3-mercaptopyruvate Sulfurtransferase Research Articles

Exercise restores endogenous H2 S synthesis and mitochondrial function in the heart of old rats.

Ageing is accompanied by a decrease in endogenous hydrogen sulphide (H2 S) synthesis and the development of mitochondrial dysfunction. The aim of our work was to study the possible participation of exercise training-induced regulation of endogenous H2 S production in the restoration of mitochondrial function in old rats. Male rats were divided into three groups: adult, old and exercise-trained old. Exercise training of old rats was performed for 4 weeks. The mRNA expression cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) were determined using reverse transcription and real-time polymerase chain reaction analysis. Mitochondrial dysfunction was determined by mPTP opening, which was investigated by spectrophotometric registration of the swelling of mitochondria isolated from the rat heart. We also studied the effect of exercise on H2 S content, oxidative stress and mtNOS activity. Exercise training in old animals significantly increased the expression of H2 S-synthesizing enzymes CSE and 3-MST and restored endogenous H2 S production in cardiac tissue and cardiac mitochondria to levels of adult animals. In addition, the training significantly reduced oxidative stress in old rats, in particular the rate of formation of •O2 - and H2 O2 , diene conjugates and malondialdehyde levels in the mitochondria of the heart. Simultaneously, in the hearts of these animals, resistance of mPTP to the inducer of its opening of calcium ions was increased. Thus, exercise training restores endogenous H2 S production, and significantly reduces oxidative stress in cardiac mitochondria of old rats that are associated with the inhibition of calcium-induced mPTP opening as an indicator of mitochondrial dysfunction.

Evidence of endogenously produced hydrogen sulfide (H2S) and persulfidation in male reproduction

Persulfidation contributes to a group of redox post-translational modifications (PTMs), which arise exclusively on the sulfhydryl group of cysteine as a result of hydrogen sulfide (H2S) action. Redox-active molecules, including H2S, contribute to sperm development; therefore, redox PTMs represent an extremely important signalling pathway in sperm life. In this path, persulfidation prevents protein damage caused by irreversible cysteine hyperoxidation and thus maintains this signalling pathway. In our study, we detected both H2S and its production by all H2S-releasing enzymes (cystathionine γ-lyase (CTH), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST)) in male reproduction, including spermatozoa. We provided evidence that sperm H2S leads to persulfidation of proteins, such as glyceraldehyde-3-phosphate dehydrogenase, tubulin, and anchor protein A-kinase. Overall, this study suggests that persulfidation, as a part of the redox signalling pathway, is tightly regulated by enzymatic H2S production and is required for sperm viability.

The expression of hydrogen sulfide-producing enzymes in primary and lung metastatic osteosarcoma.

Hydrogen sulfide (H₂S) is a novel gas transmitter signaling molecule. H₂S is synthesized by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST). There have been no reports about the roles of these enzymes in osteosarcoma and its metastases. We detected H₂S synthase expression levels in human primary osteosarcoma and lung metastatic osteosarcoma. Immunohistochemistry was performed in primary osteosarcoma (n=19), lung metastatic osteosarcoma (n=11), osteoblastoma (n=10) and bony callus (n=2). The expression of CBS, CSE, and MST was defined as negative, moderately positive and strongly positive. MST staining was moderately to strongly positive in all cases. CSE staining was negative in 94.7% (18/19) of primary osteosarcoma cases and 90.9% (10/11) of lung metastatic osteosarcoma cases. CBS staining was strongly positive in 68.4% (13/19) of primary osteosarcoma cases, moderately positive in 15.8% (3/19) of cases, and negative in 15.8% (3/19) of cases. In lung metastatic osteosarcoma, the proportions of negative, moderately positive and strongly positive cases were 63.6% (7/11), 18.2% (2/11) and 18.2% (2/11), respectively. CBS and CSE expression, especially CSE expression, decreased in both primary osteosarcoma and lung metastatic osteosarcoma, which may suggest that CBS and CSE play roles in osteoblast cell malignant transformation and osteosarcoma progression. These enzymes could be used as new prognostic assessment factors and may represent new therapeutic targets for osteosarcoma and metastasis prevention.

Pharmacological Inhibition of Endogenous Hydrogen Sulfide Attenuates Breast Cancer Progression.

Hydrogen sulfide (H2S), a gaseous signaling molecule, is associated with the development of various malignancies via modulating various cellular signaling cascades. Published research has established the fact that inhibition of endogenous H2S production or exposure of H2S donors is an effective approach against cancer progression. However, the effect of pharmacological inhibition of endogenous H2S-producing enzymes (cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MPST)) on the growth of breast cancer (BC) remains unknown. In the present study, DL-propargylglycine (PAG, inhibitor of CSE), aminooxyacetic acid (AOAA, inhibitor of CBS), and L-aspartic acid (L-Asp, inhibitor of 3-MPST) were used to determine the role of endogenous H2S in the growth of BC by in vitro and in vivo experiments. An in silico study was also performed to confirm the results. Corresponding to each enzyme in separate groups, we treated BC cells (MCF-7 and MDA-MB-231) with 10 mM of PAG, AOAA, and L-Asp for 24 h. Findings reveal that the combined dose (PAG + AOAA + L-Asp) group showed exclusive inhibitory effects on BC cells’ viability, proliferation, migration, and invasion compared to the control group. Further, treated cells exhibited increased apoptosis and a reduced level of phospho (p)-extracellular signal-regulated protein kinases such as p-AKT, p-PI3K, and p-mTOR. Moreover, the combined group exhibited potent inhibitory effects on the growth of BC xenograft tumors in nude mice, without obvious toxicity. The molecular docking results were consistent with the wet lab experiments and enhanced the reliability of the drugs. In conclusion, our results demonstrate that the inhibition of endogenous H2S production can significantly inhibit the growth of human breast cancer cells via the AKT/PI3K/mTOR pathway and suggest that endogenous H2S may act as a promising therapeutic target in human BC cells. Our study also empowers the rationale to design novel H2S-based anti-tumor drugs to cure BC.

High dietary methionine intake may contribute to the risk of nonalcoholic fatty liver disease by inhibiting hepatic H2S production

Methionine, an essential sulfur-containing amino acid, is associated with hepatic lipid accumulation; however, the underlying mechanism is unknown. This study aimed to investigate the effects of different dietary methionine levels on hepatic lipid accumulation in mice and clarify the possible mechanisms involved. The Institute of Cancer Research (ICR) mice were fed a normal diet (ND, 0.86% methionine), high-methionine diet (HMD, 2.58% methionine), or methionine-restricted diet (MRD, 0.17% methionine) for 11 consecutive weeks. Our results showed that HMD increased the liver weight and liver index, plasma and hepatic lipid profiles, and hepatic fatty infiltration area and perirenal fat volume. In addition, HMD promoted lipid synthesis, inhibited lipid catabolism and glycolysis metabolism, reduced the activities of mitochondrial respiratory chain enzyme complexes (Ⅰ and Ⅴ) and adenosine triphosphate (ATP) production, and elevated oxidative stress and inflammation in the liver. Moreover, HMD inhibited homocysteine metabolism and significantly decreased the expression and activity of cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), thereby reducing endogenous H2S production in the liver. Interestingly, MRD reversed these adverse effects, and promoted endogenous H2S production. In conclusion, inhibition of hepatic H2S production may be the mechanism behind an increased risk of nonalcoholic fatty liver disease (NAFLD) associated with high dietary methionine intake. Therefore, it is necessary to reduce methionine intake in the daily diet to prevent NAFLD and maintain good physical health.

Mitochondrial H2S Regulates BCAA Catabolism in Heart Failure.

Hydrogen sulfide (H2S) exerts mitochondria-specific actions that include the preservation of oxidative phosphorylation, biogenesis, and ATP synthesis, while inhibiting cell death. 3-MST (3-mercaptopyruvate sulfurtransferase) is a mitochondrial H2S-producing enzyme whose functions in the cardiovascular disease are not fully understood. In the current study, we investigated the effects of global 3-MST deficiency in the setting of pressure overload-induced heart failure. Human myocardial samples obtained from patients with heart failure undergoing cardiac surgeries were probed for 3-MST protein expression. 3-MST knockout mice and C57BL/6J wild-type mice were subjected to transverse aortic constriction to induce pressure overload heart failure with reduced ejection fraction. Cardiac structure and function, vascular reactivity, exercise performance, mitochondrial respiration, and ATP synthesis efficiency were assessed. In addition, untargeted metabolomics were utilized to identify key pathways altered by 3-MST deficiency. Myocardial 3-MST was significantly reduced in patients with heart failure compared with nonfailing controls. 3-MST KO mice exhibited increased accumulation of branched-chain amino acids in the myocardium, which was associated with reduced mitochondrial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism with 3,6-dichlorobenzo1[b]thiophene-2-carboxylic acid (BT2) and administration of a potent H2S donor JK-1 ameliorates the detrimental effects of 3-MST deficiency in heart failure with reduced ejection fraction. Our data suggest that 3-MST derived mitochondrial H2S may play a regulatory role in branched-chain amino acid catabolism and mediate critical cardiovascular protection in heart failure.

Stimulation of the endogenous hydrogen sulfide synthesis restores constitutive nitric oxide synthesis and endothelium-dependent vasodilation in old rats

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Hydrogen sulfide (H2S) is a gas transmitter of endogenous origin, which has an important regulatory function in the human body. Endogenous H2S is synthesized by three enzymes, cistationine-β-synthase (CBS), cistationine-γ-liase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MTS), which is coupled to the activity of cysteine aminotransferase (CAT). CAT, CBS, and CSE are pyridoxal-5- phosphate (PLP)-dependent enzymes. It is known that with age the exchange of hydrogen sulfide is disturbed against the background of increasing oxidative and nitrosative stress. At the same time there is a decrease of constitutive synthesis of nitric oxide (NO). Recent studies show that hydrogen sulfide closely interacts with the NO system. Activation of H2S biosynthesis and restoration of its pools in tissues of old animals may be a promising direction of cardiovascular disease prevention. The aim of the current work was to study the effect of H2S-synthesizing enzymes cofactor pyridoxal-5-phosphate (PLP) on the endothelium-dependent relaxation of smooth muscle vessels in old rats, as well as the H2S content and cNOS activity in aortic tissues. Methods Old rats (22-24 months old) were PLP administered per os in dose of 0,7 mg per kg daily for 2 weeks.The content of hydrogen sulfide, activity of cNOS and iNOS was measured in aortic tissues.Vascular reactions were determined in experiments on isolated aortic strips. To clarify the role of either NO or H2S in the endothelium-dependent vasorelaxation, N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma, USA, 27 mg/kg) or O-carboxymethylhydroxylamine (O-CMH) (50 mg/kg) (Sigma, USA) was injected intraperitoneally 30 min before sacrificing. Results We showed that the H2S content was 1.6 times lower in old rat aorta compared to adult ones. PLP administration for 2 weeks induced a significant increase of H2S content in aortic tissue of old rats (P<0.05). It was found that with aging, the activity of cNOS decreases by 3 times, and the stimulation of endogenous Н2S leads to an increase in the activity of this enzyme in the aortic tissues by 2.1 times as compared with those in old animals. At the same time, the activity of the iNOS enzyme with aging increases by 3.8 times, and after the introduction of PLP, it decreases almost halved. Also shown, that PLP significantly improved endothelium-dependent aortic smooth muscle relaxation in old rats. The amplitude of acetylcholine-induced relaxation which was significantly reduced in old rats was increased more than 2.5-fold. This effect was reversed by inhibitors of NO-synthase and 3-mercaptopyruvate sulfurtransferase indicating the involvement of NO and H2S in the improved endothelium-dependent vascular relaxation. Conclusions Thus, H2S acts as a regulator of the NO system that increases cNOS activity and NO synthesis in aortic tissue, which improves endothelium-dependent relaxation of aortic smooth muscle in aged rats.

Inhibition of the 3-mercaptopyruvate sulfurtransferase—hydrogen sulfide system promotes cellular lipid accumulation

H2S is generated in the adipose tissue by cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase (3-MST). H2S plays multiple roles in the regulation of various metabolic processes, including insulin resistance. H2S biosynthesis also occurs in adipocytes. Aging is known to be associated with a decline in H2S. Therefore, the question arises whether endogenous H2S deficiency may affect the process of adipocyte maturation and lipid accumulation. Among the three H2S-generating enzymes, the role of 3-MST is the least understood in adipocytes. Here we tested the effect of the 3-MST inhibitor 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) and the H2S donor (GYY4137) on the differentiation and adipogenesis of the adipocyte-like cells 3T3-L1 in vitro. 3T3-L1 cells were differentiated into mature adipocytes in the presence of GYY4137 or HMPSNE. HMPSNE significantly enhanced lipid accumulation into the maturing adipocytes. On the other hand, suppressed lipid accumulation was observed in cells treated with the H2S donor. 3-MST inhibition increased, while H2S donation suppressed the expression of various H2S-producing enzymes during adipocyte differentiation. 3-MST knockdown also facilitated adipocytic differentiation and lipid uptake. The underlying mechanisms may involve impairment of oxidative phosphorylation and fatty acid oxidation as well as the activation of various differentiation-associated transcription factors. Thus, the 3-MST/H2S system plays a tonic role in suppressing lipid accumulation and limiting the differentiation of adipocytes. Stimulation of 3-MST activity or supplementation of H2S—which has been recently linked to various experimental therapeutic approaches during aging—may be a potential experimental approach to counteract adipogenesis.

MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

Beneficial Effect of H2S-Releasing Molecules in an In Vitro Model of Sarcopenia: Relevance of Glucoraphanin.

Sarcopenia is a gradual and generalized skeletal muscle (SKM) syndrome, characterized by the impairment of muscle components and functionality. Hydrogen sulfide (H2S), endogenously formed within the body from the activity of cystathionine-γ-lyase (CSE), cystathionine- β-synthase (CBS), and mercaptopyruvate sulfurtransferase, is involved in SKM function. Here, in an in vitro model of sarcopenia based on damage induced by dexamethasone (DEX, 1 μM, 48 h treatment) in C2C12-derived myotubes, we investigated the protective potential of exogenous and endogenous sources of H2S, i.e., glucoraphanin (30 μM), L-cysteine (150 μM), and 3-mercaptopyruvate (150 μM). DEX impaired the H2S signalling in terms of a reduction in CBS and CSE expression and H2S biosynthesis. Glucoraphanin and 3-mercaptopyruvate but not L-cysteine prevented the apoptotic process induced by DEX. In parallel, the H2S-releasing molecules reduced the oxidative unbalance evoked by DEX, reducing catalase activity, O2− levels, and protein carbonylation. Glucoraphanin, 3-mercaptopyruvate, and L-cysteine avoided the changes in myotubes morphology and morphometrics after DEX treatment. In conclusion, in an in vitro model of sarcopenia, an impairment in CBS/CSE/H2S signalling occurs, whereas glucoraphanin, a natural H2S-releasing molecule, appears more effective for preventing the SKM damage. Therefore, glucoraphanin supplementation could be an innovative therapeutic approach in the management of sarcopenia.

Endogenous and exogenous hydrogen sulfide modulates urothelial bladder carcinoma development in human cell lines

The role of H2S in urothelial carcinoma (UC) is still unclear. Here we have evaluated the expression of H2S producing enzymes as well as the effect of endogenous and exogenous H2S on human bladder UC cells. In human UC cells the expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST); is significantly lower as compared to healthy cells. A modulatory role for the H2S pathway is supported by the finding that, the overexpression of CSE or CBS, but not 3-MST, inhibits cell proliferation and promotes apoptosis. A similar effect is obtained by using exogenous H2S. Diallyl trisulfide (DATS), which is a fully characterized H2S donor, inhibits the proliferation of UC cells in a time and concentration-dependent manner as well as promotes apoptosis. Moreover, DATS also induces autophagy, as determined by transcriptomic and western blot analysis. Finally, DATS inhibits mRNA expression levels of canonical markers of epithelial-mesenchymal transition by limiting migration and clonogenic ability of human UC cells in vitro. In conclusion, in urothelial carcinoma, there is an impairment of H2S pathway that involves CSE and CBS- derived hydrogen sulfide. Thus, targeting H2S signaling pathway in urothelial carcinoma could represent a novel therapeutic strategy.

Roles of the RhoA-ROCK Signaling Pathway in the Endothelial H2S Production and Vasodilation in Rat Cerebral Arteries.

Cerebral endothelial H2S protects against cerebral ischemia-reperfusion injury through vasodilation, but its cerebral vasodilation mechanism and regulation of production are poorly understood. The RhoA-ROCK pathway plays important roles in vascular function. In this study, the roles of this pathway in the endothelial H2S production and vasodilation in rat cerebral arteries were investigated. Acetylcholine significantly increased H2S-generating enzyme cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) protein expressions and H2S production in rat cerebrovascular endothelial cells (ECs), but the increases were markedly decreased by the M receptor blocker atropine or the CSE inhibitor dl-propargylglycine. Pretreatment with dl-propargylglycine or the 3-MST inhibitor l-aspartic acid markedly reduced the acetylcholine-increased H2S; CSE protein expression and H2S levels in the ECs were obviously attenuated by the RhoA agonist U46619 but increased by the RhoA inhibitor C3 transferase. U46619 also reduced 3-MST protein expression; Acetylcholine markedly inhibited RhoA protein expression and activity, but the inhibition was obviously reversed by atropine, dl-propargylglycine, and l-aspartic acid, respectively; Acetylcholine-induced endothelium-dependent vasodilation in rat cerebral basilar artery was significantly attenuated by pretreatment with dl-propargylglycine or l-aspartic acid or RhoA inhibitor CCG-1423 or ROCK inhibitor KD025, and was further decreased by co-pretreatment with dl-propargylglycine (or l-aspartic acid) and CCG-1423 (or KD025); NaHS significantly relaxed rat cerebral basilar artery vascular smooth muscle cells and inhibited ROCK1/2 activities, phosphorylated myosin light chain (MLC) protein expression, and KCl-increased [Ca2+]i, but these relaxation and inhibitions were markedly attenuated by pretreatment with C3 transferase or ROCK inhibitor Y27632. Our results demonstrated that endothelial H2S production is promoted by activation of the M receptor but inhibited by the RhoA-ROCK pathway in rat cerebral arteries; the endothelial H2S induces cerebral vasodilation by inhibiting this pathway to reduce phosphorylation of MLC and [Ca2+]i in vascular smooth muscle cells.

In silico screening, molecular dynamic simulations, and in vitro activity of selected natural compounds as an inhibitor of Leishmania donovani 3-mercaptopyruvate sulfurtransferase.

In Leishmania sp., the enzymes of de novo cysteine biosynthesis pathway require sulfide. Other organisms utilize sulfide through the sulfide reduction pathway, but Leishmania lacks the gene that encodes these enzymes. Hence, the major source of sulfide for Leishmania is believed to be from the action of 3-mercaptopyruvate sulfurtransferase (3MST) on 3-mercapto-pyruvate (3MP). There has been no effort reported in the past to screen inhibitors against L. donovani 3MST (Ld3MST). As a result, this study examines natural compounds that are potent against Ld3MST and validates it by in vitro activity and cytotoxicity tests. Initially, a library of ~ 5000 natural compounds was subjected to molecular docking approach for screening, and the best hit was validated using a long-term molecular dynamic simulation (MD). Among the docking results, quercetine-3-rutinoside (Rutin) was deemed the best hit. The results of the MD indicated that Rutin was highly capable of interacting with the varied active site segments, possibly blocking substrate access. Additionally, promastigotes and amastigotes were tested for Rutin activity and the IC50 was found to be 40.95 and 90.09 μM, respectively. Similarly, the cytotoxicity assay revealed that Rutin was not toxic even at a concentration of 819.00 μM to THP-1 cell lines. Additionally, the Ld3MST was cloned, purified, and evaluated for enzyme activity in the presence of Rutin. Reduction in the enzyme activity (~ 85%) was observed in the presence of ~ 40 μM Rutin. Thus, this study suggests that Rutin may act as a potent inhibitor of Ld3MST. With further in vivo investigations, Rutin could be a small molecule of choice for combating leishmaniasis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-022-07532-5.

Hydrogen sulfide has a role as an endogenous relaxation factor in the rat prostate

ABSTRACT Introduction Hydrogen sulfide (H2S) is the third endogenous gasotransmitter besides carbon monoxide and nitric oxide, and has a wide range of physiological functions such as neuromodulation, vasorelaxation and cytoprotection. Although in human prostate cancer cells, NaHS (a H2S donor) treatment was reported to inhibit proliferation and to elevate secretory function, physiological functions of H2S in the prostate tissue had not been investigated yet. Endogenous H2S is biosynthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). In addition, 3-mercaptopyruvate sulfurtransferase (MPST) synthesizes H2S from 3-mercaptopyruvate (3MP), which is produced from L-cysteine by cysteine aminotransferase (CAT) (CAT/MPST pathway). Recently, a new pathway for endogenous H2S biosynthesis is reported; D-cysteine is metabolized by D-amino acid oxidase (DAO) to 3MP, which is a substrate for MPST to synthesize H2S. Objective We investigated (1) tissue distribution of CBS, CSE, MPST, CAT and DAO, (2) endogenous H2S levels, and (3) effects of NaHS on contractility in the rat prostate. Methods Dorsolateral and ventral prostate (PR-D and PR-V), liver and cerebellum (L and C) were prepared from male Wistar rats (300-400 g) sacrificed with an overdose of sodium pentobarbital (80 mg/kg, ip). (1) Tissue distributions of 5 enzymes were investigated by real-time PCR, western blot and immunohistochemistry. L and C were used for positive controls. (2) H2S contents in the prostate tissues were measured by methylene blue method. (3) By using 1 x 5 mm strips of the prostate tissues, effects of NaHS (1 x 10-9 to 3 x 10-4 M) were evaluated on pre-contracted prostate strips by noradrenaline (10-5 M). Prostate strips were pretreated with propranolol (10-6 M) 30 min before the pre-contraction. Results (1) CBS, MPST and CAT were detected in the prostate. Expression levels of CBS in the PR-D were lower than those in the PR-V, and the levels of MPST in the PR-D were higher than those in the PR-V. In expression levels of CAT, there was no significant difference between two prostate tissues. On the other hand, CSE or DAO was not detected in these tissues. Immunoreactvities of these enzymes were mainly detected in the prostate glandular epithelium. (2) H2S was detected in the prostate tissues. (3) NaHS dose-dependently induced relaxation of pre-contracted PR-D and PR-V strips. There were no significant differences of the EC50 values or relaxation rate against the pre-contractions between two prostate tissues. Conclusion H2S synthesized by CBS- and CAT/MPST-mediated pathways has a role as an endogenous relaxation factor in the rat prostate. Therefore, endogenous H2S might open new avenues of therapeutic interventions for benign prostatic hyperplasia. Disclosure Work supported by industry: no.

Recovery of erectile dysfunction after administration of hydrogen sulfide donor sodium hydrogen sulfide and transplantation of adipose-derived stem cells in rats with bilateral cavernous nerve injury

ABSTRACT Introduction Radical prostatectomy causes nerve injury-related erectile dysfunction (ED), which results in a poor response to phosphodiesterase 5 inhibitors. Hydrogen sulfide (H2S), an endogenous gasotransmitter, is produced by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) and participates in erectile physiology. Adipose-derived stem cells (ADSCs) represent a promising cell source for cell replacement therapy in several degenerative diseases because of its self-renewal properties and multi-differentiation potential with low immunogenicity. Objective The current study was to evaluate the effects of H2S donor, sodium hydrogen sulfide (NaHS), and transplantation of ADSCs per se and their combination treatment on bilateral cavernous nerve injury (BCNI)-induced erectile dysfunction (ED). Methods A total of 35 Sprague-Dawley rats were divided into 5 groups, including sham-operated (Sham), BCNI (4-wk), BCNI treated with NaHS (5.6 mg/kg/day, i.p.), BCNI treated with ADSCs (1x106 cells by intracavernosal injection) and BCNI treated with NaHS and ADSCs groups. Rats in the ADSCs treatment groups were subjected to intracavernosal injection after BCNI. Erectile function was evaluated by the ratio of intracavernous pressure (ICP)/mean arterial pressure (MAP) and total ICP. The relaxant and contractile responses of corpus cavernosum (CC) were measured from in vitro studies. Protein expression and localization of nitric oxide synthases (NOS) and H2S synthesis enzymes were analyzed by Western blotting and immunohistochemistry. The smooth muscle/collagen ratio was determined using Masson trichrome staining. Results Rats with BCNI displayed significantly reduced ICP/MAP and total ICP (p<0.001 vs controls) when were partially restored by NaHS and ADSCs treatments alone. Interestingly, combined treatment completely enhanced decreased in vivo erectile responses (p<0.001 vs the NC group). Relaxation responses to acetylcholine and electrical field stimulation (EFS) as well as contractile responses to phenylephrine and EFS in the CN group were less than in controls (p< 0.05), which was normalized after all treatments groups. The combined treatment modulated changes of protein expressions and reduced ratio of smooth muscle to collagen in rats with BCNI. Conclusions The current data indicated that the combination treatment with NaHS and ADSCs is more successful than monotherapies in reversing BCNI-induced ED. H2S and cell-based therapy most probably may have a synergistic effect on erectile function via controlling molecular regulation and morphological penile alterations, which supports combined treatment for expanding clinical outcomes radical prostatectomy-induced ED. Disclosure Work supported by industry: no.

Heart and kidney H2S production is reduced in hypertensive and older rats

The prevalence of hypertension increases with age, but the mechanisms linking this phenomenon are not well understood. Hydrogen sulfide (H2S) may be involved in this process, as it plays a role in the cardiovascular system, affecting blood pressure and heart and kidney functions. The aim of this study was to evaluate the influence of hypertension and aging on sulfur-containing compounds metabolism in the hearts and kidneys of Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) of different age groups. We determined the expression and activity of four enzymes participating in H2S production: cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH), 3-mercaptopyruvate sulfurtransferase (MPST), and thiosulfate sulfurtransferase (TST). The levels of reduced/oxidized glutathione, cysteine, cystine, and cystathionine, and the ability of tissues to form hydrogen sulfide were also investigated. Tissues obtained from younger WKY rats produced the highest amounts of H2S. The effect of hypertension on the metabolism of sulfur-containing compounds was manifested by a decrease in sulfane sulfur concentrations in heart homogenates and a decrease in CTH activity in the kidneys. The hearts and kidneys of older WKY rats were characterized by lower MPST or CTH gene expression, respectively, compared to younger animals. Our study demonstrates that hypertension and aging influence cardiac and renal sulfur-containing compounds metabolism and reduce H2S production. Furthermore, we showed that MPST plays a major role in the production of hydrogen sulfide in the heart and CTH in the kidneys of rats.

Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs.

Over the last two decades, hydrogen sulfide (H2S) has emerged as an endogenous regulator of a broad range of physiological functions. H2S belongs to the class of molecules known as gasotransmitters, which typically include nitric oxide (NO) and carbon monoxide (CO). Three enzymes are recognized as endogenous sources of H2S in various cells and tissues: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). The present article reviews the regulation of these enzymes as well as the pathways of their enzymatic and nonenzymatic degradation and elimination. The multiple interactions of H2S with other labile endogenous molecules (e.g., NO) and reactive oxygen species are also outlined. Next, the various biological targets and signaling pathways are outlined, with special reference to H2S or oxidative posttranscriptional modification (persulfidation or sulfhydration) of proteins and the effect of H2S on various channels and intracellular second messenger pathways, the regulation of gene transcription and translation, and the regulation of cellular bioenergetics and metabolism. The pharmacological and molecular tools currently available to study H2S physiology are also reviewed, including their utility and limitations. In subsequent sections, the role of H2S in the regulation of various physiological and cellular functions is reviewed, including the regulation of membrane potential, endo- and exocytosis, regulation of various cell organelles (endoplasmic reticulum, Golgi, mitochondria), regulation of cell movement, cell cycle, cell differentiation, and physiological aspects of regulated cell death. Next, the physiological roles of H2S in various cell types and organ systems are overviewed, including the role of H2S in red blood cells, immune cells, the central and peripheral nervous systems (with focus on neuronal transmission, learning, and memory formation), and regulation of vascular function (including angiogenesis as well as its specialized roles in the cerebrovascular, renal, and pulmonary vascular beds) and the role of H2S in the regulation of special senses, vision, hearing, taste and smell, and pain-sensing. Finally, the roles of H2S in the regulation of various organ functions (lung, heart, liver, kidney, urogenital organs, reproductive system, bone and cartilage, skeletal muscle, and endocrine organs) are presented, with a focus on physiology (including physiological aging) but also extending to some common pathophysiological conditions. From these data, a wide array of significant roles of H2S in the physiological regulation of all organ functions emerges and the characteristic bell-shaped biphasic effects of H2S are highlighted. In addition, key pathophysiological aspects, debated areas, and future research and translational areas are identified.

Differential effects of subchronic acrylonitrile exposure on hydrogen sulfide levels in rat blood, brain, and liver.

Hydrogen sulfide (H2S), as the third gasotransmitter participates in both cellular physiological and pathological processes, including chemical-induced injuries. We recently reported acute acrylonitrile (AN) treatment inhibited endogenous H2S biosynthesis pathway in rat and astrocyte models. However, there is still no evidence to address the correlation between endogenous H2S and sub-chronic AN exposure. This study aims to explore the modulatory effects of prolonged AN exposure on endogenous H2S levels and its biosynthetic enzymes in rat blood, brain and liver. A total of 50 male Sprague-Dawley rats were randomly divided into 5 groups, including the control group and AN-treated groups at dosages of 6.25, 12.5, 25 or 50 mg/kg. Rats received one exposure/day, 5 days/week, for 4 consecutive weeks. The rat bodyweight and brain/liver organ coefficient were detected, along with liver cytochrome P450 2E1(CYP2E1) expression. In addition, the H2S contents in rat serum and plasma, and in cerebral cortex and liver tissues were measured by methylene blue method. The expression of H2S-generating enzymes, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MPST) was also measured with Western blot both in rat cerebral cortex and liver. Subchronic exposure to AN significantly inhibited bodyweight-gain and increased the liver CYP2E1 expression compared with the control. In addition, AN significantly increased H2S levels in rat plasma and serum, but not in liver. The endogenous H2S level in rat cerebral cortex was also significantly increased upon AN treatment, when expression of the major H2S-generating enzymes, CBS and 3-MPST were significantly enhanced. However, hepatic protein levels of CBS and CSE were significantly increased, whereas hepatic levels of 3-MPST were significantly decreased. This study showed that sub-chronic AN exposure increased endogenous H2S contents in rat blood and brain tissues, but not liver, which may be resulted from the distinct expression profile of H2S-producing enzymes in response to AN. The blood H2S contents may be applied as a potential novel biomarker for surveillance of chronically AN-exposed populations. Subchronic intraperitoneal exposure to acrylonitrile increased H2S content in rat blood and cerebral cortex, but not in liver.Distinct tissue expression profiles of H2S-producing enzymes contribute to the acrylonitrile-induced differential effects on the H2S level.Blood H2S level may be a biomarker for subchronic exposure to acrylonitrile.

Heparan Sulfate, Mucopolysaccharidosis IIIB and Sulfur Metabolism Disorders.

Mucopolysaccharidosis, type IIIB (MPS IIIB) is a rare disease caused by mutations in the N-alpha-acetylglucosaminidase (NAGLU) gene resulting in decreased or absent enzyme activity. On the cellular level, the disorder is characterized by the massive lysosomal storage of heparan sulfate (HS)—one species of glycosaminoglycans. HS is a sulfur-rich macromolecule, and its accumulation should affect the turnover of total sulfur in cells; according to the studies presented here, it, indeed, does. The lysosomal degradation of HS in cells produces monosaccharides and inorganic sulfate (SO42−). Sulfate is a product of L-cysteine metabolism, and any disruption of its levels affects the entire L-cysteine catabolism pathway, which was first reported in 2019. It is known that L-cysteine level is elevated in cells with the Naglu−/− gene mutation and in selected tissues of individuals with MPS IIIB. The level of glutathione and the Naglu−/− cells’ antioxidant potential are significantly reduced, as well as the activity of 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) and the level of sulfane sulfur-containing compounds. The direct reason is not yet known. This paper attempts to identify some of cause-and-effect correlations that may lead to this condition and identifies research directions that should be explored.

The Cytotoxicity of OptiBond Solo Plus and Its Effect on Sulfur Enzymes Expression in Human Fibroblast Cell Line Hs27

The aim of the study was to determine the cytotoxic concentrations and incubation times of the commonly used dental adhesive system OptiBond Solo Plus in its non-polymerized form, and to test how it relates to oxidative stress by determining the reduced and oxidized glutathione (GSH and GSSG) levels as well as to study its influence on cell number and the expression of selected sulfur enzymes, with particular emphasis on cystathionine γ-lyase (CTH) and 3-mercaptopyruvate sulfurtransferase (MPST). All investigations were conducted on an in vitro model of human fibroblast cell line Hs27. Changes in cellular plasma membrane integrity were measured by the LDH test. The expression levels were determined by RT-PCR and Western blot protocols. Changes in cell number were visualized using crystal violet staining. The RP-HPLC method was used to determine the GSH and GSSG levels. Reduced cell number was shown for all tested concentrations and times. Changes in the expression on the mRNA and protein level were demonstrated for CTH and MPST enzymes upon exposure to the tested range of OptiBond concentrations. Levels of low-molecular sulfur compounds of reduced and oxidized glutathione were also established. Cytotoxic effect of OptiBond Solo Plus may be connected with the changes of MPST and CTH sulfur enzymes in the human fibroblast Hs27 cell line. The elevated levels of these enzymes could possibly show the antioxidant response to this dental adhesive system. OptiBond Solo Plus in vitro results should be taken into consideration for further in vivo tests.