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Hydrogen sulfide (H 2 S) and polysulfides (H 2 S n , n ≥ 2) produced by enzymes play a role as signalling molecules regulating neurotransmission, vascular tone, cytoprotection, inflammation, oxygen sensing, and energy formation. H 2 S n , which have additional sulfur atoms to H 2 S, and other S-sulfurated molecules such as cysteine persulfide and S-sulfurated cysteine residues of proteins, are produced by enzymes including 3-mercaptopyruvate sulfurtransferase (3MST). H 2 S n are also generated by the chemical interaction of H 2 S with NO, or to a lesser extent with H 2 O 2 . S-sulfuration (S-sulfhydration) has been proposed as a mode of action of H 2 S and H 2 S n to regulate the activity of target molecules. Recently, we found that H 2 S/H 2 S 2 regulate the release of neurotransmitters, such as GABA, glutamate, and D-serine, a co-agonist of N-methyl-D-aspartate (NMDA) receptors. H 2 S facilitates the induction of hippocampal long-term potentiation, a synaptic model of memory formation, by enhancing the activity of NMDA receptors, while H 2 S 2 achieves this by activating transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes, potentially leading to the activation of nearby neurons. The recent findings show the other aspects of TRPA1 channels-that is, the regulation of the levels of sulfur-containing molecules and their metabolizing enzymes. Disturbance of the signalling by H 2 S/H 2 S n has been demonstrated to be involved in various diseases, including cognitive and psychiatric diseases. The physiological and pathophysiological roles of these molecules will be discussed.

Hydrogen sulfide (H 2 S) and polysulfides (H 2 S n , 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 H 2 S/H 2 S n on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H 2 S/H 2 S 2 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 H 2 S 2 , transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H 2 S/H 2 S 2 , 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. H 2 S/H 2 S n , 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits., (© 2023. Springer Nature Limited.)

Since the first description of hydrogen sulfide (H 2 S) as a toxic gas in 1713 by Bernardino Ramazzini, most studies on H 2 S have concentrated on its toxicity. In 1989, Warenycia et al. demonstrated the existence of endogenous H 2 S in the brain, suggesting that H 2 S may have physiological roles. In 1996, we demonstrated that hydrogen sulfide (H 2 S) is a potential signaling molecule, which can be produced by cystathionine β-synthase (CBS) to modify neurotransmission in the brain. Subsequently, we showed that H 2 S relaxes vascular smooth muscle in synergy with nitric oxide (NO) and that cystathionine γ-lyase (CSE) is another producing enzyme. This study also opened up a new research area of a crosstalk between H 2 S and NO. The cytoprotective effect, anti-inflammatory activity, energy formation, and oxygen sensing by H 2 S have been subsequently demonstrated. Two additional pathways for the production of H 2 S with 3-mercaptopyruvate sulfurtransferase (3MST) from l- and d-cysteine have been identified. We also discovered that hydrogen polysulfides (H 2 S n , n ≥ 2) are potential signaling molecules produced by 3MST. H 2 S n regulate the activity of ion channels and enzymes, as well as even the growth of tumors. S -Sulfuration ( S -sulfhydration) proposed by Snyder is the main mechanism for H 2 S/H 2 S n underlying regulation of the activity of target proteins. This mini review focuses on the key findings on H 2 S/H 2 S n signaling during the first 25 years.

Hydrogen sulfide (H 2 S) together with polysulfides (H 2 S n , n > 2) are signalling molecules like NO with various physiological roles including regulation of neuronal transmission, vascular tone, inflammation and oxygen sensing. H 2 S and H 2 S n diffuse to the target proteins for S-sulfurating their cysteine residues that induces the conformational changes to alter the activity. On the other hand, 3-mercaptopyruvate sulfurtransferase transfers sulfur from a substrate 3-mercaptopyruvate to the cysteine residues of acceptor proteins. A similar mechanism has also been identified in S-nitrosylation. S-sulfuration and S-nitrosylation by enzymes proceed only inside the cell, while reactions induced by H 2 S, H 2 S n and NO even extend to the surrounding cells. Disturbance of signalling by these molecules as well as S-sulfuration and S-nitrosylation causes many nervous system diseases. This review focuses on the signalling by H 2 S and H 2 S n with S-sulfuration comparing to that of NO with S-nitrosylation and discusses on their roles in physiology and pathophysiology., (© 2020 The British Pharmacological Society.)

Hydrogen sulfide (H 2 S) is a signalling molecule that regulates neuronal transmission, vascular tone, cytoprotection, inflammatory responses, angiogenesis, and oxygen sensing. Some of these functions have recently been ascribed to its oxidized form polysulfides (H 2 S n ), which can be produced by 3-mercaptopyruvate sulfurtransferase (MPST), also known as a H 2 S-producing enzyme. H 2 S n activate ion channels, tumour suppressors, transcription factors, and protein kinases. H 2 S n S-sulfurate (S-sulfhydrate) cysteine residues of these target proteins to modify their activity by inducing conformational changes through the formation of a disulfide bridge between the two cysteine residues involved. The chemical interaction between H 2 S and NO also generates H 2 S n , which may be a chemical entity that exerts the synergistic effect of H 2 S and NO. MPST also produces redox regulators cysteine persulfide (CysSSH), GSH persulfide (GSSH), and persulfurated proteins. In addition to MPST, haemoproteins such as haemoglobin, myoglobin, neuroglobin, and catalase as well as SOD can produce H 2 S n , and sulfide quinone oxidoreductase and cysteinyl tRNA synthetase can make GSSH and CysSSH. This review focuses on the recent progress in the study of the production and physiological roles of these persulfurated and polysulfurated molecules. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc., (© 2019 The British Pharmacological Society.)

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 (H 2 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 H 2 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 H 2 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 H 2 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 H 2 S/polysulfides production., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Hydrogen sulfide (H 2 S) is a signaling molecule used to modify neuronal transmission, regulate vascular tone, protect tissues from oxidative stress, sense oxygen, and generate ATP. Hydrogen polysulfides (H 2 S n ) have recently been identified as signaling molecules that mediate the activation of ion channels, regulation of tumor growth, and the transcriptional regulation of oxidative stress; some of which were previously ascribed to H 2 S. Cystathionine β-synthetase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are known as H 2 S-producing enzymes. 3MST also produces H 2 S n and other persulfurated molecules such as cysteine persulfide, glutathione persulfide, and persulfurated proteins. The chemical interaction of H 2 S and nitric oxide (NO) also produces H 2 S n , which may be the mechanism underlying the synergistic effect of H 2 S and NO that was initially reported on vascular relaxation. H 2 S n and other persulfurated molecules elicit their effect via S-sulfuration (S-sulfhydration) of specific cysteine residues of the target proteins. This review article focuses on the production and roles of H 2 S n as well as H 2 S in the central nervous system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

More than twenty years have passed since the demonstration of hydrogen sulfide (H 2 S) as a signaling molecule. Various roles of this molecule have been reported including neuromodulation, vascular relaxation, cytoprotection, anti-inflammation, and oxygen sensing. During the study of its effect on neuromodulation, we found TRP channels as a target of H 2 S, and later identified polysulfides (H 2 S n ) as chemical entity of the ligand. We found that H 2 S relaxes vasculatures in synergy with NO, and recently identified H 2 S n as products produced by the chemical interaction between H 2 S and NO to exert the effect, suggesting that it may be a mechanism for the synergy between the two molecules. It has attracted attention that sulfite, a further metabolite of H 2 S and H 2 S n , protects neurons from oxidative stress by a mechanism different from that by H 2 S and H 2 S n .

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H 2 S n revealed that H 2 S 2 first appeared, and then H 2 S and H 2 S 3 did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H 2 S n ), have various physiological functions in mammalian cells. Although H 2 S n molecules have been considered as secondary metabolites derived from hydrogen sulfide (H 2 S) 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 H 2 S, H 2 S 2 , H 2 S 3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H 2 S and H 2 S n standards under alkaline conditions (pH 9.5) induced significant decreases in H 2 S 2 and H 2 S 3 levels and a significant increase in the H 2 S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H 2 S 2 and H 2 S 3 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 H 2 S, H 2 S 2 , and H 2 S 3 in mouse brain under physiological pH conditions. The concentrations of H 2 S and H 2 S 2 were 0.030 ± 0.004μmol/g protein and 0.026 ± 0.002μmol/g protein, respectively. Although the level of H 2 S 3 was below the quantification limit of this method, H 2 S 3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H 2 S 2 and H 2 S 3 in mammalian brain tissues. H 2 S 2 and H 2 S 3 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H 2 S) has been demonstrated to have physiological roles such as neuromodulation, vascular tone regulation, cytoprotection, oxygen sensing, inflammatory regulation, and cell growth. Recently, hydrogen polysulfides (H 2 S n ) have been found to be produced by 3-mercaptopyruvate sulfurtransferase and to regulate the activity of ion channels, tumor suppressers, and protein kinases. Furthermore, some of the effects previously reported to be mediated by H 2 S are now ascribed to H 2 S n . Cysteine persulfide and cysteine polysulfide may also be involved in cellular redox regulation. The chemical interaction between H 2 S and nitric oxide (NO) can also produce H 2 S n , nitroxyl, and nitrosopersulfide, suggesting their involvement in the reactions previously thought to be mediated by NO alone. This Forum focuses on and critically discusses the recent progress in the study of H 2 S n , H 2 S, and NO as well as other per- or polysulfide species. Antioxid. Redox Signal. 00, 000-000.

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 (H 2 S 2 and H 2 S 3 ). 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-SS n H and GSS n H, 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.

Hydrogen sulfide (H 2 S) 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 H 2 S and NO generates polysulfides (H 2 S n ), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca 2+ and H 2 S n , showed that H 2 S n 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 H 2 S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H 2 S n are products of synergy between H 2 S and NO and provides a new insight into the signaling mechanisms.

Hydrogen sulfide (H 2 S) is a physiological mediator with various roles, including neuro-modulation, vascular tone regulation, and cytoprotection against ischemia-reperfusion injury, angiogenesis, and oxygen sensing. Hydrogen polysulfide (H 2 S n ), which possesses a higher number of sulfur atoms than H 2 S, recently emerged as a potential signaling molecule that regulates the activity of ion channels, a tumor suppressor, transcription factors, and protein kinases. Some of the previously reported effects of H 2 S are now attributed to the more potent H 2 S n . H 2 S n is produced by 3-mercaptopyruvate sulfurtransferase (3MST) from 3-mercaptopyruvate (3MP) and is generated by the chemical interaction of H 2 S with nitric oxide (NO). H 2 S n sulfhydrates (sulfurates) cysteine residues of target proteins and modifies their activity, whereas H 2 S sulfurates oxidized cysteine residues as well as reduces cysteine disulfide bonds. This review focuses on the recent progress made in studies concerning the production and physiological roles of H 2 S n and H 2 S.

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

It has been almost two decades since the first demonstration of hydrogen sulfide (H2S) as a physiological mediator of cognitive function and vascular tone. H2S is physiologically important because it protects various organs from ischemia-reperfusion injury besides regulating inflammation, oxygen sensing, cell growth, and senescence. The production, metabolism, and regulation of H2S have been studied extensively. H2S modulates target proteins through sulfhydration (or sulfuration) or by the reduction of cysteine disulfide bonds. A large number of novel H2S-donating compounds are being developed owing to the therapeutic potential of H2S. Recently, polysulfides, rather than H2S, have been identified as molecules that sulfhydrate (or sulfurate) their target proteins.

Significance: Hydrogen sulfide (H2S) has been recognized as a signaling molecule as well as a cytoprotectant. It modulates neurotransmission, regulates vascular tone, and protects various tissues and organs, including neurons, the heart, and kidneys, from oxidative stress and ischemia-reperfusion injury. H2S is produced from l-cysteine by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase., Recent Advances: In addition to these enzymes, we recently identified a novel pathway to produce H2S from d-cysteine, which involves d-amino acid oxidase (DAO) along with 3MST. These enzymes are localized in the cytoplasm, mitochondria, and peroxisomes. However, some enzymes translocate to organelles under specific conditions. Moreover, H2S-derived potential signaling molecules such as polysulfides and HSNO have been identified., Critical Issues: The physiological stimulations, which trigger the production of H2S and its derivatives and maintain their local levels, remain unclear., Future Directions: Understanding the regulation of the H2S production and H2S-derived signaling molecules and the specific stimuli that induce their release will provide new insights into the biology of H2S and therapeutic development in diseases involving these substances.

Hydrogen sulfide (H2S) has been recognized as a signaling molecule as well as a cytoprotective molecule. H2S modulates neurotransmission, regulates vascular tone, protects various tissues and organs, regulates inflammation, induces angiogenesis, and detects cellular oxygen levels. H2S is produced from L-cysteine by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) together with cysteine aminotransferase (CAT). Recently, a novel pathway for the production of H2S from D-cysteine was identified, involving D-amino acid oxidase (DAO) together with 3MST. Sulfuration (also called sulfhydration), which adds sulfur atoms to the cysteine residues of target proteins to modify protein activity, has been extensively studied as a mode of H2S action. Recently, hydrogen polysulfides (H2Sn, where n=3-7; n=2 is termed as persulfide) have been found to sulfurate target proteins in the brain, including transient receptor potential ankyrin 1 (TRPA1) channels, Kelch-like ECH-associating protein 1 (Keap1), and phosphatase and tensin homolog (PTEN), much more potently than H2S. The physiological stimuli that trigger the production of H2S and polysulfides, and the mechanisms maintaining their local levels, remain unknown. Understanding the regulation of H2Sn (including H2S) production, and the specific stimuli that induce their release, will provide new insight into the biology of H2S and will provide novel avenues for therapeutic development in diseases involving H2S-related substances.

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.

Hydrogen sulfide (H2S) is recognized as a biological mediator with various roles such as neuromodulation, regulation of the vascular tone, cytoprotection, anti-inflammation, oxygen sensing, angiogenesis, and generation of mitochondrial energy. It is produced by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). The activity of CBS is enhanced by S-adenosyl methionine (SAM) and glutathionylation, while it is inhibited by nitric oxide (NO) and carbon monoxide (CO). The activity of CSE and cysteine aminotransferase (CAT), which produces the 3MST substrate 3-mercaptopyruvate (3MP), is regulated by Ca2+. H2S is oxidized to thiosulfate in mitochondria through the sequential action of sulfide quinone oxidoreductase (SQR), sulfur dioxygenase, and rhodanese. The rates of the production and clearance of H2S determine its cellular concentration. Polysulfides (H2Sn) have been found to occur in the brain and activate transient receptor potential ankyrin 1 (TRPA1) channels, facilitate the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus, and suppress the activity of phosphatase and tensin homolog (PTEN) by sulfurating (sulfhydrating) the target cysteine residues. A cross talk between H2S and NO also plays an important role in cardioprotection as well as regulation of the vascular tone. H2S, polysulfides, and their cross talk with NO may mediate various physiological and pathophysiological responses.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Significance: Hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions. It regulates synaptic transmission, vascular tone, inflammation, transcription, and angiogenesis; protects cells from oxidative stress and ischemia-reperfusion injury; and promotes healing of ulcers., Recent Advances: In addition to cystathionine β-synthase and cystathionine γ-lyase, 3-mercaptopyruvate sulfurtransferase along with cysteine aminotransferase was recently demonstrated to produce H2S. Even in bacteria, H2S produced by these enzymes functions as a defense against antibiotics, suggesting that the cytoprotective effect of H2S is a universal defense mechanism in organisms from bacteria to mammals., Critical Issues: The functional form of H2S-undissociated H2S gas, dissociated HS ion, or some other form of sulfur-has not been identified., Future Directions: The regulation of H2S production by three enzymes may lead to the identification of the physiological signals that are required to release H2S. The identification of the physiological functions of other forms of sulfur may also help understand the biological significance of H2S.

Hydrogen sulfide (H2S) is a well-known toxic gas that has the smell of rotten eggs. This pungent gas was considered as a physiological mediator, after 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), and 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT). We recently identified a fourth pathway, where H2S is produced from D-cysteine by the enzyme D-amino acid oxidase (DAO) along with 3MST. We demonstrated that H2S is a neuromodulator that facilitates hippocampal long-term potentiation (LTP) by enhancing the activity of N-methyl-D-aspartate (NMDA) receptors. It also induces Ca(2+) influx in the astrocytes by activating the transient receptor potential ankyrin-1 (TRPA1) channels. In addition to being a signaling molecule, it also functions as a neuroprotective agent by enhancing the production of glutathione, a major intracellular antioxidant that scavenges the reactive oxygen species (ROS) in the mitochondria. H2S regulates the activity of the enzymes by incorporating the bound sulfane sulfur to cysteine residues. This modification is known as sulfhydration or sulfuration. The neuroprotective ubiquitin E3 ligase, parkin, enhances its neuroprotective activity by this modification. This review is focused on the functional role of H2S as a signaling molecule and as a cytoprotectant in the nervous system. In addition, this review shows the recent findings that indicate that the H2S-derived polysulfides found in the brain activate TRPA1 channels more potently than parental H2S., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S 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 μM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 μM, EC50 116 μM). 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 H2S-derived signaling molecules that stimulate TRP channels in the brain.

Hydrogen sulfide (H(2)S) is recognized as a physiologic mediator produced in a variety of tissues. It is produced by three enzymes, cystathionine β-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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L-cysteine by cystathionine β-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.

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.

Aims: The role of hydrogen sulfide (H(2)S) in endotoxin (lipopolysaccharide [LPS])-induced inflammation is incompletely understood. We examined the impact of H(2)S breathing on LPS-induced changes in sulfide metabolism, systemic inflammation, and survival in mice., Results: Mice that breathed air alone exhibited decreased plasma sulfide levels and poor survival rate at 72 h after LPS challenge. Endotoxemia markedly increased alanine aminotransferase (ALT) activity and nitrite/nitrate (NOx) levels in plasma and lung myeloperoxidase (MPO) activity in mice that breathed air. In contrast, breathing air supplemented with 80 ppm of H(2)S for 6 h after LPS challenge markedly improved survival rate compared to mice that breathed air alone (p<0.05). H(2)S breathing attenuated LPS-induced increase of plasma ALT activity and NOx levels and lung MPO activity. Inhaled H(2)S suppressed LPS-induced upregulation of inflammatory cytokines, while it markedly induced anti-inflammatory interleukin (IL)-10 in the liver. Beneficial effects of H(2)S inhalation after LPS challenge were associated with restored sulfide levels and markedly increased thiosulfate levels in plasma. Increased thiosulfate levels after LPS challenge were associated with upregulation of rhodanese, but not cystathionine-γ-lyase (CSE), in the liver. Administration of sodium thiosulfate dose-dependently improved survival after LPS challenge in mice., Innovation: By measuring changes in plasma levels of sulfide and sulfide metabolites using an advanced analytical method, this study revealed a critical role of thiosulfate in the protective effects of H(2)S breathing during endotoxemia., Conclusion: These observations suggest that H(2)S breathing prevents inflammation and improves survival after LPS challenge by altering sulfide metabolism in mice.

Cystathionine γ-lyase (CSE) is one of the major enzymes for the production of hydrogen sulphide (H(2)S), a multifunctional gasotransmitter in the pancreatic β-cell. We examined the mechanisms by which glucose induces CSE expression in mouse pancreatic islets and the insulin-secreting cell line MIN6. CSE expression was increased by anti-diabetic sulphonylureas, and decreased by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+) channel blocker nitrendipine. Application of the synthetic inhibitors of protein kinases revealed the involvement of Ca(2+)/calmodulin-dependent protein kinase (CaMK) II and extracellular signal-regulated protein kinase (ERK) in glucose- and thapsigargin-induced CSE expression. The CaMK IIδ knockdown also suppressed CSE expression. Knockdown of the transcription factors Sp1 and Elk1, both of which can be phosphorylated by ERK, blunted CSE expression. By a reporter assay, we found Sp1 may directly and Elk1 may indirectly regulate CSE expression. These findings suggest Ca(2+)-dependent CSE expression may be mediated via protein phosphorylation of Sp1 and Elk1 in pancreatic β-cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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 (H(2)S) has recently been recognized as a signaling molecule as well as a cytoprotectant. Cystathionine β-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.

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.

Endogenous levels of sulfide in the brain have been measured in rats, humans and bovines in 1989 and 1990, suggesting that H(2)S may have a physiological function. We demonstrated in 1996 that cystathionine β-synthase can produce H(2)S in the brain and that H(2)S facilitates the induction of hippocampal long-term potentiation by enhancing the activity of NMDA receptors. The following year, we showed that another H(2)S-producing enzyme, cystathionine γ-lyase, is expressed in the thoracic aorta, portal vein and ileum and that H(2)S relaxes these tissues. We proposed that H(2)S may be a neuromodulator as well as a smooth muscle relaxant. In addition to a function as a signalling molecule, we demonstrated another function as a cytoprotectant in 2004. Hydrogen sulfide protects neurons from oxidative stress by reinstating the reduced glutathione levels. We recently demonstrated that a third H(2)S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST), is expressed in neurons and vascular endothelium. In addition to reinstating glutathione levels, H(2)S produced by 3MST, which is mainly localized to mitochondria, reduces reactive oxygen species generated in these organelles.

Hydrogen sulfide (H(2)S), which is a well-known toxic gas, has been recognized as a signal molecule as well as a cytoprotectant. It is produced by three enzymes, cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase along with cysteine aminotransferase. In addition to an immediate release of H(2)S from producing enzymes, it can be stored as bound sulfane sulfur, which may release H(2)S in response to physiological stimuli. As a signal molecule, it modulates neuronal transmission, relaxes smooth muscle, regulates release of insulin and is involved in inflammation. Because of its reputation as a toxic gas, the function as a cytoprotectant has been overlooked: the nervous system and cardiovascular system are protected from oxidative stress. In this review, enzymatic production, release mechanism and functions of H(2)S are focused on.

Hydrogen sulfide (H(2)S) has emerged as the third endogenous gaseous mediator in the central and peripheral nervous system. H(2)S is generated by three enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). In the CNS, H(2)S, generated mainly by CBS in astrocytes and 3MST in neurons, appears to participate in cognition, memory, regulation of the cardiopulmonary functions and neuroprotection. In the peripheral nervous system, evidence suggests that H(2)S may be involved in autonomic control of the cardiopulmonary and gastrointestinal functions as well pain and inflammation.

Three hundred years have passed since the first description of the toxicity of hydrogen sulfide (H(2)S). Three papers in 1989 and 1990 described relatively high concentrations of sulfide in the brain. In 1996 we demonstrated that cystathionine beta-synthase (CBS) is a H(2)S producing enzyme in the brain and that H(2)S enhances the activity of NMDA receptors and facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory. In the following year, we demonstrated that another H(2)S producing enzyme, cystathionine gamma-lyase is in the thoracic aorta, portal vein, and the ileum, and that H(2)S relaxes these tissues. Based on these observations we proposed H(2)S as a neuromodulator as well as a smooth muscle relaxant. We recently demonstrated that the third H(2)S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) produces H(2)S in the brain as well as in vascular endothelium. Various functions in many tissues have been proposed. H(2)S protects neurons and cardiac muscle from oxidative stress. H(2)S has pro- and anti-inflammatory effects, nociceptive effects, the regulatory function of insulin release, and is even involved in longevity. Recent progress in the studies of physiological functions of H(2)S in neurons and smooth muscle was described.

Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant in the brain. We recently showed that H(2)S protects neurons from oxidative stress by increasing the levels of glutathione (GSH), a major cellular antioxidant, by more than twice that of a control through enhancing the cystine transport. Here we show that H(2)S enhances the transport of cysteine to increase GSH production more than cystine transport and to redistribute the localization of GSH to mitochondria. The efficiency of GSH production enhanced by H(2)S is even greater by fourfold under oxidative stress by glutamate. H(2)S reinstated GSH levels in the fetal brain decreased by ischemia/reperfusion in utero. In addition, Neuro2a cells expressing a mitochondrial H(2)S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST), along with cysteine aminotransferase (CAT), showed significant resistance to oxidative stress. The present study shows that H(2)S protects cells from oxidative stress by two mechanisms. It enhances the production of GSH by enhancing cystine/cysteine transporters and redistributes GSH to mitochondria. H(2)S produced in mitochondria also may directly suppress oxidative stress. It provides a new mechanism of neuroprotection from oxidative stress by H(2)S.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant. Currently, pyridoxal-5'-phosphate (PLP)-dependent cystathionine beta-synthase (CBS) is thought to be the major H(2)S-producing enzyme in the brain. We recently found that brain homogenates of CBS-knockout mice, even in the absence of PLP, produce H(2)S at levels similar to those of wild-type mice, suggesting the presence of another H(2)S-producing enzyme. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) in combination with cysteine aminotransferase (CAT) produces H(2)S from cysteine. In addition, 3MST is localized to neurons, and the levels of bound sulfane sulfur, the precursor of H(2)S, are greatly increased in the cells expressing 3MST and CAT but not increased in cells expressing functionally defective mutant enzymes. These data present a new perspective on H(2)S production and storage in the brain.

Hydrogen sulfide (H2S) is recognized as a neuromodulator as well as neuroprotectant in the brain. H2S can be produced from cysteine by enzymes such as cystathionine beta-synthase. However, a mechanism for releasing H2S under physiologic conditions has not been identified. Here we show that H2S is released from bound sulfur, an intracellular store of sulfur, in neurons and astrocytes of mice and rats in the presence of physiologic concentrations of endogenous reducing substances glutathione and cysteine. The highest pH to release H2S from another sulfur store, acid-labile sulfur, which is localized mainly in mitochondria, is 5.4. Because mitochondria are not in the acidic condition, acid-labile sulfur may not be a physiologic source of H2S. Free H2S is immediately absorbed and stored as bound sulfur. Our novel method, using silver particles to measure free H2S, shows that free H2S is maintained at a low level in basal conditions. Alkalinization of the cytoplasm is required for effective release of H2S from bound sulfur, and this condition is achieved in astrocytes by the high concentrations of extracellular K+ that are normally present when nearby neurons are excited. These data present a new perspective on the regulation of H2S in the brain.

We examined the expression of the major H(2)S-producing enzymes, cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. L-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with L-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor DL-propargylglycine antagonized these L-cysteine effects. We suggest H(2)S may function as an 'intrinsic brake' which protects beta-cells from glucotoxicity.

Hydrogen sulfide (H2S) can enzymatically be produced from cysteine in the brain. H2S functions as a synaptic modulator as well as a neuroprotectant from oxidative stress in the brain. Here we show that H2S specifically enhances the reducing activity in neurons and mouse neuroblastoma Neuro2a cells. An inhibitor of protein tyrosine kinase, genistein, suppresses the effect of H2S, suggesting that tyrosine kinase may be involved in the enhancement of reducing activity by H2S. The H2S-specific enhancement of the reducing activity in neurons may lead to a neurotrophic role in the brain.

The recent discovery that hydrogen sulfide (H(2)S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H(2)S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H(2)S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemia-reperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H(2)S by cardiac-specific overexpression of cystathionine gamma-lyase (alpha-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H(2)S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H(2)S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Hydrogen sulfide (H2S) enhances the induction of hippocampal long-term potentiation (LTP) and induces calcium waves in astrocytes. Based on these observations, H2S has been proposed to be a synaptic modulator in the brain. Here we show that differentiated astrocytes acquire sensitivity to H2S that is diminished by their transformation into reactive astrocytes. Although sodium hydrosulfide hydrate (NaHS), a donor of H2S, did not increase the intracellular concentration of Ca2+ in progenitors, exposure of progenitors to leukemia inhibitory factor (LIF), which induces differentiation into glial fibrillary acidic protein (GFAP)-positive astrocytes, greatly increased the sensitivity to NaHS. In contrast, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), dibutyryl cyclic AMP (db cAMP) and interleukin-1beta (IL-1beta) induced the conversion to reactive astrocytes with diminished sensitivity to NaHS. This suppressive effect of EGF on the sensitivity to NaHS was inhibited by cycloheximide, indicating that de novo protein synthesis was required for the suppression of H2S sensitivity.

Background & Aims: Hydrogen sulfide (H(2)S) has been suggested as a novel gasomediator. We explored its unknown neuromodulatory role in human and guinea-pig colon., Methods: We used immunohistochemistry to detect H(2)S-producing enzymes cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) in enteric neurons, Ussing chambers to measure mucosal ion secretion, and neuroimaging with voltage- and Ca(++)-sensitive dyes to record H(2)S effects on guinea-pig and human enteric neurons., Results: More than 90% of guinea-pig and human submucous and myenteric neurons were colabeled for CSE and CBS. Myenteric interstitial cells of Cajal were CSE-immunoreactive. The exogenous H(2)S donor NaHS (0.2-2.5 mmol/L) concentration-dependently increased chloride secretion in human and guinea-pig submucosa/mucosa preparations, but not in the colonic epithelial cell line T84. The secretory response was reduced significantly by tetrodotoxin (0.5 micromol/L), capsaicin desensitization (10 micromol/L), and the transient receptor potentials vanilloid receptor 1 antagonist capsazepine (10 micromol/L). The endogenous H(2)S donor L-cysteine also induced secretion that was diminished significantly by capsaicin desensitization, the CBS inhibitor amino-oxyacetic acid, and the CSE inhibitor propargylglycine. NaHS increased spike discharge in 23% of guinea-pig and 36% of human submucous neurons, but had no effect on Ca(++) mobilization in cultured guinea-pig enteric neurons. This excitatory response was reduced significantly by capsaicin desensitization and capsazepine, but not by glibenclamide (10 micromol/L)., Conclusions: The presence of H(2)S-producing enzymes in human and guinea-pig enteric neurons, the excitatory action on enteric neurons, and the prosecretory effects of NaHS suggest H(2)S as a novel gut-signaling molecule. Its action mainly involves transient receptor potentials vanilloid receptor 1 receptors on extrinsic afferent terminals, which in turn activate enteric neurons.