Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity.

Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) are signaling molecules produced by 3-mercaptopyruvate sulfurtransferase (3MST) that play various physiological roles, including the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation, by enhancing N-methyl-D-aspartate (NMDA) receptor activity. However, the presynaptic action of H2S/H2Sn on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H2S/H2S2 applied to the rat hippocampus by in vivo microdialysis induces the release of GABA, glutamate, and D-serine, a co-agonist of NMDA receptors. Animals with genetically knocked-out 3MST and the target of H2S2, transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H2S/H2S2, 3MST, and TRPA1 activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia. H2S/H2Sn, 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits.

Excess hydrogen sulfide and polysulfides production underlies a schizophrenia pathophysiology.

Mice with the C3H background show greater behavioral propensity for schizophrenia, including lower prepulse inhibition (PPI), than C57BL/6 (B6) mice. To characterize as-yet-unknown pathophysiologies of schizophrenia, we undertook proteomics analysis of the brain in these strains, and detected elevated levels of Mpst, a hydrogen sulfide (H2 S)/polysulfide-producing enzyme, and greater sulfide deposition in C3H than B6 mice. Mpst-deficient mice exhibited improved PPI with reduced storage sulfide levels, while Mpst-transgenic (Tg) mice showed deteriorated PPI, suggesting that "sulfide stress" may be linked to PPI impairment. Analysis of human samples demonstrated that the H2 S/polysulfides production system is upregulated in schizophrenia. Mechanistically, the Mpst-Tg brain revealed dampened energy metabolism, while maternal immune activation model mice showed upregulation of genes for H2 S/polysulfides production along with typical antioxidative genes, partly via epigenetic modifications. These results suggest that inflammatory/oxidative insults in early brain development result in upregulated H2 S/polysulfides production as an antioxidative response, which in turn cause deficits in bioenergetic processes. Collectively, this study presents a novel aspect of the neurodevelopmental theory for schizophrenia, unraveling a role of excess H2 S/polysulfides production.

Sulfite protects neurons from oxidative stress.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S) and polysulfides (H2 Sn ) are signalling molecules that mediate various physiological responses including cytoprotection. Their oxidized metabolite sulfite (SO3 2- ) is found in blood and tissues. However, its physiological role remains unclear. In this study, we investigated the cytoprotective effect of sulfite on neurons exposed to oxidative stress caused by high concentrations of the neurotransmitter glutamate, known as oxytosis. EXPERIMENTAL APPROACH: Concentrations of sulfite as well as those of cysteine and GSH in rats were measured by HPLC. Cytoprotective effects of sulfite on primary cultures of rat neurons against oxytosis was examined by WST-8 cytoprotective and LDH cytotoxicity assays and compared with that of H2 S, H2 Sn and thiosulfate. KEY RESULTS: Free sulfite, present at approximately 2 µM in the rat brain, converts cystine to cysteine more efficiently than H2 S and H2 Sn and facilitates transport of cysteine into cells. Physiological concentrations of sulfite protected neurons from oxytosis and were accompanied by increased intracellular concentrations of cysteine and GSH probably due to converting extracellular cystine to cysteine, more efficiently than H2 S and H2 Sn . In contrast, thiosulfate only slightly protected neurons from oxytosis. CONCLUSIONS AND IMPLICATIONS: Our present data have shown sulfite to be a novel cytoprotective molecule against oxytosis, through maintaining cysteine levels in the extracellular milieu, leading to increased intracellular cysteine and GSH. Although there may be adverse clinical effects in sensitive individuals, our results provide a new insight into the therapeutic application of sulfite to neuronal diseases caused by oxidative stress. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

[Production of H2S, H2Sn, and persulfide species (CysSSH and GSSH) by 3-mercaptopyruvate sulfurtransferase].

Accumulating evidence shows that hydrogen sulfide (H2S) has physiological roles in various tissues and organs, including the regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney, and brain from ischemic insult. H2S is produced from l-cysteine by pyridoxal 5'-phosphate (PLP)-dependent enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). 3-Mercaptopyruvate sulfurtransferase (3MST) is the third H2S-producing enzyme, and its substrate 3-mercaptopyruvate (3MP) is provided from l-cysteine and α-ketoglutarate (α-KG) by a PLP-dependent cysteine aminotransferase (CAT). An additional pathway for the production of H2S from d-cysteine metabolized by d-amino acid oxidase (DAO) together with 3MST has been identified. Recent studies have shown that hydrogen polysulfides (H2Sn) have been found to stimulate transient receptor potential ankyrin1 (TRPA1) channel, much more potently than does H2S. 3MST produces cysteine-persulfide (CysSSH) and its glutathione counterpart (GSSH), potential redox regulators, together with the potential signaling molecules H2Sn. In addition, the interaction between H2S and nitric oxide (NO) also generates H2Sn. These observations provide new insights into the production and physiological roles of these molecules.

Analysis of endogenous H2S and H2Sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS－ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004µmol/g protein and 0.026 ± 0.002µmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.

3-Mercaptopyruvate sulfurtransferase produces potential redox regulators cysteine- and glutathione-persulfide (Cys-SSH and GSSH) together with signaling molecules H2S2, H2S3 and H2S.

Cysteine-persulfide (Cys-SSH) is a cysteine whose sulfhydryl group is covalently bound to sulfur (sulfane sulfur). Cys-SSH and its glutathione (GSH) counterpart (GSSH) have been recognized as redox regulators, some of which were previously ascribed to cysteine and GSH. However, the production of Cys-SSH and GSSH is not well understood. Here, we show that 3-mercaptopyruvate sulfurtransferase (3MST) produces Cys-SSH and GSSH together with the potential signaling molecules hydrogen per- and tri-sulfide (H2S2 and H2S3). Cys-SSH and GSSH are produced in the brain of wild-type mice but not in those of 3MST-KO mice. The levels of total persulfurated species in the brain of 3MST-KO mice are less than 50% of that in the brain of wild-type mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST showed that Cys-SSH and GSSH were produced in the presence of physiological concentrations of cysteine and glutathione, while those with longer sulfur chains, Cys-SSnH and GSSnH, were produced in the presence of lower than physiological concentrations of cysteine and glutathione. The present study provides new insights into the production and physiological roles of these persulfurated species as well as the therapeutic targets for diseases in which these molecules are involved.

Polysulfides (H2Sn) produced from the interaction of hydrogen sulfide (H2S) and nitric oxide (NO) activate TRPA1 channels.

Hydrogen sulfide (H2S) exerts synergistic effects with another gaseous signaling molecule nitric oxide (NO) on ion channels and vasculature. However, the mechanism of the synergy is not well understood. Here, we show that the interaction between H2S and NO generates polysulfides (H2Sn), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca2+ and H2Sn, showed that H2Sn and their effects were abolished by cyanolysis and by reducing substances such as dithiothreitol (DTT), cysteine, and glutathione (GSH). However, the effects of nitroxyl or nitrosopersulfide, other potential products of H2S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H2Sn are products of synergy between H2S and NO and provides a new insight into the signaling mechanisms.

Discovery and Mechanistic Characterization of Selective Inhibitors of H2S-producing Enzyme: 3-Mercaptopyruvate Sulfurtransferase (3MST) Targeting Active-site Cysteine Persulfide.

Very recent studies indicate that sulfur atoms with oxidation state 0 or -1, called sulfane sulfurs, are the actual mediators of some physiological processes previously considered to be regulated by hydrogen sulfide (H2S). 3-Mercaptopyruvate sulfurtransferase (3MST), one of three H2S-producing enzymes, was also recently shown to produce sulfane sulfur (H2Sn). Here, we report the discovery of several potent 3MST inhibitors by means of high-throughput screening (HTS) of a large chemical library (174,118 compounds) with our H2S-selective fluorescent probe, HSip-1. Most of the identified inhibitors had similar aromatic ring-carbonyl-S-pyrimidone structures. Among them, compound 3 showed very high selectivity for 3MST over other H2S/sulfane sulfur-producing enzymes and rhodanese. The X-ray crystal structures of 3MST complexes with two of the inhibitors revealed that their target is a persulfurated cysteine residue located in the active site of 3MST. Precise theoretical calculations indicated the presence of a strong long-range electrostatic interaction between the persulfur anion of the persulfurated cysteine residue and the positively charged carbonyl carbon of the pyrimidone moiety of the inhibitor. Our results also provide the experimental support for the idea that the 3MST-catalyzed reaction with 3-mercaptopyruvate proceeds via a ping-pong mechanism.

Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.

Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn, and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

Polysulfide promotes neuroblastoma cell differentiation by accelerating calcium influx.

Polysulfides are a typical type of bound sulfur, which is physiologically stable form of sulfur species, derived from the hydrogen sulfide (H2S) that is generated endogenously in cells. We previously reported that bound sulfur protects neuronal cells from oxidative injury. In the present study, we demonstrated that polysulfides inhibited cell growth and promoted neurite outgrowth in mouse neuroblastoma Neuro2A (N2A) cells. However, Na2S showed no effect on neurite outgrowth in N2A cells. Furthermore, 2-APB and SKF96365, which are typical transient receptor potential (TRP) channel inhibitors, suppressed the neurite outgrowth induced by Na2S4. These new findings suggest that bound sulfur could induce neurite outgrowth and cell differentiation of N2A cells by accelerating calcium influx.

Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells.

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.

Production of hydrogen sulfide from d-cysteine and its therapeutic potential.

Accumulating evidence shows that H2S has physiological functions in various tissues and organs. It includes regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney, and brain from ischemic insult. H2S is produced by enzymes from l-cysteine; cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase. We recently discovered an additional pathway for the production of H2S from d-cysteine. d-Amino acid oxidase provides 3-mercaptopyruvate for 3MST to produce H2S. d-Cysteine protects cerebellar neurons from oxidative stress and attenuates ischemia-reperfusion injury caused in the kidney more effectively than l-cysteine. This review focuses on a novel pathway for the production of H2S and its therapeutic application especially to the renal diseases.

A novel method for the analysis of 3-mercaptopyruvate using high-performance liquid chromatography with fluorescence detection.

3-Mercaptopyruvate (3-MP) is a metabolite of cysteine present in mammalian tissues and is known to be a substrate of 3-mercaptopyruvate sulfurtransferase (3MST, EC.3.4.1.2). The physiological relevance of the 3-MP pathway has not been fully recognized because the metabolic behavior of 3-MP remains unclear. Here, we describe a novel method using high-performance liquid chromatography with fluorescence detection to measure 3-MP formation from cysteine. To demonstrate the practical value of the present method, we applied it to analyze the 3-MP produced in biological samples from mouse tissue.

Hydrogen sulfide is produced by cystathionine γ-lyase at the steady-state low intracellular Ca(2+) concentrations.

Hydrogen sulfide (H(2)S) is recognized as a physiologic mediator produced in a variety of tissues. It is produced by three enzymes, cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). However, the regulation of H(2)S production by CSE has not well been understood. Here we show that H(2)S producing activity of CSE is regulated by intracellular Ca(2+) concentrations. In the presence of pyridoxal 5'-phosphate (PLP) CSE efficiently produces H(2)S at the steady-state low Ca(2+) concentrations but is suppressed at high Ca(2+) concentrations. In the absence of PLP H(2)S production maintains the suppressed levels at high Ca(2+) concentrations and decreased further at low Ca(2+) concentrations. These observations suggest that CSE produces H(2)S at the steady-state in cells and that the production is suppressed when the intracellular Ca(2+) concentrations are increased.

A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells.

In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L-cysteine by cystathionine ß-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase coupled with cysteine aminotransferase. Here we report an additional biosynthetic pathway for the production of hydrogen sulphide from D-cysteine involving 3-mercaptopyruvate sulfurtransferase and D-amino acid oxidase. Unlike the L-cysteine pathway, this D-cysteine-dependent pathway operates predominantly in the cerebellum and the kidney. Our study reveals that administration of D-cysteine protects primary cultures of cerebellar neurons from oxidative stress induced by hydrogen peroxide and attenuates ischaemia-reperfusion injury in the kidney more than L-cysteine. This study presents a novel pathway of hydrogen sulphide production and provides a new therapeutic approach to deliver hydrogen sulphide to specific tissues.

Hydrogen sulfide is a signaling molecule and a cytoprotectant.

SIGNIFICANCE: Accumulating evidence shows that hydrogen sulfide may function as a signaling molecule in processes such as neuromodulation in the brain and smooth muscle relaxation in the vascular system. It also has a cytoprotective effect, since it can protect neurons and cardiac muscle from oxidative stress and ischemia-reperfusion injury, respectively. Hydrogen sulfide can also modulate inflammation, insulin release, and angiogenesis. RECENT ADVANCES: The regulation of the activity of 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT), one of the H(2)S producing pathways, has been demonstrated. The production of H(2)S by the pathway, which is regulated by Ca(2+) and facilitated by thioredoxin and dihydrolipoic acid, is also involved in H(2)S signaling as well as cytoprotection. Sulfur hydration of proteins by H(2)S has been proposed to modulate protein functions. H(2)S-sensitive fluorescent probes, which enable us to measure the localization of H(2)S in real time, have been developed. CRITICAL ISSUES: The basal concentrations of H(2)S have recently been measured and found to be much lower than those initially reported. However, the concentration of H(2)S reached in stimulated cells, as well as the regulation of H(2)S producing enzymes is not well understood. It has been proposed that some of the effects of H(2)S on the regulation of enzymes and receptors might be explained through the properties of sulfane sulfur (S(0)), another form of active sulfur. FUTURE DIRECTIONS: The determination of H(2)S concentrations in activated cells using new methods including H(2)S-sensitive fluorescent probes, as well as the investigation of the effects of H(2)S using specific inhibitors, may provide better understanding of the physiological function of this molecule. Clarifying mechanisms of H(2)S activity may also facilitate the development of new therapeutic compounds.

Development of a highly selective fluorescence probe for hydrogen sulfide.

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

Hydrogen sulfide protects the retina from light-induced degeneration by the modulation of Ca2+ influx.

Hydrogen sulfide (H(2)S) has recently been recognized as a signaling molecule as well as a cytoprotectant. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are well-known as H(2)S-producing enzymes. We recently demonstrated that 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) produces H(2)S in the brain and in vascular endothelium. However, the cellular distribution and regulation of these enzymes are not well understood. Here we show that 3MST and CAT are localized to retinal neurons and that the production of H(2)S is regulated by Ca(2+); H(2)S, in turn, regulates Ca(2+) influx into photoreceptor cells by activating vacuolar type H(+)-ATPase (V-ATPase). We also show that H(2)S protects retinal neurons from light-induced degeneration. The excessive levels of light exposure deteriorated photoreceptor cells and increased the number of TUNEL- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells. Degeneration was greatly suppressed in the retina of mice administered with NaHS, a donor of H(2)S. The present study provides a new insight into the regulation of H(2)S production and the modulation of the retinal transmission by H(2)S. It also shows a cytoprotective effect of H(2)S on retinal neurons and provides a basis for the therapeutic target for retinal degeneration.

Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide.

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.