roles in processes such as neuromodulation, vascular tone regulation, cytoprotection against oxidative stress, angiogenesis, anti-inflammation, and oxygen sensing. Enzymes that produce H2S, such as cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase have been studied intensively and well characterized. Polysulfides, which have a higher number of inner sulfur atoms than that in H2S, were recently identified as potential signaling molecules that can activate ion channels, transcription factors, and tumor suppressors with greater potency than that of H2S. This article focuses on our contribution to the discovery of these molecules and their metabolic pathways and mechanisms of action.

Accumulating evidence shows that hydrogen sulfide (H 2 S) has a variety of physiological functions. H 2 S is produced from cysteine by 3 sulfurtransferases. H 2 S, in turn, generates polysulfides, the functions of which are not well understood. H 2 S induces Ca 2+ influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca 2+ influx by activating transient receptor potential (TRP) A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77 ± 0.26) and that the maximum response was induced at 0.5 microM, which is 1/320 of the concentration of H 2 S required to achieve a response of similar magnitude (160 microM, EC50 116 microM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca 2+ influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H 2 S -derived signaling molecules that stimulate TRP channels in the brain.

Introduction H 2 S plays key roles in various physiological processes. So, compounds which can regulate activities of H 2 S producing enzymes are expected to be useful as chemical tools for life sciences and medicine. These compounds also would be able to contribute to the progress of H 2 S research. Here we show the discovery of small molecule inhibitors for 3-mercaptopyruvate sulfurtransferase (3MST) and cystathionine gamma lyase (CSE) by high-throughput screening (HTS). Method For screening, we used a novel fluorescence probe for H 2 S (HSip-1), which has been developed by our group (J. Am. Chem. Soc. 133 (2011) 18003). By using this probe, we established an assay system. In the assay system, the increase of the fluorescence intensity is suppressed, when the enzyme activity is inhibited by a compound and the production of H 2 S decreases. We then performed 3MST and CSE inhibitor screening of a chemical library containing about 160,000 compounds. Results We selected compounds which could suppress the fluorescence increase of HSip-1. After the repeatability test, we excluded HS − reactive compound as false positive compounds, and then found hit compounds. These compounds were selective against targeted H 2 S producing enzymes. Conclusion As a result of screening, we could discover both selective 3MST and CSE inhibitors. We will examine the utility of these compounds by applying them to living cells and we expect these compounds would be used as research tools.

Hydrogen sulfide (H 2 S) acts as a signaling molecule and cytoprotectant. H 2 S is known to be produced from l -cysteine by two pyridoxal 5′-phosphate (PLP)-dependent enzymes, cystathionine beta-synthase and cystathionine gamma-lyase. We recently demonstrated that 3-mercaptopyruvate sulfurtransferase (3MST) produces H 2 S from 3-mercaptopyruvate (3MP), which is provided from l -cysteine and alpha-ketoglutarate by PLP-dependent cysteine aminotransferase (CAT). We also found an additional pathway for the production of H 2 S from d -cysteine involving 3MST and d -amino acid oxidase (DAO). The enzymatic activities associated with d -cysteine are distinct from those of l -cysteine with respect to the optimal pH and dependency on PLP. Unlike the l -cysteine pathway, this d -cysteine-dependent pathway seems to operate predominantly in the cerebellum and the kidney. Administration of d -cysteine protected primary cultures of cerebellar neurons from oxidative stress induced by H 2 O 2 and attenuated ischemia–reperfusion injury in the kidney more than l -cysteine. The present study presents a novel pathway of H 2 S production and provides a new therapeutic approach to deliver H 2 S to specific tissues.

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.

Hydrogen sulfide (H2S) is a toxic gas that emits an unpleasant smell of rotten eggs. About 20 years ago, the discovery of endogenous sulfide in mammalian brain urged us to study the role of H2S in neuronal transmission. We found a neuromodulatory role with cystathionine beta–synthase (CBS) as H2S-producing enzyme in the brain. In the following year we demonstrated that H2S relaxes vascular smooth muscle in synergy with nitric oxide (NO) with cystathionine gamma–lyase (CSE) as another enzyme. Our finding of a cytoprotective effect of H2S on neurons from oxidative stress led to the identification of protecting various tissues and organs from ischemia–reperfusion injury. In addition to the H2S-producing pathway with 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT), we recently found a novel pathway with 3MST and d -amino acid oxidase (DAO) that produces H2S from d -cysteine. This pathway is more efficient than l -cysteine pathways in the kidney, and promises the therapeuric application of d -cysteine to the renal ischemic diseases. H2S is oxidized to polysufides that exist in the brain and activate transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes more efficiently than parental H2S. H2S exerts its effect by reducing a target such as a cysteine disulfide bond, whereas polysulfides by adding bound sulfane sulfur to protein thiols (sulfhydration or sulfuration). The physiological roles of H2S and polysulfides will be discussed.

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 sulfides-related algesic action, especially under inflammatory conditions.

Over 20 years ago, we developed a sensitive method for the determination of H 2 S 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 H 2 S. 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 H 2 S. 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.

The relatively high concentrations of endogenous sulfide in the mammalian brain were measured in 1989, suggesting that hydrogen sulfide (H2S) might have a physiological function. In 1996 we demonstrated that cystathionine β-synthase (CBS) is a H2S producing enzyme in the brain and that H2S facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory, by enhancing the activity of N-methyl d -aspartate (NMDA) receptors. The following year we demonstrated that another H2S producing enzyme, cystathionine γ-lyase (CSE) is found in tissues including vasculature and that H2S relaxes them. Based on these observations we proposed that H2S is a neuromodulator and a smooth muscle relaxant. In addition to the function as a signaling molecule, we and others found a cytoprotective effect of this molecule; H2S protects neurons from oxidative stress. This finding led to the identification of the protection of various organs including the heart, pancreas, retina, and the kidney against ischemia–reperfusion injury. From our finding that the brains of CBS knockout mice was still able to produce H2S, we found another pathway; 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT). 3MST produces H2S in the presence of thioredoxin or dihydrolipoic acid (DHLA). We recently found a novel pathway to produce H2S from d -cysteine, a negative control. d -Amino acid oxidase (DAO) metabolizes d -cysteine to an achiral α–keto acid, 3-mercaptopyruvate (3MP), which is further metabolized to H2S by 3MST. This pathway is mainly localized in the cerebellum and the kidney. The production of H2S from d -cysteine is 80 times more efficient than that from l -cysteine in the kidney, and the administration of d -cysteine to mice ameliorates renal ischemia–reperfusion injury more effectively than l -cysteine. These results show a therapeutic potential of d -cysteine to the renal diseases and even to the kidney transplantation. Our additional contribution to this field is the discovery of H2S-derived polysulfides, which exist in the brain and activate transient receptor potential ankyrin-1 (TRPA1) channels 300 times more potently than H2S. TRPA1 channels mediate the sensory transduction and respond to a variety of stimuli, including cold temperature, pungent compounds and environmental irritants, but its endogenous ligand has not been identified. The sulfane sulfur of polysulfides is reactive electrophile and readily transferred to a nucleophilic protein thiolate, to generate the protein persulfide (sulfhydration). The sulfhydration activity of polysulfides is much greater than H2S. The production of H2S and the physiological function of H2S and polysulfides will be discussed.

Recently, we have reported a novel fluorescence probe for H 2 S, HSip-1, based on azamacrocyclic Cu 2+ complex chemistry (J. Am. Chem. Soc., 133 (2011) 18003) (Figure A). HSip-1 can selectively detect H 2 S in aqueous solution with high selectivity over biothiols, inorganic sulfur compounds, ROS and RNS, and has excellent photophysical properties for biological applications (Φ fl = 0.019 and 0.78 in the absence or the presence of H 2 S, respectively), deriving from its fluorescein scaffold. We also confirmed that HSip-1 could be used both for detection of pseudoenzymatic H 2 S release in a cuvette and of H 2 S production by 3-mercaptopyruvate sulfurtransferase (3MST) or lysate of 3MST-expressing cells, and 3-mercaptopyruvate, i.e., H 2 S-producing enzyme and its substrate. So, we anticipated that HSip-1 would be useful for not only fluorescence imaging, but also high-throughput screening of CBS (cystathionine β -synthase), CSE (cystathionine γ -lyase) and 3MST inhibitors, which should be required for detailed investigation of a wide range of biological functions of H 2 S. To investigate whether the probe can be used to monitor recombinant enzymatic H 2 S generation, we examined its efficacy with recombinant human CSE, which was purchased from Cayman and 3MST produced by Escherichia coli BL21 strain using a plasmid (3MST/pGEX) expressing 3MST with GST tag. H 2 S production by CSE (20 μg/mL) was readily detected in the presence of millimolar concentrations of the thiol substrate (cysteine; 2.5 mM) and pyridoxal phosphate (50 μM) in 30 mM HEPES buffer (pH 7.4). The fluorescence increment was suppressed by pretreatment with PAG (propargylglycine; 1 mM), which is an inhibitor of CSE (Figure B). Moreover, H 2 S production by 3MST (3.8 μg/mL) was also detected in the presence of thiol substrates (50 μM 3MP and 40 μM DTT) in 30 mM HEPES buffer (pH 7.4) (Figure C). We further confirmed that these systems could be applied to 384-well plate assay. By utilizing HSip-1 as an indicator for H 2 S, we then performed 3MST inhibitor screening of a chemical library containing about 150,000 compounds obtained from Open Innovation Center for Drug Discovery, The University of Tokyo. In this conference, we will show the detailed protocols and results in our poster.