Addition Of NaHS Research Articles

Hydrogen sulfide inhibits early development of atherosclerosis by modulating macrophage uptake of oxidized lipoproteins.

Atherosclerosis, a major cause of cardiovascular diseases, is characterized by the accumulation of oxidized lipoproteins (ox-LDL) within arterial walls, leading to inflammation and plaque formation. Hydrogen sulfide (H2S) has demonstrated anti-inflammatory and vascular protective properties, but its role in modulating macrophage endocytosis of ox-LDL and its impact on early atherosclerosis development remains unclear. Macrophage cultures were utilized for ox-LDL uptake experiments. Macrophages were pretreated with sodium hydrosulfide (NaHS) (50 μmol/L) or propargylglycine (PPG, 3 mmol/L) for 1 h, followed by incubation with DiI-ox-LDL (10 μg/mL) for an additional 2 h. DiI-ox-LDL uptake was visualized using live-cell imaging. The expression of scavenger receptors CD36 and SR-A was assessed through immunofluorescent staining and western blot analysis. To determine the intracellular signal transduction pathways involved, macrophages were pretreated with NF-κB pathway blocker pyrrolidine dithiocarbamate or MAPK inhibitor PD98059 before the addition of NaHS. NaHS significantly inhibited ox-LDL uptake by macrophages, while PPG treatment markedly increased this process. Immunocytochemistry and western blot analysis revealed that the expressions of CD36 and SR-A were induced by ox-LDL but inhibited by NaHS in a concentration- and time-dependent manner. Furthermore, H2S down-regulated ox-LDL receptors CD36 and SR-A through the NF-κB signal pathway. H2S inhibits early atherosclerosis development by modulating macrophage uptake of ox-LDL through the down-regulation of CD36 and SR-A receptors via the NF-κB signaling pathway. These findings provide new evidence for the role of H2S in atherosclerosis and its potential therapeutic value.

KRAS G12D mutation eliminates reactive oxygen species through the Nrf2/CSE/H 2S axis and contributes to pancreatic cancer growth.

In pancreatic cancer, KRAS G12D can trigger pancreatic cancer initiation and development. Rapid tumor growth is often accompanied by excess intracellular reactive oxygen species (ROS) production, which is unfavorable to tumor. However, the regulation of intracellular ROS levels in KRAS mutant pancreatic cancer remains unclear. In this study, we establish BxPC3 stable cell strains expressing KRAS wild type (WT) and G12D mutation and find unchanged ROS levels despite higher glycolysis and proliferation viability in KRAS mutant cells than KRAS WT cells. The key hydrogen sulfide (H 2S)-generating enzyme cystathionine-γ-lyase (CSE) is upregulated in KRAS mutant BxPC3 cells, and its knockdown significantly increases intracellular ROS levels and decreases cell glycolysis and proliferation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is activated by KRAS mutation to promote CSE transcription. An Nrf2 binding site (‒47/‒39 bp) in the CSE promoter is verified. CSE overexpression and the addition of NaHS after Nrf2 knockdown or inhibition by brusatol decreases ROS levels and rescues cell proliferation. Our study reveals the regulatory mechanism of intracellular ROS levels in KRAS mutant pancreatic cancer cells, which provides a potential target for pancreatic cancer therapy.

The effect of sodium hydrosulfide on molybdenite flotation in seawater and diluted seawater

Seawater is used in copper-molybdenum flotation plants when fresh water resources are limited. To minimize the negative effect of process seawater on molybdenite flotation, diluting process seawater with fresh water is a common practice. This study was designed to identify whether the dilution of process seawater in copper-molybdenum flotation plants benefits molybdenite flotation where NaHS is added to depress chalcopyrite. Seawater was prepared based on the concentrations of salts in the process seawater used in a copper-molybdenum flotation plant, and diluted with different proportions of de-ionized water. The concentrations of NaHS chosen to test in molybdenite flotation were determined by conducting chalcopyrite flotation to depress chalcopyrite. It was found that the concentration of NaHS which maximumly depressed chalcopyrite also depressed molybdenite to some extent in both seawater and diluted seawater. Water chemical equilibrium calculation, Cryo-XPS analysis and SEM measurement indicated that the depression mechanisms of NaHS on molybdenite were different in seawater and diluted seawater. Significant dilution of seawater weakened the buffer effect of seawater on pH and raised the pH in flotation significantly after the addition of NaHS so that Mg(OH)2 and CaCO3 precipitates formed to depress molybdenite. Insignificant dilution of seawater had little effect on flotation pH after the addition of NaHS, but the adsorption of HS− and the formation of SO42− on micro-edges of basal plane surfaces of molybdenite at a high NaHS concentration decreased the hydrophobicity of the basal plane surfaces, leading to reduced molybdenite flotation recovery.

Comparative Proteomic Analysis Reveals the Regulatory Effects of H2S on Salt Tolerance of Mangrove Plant Kandelia obovata.

As a dominant mangrove species, Kandelia obovata is distributed in an intertidal marsh with an active H2S release. Whether H2S participates in the salt tolerance of mangrove plants is still ambiguous, although increasing evidence has demonstrated that H2S functions in plant responses to multiple abiotic stresses. In this study, NaHS was used as an H2S donor to investigate the regulatory mechanism of H2S on the salt tolerance of K. obovata seedlings by using a combined physiological and proteomic analysis. The results showed that the reduction in photosynthesis (Pn) caused by 400 mM of NaCl was recovered by the addition of NaHS (200 μM). Furthermore, the application of H2S enhanced the quantum efficiency of photosystem II (PSII) and the membrane lipid stability, implying that H2S is beneficial to the survival of K. obovata seedlings under high salinity. We further identified 37 differentially expressed proteins by proteomic approaches under salinity and NaHS treatments. Among them, the proteins that are related to photosynthesis, primary metabolism, stress response and hormone biosynthesis were primarily enriched. The physiological and proteomic results highlighted that exogenous H2S up-regulated photosynthesis and energy metabolism to help K. obovata to cope with high salinity. Specifically, H2S increased photosynthetic electron transfer, chlorophyll biosynthesis and carbon fixation in K. obovata leaves under salt stress. Furthermore, the abundances of other proteins related to the metabolic pathway, such as antioxidation (ascorbic acid peroxidase (APX), copper/zinc superoxide dismutase (CSD2), and pancreatic and duodenal homeobox 1 (PDX1)), protein synthesis (heat-shock protein (HSP), chaperonin family protein (Cpn) 20), nitrogen metabolism (glutamine synthetase 1 and 2 (GS2), GS1:1), glycolysis (phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI)), and the ascorbate–glutathione (AsA–GSH) cycle were increased by H2S under high salinity. These findings provide new insights into the roles of H2S in the adaptations of the K. obovata mangrove plant to high salinity environments.

Regulation of high-efficient regeneration of sodium borohydride by magnesium-aluminum alloy

Sodium borohydride (NaBH4) is considered to be an excellent hydrogen-generation material via hydrolysis. However, it remains challenge to regenerate it after hydrolysis in practical applications. Therefore, a low-cost regeneration method from hydrolysis by-products for recycling is the key issue. In this study, NaBH4 was successfully regenerated by direct high-energy ball milling of magnesium alloy (Mg17Al12) and hydrolysis by-product NaBO2·2H2O with addition of a small amount of NaH. Notably, unlike traditional method, here there is no need of adding extra hydrogen and the H− in the regenerated NaBH4 is mainly from the H+ in the coordinated water. It was found that through adding a small amount of NaH, the highest yield may reach 57% after optimizing the regeneration process. In addition, it is the first time we found that not only Mg can participate in the reaction, but also Al; and the addition of NaH can promote the reaction. Furthermore, the regeneration mechanism of NaBH4 was analyzed and the transition pattern [B(OH)4]−/[BH3(OH)]−/[BH4]− was successfully observed during the regeneration process. It is worth emphasizing that NaBH4 can be regenerated through ball milling method, which is energy-saving and suitable for up-scaling in the future.

P025 CYSTATHIONINE-GAMMA-LYASE REGULATES MACROPHAGE INTERLEUKIN-10 (IL-10) PRODUCTION IN COLITIS

Cystathionine-β-synthase (CBS) & cystathionine-γ-lyase (CSE), convert homocysteine into cysteine & produce H2S, an anti-inflammatory mediator. In rodent colitis models, CBS & CSE activity is elevated and inhibition or knockout of CSE worsens colitis & decreases levels of anti-inflammatory cytokine IL-10. IL-10 KO mice develop spontaneous colitis accompanied by homocysteinemia & decreased H2S production, which are reversed by IL-10 administration. However, the cellular source of CSE-dependent IL-10 production is unknown. We hypothesize that macrophage-derived CSE is critical for IL-10 production. Human colitis & normal colon specimens were collected. CSE knockout (CSE KO) mice and wild-type (WT) controls received either 7 days of colitis-inducer DSS (2% in drinking water) or vehicle. CSE & cell-type specific markers were evaluated in human/mouse colons by immunofluorescent microscopy & flow cytometry. Peritoneal macrophages from CSE KO & WT mice (N = 6) were isolated & treated with CSE inhibitor propargyl glycine (PAG), H2S donor NaHS or vehicle. IL-10 & β-actin expression levels were determined by qRT-PCR. Confocal microscopy of colitis & normal human colon tissue localized CSE to cells of the lamina propria expressing CD45, a marker of hematopoietic cells. Colonic tissue from mice receiving either DSS or vehicle also showed CSE staining in the lamina propria co-localized with macrophage marker F4/80. Flow cytometry showed co-expression of CSE with F4/80 but not Treg marker FOXP3. Comparison of macrophages from CSE KO to WT mice revealed a 6-fold reduction of IL-10 mRNA. Further, treatment of macrophages from WT mice with PAG inhibited IL-10 expression compared to controls. Addition of NaHS did not alter CSE KO macrophage expression of IL-10. We demonstrate that in macrophages CSE & IL-10 expression are linked in an H2S-independent manner. This suggests that CSE regulates inflammation through both H2S & IL-10.

Exogenous H2S Inhibits Autophagy in Unilateral Ureteral Obstruction Mouse Renal Tubule Cells by Regulating the ROS-AMPK Signaling Pathway

Background/Aims: The induction of excessive autophagy by increased levels of oxidative stress is one of the main mechanisms underlying unilateral ureteral obstruction (UUO)-induced vascular endothelial cell dysfunction. Hydrogen sulfide (H2S) has been shown to have an anti-oxidative effect, but its mode of action on excessive autophagy in vascular endothelial cells is unclear. Methods: Surgery was used to induce UUO in male C57BL/6 mice as an in vivo model. Human renal epithelial cells (HK-2) were treated with H2O2 as an in vitro model. NaHS was used as an exogenous H2S donor. Transmission electron microscopy was applied to observe the structure of renal autophagosomes. The expression of proteins related to autophagy and apoptosis was detected by western blot analysis in vivo and in vitro. Flow cytometry (DCFH-DA) was used to examine the levels of intracellular reactive oxygen species (ROS). The terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to detect cell apoptosis. Compound C was used to analyze the association of AMPK with autophagy. Results: Compared with the sham group, in which the ureter was exposed but not ligated, the cell apoptosis index, number of autophagosomes, protein expression of microtubule-associated protein 1 light-chain 3 (LC3)-II/I, beclin-1, and p-AMPK/AMPK were significantly increased in the UUO group. On the other hand, p62, cystathionine β-synthase, and cystathionine γ-lyase protein expression levels and H2S concentration were significantly decreased (p < 0.05). These alterations were ameliorated by the addition of NaHS (p < 0.05). Similar results were observed in vitro. By using the AMPK inhibitor compound C, it was indicated that AMPK was involved in ROS-induced autophagy. In addition, using tissue from patients with obstructive nephropathy, excessive autophagy was observed by an increased LC3-II/LC3-I ratio. Conclusion: NaHS-treatment may exert a protective effect on mouse kidney against UUO by suppressing the ROS-AMPK pathway. ROS-AMPK-mediated autophagy may represent a promising therapeutic target for obstructive nephropathy.

Exogenous H2S restores ischemic post-conditioning-induced cardioprotection through inhibiting endoplasmic reticulum stress in the aged cardiomyocytes

BackgroundA gasotransmitter hydrogen sulfide (H2S) plays an important physiological and pathological role in cardiovascular system. Ischemic post-conditioning (PC) provides cardioprotection in the young hearts but not in the aged hearts. Exogenous H2S restores PC-induced cardioprotection by inhibition of mitochondrial permeability transition pore opening and oxidative stress and increase of autophagy in the aged hearts. However, whether H2S contributes to the recovery of PC-induced cardioprotection via down-regulation of endoplasmic reticulum stress (ERS) in the aged hearts is unclear.MethodsThe aged H9C2 cells (the cardiomyocytes line) were induced using H2O2 and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. Related protein expressions were detected through Western blot.ResultsIn the present study, we found that 30 μM H2O2 induced H9C2 cells senescence but not apoptosis. Supplementation of NaHS protected against H/R-induced apoptosis, the expression of cleaved caspase-3 and cleaved caspase-9 and the release of cytochrome c. The addition of NaHS also counteracted the reduction of cell viability caused by H/R and decreased the expression of GRP 78, CHOP, cleaved caspase-12, ATF 4, ATF 6 and XBP-1 and the phosphorylation of PERK, eIF 2α and IRE 1α. Additionally, NaHS increased Bcl-2 expression. PC alone did not provide cardioprotection in H/R-treated aged cardiomyocytes, which was significantly restored by the supplementation of NaHS. The beneficial role of NaHS was similar to the supply of 4-PBA (an inhibitor of ERS), GSK2656157 (an inhibitor of PERK), STF083010 (an inhibitor of IRE 1α), respectively, during PC.ConclusionOur results suggest that the recovery of myocardial protection from PC by exogenous H2S is associated with the inhibition of ERS via down-regulating PERK-eIF 2α-ATF 4, IRE 1α-XBP-1 and ATF 6 pathways in the aged cardiomyocytes.

A near-infrared fluorescent probe for rapid and selective detection of hydrosulfide and imaging in live cells

A heptamethine cyanine-based near-infrared (NIR) fluorescent probe Cy-1 for hydrosulfide detection was developed, with the response mechanism based on nucleophilic substitution of acryloyl moiety by HS−. Upon addition of NaHS, Cy-1 displayed a rapid fluorescence quenching (excited at 700 nm) and a quick fluorescence enhancement (excited at 544 nm) as well as obvious changes in absorption spectra and color. The response was fast within tens of seconds and could be observed by naked eyes. By contrasting Cy-1 with the reported probe CyAC, the superiority of Cy-1 in selectivity over Cys was demonstrated. Besides, it also exhibited excellent selectivity and sensitivity to HS− over various anions (0.33-μM detection limit). Additionally, this probe was successfully applied in fetal bovine serum samples and imaging of hydrosulfide in living cells.

INFLUENCE HYDROGEN SULFIDE ON CA2 + -DEPENDENT POTASSIUM PERMEABILITY OF THE MEMBRANE OF HUMAN ERYTHROCYTES

The purpose of the research was to study the influence of hydrogen sulfide donor NaHS on red blood cells membrane potential changes caused by the activation of Ca2+ dependent K+ channels in the presence of calcium ionophore or artificial electron donor system ascorbate phenazine methosulfate. Materials and methods. We used the packed erythrocytes obtained from venous blood of 25 healthy volunteers at the age of 20-27 years. Registration of the membrane potential of erythrocytes in the presence of Ca2+ ionophore (A23187) or artificial electron donor system ascorbate – FMS was performed with potentiometric method based on the fact that in the presence of the protonophore distribution of H + depends on the membrane potential Em like as Em = RT/F (pHi – pH0 ). Here pHi and pH0 are pH of the incubation medium and cytoplasm, respectively. An amplitude of hyperpolarizing response (HR) was calculated as an integral characteristic of Ca2+-dependent permeability for K+. Results. The addition of NaHS at concentrations from 0.2 mM to 0.005 mM into the cells incubation medium caused a change in the amplitude of hyperpolarizing response of erythrocyte membranes induced by both methods. In the presence of 0.005 mM NaHS the amplitude of A23187-dependent HR increased significantly, whereas the amplitude of the redox-dependent HR decreased. Suppression of A23187-dependent HR in the presence of higher concentrations of NaHS was more pronounced than the suppression of the amplitude of redox-dependent response. The amplitude of A23187-dependent HR increased under the joint action of hydrogen sulfide and the blocker of Na+, K+, 2Cl- -cotransport (NKCC) bumetanide, and the amplitude of redox-dependent HR decreased in comparison with the parameters obtained in the absence of a blocker. Conclusion. It is found that hydrogen sulfide exerts a modulatory effect on K (Ca2+) - channels of erythrocyte membrane. The effect of H2 S depends on the method of activation of the studied channels. A23187-dependent HR was more sensitive to H2S in comparison to redox-induced HR of erythrocyte membrane. The effect of NaHS on the amplitude of the HR in the presence of NKCC blocker bumetanide also depended on the method of stimulation of channels.

Exogenous H2S contributes to recovery of ischemic post-conditioning-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2-STAT3 pathways in the aging cardiomyocytes.

BackgroundHydrogen sulfide (H2S), a third member of gasotransmitter family along with nitric oxide and carbon monoxide, generated from mainly catalyzed by cystathionine-lyase, possesses important functions in the cardiovascular system. Ischemic post-conditioning (PC) strongly protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes. However, PC protection is ineffective in the aging cardiomyocytes. Whether H2S restores PC-induced cardioprotection by decrease of reactive oxygen species (ROS) level in the aging cardiomyocytes is unknown.MethodsThe aging cardiomyocytes were induced by treatment of primary cultures of neonatal cardiomyocytes using d-galactose and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. ROS level was analyzed using spectrofluorimeter. Related protein expressions were detected through Western blot.ResultsTreatment of NaHS (a H2S donor) protected against H/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The supplementation of NaHS also decreased the activity of LDH and CK, MDA contents, ROS levels and the phosphorylation of IκBα, NF-κB, JNK2 and STAT3, and increased cell viability, the expression of Bcl-2, the activity of SOD, CAT and GSH-PX. PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS. The beneficial role of NaHS was similar to the supply of N-acetyl-cysteine (NAC, an inhibitor of ROS), Ammonium pyrrolidinedithiocarbamate (PDTC, an inhibitor of NF-κB) and AG 490 (an inhibitor of JNK2), respectively, during PC.ConclusionOur results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

Hydrogen sulfide as a signal molecule in hematin-induced heat tolerance of tobacco cell suspension

Carbon monoxide (CO) is considered as a new emerging cell signal molecule which is involved in plant growth, development, and acquisition of stress tolerance. In recent years, hydrogen sulfide (H2S) has been found to have similar functions, but crosstalk between CO and H2S in the acquisition of heat tolerance in plants is not clear. In this study, pretreatment of tobacco (Nicotiana tabaccum L.) cells cultured in a suspension with a CO donor hematin significantly increased survival percentage of cells under a heat stress and regrowth ability after the heat stress, alleviated a decrease in cell vitality, and accumulation of malondialdehyde. In addition, treatment with hematin enhanced the activity of L-cysteine desulfhydrase, a key enzyme in H2S biosynthesis, which in turn induced accumulation of endogenous H2S in tobacco cells. Interestingly, hematin-induced heat tolerance was enhanced by addition of NaHS, a H2S donor, but weakened by specific inhibitors of H2S biosynthesis DL-propargylglycine or its scavenger hypotaurine. Furthermore, pretreatment with hemoglobin (a CO scavenger) and zinc protoporphyrin IX (a CO specific synthetic inhibitor) had no significant effect on NaHS-induced heat tolerance of tobacco cells. These results suggest that CO pretreatment could improve the heat tolerance of tobacco suspension cultured cells, and H2S might exert its signal role downstream to CO-induced heat tolerance.

Hydrogen sulfide partly mediates abscisic acid-induced heat tolerance in tobacco (Nicotiana tabacum L.) suspension cultured cells

Abscisic acid (ABA), a plant hormone, plays crucial roles in plant growth, development and response to environmental stress. Recently, hydrogen sulfide (H2S) also has emerged similar functions, but interaction between ABA and H2S in the acquisition of heat tolerance is not clear. In this study, pretreatment of tobacco suspension cultured cells with ABA improved the survival percentage and regrowth ability, alleviated a decrease in cell vitality, increase in malondialdehyde content and electrolyte leakage of cells under heat stress, suggesting that ABA pretreatment could improve the heat tolerance of tobacco cells. In addition, treatment with ABA induced the accumulation of endogenous H2S in both tobacco cells and culture medium, increased the activity of l-cysteine desulfhydrase, a key enzyme in H2S biosynthesis. Also, ABA-induced heat tolerance was enhanced by addition of NaHS, a H2S donor, but weakened by dl-propargylglycine (specific inhibitor of H2S biosynthesis) and hypotaurine (H2S scavenger) respectively. These results suggest that ABA pretreatment could improve the heat tolerance of tobacco suspension cultured cells, and H2S, at least partly, mediated the acquisition of heat tolerance induced by ABA.

Exogenous hydrogen sulfide restores cardioprotection of ischemic post-conditioning via inhibition of mPTP opening in the aging cardiomyocytes.

The physiological and pathological roles of hydrogen sulfide (H2S) in the regulation of cardiovascular functions have been recognized. H2S protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes, and ischemic post-conditioning (PC) plays an important role in cardioprotection from H/R injury in neonatal cardiomyocytes but not in aging cardiomyocytes. Whether H2S is involved in the recovery of PC-induced cardioprotection in aging cardiomyocytes is unclear. In the present study, we found that both H/R and PC decreased cystathionine-γ-lyase (CSE) expression and the production rate of H2S. Supplementation of NaHS protected against H/R-induced apoptosis, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c), and mPTP opening. The addition of NaHS also counteracted the reduction of cell viability caused by H/R and increased the phosphorylation of ERK1/2, PI3K, Akt, GSK-3β and mitochondrial membrane potential. Additionally, NaHS increased Bcl-2 expression, promoted PKC-ε translocation to the cell membrane, and activated mitochondrial ATP-sensitive K channels (mitoKATP). PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the supplementation of NaHS. In conclusion, our results suggest that exogenous H2S restores PC-induced cardioprotection via the inhibition of mPTP opening by the activation of the ERK1/2-GSK-3β, PI3K-Akt-GSK-3β and PKC-ε-mitoKATP pathways in aging cardiomyocytes. These findings provide a novel target for the treatment of aging ischemic cardiomyopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0035-9) contains supplementary material, which is available to authorized users.

Hydrogen sulfide regulates the colonic motility by inhibiting both L-type calcium channels and BKCa channels in smooth muscle cells of rat colon.

ObjectiveTo examine the hypothesis that hydrogen sulfide (H2S) regulates the colonic motility by modulating both L-type voltage-dependent calcium channels and large conductance Ca2+-activated K+ (BKCa) channels.MethodsImmunohistochemistry was performed on rat colonic samples to investigate the localization of the H2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record both L-type calcium currents (I Ca,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from male Wistar rats.ResultsImmunohistochemistry revealed the presence of CBS and CSE in mucosa, smooth muscle cells and myenteric neurons. The H2S donor NaHS inhibited spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner, and the inhibitory effects were not blocked by tetrodotoxin. NaHS inhibited the peak I Ca,L in colonic SMCs at a membrane potential of 0 mV. The current-voltage (I-V) relationship of L-type calcium channels was modified by NaHS, and the peak of the I-V curve was shifted to the right. NaHS (200μΜ) evoked a significant rightward shift of the steady-state activation curve and inhibited the inactivation of L-type calcium channels. Furthermore, NaHS reversibly decreased the peak I Ca,L in a dose-dependent manner. Likewise, BKCa channels were significantly inhibited by NaHS, and the addition of NaHS caused a time- and dose-dependent reduction in the BKCa current.ConclusionThe relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. H2S may be involved in the regulation of calcium homeostasis in colonic SMCs of rat colon.

Hydrogen sulfide acts as a downstream signal molecule in salicylic acid-induced heat tolerance in maize (Zea mays L.) seedlings

Salicylic acid (SA), 2-hydroxy benzoic acid, is a small phenolic compound with multifunction that is involved in plant growth, development, and the acquisition of stress tolerance. In recent years, hydrogen sulfide (H2S) has been found to have similar functions, but cross talk between SA and H2S in the acquisition of heat tolerance is not clear. In this study, pretreatment of maize seedlings with SA improved the survival percentage of seedlings under heat stress, indicating that SA pretreatment could improve the heat tolerance of maize seedlings. In addition, treatment with SA enhanced the activity of L-cysteine desulfhydrase (L-DES), a key enzyme in H2S biosynthesis, which in turn induced accumulation of endogenous H2S. Interestingly, SA-induced heat tolerance was enhanced by addition of NaHS, a H2S donor, but weakened by specific inhibitors of H2S biosynthesis DL-propargylglycine (PAG) and its scavenger hydroxylamine (HT). Furthermore, pretreatment with paclobutrazol (PAC) and 2-aminoindan-2-phosphonic acid (AIP), inhibitors of SA biosynthesis, had no significant effect on NaHS-induced heat tolerance of maize seedlings. Similarly, significant change in the activities of phenylalanine ammonia lyase (PAL) and benzoic-acid-2-hydroxylase (BA2H), the key enzymes in SA biosynthesis, and the content of endogenous SA, was not observed in maize seedlings by NaHS treatment. All of the above-mentioned results suggest that SA pretreatment could improve the heat tolerance of maize seedlings, and H2S might be a novel downstream signal molecule in SA-induced heat tolerance.

P86 Role of hydrogen sulfide as an oxygen sensor in the human pulmonary vasculature

Introduction Animal studies have shown that H 2 S can act as an oxygen sensor in the vasculature. To date the role of H 2 S as an oxygen sensor has not been investigated in human vascular preparations. The aim of this study was to compare the effects of H 2 S and hypoxia on pulmonary vascular tone in human pulmonary arteries and veins. Methods Human pulmonary arteries and veins were obtained at resection for cancer and mounted in organ baths to measure isometric tension. The effects of H 2 S on resting tension and active tension to ET-1 were determined by cumulative addition of NaHS. In the same vessels the effects of hypoxia on resting and active tension were determined by changing the aerating gas to 95% N 2 : 5% CO 2 . Results NaHS caused a dose dependent (0.1–3 mM) relaxation of human pulmonary arteries ( n = 8) and veins ( n = 8) from resting and active tension. Hypoxia also caused a relaxation of arteries and veins and the magnitude of relaxation to hypoxia was comparable to the maximum relaxation to NaHS. However, the effects of hypoxia and NaHS were not consistent in all vessels. In some vessels hypoxia caused a relaxation whereas NaHS had no effect and in others NaHS caused a relaxation and hypoxia had no effect. Conclusion It has been shown for the first time that H 2 S causes a dose dependent relaxation of human pulmonary vessels. Similarities in the effect of H 2 S and hypoxia suggest a causal relationship, however, some inconsistencies in the results warrant further investigation.

Sensitization of sodium channels by cystathionine β‐synthetase activation in colon primary sensory neurons contributes to visceral hypersensitivity in adult rats with neonatal maternal deprivation (651.20)

Aims: This study was designed to determine roles for sodium channels and endogenous H2S signaling in a rat model of chronic visceral hypersensitivityinduced by neonatal maternal deprivation (NMD). Methods: Neonatal male rats were exposed to a 3 hour period of daily maternal separation. Colon‐specific DRG neurons were acutely dissociated for measuring excitability and sodium channel currents under whole‐cell patch clamp configurations. Expression of NaV1.8 was analyzed by western blot. Results: NMD significantly increased AWR scores. TTX‐resistant sodium current density was greatly enhanced in colon DRG neurons, activation curves showed a leftward shift in NMD rats. Furthermore, NMD remarkably enhanced expression of NaV1.8 at protein levels. AOAA, an inhibitor of CBS, significantly suppressed the expression of NaV1.8. AOAA treatment also reduced the TTX‐R sodium current density, and reversed hyperexcitability of colon‐specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H2S, greatly enhanced TTX‐R sodium current density, leftshifted activation curve and enhanced excitability of colon DRG neurons in age‐matched healthy rats. Conclusion: Our data suggest that NMD enhances TTX‐resistant sodium activity of colon DRG neurons, which is most likely mediated by CBS‐H2S signaling, thus contributing to chronic visceral hypersensitivity.Grant Funding Source: Supported by National Natural Science Foundation of China

Heme oxygenase-1 is involved in sodium hydrosulfide-induced lateral root formation in tomato seedlings

By using pharmacological and molecular approaches, we discovered the involvement of HO-1 in NaHS-induced lateral root formation in tomato seedlings. Heme oxygenase-1 (HO-1) and hydrogen sulfide (H2S) regulate various responses to abiotic stress and root development, but their involvement in the simultaneous regulation of plant lateral root (LR) formation is poorly understood. In this report, we observed that the exogenously applied H2S donor sodium hydrosulfide (NaHS) and the HO-1 inducer hemin induce LR formation in tomato seedlings by triggering intracellular signaling events involving the induction of tomato HO-1 (SlHO-1), and the modulation of cell cycle regulatory genes, including the up-regulation of SlCDKA;1 and SlCYCA2;1, and simultaneous down-regulation of SlKRP2. The response of NaHS in the induction of LR formation was impaired by the potent inhibition of HO-1, which was further blocked when 50 % saturation of carbon monoxide (CO) aqueous solution, one of the catalytic by-products of HO-1, was added. Further molecular evidence revealed that the NaHS-modulated gene expression of cell cycle regulatory genes was sensitive to the inhibition of HO-1 and reversed by cotreatment with CO. The impairment of LR density and length as well as lateral root primordia number, the decreased tomato HO-1 gene expression and HO activity caused by an H2S scavenger hypotaurine were partially rescued by the addition of NaHS, hemin and CO (in particular). Together, these results revealed that at least in our experimental conditions, HO-1 might be involved in NaHS-induced tomato LR formation. Additionally, the use of NaHS and hemin compounds in crop root organogenesis should be explored.

Sensitization of sodium channels by cystathionine β-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation

BackgroundThe pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS–H2S signaling in sensitization of sodium channels in a previously validated rat model of IBS. MethodsNeonatal male rats (postnatal days 2–15) were exposed to a 3hour period of daily maternal separation with temperature maintained at ~33°C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. The expression of NaV1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H2S producing enzyme CBS antagonist was injected intraperitoneally. ResultsWe showed that CBS was colocalized with NaV1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of NaV1.8 in NMD rats. AOAA treatment also inhibited the TTX-R sodium current density, right-shifted the V1/2 of activation curve, and reversed hyperexcitability of colon-specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H2S, greatly enhanced TTX-R sodium current density, left shifted the activation curve and enhanced excitability of colon DRG neurons in age-matched healthy rats. Furthermore, application of H-89, an inhibitor of protein kinase A, markedly attenuated the potentiation of TTX-R sodium current density by NaHS. ConclusionThese data suggest that sensitization of sodium channels of colon DRG neurons in NMD rats is most likely mediated by CBS–H2S signaling, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.