Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H₂S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of K_ATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H₂S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of K_ATP-channels.     

Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells

Hydrogen sulfide (H₂S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H₂S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Ca_v3.2 T-type Ca²⁺ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Ca_v3.2. In the present study, we asked if endogenous and exogenous H₂S participates in regulation of the channel functions in Ca_v3.2-transfected HEK293 (Ca_v3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Ca_v3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Ca_v3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5 mM, but not 0.1–0.3 mM, increased T-currents in Ca_v3.2-HEK293 cells. Similarly, Na₂S, another H₂S donor, at 0.1–0.3 mM significantly increased T-currents in the presence, but not absence, of PPG in Ca_v3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na₂S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na₂S was higher than NaHS. These results suggest that the function of Ca_v3.2 T-channels is tonically enhanced by endogenous H₂S synthesized by CSE in Ca_v3.2-HEK293 cells, and that exogenous H₂S is capable of enhancing Ca_v3.2 function when endogenous H₂S production by CSE is inhibited. In addition, Na₂S is considered a more potent H₂S donor than NaHS in vitro as well as in vivo.     

Endogenous production of hydrogen sulfide in isolated bovine eye

Hydrogen sulfide (H₂S) is a novel gasotransmitter with physiological and pathological functions in vascular homeostasis, cardiovascular system and central nervous system. In the present study, we determined the endogenous levels of H₂S in various tissues of the bovine eye. We also examined the basal levels of H₂S in response to donors (sodium hydrosulfide, NaHS and sodium sulfide, Na₂S), substrate (l-cysteine), inhibitors (propargylglycine, PAG and aminooxyacetic acid, AOA) and activator (S-adenosyl-l-methionine, SAM) of this gas in the bovine retina. H₂S was measured using a well established spectrophotometric method. The highest concentration of endogenous H₂S was detected in cornea (19 ± 2.85 nmoles/mg protein, n = 6) and retina (17 ± 2.1 nmoles/mg protein, n = 6). Interestingly, H₂S was not present in vitreous humor. The inhibitors of CSE and CBS; PAG (1 mM) and AOA (1 mM), significantly attenuated the production of H₂S in the bovine retina by 56.8 and 42%, respectively. On the other hand the activator of CBS; SAM (100 μM), H₂S donors; NaHS (1 μM) and Na₂S (100 μM), significantly increased endogenous levels of H₂S in bovine retina. l-cysteine (10–300 μM) produced a significant (P < 0.05) concentration-dependent increase in H₂S levels reaching a maximal at 300 μM. We conclude that H₂S is endogenously produced in various tissues of the isolated bovine eye. Moreover, endogenous levels of H₂S are enhanced in the presence of substrate (l-cysteine), an activator of CBS (SAM) and H₂S donors but are blocked by inhibitors of enzymes that synthesize this gas in neural retina.     

T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Exogenous hydrogen sulfide attenuates diabetic myocardial injury through cardiac mitochondrial protection

In the study, we investigated how exogenous H₂S (hydrogen sulfide) influenced streptozotocin (STZ)-induced diabetic myocardial injury through cardiac mitochondrial protection and nitric oxide (NO) synthesis in intact rat hearts and primary neonatal rat cardiomyocytes. Diabetes was induced by STZ (50?mg/kg) and the daily administration of 100?μM NaHS (sodium hydrosulfide, an H₂S donor) in the diabetes?+?NaHS treatment group. At the end of 4, 8, and 12?weeks, the morphological alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the level of NO were measured. The expressions of cystathionine-γ-lyase (CSE), caspase-3 and -9, the mitochondrial NOX4 and cytochrome c were analyzed by western blotting. The results showed the cardiac function injured, morphological changes and the apoptotic rate increased in the diabetic rat hearts. In the primary neonatal rat cardiomyocytes of high glucose group, ROS production was increased markedly, whereas the expression of CSE and the level of NO was decreased. However, treatment with NaHS significantly reversed the diabetic rat hearts function, the morphological changes and decreased the levels of ROS and NO in the primary neonatal rat cardiomyocytes administrated with high glucose group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and -9 and inhibited the release of cytochrome c from mitochondria in the primary neonatal rat cardiomyocytes. In conclusion, H₂S is involved in the attenuation of diabetic myocardial injury through the protection of cardiac mitochondria.     

Effects of pH and Lactate on Hydrogen Sulfide Production by Oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H₂S), one of the major components of oral malodor. However, there is little information on the physiological properties of H₂S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H₂S production. Thus, in the present study, the H₂S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H₂S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H₂S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H₂S. H₂S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H₂S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H₂S, ammonia was produced in cell extracts of all the strains, indicating that H₂S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H₂S from l-cysteine and that their H₂S production can be regulated by oral environmental factors, namely, pH and lactate.     

Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in Medicago sativa by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H₂S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H₂S production as well as the activation of two H₂S-synthetic enzymes activities, including _L-cysteine desulfhydrase (LCD) and _D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H₂S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H₂S production triggered by Cd, but also alleviated Cd toxicity in a H₂S-dependent fashion. By contrast, the inhibition of H₂S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H₂S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.     

Sodium Hydrosulphide Alleviates Remote Lung Injury Following Limb Traumatic Injury in Rats

Hydrogen sulphide (H₂S) was found to attenuate ventilator or oleic acid induced lung injury. The aim of this study was to explore the effects of exogenous H₂S donor, sodium Hydrosulphide (NaHS), on lung injury following blast limb trauma and the underlying mechanisms. For in vitro experiments, pulmonary micro-vessel endothelial cells (PMVECs) were cultured and treated with NaHS or vehicle in the presence of TNF-α. For in vivo, blast limb traumatic rats, induced by using chartaceous electricity detonators, were randomly treated with NaHS, cystathionine gamma-lyase inhibitor (PAG) or vehicle. In vitro, NaHS (100 µM) treatment increased PMVECs viability and decreased LDH release into culture media after tumor necrosis factor (TNF) α challenge. In addition, NaHS treatment prevented the increase of nitric oxide, Intercellular Adhesion Molecule 1(ICAM-1) and interleukin (IL)-6 production and inducible nitric oxide synthase activation induced by TNF-α. Knock-down of NF-E2-Related Factor 2 (Nrf2) partially abolished the protective effect of NaHS. In vivo, NaHS treatment significantly alleviated lung injury following blast limb trauma, demonstrated by a decreased histopathological score and lung water content. Furthermore, NaHS treatment reversed the decrease of H₂S concentration in plasma, prevented the increase of TNF-α, IL-6, malondialdehyde and myeloperoxidase, increased the Nrf2 downstream effector glutathione in both plasma and lungs, and reversed the decrease of superoxide dismutase in both plasma and lungs induced by blast limb trauma. Our data indicated that NaHS protects against lung injury following blast limb trauma which is likely associated with suppression of the inflammatory and oxidative response and activation of Nrf2 cellular signal.     

Effect of Hydrogen Sulfide on Cyclic AMP Production in Isolated Bovine and Porcine Neural Retinae

Hydrogen sulfide (H₂S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H₂S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na₂S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 μM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM–100 μM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na₂S (10 nM–100 μM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na₂S (100 μM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 μM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na₂S. We conclude that H₂S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H₂S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H₂S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.     

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells

Hydrogen sulfide (H₂S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H₂S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca²⁺ imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H₂S donor, activates cytosolic calcium (Ca_c) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1 nM-200 μM) evoked Ca_c signals also in “normal” human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1-10 μM). Remarkably H₂S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5 mM, 30 min), an inhibitor of the H₂S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Ca_c signals induced by VEGF (20 ng/ml). We conclude that H₂S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment.     

Hydrogen sulphide increases pulmonary veins and atrial arrhythmogenesis with activation of protein kinase C

Hydrogen sulphide (H₂S), one of the most common toxic air pollutants, is an important aetiology of atrial fibrillation (AF). Pulmonary veins (PVs) and left atrium (LA) are the most important AF trigger and substrate. We investigated whether H₂S may modulate the arrhythmogenesis of PVs and atria. Conventional microelectrodes and whole‐cell patch clamp were performed in rabbit PV, sinoatrial node (SAN) or atrial cardiomyocytes before and after the perfusion of NaHS with or without chelerythrine (a selective PKC inhibitor), rottlerin (a specific PKC δ inhibitor) or KB‐R7943 (a NCX inhibitor). NaHS reduced spontaneous beating rates, but increased the occurrences of delayed afterdepolarizations and burst firing in PVs and SANs. NaHS (100 μmol/L) increased I_KATP and I_NCX in PV and LA cardiomyocytes, which were attenuated by chelerythrine (3 μmol/L). Chelerythrine, rottlerin (10 μmol/L) or KB‐R7943 (10 μmol/L) attenuated the arrhythmogenic effects of NaHS on PVs or SANs. NaHS shortened the action potential duration in LA, but not in right atrium or in the presence of chelerythrine. NaHS increased PKC activity, but did not translocate PKC isoforms α, ε to membrane in LA. In conclusion, through protein kinase C signalling, H₂S increases PV and atrial arrhythmogenesis, which may contribute to air pollution‐induced AF.     

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 Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

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.     

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.     

Alpha-lipoic acid regulates the autophagy of vascular smooth muscle cells in diabetes by elevating hydrogen sulfide level

Dysfunctional vascular smooth muscle (VSM) plays a vital role in the process of atherosclerosis in patients with type 2 diabetes mellitus (T2DM). Alpha-lipoic acid (ALA) can prevent the altered VSM induced by diabetes. However, the precise mechanism underlying the beneficial effect of ALA is not well understood. This study aimed to determine whether ALA ameliorates VSM function by elevating hydrogen sulfide (H₂S) level in diabetes and whether this effect is associated with regulation of autophagy of VSM cells (VSMCs). We found decreased serum H₂S levels in Chinese patients and rats with type 2 diabetes mellitus (T2DM). ALA treatment could increase H₂S level, which reduced the autophagy-related index and activation of the 5′-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, thereby protecting vascular function in rats with T2DM. Propargylglycine (PPG), a cystathionine-γ-lyase inhibitor, could weaken the ALA effect. In cultured VSMCs, high glucose level also reduced H₂S level, upregulated the autophagy-related index and activated the AMPK/mTOR pathway, which were reversed by concomitant application of sodium hydrosulfide (NaHS, an H₂S donor) or ALA. The protective effect of NaHS or ALA was attenuated by rapamycin (an autophagy activator), 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (an AMPK activator) or PPG. In contrast, Compound C (an AMPK inhibitor) enhanced the effect of ALA or NaHS. ALA may have a protective effect on VSMCs in T2DM by elevating H₂S level and downregulating autophagy via the AMPK/mTOR pathway. This study provides a new target for addressing diabetic macroangiopathy.     

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.     

Hydrogen Sulfide Regulates Cardiovascular Function by Influencing the Excitability of Subfornical Organ Neurons

Hydrogen sulfide (H₂S), a gasotransmitter endogenously found in the central nervous system, has recently been suggested to act as a signalling molecule in the brain having beneficial effects on cardiovascular function. This study was thus undertaken to investigate the effect of NaHS (an H₂S donor) in the subfornical organ (SFO), a central nervous system site important to blood pressure regulation. We used male Sprague-Dawley rats for both in vivo and in vitro experiments. We first used RT-PCR to confirm our previous microarray analyses showing that mRNAs for the enzymes required to produce H₂S are expressed in the SFO. We then used microinjection techniques to investigate the physiological effects of NaHS in SFO, and found that NaHS microinjection (5 nmol) significantly increased blood pressure (mean AUC = 853.5±105.7 mmHg*s, n = 5). Further, we used patch-clamp electrophysiology and found that 97.8% (88 of 90) of neurons depolarized in response to NaHS. This response was found to be concentration dependent with an EC₅₀ of 35.6 µM. Coupled with the depolarized membrane potential, we observed an overall increase in neuronal excitability using an analysis of rheobase and action potential firing patterns. This study has provided the first evidence of NaHS and thus H₂S actions and their cellular correlates in SFO, implicating this brain area as a site where H₂S may act to control blood pressure.     

Hydrogen sulphide triggers VEGF-induced intracellular Ca signals in human endothelial cells but not in their immature progenitors

Hydrogen sulphide (H₂S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is central to endothelial proliferation and may be triggered by both VEGF and H₂S. Albeit VEGFR-2 might serve as H₂S receptor, the mechanistic relationship between VEGF- and H₂S-induced Ca²⁺ signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H₂S donor, stimulates pro-angiogenic Ca²⁺ signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca²⁺]_i in Ea.hy926 cells. NaHS-induced Ca²⁺ signals in Ea.hy926 cells did not require extracellular Ca²⁺ entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP₃) signalling pathway. Moreover, the Ca²⁺ response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca²⁺ signals were suppressed by dl-propargylglycine (PAG), which blocks the H₂S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca²⁺]_i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca²⁺ oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H₂S-evoked elevation in [Ca²⁺]_i is essential to trigger the pro-angiogenic Ca²⁺ response to VEGF in mature endothelial cells, but not in their immature progenitors.