Effect Of NaHS Research Articles

OP24 Characterization of TRPA1-mediated and TRPA1-independent microcirculatory responses to hydrogen sulfide in mice

Capsaicin-sensitive sensory neurons express several ion channels of the TRP family, such as transient receptor potential ankyrin 1 (TRPA1) receptors. TRPA1 can be activated by several chemical stimuli, such as allylisothiocyanate (AITC) or cinnamaldehyde resulting in the release of inflammatory neuropeptides, e.g. calcitonin gene-related peptide (CGRP) [1] . Recently, the gaseous mediator hydrogen sulfide (H2S) was suggested to have effect on capsaicin-sensitive sensory neurons acting via TRPA1 receptors. Microcirculatory changes in the mouse ear and hind paw have been investigated in this study. Balb/c, C57BL/6, TRPA1 KO and TRPV1 KO mice (20–30 g) were treated with sodium hydrogen sulfide (NaHS) or AITC. Ipsilateral ears of the animals were treated with 2% AITC or 5% NaHS. Contralateral ears received respective vehicles. Balb/c mice received selective TRPA1 receptor antagonist HC-030031 (i.p., 30 or 100 mg/kg). NaHS (0.5 μmol/10 μL Tyrode) was injected s.c. into hind paws of C57BL/6 and TRPA1 KO mice. Skin blood flow was detected by laser Doppler imaging. AITC treatment of mouse ears led to 29.8 ± 2.8% increase in blood flow. NaHS elevated cutaneous blood flow of the mouse ears by 61 ± 4.5%. Effect of NaHS on microcirculation was ameliorated by HC-030031. Blood flow of TRPA1 KO mice showed significantly smaller increase in response to NaHS compared to the wild-type counterparts. Vasodilatation in TRPV1 KO animals did not differ from control. CGRP8−37 peptide antagonist of CGRP only partly inhibited the response. Intraplantar administration of NaHS elevated blood flow of murine hind paws by 34.1 ± 2.8%. The plantar microcirculatory response to NaHS was unaffected by genetic lack of TRPA1 receptor or resiniferatoxin pretreatment. We conclude that NaHS-induced vasodilatation in the mouse ear is partly mediated by TRPA1 receptors, but is independent of sensory nerves in the skin of the hind paw.

Sa2024 Involvement of Gastric Secretion and Primary Capsaicin-Sensitive Afferent Nerves in the Gastroprotection Induced by Hydrogen Sulfide in Rat Stomach

Hydrogen sulfide (H2S) modulates a number of physiological actions including vasodilatation, inhibition of oxidant stress and apoptosis. H2S which is formed in gastric epithelium by the activity of two enzymes cysthationine γ-lyase (CSE) and cystathionine β-synthetase (CBS) has been shown to protect the gastric mucosa from ethanol-induced gastric lesions but whether its efficacy against acid-dependent stress-induced damage has been little studied. It also remains unknown how the expression of CSE and CBS contribute to the mucosal damage induced by stress in the presence or absence of H2S. We studied the effect of NaHS, a H2S-donor and L-cysteine, a H2S precursor on 1) gastric acid secretion in rats equipped with chronic gastric fistulas (GF) and water immersion restraint stress (WRS)-induced gastric lesions with intact and capsaicin-denervated sensory nerves (large dose 125 mg/kg s.c for 3 days). Rats were pretreated 30 min before the WRS with A) NaHS (5 mg/kg i.g); B) Lcysteine (10 mg/kg i.g.) with or without the combination with inhibitors of CSE activity, beta-cyano-L-alanine (BCA, 50 mg/kg i.g.) and D,L-propargylglycine (PAG 15 80 mg/kg i.g.). The number of gastric lesions was measured by planimetry, the gastric blood flow (GBF) by H2-gas clearance technique and the mRNA expression of CSE, CBS, HIF-1 α and antioxidizing enzymes SOD and GPx was assessed by RT-PCR and Western Blot. NaHS (120 mg/kg) and cysteine (5-40 mg/kg) dose-dependently inhibited basal and histaminestimulated gastric acid secretion in GF rats. Exposure to WRS caused mucosal hemorrhagic lesions accompanied by the fall in GBF and upregulation of mRNA expression of CSE and CBS. Pretreatment with NaHS and L-cysteine significantly reduced WRS-induced gastric damage and significantly raised GBF and these effects were completely lost in animals with capsaicin denervation. BCA and PAG which dose-dependently augmented the number of WRS lesions, reversed the NaHS and L-cysteine-induced protection and hyperemia against WRS-induced gastric damage. Upregulation of mRNA expression for HIF-1 α mRNA, CSE and CBS in the gastric mucosa exposed to WRS was diminished by NaHS and L-cysteine and these effects were further enhanced in capsaicin-denervated rats treated with NaHS and L-cysteine. The increased expression of SOD and GPx mRNA was observed in NaHSand L-cysteine-pretreated rats but not in those with capsaicin-denervation. We conclude that: 1) an increase in the expression of CSE and CBS, key enzymes in H2S biosynthesis, could compensate for the stress-induced impairment of gastric mucosal defense; 2) antisecretory activity of H2S donors contributes to the attenuation of acid-dependent injury caused by WRS, and 3) sensory mediators and antioxidizing enzymes SOD and GPx play an important role in H2S-induced gastroprotection against WRS-induced ulcerogenesis.

Hydrogen Sulfide Treatment Promotes Glucose Uptake by Increasing Insulin Receptor Sensitivity and Ameliorates Kidney Lesions in Type 2 Diabetes

To examine if hydrogen sulfide (H2S) can promote glucose uptake and provide amelioration in type 2 diabetes. 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. 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.

The Inhibitory Role of Hydrogen Sulfide in Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

Cystathionine γ-lyase (CSE) is one of the major enzymes producing hydrogen sulfide (H2S) in lungs, participating in the regulation of respiratory functions. The role of CSE-derived H2S in eosinophil-dominant inflammation in allergic diseases has been unclear. The objective of this study was to explore the protective role of H2S against allergen-induced airway hyperresponsiveness (AHR) and inflammation. CSE expression and H2S production rate were assessed in mouse lung tissues with ovalbumin (OVA)-induced acute asthma. AHR, airway inflammation, and Th2 response in wild-type (WT) mice were compared with those in CSE gene knockout (KO) mice. The effect of NaHS, an exogenous H2S donor, was also evaluated on these parameters. CSE expression was absent and H2S production rate was significantly lower in the lungs of CSE KO mice when compared with WT littermates. OVA challenge decreased lung CSE expression and H2S production in WT mice. CSE deficiency resulted in aggravated AHR, increased airway inflammation, and elevated levels of Th2 cytokines such as IL-5, IL-13, and eotaxin-1 in bronchoalveolar lavage fluid after OVA challenge. The aforementioned alterations were reversed by exogenous H2S treatment. More importantly, NaHS supplement rescued CSE KO mice from the aggravated pathological process of asthma. The CSE/H2S system plays a critical protective role in the development of asthma. A new therapeutic potential for asthma via targeting CSE/H2S metabolism is indicated.

Hydrogen sulfide in regulation of frog myocardium contractility

Hydrogen sulfide (H2S) is an endogenously synthesized gaseous molecule which, along with nitric oxide and carbon monoxide, induces a number of effects in cardiovascular system under normal and pathological conditions. In the present work, the effects and underlying mechanisms of the H2S donor sodium hydrosulfide (NaHS) on the isometric force of frog myocardium contraction have been studied. NaHS at the concentration of 100 μM induced negative inotropic effect and reduced the maximum velocity of the contraction and relaxation of the isolated ventricle strips. The substrate of H2S synthesis, L-cysteine (200 μM and 1 mM), induced the same effect, while the inhibitors of cystathionin-γ-lyase, the H2S-producing enzyme in heart, β-cyanoalanine (500 μM) and propargylglycine (500 μM), increased the amplitude of contraction. Inhibition of cystathionin-γ-lyase by β-cyanoalanine prevented the negative inotropic effect of L-cysteine. After the inhibition of adenylate cyclase by MDL-12,330A (3 μM) or phosphodiesterases by IBMX (200 μM), the effect of NaHS was less than that in the control. In the presence of membrane-penetrating analogous of cAMP, 8Br-cAMP (100 μM) and pCPT-cAMP (100 μM), the negative inotropic effect of NaHS was completely retained. The effect of NaHS significantly decreased after preliminary application of the NO donor, SNAP (10 μM), and did not change after the inhibition of NO synthases by L-NAME (100 μM). The results suggest the possibility of endogenous synthesis of H2S in frog myocardium and regulation of its contractility by the activation of phosphodiesterases hydrolyzing cAMP, which leads to a decrease in the activation of cAMP-dependent protein kinases and phosphorylation of voltage-dependent L-type Ca channels. As a result, the reduction of calcium entry into cardiomyocytes decreases the contractility of frog myocardium.

Hydrogen sulfide suppresses outward rectifier potassium currents in human pluripotent stem cell-derived cardiomyocytes.

AimHydrogen sulfide (H2S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H2S.Methods and ResultsCardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H2S, on action potential (AP), outward rectifier potassium currents (I Ks and I Kr), L-type Ca2+ currents (I CaL) and hyperpolarization-activated inward current (I f) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I K (I Ks and I Kr), I CaL and I f were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties.ConclusionsThis is the first demonstration of effects of H2S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H2S on I CaL and revealed additional novel inhibitory effects on I f, I Ks and I Kr currents in human cardiomyocytes.

P52 Perfusion of isolated carotid sinus with hydrogen sulfide attenuated the renal sympathetic nerve activity in anesthetized male rats

The present study was to define the effect of H 2 S on central region which regulates sympathetic outflow by perfusion of isolated carotid sinus with H 2 S. Sodium hydrogen sulfide (NaHS), a H 2 S donor, was perfused into isolated carotid sinus; the functional curve of the carotid sinus baroreflex was measured by recording changes in renal sympathetic nerve activity (RSNA) in anesthetized male rats. (1) Perfusion of isolated carotid sinus with NaHS (25, 50, and 100 μmol/L) dose-dependently inhibited sympathetic outflow by enhancing the response of RSNA to the increased intrasinus pressure (ISP). RSNA was decreased to 84.95 ± 3.58% ( P < 0.01), 63.89 ± 2.53% ( P < 0.01) and 48.70 ± 4.16% ( P < 0.01) compare with control. (2) Pretreatment with glibenclamide (20 μmol/L), a K ATP channels blocker, the above effect of NaHS was abolished. (3) Prior perfusion of Bay K8644 (500 nmol/L), an agonist of calcium channels, the effect of NaHS was eliminated. (4) Perfusion of the cystathionine γ-lyase (CSE) inhibitor, DL -propargylglycine (PPG, 200 μmol/L) increased sympathetic outflow. The results suggest that perfusion of isolated carotid sinus with NaHS inhibits sympathetic outflow as evidenced by the decrease of RSNA. The effect is mediated by opening a K ATP channels and further closing the calcium channel in smooth muscle cells. Endogenous H 2 S may tonically suppress the sympathetic vasomotor by activating the activity of the carotid sinus baroreflex (CSB).

P36 Hydrogen sulfide inhibits RANKL-induced ROS production and osteoclast differentiation

Osteoclasts (OC), the bone resorbing cells, are multinucleated cells arising from the fusion of circulating monocytic precursors. Once differentiated, they settle into bone and break down bone mineral matrix, thereby playing a vital role in physiological bone turnover.In post-menopausal osteoporosis or in inflammatory bone disease, OC are activated leading to an excess bone erosion which in not balanced by an equal bone formation by osteoblasts, a process that leads to a loss of bone mass, increased bone fragility and fracture risk. The rate of monocyte differentiation into OC has been shown to be a critical step in pathological bone erosion.Here we sought to investigate whether hydrogen sulfide affect OC differentiation and function. Human osteoclast precursors were isolated from peripheral blood obtained from healthy donors by immunomagnetic positive selection of CD11b+ cells. Mature OC were obtained in vitro by culturing CD11b+ cells for 8–10 days in the presence of M-CSF (10 ng/ml) and RANKL (75 ng/ml). The effect of hydrogen sulfide was evaluated by treating the cells with NaHS in the range of concentrations between 50 and 300 μg/ml.We first assessed whether NaHS affect cell survival and proliferation. LDH activity assay revealed no cytotoxic effect of NaHS at 50, 10, 200 or 300 μg/ml at 24 h after stimulation, while 72 h after stimulation the concentration of 300 μg/ml induced a 25% increase in cell death. Cell proliferation was evaluated by 3 H-timidine uptake: consistent with citotoxicity data, NaHS did not affect the proliferation of CD11b+ cells in response to M-CSF stimulation up to a concentration of 200 μg/ml. Evaluation of apoptosis by Annexin V/PI further confirmed that NaHS did not induced cell death at 50, 100 or 200 μg/ml while induced a 30% increase of apoptosis at the concentration of 300 μg/ml. To assess the effect of NaHS on OC differentiation, TRAP + mature OC were counted at the end of the culture. NaHS dose-dependently inhibited OC formation, leading to a significant decrease in OC number starting a 100 μg/ml. Functional Pit Assay confirmed that NaHS significantly inhibits the ability of OC to break down a bone-like mineral matrix. Attesting to specificity, similar data were obtained in the presence of the H 2 S-donor GYY4137. Therefore, hydrogen sulfide inhibits OC differentiation and function in a range of concentration which does not affect cell survival. As RANKL signaling is critically dependent on ROS generation in OC progenitor cells, we evaluated the effect of NaHS on RANKL-dependent ROS generation by Dichlorofluorescein staining. As expected, RANKL (100 μg/ml, 30 min) induced a significant production of intracellular ROS in OC precursors cells; however, NaHS (200 μg/ml), significantly inhibited RANKL-induced ROS generation.In conclusion, Hydrogen sulfide dose-dependently prevents OC differentiation and function and this effect is associated to a significant inhibition of RANKL-induced ROS production. These findings suggest a possible therapeutical role for hydrogen sulfide donors in the treatment of pathological bone loss.

P26 Modulation of hydrogen sulfide and nitric oxide on tension of isolated human greater omentum artery

Background Hydrogen Sulfide has been considered the third gaseous transmitter after nitric oxide and carbon monoxide. Increasing evidence indicates that hydrogen Sulfide has protective effect on cardiac and hepatic I/R injury or protective effect on remote organs of limbs I/R injury in rats. In this study, we investigate the modulation of hydrogen sulfide and nitric oxide on tension of isolated human greater omentum artery. Methods The specimens of small arteries within greater omentum were harvested from 15 patients suffering from gastric cancer or colon-rectal cancer without local or distal transmission during open radical operation. The arteries were cut into about 2 mm artery rings. The dose–response curves of hydrogen sulfide donor NaHS (10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 mol/L) and nitric oxide donor sodium nitroprusside (10 −5 , 10 −6 , 10 −7 , 10 −8 mol/L) to artery rings were recorded with Powerlab and DMT system. Glibenclamide (10 −4 mol/L), nitrendipine (10 −4 mol/L) and Nω -nitro- l -arginine methyl ester (L-NAME) (10 −4 mol/L) were used to study on the relaxation effect of NaHS (10 −3 mol/L). Results NaHS and sodium nitroprusside had a dose-dependent relaxation on isolated human greater omentum artery. Glibenclamide and Nω -nitro- l -arginine methyl ester (L-NAME) could make an inhibition of relaxation effect induced by NaHS, whereas nitrendipine could make a slightly enhancement. Interestingly, NaHS (10 −3 M) could make a brief contraction to achieve maximal relaxation followed by another contraction. Conclusion Hydrogen sulfide and nitric oxide could make an independent and collaborative relaxation on human greater omentum artery, but hydrogen sulfide at a specific concentration can cause a short contraction on the artery.

P45 Hydrogen sulfide protects coronary artery vasospasm after myocardial infarction and eliminates myocardial infarction zone by promoting to growth new vessels

Hydrogen sulfide (H 2 S) has recently been identified as a regulator of various physiological events including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. H 2 S leads to down-regulation of inflammatory responses and provides myocardial protection during acute myocardial infarction (MI). We previously reported that H 2 S inhibits antiangiogenic factors such as endostatin and angiostatin, leading to development of new vessels. We have shown that coronary vessels elicited vasoconstriction to acetylcholine (Ach) and revealed a phenomenon of vasospasm; we examined the role of hyperhomocysteinemia in coronary endothelial dysfunction, vessel wall thickness, and lumen narrowing, which leads to acute/chronic coronary vasospasm. We hypothesize that H 2 S eliminates vessel damage and improves endothelial function, which causes the elimination of coronary vaso-constriction. To verify this, MI was created in wild-type mice, which were treated with sodium hydrosulfide (NaHS, H 2 S donor) in drinking water and compared with untreated control mice. The echocardiography in mice treated with H 2 S showed improvement of heart function compared to untreated mice. The X-ray and Doppler blood flow measurements showed enhancement of cardiac-angiogenesis in mice treated with H 2 S. This observed cardio-protection was associated with an inhibition of anti-angiogenic proteins and stimulation of angiogenic factors. The endothelium-impaired arteries from MI mice were constricted in response to Ach, and this vasoconstriction was mitigated with NaHS supplementation. The level of endothelial nitric oxide synthase (eNOS) was lower in coronary artery in MI than of MI + NaHS mice. Treatment with NaHS increased the levels of Ach-induced NO generation in the coronary artery of MI mice. The results suggest that Ach induced coronary vasoconstriction in MI mice and this vasoconstriction was ameliorated by NaHS treatment. The mechanisms for the impairment of vascular function and therapeutic effects of NaHS may be related to the regulation of eNOS expression, NO availability and tissue homocysteine. We established that administration of NaHS at the time of MI ameliorated infarct size and preserved LV function during development of MI in mice, which also improved coronary endothelial function and promoted new vessel growth.

Mechanism of action of hydrogen sulfide on cyclic AMP formation in rat retinal pigment epithelial cells

Hydrogen sulfide (H2S), a colorless gas with the pungent odor of rotten eggs has been reported to produce pharmacological actions in ocular and non-ocular tissues. We have evidence that H2S, using sodium hydrosulfide (NaHS) and sodium sulfide (Na2S) as donors can increase cyclic AMP (cAMP) production in neural retina. In the present study, we investigated the mechanism of action of H2S on cyclic nucleotide production in rat retinal pigment epithelial cells (RPE-J). Cultured RPE-J cells were incubated for 30min in culture medium containing the cyclic nucleotide phosphodiesterase (PDE) inhibitor, IBMX (2mM). Cells were exposed to varying concentrations of NaHS, the H2S substrate (l-cysteine), cyclooxygenase (COX) inhibitors or the diterpene activator of adenylate cyclase, forskolin in the presence or absence of H2S biosynthetic enzymes or the ATP-sensitive potassium (KATP) channel antagonist, glibenclamide. Following drug-treatment at different time intervals, cell homogenates were prepared for cAMP assay using a well established methodology. In RPE-J cells, NaHS (10nM–1μM) produced a time-dependent increase in cAMP concentrations over basal levels which reached a maximum at 20min. At this time point, both NaHS (1nM–100μM) and l-cysteine (1nM–10μM) produced a concentration-dependent significant (p<0.05) increase in cAMP concentrations over basal level. The effects of NaHS on cAMP levels in RPE-J cells was enhanced significantly (p<0.01) in the presence of the COX inhibitors, indomethacin and flurbiprofen. In RPE-J cells, the effects caused by forskolin (10μM) on cAMP production were potentiated by addition of low concentrations of NaHS. Both the inhibitor of cystathionine β-synthase (CBS), aminooxyacetic acid (AOA, 1mM) and the inhibitor of cystathionine γ-lyase (CSE), proparglyglycine (PAG, 1mM) significantly attenuated the increased effect of l-cysteine on cAMP production. The KATP channel antagonist, glibenclamide (100μM) caused inhibition of NaHS induced-increase of cAMP formation in RPE-J cells. We conclude that, H2S (using H2S donor and substrate) can increase cAMP production in RPE-J cells, and removal of the apparent inhibitory effect of prostaglandins unmasks an excitatory activity of H2S on cAMP. Effects elicited by the H2S substrate on cAMP formation are dependent on biosynthesis of H2S catalyzed by the biosynthetic enzymes, CBS and CSE. In addition to the adenylyl cylcase pathway, KATP channels are involved in mediating the observed effects of the H2S on cAMP production.

Role of ATP sensitive potassium channel in protective effects of post-conditioning with hydrogen sulfide against hepatic ischemia-reperfusion injury in rats

Objective To investigate the role of ATP sensitive potassium (KATP ) channel in protective effects of post-conditioning with hydrogen sulfide against hepatic ischemia-reperfusion (I/R) injury in rats.Methods Thirty adult male SD rats weighing 220-250 g were randomly divided into 5 groups ( n =6 each):sham operation gorup (group S) ; hepatic I/R group; NaHS group; glibenclamide + NaHS group (group G) and 5-hydroxydecanoate (5-HD) + NaHS group (group 5-HD).Hepatic ischemia was produced by 60 min occlusion of left and middle lobe blood flow (about 70％ of the whole liver blood flow) followed by 6 h reperfusion in groups I/R,NaHS,G and 5-HD.NaH S 28 μmol/kg was administered intraperitoneally (IP) at 5 min before reperfusion in groups NaHS,G and 5-HD.Glibenclamide 6 mg/kg and 5-HD 10 mg/kg (KATP channel blockers) were administered IP at 5 min before IP NaHS in groups G and 5-HD respectively.Blood samples were collected at the end of 6 h of reperfusion for measurement of ALT and AST activities and liver specimens were obtained for microscopic examination and measurement of TNF-α content and MPO activity.Results Liver I/R significantly increased ALT,AST and MPO activities and TNF-α concentration in group I/R as compared with group S.NaHS administered at 5 min before reperfusion significantly attenuated hepatic I/R injury.Glibenclamide and 5-HD administered before NaHS could block the protective effect of NaHS against hepatic I/R injury.Conclusion Hydrogen sulfide postconditioning can protect the liver from I/R injury by activating KATP channel. Key words: KATP channels; Hydrogen sulfide; Reperfusion injury; Livers

Electrophysiological effects of hydrogen sulfide on human atrial fibers.

It has been reported that endogenous or exogenous hydrogen sulfide (H(2)S) exerts physiological effects in the vertebrate cardiovascular system. We have also demonstrated that H(2)S acts as an important regulator of electrophysiological properties in guinea pig papillary muscles and on pacemaker cells in sinoatrial nodes of rabbits. This study was to observe the electrophysiological effects of H(2)S on human atrial fibers. Human atrial samples were collected during cardiac surgery. Parameters of action potential in human atrial specialized fibers were recorded using a standard intracellular microelectrode technique. NaHS (H(2)S donor) (50, 100 and 200 µmol/L) decreased the amplitude of action potential (APA), maximal rate of depolarization (V(max)), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF), and shortened the duration of 90% repolarization (APD(90)) in a concentration-dependent manner. ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide (Gli, 20 µmol/L) partially blocked the effects of NaHS (100 µmol/L) on human atrial fiber cells. The L-type Ca(2+) channel agonist Bay K8644 (0.5 µmol/L) also partially blocked the effects of NaHS (100 µmol/L). An inhibitor of cystathionine γ-lyase (CSE), DL-propargylglycine (PPG, 200 µmol/L), increased APA, V(max), VDD and RPF, and prolonged APD(90). H(2)S exerts a negative chronotropic action and accelerates the repolarization of human atrial specialized fibers, possibly as a result of increases in potassium efflux through the opening of K(ATP) channels and a concomitant decrease in calcium influx. Endogenous H(2)S may be generated by CSE and act as an important regulator of electrophysiological properties in human atrial fibers.

Role of Calcium and Potassium Channels in Effects of Hydrogen Sulfide on Frog Myocardial Contractility

The effects of sodium hydrosulfide NaHS, a donor of hydrogen sulfide H2S, on the force of muscle contraction were examined on isolated myocardial strips from frog ventricles. NaHS decreased the amplitude of muscle contractions in a dose-dependent manner under normal conditions and during inhibition of Ca channels with nifedipine. In contrast, under conditions of blockade of ATP-dependent potassium channels with glibenclamide, NaHS exerted a positive inotropic effect from the first minute of application. Neither blockade, nor activation of ATP-dependent K-channels with glibenclamide modulated the negative inotropic effect of NaHS. Inhibition of K-channels with tetraethylammonium (TEA) (3, 5, 10 mM) or 4-aminopyridine increased the amplitude of myocardial contractions. Preliminary application of 4-aminopyridine or TEA (3 mM) did not eliminate NaHS-induced negative inotropic effect, although higher TEA concentrations (5 or 10 mM) prevented it. The data indicate that the targets of H(2)S in frog myocardium are ATP-dependent, Ca-activated, and voltage-dependent K-channels.

Hydrogen sulfide inhibits the spontaneous and oxytocin-induced contractility of human pregnant myometrium

Hydrogen sulfide (H2S) is a novel endogenous gaseous signaling transmitter in mammalian tissues including smooth muscle tissues. We investigated the effect of sodium hydrosulfide (NaHS), a H2S donor, on the contractility of isolated human myometrium strips from term pregnant women who were undergoing labor. Cumulative effects of NaHS on spontaneous and oxytocin-induced contractility were evaluated by using isometric tension recordings. NaHS (0.1 μM–1 mM) concentration dependently inhibited spontaneous contractility of laboring myometrium, with a decrease in amplitude and frequency. NaHS (0.1 μM–1 mM) decreased the frequency but not the amplitude of oxytocin (1 μM)-induced contractions. NaHS-induced relaxation could be prevented by pretreatment with glibenclamide, an inhibitor of K+ATP channels. Thus, NaHS evokes relaxation of human pregnant myometrium, suggesting a possible role of H2S during human pregnancy.

Hydrogen sulfide reduces mRNA and protein levels of beta-site amyloid precursor protein cleaving enzyme 1 in PC12 cells

Hydrogen sulfide (H 2S) is now identified as a new neuromodulator. Increasing evidence suggest that H 2S may play an important role in the progression of Alzheimer's disease (AD). The aim of the present study is to investigate the effects of H 2S on beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) expression and amyloid beta (Aβ) secretion in PC12 cells. The levels of BACE-1 mRNA were measured by quantitative polymerase chain reaction analysis. BACE-1 protein levels were assessed by Western blot. Cellular culture medium levels of Aβ1-42 were analyzed by ELISA. We found that sodium hydrosulfide (NaHS), a H 2S donor, decreased BACE-1 mRNA and protein levels and Aβ1-42 release. Furthermore, NaHS promoted the phosphorylation of Akt and ERK but not JNK or p38 MAPK. However, the effects of NaHS on BACE-1 expression and Aβ1-42 secretion were abolished by inhibitors of phosphatidylinositol 3-kinase (PI3-K), but not of mitogen-activated protein kinase kinases (MEK). Our data indicate that H 2S reduces BACE-1 expression in PC12 cells via activation of PI3-K/Akt signaling pathways. H 2S releasing drugs may have therapeutic potential in AD patients.

Hydrogen sulfide promotes wheat seed germination under osmotic stress

Effects of NaHS, H2S donor, on germination and antioxidant metabolism in wheat (Triticum aestivum L.) seeds under osmotic stress were investigated. With the enhancement of osmotic stress, which was mimicked by PEG-6000, the seed germination dropped gradually. NaHS treatment could promote wheat seed germination against osmotic stress in a dose-dependent manner; while Na+ and other sulfur-containing components, such as S2−, SO42−, SO32−, HSO4− and HSO3−, were not able to improve seed germination as NaHS did, confirming H2S or HS− derived from NaHS contribute to the protective roles. Further experiments showed that NaHS treatment combined with PEG enhanced the activities of amylase and esterase in comparison to PEG treatment alone. Alternatively, NaHS treatment significantly reduced malondialdehyde and hydrogen peroxide accumulation in seeds. Significant enhancement of catalase and ascorbate peroxidase activities and decrease in lipoxygenase activity were observed in NaHS treated seeds, while peroxidase and superoxide dismutase activities were not affected as compared with the control. Furthermore, the H2S donor treatment could retain higher levels of endogenous H2S in wheat seeds under osmotic stress. These data indicated that H2S played a protective role in wheat seed against osmotic stress.

4-Aminopyridine-sensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery

The present study aimed at examining the role of potassium channels and endothelium in relaxations induced by sodium hydrogen sulphide (NaHS), which is the donor of gaseous hydrogen sulphide (H2S) and the effect of NaHS on endothelium-dependent relaxations in rat coronary arteries. Rat coronary arteries were suspended in a myograph for force measurement and changes of the membrane potential in arteries were determined by membrane potential-sensitive fluorescence dye. NaHS relaxed coronary arteries pre-contracted by U46619 and the relaxation was significantly less in high KCl-contracted rings. NaHS-induced relaxations were reduced by 4-aminopyridine (4-AP) but unaffected by glibenclamide, iberiotoxin, NG-nitro-L-arginine methyl ester, ODQ, indomethacin or by endothelium removal. The inhibitory effect of 4-AP was absent in NaHS-induced relaxations in high KCl-contracted rings. Addition of NaHS caused membrane hyperpolarization and this effect was inhibited by 4-AP but not by glibenclamide. NaHS causes endothelium-independent relaxations in rat coronary arteries partially through activation of 4-AP-sensitive potassium channel and ensuring hyperpolarization. Other potassium channels, Na+–K+ pump or endothelium-derived relaxing factors play little role.

Role of cyclic nucleotides in effects of hydrogen sulfide on the mediator release in frog neuromuscular junction

The role of cyclic nucleotides in the effects of hydrogen sulfide (H2S) on transmitter release in the frog neuromuscular junction was studied using focal microelectrode recording of synaptic signals. A donor of H2S, sodium hydrogen sulfide (NaHS) (100 µM) reversibly increased the amplitude and quantum content of end-plate currents (EPCs) without changing the nerve ending response. The effect of H2S on evoked transmitter release was decreased by preliminary application of the membrane-permeable cAMP analogue pCPT-cAMP (100 µM). Inhibition of adenylate cyclase by MDL-12330A (0.8 µM) did not alter the effect of NaHS on transmitter release. Membrane-permeable cGMP analogue pCPT-cGMP (50 µM) and inhibitor of guanylate cyclase ODQ (0.1 µM) did not change the effect of NaHS. Therefore, we conclude that the H2S-induced increase in the transmitter release was not a result of guanylate cyclase activity. It was concluded that the H2S effects are related to the functioning of cAMP-dependent mechanisms; however, these effects are not associated with the direct action of H2S on the adenylate cyclase activity.

T1791 Effect of NaHS On Circular Muscle Tone and Spontaneous Inhibitory Junction Potentials (IJPs) in the Mouse Colon

T1791 Effect of NaHS On Circular Muscle Tone and Spontaneous Inhibitory Junction Potentials (IJPs) in the Mouse Colon