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Hydrogen Sulfide: A New Tool to Design and Develop Drugs
Abstract
Abstract:
Hydrogen sulfide (H2S) is a gaseous molecule that is produced by the body. Elevated levels of H2S can result in a number of physiological responses. The specific mechanisms in which H2S directly facilitates these responses have not yet been well characterized, however, it has recently been established that sulfhydration of proteins is integral to the effects of H2S. A growing field of research is the development of new drugs which suppress endogenous hydrogen sulfide synthesis, as well as novel H2S donors. Evidence suggests that modulation of the H2S signaling system may be an excellent therapeutic approach for a range of clinical conditions including heart failure, inflammatory diseases, hypertension, acute myocardial infarction, gastrointestinal diseases and cancer. In this review, we describe an overview of the physiological and pathophysiological roles of H2S. We further discuss the current research of H2S modulating drugs and suggest potential approaches for the continued development of novel drugs.
Hydrogen sulfide (H2 S), together with nitric oxide (NO) and carbon monoxide (CO), belongs to the gasotransmitter family and plays important roles in mammals as a signaling molecule. Many studies have also shown the various therapeutic effects of H2 S, which include protection against myocardial ischemia injury, cytoprotection against oxidative stress, mediation of neurotransmission, inhibition of insulin signaling, regulation of inflammation, inhibition of the hypoxia-inducible pathway, and dilation of blood vessels. One major challenge in the development of H2 S-based therapeutics is its delivery. In this manuscript, we assess the various drug delivery strategies in the context of being used research tools and eventual developability as therapeutic agents.
Hydrogen sulfide is synthesized endogenously in mammals and has been shown to have both physiological and pathological functions. So far there has been little agreement as to the actual levels of endogenous sulfide under physiological or pathological conditions; this is partly due to the complexity involved in measuring free sulfides due to H2S volatility, oxidation, reactivity and the presence of bound labile sulfur in tissues. In this report we describe a method of measuring free tissue sulfides using a zinc sulfide precipitation and wash method. It is an indirect method that measures the sulfide difference between samples prepared at pH 9 and pH 6, assuming that at pH 9 free sulfides would be retained in solution, while at pH 6 free sulfides would volatilize during sample preparation. Using this approach we were able to measure appreciable amounts of free sulfides in mouse: lung, pancreas, liver and kidney at 0.036 + 0.006, 0.082 + 0.009, 0.215 + 0.016 and 0.323 + 0.031 nmole per mg of tissue respectively (n = 6).
Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.
A series of O-aryl- and alkyl-substituted phosphorodithioates were designed and synthesized as hydrogen sulfide (H2S) donors. H2S releasing capability of these compounds was evaluated using fluorescence methods. O-aryl substituted donors showed slow and sustained H2S release while O-alkylated compounds showed very weak H2S releasing capability. We also evaluated donors' protective effects against hydrogen peroxide (H2O2)-induced oxidative damage in myocytes and donors' toxicity toward B16BL6 mouse melanoma cells.
The physiological functions of hydrogen sulfide (H2S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H2S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H2S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H2S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H2S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H2S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H2S as a tumor growth factor and anticancer drug target.
Background
Hydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role and mechanism for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study is to investigate distribution of endogenous H2S synthesizing enzyme cystathionine-β-synthetase (CBS) expression and role of H2S on excitability and voltage-gated potassium channels of TG neurons.
Methods
Immunofluorescence studies were carried out to determine whether CBS was co-expressed in Kv1.1 or Kv1.4-positive TG neurons. Whole cell patch clamp recordings were employed on acutely isolated TG neurons from adult male Sprague Dawley rats (6–8 week old). von Frey filaments were used to examine the pain behavioral responses in rats following injection of sodium hydrosulfide.
Results
In rat TG, 77.3±6.6% neurons were immunoreactive for CBS, 85.1±3.8% for Kv1.1 and 97.8±1.1% for Kv1.4. Double staining showed that all CBS labeled cells were Kv1.1 and Kv1.4 positive, but only 92.2±6.1% of Kv1.1 and 78.2±9.9% of Kv1.4 positive cells contained CBS. Application of H2S donor NaHS (250 μM) led to a significant depolarization of resting membrane potential recorded from TG neurons. NaHS application also resulted in a dramatic reduction in rheobase, hyperpolarization of action potential threshold, and a significant increase in the number of action potentials evoked at 2X and 3X rheobase stimulation. Under voltage-clamp conditions, TG neurons exhibited transient A-type (IA) and sustained outward rectifier K+ currents (IK). Application of NaHS did suppress IK density while did not change IA density of TG neurons (n=6). Furthermore, NaHS, a donor of hydrogen sulfide, produced a significant reduction in escape threshold in a dose dependent manner.
Conclusion
These data suggest that endogenous H2S generating enzyme CBS was co-localized well with Kv1.1 and Kv1.4 in TG neurons and that H2S produces the mechanic pain and increases neuronal excitability, which might be largely mediated by suppressing IK density, thus identifying for the first time a specific molecular mechanism underlying pain and sensitization in TG.
Aims:
H2S, a third member of gasotransmitter family along with nitric oxide and carbon monoxide, exerts a wide range of cellular and molecular actions in our body. Cystathionine gamma-lyase (CSE) is a major H2S-generating enzyme in our body. Aging at the cellular level, known as cellular senescence, can result from increases in oxidative stress. The aim of this study was to investigate how H2S attenuates oxidative stress and delays cellular senescence.
Results:
Here we showed that mouse embryonic fibroblasts isolated from CSE knockout mice (CSE KO-MEFs) display increased oxidative stress and accelerated cellular senescence in comparison with MEFs from wild-type mice (WT-MEFs). The protein expression of p53 and p21 was significantly increased in KO-MEFs, and knockdown of p53 or p21 reversed CSE deficiency-induced senescence. Incubation of the cells with NaHS (a H2S donor) significantly increased the glutathione (GSH) level and rescued KO-MEFs from senescence. Nrf2 is a master regulator of the antioxidant response, and Keap1 acts as a negative regulator of Nrf2. NaHS S-sulfhydrated Keap1 at cysteine-151, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream genes, such as glutamate-cysteine ligase and GSH reductase.
Innovation:
These results provide a mechanistic insight into how H2S signaling mediates cellular senescence induced by oxidative stress.
Conclusion:
H2S protects against cellular aging via S-sulfhydration of Keap1 and Nrf2 activation in association with oxidative stress.
Aims:
The signaling molecule hydrogen sulfide (H2S) protects cells against oxidative stress and activates NF-E2 p45-related factor 2 (Nrf2), a transcription factor that regulates antioxidant genes. We sought to establish whether H2S requires Nrf2 to protect against oxidative stress, and whether activation of Nrf2 by H2S involves antagonism of Kelch-like ECH-associated protein-1 (Keap1), a redox-sensitive ubiquitin ligase substrate adaptor that represses Nrf2 under normal homeostatic conditions.
Results:
H2S stabilizes Nrf2 protein and induces Nrf2-target genes via an antioxidant-/electrophile-response element. In mouse embryonic fibroblasts, the ability of H2S to protect against cell death caused by the redox-cycling agent menadione is dependent on Nrf2. Moreover, Nrf2 regulates murine genes involved in the production of H2S (Cystathionine-beta-synthase [Cbs] and Cystathionine-gamma-lyase [Cse]) and the degradation of H2S (Sulfide:quinone reductase-like [yeast] [Sqrdl]). We found that H2S stabilizes Nrf2 through inhibition of Keap1, an event that requires covalent modification of amino acids C226 and C613 in the substrate adaptor. Upregulation of Nrf2 by H2S partially involves the production of H2O2, which inhibits Keap1 by stimulating the formation of an intramolecular disulfide bond between C226 and C613. The Keap1 C226 and C613 residues are also S-sulfhydrated by H2S, and this may entail reduction of the C226-C613 disulfide bridge formed by H2O2.
Innovation:
Upregulation of Nrf2 by H2S and H2O2 involves inactivation of Keap1 through modification of C226 and C613.
Conclusion:
Inhibition of Keap1 by H2S leads to Nrf2-mediated induction of cytoprotective genes. Nrf2 controls Cbs, Cse, and Sqrdl, suggesting that a feedback loop exists between Nrf2 and H2S.
In this study, we have examined the effects of chronic L-3,4-dihydroxyphenylalanine (L-DOPA) administration on the remaining dopaminergic neurons in rats with 6-hydroxydopamine (6-OHDA) or buffered FeCl3 partial lesions to the nigrostriatal tract. L-DOPA administration increased the turnover of dopamine in the striatum. L-DOPA administration for 1 week produced an increase in the level of striatal RTI-121 binding, a specific marker for dopamine uptake sites on the dopaminergic nerve terminals in the striatum. However, longer periods of L-DOPA treatment decreased the level of RTI-121 binding in the striatum. In the partial 6-OHDA lesion model, L-DOPA treatment had a time-dependent effect on the number of neurons demonstrating a dopaminergic phenotype i.e., neurons that are tyrosine hyrdoxylase (TH)-immunopositive, on the lesioned side of the brain. In the first few weeks of treatment, L-DOPA decreased the number of TH-positive neurons but with long-term treatment, i.e., 24 weeks, L-DOPA increased the number of neurons demonstrating a dopaminergic phenotype. Even in the buffered FeCl3 infusion model, where the levels of iron were increased, L-DOPA treatment did not have any detrimental effects on the number of TH-positive neurons on the lesioned side of the brain. Consequently, chronic L-DOPA treatment does not have any detrimental effects to the remaining dopaminergic neurons in rats with partial lesions to the nigrostriatal tract; indeed in the 6-OHDA lesion model, long-term L-DOPA may increase the number of neurons, demonstrating a dopaminergic phenotype. © 2001 Movement Disorder Society.
Introduction:
The view of hydrogen sulfide has changed from a toxic by-product to a crucial signaling molecule, with enormous potential as a pharmacological target for diseases ranging from heart disease to sepsis. Despite this progression of ideas, there is still a large amount that is unknown about this gaseous signaling molecule. Hydrogen sulfide has been implicated as a tissue protectant in many pathological conditions, the mechanisms of tissue protection is a point of controversy, particularly distinguishing the direct actions from the indirect downstream effects of hydrogen sulfide. This point of controversy is particularly pertinent in inflammation research.
Areas covered:
Current research into the pathways in which hydrogen sulfide can act as a pro-inflammatory molecule and as an anti-inflammatory molecule.
Expert opinion:
How controversies regarding hydrogen sulfide may have occurred is discussed. Addressed are the direct and indirect pathways of hydrogen sulfide on inflammation, the effects of different concentrations of hydrogen sulfide and how the effects of hydrogen sulfide on the immune system vary with different delivery mechanisms. Furthermore, there is a discussion on what key gaps exist in current knowledge and must be addressed before hydrogen sulfide can be considered a valid pharmacological target.
Hydrogen sulfide (H(2)S), a novel gaseous messenger, is synthesized endogenously from L-cysteine by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). S-propargyl-cysteine (SPRC) is a slow H(2)S releasing drug that provides cysteine, a substrate of CSE. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 µg/kg) for 10 hours. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered either 12 h before or 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H(2)S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC given 3 h prior to the induction of AP. Furthermore, the beneficial effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. SPRC administered 12 h before AP induction did not cause significant improvement in pancreatic and lung inflammation. Plasma H(2)S concentration showed significant difference in H(2)S levels between control, vehicle and SPRC (administered 3 h before AP) treatment groups. In conclusion, these data provide evidence for protective effects of SPRC in AP possibly by virtue of its slow release of endogenous H(2)S.
Hydrogen sulfide (H(2)S) displays vasodilative, anti-oxidative, anti-inflammatory and cytoprotective activities. Impaired production of H(2)S contributes to the increased intrahepatic resistance in cirrhotic livers. The study aimed to investigate the roles of H(2)S in carbon tetrachloride (CCl(4))-induced hepatotoxicity, cirrhosis and portal hypertension.
Sodium hydrosulfide (NaHS), a donor of H(2)S, and DL-propargylglycine (PAG), an irreversible inhibitor of cystathionine γ-lyase (CSE), were applied to the rats to investigate the effects of H(2)S on CCl(4)-induced acute hepatotoxicity, cirrhosis and portal hypertension by measuring serum levels of H(2)S, hepatic H(2)S producing activity and CSE expression, liver function, activity of cytochrome P450 (CYP) 2E1, oxidative and inflammatory parameters, liver fibrosis and portal pressure. CCl(4) significantly reduced serum levels of H(2)S, hepatic H(2)S production and CSE expression. NaHS attenuated CCl(4)-induced acute hepatotoxicity by supplementing exogenous H(2)S, which displayed anti-oxidative activities and inhibited the CYP2E1 activity. NaHS protected liver function, attenuated liver fibrosis, inhibited inflammation, and reduced the portal pressure, evidenced by the alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), albumin, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and soluble intercellular adhesion molecule (ICAM)-1, liver histology, hepatic hydroxyproline content and α-smooth muscle actin (SMA) expression. PAG showed opposing effects to NaHS on most of the above parameters.
Exogenous H(2)S attenuates CCl(4)-induced hepatotoxicity, liver cirrhosis and portal hypertension by its multiple functions including anti-oxidation, anti-inflammation, cytoprotection and anti-fibrosis, indicating that targeting H(2)S may present a promising approach, particularly for its prophylactic effects, against liver cirrhosis and portal hypertension.
The reaction of hydrogen sulfide (H2S) with peroxynitrite (a key mediator in numerous pathological states) was studied in vitro and in different cellular models. The results show that H2S can scavenge peroxynitrite with a corresponding second order rate constant of 3.3 ± 0.4 × 10³ M⁻¹·s⁻¹ at 23°C (8 ± 2 × 10³ M⁻¹·s⁻¹ at 37°C). Activation parameters for the reaction (ΔH‡, ΔS‡ and ΔV‡) revealed that the mechanism is rather associative than multi-step free-radical as expected for other thiols. This is in agreement with a primary formation of a new reaction product characterized by spectral and computational studies as HSNO₂ (thionitrate), predominantly present as sulfinyl nitrite, HS(O)NO. This is the first time a thionitrate has been shown to be generated under biologically relevant conditions. The potential of HS(O)NO to serve as a NO donor in a pH-dependent manner and its ability to release NO inside the cells has been demonstrated. Thus sulfide modulates the chemistry and biological effects of peroxynitrite by its scavenging and formation of a new chemical entity (HSNO₂) with the potential to release NO, suppressing the pro-apoptotic, oxidative and nitrative properties of peroxynitrite. Physiological concentrations of H₂S abrogated peroxynitrite-induced cell damage as demonstrated by the: (i) inhibition of apoptosis and necrosis caused by peroxynitrite; (ii) prevention of protein nitration; and (iii) inhibition of PARP-1 [poly(ADP-ribose) polymerase 1] activation in cellular models, implying that a major part of the cytoprotective effects of hydrogen sulfide may be mediated by modulation of peroxynitrite chemistry, in particular under inflammatory conditions.
Cisplatin (CP)-induced renal damage is associated with inflammation. Hydrogen sulphide (H2S) is involved in models of inflammation. This study evaluates the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H2S formation, on the renal damage induced by CP.
The rats were injected with CP (5 mg/kg, i.p.) or PAG (5 mg/kg twice a day, i.p.) for 4 days, starting 1 h before CP injection. Control rats were injected with 0.15 M NaCl or PAG only. Blood and urine samples were collected 5 days after saline or CP injections for renal function evaluation. The kidneys were removed for tumour necrosis factor (TNF)-α quantification, histological, immunohistochemical and Western blot analysis. The cystathionine γ-lyase (CSE) activity and expression were assessed. The direct toxicity of H(2)S in renal tubular cells was evaluated by the incubation of these cells with NaHS, a donor of H2S.
CP-treated rats presented increases in plasma creatinine levels and in sodium and potassium fractional excretions associated with tubulointerstitial lesions in the outer medulla. Increased expression of TNF-α, macrophages, neutrophils and T lymphocytes, associated with increased H2S formation rate and CSE expression, were also observed in the outer medulla from CP-injected rats. All these alterations were reduced by treatment with PAG. A direct toxicity of NaHS for renal tubular epithelial cells was not observed.
Treatment with PAG reduces the renal damage induced by CP. This effect seems to be related to the H2S formation and the restriction of the inflammation in the kidneys from PAG + CP-treated rats.
S) is naturally synthesized in many types of mammalian cells from L-cysteine in the reactions catalyzed by cystathionine-β-synthase and cystathionine-γ-lyase (CSE). H 2 S has been demonstrated to play a proinflammatory role in various animal models of hindpaw edema, acute pancreatitis, lipopolysaccharide-induced endotoxemia and cecal ligation, and puncture–induced sepsis. Full-thickness burns that exceed 25 % of the total body surface area (TBSA) produce a profound systemic inflammatory reaction characterized by leukocyte activation and plasma leakage in the microvasculature of tissues and organs remote from the wound. The aim of this study was to investigate the effect of local burn injury on induced distant organ endogenous H 2 S release and expression of CSE. Male BALB/c mice were subjected to 30 % TBSA full-thickness burn and treated with saline (administered intraperitoneally [i.p.]); DL-propargylglycine (PAG, 50 mg/kg i.p.), which is a CSE inhibitor; or sodium hydrosulfide (NaHS, 10 mg/kg i.p.), which is an H 2 S donor. PAG was administered either 1 h before or 1 h after the burn injury, whereas NaHS was given at the same time as the burn injury. Measurements of liver myeloperoxidase (MPO) activities, liver
The main lesion in Parkinson disease (PD) is loss of substantia nigra dopaminergic neurons. Levodopa (l-DOPA) is the most widely used therapy, but it does not arrest disease progression. Some possible contributing factors to
the continuing neuronal loss are oxidative stress, including oxidation of l-DOPA, and neurotoxins generated by locally activated microglia and astrocytes. A possible method of reducing these factors
is to produce l-DOPA hybrid compounds that have antioxidant and antiinflammatory properties. Here we demonstrate the properties of four such
l-DOPA hybrids based on coupling l-DOPA to four different hydrogen sulfide-donating compounds. The donors themselves were shown to be capable of conversion
by isolated mitochondria to H2S or equivalent SH− ions. This capability was confirmed by in vivo results, showing a large increase in intracerebral dopamine and glutathione after iv administration in rats. When human microglia,
astrocytes, and SH-SY5Y neuroblastoma cells were treated with these donating agents, they all accumulated H2S intracellularly as did their derivatives coupled to l-DOPA. The donating agents and the l-DOPA hybrids reduced the release of tumor necrosis factor-α, interleukin-6, and nitric oxide from stimulated microglia, astrocytes
as well as the THP-1 and U373 cell lines. They also demonstrated a neuroprotective effect by reducing the toxicity of supernatants
from these stimulated cells to SH-SY5Y cells. l-DOPA itself was without effect in any of these assays. The H2S-releasing l-DOPA hybrid molecules also inhibited MAO B activity. They may be useful for the treatment of PD because of their significant
antiinflammatory, antioxidant, and neuroprotective properties.
Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation.
To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice.
Sepsis was induced by cecal ligation and puncture (CLP). Measurements and Main Results: The pretreatments of mice with H(2)S donors (NaHS or Lawesson's reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80%. Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed. Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and l-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis. Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to approximately 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker).
These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.
Hydrogen sulfide (H(2)S), a gaseous mediator plays an important role in a wide range of physiological and pathological processes. H(2)S has been extensively studied for its various roles in cardiovascular and neurological disorders. However, the role of H(2)S in inflammation is still controversial. The current study was aimed to investigate the therapeutic potential of sodium hydrosulfide (NaHS), an H(2)S donor in in vivo model of acute pancreatitis in mice.
Acute pancreatitis was induced in mice by hourly caerulein injections (50 mug/kg) for 10 hours. Mice were treated with different dosages of NaHS (5 mg/kg, 10 mg/kg or 15 mg/kg) or with vehicle, distilled water (DW). NaHS or DW was administered 1 h before induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and were processed to measure the plasma amylase, myeloperoxidase (MPO) activities in pancreas and lung and chemokines and adhesion molecules in pancreas and lung.
It was revealed that significant reduction of inflammation, both in pancreas and lung was associated with NaHS 10 mg/kg. Further the anti-inflammatory effects of NaHS 10 mg/kg were associated with reduction of pancreatic and pulmonary inflammatory chemokines and adhesion molecules. NaHS 5 mg/kg did not cause significant improvement on inflammation in pancreas and associated lung injury and NaHS 15 mg/kg did not further enhance the beneficial effects seen with NaHS 10 mg/kg.
In conclusion, these data provide evidence for anti-inflammatory effects of H(2)S based on its dosage used.
Hydrogen sulfide (H2S), a messenger molecule generated by cystathionine gamma-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H2S signals have been elusive. We now show that H2S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.
Edema is a significant post‐surgery complication in transplanted lungs and may be attributed in part to ischemia‐reperfusion injury. Endogenous hydrogen sulfide (H 2 S) may mediate beneficial effects in the heart, but effects in the lung are unknown. In the present study we investigated the role of H 2 S on ischemia‐reperfusion induced edema formation in rat lungs exposed to 90 min warm ischemia followed by 30 min of ventilation and perfusion. Lungs were perfused in situ using a peristaltic pump at constant flow to produce physiological (19–24 mmHg) pressure waves at 37°C. Ventilation was initiated at 3 cm H 2 O positive end‐expiratory pressure (PEEP), and lungs were inflated to 10 cm H 2 O PEEP and not ventilated during warm ischemia. After a final 30 min of ventilation and reperfusion, isolated right lobes were blotted dry, weighed, and dried in an oven for 24 hr at 80°C. Exogenous hydrogen sulfide (Na 2 S, 200 μM) and inhibition of the H 2 S producing enzyme cystathionine β‐synthase with amino oxyacetic acid did not affect edema formation, whereas inhibition of the H 2 S producing enzyme cystathionine γ‐lyase (CSE) with propargylglycine and inhibition of pyridoxyl‐5‐phosphate with hydroxylamine significantly reduced edema formation. These findings suggest that H 2 S may facilitate post‐ischemic edema formation in rat lungs via the CSE pathway. Supported by the MedCen Foundation of the Medical Center of Central Georgia.
The actin cytoskeleton network forms a key link between T-cell antigen receptor (TCR) stimulation and T-cell effector functions, providing a structural basis for T-cell morphological changes and signal transduction. Accumulating evidence positions the Wiskott-Aldrich syndrome protein (WASp), a scaffolding protein that promotes actin polymerization, at the center of actin cytoskeleton-dependent T-cell function. During the past decade, we and others have utilized multidisciplinary technologies, including live-cell imaging, biochemical, and biophysical analyses, to gain insight into the mechanisms by which WASp and other cytoskeletal proteins control actin homeostasis. Following TCR engagement, WASp is rapidly activated and recruited to TCR microclusters, as part of multiprotein complexes, where it promotes actin remodeling. Late in the activation process, WASp is internalized and eventually degraded. In this review, we describe the dynamic interactions of WASp with signaling proteins, which regulate its activation and recruitment to the TCR and to actin-rich sites. Finally, we present the molecular mechanism of WASp downregulation. Some of the signaling proteins that mediate WASp activation eventually lead to its degradation. Thus, we focus here on the regulation of WASp expression and function and the mechanisms whereby they control actin machinery and T-cell effector functions.
Aims:
The best-established mechanism of opioid dependence is the up-regulation of adenylate cyclase (AC)/cAMP pathway, which was reported to be negatively regulated by hydrogen sulfide (H2S), a novel endogenous neuromodulator. The present study was, therefore, designed to determine whether H2S is able to attenuate the development of opioid dependence via down-regulating AC/cAMP pathway.
Results:
We demonstrated that application of sodium hydrosulphide (NaHS) and GYY4137, two donors of H2S, significantly alleviated naloxone-induced robust withdrawal jumping (the most sensitive and reliable index of opioid physical dependence) in morphine-treated mice. Repeated treatment with NaHS inhibited the up-regulated protein expression of AC in the striatum of morphine-dependent mice. Furthermore, NaHS also attenuated morphine/naloxone-elevated mRNA levels of AC isoform 1 and 8, production of cAMP, and phosphorylation of cAMP response element-binding protein (CREB) in mice striatum. These effects were mimicked by the application of exogenous H2S or over-expression of cystathione-β-synthase, an H2S -producing enzyme, in SH-SY5Y neuronal cells on treatment with [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin, a selective μ-opioid receptor agonist. Blockade of extracellular-regulated protein kinase 1/2 (ERK1/2) with its specific inhibitor attenuated naloxone-induced CREB phosphorylation. Pretreatment with NaHS or stimulation of endogenous H2S production also significantly suppressed opioid withdrawal-induced ERK1/2 activation in mice striatum or SH-SY5Y cells.
Innovation:
H2S treatment is important in prevention of the development of opioid dependence via suppression of cAMP pathway in both animal and cellular models.
Conclusion:
Our data suggest a potential role of H2S in attenuating the development of opioid dependence, and the underlying mechanism is closely related to the inhibition of AC/cAMP pathway.
Background:
Hydrogen sulfide (H2S) is considered an important neuromodulator in the central nervous system. We designed the present study to investigate the effects of exogenous H2S in a rat model of traumatic brain injury (TBI) and the mechanism(s) that underlie this effect.
Methods:
We induced a TBI model by controlled cortical impact injury. We intraperitoneally administered sodium hydrosulfide (NaHS) (an H2S donor) (3 mg/kg) or vehicle alone at 5 min after a TBI operation. We then measured the H2S level, brain edema, blood-brain barrier integrity, neurologic dysfunction, and lesion volume in all animals. Moreover, we assessed the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP)channels by intraperitoneal injection of the selective blocker 5-hydroxydecanoate before NaHS administration. In addition, we detected the levels of oxidative products and the activities of antioxidant enzymes in brain tissue.
Results:
Administration of NaHS significantly increased the H2S level of brain tissue in TBI-challenged rats. The TBI-challenged animals exhibited significant brain injuries, characterized by an increase of blood-brain barrier permeability, brain edema, and lesion volume, as well as neurologic dysfunction, which were significantly ameliorated by NaHS treatment. However, the protective effects of H2S in TBI could be abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate. Moreover, we found that NaHS treatment increased endogenous antioxidant enzymatic activities and decreased oxidative product levels in brain tissue of TBI-challenged rats.
Conclusions:
Exogenous H2S administered at an appropriate dose can exert a protective effect against TBI via activation of mitoK(ATP) channels and reduction of oxidative stress.
Aims:
To examine if hydrogen sulfide (H2S) can promote glucose uptake and provide amelioration in type 2 diabetes.
Results:
Treatment with sodium hydrosulfide (NaHS, an H2S donor) increased glucose uptake in both myotubes and adipocytes. The H2S gas solution showed similar effects. The NaHS effects were blocked by an siRNA-mediated knockdown of the insulin receptor (IR). NaHS also increased phosphorylation of the IR, PI3K, and Akt. In Goto-Kakizaki (GK) diabetic rats, chronic NaHS treatment (30 μmol·kg(-1)·day(-1)) decreased fasting blood glucose, increased insulin sensitivity, and increased glucose tolerance with increased phosphorylation of PI3K and Akt in muscles. Similar insulin-sensitizing effects of NaHS treatment were also observed in Wistar rats. Moreover, glucose uptake was reduced in the cells with siRNA-mediated knockdown of the H2S-generating enzyme cystathionine γ-lyase in the presence or absence of exogenous H2S. Moreover, chronic NaHS treatment reduced oxygen species and the number of crescentic glomeruli in the kidney of GK rats.
Innovation and conclusion:
This study provides the first piece of evidence for the insulin-sensitizing effect of NaHS/H2S in the both in vitro and in vivo models of insulin resistance. REBOUND TRACK: This work was rejected during a standard peer review and rescued by the Rebound Peer Review (Antoxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Jin-Song Bian, Samuel Dudley, Hideo Kimura, and Xian Wang.
Recent studies show that endogenous hydrogen sulfide (H(2)S) plays an anti-inflammatory role in the pathogenesis of airway inflammation. This study investigated whether exogenous H(2)S may counteract oxidative stress-mediated lung damage in allergic mice. Female BALB/c mice previously sensitized with ovalbumin (OVA) were treated with sodium hydrosulfide (NaHS) 30min before OVA challenge. Forty eight hours after antigen-challenge, the mice were killed and leukocyte counting as well as nitrite plus nitrate concentrations were determined in the bronchoalveolar lavage fluid, and lung tissue was analysed for nitric oxide synthase (NOS) activity, iNOS expression, superoxide dismutase (SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (GPx) activities, thiobarbituric acid reactive species and 3-nitrotyrosine containing proteins (3-NT). Pre-treatment of OVA-sensitized mice with NaHS resulted in significant reduction of both eosinophil and neutrophil migration to the lungs, and prevented the elevation of iNOS expression and activity observed in the lungs from the untreated allergic mice, although it did not affect 3-NT. NaHS treatment also abolished the increased lipid peroxidation present in the allergic mouse lungs and increased SOD, GPx and GR enzyme activities. These results show, for the first time, that the beneficial in vivo effects of the H(2)S-donor NaHS on allergic airway inflammation involve its inhibitory action on leukocyte recruitment and the prevention of lung damage by increasing endogenous antioxidant defenses. Thus, exogenous administration of H(2)S donors may be beneficial in reducing the deleterius impact of allergic pulmonary disease, and might represent an additional class of pharmacological agents for treatment of chronic pulmonary diseases.
Hydrogen sulfide (H(2)S) was recently discovered to be synthesized in mammalian tissues by several different enzymes. Numerous studies have shown that H(2)S has vasodilator and antihypertensive effects in the cardiovascular system. However, intracellular mechanisms of the H(2)S-induced vasodilation and its interactions with other endothelium-derived relaxing factors, such as nitric oxide (NO), remain unclear. We investigated whether H(2)S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. NaHS, a H(2)S donor, dose-dependently increased NO production in cultured endothelial cells. This effect was abolished by a calcium chelator (BAPTA-AM), but not by the absence of extracellular calcium. The NaHS-induced NO production was partially blocked by inhibitors of ryanodine receptor (dantrolene) or inositol 1,4,5-triphosphate receptor (xestospongin C). NaHS significantly increased intracellular calcium concentrations, and this effect was attenuated by dantrolene or xestospongin C. NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. The NaHS-induced eNOS phosphorylation and NO production were not affected by a PI3K/Akt inhibitor (wortmannin). The data of this study suggest that H(2)S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function.
The present study was undertaken to study the effects of exogenous hydrogen sulfide (H(2)S) on global cerebral ischemia-reperfusion(I/R) and the underlying mechanisms. After a 24h I/R, administration of NaHS, an exogenous donor for H(2)S, at the dose of 0.2 or 0.4μmol/kg significantly decreased the apoplexy index, neurological symptom scoring, and brain infarcted area as compared to the I/R group in a dose dependent manner. At the same time, NaHS-treated rats displayed significant reduction of MDA content, and striking increase of SOD activity in the brain tissues compared with I/R group. The up-regulated mRNA levels of p47(phox) and gp91(phox) subunits of NADPH oxidase were also suppressed by NaHS treatment. In this study, the pro-inflammatory markers TNF-α and MCP-1 in I/R group were markedly increased by 24h I/R, which were significantly attenuated by NaHS in a dose-dependent manner. In contrast, the anti-inflammatory factor IL-10 was markedly induced by NaHS administration. In addition, the expression of the anti-apoptotic protein Bcl-2 was significantly decreased in I/R group compared with the sham-operated group. This reduction was significantly blunted in NaHS-treated group. On the contrary, the pro-apoptotic protein Bax content in brain tissues of I/R group was markedly elevated compared with sham-operated animals. And such an induction of Bax content was significantly ameliorated by NaHS. Taken together, our results suggest that hydrogen sulfide has potent protective effect against a severe cerebral injury induced by a global I/R possibly through the inhibition of oxidative stress, inflammation and apoptosis.
Hydrogen sulfide, H(2)S, is a colorless gas with a strong odor that until recently was only considered to be a toxic environmental pollutant with little or no physiological significance. However, the past few years have demonstrated its role in many biological systems and it is becoming increasingly clear that H(2)S is likely to join nitric oxide (NO) and carbon monoxide (CO) as a major player in mammalian biology. In this review, we have provided an overview of the chemistry and biology of H(2)S and have summarized the chemistry and biological activity of some natural and synthetic H(2)S-donating compounds. The naturally occurring compounds discussed include, garlic, sulforaphane, erucin, and iberin. The synthetic H(2)S donors reviewed include, GYY4137; cysteine analogs; S-propyl cysteine, S-allyl cysteine, S-propargyl cysteine, and N-acetyl cysteine. Dithiolethione and its NSAID and other chimeras such as, L-DOPA, sildenafil, aspirin, diclofenac, naproxen, ibuprofen, indomethacin, and mesalamine have also been reviewed in detail. The newly reported NOSH-aspirin that releases both NO and H(2)S has also been discussed.
Nitric oxide is a small messenger molecule utilized by nature in cell signalling and the non-specific immune response. At present, nitric oxide releasing prodrugs cannot be efficiently targeted towards a specific body compartment, which restricts their therapeutic applications. To address this limitation, we have designed two photolabile nitric oxide releasing prodrugs, tert-butyl S-nitrosothiol and tert-dodecane S-nitrosothiol, which are based on the S-nitrosothiol functionality. By modulating the prodrugs' hydrophobicity, we postulated that we could increase their stability within the cell by preventing their interaction with hydrophilic thiols and metal ions; processes that are known to inactivate this prodrug class. Our data demonstrate that these prodrugs have improved nitric oxide release kinetics compared to currently available S-nitrosothiols, as they are highly stable in vitro in the absence of irradiation (t(1/2) > 3 h), while their rate of decomposition can be regulated by controlling the intensity or duration of the photostimulus. Nitric oxide release can readily be achieved using non-laser based light sources, which enabled us to characterize photoactivation as a trigger mechanism for nitric oxide release in A549 lung carcinoma cells. Here we confirmed that irradiation induced highly significant increases in cytotoxicity within a therapeutic drug range (1-100 μm), and the utility of this photoactivation switch opens up avenues for exploring the applications of these prodrugs for chemical biology studies and chemotherapy.
Inhibition of oxidative stress has been reported to be involved in the cardioprotective effects of hydrogen sulfide (H(2)S) during ischemia/reperfusion (I/R). However, the mechanism whereby H(2)S regulates the level of cardiac reactive oxygen species (ROS) during I/R remains unclear. Therefore, we investigated the effects of H(2)S on pathways that generate and scavenge ROS. Our results show that pretreating rat neonatal cardiomyocytes with NaHS, a H(2)S donor, reduced the levels of ROS during the hypoxia/reoxygenation (H/R) condition. We found that H(2)S inhibited mitochondrial complex IV activity and increased the activities of superoxide dismutases (SODs), including Mn-SOD and CuZn-SOD. Further studies indicated that H(2)S up-regulated the expression of Mn-SOD but not CuZn-SOD. Using a cell-free system, we showed that H(2)S activates CuZn-SOD. An isothermal titration calorimetry (ITC) analysis indicated that H(2)S directly interacts with CuZn-SOD. Taken together, H(2)S inhibits mitochondrial complex IV and activates SOD to decrease the levels of ROS in cardiomyocytes during I/R.
Renal protection of s-allyl cysteine (SAC) and s-propyl cysteine (SPC) in diabetic mice against inflammatory injury was examined. Each agent at 0.5 and 1 g/L was added to the drinking water for 10 weeks. SAC or SPC intake significantly reduced the plasma blood urea nitrogen level and increased creatinine clearance (P < 0.05). These treatments significantly lowered the renal level of reactive oxygen species, nitric oxide, interleukin-6, tumor necrosis factor-α, and prostaglandin E(2) in diabetic mice (P < 0.05). Renal mRNA expression of inducible nitric oxide synthase, cyclooxygenase-2, protein kinase C (PKC)-α, PKC-β, and PKC-γ was enhanced in diabetic mice (P < 0.05); however, SAC or SPC treatments dose dependently declined mRNA expression of these factors (P < 0.05). Nuclear factor κB (NF-κB) activity, mRNA expression, and protein production in kidney of diabetic mice were significantly increased (P < 0.05). SAC or SPC intake dose dependently suppressed NF-κB activity, NF-κB p65 mRNA expression, and protein level (P < 0.05). Diabetes also enhanced renal protein expression of mitogen-activated protein kinase (P < 0.05). SAC and SPC, only at a high dose, significantly suppressed protein production of p-p38 and p-ERK1/2 (P < 0.05). Renal mRNA expression and protein generation of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ were significantly down-regulated in diabetic mice (P < 0.05), but the intake of SAC or SPC at high dose up-regulated PPAR-α and PPAR-γ (P < 0.05). These findings support that SAC and SPC are potent anti-inflammatory agents against diabetic kidney diseases.
Hydrogen sulfide is an endogenously generated molecule with many reported physiological functions. Although several biological targets have been proposed, the biochemical mechanisms by which it elicits activity are not established. Thus, in an effort to begin to delineate the fundamental biological chemistry of H(2)S, we have examined the reaction of H(2)S with oxidized thiols and thiol proteins in order to determine whether persulfide formation occurs, is stable and how this may affect protein function. We have found that persulfides are easily generated, relatively stable and can alter enzyme activity. Moreover, we have begun to develop methodology for in situ generation of persulfides to facilitate further study of this potentially important species.
Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H(2)S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine.
The purpose of this study was to determine if H(2)S is a major physiological EDHF.
We now show that H(2)S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H(2)S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H(2)S-elicited hyperpolarization. The endothelial intermediate conductance (IK(Ca)) and small conductance (SK(Ca)) potassium channels mediate in part the effects of H(2)S, as selective IK(Ca) and SK(Ca) channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H(2)S-induced vasorelaxation.
H(2)S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H(2)S biosynthesis offer therapeutic potential.
This study was designed to investigate the protective effects of exogenous hydrogen sulfide (H(2)S) on trauma-hemorrhagic shock (T-H).
Forty-eight male Sprague-Dawley rats were anesthetized, while 32 were subjected to both midline laparotomy and hemorrhagic shock (35-40 mmHg for 90 min) by bleeding them from the femoral artery. One hour later, resuscitation was initiated with Ringer lactate. NaHS (28 μmol/kg) or vehicle alone was administered intraperitoneally at the onset of resuscitation. Two hours later, eight animals from each group were re-anesthetized to determine cardiac function, blood gas concentrations, and hepatic and renal function. Superoxide dismutase activity (SOD), malondialdehyde concentrations (MDA), and the activity of myeloperoxidase (MPO) in the serum were measured and pulmonary wet/dry (W/D) ratio and histopathologic evaluations performed.
NaHS resulted in an increase in mean arterial blood pressure, left ventricular pressure and positive (+dP/dt(max)) and negative (-dP/dt(max)) first derivatives of pressure as compared with the vehicle only group. The pH, PaO(2) and base excess (BE) were increased in the NaHS-treated group compared with the vehicle-treated group. Aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and serum creatinine were reduced in the NaHS-treated group. NaHS also significantly reduced the high mortality rate at 24 h otherwise caused by T-H. The NaHS-treated group showed a remarkable decrease in MDA and MPO concentrations in plasma and an increase in SOD as compared with the vehicle-treated group. Histopathologic analysis indicated less edema, congestion, inflammatory cell infiltration and necrosis in heart, lung, liver and kidney tissue in NaHS-treated group.
The present study demonstrates that exogenous H(2)S administered at an appropriate dose confers protective effects after T-H and resuscitation, by preventing a decrease in the antioxidant defense system.
Hydrogen sulfide (H2S) is a novel gaseous mediator produced by cystathionine-beta-synthase and cystathionine-gamma-lyase in the cardiovascular system, including the heart. Using a rat model of regional myocardial ischemia/reperfusion, we investigated the effects of an H2S donor (sodium hydrogen sulfide [NaHS]) on the infarct size and apoptosis caused by ischemia (25 min) and reperfusion (2 h). Furthermore, we investigated the potential mechanism(s) of the cardioprotective effect(s) afforded by NaHS. Specifically, we demonstrate that NaHS (1) attenuates the increase in caspase 9 activity observed in cardiac myocytes isolated from the area at risk (AAR) of hearts subjected in vivo to regional myocardial I/R and (2) ameliorates the decrease in expression of Bcl-2 within the AAR obtained from rat hearts subjected to regional myocardial I/R. The cardioprotective effects of NaHS were abolished by 5-hydroxydeconoate, a putative mitochondrial adenosine triphosphate-sensitive potassium channel blocker. Furthermore, NaHS attenuated the increase in the I/R-induced (1) phosphorylation of p38 mitogen-activated protein kinase and Jun N-terminal kinase, (2) translocation from the cytosol to the nucleus of the p65 subunit of nuclear factor-kappaB, (3) intercellular adhesion molecule 1 expression, (4) polymorphonuclear leukocyte accumulation, (5) myeloperoxidase activity, (6) malondialdehyde levels, and (7) nitrotyrosine staining determined in the AAR obtained from rat hearts subjected to regional myocardial I/R. In conclusion, we demonstrate that the cardioprotective effect of NaHS is secondary to a combination of antiapoptotic and anti-inflammatory effects. The antiapoptotic effect of NaHS may be in part due to the opening of the putative mitochondrial adenosine triphosphate-sensitive potassium channels.
Hydrogen sulfide (H(2)S) displays anti-inflammatory and cytoprotective activities to attenuate myocardial ischemia-reperfusion (MIR)-induced injury, but its role in MIR in diabetics is not known. This study was undertaken to investigate whether H(2)S plays a protective role in MIR in diabetic rats.
Diabetes was induced by streptozocin in Wistar rats, which were subjected to myocardial ischemia by blocking the left circumflex artery for 30 min, followed by 2h reperfusion. dl-propargylglycine (PAG) and sodium hydrosulfide (NaHS) were administered to the rats to investigate their effects on severity of MIR-induced injury.
Diabetic rats had smaller myocardial infarct sizes and higher serum levels of H(2)S (both P < 0.05) than non-diabetics when they underwent MIR. MIR significantly increased the serum level of H(2)S (49.5 ± 7.1 μM), H(2)S-synthesizing activity (7.4 ± 1.6 nmol/mg) and the myocardial infarct size (44.0 ± 7.2%), compared with sham-operated diabetic rats (21.7 ± 2.1 μM, 0.15 ± 0.4 nmol/mg and 1.2 ± 0.4%, respectively). Administration of NaHS increased the H(2)S level (65.8 ± 6.9 μM) and had little effect on H(2)S production activity (6.5 ± 2.2 nmol/mg), while PAG reduced both the H(2)S level (29.2 ± 5.0 μM) and H(2)S-synthesizing activity (2.2 ± 1.8 nmol/mg). NaHS significantly reduced the myocardial infarct size (31.2 ± 4.7%), inhibited the production of lipid peroxidation, MPO activity, and cell apoptosis, and downregulated expression of caspase-3, Fas, FasL, and TNF-α, which had been elevated by MIR, while PAG further increased the myocardial infarct size (58.3 ± 5.9%), and displayed opposite effects.
The study indicates that H(2)S may play a protective role in MIR-induced myocardial injury in diabetics by its anti-apoptotic, anti-oxidative and anti-inflammatory activities.
Hydrogen sulphide (H₂S), a potentially toxic gas, is also involved in the neuroprotection, neuromodulation, cardioprotection, vasodilatation and the regulation of inflammatory response and insulin secretion. We have recently reported that H₂S suppresses pancreatic β-cell apoptosis induced by long-term exposure to high glucose. Here we examined the protective effects of sodium hydrosulphide (NaHS), an H₂S donor, on various types of β-cell damage.
Isolated islets from mice or the mouse insulinoma MIN6 cells were cultured with palmitate, cytokines (a mixture of tumour necrosis factor-α, interferon-γ and interleukin-1β), hydrogen peroxide, thapsigargin or tunicamycin with or without NaHS. We examined DNA fragmentation, caspase-3 and -7 activities and reactive oxygen species (ROS) production in the treated cells thereafter. Apoptotic cell death in isolated islets was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method.
NaHS suppressed DNA fragmentation and the activities of caspase-3 and -7 induced by palmitate, the cytokines or hydrogen peroxide. In contrast, NaHS failed to protect islets and MIN6 cells from apoptosis induced by thapsigargin and tunicamycin, both of which cause endoplasmic reticulum stress. NaHS suppressed ROS production induced by cytokines or hydrogen peroxide but it had no effect on ROS production in thapsigargin-treated cells. NaHS increased Akt phosphorylation in MIN6 cells treated with cytokines but not in cells treated with thapsigargin. Treatment with NaHS decreased TUNEL-positive cells in cytokine-exposed islets.
H₂S may prevent pancreatic β-cells from cell apoptosis via an anti-oxidative mechanism and the activation of Akt signalling.
We investigated the effects of the hydrogen sulfide (H₂S)-releasing derivatives of aspirin (ACS14) and salicylic acid (ACS21) in a rat model of metabolic syndrome induced by glutathione (GSH) depletion, causing hypertension and other pathological cardiovascular alterations. GSH depletion was induced in normal rats by the GSH-synthase inhibitor buthionine sulfoximine (BSO, 30 mmol/L day for seven days in the drinking water). Systolic blood pressure and heart rate were measured daily by the tail-cuff method, and plasma thromboxane B₂, 6-keto-prostaglandin F(2α), 8-isoprostane, GSH, insulin and glucose were determined at the end of the seven-day BSO schedule. In addition, ischemia/reperfusion-induced myocardial dysfunction and endothelial dysfunction were assayed on isolated heart and aortic rings, respectively. Unlike aspirin and salicylic acid, ACS14 and ACS21 reduced BSO-induced hypertension, also lowering plasma levels of thromboxane B₂, 8-isoprostane and insulin, while GSH remained in the control range. Neither ACS14 nor ACS21 caused gastric lesions. Both restored the endothelial dysfunction observed in aortic rings from BSO-treated rats, and in ischemia/reperfusion experiments they lowered left ventricular end-diastolic pressure, consequently improving the developed pressure and the maximum rise and fall of left ventricular pressure. Together with this improvement of heart mechanics there were reductions in the activity of creatine kinase and lactate dehydrogenase in the cardiac perfusate. This implies that H₂S released by both ACS14 and ACS21 was involved in protecting the heart from ischemia/reperfusion, and significantly limited vascular endothelial dysfunction in aortic tissue and the related hypertension.
Endogenously produced hydrogen sulfide is thought to function as an intracellular messenger. There is, however, little information on tissue concentrations of free hydrogen sulfide, the putative messenger form of this molecule, versus that of the bound (acid-labile) form. The present report describes the application of a novel technique to measure free and acid-labile hydrogen sulfide in mouse tissues. Very low free hydrogen sulfide concentrations (<0.050 μmol/kg) were observed in brain, liver, blood, heart, kidney, striated muscle, and esophagus. Aortic concentrations of free hydrogen sulfide were 20 to 100 times greater than that of the other tissues. Acid-labile hydrogen sulfide concentrations were multiple orders of magnitude greater than that of the free form in every tissue other than aorta. Previous reports of tissue hydrogen sulfide concentrations of 30 to >100 μmol/kg measured bound rather than free hydrogen sulfide, the observation that aorta contains anomalously high free hydrogen sulfide concentrations lends support for a vasodilator function for this molecule, and the very low free hydrogen sulfide concentrations in most tissues seemingly requires intermediation of a yet to be described receptor-like mechanism if this molecule is to serve as a gasotransmitter.
Hydrogen sulphide (H(2)S) is a labile, endogenous metabolite of cysteine, with multiple biological roles. The development of sulphide-based therapies for human diseases will benefit from a reliable method of quantifying H(2)S in blood and tissues.
Concentrations of reactive sulphide in saline and freshly drawn whole blood were quantified by reaction with the thio-specific derivatization agent monobromobimane, followed by reversed-phase fluorescence HPLC and/or mass spectrometry. In pharmacokinetic studies, male rats were exposed either to intravenous infusions of sodium sulphide or to H(2)S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H(2)S/HS(-) were concomitantly measured using an amperometric sensor.
Monobromobimane was found to rapidly and quantitatively derivatize sulphide in saline or whole blood to yield the stable small molecule sulphide dibimane. Extraction and quantification of this bis-bimane derivative were validated via reversed-phase HPLC separation coupled to fluorescence detection, and also by mass spectrometry. Baseline levels of sulphide in blood were in the range of 0.4-0.9 microM. Intravenous administration of sodium sulphide solution (2-20 mg x kg(-1) x h(-1)) or inhalation of H(2)S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg x kg(-1) x h(-1) of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H(2)S gas inhalation.
The monobromobimane derivatization method is a sensitive and reliable means to measure reactive sulphide species in whole blood. Using this method, we have established a bioequivalence between infused sodium sulphide and inhaled H(2)S gas.
The paradigm of using nanoparticulate pharmaceutical carriers has been well established over the past decade, both in pharmaceutical research and in the clinical setting. Drug carriers are expected to stay in the blood for long time, accumulate in pathological sites with affected and leaky vasculature (tumors, inflammations, and infarcted areas) via the enhanced permeability and retention (EPR) effect, and facilitate targeted delivery of specific ligand-modified drugs and drug carriers into poorly accessible areas. Among various approaches to specifically target drug-loaded carrier systems to required pathological sites in the body, two seem to be most advanced--passive (EPR effect-mediated) targeting, based on the longevity of the pharmaceutical carrier in the blood and its accumulation in pathological sites with compromised vasculature, and active targeting, based on the attachment of specific ligands to the surface of pharmaceutical carriers to recognize and bind pathological cells. Here, we will consider and discuss these two targeting approaches using tumor targeting as an example.
Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H(2)S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen-glucose deprivation or Na(2)S(2)O(4) (an oxygen scavenger) treatment. We found that treatment with NaHS (an H(2)S donor, 10-100 microM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H(2)S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-beta-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H(2)S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K(ATP)/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H(2)S in the brain during cerebral hypoxia.
Hydrogen sulphide is an important mediator of gastric mucosal defence. The use of non-steroidal anti-inflammatory drugs continues to be limited by their toxicity, particularly in the upper gastrointestinal tract. We evaluated the gastrointestinal safety and anti-inflammatory efficacy of a novel hydrogen sulphide-releasing derivative of naproxen, ATB-346 [2-(6-methoxy-napthalen-2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester].
The ability of ATB-346 versus naproxen to cause gastric damage was evaluated in healthy rats and in rats with compromised gastric mucosal defence. Effects on the small intestine and on the healing of ulcers were also assessed. The ability of ATB-346 to inhibit cyclooxygenase-1 and 2 and to reduce inflammation in vivo was also evaluated.
ATB-346 suppressed gastric prostaglandin E(2) synthesis as effectively as naproxen, but produced negligible damage in the stomach and intestine. In situations in which the gastric mucosa was rendered significantly more susceptible to naproxen-induced damage (e.g. ablation of sensory afferent nerves, inhibition of endogenous nitric oxide or hydrogen sulphide synthesis, co-administration with aspirin, antagonism of K(IR)6.x channels), ATB-346 did not cause significant damage. Unlike naproxen and celecoxib, ATB-346 accelerated healing of pre-existing gastric ulcers. In a mouse airpouch model, ATB-346 suppressed cyclooxygenase-2 activity and inhibited leukocyte infiltration more effectively than naproxen. ATB-346 was as effective as naproxen in adjuvant-induced arthritis in rats, with a more rapid onset of activity. Unlike naproxen, ATB-346 did not elevate blood pressure in hypertensive rats.
ATB-346 exhibits anti-inflammatory properties similar to naproxen, but with substantially reduced gastrointestinal toxicity.
Hydrogen sulfide (H(2)S) is a gaseous mediator involved in a multitude of physiological functions; however the role of H(2)S in the gut is far from being understood completely. The aim of this study was to determine the effect of d-l-propargylglycine (PAG), an inhibitor of H(2)S synthesis, on ethanol-induced gastric injury in rat and to examine the role of l-cysteine, exogenous H(2)S, prostaglandins, non-protein sulphydryls groups, nitric oxide and K(ATP) channels in the gastroprotective effect of PAG. Administration of PAG (3.12 to 75mg/kg i.p.) or l-cysteine (0.3 to 300mg/kg, p.o.) exhibited a dose-dependent protective effect after intragastric administration of 1ml of ethanol to induce gastric injury. The gastroprotective effect of PAG (25mg/kg i.p.) was maintained after post-treatment with l-cysteine (10mg/kg p.o.), while NaHS (8.4mg/kg p.o.) inhibited this effect. The levels of gastric hydrogen sulfide were increased after ethanol-induced gastric damage and they were reverted by PAG while prostaglandin E(2) levels in gastric tissue were decreased by ethanol and PAG did not revert to this effect. Pretreatment with indomethacin (10mg/kg i.p.) and N-ethylmaleimide (NEM, 10mg/kg s.c.) resulted in a reversion of the gastroprotective effect of PAG while N(G)-nitro-l-arginine methyl ester (L-NAME, 70mg/kg s.c.), glibenclamide (1mg/kg i.p.) or diazoxide (3mg/kg i.p.) did not induce any changes. These results suggest that ethanol-induced gastric injury is related with an increment of endogenous H(2)S levels, and therefore a decrement of H(2)S levels by PAG is a benefit to protect gastric injury caused by ethanol.
Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant in the brain. We recently showed that H(2)S protects neurons from oxidative stress by increasing the levels of glutathione (GSH), a major cellular antioxidant, by more than twice that of a control through enhancing the cystine transport. Here we show that H(2)S enhances the transport of cysteine to increase GSH production more than cystine transport and to redistribute the localization of GSH to mitochondria. The efficiency of GSH production enhanced by H(2)S is even greater by fourfold under oxidative stress by glutamate. H(2)S reinstated GSH levels in the fetal brain decreased by ischemia/reperfusion in utero. In addition, Neuro2a cells expressing a mitochondrial H(2)S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST), along with cysteine aminotransferase (CAT), showed significant resistance to oxidative stress. The present study shows that H(2)S protects cells from oxidative stress by two mechanisms. It enhances the production of GSH by enhancing cystine/cysteine transporters and redistributes GSH to mitochondria. H(2)S produced in mitochondria also may directly suppress oxidative stress. It provides a new mechanism of neuroprotection from oxidative stress by H(2)S.
The current study was designed to evaluate the pharmacologic effects of three novel cysteine-containing compounds: S-propyl-l-cysteine (SPC), S-allyl-l-cysteine (SAC), and S-propargyl-l-cysteine (SPRC) on H(2)S production and antioxidant defenses in an acute myocardial infarction (MI) rat model. The enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as glutathione redox status and malonaldehyde (MDA) content, also were determined. All three compounds were found to preserve SOD and GPx activities and also tissue GSH levels while reducing the formation of the lipid peroxidation product MDA in ventricular tissues. With immunfluorescence assays, we observed the expression of CSE and Mn-SOD. The morphologic changes of the cardiac cells are seen with both light and electron microscopy. The corresponding pathologic alterations were characterized mainly as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. Propargylglycine, a selective inhibitor of CSE, abolished the protective effects of each compound used in the current model. Our study provides novel evidence that SPC, SAC, and SPRC have cardioprotective effects in MI by reducing the deleterious effects of oxidative stress by modulating the endogenous levels of H(2)S and preserving the activities of antioxidant defensive enzymes like SOD.