Endogenous Prostaglandins and Afferent Sensory Nerves in Gastroprotective Effect of Hydrogen Sulfide against Stress-Induced Gastric Lesions

Hydrogen sulfide (H₂S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H₂S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H₂S precursor L-cysteine, H₂S-donor NaHS, the H₂S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H₂S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H₂S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H₂S and or L-cysteine, the H₂S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H₂S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H₂S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.     

Upregulation of Cystathionine β-Synthetase Expression Contributes to Visceral Hyperalgesia Induced by Heterotypic Intermittent Stress in Rats

Background

Hydrogen sulfide (H₂S) functions as a neuromodulator, but whether it modulates visceral pain is not well known. This study was designed to determine the role for the endogenous H₂S producing enzyme cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CSE) in a validated rat model of visceral hyperalgesia (VH).

Methods

VH was induced by nine-day heterotypic intermittent stress (HIS). Abdominal withdrawal reflex (AWR) scores were determined by measuring the visceromoter responses to colorectal distension (CRD). Dorsal root ganglia (DRG) neurons innervating the colon were labeled by injection of DiI (1,1''-dioleyl-3,3,3'',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall. Patch clamp recording techniques were employed to examine excitability and sodium channel currents of colon specific DRG neurons. Tissues from colon related thoracolumbar DRGs were analyzed for CBS, CSE and sodium channel expression.

Results

HIS significantly increased the visceromotor responses to CRD in association with an upregulated expression of CBS not CSE proteins in colon related DRGs. Administration of O-(Carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, attenuated the AWR scores in HIS-treated rats, in a dose dependent fashion. In contrast, AOAA did not produce any effect on AWR scores in healthy control rats. AOAA reversed the potentiation of sodium channel current densities of colon specific DRG neurons of HIS rats. To further confirm the role for CBS-H₂S signaling, NaHS was used to mimic the production of H₂S by CBS. Application of NaHS significantly enhanced neuronal excitability and potentiated sodium channel current densities of colon DRG neurons from healthy control rats. Furthermore, AOAA reversed the upregulation of Na_V1.7 and Na_V1.8 in colon related DRGs of HIS rats.

Conclusion

Our results suggest that upregulation of CBS expression might play an important role in developing VH via sensitization of sodium channels in peripheral nociceptors, thus identifying a specific neurobiological target for the treatment of VH in functional bowel syndromes.     

Hydrogen Sulfide Plays a Key Role in the Inhibitory Neurotransmission to the Pig Intravesical Ureter

According to previous observations nitric oxide (NO), as well as an unknown nature mediator are involved in the inhibitory neurotransmission to the intravesical ureter. This study investigates the hydrogen sulfide (H₂S) role in the neurogenic relaxation of the pig intravesical ureter. We have performed western blot and immunohistochemistry to study the expression of the H₂S synthesis enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), measurement of enzymatic production of H₂S and myographic studies for isometric force recording. Immunohistochemical assays showed a high CSE expression in the intravesical ureter muscular layer, as well as a strong CSE-immunoreactivity within nerve fibres distributed along smooth muscle bundles. CBS expression, however, was not consistently observed. On ureteral strips precontracted with thromboxane A₂ analogue U46619, electrical field stimulation (EFS) and the H₂S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked frequency- and concentration-dependent relaxations. CSE inhibition with DL-propargylglycine (PPG) reduced EFS-elicited responses and a combined blockade of both CSE and NO synthase (NOS) with, respectively, PPG and N^G-nitro-L-arginine (L-NOARG), greatly reduced such relaxations. Endogenous H₂S production rate was reduced by PPG, rescued by addition of GYY4137 and was not changed by L-NOARG. EFS and GYY4137 relaxations were also reduced by capsaicin-sensitive primary afferents (CSPA) desensitization with capsaicin and blockade of ATP-dependent K⁺ (K_ATP) channels, transient receptor potential A1 (TRPA₁), transient receptor potential vanilloid 1 (TRPV₁), vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide (VIP/PACAP) and calcitonin gene-related peptide (CGRP) receptors with glibenclamide, {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031, AMG9810, PACAP_6–38 and CGRP_8–37, respectively. These results suggest that H₂S, synthesized by CSE, is involved in the inhibitory neurotransmission to the pig intravesical ureter, through an NO-independent pathway, producing smooth muscle relaxation via K_ATP channel activation. H₂S also promotes the release of inhibitory neuropeptides, as PACAP 38 and/or CGRP from CSPA through TRPA₁, TRPV₁ and related ion channel activation.     

Role of Hydrogen Sulfide in Early Blood-Brain Barrier Disruption following Transient Focal Cerebral Ischemia

We determined the role of endogenous hydrogen sulfide (H₂S) in cerebral vasodilation/hyperemia and early BBB disruption following ischemic stroke. A cranial window was prepared over the left frontal, parietal and temporal cortex in mice. Transient focal cerebral Ischemia was induced by directly ligating the middle cerebral artery (MCA) for two hours. Regional vascular response and cerebral blood flow (CBF) during ischemia and reperfusion were measured in real time. Early BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. Topical treatment with DL-propargylglycine (PAG, an inhibitor for cystathionine γ-lyase (CSE)) and aspartate (ASP, inhibitor for cysteine aminotransferase/3-mercaptopyruvate sulfurtransferase (CAT/3-MST)), but not O-(Carboxymethyl)hydroxylamine hemihydrochloride (CHH, an inhibitor for cystathionine β-synthase (CBS)), abolished postischemic cerebral vasodilation/hyperemia and prevented EB and Na-F extravasation. CSE knockout (CSE^-/-) reduced postischemic cerebral vasodilation/hyperemia but only inhibited Na-F extravasation. An upregulated CBS was found in cerebral cortex of CSE^-/- mice. Topical treatment with CHH didn’t further alter postischemic cerebral vasodilation/hyperemia, but prevented EB extravasation in CSE^-/- mice. In addition, L-cysteine-induced hydrogen sulfide (H₂S) production similarly increased in ischemic side cerebral cortex of control and CSE^-/- mice. Our findings suggest that endogenous production of H₂S by CSE and CAT/3-MST during reperfusion may be involved in postischemic cerebral vasodilation/hyperemia and play an important role in early BBB disruption following transient focal cerebral ischemia.     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H₂S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H₂S-producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H₂S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPKα1-shRNA. In summary, we show that H₂S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H₂S signaling might be further investigated as a novel target for anti-osteoporosis treatment.     

A New Slow Releasing,H2S Generating Compound,GYY4137 Relaxes Spontaneous and Oxytocin-Stimulated Contractions of Human and Rat Pregnant Myometrium

Better tocolytics are required to help prevent preterm labour. The gaseotransmitter Hydrogen sulphide (H₂S) has been shown to reduce myometrial contractility and thus is of potential interest. However previous studies used NaHS, which is toxic and releases H₂S as a non-physiological bolus and thus alternative H₂S donors are sought. GYY4137 has been developed to slowly release H₂S and hence better reflect endogenous physiological release. We have examined its effects on spontaneous and oxytocin-stimulated contractility and compared them to NaHS, in human and rat myometrium, throughout gestation. The effects on contractility in response to GYY4137 (1 nM–1 mM) and NaHS (1 mM) were examined on myometrial strips from, biopsies of women undergoing elective caesarean section or hysterectomy, and from non-pregnant, 14, 18, 22 day (term) gestation or labouring rats. In pregnant rat and human myometrium dose-dependent and significant decreases in spontaneous contractions were seen with increasing concentrations of GYY4137, which also reduced underlying Ca transients. GYY4137 and NaHS significantly reduced oxytocin-stimulated and high-K depolarised contractions as well as spontaneous activity. Their inhibitory effects increased as gestation advanced, but were abruptly reversed in labour. Glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of GYY4137. These data suggest (i) H₂S contributes to uterine quiescence from mid-gestation until labor, (ii) that H₂S affects L-type calcium channels and K_ATP channels reducing Ca entry and thereby myometrial contractions, (iii) add to the evidence that H₂S plays a physiological role in relaxing myometrium, and thus (iv) H₂S is an attractive target for therapeutic manipulation of human myometrial contractility.     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

Hydrogen Sulfide in Paraventricular Nucleus Enhances Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats

Background

Intracerebroventricular infusion of NaHS, a hydrogen sulfide (H₂S) donor, increased mean arterial pressure (MAP). This study was designed to determine the roles of H₂S in the paraventricular nucleus (PVN) in modulating sympathetic activity and cardiac sympathetic afferent reflex (CSAR) in chronic heart failure (CHF).

Methodology/Principal Findings

CHF was induced by left descending coronary artery ligation in rats. Renal sympathetic nerve activity (RSNA) and MAP were recorded under anesthesia. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of low doses of a H₂S donor, GYY4137 (0.01 and 0.1 nmol), had no significant effects on RSNA, MAP and CSAR. High doses of GYY4137 (1, 2 and 4 nmol) increased baseline RSNA, MAP and heart rate (HR), and enhanced CSAR. The effects were greater in CHF rats than sham-operated rats. A cystathionine-β-synthase (CBS) inhibitor, hydroxylamine (HA) in PVN had no significant effect on the RSNA, MAP and CSAR. CBS activity and H₂S level in the PVN were decreased in CHF rats. No significant difference in CBS level in PVN was found between sham-operated rats and CHF rats. Stimulation of cardiac sympathetic afferents with capsaicin decreased CBS activity and H₂S level in the PVN in both sham-operated rats and CHF rats.

Conclusions

Exogenous H₂S in PVN increases RSNA, MAP and HR, and enhances CSAR. The effects are greater in CHF rats than those in sham-operated rats. Endogenous H₂S in PVN is not responsible for the sympathetic activation and enhanced CSAR in CHF rats.     

The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo

The slow-releasing hydrogen sulfide (H₂S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H₂S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H₂S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H₂S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G₂/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H₂S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H₂S donors should be investigated further as potential anti-cancer agents.     

Effect of Hydrogen Sulfide on Sympathetic Neurotransmission and Catecholamine Levels in Isolated Porcine Iris-Ciliary Body

In the present study, we investigated the pharmacological action of hydrogen sulfide (H₂S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na₂S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H₂S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [³H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na₂S caused a concentration-dependent inhibition of electrically evoked [³H]NE release from porcine iris-ciliary body without affecting basal [³H]NE efflux. The inhibitory action of H₂S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H₂S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H₂S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.     

Hydrogen sulfide attenuates carbon tetrachloride-induced hepatotoxicity, liver cirrhosis and portal hypertension in rats

Background

Hydrogen sulfide (H₂S) displays vasodilative, anti-oxidative, anti-inflammatory and cytoprotective activities. Impaired production of H₂S contributes to the increased intrahepatic resistance in cirrhotic livers. The study aimed to investigate the roles of H₂S in carbon tetrachloride (CCl₄)-induced hepatotoxicity, cirrhosis and portal hypertension.

Methods and Findings

Sodium hydrosulfide (NaHS), a donor of H₂S, and DL-propargylglycine (PAG), an irreversible inhibitor of cystathionine γ-lyase (CSE), were applied to the rats to investigate the effects of H₂S on CCl₄-induced acute hepatotoxicity, cirrhosis and portal hypertension by measuring serum levels of H₂S, hepatic H₂S producing activity and CSE expression, liver function, activity of cytochrome P450 (CYP) 2E1, oxidative and inflammatory parameters, liver fibrosis and portal pressure. CCl₄ significantly reduced serum levels of H₂S, hepatic H₂S production and CSE expression. NaHS attenuated CCl₄-induced acute hepatotoxicity by supplementing exogenous H₂S, 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.

Conclusions

Exogenous H₂S attenuates CCl₄-induced hepatotoxicity, liver cirrhosis and portal hypertension by its multiple functions including anti-oxidation, anti-inflammation, cytoprotection and anti-fibrosis, indicating that targeting H₂S may present a promising approach, particularly for its prophylactic effects, against liver cirrhosis and portal hypertension.     

GYY4137 ameliorates sepsis-induced cardiomyopathy via NLRP3 pathway

Sepsis-induced cardiomyopathy (SICM) has a poor prognosis, with no effective therapeutic strategy currently. This study aimed to explore the mechanism underlying SICM and investigate the protective role of the hydrogen sulfide (H₂S) donor GYY4137. This study included patients with SICM and animal models of SICM with wild-type and Nlrp3^?/? mice, which were treated with or without GYY4137. Echocardiography, ELISA, TUNEL staining, and immunofluorescence were used to investigate phenotypic alterations. Serum levels of H₂S and cytokines were measured. Inflammatory cell infiltration in the myocardial tissue was identified using immunohistochemistry and immunofluorescence. RNA expression profiles were identified using RNA sequencing. The protective mechanism of GYY4137 was further validated in the crosstalk between macrophages and cardiomyocytes using immunoblotting, real-time polymerase chain reaction (RT-PCR), and immunofluorescence when conditional medium of macrophages boosted by LPS were co-cultured with cardiomyocytes. Patients and animal models of SICM presented with lower serum H₂S levels and heart dysfunction. GYY4137 reduced macrophage infiltration in septic heart tissue. GO analysis suggested that GYY4137 was involved in the inflammatory process. GYY4137 inhibited NLRP3 inflammasome activity in macrophages, reduced the secretion of inflammatory factors, and decreased the production of reactive oxygen species (ROS) in cardiomyocytes, thus exerting protective effects against SICM. We further found that the protective effects of GYY4137 were absent in Nlrp3-knockout models. GYY4137 ameliorates myocardial injury in SICM via the NLRP3 pathway by inhibiting the inflammatory response and reducing the production of myocardial ROS.     

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H₂S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H₂S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund''s adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO₂⁻), PGE₂, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.     

Differential Effects of Cystathionine-γ-lyase-Dependent Vasodilatory H(2)S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background

Hydrogen sulfide (H₂S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H₂S are not fully understood. We tested the hypotheses that (1) H₂S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K_v) K⁺ channels and (2) that H₂S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Methodology/Principal Findings

Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H₂S donors. KCNQ-type K_v channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H₂S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition.

Conclusions/Signficance

Our results demonstrate potent vasorelaxant effects of H₂S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H₂S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H₂S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H₂S/KCNQ pathways to influence states of vascular dysfunction may be possible.     

Altered gene expression of hydrogen sulfide-producing enzymes in the liver and muscles tissues of hyperthyroid rats

Thyroid hormones have a role in the regulation of hydrogen sulfide (H₂S) biosynthesis. In this study, we determined the effects of hyperthyroidism on H₂S levels in various tissues and messenger RNA (mRNA) expression of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in the liver and muscles of the rat. Sixteen male Wistar rats were divided into the hyperthyroid and the control groups. Hyperthyroidism was induced by adding l -thyroxine (12 mg/L) to drinking water for a period of 21 days. H₂S concentrations in serum, liver, aorta, heart, and soleus muscles, as well as mRNA expressions of CBS, CSE, and 3-MST in these tissues were measured at Day 21. Hyperthyroid rats had lower H₂S levels in the serum compared with controls (14.7 ± 1.4 vs. 25.7 ± 1.6 µmol/L, p < 0.001). Compared with controls, hyperthyroid rats had lower levels of H₂S in the aorta (89%), heart (80%), and soleus (103%) muscles, but higher levels in the liver (35%). Hyperthyroidism decreased the ratio of CBS/CSE mRNA expression in the liver and the CSE/CBS mRNA expression in the muscles by decreasing CBS levels in liver (34% cf. controls) and CSE levels in the aorta, heart, and soleus muscles (respectively, 51%, 7%, and 52% cf.). In addition, hyperthyroidism decreased the mRNA expression of 3-MST in the liver (51%) and aorta (33%), and increased it in the heart (300%) and soleus muscle (182%). In conclusion, hyperthyroidism increased H₂S levels in the liver and decreased it in muscles; these effects are at least in part due to increases and decreases in expression of CSE in the liver and muscles, respectively. These data indicate an association between thyroid hormone status and gene expression of the H₂S-producing enzymes in the rat.     

Cystathionine γ lyase–hydrogen sulfide increases peroxisome proliferator-activated receptor γ activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes

Adipocytes express the cystathionine γ lyase (CSE)–hydrogen sulfide (H₂S) system. CSE–H₂S promotes adipogenesis but ameliorates adipocyte insulin resistance. We investigated the mechanism of how CSE–H₂S induces these paradoxical effects. First, we confirmed that an H₂S donor or CSE overexpression promoted adipocyte differentiation. Second, we found that H₂S donor inhibited but CSE inhibition increased phosphodiesterase (PDE) activity. H₂S replacing isobutylmethylxanthine in the differentiation program induced adipocyte differentiation in part. Inhibiting PDE activity by H₂S induced peroxisome proliferator activated receptor γ (PPARγ) protein and mRNA expression. Of note, H₂S directly sulfhydrated PPARγ protein. Sulfhydrated PPARγ increased its nuclear accumulation, DNA binding activity and adipogenesis gene expression, thereby increasing glucose uptake and lipid storage, which were blocked by the desulfhydration reagent DTT. H₂S induced PPARγ sulfhydration, which was blocked by mutation of the C139 site of PPARγ. In mice fed a high-fat diet (HFD) for 4 weeks, the CSE inhibitor decreased but H₂S donor increased adipocyte numbers. In obese mice fed an HFD for 13 weeks, H₂S treatment increased PPARγ sulfhydration in adipose tissues and attenuated insulin resistance but did not increase obesity. In conclusion, CSE–H₂S increased PPARγ activity by direct sulfhydration at the C139 site, thereby changing glucose into triglyceride storage in adipocytes. CSE–H₂S-mediated PPARγ activation might be a new therapeutic target for diabetes associated with obesity.     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.     

H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal KATP channel

Here we explored the impact of hydrogen sulfide (H₂S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10 mM). GYY4137 slowly released appreciable levels of H₂S in the range of 10–275 μM, and H₂S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24 h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H₂S synthesizing enzyme) and ATP-sensitive potassium (K_ATP) channels. The K_ATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the K_ATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H₂S causes the relaxation of human ASM and implicate as well the role for sarcolemmal K_ATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H₂S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.