Cystathionine Γ-lyase Inhibitor Research Articles

Folic acid mitigated cardiac dysfunction by normalizing the levels of tissue inhibitor of metalloproteinase and homocysteine-metabolizing enzymes postmyocardial infarction in mice

Myocardial infarction (MI) results in significant metabolic derangement, causing accumulation of metabolic by product, such as homocysteine (Hcy). Hcy is a nonprotein amino acid generated during nucleic acid methylation and demethylation of methionine. Folic acid (FA) decreases Hcy levels by remethylating the Hcy to methionine, by 5-methylene tetrahydrofolate reductase (5-MTHFR). Although clinical trials were inconclusive regarding the role of Hcy in MI, in animal models, the levels of 5-MTHFR were decreased, and FA mitigated the MI injury. We hypothesized that FA mitigated MI-induced injury, in part, by mitigating cardiac remodeling during chronic heart failure. Thus, MI was induced in 12-wk-old male C57BL/J mice by ligating the left anterior descending artery, and FA (0.03 g/l in drinking water) was administered for 4 wk after the surgery. Cardiac function was assessed by echocardiography and by a Millar pressure-volume catheter. The levels of Hcy-metabolizing enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 5-MTHFR, were estimated by Western blot analyses. The results suggest that FA administered post-MI significantly improved cardiac ejection fraction and induced tissue inhibitor of metalloproteinase, CBS, CSE, and 5-MTHFR. We showed that FA supplementation resulted in significant improvement of myocardial function after MI. The study eluted the importance of homocysteine (Hcy) metabolism and FA supplementation in cardiovascular disease.

H 2 S mediates O 2 sensing in the carotid body

Gaseousmessengers, nitric oxide and carbon monoxide, have been implicated in O2 sensing by the carotid body, a sensory organ that monitors arterial blood O2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H2S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine gamma-lyase (CSE), an H2S-generating enzyme, with hypoxia increasing H2S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H2S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H2S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.

Upregulation of Ca v3.2 T-type calcium channels targeted by endogenous hydrogen sulfide contributes to maintenance of neuropathic pain

Hydrogen sulfide (H 2S) formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE) is now considered a gasotransmitter in the mammalian body. Our previous studies have shown that H 2S activates/sensitizes Ca v3.2 T-type Ca 2+ channels, leading to facilitation of somatic and visceral nociception, and that CSE-derived endogenous H 2S participates in inflammatory pain. Here, we show novel evidence for involvement of the endogenous H 2S–Ca v3.2 pathway in neuropathic pain. In the rat subjected to the right L5 spinal nerve cutting (L5SNC), a neuropathic pain model, i.p. administration of d l-propargylglycine (PPG) and β-cyanoalanine, irreversible and reversible CSE inhibitors, respectively, strongly suppressed the neuropathic hyperalgesia/allodynia. The anti-hyperalgesic effect of PPG was reversed by intraplantar administration of NaHS, a donor for H 2S, in the L5SNC rat. Intraplantar administration or topical application of mibefradil, a T-type Ca 2+ channel blocker, reversed hyperalgesia in the L5SNC rat. The protein levels of Ca v3.2, but not CSE, in the ipsilateral L4, L5 and L6 dorsal root ganglia were dramatically upregulated in the L5SNC rat. Finally, silencing of Ca v3.2 in DRG by repeated intrathecal administration of Ca v3.2-targeting siRNA significantly attenuated the neuropathic hyperalgesia in the L5SNC rat. In conclusion, our data suggest that Ca v3.2 T-type Ca 2+ channels in sensory neurons are upregulated and activated/sensitized by CSE-derived endogenous H 2S after spinal nerve injury, contributing to the maintenance of neuropathic pain. We thus propose that Ca v3.2 and CSE could be targets for the development of therapeutic drugs for the treatment of neuropathic pain.

A Crucial Role for Hydrogen Sulfide in Oxygen Sensing via Modulating Large Conductance Calcium-Activated Potassium Channels

Hydrogen sulfide (H(2)S) is an important signaling molecule produced from L-cysteine by cystathionine beta-synthetase (CBS) or cystathionine gamma-lyase (CSE). Here we examined the role of H(2)S in the oxygen-sensing function of the carotid body chemoreceptors, where the large conductance Ca(2+)-activated potassium channel (BK(Ca)) plays a key role. In the isolated mouse carotid body/sinus nerve preparations, the H(2)S donor, NaHS, excited the chemoreceptor afferent nerves in a concentration-dependent manner that was reversed by carbon monoxide donor. The NaHS-evoked excitation was abolished by removing extracellular Ca(2+), or using Cd(2+), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and hexomethonium, suggesting that H(2)S evokes release of ATP/ACh from type I glomus cells of the carotid body. The chemoreceptor afferent activation by hypoxia was decreased remarkably using CBS inhibitors, amino oxyacetic acid (AOAA) and hydroxylamine, but not CSE inhibitors, propargylglycine and beta-cyano-L-alanine, despite expression of both enzymes in type I glomus cells. In these cells, the BK(Ca) currents were inhibited by hypoxia and such inhibition was mimicked by NaHS and diminished by AOAA. Finally, mice hyperventilated in response to hypoxia, which was prevented by CBS inhibitors. These data suggest that H(2)S plays a crucial role in mediating the response of carotid body chemoreceptors to hypoxia via modulating the BK(Ca) channels.

Human pharmacology of hydrogen sulfide, putative gaseous mediator

Nitric oxide (NO), the first low molecular weight inorganic gas to be established as a biological mediator, had previously been regarded merely as a toxic pollutant. It was joined, similarly implausibly to the minds of a generation brought up with the hazards of coal gas as domestic heating, by carbon monoxide (CO) – potentially lethal as an exhaust gas, but also formed in mammalian tissues together with biliverdin by inducible and/ or constitutive forms of haem oxygenase, and implicated subsequently as a signalling molecule, not only in the central nervous system (especially olfactory pathways) and cardiovascular system but also in respiratory, gastrointestinal, endocrine and reproductive functions [1]. Hydrogen sulfide (H2S) was known to generations of schoolboys in one biological context – as the source of the odour of rotten eggs – but even so, the proposal in the early 2000s [2] that it too is a gaseous mediator was met with some scepticism.The toxicology of H2S had previously been studied [3], and actions on enzymes including monoamine oxidase and carbonic anhydrase identified, but more recent work has also shown a rich and diverse pharmacology consistent with functions as a signalling molecule under physiological conditions. It has also been implicated both as a harmful and also as a protective factor in the pathophysiology of a range of experimental models of disease [4], and its therapeutic potential has attracted comment [5]. There are striking similarities between these three gaseous mediators, as well as contrasts, that underlie complex and imperfectly understood functional interactions between them. All three are highly diffusible labile molecules that are rapidly eliminated from the body: NO as nitrite and nitrate in urine as well as NO in exhaled air; CO in exhaled air; H2S as thiosulfate, sulfite and sulfate in urine (Figure 1) as well as (it emerges, see below) in exhaled breath. All three react with haemoglobin, yielding methaemoglobin or nitrosylhaemoglobin (respectively inactive and active metabolites) from distinct reactions of haem and of globin with NO; carboxyhaemoglobin or sulfhaemoglobin from CO or H2S. All three affect cellular energetics via actions on cytochrome c oxidase. All three have vasodilator effects, and all have anti-inflammatory and cytoprotective effects at low concentrations in contrast to causing cellular injury at higher concentrations – consistent with Paracelsus’s aphorism that the distinction between drugs and poisons is determined exclusively by the dose.

Role of Hydrogen Sulfide Production in Inhibitory Action of L-Cysteine on Isolated Porcine Irides

Purpose: To investigate the direct pharmacological actions of L-cysteine, a substrate for the production of H2S, on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we examined the underlying mechanism of action of L-cysteine in this smooth muscle.Methods: Isolated porcine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer solution at 37°C. Longitudinal isometric tension was recorded via a grass FT03 Force-Displacement Transducer and analyzed using the PolyView computer software. The relaxant action of L-cysteine on carbachol-induced tone was studied in the absence and presence of inhibitors of enzymes of the biosynthetic pathways for H2S, and prostanoids. In addition, we also examined the effect of ATP-sensitive K+ (KATP) channel antagonist, glibenclamide on relaxations induced by L-cysteine.Results: L-cysteine (30 nM–1 mM) evoked concentration-dependent relaxations of carbachol-induced tone in isolated porcine irides, reaching a maximum inhibition of 43% at 1 mM. This response was enhanced significantly (P < 0.001) in the presence of the COX inhibitor, flurbiprofen (3 μM). Additionally,in the presence of flurbiprofen, the H2S donors, NaHS and Na2S, mimicked the relaxations produced by L-cysteine, yielding IC50 values of 5.8 μM and 180 μM, espectively. Both the inhibitor of cystathionine β-synthase, AOA (30 μM) and the KATP channel antagonist, glibenclamide (100 μM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to L-cysteine and attenuated the maximum inhibitory response. Conversely, the inhibitor of cystathionine γ-lyase, PAG (1 mM) blocked only relaxations caused by high concentrations of L-cysteine (> 100 μM).Conclusions: The inhibitory action of L-cysteine in isolated porcine irides is dependent on the endogenous production of H2S by cystathionine γ-lyase and cystathionine β-synthase. Furthermore, prostanoids and KATP channels are involved in the inhibitory action of L-cysteine in this tissue.

Role of Hydrogen Sulfide in Severe Burn Injury-Induced Inflammation in Mice

Endogenous hydrogen sulfide (H(2)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 H(2)S-synthesizing activity, plasma H(2)S level and liver and lung CSE mRNA expression and histological examination of tissues were performed after burn injury. Burn injury significantly increased the plasma H(2)S level and liver H(2)S synthesis 8 h after burn compared with the sham group. Burn injury also resulted in a significant upregulation of CSE mRNA in liver and lung. Prophylactic as well as therapeutic administration of PAG significantly reduced burn-associated systemic inflammation, as evidenced by MPO activity and histological changes in liver and lung. Injection of NaHS significantly aggravated burn-associated systemic inflammation. Therefore, our findings show for the first time the role of H(2)S in contributing to inflammatory damage after burn injury.

Intermittent Hypoxia in Rats Reduces Hydrogen Sulfide Dependent Dilation to Enhance Myogenic Tone in Mesenteric Arteries

Sleep apnea is an independent risk factor for hypertension and rats exposed to intermittent hypoxia (IH) develop hypertension. We have shown that exposing rats for 14 days to IH enhances myogenic tone (MT) in small mesenteric arteries (MA) compared to MA from control (Sham) rats. Endothelial disruption enhanced MT in Sham arteries but had no effect in IH arteries. H2S is an endogenous vasodilator produced by cystathionine‐γ‐lyase (CSE) in the vasculature. We hypothesized that H2S produced by CSE in the endothelium reduces MT in MA from Sham rats and that H2S production in MA is impaired after 14 days of IH. Vessel wall [Ca2+]i and inner diameter were recorded in pressurized, fura‐2 loaded MA from IH and Sham rats. The CSE inhibitor β cyano L‐alanine (BCA) enhanced MT in Sham arteries compared to vehicle (20.2 ± 6.5% vs. 8.6 ± 1.0) and had no effect in IH arteries (24.0 ± 5.2 vs. 23.6 ± 7.9%). The H2S scavenger Bi(III) subsalicylate enhanced MT in Sham arteries similarly to BCA (20.4 ± 3.4%). NaHS (H2S donor) dilated both Sham and IH endothelium disrupted arteries (42.7 ± 5.7 vs. 57.5 ± 14.4%) with MT. These results suggest that H2S produced by CSE reduces MT in small endothelium intact MA and IH abolishes this effect of H2S. Dilation to NaHS is unaffected in IH endothelium disrupted arteries suggesting that enhanced MT is due to lower levels of endogenous H2S in MA.This work was supported by NIH grants HL82799 (NLK) and HL07736 (OJW).

CHRONIC BLOCKADE OF ENDOGENOUS FORMATION OF HYDROGEN SULFIDE CAUSED INCREASE IN SYSTEMIC BLOOD PRESSURE IN RATS

Hydrogen sulfide (H2S), an endogenous vasodilator, is produced by two enzymes; cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS). In this study, the effects of chronic inhibition of CSE by DL‐propargylglycine (37.5 mg/kg/day; ip) and CBS by aminoxyacetic acid (8.8 mg/kg/day; ip) given either alone or in combination for 4 weeks on systemic blood pressure (SBP) and renal function were examined in rats (n=6 in each group). Prior to the start and then every 7th day during treatment period, SBP was measured by tail‐cuff method and 24 hr urine collections were made using metabolic cages. Urinary excretion rate of sulfate (USV; marker for endogenous H2S level) was determined by a colorimetric assay. At the end of treatment period, rats were subjected to acute experiments under anesthesia for determination of renal blood flow (RBF; Transonic flow‐probe) and glomerular filtration rate (GFR; inulin clearance). Compared to vehicle treated group, only the combined therapy group showed a decrease in USV (553±71 vs 248±50 nmol/24hr) and an increase in mean SBP (107±3 vs 130±2 mmHg) with its consequent increase in sodium excretion (4±1 vs 13±3 mmol/24hr). This group with combined therapy also showed a decrease in RBF (7±0.4 vs 4±0.3 mL/min/g) but not appreciably in GFR (1.0±0.1 vs 0.9±0.03 mL/min/g). These data suggest that a decrease in endogenous H2S production exerts direct vascular effect leading to an increase in SBP.

L-Cysteine Stimulates Hydrogen Sulfide Synthesis in Myocardium Associated With Attenuation of Ischemia-Reperfusion Injury

Hydrogen sulfide (H( 2)S) is a biological mediator produced by enzyme-regulated pathways from L-cysteine, which is a substrate for cystathionine-gamma-lyase (CSE). In myocardium, endogenously and exogenously administered H(2)S has been shown to protect against ischemia-reperfusion injury. We hypothesized that L-cysteine exerts its protective action through stimulation of H(2)S production. Rat isolated hearts were Langendorff-perfused and underwent 35-minute regional ischemia and 120-minute reperfusion. L-cysteine perfusion from 10 minutes before ischemia until 10 minutes after reperfusion limited infarct size in a concentration-dependent manner, maximal at 1 mmol/L (control 36.4% +/- 2.4% vs L-cysteine 24.3% +/- 3.4%, P < .05). This protective action was attenuated by the CSE inhibitor, DL-propargylglycine (PAG) 1 mmol/L (31.4 +/- 5.9%, not significant vs control) but administration of the CSE cofactor pyridoxal-5'-phosphate (PLP) 50 mumol/L did not enhance the effect of L-cysteine. Ten minutes normoxic perfusion with L-cysteine 1 mmol/L caused a 3-fold increase in myocardial H(2)S concentration (0.64 +/- 0.16 vs 2.01 +/- 0.07 mumol/g protein, P < .01), an effect that was significantly attenuated by PAG (1.17 +/- 0.15 mumol/g protein). These data provide evidence that exogenous L-cysteine administration limits ischemia-reperfusion injury through a mechanism that appears to be at least partially dependent on H(2)S synthesis.

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

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

Rhodanese, but not cystathionine-γ-lyase, is associated with dextran sulfate sodium-evoked colitis in mice: A sign of impaired colonic sulfide detoxification?

Clinical studies suggest that colonic luminal hydrogen sulfide (H 2S), produced by sulfate-reducing bacteria or through other pathways, might be involved in the pathogenesis of inflammatory bowel disease (IBD). Nonetheless, this hypothesis has been poorly investigated by basic studies using laboratory animals. We thus focused on two enzymes, cystathionine-γ-lyase (CSE) that generates H 2S from l-cysteine, and rhodanese that directly or indirectly detoxifies H 2S, particularly in relation to the colitis induced by dextran sulfate sodium (DSS) in mice. CSE was a major H 2S-forming enzyme in colonic and renal homogenates from mice and rats, and the rhodanese activity was also detectable in both tissues. Colitis-related symptoms including decreased body weight gain, diarrhea, hematochezia and shortening of colon length were observed in the mice drinking DSS. Those symptoms were not or only slightly attenuated by repeated administration of a CSE inhibitor. CSE activity and protein levels in the colonic tissue did not notably change in the mice with colitis. In contrast, the activity and protein/mRNA levels of rhodanese in the colon, but not kidney, significantly decreased nearly in parallel with the development of colitis, followed by elevation of rhodanese activity in red blood cells (RBCs). These data show that rhodanese, but not CSE, is associated with DSS-induced colitis in mice, leading to a hypothesis that impaired detoxification of H 2S due to down-regulation or suppression of colonic rhodanese is involved in IBD. The delayed enhancement of rhodanese activity in RBCs, a possible compensative event, might be available as a disease marker for IBD.

Hydrogen sulfide as a mediator of human corpus cavernosum smooth-muscle relaxation

Hydrogen sulfide (H(2)S) is synthesized by 2 enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). L-Cysteine (L-Cys) acts as a natural substrate for the synthesis of H(2)S. Human penile tissue possesses both CBS and CSE, and tissue homogenates efficiently convert L-Cys to H(2)S. CBS and CSE are localized in the muscular trabeculae and the smooth-muscle component of the penile artery, whereas CSE but not CBS is also expressed in peripheral nerves. Exogenous H(2)S [sodium hydrogen sulfide (NaHS)] or L-Cys causes a concentration-dependent relaxation of strips of human corpus cavernosum. L-Cys relaxation is inhibited by the CBS inhibitor, aminoxyacetic acid (AOAA). Electrical field stimulation of human penile tissue, under resting conditions, causes an increase in tension that is significantly potentiated by either propargylglycine (PAG; CSE inhibitor) or AOAA. In rats, NaHS and L-Cys promote penile erection, and the response to L-Cys is blocked by PAG. Our data demonstrate that the L-Cys/H(2)S pathway mediates human corpus cavernosum smooth-muscle relaxation.

Hydrogen sulfide as a novel mediator for pancreatic pain in rodents

Objective:Hydrogen sulfide (H2S) is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H2S in...

Structural Basis for the Inhibition Mechanism of Human Cystathionine γ-Lyase, an Enzyme Responsible for the Production of H2S

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

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H 2S-producing enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. l-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with l-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl-propargylglycine antagonized these l-cysteine effects. We suggest H 2 S may function as an ‘intrinsic brake’ which protects beta-cells from glucotoxicity.

Mechanisms responsible for pulmonary hypertension

Mechanisms responsible for pulmonary hypertension

Inhibitory action of hydrogen sulfide on muscarinic receptor-induced contraction of isolated porcine irides

We investigated the pharmacological actions of hydrogen sulfide (H 2S) using sodium hydrosulfide (NaHS) and sodium sulfide (Na 2S) as donors on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we also investigated the mechanism of action of H 2S in this smooth muscle. Isolated porcine iris muscle strips were set up in organ baths and prepared for measurement of longitudinal isometric tension. The relaxant action of NaHS or Na 2S on carbachol-induced tone was studied in the absence and presence of a K +-channel inhibitor and inhibitors/activators of enzymes of the biosynthetic pathways for H 2S, prostanoid and nitric oxide production. In the concentration range, 10 nM to 100 μM, NaHS produced a concentration-dependent relaxation of carbachol-induced tone reaching a maximum of inhibition of 28% at 30 μM. The cyclooxygenase inhibitor, flurbiprofen (1 μM), enhanced relaxations induced by both NaHS and Na 2S yielding IC 50 values of 7 μM and 70 μM, respectively. With exception of l-NAME (300 μM) inhibitors of cystathionine γ-lyase, propargylglycine, (PAG) (1 mM) and β-cyanoalanine, (BCA) (1 mM) and inhibitors of cystathionine β-synthase, aminooxyacetic acid (AOA) (30 μM) and hydroxylamine (HOA) (30 μM) caused significant ( P < 0.001) rightward shifts in the concentration–response curves to NaHS. An activator of cystathionine β-synthase, SAM (100 μM), enhanced relaxations elicited by low concentrations of NaHS but attenuated responses caused by the higher concentrations of this H 2S donor. The inhibitor of K ATP channel, glibenclamide (100 and 300 μM), blocked relaxations induced by NaHS. We conclude that the observed inhibitory action of NaHS and Na 2S in isolated porcine irides is dependent on endogenous production of prostanoids and the biosynthesis of H 2S by cystathionine γ-lyase and cystathionine β-synthase. Furthermore, relaxation induced by H 2S is mediated, at least in part, by K ATP channels. Nitric oxide is not involved in the relaxation induced by this gas in the isolated porcine irides.

Hydrogen Sulphide Is Involved in Testosterone Vascular Effect

BackgroundTestosterone (T) induces a rapid relaxation in vascular tissues of different species due to a nongenomic effect of this steroid on vessels. Different mechanisms have been proposed to explain T-induced vasodilatation but the effective mechanism(s) and the mediators involved are still a matter of debate. ObjectivesWe have evaluated if H2S pathway is involved in T vascular effects. Design and settingMale Wistar rats were sacrificed and thoracic aorta was rapidly dissected and cleaned from fat and connective tissue. Rings of 2–3mm length were cut and placed in organ baths filled with oxygenated Krebs solution at 37°C and mounted to isometric force transducers. H2S determination was performed on thoracic aortic rings incubated with T or vehicle and in presence of inhibitors. H2S concentration was calculated against a calibration curve of NaHS (3–250μM). Results were expressed as nmoles/mg protein. MeasurementsVascular reactivity was evaluated by using isometric transducers. H2S determination was performed by using a cystathionine β-synthetase (CBS) and cystathionine γ lyase (CSE) activity assay. CSE and CBS protein levels were assessed by Western blot analysis. Statistical analysis was performed by using two-way ANOVA and unpaired Student's t-test where appropriate. ResultsT significantly increased conversion of L-cysteine to H2S. This effect was significantly reduced by PGG and BCA, two specific inhibitors of CSE. T (10nM–10μM) induced a concentration-dependent vasodilatation of rat aortic rings in vitro that was significantly and concentration-dependent inhibited by PGG, BCA, and glybenclamide. Incubation of aorta with T up to 1h did not change CBS/CSE expression, suggesting that T modulates enzymatic activity. ConclusionsHere we demonstrate that T vasodilator effect involves H2S, a novel gaseous mediator. T modulates H2S levels by increasing the enzymatic conversion of L-cysteine to H2S.

Pro‐inflammatory effects of hydrogen sulphide on substance P in caerulein‐induced acute pancreatitis

Hydrogen sulphide (H2S), a novel gasotransmitter, has been recognized to play an important role in inflammation. Cystathionine-γ-lyase (CSE) is a major H2S synthesizing enzyme in the cardiovascular system and DL-propargylglycine (PAG) is an irreversible inhibitor of CSE. Substance P (SP), a product of preprotachykinin-A (PPT-A) gene, is a well-known pro-inflammatory mediator which acts principally through the neurokinin-1 receptor (NK-1R). We have shown an association between H2S and SP in pulmonary inflammation as well as a pro-inflammatory role of H2S and SP in acute pancreatitis. The present study was aimed to investigate the interplay between pro-inflammatory effects of H2S and SP in a murine model of caerulein-induced acute pancreatitis. Acute pancreatitis was induced in mice by 10 hourly intraperitoneal injections of caerulein (50 (g/kg). PAG (100 mg/kg, i.p.) was administered either 1 hr before (prophylactic) or 1 hr after (therapeutic) the first caerulein injection. PAG, given prophylactically as well as therapeutically, significantly reduced plasma H2S levels and pancreatic H2S synthesizing activities as well as SP concentrations in plasma, pancreas and lung compared with caerulein-induced acute pancreatitis. Furthermore, prophylactic as well as therapeutic administration of PAG significantly reduced PPT-A mRNA expression and NK-1R mRNA expression in both pancreas and lung when compared with caerulein-induced acute pancreatitis. These results suggest that the pro-inflammatory effects of H2S may be mediated by SP-NK-1R pathway in acute pancreatitis.