Search

Caloric restriction (CR) is one of the most effective interventions to prolong lifespan and promote health. Recently, it has been suggested that hydrogen sulfide (H2S) may play a pivotal role in mediating some of these CR-associated benefits. While toxic at high concentrations, H2S at lower concentrations can be biologically advantageous. H2S levels can be artificially elevated via H2S-releasing donor drugs. In this study, we explored the function of a novel, slow-releasing H2S donor drug (FW1256) and used it as a tool to investigate H2S in the context of CR and as a potential CR mimetic. We show that exposure to FW1256 extends lifespan and promotes health in Caenorhabditis elegans (C. elegans) more robustly than some previous H2S-releasing compounds, including GYY4137. We looked at the extent to which FW1256 reproduces CR-associated physiological effects in normal-feeding C. elegans. We found that FW1256 promoted healthy longevity to a similar degree as CR but with fewer fitness costs. In contrast to CR, FW1256 actually enhanced overall reproductive capacity and did not reduce adult body length. FW1256 further extended the lifespan of already long-lived eat-2 mutants without further detriments in developmental timing or fertility, but these lifespan and healthspan benefits required H2S exposure to begin early in development. Taken together, these observations suggest that FW1256 delivers exogenous H2S efficiently and supports a role for H2S in mediating longevity benefits of CR. Delivery of H2S via FW1256, however, does not mimic CR perfectly, suggesting that the role of H2S in CR-associated longevity is likely more complex than previously described.

Introduction His bundle pacing (HBP) aims to deliver physiological activation of the ventricles via the native His-Purkinje conduction system. A challenge of HBP is the limited market of implantation tools, pacing leads and specifically designed pacing algorithms. Purpose Over the last three years both Medtronic (MDT) and Boston Scientific (BSC) generators have been used for HBP in a large tertiary centre. We examined whether there was any difference between lead parameters and battery longevity depending on the type of manufacturer used. Methods Patients implanted with a MDT Select Secure model 3830 lead were included in this retrospective study. Data collected included HBP thresholds (analysed at 1ms pulse width) at implant and at the most recent device check, HBP percentages and battery longevity (months). Battery longevity were calculated by adding duration of follow up and estimated battery life at last follow up. Results A total of 31 patients were included for data analysis (58% male and mean age 72 years). 18 patients had MDT generators of which 3 were PPMs, 5 were CRT-Ps and 10 were CRT-Ds. 13 patients had BSC generators of which 5 were PPMs, 5 were CRT-Ps and 3 were CRT-Ds. Mean follow up of the cohort was 12.7±9.02 months. Mean HBP percentages were 77±37% and 72.2±42.1% for MDT and BSC, respectively (p=0.430). Mean HBP threshold (Volts) at implant was significantly lower with BSC vs MDT (0.85±0.58 vs 1.84±1.06, p=0.01), and similar after follow up (1.01±0.91 vs 1.32±0.73, p=NS). There were no statistically significant differences between mean HBP threshold at implant compared to follow up for both manufacturers. Interestingly, mean battery longevity for BSC vs MDT generators was significantly higher (136±29.3 vs 90.5±29.1, p Conclusion Our results suggest HBP with MDT Select Secure 3830 lead is feasible with BSC generators. There is potentially greater battery longevity with BSC compared to MDT generators. Further studies are needed with greater numbers and longer follow up to confirm this finding. Funding Acknowledgement Type of funding sources: None.

Hydrogen sulfide (H

Hydrogen sulfide (H2S) is the "third gasotransmitter" recognized alongside nitric oxide (NO) and carbon monoxide (CO). H2S exhibits an array of biological effects in mammalian cells as revealed by studies showing important roles in the cardiovascular system, in cell signalling processes, post-translational modifications and in the immune system. Regarding the latter, using pharmacological and genetic approaches scientists have shown this molecule to have both pro- and anti-inflammatory effects in mammalian systems. The anti-inflammatory effects of H2S appeared to be due to its inhibitory action on the nuclear factor kappa beta signalling pathway; NF-kB representing a transcription factor involved in the regulation pro-inflammatory mediators like nitric oxide, prostaglandins, and cytokines. In contrast, results from several animal model describe a more complicated picture and report on pro-inflammatory effects linked to exposure to this molecule; linked to dosage used and point of administration of this molecule. Overall, roles for H2S in several inflammatory diseases spanning arthritis, atherosclerosis, sepsis, and asthma have been described by researchers. In light this work fascinating research, this chapter will cover H2S biology and its many roles in the immune system.

Significance: In recent times, it has emerged that some dietary sulfur compounds can act on mammalian cell signaling systems via their propensity to release hydrogen sulfide (H2S). H2S plays important biochemical and physiological roles in the heart, gastrointestinal tract, brain, kidney, and immune systems of mammals. Reduced levels of H2S in cells and tissues correlate with a spectrum of pathophysiological conditions, including heart disease, diabetes, obesity, and altered immune function. Recent Advances: In the last decade, researchers have now begun to explore the mechanisms by which dietary-derived sulfur compounds, in addition to cysteine, can act as sources of H2S. This research has led to the identified several compounds, organic sulfides, isothiocyanates, and inorganic sulfur species including sulfate that can act as potential sources of H2S in mammalian cells and tissues. Critical Issues: We have summarised progress made in the identification of dietary factors that can impact on endogenous H2S levels in mammals. We also describe current research focused on how some sulfur molecules present in dietary plants, and associated chemical analogues, act as sources of H2S, and discuss the biological properties of these molecules as studied in a range of in vitro and in vivo systems. Future Directions: The identification of sulfur compounds in edible plants that can act as novel H2S releasing molecules is intriguing. Research in this area could inform future studies exploring the impact of diet on H2S levels in mammalian systems. Despite recent progress, additional work is needed to determine the mechanisms by which H2S is released from these molecules following ingestions of dietary plants in humans, whether the amounts of H2S produced is of physiological significance following the metabolism of these compounds in vivo, and if diet could be used to manipulated H2S levels in humans. Importantly, this will lead to a better understanding of the biological significance of H2S generated from dietary sources, and this information could be used in the development of plant breeding initiatives to increase the levels of H2S releasing sulfur compounds in crops, or inform dietary intervention strategies that could be used to alter the levels of H2S in humans.

Hydrogen sulfide (H2S) is recognized as an endogenous gaseous signaling molecule generated by cystathionine &gamma, lyase (CSE) in cardiovascular tissues. H2S up-regulation has been shown to reduce ischemic injury, and H2S donors are cardioprotective in rodent models when administered concurrent with myocardial ischemia. We evaluated the potential utility of H2S therapy in ameliorating cardiac remodeling with administration delayed until 2 h post-infarction in mice with or without cystathionine &gamma, lyase gene deletion (CSE&ndash, /&ndash, ). The slow-release H2S donor, GYY4137, was administered from 2 h after surgery and daily for 28 days following myocardial infarction (MI) induced by coronary artery ligation, comparing responses in CSE&ndash, with wild-type (WT) mice (n = 5&ndash, 10/group/genotype). Measures of cardiac function and expression of key genes associated with cardiac hypertrophy, fibrosis, and apoptosis were documented in atria, ventricle, and kidney tissues. Post-MI GYY4137 administration reduced infarct area and restored cardiac function, accompanied by reduction of the elevated ventricular expression of genes mediating cardiac remodeling to near-normal levels. Few differences between WT and CSE&ndash, mice were observed, except CSE&ndash, mice had higher blood pressures, and higher atrial Mir21a expression across all treatment groups. These findings suggest endogenous CSE gene deletion does not substantially exacerbate the long-term response to MI. Moreover, the H2S donor GYY4137 administered after onset of MI preserves cardiac function and protects against adverse cardiac remodeling in both WT and CSE-deficient mice.

Nitroxyl (HNO), one-electron reduced and protonated sibling of nitric oxide (NO), is a potential regulator of cardiovascular functions. It produces positive inotropic, lusitropic, myocardial anti-hypertrophic and vasodilator properties. Despite of these favorable actions, the significance and the possible mechanisms of HNO in diabetic hearts have yet to be fully elucidated. H9c2 cells or primary neonatal mouse cardiomyocytes were incubated with normal glucose (NG) or high glucose (HG). Male C57BL/6 mice received intraperitoneal injection of streptozotocin (STZ) to induce diabetes. Here, we demonstrated that the baseline fluorescence signals of HNO in H9c2 cells were reinforced by both HNO donor Angeli's salt (AS), and the mixture of hydrogen sulfide (H2S) donor sodium hydrogen sulfide (NaHS) and NO donor sodium nitroprusside (SNP), but decreased by HG. Pretreatment with AS significantly reduced HG-induced cell vitality injury, apoptosis, reactive oxygen species (ROS) generation, and hypertrophy in H9c2 cells. This effect was mediated by induction of caveolin-3 (Cav-3)/endothelial nitric oxide (NO) synthase (eNOS) complex. Disruption of Cav-3/eNOS by pharmacological manipulation or small interfering RNA (siRNA) abolished the protective effects of AS in HG-incubated H9c2 cells. In STZ-induced diabetic mice, administration of AS ameliorated the development of diabetic cardiomyopathy, as evidenced by improved cardiac function and reduced cardiac hypertrophy, apoptosis, oxidative stress and myocardial fibrosis without affecting hyperglycemia. This study shed light on how interaction of NO and H2S regulates cardiac pathology and provide new route to treat diabetic cardiomyopathy with HNO.

Aims: Cisplatin is a major therapeutic drug for solid tumors, but can cause severe nephrotoxicity. However, the role and therapeutic potential of hydrogen sulfide (H2S), an endogenous gaso...

Aims In patients with heart failure and a pathologically prolonged PR interval, left ventricular (LV) filling can be improved by shortening atrioventricular delay using His-bundle pacing. His-bundle pacing delivers physiological ventricular activation and has been shown to improve acute haemodynamic function in this group of patients. In the HOPE-HF (His Optimized Pacing Evaluated for Heart Failure) trial, we are investigating whether these acute haemodynamic improvements translate into improvements in exercise capacity and heart failure symptoms. Methods and results This multicentre, double-blind, randomized, crossover study aims to randomize 160 patients with PR prolongation (≥200 ms), LV impairment (EF ≤ 40%), and either narrow QRS (≤140 ms) or right bundle branch block. All patients receive a cardiac device with leads positioned in the right atrium and the His bundle. Eligible patients also receive a defibrillator lead. Those not eligible for implantable cardioverter defibrillator have a backup pacing lead positioned in an LV branch of the coronary sinus. Patients are allocated in random order to 6 months of (i) haemodynamically optimized dual chamber His-bundle pacing and (ii) backup pacing only, using the non-His ventricular lead. The primary endpoint is change in exercise capacity assessed by peak oxygen uptake. Secondary endpoints include change in ejection fraction, quality of life scores, B-type natriuretic peptide, daily patient activity levels, and safety and feasibility assessments of His-bundle pacing. Conclusions Hope-HF aims to determine whether correcting PR prolongation in patients with heart failure and narrow QRS or right bundle branch block using haemodynamically optimized dual chamber His-bundle pacing improves exercise capacity and symptoms. We aim to complete recruitment by the end of 2018 and report in 2020.

Garlic (Allium sativum) and allied plant species are rich sources of sulfur compounds. Major roles for garlic and its sulfur constituents include the regulation of vascular homeostasis and the control of metabolic systems linked to nutrient metabolism. Recent studies have indicated that some of these sulfur compounds, such as diallyl trisulfide (DATS), alter the levels of gaseous signalling molecules including nitric oxide (NO), hydrogen sulfide (H2S), and perhaps carbon monoxide (CO) in mammalian tissues. These gases are important in cellular processes associated with the cardiovascular system, inflammation, and neurological functions. Importantly, these studies build on the known biological effects of garlic and associated sulfur constituents. This review highlights our current understanding of the health benefits attributed to edible plants like garlic.

Cystathionine-γ-lyase (CSE), an enzyme associated with hydrogen sulfide (H(2)S) production, is an important endogenous regulator of inflammation. Jumonji domain-containing protein 3 (JMJD3) is implicated in the immune response and inflammation. Here, we investigated the potential contribution of JMJD3 to endogenous CSE-mediated inflammation in rheumatoid arthritis (RA). Upregulated CSE and JMJD3 were identified in synovial fibroblasts (SFs) from RA patients as well as in the joints of arthritic mice. Knocking down CSE augmented inflammation in IL-1β-induced SFs by increasing JMJD3 expression. In addition, CSE(−/−) mice with collagen-induced arthritis (CIA) developed severe joint inflammation and bone erosion. Conversely, overexpressing CSE inhibited JMJD3 expression by the transcription factor Sp-1 and was accompanied by reduced inflammation in IL-1β-treated SFs. Furthermore, JMJD3 silencing or the administration of the JMJD3 inhibitor GSK-J4 significantly decreased the inflammatory response in IL-1β-treated SFs, mainly by controlling the methylation status of H3K27me3 at the promoter of its target genes. GSK-J4 markedly attenuated the severity of arthritis in CIA mice. In conclusion, suppressing JMJD3 expression by the transcription factor Sp-1 is likely responsible for the ability of CSE to negatively modulate the inflammatory response and reduce the progression of RA.

Caloric restriction (CR) is a dietary regimen that aims to reduce the intake of total calories while maintaining adequate supply of key nutrients so as to avoid malnutrition. CR is one of only a small number of interventions that show promising outcomes on health span and lifespan across different species. There is growing interest in the development of compounds that might replicate CR-related benefits without actually restricting food intake. Hydrogen sulfide (H2S) is produced inside the bodies of many animals, including humans, by evolutionarily conserved H2S synthesizing enzymes. Endogenous H2S is increasingly recognized as an important gaseous signalling molecule involved in diverse cellular and molecular processes. However, the specific role of H2S in diverse biological processes remains to be elucidated and not all its biological effects are beneficial. Nonetheless, recent evidence suggests that the biological functions of H2S intersect with the network of evolutionarily conserved nutrient sensing and stress response pathways that govern organismal responses to CR. Induction of H2S synthesizing enzymes appears to be a conserved and essential feature of the CR response in evolutionarily distant organisms, including nematodes and mice. Here we review the evidence for a role of H2S in CR and lifespan modulation. H2S releasing drugs, capable of controlled delivery of exogenous H2S, are currently in clinical development. These findings suggest such H2S releasing drugs as a promising novel avenue for the development of CR mimetic compounds.

Significance: Hydrogen sulfide (H2S) is regarded as the third gasotransmitter along with nitric oxide and carbon monoxide. Extensive studies have demonstrated a variety of biological roles for H2S in neurophysiology, cardiovascular disease, endocrine regulation, and other physiological and pathological processes. Recent Advances: Novel H2S donors have proved useful in understanding the biological functions of H2S, with morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) being one of the most common pharmacological tools used. One advantage of GYY4137 over sulfide salts is its ability to release H2S in a slow and sustained manner akin to endogenous H2S production, rather than the delivery of H2S as a single concentrated burst. Critical Issues: Here, we summarize recent progress made in the characterization of the biological activities and pharmacological effects of GYY4137 in a range of in vitro and in vivo systems. Recent developments in the structural modification of GYY4137 to generate new compounds and their biological effects are also discussed. Future Directions: Slow-releasing H2S donor, GYY4137, and other phosphorothioate-based H2S donors are potent tools to study the biological functions of H2S. Despite recent progress, more work needs to be performed on these new compounds to unravel the mechanisms behind H2S release and pace of its discharge, as well as to define the effects of by-products of donors after H2S liberation. This will not only lead to better in-depth understanding of the biological effects of H2S but will also shed light on the future development of a new class of therapeutic agents with potential to treat a wide range of human diseases.

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly worldwide. Mitochondrial dysfunction has been proposed as a key event in the etiology of AD. We have previously modeled amyloid-beta (Aβ)-induced mitochondrial dysfunction in a transgenic Caenorhabditis elegans strain by expressing human Aβ peptide specifically in neurons (GRU102). Here, we focus on a deeper analysis of these metabolic changes associated with Aβ-induced mitochondrial dysfunction. Integrating metabolomics, transcriptomics, biochemical studies and computational modeling, we identify alterations in Tricarboxylic Acid (TCA) cycle metabolism following even low-level Aβ expression. In particular, GRU102 show reduced activity of a rate-limiting TCA cycle enzyme, alpha-ketoglutarate dehydrogenase. These defects are associated with elevation of protein carbonyl content specifically in mitochondria. Importantly, metabolic failure occurs before any significant increase in global protein aggregate is detectable. Treatment with an antidiabetes drug, Metformin, reverses Aβ-induced metabolic defects, reduces protein aggregation and normalizes the lifespan of GRU102. Our results point to metabolic dysfunction as an early and causative event in AD pathology and a promising target for intervention.

Significance: Hydrogen sulfide (H(2)S) has been recognized as the third gaseous transmitter alongside nitric oxide and carbon monoxide. In the past decade, numerous studies have demonstrated an active role of H(2)S in the context of cancer biology. Recent Advances: The three H(2)S-producing enzymes, namely cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3MST), have been found to be highly expressed in numerous types of cancer. Moreover, inhibition of CBS has shown anti-tumor activity, particularly in colon cancer, ovarian cancer, and breast cancer, whereas the consequence of CSE or 3MST inhibition remains largely unexplored in cancer cells. Intriguingly, H(2)S donation at high amounts or a long time duration has also been observed to induce cancer cell apoptosis in vitro and in vivo while sparing noncancerous fibroblast cells. Therefore, a bell-shaped model has been proposed to explain the role of H(2)S in cancer development. Specifically, endogenous H(2)S or a relatively low level of exogenous H(2)S may exhibit a pro-cancer effect, whereas exposure to H(2)S at a higher amount or for a long period may lead to cancer cell death. This indicates that inhibition of H(2)S biosynthesis and H(2)S supplementation serve as two distinct ways for cancer treatment. This paradoxical role of H(2)S has stimulated the enthusiasm for the development of novel CBS inhibitors, H(2)S donors, and H(2)S-releasing hybrids. Critical Issues: A clear relationship between H(2)S level and cancer progression remains lacking. The possibility that the altered levels of these byproducts have influenced the cell viability of cancer cells has not been excluded in previous studies when modulating H(2)S producing enzymes. Future Directions: The consequence of CSE or 3MST inhibition in cancer cells need to be examined in the future. Better portrayal of the crosstalk among these gaseous transmitters may not only lead to an in-depth understanding of cancer progression but also shed light on novel strategies for cancer therapy.

Introduction The European Society of Cardiology 2016 atrial fibrillation (AF) guidance suggests atrioventricular node (AVN) ablation and pacemaker implantation in patients with AF unresponsive to rate and rhythm control therapies.1 For patients with conventional cardiac resynchronisation therapy (CRT) indications and AF, the recommendations are clear. However, in patients with normal left ventricular (LV) function and a pace and ablate strategy, there is limited evidence to support biventricular pacing (CRT) in favour of conventional right-ventricular permanent pacemakers (PPM). We audited our practice of AVN ablation to evaluate the current practice in our institution, and to observe for differences between pacing groups. Methods Our devices database was searched for patients who underwent AV node ablation between June 2016 to September 2018. Descriptive and statistical analysis were performed, using t-test and Chi-squared methods. Results 142 patients underwent AV node ablation, and 56 patients were identified with preserved LV function (LVEF > 50%) with a pace and ablate management strategy. Of these patients, 35 had a PPM, and 21 a CRT implanted prior to ablation. Comparing these groups, patients undergoing pacemaker implantation were significantly older (PPM 76± 14 vs CRT 67± 11, p= 0.014). There was also a significant difference in male gender, (PPM 8 (22.9%) vs CRT 13 (61.9%), p= 0.0035). Mean LVEF was similar between PPM and CRT groups (56±4% vs. 53±3%). There was a trend in higher background ischaemic heart disease in the CRT group (CRT 3 (14.2%) vs. PPM 1 (2.9%), p=0.108). At initial device implantation, two patients intended for CRT had a failed attempt at LV lead placement (5.7%), therefore a PPM was implanted. Device procedure time appears longer in the CRT group (CRT 132.1 mins ± 40.9, vs. PPM 85.8 mins ± 34.8). Total mean follow-up period was 345 days (± 197). Two patients (5.7%) in the PPM group required lead revisions, compared to none in the CRT group. A total of two patients required repeat procedures for failed ablations, with no other procedural complications. There was a trend in higher all cause mortality in the PPM group compared with the CRT group (4 (11.4%) vs. 1 (4.8%), p= 0.397). Discussion and conclusion The BLOCK HF trial demonstrated that biventricular pacing was superior to right-ventricular pacing in patients with an LVEF Our study describes the variability in our clinical practice given the split in PPM vs CRT in patients with near normal LV function. The increased age and mortality trend in the PPM group may reflect an increased frailty or co-morbidity compared with the CRT group. However, long term clinical outcomes are required in order to provide clarity on this important clinical decision. Conflict of Interest None

Hydrogen sulfide (H2S) has profound biological effects within living organisms and is now increasingly being considered alongside other gaseous signalling molecules, such as nitric oxide (NO) and carbon monoxide (CO). Conventional use of pharmacological and molecular approaches has spawned a rapidly growing research field that has identified H2S as playing a functional role in cell-signalling and post-translational modifications. Recently, a number of laboratories have reported the use of siRNA methodologies and genetic mouse models to mimic the loss of function of genes involved in the biosynthesis and degradation of H2S within tissues. Studies utilising these systems are revealing new insights into the biology of H2S within the cardiovascular system, inflammatory disease, and in cell signalling. In light of this work, the current review will describe recent advances in H2S research made possible by the use of molecular approaches and genetic mouse models with perturbed capacities to generate or detoxify physiological levels of H2S gas within tissues.

Testis and sperm are particularly susceptible to inflammation and oxidative stress. Although hydrogen sulfide (HWe found that both subfertile and infertile patients, especially asthenospermic patients, exhibited decreased concentration of HThese results provide the first indication that HH

Oxidative stress is a key contributor to endothelial dysfunction and associated cardiovascular pathogenesis. Hydrogen sulfide (H2S) is an antioxidant gasotransmitter that protects endothelial cells against oxidative stress. Sirtuin3 (SIRT3), which belongs to the silent information regulator 2 (SIR2) family, is an important deacetylase under oxidative stress. H2S is able to regulate the activity of several sirtuins. The present study aims to investigate the role of SIRT3 in the antioxidant effect of H2S in endothelial cells. Results: Cultured EA.hy926 endothelial cells were exposed to hydrogen peroxide (H2O2) as a model of oxidative stress-induced cell injury. GYY4137, a slow-releasing H2S donor, improved cell viability, reduced oxidative stress and apoptosis, and improved mitochondrial function following H2O2 treatment. H2S reversed the stimulation of MAPK phosphorylation, downregulation of SIRT3 mRNA and reduction of the superoxide dismutase 2 and isocitrate dehydrogenase 2 expression which were induced by H2O2. H2S also increased activator protein 1 (AP-1) binding activity with SIRT3 promoter and this effect was absent in the presence of the specific AP-1 inhibitor, SR11302 or curcumin. Paraquat administration to mice induced a defected endothelium-dependent aortic vasodilatation and increased oxidative stress in both mouse aorta and small mesenteric artery, which were alleviated by GYY4137 treatment. This vasoprotective effect of H2S was absent in SIRT3 knockout mice. Innovation: The present results highlight a novel role for SIRT3 in the protective effect of H2S against oxidant damage in the endothelium both in vitro and in vivo. Conclusion: H2S enhances AP-1 binding activity with the SIRT3 promoter, thereby upregulating SIRT3 expression and ultimately reducing oxidant-provoked vascular endothelial dysfunction.

Hydrogen sulfide (H2S) is a gasotransmitter that regulates cardiovascular functions. The present study aimed to determine the protective effect of slow-releasing H2S donor GYY4137 on myocardial ischemia and reperfusion (I/R) injury and to investigate the possible signaling mechanisms involved. Male Sprague-Dawley rats were treated with GYY4137 at 12.5 mg/(kg·day), 25 mg/(kg·day) or 50 mg/(kg·day) intraperitoneally for 7 days. Then, rats were subjected to 30 minutes of left anterior descending coronary artery occlusion followed by reperfusion for 24 hours. We found that GYY4137 increased the cardiac ejection fraction and fractional shortening, reduced the ischemia area, alleviated histological injury and decreased plasma creatine kinase after myocardial I/R. Both H2S concentration in plasma and cystathionine-γ-lyase (CSE) activity in the myocardium were enhanced in the GYY4137 treated groups. GYY4137 also decreased malondialdehyde and myeloperoxidase levels in serum, attenuated superoxide anion level and suppressed phosphorylation of mitogen activated protein kinases in the myocardium after I/R. Meanwhile, GYY4137 increased the expression of Bcl-2 but decreased the expression of Bax, caspase-3 activity and apoptosis in the myocardium. The data suggest that GYY4137 protects against myocardial ischemia and reperfusion injury by attenuating oxidative stress and apoptosis.

Hydrogen sulfide (H2S) has recently emerged as an important biological gasomediator as a result of numerous insightful studies. The use of H2S releasing compounds has attracted much attention as they can exert crucial effects on a wide range of cellular signaling processes. Some of these effects are potentially exploitable in terms of anti-inflammatory and anti-tumor effects, as well as precise ion-channel regulation, cardiovascular protection and oxidation resistance. Unfortunately, the potential therapeutic effects of H2S are controversial due to conflicting published results regarding its effects on cellular activities arising, perhaps in part, from the use of different H2S donors. Therefore it is essential to review the most commonly used H2S releasing compounds, some of which are currently in clinical trials along with their associated in vitro and/or in vivo biological effects.

Background and Purpose Atherosclerosis is associated with reduced vascular hydrogen sulfide (H2S) biosynthesis. GYY4137 is a novel slow-releasing H2S compound that may effectively mimic the time course of H2S release in vivo. However, it is not known whether GYY4137 affects atherosclerosis. Experimental Approach RAW 264.7 cells and human blood monocyte-derived macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with/without GYY4137. ApoE−/− mice were fed a high-fat diet for 4 weeks and administered GYY4137 for 30 days. Lipid and atherosclerotic lesions were measured by oil red O staining. Endothelium-dependent relaxation was assessed in response to acetylcholine. Superoxide production was detected by dihydroethidium staining. Expression of mRNA and protein were evaluated by quantitative real-time PCR and Western blot. Key Results GYY4137 inhibited ox-LDL-induced foam cell formation and cholesterol esterification in cultured cells. GYY4137 decreased the expression of lectin-like ox-LDL receptor-1, iNOS, phosphorylated IκBα, NF-κB, ICAM-1, VCAM-1 and chemokines, including CXCL2, CXCR4, CXCL10 and CCL17, but increased the scavenger protein CD36, in ox-LDL-treated RAW 264.7 cells. In vivo, GYY4137 decreased aortic atherosclerotic plaque formation and partially restored aortic endothelium-dependent relaxation in apoE−/− mice. GYY4137 decreased ICAM-1, TNF-α and IL-6 mRNA expression as well as superoxide (O2−) generation in aorta. In addition, GYY4137 increased aortic eNOS phosphorylation and expression of PI3K, enhanced Akt Ser473 phosphorylation and down-regulated the expression of LOX-1. Conclusion and Implications GYY4137 inhibits lipid accumulation induced by ox-LDL in RAW 264.7 cells. In vivo, GYY4137 decreased vascular inflammation and oxidative stress, improved endothelial function and reduced atherosclerotic plaque formation in apoE−/− mice.

Hydrogen sulfide (H2S) has been shown to have powerful antioxidative and anti-inflammatory properties that can regulate multiple cardiovascular functions. However, its precise role in diabetes-accelerated atherosclerosis remains unclear. We report here that H2S reduced aortic atherosclerotic plaque formation with reduction in superoxide (O2 -/-) generation and the adhesion molecules in streptozotocin (STZ)-induced LDLr-/- mice but not in LDLr-/-Nrf2-/- mice. In vitro, H2S inhibited foam cell formation, decreased O2 - generation, and increased nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and consequently heme oxygenase 1 (HO-1) expression upregulation in high glucose (HG) plus oxidized LDL (ox-LDL)-treated primary peritoneal macrophages from wild-type but not Nrf2-/- mice. H2S also decreased O2 -/- and adhesion molecule levels and increased Nrf2 nuclear translocation and HO-1 expression, which were suppressed by Nrf2 knockdown in HG/ox-LDL-treated endothelial cells. H2S increased S-sulfhydration of Keap1, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and inhibited O2 -/- generation, which were abrogated after Keap1 mutated at Cys151, but not Cys273, in endothelial cells. Collectively, H2S attenuates diabetes-accelerated atherosclerosis, which may be related to inhibition of oxidative stress via Keap1 sulfhydrylation at Cys151 to activate Nrf2 signaling. This may provide a novel therapeutic target to prevent atherosclerosis in the context of diabetes.

Background Hydrogen sulfide (H 2 S) is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel‐like factor 5 (KLF5) exerts diverse functions in the cardiovascular system. Whether and how H 2 S regulates KLF5 in myocardial hypertrophy is unknown. Methods and Results In our study, hypertrophic myocardial samples in the clinic were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats and neonatal rat cardiomyocytes were studied for functional and signaling responses to GYY4137, an H 2 S‐releasing compound. Expression of cystathionine γ‐lyase, a principal enzyme for H 2 S generation in heart, decreased in human hypertrophic myocardium, whereas KLF5 expression increased. After GYY4137 administration for 4 weeks, myocardial hypertrophy was inhibited in spontaneously hypertensive rats, as demonstrated by improvement in cardiac structural parameters, heart mass, size of cardiac myocytes, and expression of atrial natriuretic peptide. H 2 S diminished expression of KLF5 in myocardium of spontaneously hypertensive rats and in hypertrophic cardiomyocytes. H 2 S also inhibits platelet‐derived growth factor A promoter activity, decreased recruitment of KLF5 to the platelet‐derived growth factor A promoter, and reduced atrial natriuretic peptide expression in angiotensin II–stimulated cardiomyocytes, and these effects are suppressed by KLF5 knockdown. KLF5 promoter activity and KLF5 expression was also reversed by H 2 S. H 2 S increased the S‐sulfhydration on specificity protein 1 in cardiomyocytes. Moreover, H 2 S decreased KLF5 promoter activity; reduced KLF5 mRNA expression; attenuated specificity protein 1 binding activity with KLF5 promoter; and inhibited hypertrophy after specificity protein 1 mutated at Cys659, Cys689, and Cys692 but not Cys664 overexpression. Conclusions These findings suggest that H 2 S regulates KLF5 transcription activity via specificity protein 1 S‐sulfhydration at Cys664 to prevent myocardial hypertrophy.

Hydrogen sulfide (H 2 S) has been investigated for its potential in therapy. Recently, we reported novel H 2 S donor molecules based on a thiophosphorus core, which slowly release H 2 S and have improved anti-proliferative activity in cancer cell lines compared to the most widely studied H 2 S donor GYY4137 ( 1 ). Herein, we have prepared new thiophosphorus organic H 2 S donors with different ring sizes and evaluated them in two solid tumor cell lines and one normal cell line. A seven membered ring compound, 17 , was found to be the most potent with sub-micromolar IC 50s in breast (0.76 μM) and ovarian (0.76 μM) cancer cell lines. No significant H 2 S release was detected in aqueous solution for this compound. However, confocal imaging showed that H 2 S was released from 17 inside cells at a similar level to the widely studied H 2 S donor GYY4137, which was shown to release 10 μM H 2 S after 12 h at a concentration of 400 μM. Comparison of 17 with its non-sulfur oxygen analogue, 26 , provided evidence that the sulfur atom is important for its potency. However, the significant potency observed for 26 (5.94–11.0 μM) indicates that the high potency of 17 is not entirely due to release of H 2 S. Additional mechanism(s) appear to be responsible for the observed activity, hence more detailed studies are required to better understand the role of H 2 S in cancer with potent thiophosphorus agents.

Hypometabolism may play an important role in the pathogenesis of ageing and ageing-related diseases. The nematode Caenorhabditis elegans offers the opportunity to study “living mitochondria” in a small (~1 mm) animal replete with a highly stereotypical, yet complex, anatomy and physiology. Basal oxygen consumption rate is often employed as a proxy for energy metabolism in this context. This parameter is traditionally measured using single-chamber Clark electrodes without the addition of metabolic modulators. Recently, multi-well oxygen electrodes, facilitating addition of metabolic modulators and hence study of respiratory control during different mitochondrial respiration states, have been developed. However, only limited official protocols exist for C. elegans, and key limitations of these techniques are therefore unclear. Following modification and testing of some of the existing protocols, we used these methods to explore mitochondrial bioenergetics in live nematodes of an electron transfer chain Complex II mutant strain, mev-1, and identified a previously undetected metabolic defect. We find that mev-1 mutants cannot respond adequately to increased energy demands, suggesting that oxidative phosphorylation is more severely impaired in these animals than has previously been appreciated.

Macrophages are of key importance for tissue repair after myocardial infarction (MI). Hydrogen sulfide (H2S) has been shown to exert cardioprotective effects in MI. However, the mechanisms by which H2S modulates cardiac remodeling and repair post-MI remain to be clarified.In our current study, we showed that H2S supplementation ameliorated pathological remodeling and dysfunction post-MI in wild-type (WT) and CSE KO mice, resulting in decreased infarct size and mortality, accompanied by an increase in the number of M2-polarized macrophages at the early stage of MI. Strikingly, adoptive transfer of NaHS-treated bone marrow-derived macrophages into WT and CSE KO mice with depleted macrophages also ameliorated MI-induced cardiac functional deterioration. Further mechanistic studies demonstrated that NaHS-induced M2 polarization was achieved by enhanced mitochondrial biogenesis and fatty acid oxidation.Our study shows (for the first time) that H2S may have the potential as a therapeutic agent for MI via promotion of M2 macrophage polarization. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Hideo Kimura, Chaoshu Tang, Xiaoli Tian, and Kenneth Olson. Antioxid. Redox Signal. 25, 268-281.

Exogenous hydrogen sulfide (H2S) is known to exert anti-inflammatory effects both in macrophages and in animal models. In this study, we first showed that NaHS caused a concentration dependent reduction in TNFα and IL-6 secretion in LPS-stimulated RAW264.7 macrophages in the absence of cell death. Thereafter, we screened a series of novel slow H2S donors for similar activity. One such compound, FW1256, concentration dependently decreased TNFα, IL-6, PGE2 and NO generation in LPS-stimulated RAW264.7 macrophages and BMDMs. FW1256 also significantly reduced IL-1β, COX-2 and iNOS mRNA and protein in LPS-stimulated RAW264.7 macrophages. Mechanistically, FW1256 decreased NFκB activation as evidenced by reduced cytosolic phospho-IκBα levels and reduced nuclear p65 levels in LPS-stimulated RAW264.7 macrophages treated with FW1256. Using a H2S fluorescent probe in FW1256-treated RAW264.7 macrophages, H2S release from FW1256 was apparent over a period of 24h in these cells. Moreover, the effect of FW1256 on TNFα and IL-6 by FW1256 in LPS-stimulated RAW264.7 macrophages was reversed by treatment with the H2S scavenger, vitamin B12a. FW1256 had no cytotoxic effect on LPS-stimulated RAW264.7 macrophages or BMDMs. In vivo, FW1256 administration also reduced IL-1β, TNFα, nitrate/nitrite and PGE2 levels in LPS-treated mice. We show here a novel slow H2S-releasing compound that exerts anti-inflammatory effects in macrophages and in vivo. FW1256 may be a useful tool to study the biological effects of exogenous H2S and could also have future therapeutic value in inflammatory conditions.

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8–24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.

Hyperhomocysteinemia (HHcy) is a metabolic disorder marked by an excess amount of the amino acid homocysteine (Hcy) in the blood stream. Hcy is a H(2)S precursor-formed from the metabolism of methionine. Elevated Hcy levels have been associated with higher blood pressure. However, the precise contribution of H(2)S to blood pressure in HHcy is not known. In the current study, we have examined a novel link between H(2)S, blood pressure and HHcy. Male Sprague-Dawley rats were injected with PAG, NaHS, L-NAME+PAG and saline. HHcy condition was induced by providing methionine (1 g/kg) in drinking water for 8 weeks. After 8 weeks, plasma Hcy and H(2)S were measured. The treated rats were anaesthetized with a mixture of ketamine hydrochloride and medetomidine. Blood pressures were measured by intra-carotid artery catheterization and to further investigate the immediate effect of NO and H(2)S, exogenous drugs namely NaHS, SNP, Ach and NA were administered. Plasma Hcy levels were higher in HHcy groups and this group exhibited hypertension. We observed high blood pressure at low levels of H(2)S and vice versa. Endogenous H(2)S in HHcy condition facilitated a mild decrease in MAP (Mean Arterial Pressure). Exogenous SNP (NO donor) showed a greater pressure decrease in HHcy group. The underlying mechanism is yet to be exploited. High levels of Hcy play an important role in the pathogenesis of hypertension. The results suggest that both endogenous and exogenous H(2)S may play a vital role in regulating blood pressure in HHcy.

The role of hydrogen sulfide (H(2)S) in inflammation is controversial, with both pro- and antiinflammatory effects documented. Many studies have used simple sulfide salts as the source of H(2)S, which give a rapid bolus of H(2)S in aqueous solutions and thus do not accurately reflect the enzymatic generation of H(2)S. We therefore compared the effects of sodium hydrosulfide and a novel slow-releasing H(2)S donor (GYY4137) on the release of pro- and antiinflammatory mediators in lipopolysaccharide (LPS)-treated murine RAW264.7 macrophages. For the first time, we show that GYY4137 significantly and concentration-dependently inhibits LPS-induced release of proinflammatory mediators such as IL-1beta, IL-6, TNF-alpha, nitric oxide (*NO), and PGE(2) but increased the synthesis of the antiinflammatory chemokine IL-10 through NF-kappaB/ATF-2/HSP-27-dependent pathways. In contrast, NaHS elicited a biphasic effect on proinflammatory mediators and, at high concentrations, increased the synthesis of IL-1beta, IL-6, NO, PGE(2) and TNF-alpha. This study clearly shows that the effects of H(2)S on the inflammatory process are complex and dependent not only on H(2)S concentration but also on the rate of H(2)S generation. This study may also explain some of the apparent discrepancies in the literature regarding the pro- versus antiinflammatory role of H(2)S.

In recent years, increased interest has been directed towards hydrogen sulfide (H2S) as the third gasotransmitter and its role in various diseases. Cystathionine-gamma-lyase (CSE) is one of the enzymes responsible for the endogenous production of H2S in mammals. With the aid of the crystal structures of human CSE and site-directed mutagenesis studies, we have identified several amino acid residues in CSE that are actively involved in the catalysis of H2S production. Contrary to reports suggesting that Tyr114 is required for substrate binding, our results reveal a significant increase in the production of H2S upon mutation of Tyr114 to phenylalanine. This is attributed to an increased rate of pyridoxal 5'-phosphate (PLP) regeneration due to weakened pi-stacking interactions between Phe114 and PLP. Thr189 is also identified as a crucial residue where hydrogen bonding to Asp187 keeps the latter in an optimal position for hydrogen bonding to the pyridoxal nitrogen of PLP. Furthermore, mutation of Glu339 to lysine, alanine or tyrosine reveals the importance of the hydrophobicity of the 339th amino acid in determining the specificity of the enzyme for the catalysis of alpha,gamma-elimination or alpha,beta-elimination reaction. Our study also shows that the rate of H2S production is increased with increasing exogenous PLP concentration, hence supporting our hypothesis that apo-CSE is formed during the catalysis of H2S production. Taken together, these findings suggest novel routes towards the design of activators or inhibitors that modulate the production of H2S; these modulators may also serve as lead compounds in the development of drugs or mechanistic probes in the study of various H2S-related diseases.

Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) together make up a family of biologically active gases (the so-called ‘gaseous triumvirate’) with an increasingly well defined range of physiological effects plus roles to play in a number of disease states. Over the years, most researchers have concentrated their attention on understanding the part played by a single gas in one or more body systems. It is becoming more clear that all three gases are synthesised naturally in the body, often by the same cells within the same organs, and that all three gases exert essentially similar biological effects albeit via different mechanisms. Within the cardiovascular system, for example, all are vasodilators, promote angiogenesis and vascular remodelling and are protective towards tissue damage in for example, ischaemia-reperfusion injury in the heart. Similarly, all exhibit complex effects in inflammation with both pro- and anti-inflammatory effects recognised. It seems likely that cell function is controlled not by the activity of single gases working in isolation but by the concerted activity of all three of these gases working together.

Introduction In our male animal models, hydrogen sulfide (H 2 S) displayed significant vasodilatory and smooth muscle relaxant effects suggestive of an endogenous physiological role in erectile process. Aim In this first exploratory study, we aimed to identify the existence and mechanism of H 2 S pathway in female sexual physiology. Methods Vaginal and clitoral cavernosal smooth muscle strips from New Zealand white rabbits (N = 12) were exposed to stable H 2 S donor, sodium hydrosulfide hydrate (NaHS.xH 2 O, 100 µM–1.6 mM), in isometric tension studies. The NaHS responses were repeated after incubations with (i) N ω -nitro-L-arginine (50 µM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM) or cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine (MDL 12,330A) (10 µM); and (ii) potassium chloride medium (high K + 60 mM/low K + 10 mM), tetraethylammonium (10 mM) or glibenclamide (100 µM). Relaxant effect of NaHS was also compared with those of nitroglycerine (0.18–78.2 µM) and sildenafil (0.084–25.3 µM). Additionally, samples (N = 16) were collected for estimations of plasma and tissue H 2 S and expression levels of cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) proteins. Main Outcome Measures In vitro evidences for H 2 S formation and its physiopharmacological effects. Results NaHS produced significant concentration-dependent relaxation of vaginal and cavernosal smooth muscles with inhibitions by combination of ODQ and MDL 12,330A (26.4%), N ω -nitro-L-arginine (22.2%), high K + (15.1%) or glibenclamide (10.1%). Based on molar potency, NaHS was 18.3 and 6.3 times weaker than nitroglycerine and sildenafil, respectively. Quantitative assays indicated that plasma H 2 S level was 16.5 ± 2.58 µM, and H 2 S was synthesized in the clitoral and vaginal smooth muscles (1.8 and 3.9 nmol/mg soluble protein compared with 26.5 nmol/mg in the liver: positive control). Similarly, western blotting identified the protein expression bands of CSE (44.5 kDa) and CBS (63 kDa) in these genital tissue samples. Conclusion These pilot studies clearly indicate the smooth muscle relaxant effect of H 2 S in female genital tract, mediating through cyclic adenosine 3′:5′-monophosphate, nitric oxide-cyclic guanosine monophosphate and K + ATP channels. Taken together with biochemical and molecular evidences for endogenous formation, H 2 S pathway could be a contributing factor in female sexual responses. Srilatha B, Lingxu H, Adaikan GP, and Moore PK. Initial characterization of hydrogen sulfide effects in female sexual function. J Sex Med 2009;6:1875–1884.

Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Dexamethasone (1 mg/kg, i.p.) administered either 1 hr before or 1 hr after E. coli lipopolysaccharide (LPS, 4 mg/kg, i.p.) in conscious rats inhibited the subsequent (4 hrs) rise in plasma cytokine (interleukin [IL]-1beta, tumour necrosis factor [TNF]-alpha), nitrate/nitrite (NO(x)), soluble intercellular adhesion molecule-1 (sICAM-1) concentration and lung/liver myeloperoxidase activity indicative of an anti-inflammatory effect. Dexamethasone also reduced the LPS-evoked rise in plasma hydrogen sulphide (H(2)S) concentration, liver H(2)S synthesizing activity and expression of cystathionine gamma lyase (CSE) and inducible nitric oxide synthase (iNOS). Mifepristone (RU-486) inhibited these effects. Dexamethasone (1-10 microM) reduced the LPS-evoked release of IL-1beta, TNF-alpha and L-selectin, decreased expression of CSE and iNOS and diminished nuclear factor kappaB (NF-kappaB)-DNA binding in isolated rat neutrophils. In contrast, NaHS (100 microM) increased L-selectin release from rat neutrophils. Dexamethasone also reduced LPS-induced up-regulation of CSE in foetal liver cells. 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ, 10 nM), a selective inhibitor of transcription via the NF-kappaB pathway, abolished LPS-induced up-regulation of CSE expression. We propose that inhibition of CSE expression and reduction in formation of the pro-inflammatory component of H(2)S activity contributes to the anti-inflammatory effect of dexamethasone in endotoxic shock. Whether H(2)S plays a part in the anti-inflammatory effect of this steroid in other forms of inflammation such as arthritis or asthma warrants further study.

Dear Editor: In recent years, it has become clear that hydrogen sulphide (H2S) plays a number of biological roles and may function as a novel gasotransmitter in the body alongside nitric oxide (NO) and carbon monoxide (CO) [1]. H2S is synthesized naturally from L-cysteine in mammalian tissues in a reaction catalysed by two enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) [2]. Recently, attention has been focused on the potential physiological and pathophysiological role of H2S in the body. It has been shown, for example, H2S plays a role in the regulation of vascular function both in health and disease [3–5]. Interestingly, H2S is also produced naturally at sites of inflammation and it is known that chronic inflammation is associated with uncontrolled cellular proliferation. However, in contrast to other gasotransmitter (e.g. NO) very little information exists on the mechanism by which H2S influences cell growth. In the present study, we have attempted to assess the biological effects of H2S in normal human lung fibroblast (MRC-5) cells. Recently, we have reported that H2S treatment increased cell death, formation of micronuclei (MN) and alteration in cell cycle [6]. Broadly, similar conclusions were also reported using single cell gel electrophoresis (SCGE) to show that sodium sulphide (Na2S: 250 μM/L) caused radical-associated DNA damage in the Chinese hamster ovary (CHO) cells [7]. Together, these data indicate that H2S is a potent clastogenic agent and suggest that it has a role in DNA damage-induced responses. In the present study, we have intended to understand the mechanism(s) involved in the genomic instability caused by H2S. The tumour suppressor protein p53 plays a key role in maintaining genomic integrity by controlling cell-cycle progression and cell survival [8]. Cells under stresses such as DNA damage, hypoxia and aberrant oncogene signals trigger the tumour suppressor protein p53, which transcribes genes that induce cell-cycle arrest, DNA repair and apoptosis [8, 9]. The mechanisms by which H2S up-regulates p53 and thereby induces DNA damage and alters cell-cycle progression remain unclear. In the present study, we report that the up-regulation of both the inducer protein p53 and the effector protein p21 in normal lung fibroblast cells several hours after 50 μM of NaHS (donor of H2S) treatment followed by the key proteins involved in cell cycle, i.e. Cyclin A, Cyclin E (a trend for CDC-6, p16 to increase) and decrease in Cyclin D, with a trend for p27 to decline (Fig.​(Fig.1A).1A). Interestingly, down-regulation of p27 and Cyclin D coincided with that of the H2S-induced growth arrest reported in our previous study, suggesting that these proteins may have a role in mediating the H2S induced cell cycle arrest. Furthermore, in the p53∼/_ MEF cells showed up-regulation of Cyclin D following the NaHS treatment (Fig.​(Fig.1B)1B) suggests that p53 is essential to maintain the genomic integrity of the NaHS treated cells. In addition, immunofluorescence studies were performed to better understanding the activation of p53 by H2S. Cells were stained with a specific antibody of p53. The results showed a higher expression of p53 in the nucleus of the H2S treated cells (Fig.​(Fig.1F1F). Figure 1 H2S-induced p53 is transcriptionally active. In the Western blot studies, whole cell or subcellular extracts were prepared at the indicated time-points and thereafter equal amount of proteins (25-50 μg) were separated by 4-20% SDS-PAGE, transferred ... Further, we determined whether the activation of p53 and p21 was attributed to the increase in protein stability. Cells were either treated or untreated with NaHS. Six hours after NaHS treatment, cells were treated with cycloheximide (CHX, 10 μg/ml) to inhibit the de novo protein synthesis. p53 and p21 steady state levels were monitored at various time-points after CHX addition (Fig.​(Fig.1C).1C). The rate at which p53 and p21 levels decreased under these conditions was measured as protein stability. In the untreated cells, p53 and p21 levels were decreased following CHX treatment. In contrast to H2S-treated cells, p53 level is elevated for up to 6 hrs following CHX treatment. However, CHX treatment blocks the p21 expression. In the present study, we investigated the subcellular expression of p53 followed by the H2S treatment. Cells treated earlier with NaHS and followed by CHX treatment were treated earlier with NaHS for 6 hrs were sampled at the indicated time-points and subjected to cytoplasmic (Fig.​(Fig.1D)1D) and nuclear (Fig.​(Fig.1E)1E) fractions as explained earlier [10, 11]. In the unstressed cells, cytoplasm is the exclusive site of p53 degradation, and therefore nuclear export of p53 is prerequisite for its delivery to cytoplasmic proteasomes. In the present study, steady state decrease in p53 level was only evident in nuclear fraction, indicating that NaHS treatment accumulates p53 in the nucleus. In conclusion, we demonstrate for the first time that the H2S-induced increase in p53 level is due to an increase in p53 protein stability. These results are of significance as they uncover a hitherto unknown and a potentially fundamental role for H2S in determining the cell fate and may provide for development of a novel therapeutic approach to the regulation of cell growth.

Background— The potential biological significance of hydrogen sulfide (H 2 S) has attracted growing interest in recent years. The aim of this study was to characterize a novel, water-soluble, slow-releasing H 2 S compound [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate (GYY4137)] and evaluate its use as a tool to investigate the cardiovascular biology of this gas. Methods and Results— The acute vasorelaxant effect of drugs was assessed in rat aortic rings and perfused rat kidney in vitro and in the anesthetized rat in vivo. The chronic effect of GYY4137 on blood pressure in normotensive and spontaneously hypertensive rats was determined by tail-cuff plethysmography. GYY4137 released H 2 S slowly both in aqueous solution in vitro and after intravenous or intraperitoneal administration in anesthetized rats in vivo. GYY4137 caused a slow relaxation of rat aortic rings and dilated the perfused rat renal vasculature by opening vascular smooth muscle K ATP channels. GYY4137 did not affect rat heart rate or force of contraction in vitro. GYY4137 exhibited antihypertensive activity as evidenced by ability to reduce N G -nitro- l -arginine methyl ester–evoked hypertension in the anesthetized rat and after chronic (14-day) administration in spontaneously hypertensive rats. Conclusions— These results identify GYY4137 as a slow-releasing H 2 S compound with vasodilator and antihypertensive activity. GYY4137 is likely to prove useful in the study of the many and varied biological effects of H 2 S. GYY4137 may also prove of therapeutic value in cardiovascular disease.

Objectives The present study investigated whether chemokines are involved in hydrogen sulfide (H2S)-associated pathogenesis of acute pancreatitis and associated lung injury. Methods We have examined the effect of DL-propargylglycine, a cystathionine gamma-lyase inhibitor, on the synthesis of CC chemokine monocyte chemotactic protein 1, Regulated upon Activation, Normal T-cell Expressed, and Secreted, and macrophage inflammatory protein-1alpha (MIP-1alpha), and CXC chemokine MIP-2 in an in vitro and in vivo model of cerulein-induced acute pancreatitis and associated lung injury. In addition, the pancreatic acinar cells were treated with H2S donor drug, sodium hydrosulfide. The expression of these chemokines in the pancreatic acini, pancreas, and lungs was determined by quantitative real-time reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Results After treatment with DL-propargylglycine, reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay demonstrated down-regulation of cerulein-induced increase in monocyte chemotactic protein 1, MIP-1alpha, and MIP-2 expression but had no apparent effect on Regulated upon Activation, Normal T-cell Expressed, and Secreted expression. Conclusions These results suggest that the proinflammatory effect of H2S may be mediated by chemokines.

Hydrogen sulfide (H2S) is a naturally occurring gas that may act as an endogenous signaling molecule. In the brain, H2S is mainly produced by cystathionine beta-synthase (CBS) and its cellular effects have been attributed to interactions with N-methyl-D-aspartate (NMDA) receptors and cyclic adenosine 3',5'-monophosphate (cAMP). In contrast, direct vasodilator actions of H2S are most probably mediated by opening smooth muscle ATP-sensitive K+ (K(ATP)) channels. In the hypothalamus, K(ATP) channel-dependent mechanisms are involved in CNS-mediated regulation of blood pressure. In this report, we investigated the hypothesis that H2S may act via K(ATP) channels in the hypothalamus to regulate blood pressure. Mean arterial blood pressure (MAP) and heart rate were monitored in freely moving rats via a pressure transducer placed in the femoral artery. Drugs were infused via a cannula placed in the posterior hypothalamus. Infusion of 200 microM sodium hydrogen sulfide (NaHS), an H2S donor, into the hypothalamus of freely moving rats reduced MAP and heart rate. Infusion of 300 nM to 3 microM gliclazide dose-dependently blocked the effect of 200 microM NaHS. Infusion of the CBS activator, s-adenosyl-L-methionine (0.1 mM and 1 mM), likewise decreased MAP. Infusion of the CBS inhibitors aminooxyacetic acid (10 mM) and hydroxylamine (20 mM) increased MAP but did not block the effects of infusion of 200 microM NaHS. These data indicate that actions of H2S in the hypothalamus decrease blood pressure and heart rate in freely moving rats. This effect appears to be mediated by a K(ATP) channel-dependent mechanism and mimicked by endogenous H2S.