Sulfide Pathway Research Articles

OP13 The hydrogen sulfide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia

Hyperhomocysteinemia is a risk factor for neurovascular and cardiovascular disease associated to endothelial dysfunction and accelerated atherosclerosis [1] , [2] , [3] . Many clinical and epidemiological studies have demonstrated a positive correlation among homocysteine (Hcy) plasma levels and cardiovascular disorders [4] leading to the general conclusion that Hcy is a pro-thrombotic factor [4] , [5] . Hcy is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR) [6] . Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulfide (H 2 S), through multiple steps involving cystathionine β -synthase (CBS) and cystathionine γ -lyase (CSE) [7] . To date, the influence of H 2 S on platelet function has been poorly explored. Here we have evaluated the involvement of H 2 S in platelet reactivity by using platelets harvested from healthy volunteers or patients firstly diagnosed with hyperhomocysteinemia (MTHFR++ carriers). Sodium hydrogen sulfide (NaHS) or l -cysteine were used as exogenous or endogenous source of H 2 S, respectively. NaHS (0.1–100 μM) or l -cysteine (0.1–100 μM) did not cause per se platelet aggregation. However both NaHS and l -cysteine significantly increased platelet aggregation induced by the thrombin receptor activator peptide-6 amide (TRAP-6, 2 μM) in a concentration-dependent manner in platelets harvested from healthy volunteers. When platelets harvested from MTHFR++ carriers were used platelet aggregation was further potentiated. The potentiating effect present in this latter group was reverted by the inhibition of either CBS or CSE. Therefore the l -cysteine/H 2 S pathway is involved in the increased responsivity in MTHFR++ carriers. The role played by this pathway is further supported by the finding that H 2 S levels were significantly higher in both platelets and plasma. To gain further insights into the downstream signaling we evaluated the involvement of thromboxaneA 2 (TXA 2 ). TXA 2 levels were markedly increased in response to both NaHS or l -cysteine in platelets of healthy volunteers. Inhibition of phospholipase A 2 , cyclooxygenase or blockade of thromboxane receptor markedly reduced TXA 2 production triggered by H 2 S. These results confirmed that H 2 S triggers the arachidonic acid cascade and that the main downstream signal is thromboxane. Since phospholipase A 2 is the rate limiting enzyme of the arachidonic cascade we evaluated if H 2 S can activate phospholipase A 2 by measuring its phosphorylation. Phospholipase A 2 phosphorylation was significantly higher in MTHFR++ carriers when compared to healthy volunteers. Our data suggest that in MTHFR++ carriers there is an increase in platelet activity dependent upon H 2 S generated within the platelet that boosts the arachidonic acid cascade. Therefore in MTHFR++ carriers the activation of the l -cysteine/H 2 S pathway within the platelets primes the cells making them more responsive to endogenous stimuli that normally do not activate healthy platelets.

Dysregulation of cystathionine γ-lyase (CSE)/hydrogen sulfide pathway contributes to ox-LDL-induced inflammation in macrophage

Hydrogen sulfide (H2S), mainly produced by cystathionine γ-lyase (CSE) in vascular system, emerges as a novel gasotransmitter exerting anti-inflammatory and anti-atherosclerotic effects. Alterations of CSE/H2S pathway may thus be involved in atherosclerosis pathogenesis. However, the underlying mechanisms are poorly understood. The present study showed that the levels of CSE mRNA and protein expression, as well as H2S production were decreased in ox-LDL-treated macrophage. CSE overexpression reduced the ox-LDL-stimulated tumor necrosis factor-α (TNF-α) generation in Raw264.7 and primary macrophage while CSE knockdown enhanced it. Exogenous supplementation of H2S with NaHS and Na2S also decreased the production of TNF-α and intercellular adhesion molecule-1 (ICAM-1) in ox-LDL-stimulated macrophage, and alleviated the adhesion of macrophage to endothelial monolayer. Cysteine, a CSE preferential substrate for H2S biosynthesis, produced similar effects on the pro-inflammatory cytokine generation, which were reversed by CSE inhibitors PAG and BCA, respectively. Moreover, NaHS and Na2S attenuated the phosphorylation and degradation of IκBα and p65 nuclear translocation, as well as JNK activation caused by ox-LDL. The JNK inhibitor suppressed the NF-κB transcription activity in ox-LDL-treated cells. Furthermore, inhibitors of NF-κB (PDTC), ERK (U0126 and PD98059) and JNK (SP600125) partially blocked the suppression by ox-LDL on the CSE mRNA levels. Taken together, the findings demonstrate that ox-LDL may down-regulate the CSE/H2S pathway, which plays an anti-inflammatory role in ox-LDL-stimulated macrophage by suppressing JNK/NF-κB signaling. The study reveals new therapeutic strategies for atherosclerosis, based on modulating CSE/H2S pathway.

Sulfur dioxide upregulates the inhibited endogenous hydrogen sulfide pathway in rats with pulmonary hypertension induced by high pulmonary blood flow

Pulmonary hypertension (PH) is an important pathophysiological process in the development of many diseases. However, the mechanism responsible for the development of PH remains unknown. The objective of the study was to explore the possible impact of sulfur dioxide (SO2) on the endogenous hydrogen sulfide (H2S) pathway in rats with PH induced by high pulmonary blood flow. Compared with sham group, the systolic pulmonary artery pressure (SPAP) in the shunt group was significantly increased, along with the increased percentage of muscularized arteries and partially muscularized arteries of small pulmonary arteries. Compared with the shunt group, SPAP in the shunt+SO2 group was significantly decreased, and the percentage of muscularized pulmonary arteries was also decreased. Additionally, rats that developed PH had significantly lower levels of SO2 concentration, aspartate aminotransferase (AAT) activity, protein and mRNA expressions of AAT2 in pulmonary tissues. Administration of an SO2 donor could alleviate the elevated pulmonary arterial pressure and decrease the muscularization of pulmonary arteries. At the same time, it increased the H2S production, protein expression of cystathionine-γ-lyase (CSE), mRNA expression of CSE, mercaptopyruvate transsulphurase (MPST) and cystathionine-β-synthase (CBS) in the pulmonary tissue of the rats. The results suggested that endogenous SO2/AAT2 pathway and the endogenous H2S production were downregulated in rats with PH induced by high pulmonary blood flow. However, SO2 could reduce pulmonary arterial pressure and improve the pulmonary vascular pathological changes in association with upregulating endogenous H2S pathway.

P19 Imbalance of endogenous hydrogen sulfide and homocysteine pathway in chronic obstructive pulmonary disease combined with cardiovascular disease

Background Chronic obstructive pulmonary disease (COPD) is a multi-system disease. The pathogenesis of COPD combined with cardiovascular disease (CVD) is not fully identified. Endogenous hydrogen sulfide (H2S) is a third gaseous transmitter playing an important role in the pathophysiology of COPD. Homocysteine (Hcy) is an important substrate for the endogenous H2S production. Studies showed that hyperhomocysteinemia is a risk factor in the pathogenesis of CVD. The present study is to explore the imbalance of endogenous H2S and Hcy metabolic pathway in patients with COPD combined with CVD. Methods 51 patients with stable COPD with (n = 15) and without CVD (n = 36) were prospectively enrolled in the study. We collected demographics, clinical variables including modified Medical Research Council (MMRC) dyspnea score, St. George Respiratory Questionnaire (SGRQ) score, COPD assessment test (CAT) score, pulmonary function, sputum cell differential counts. Serum H2S and Hcy were measured by sulfide sensitive electrode and ELISA, respectively. We also measured other inflammatory mediators such as serum C-reactive protein (CRP) and tumor necrosis factor-a (TNF-a). Results Patients had similar age, gender distribution and smoking history in COPD + CVD group and COPD group. BMI was higher in COPD + CVD group than COPD group (24.7 ± 2.1 vs. 22.3 ± 3.2 kg/m2, p Conclusion Imbalance of endogenous H2S and Hcy pathway may be involved in the pathogenesis in COPD combined with CVD. Supported by National Natural Science Foundation of China (Nos. 30871127 and 81170012).

Sildenafil Effect on the Human Bladder Involves the L-cysteine/Hydrogen Sulfide Pathway: A Novel Mechanism of Action of Phosphodiesterase Type 5 Inhibitors

BackgroundPhosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H2S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). ObjectiveTo evaluate whether the L-cysteine/H2S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome. Design, setting, and participantsSamples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H2S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30μM), 8-bromo–cyclic guanosine monophosphate (8-bromo–cGMP; 100μM) or dibutyryl–cyclic adenosine monophosphate (dibutyryl-cAMP; 100μM). A curve concentration effect of sodium hydrosulfide (NaHS), H2S donor (0.1μM to 10mM), L-cysteine (0.1μM to 10mM), and sildenafil (0.1–10μM) was performed on precontracted detrusor dome strips. To investigate H2S signaling in a sildenafil effect, CBS and CSE inhibitors were used. Outcome measurements and statistical analysisAnalysis of variance was used, followed by the Bonferroni post hoc test. Results and limitationsCBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H2S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H2S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H2S production. ConclusionsThe sildenafil relaxant effect on the human bladder involves the H2S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H2S pathway in the human bladder may open new therapeutic approaches.

Endogenous hydrogen sulfide insufficiency as a predictor of sexual dysfunction in aging rats

Objective: Our earlier studies showed that endogenous hydrogen sulfide (H2S) pathway contributed significantly to erectile function. In this study, we tested the hypothesis that age-dependent changes in the bioavailability of H2S increased the risk of erectile dysfunction (ED). Methods: Young, adult (3-month) and older (18-month) male Sprague-Dawley rats (n = 6−8/group) were treated daily with sodium hydrosulfide hydrate (NaHS), DL-propargylglycine, sildenafil or l-NAME for 10 weeks. Subsequent to cavernous nerve electrical stimulation, intracavernosal pressure (ICP) responses were determined, and the samples were collected and processed for hormonal (plasma) and gaseous parameters (plasma and erectile corpus cavernosum [CC]) using standard assay protocols. Results: Aging significantly reduced the ICP response (35.9 ± 2.0 mmHg vs. 45.2 ± 1.9 mmHg in young controls), which was countered by NaHS (53.5 ± 6.0) or sildenafil (52.8 ± 9.8) treatment. In these rats, marked increments to testosterone (T) or estradiol resulted from NaHS supplementation. Similar to age-dependent decline in NO, the plasma and CC level of H2S was significantly lower in senescent rats when compared with young animals (p < 0.05). Conclusion: Our results confirm that ED with aging may be linked to a derangement in the H2S pathway accompanied by low T levels. It is likely that a pharmacologic intervention delivering H2S will provide additional benefits to sexual function from an improved T milieu.

Role of cystathionine γ-lyase/hydrogen sulfide pathway in cardiovascular disease: a novel therapeutic strategy?

Hydrogen sulfide (H(2)S) has traditionally been considered a toxic environmental pollutant. In the late 1990s, the presumed solely harmful role of H(2)S has been challenged because H(2)S may also be involved in the maintenance and preservation of cardiovascular homeostasis. The production of endogenous H(2)S has been attributed to three key enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase. The recognition of H(2)S as the third gaseous signaling molecule has stimulated research on a multitude of pathophysiologic events in the cardiovascular system. In particular, important roles in cardiovascular disorder processes are ascribed to the CSE/H(2)S pathway, such as atherosclerosis, myocardial infarction, hypertension, and shock. Many biological activities and molecular mechanisms of H(2)S in the cardiovascular system have been demonstrated in studies using different tools, such as the genetic overexpression of CSE, the direct administration of H(2)S donors, or the use of H(2)S-releasing pro-drugs. Unfortunately, the role of the CSE/H(2)S pathway in cardiovascular disease remains controversial in numerous areas, and many questions regarding the gaseous molecule still remain unanswered. Advances in basic research indicate that the CSE/H(2)S pathway may provide potential therapeutic targets for treating cardiovascular disorders. But the molecular targets of H(2)S still need to be identified.

Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H(2)S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H(2)S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

Proteomics Reveals that Redox Regulation Is Disrupted in Patients with Ethylmalonic Encephalopathy

Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide.

Therapeutic applications of organosulfur compounds as novel hydrogen sulfide donors and/or mediators

Hydrogen sulfide, once considered as toxic gas, is now recognized as an important biological mediator. The deficiency of hydrogen sulfide could lead to various pathological changes, such as arterial and pulmonary hypertension, Alzheimer’s disease, gastric mucosal injury and liver cirrhosis. However, excessive production of hydrogen sulfide, by using inorganic hydrogen sulfide donors such as NaHS, may contribute to the pathogenesis of inflammatory diseases, septic shock, cerebral stroke and mental retardation in patients with Down syndrome. Therefore, an increasing interest in organic molecules that are capable of regulating the formation of hydrogen sulfide has extended in recent years. Allium vegetables are one natural source of organic sulfur-containing compounds and have been widely investigated regarding their therapeutic applications, and it has been proven that the ingredients of garlic, such as diallyl disulfide, diallyl trisulfide and S-ally cysteine act as hydrogen sulfide donors or mediators in pharmaceutical studies. In addition, S-propargyl cysteine (ZYZ-802) and S-propyl cysteine, two synthetic cysteine analogs, have been examined and could be used to treat ischemic heart disease via modulation of the hydrogen sulfide pathway. In addition, drugs containing hydrogen sulfide-releasing moieties have been synthesized and widely reported in recent years, such as S-nonsteroidal anti-inflammatory drugs and the derivative of Lawesson’s reagents, which exhibit varied biological effects in experiments. As cystathionine β-synthase and cystathionine γ-lyase are the enzymes that are able to catalyze the production of endogenous hydrogen sulfide from cysteine, their inhibitors, such as dl-propylargylglycine and β-cyanoalanine, have been frequently used in studies on the biological mechanism of hydrogen sulfide. All these hydrogen sulfide donors, mediators and inhibitors have provided useful tools in the research of a variety of biological effects and are promising drug candidates of hydrogen sulfide.

Hydrogen sulfide attenuates epithelial–mesenchymal transition of human alveolar epithelial cells

We previously reported that the endogenous cystathionine γ-lyase (CSE)/hydrogen sulfide (H 2S) pathway is implicated in the pathogenesis of bleomycin-induced pulmonary fibrosis in rats, but the exact cellular mechanisms are not well characterized. Epithelial–mesenchymal transition (EMT), induced by transforming growth factor β1 (TGF-β1) in alveolar epithelial cells, plays an important role in the pathogenesis of pulmonary fibrosis. We studied whether H 2S could attenuate EMT in cultured alveolar epithelial cells and TGF-β1 treatment suppressed CSE expression in A549 cells. Inhibition of endogenous CSE by dl-propargylglycine led to spontaneous EMT, as manifested by decreased E-cadherin level, increased vimentin expression and fibroblast-like morphologic features. Exogenous H 2S applied to TGF-β1-treated A549 cells decreased vimentin expression, increased E-cadherin level and retained epithelial morphologic features. In addition, preincubation with H 2S decreased Smad2/3 phosphorylation in A549 cells stimulated by TGF-β1, and H 2S-inhibited alveolar EMT was mimicked by treatment with SB505124, a Smad2/3 inhibitor, but not pinacidil, an ATP-sensitive K + channel (K ATP) opener. H 2S serves a critical role in preserving an epithelial phenotype and in attenuating EMT in alveolar epithelial cells, mediated, at least in part, by decreased Smad2/3 phosphorylation and not dependent on K ATP channel opening.

The dual role of the cystathionine γ-lyase/hydrogen sulfide pathway in CVB3-induced myocarditis in mice

The present study found that serum H2S level, H2S production rate, CSE mRNA and CSE protein levels were increased in CVB3-induced myocarditis. dl-proparglygylcine (PAG), an irreversible CSE inhibitor, decreased the infected myocardium titers on postinfection day 4, while NaHS, a H2S donor, alleviated myocardial injury and necrosis, inflammatory cell infiltration and interstitial edema on postinfection day 10. These data reveal that the CSE/H2S pathway is upregulated in the heart in a murine model of CVB3-induced myocarditis and that inhibition of endogenous H2S is beneficial to treatment early in the disease while administration of exogenous H2S is protective to infected myocardium during the later stage.

Hydrogen sulfide attenuates the pathogenesis of pulmonary fibrosis induced by bleomycin in rats

The present study investigated the role of the endogenous cystathionine gamma-lyase (CSE) / hydrogen sulfide pathway in the pathogenesis of pulmonary fibrosis. Rats treated with intratracheal bleomycin were exposed either to the H2S donor NaHS or to saline. The results on day 7 showed that plasma H2S concentration and pulmonary CSE activity (H2S production rate) were significantly lower in rats treated with bleomycin and saline (fibrosis-alone) than in controls, whereas on day 28 plasma H2S concentration was higher and pulmonary CSE activity was the same as that of controls. The relative CSE mRNA level in the lungs of rats treated with bleomycin was significantly higher than control values on days 7 and 28. After exposure to NaHS, the total lung hydroxyproline content and the malondialdehyde (MDA) content were both significantly lower, with no difference observed between NaHS high-dose and low-dose treatments. Further, MDA formation stimulated by the free radical-generating system (FRGS) in vitro was lower in lung tissue incubated with NaHS than it was in tissue incubated with FRGS alone. These results suggest that NaHS administration ameliorated the pulmonary fibrosis induced by bleomycin in rats and that this protective effect of H2S may be mediated by its antioxidative action.

Inhibition of the cystathionine-γ-lyase/hydrogen sulfide pathway in rat vascular smooth muscle cells by cobalt-60 gamma radiation

Background Radiation is a promising treatment for in stent restenosis and restenosis following percutaneous transluminal coronary angioplasty, which has troubled interventional cardiologists for a long time. It inhibits neointima hyperplasia, vascular remodeling, and increases the mean luminal diameter. The mechanism of intracoronary brachytherapy for restenosis is not well understood. Endogenous gaseous transmitters including nitric oxide and carbon monoxide are closely related to restenosis. Hydrogen sulfide, a new endogenous gaseous transmitter, is able to inhibit the proliferation of vascular smooth muscle cells and vascular remodeling. This study aimed to clarify the effect of radiation on cystathionine-γ-lyase/hydrogen sulfide pathway in rat smooth muscle cells. Methods We studied the effect of radiation on the cystathionine-γ-lyase/hydrogen sulfide pathway. Rat vascular smooth muscle cells were radiated with 60Co γ at doses of 14 Gy and 25 Gy respectively. Then the mRNA level of cystathionine-γ-lyase was studied by quantitative reverse-transcription competitive polymerase chain reaction. Hydrogen sulfide concentration in culture medium was determined by methylene blue spectrophotometry. Cystathionine-γ-lyase activity in vascular smooth muscle cells was also studied. Results 60Co γ radiation at a dose of 1 Gy did not affect the cystathionine-γ-lyase/hydrogen sulfide pathway significantly. However, 60Co γ radiation at doses of 14 Gy and 25 Gy decreased the hydrogen sulfide synthesis by 21.9% (P &lt;0.05) and 26.8% (P&lt;0.01) respectively. At the same time, they decreased the cystathionine-γ-lyase activity by 15.1% (P&lt;0.05) and 20.5% (P &lt;0.01) respectively, and cystathionine-γ-lyase mRNA expression by 29.3% (P &lt;0.01) and 38.2% (P &lt;0.01) respectively. Conclusion Appropriate 60Co γ radiation inhibits the H2S synthesis by inhibiting the gene expression of cystathionine-γ-lyase and the cystathionine-γ-lyase activity.

Hydrogen sulfide removal from biogas by zeolite adsorption. Part II. MD simulations

Coupled Grand Canonical-Canonical Monte Carlo and molecular dynamics (MD) simulation techniques have been used to investigate in details the adsorption of low-pressure hydrogen sulfide (H2S) in zeolites, and the selective adsorption behavior towards carbon dioxide and methane, the main biogas constituents. Results from Monte Carlo (MC) simulations indicated, among many others, zeolite NaY as the best option for H2S removal. Afterwards, deterministic simulations have been performed to investigate hydrogen sulfide pathway inside NaY, with respect to other adsorbed molecules (methane and carbon dioxide), as a function of zeolite loading and H2S partial pressure (i.e., biogas composition). Thermodynamic evaluations for 2D molecular dynamic simulations in terms of binding energy evolution vs. time confirm and reinforce the results obtained from Monte Carlo simulations, testifying the greater affinity for H2 St o NaY zeolite framework. Results give also new quantitative insights in terms of pathways, binding energies, and equilibration time inside zeolite pores for stabilization.

Denitrification in a binary culture and thiocyanate metabolism in Thiohalophilus thiocyanoxidans gen. nov. sp. nov. – a moderately halophilic chemolithoautotrophic sulfur-oxidizing Gammaproteobacterium from hypersaline lakes

Anaerobic enrichment culture with thiocyanate as electron donor and nitrate as electron acceptor at 2 M NaCl inoculated with a mixture of sediments from hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in a selection of a binary consortium of moderately halophilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) capable of complete denitrification of nitrate with thiosulfate as the electron donor. One consortium member, strain HRhD 3sp, was isolated into pure culture with nitrate and thiosulfate using a density gradient. This strain was responsible for the reduction of nitrate to nitrite in the consortium, while a second strain, HRhD 2, isolated under microoxic conditions with thiosulfate as substrate, was capable of anaerobic growth with nitrite and thiosulfate. Nitrite, either as substrate or as product, was already toxic at very low concentrations for both strains. As a result, optimal growth under anaerobic conditions could only be achieved within the consortium. On the basis of phylogenetic analysis, both organisms were identified as new lineages within the Gammaproteobacteria. As well as thiosulfate, strain HRhD 2 can also use thiocyanate as electron donor, representing a first halophilic SOB capable of growth with thiocyanate at 2-4 M NaCl. Product and enzymatic analysis identified the "carbonyl sulfide (COS) pathway" of primary thiocyanate degradation in this new species. On the basis of phenotypic and genetic analysis, strain HRhD 2 is proposed to be assigned to a new genus and species Thiohalophilus thiocyanoxidans.

Hydrogen sulfide ameliorates vascular calcification induced by vitamin D3 plus nicotine in rats1

To investigate the role of the endogenous cystathionine gamma-synthase (CSE)/hydrogen sulfide (H2S) pathway in vascular calcification in vivo. A rat vascular calcification model was established by administration of vitamin D3 plus nicotine (VDN). The amount of CSE and osteopontin (OPN) mRNA was determined by using semi-quantitative reverse-transcription polymerase chain reaction. The calcium content, 45Ca2+ accumulation and alkaline phosphatase (ALP) activity were measured. H2S production and CSE activity were measured. von Kossa staining produced strong positive black/brown staining in areas among the elastic fibers of the medial layer in the calcified aorta. The calcium content, 45Ca2+ accumulation and ALP activity in calcified arteries increased by 6.77-, 1.42-, and 1.87-fold, respectively, compared with controls. The expression of the OPN gene was upregulated (P<0.01). Expression of the CSE gene was downregulated. However, calcium content, 45Ca2+ uptake and ALP activity in the VDN plus NaHS group was lower than that in the VDN group. The content of calcium and 45Ca2+ accumulation and activity of ALP in the aorta were 34.8%, 40.75% and 63.5% lower in the low-dosage NaHS group than in the VDN group, respectively (P<0.01), and the calcium content and deposition of 45Ca2+ and activity of ALP was 83.9%, 37.8 % and 46.2% lower in the aorta in the high-dosage NaHS group than in the VDN group, respectively (P<0.01). The expression of the OPN gene was downregulated. The production of H2S, and CSE activity were decreased and CSE gene expression was downregulated in rats with vascular calcification. H2S can ameliorate vascular calcification, suggesting that the H2S/CSE pathway plays a regulatory role in the pathogenesis of vascular calcification.

Down-Regulation of Endogenous Hydrogen Sulfide Pathway in Pulmonary Hypertension and Pulmonary Vascular Structural Remodeling Induced by High Pulmonary Blood Flow in Rats

The mechanisms responsible for the development of pulmonary hypertension (PH) and pulmonary vascular structural remodeling induced by high pulmonary blood flow are not fully understood. The present study was designed to explore the possible changes in endogenous hydrogen sulfide (H2S), a novel gasotransmitter, on the pathogenesis of PH and pulmonary vascular structural remodeling induced by high pulmonary blood flow. Twenty-two male Sprague-Dawley rats were randomly divided into a shunting group (n=11) and control group (n=11). Rats in the shunting group underwent an abdominal aorta-inferior cava vein shunting operation. After 11 weeks of shunting, the plasma level of H2S and lung tissue H2S producing rate were much lower than those of the control group (p<0.01). In situ hybridization analysis showed that the expression of cystathionine gamma-lyase (CSE) mRNA was down-regulated in the pulmonary arteries of the shunting rats compared with the control group (p<0.01), and competitive quantitative reverse transcription-polymerase chain reaction showed that the relative amount of CSEmRNA in lung tissue was decreased significantly (p<0.01). The endogenous H2S pathway is down-regulated in PH and pulmonary vascular structural remodeling is induced by high pulmonary blood flow.

Sulfur isotope partitioning during experimental formation of pyrite via the polysulfide and hydrogen sulfide pathways: implications for the interpretation of sedimentary and hydrothermal pyrite isotope records

We show that the sulfur isotopic composition of sedimentary and hydrothermal pyrite is a good approximation of the average sulfur isotopic composition of the dissolved sulfide sources from which the pyrite formed. Consequently, pyrite sulfur isotope systematics normally provide little evidence of the pyrite-forming mechanism in most natural systems. Stable sulfur isotope partitioning during pyrite (FeS 2) synthesis via the polysulfide and H 2S pathways was investigated between 80 and 120 °C. Iron monosulfide (FeS) was reacted with hydrogen sulfide (H 2S) or tetrasulfide (S 4 2−) in aqueous solution under strictly anoxic conditions. The results provide independent confirmation of the hydrogen sulfide and polysulfide mechanisms. The measured isotopic composition of the synthesized pyrite is compared with (1) isotopic mixing models of the reactant reservoirs and (2) predictions based on the suggested mechanisms for the hydrogen sulfide and polysulfide pathways for pyrite formation. The isotopic composition of the pyrite product is consistent with the result predicted from the reaction mechanisms. Pyrite produced via the H 2S pathway has a composition reflecting isotopic contributions from both FeS and H 2S reservoirs. Pyrite formed via the polysulfide pathway inherits an isotopic composition dominated by the polysulfide reservoir. In both cases, solubility driven isotope exchange between FeS and aqueous S species contribute to the final pyrite composition. We show that published experimental sulfur isotope data for pyrite formation which apparently support conflicting pyrite-forming pathways, are consistent with pyritization via the polysulfide and H 2S pathways. Formation rates of natural pyrite, however, may be too slow compared to solubility exchange for the influence of the reaction pathway on the isotopic composition to be significant.

Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol

Previous work has shown that the endogenous cystathionine γ-synthase (CSE)/hydrogen sulfide (H 2S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H 2S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H 2S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H 2S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H 2S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.