Exogenous administration of hydrogen sulfide alleviates homocysteine induced inflammation in ARPE-19 cells.

Plasma homocysteine (Hcy) is an independent risk factor for Age related macular degeneration (AMD) and an inducer of inflammation. Homocysteine catabolism releases hydrogen sulfide (H2S). H2S has controversial effects on inflammation. In this study we have analysed the endogenous and exogenous H2S in modulating inflammation using adult retinal pigment epithelial (ARPE-19) cells as an in vitro model for AMD. ARPE-19 cells were treated with various concentrations of Hcy (15, 30 and 50 µM) for 3 h. Expression of Hcy transulfuration genes (CBS, CSE) by qPCR and western blot. H2S levels were measured using Free Radical Analyzer System (WPI, USA). The inflammatory markers (IL-6 and IL-8) were evaluated using real-time PCR and ELISA. Hcy exposure increased CBS protein expression, hydrogen sulfide levels and pro-inflammatory cytokines, modulating CBS by silencing did not alter H2S levels, but inhibition of CSE with PAG inhibited H2S production and decreased cytokine (IL-6 and IL-8) levels. On the contrary exogenous supply of hydrogen sulfide with NaHS and by compound 1c showed anti-inflammatory effects even in the presence of Hcy. This study shows that exogenous delivery of H2S decreases inflammation in retinal pigment epithelial cells on exposure to Hcy in ARPE-19 cells.

Cystathionine ß-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine ß-synthase/hydrogen sulfide (CBS/H2S) axis is involved in the regulation of inflammation. However, roles of CBS in HCC development and immune evasion have not been systematically investigated, and their underlying mechanisms remain elusive. Here, we investigated the roles of CBS in tumor cells and tumor microenvironment of HCC. METHODS: 236 HCC samples were collected to detect the expression of CBS, cleaved Caspase-3 and paired related homeobox 2 (PRRX2) and the number of immune cells. HCC cell lines were employed to examine the effects of CBS on cellular viability, apoptosis and signaling in vitro. Cbs heterozygous knockout mice, C57BL/6 mice, nude mice and non-obese diabetic severe combined immunodeficiency mice were used to investigate the in vivo functions of CBS. RESULTS: Downregulation of CBS was observed in HCC, and low expression of CBS predicted poor prognosis in HCC patients. CBS overexpression dramatically promoted cellular apoptosis in vitro and inhibited tumor growth in vivo. Activation of the Cbs/H2S axis also reduced the abundance of tumor-infiltrating Tregs, while Cbs deficiency promoted Tregs-mediated immune evasion and boosted tumor growth in Cbs heterozygous knockout mice. Mechanistically, CBS facilitated the expression cleaved Caspase-3 in tumor cells, and on the other hand, suppressed Foxp3 expression in Tregs via inactivating IL-6/STAT3 pathway. As a transcription factor of IL-6, PRRX2 was reduced by CBS. Additionally, miR-24-3p was proven to be an upstream suppressor of CBS in HCC. CONCLUSIONS: Our results indicate the antitumor function of CBS in HCC by inactivation of the PRRX2/IL-6/STAT3 pathway, which may serve as a potential target for HCC clinical immunotherapy.

Clinical Significance of CBS and CCL21 in Gallbladder Adenocarcinomas and Squamous Cell/Adenosquamous Carcinomas.

Gallbladder cancer (GBC) is a rare disease with high mortality. However, no biomarkers for the carcinogenesis, progression, prognosis, and early diagnosis are clinically available. This study investigated the expressions of cystathionine-ß-synthase (CBS) and C-C chemokine receptor 7 (CCR7) protein and their clinical and pathologic significances in gallbladder squamous cell/adenosquamous carcinomas (SC/ASC) and adenocarcinomas (AC). CBS and chemokine ligand 21 (CCL21) expression was measured using immunohistochemistry in 69 SC/ASCs and 146 ACs. A significantly high percentage of patients with an age above 45 years, lymph node metastasis, and invasion was observed in the SCs/ASCs compared with ACs (P<0.05). Both AC and SC/ASC patients with positive CBS and CCL21 expression exhibited a high tumor-lymph node-metastasis stage, lymph node metastasis, and invasion compared with patients with negative CBS and CCL21 expression (P<0.05 or P<0.01). SC/ASC patients with positive CBS expression was prone to have a larger tumor size than those with negative expression (P<0.05). Positive CBS and CCL21 expression correlated with poor differentiation and larger tumor size in AC patients. Positive CBS and CCL21 are closely associated with a decreased overall survival in SC/ASC and AC patients (P<0.05 or P<0.01) and were independent factors for a poor-prognosis. Both CBS and CCL21 showed a good overall diagnostic performance for SC/ASC (AUC=0.742 and AUC=0.764, respectively) and AC (AUC=0.734 and AUC=0.718, respectively). In conclusion, positive CBS and CCL21 expression are closely associated with the clinical severity and poor prognosis in GBC, and can be a marker for the diagnosis of AC and SC/ASC type of GBC.

Analysis of Vascular Hydrogen Sulfide Biosynthesis.

With potent vasodilatory and pro-angiogenic properties, hydrogen sulfide (H2S) is now accepted as the third gasotransmitter after nitric oxide (NO) and carbon monoxide. Endogenous H2S is mainly synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). Akin to previous studies showing hormonal regulation of NO biosynthesis, we first reported that uterine and systemic artery H2S biosynthesis is regulated by exogenous estrogens in an ovariectomized sheep model of estrogen replacement therapy, specifically stimulating CBS, but not CSE, expression, in uterine (UA) and mesenteric (MA), but not carotid (CA), arteries in ovariectomized nonpregnant sheep. We have found significantly elevated H2S biosynthesis due to CBS upregulation under estrogen-dominant physiological states, the proliferative phase of menstrual cycle and pregnancy in primary human UAs. Our studies have pioneered the role of H2S biology in uterine hemodynamics regulation although there is still much that needs to be learned before a thorough elucidation of a role that H2S plays in normal physiology of uterine hemodynamics and its dysregulation under pregnancy complications can be determined. In this chapter we describe a series of methods that we have optimized for analyzing vascular H2S biosynthesis, including (1) real-time quantitative PCR (qPCR) for assessing tissue and cellular levels of CBS and CSE mRNAs, (2) immunoblotting for assessing CBS and CSE proteins, (3) semiquantitative immunofluorescence microscopy to specifically localize CBS and CSE proteins on vascular wall and to quantify their cellular expression levels, and (4) methylene blue assay for assessing H2S production in the presence of selective CBS and CSE inhibitors.

Cystathione ß-Synthase Is Increased in Thyroid Malignancies.

BACKGROUND: Cystathione ß-synthase (CBS) catalyzes the conversion of homocysteine and cysteine to hydrogen sulfide (H2S) and cystathione, via the trans-sulfuration pathway. CBS protein expression levels are increased in several different human malignancies, with increased protein expression correlating with parameters such as tumor stage, anaplasia, metastases, and chemotherapy resistance. MATERIALS AND METHODS: This study employed tissue microarrays to examine CBS expression in benign thyroid tissue, thyroid oncocytomas, thyroid follicular adenomas, and in follicular, papillary, anaplastic, and medullary thyroid carcinomas. RESULTS: CBS expression was increased in all thyroid carcinomas types compared to benign thyroid tissue, but not in thyroid follicular adenomas or oncocytomas. A similar pattern was observed for nicotinamide phosphoribosyltransferase (NAMPT) tissue microarray analysis comparing thyroid adenomas and follicular carcinomas. CONCLUSION: For the first time, we showed that an H2S-syntheszing enzyme plays a role in thyroid malignancies. Additionally, our data suggest that CBS and NAMPT immunohistochemistry may be useful in differentiating follicular adenomas from follicular carcinomas.

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (ß-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

Hydrogen Sulfide: A Novel Player in Airway Development, Pathophysiology of Respiratory Diseases, and Antiviral Defenses.

Hydrogen sulfide (H2S) is a biologically relevant signaling molecule in mammals. Along with the volatile substances nitric oxide (NO) and carbon monoxide (CO), H2S is defined as a gasotransmitter. It plays a physiological role in a variety of functions, including synaptic transmission, vascular tone, angiogenesis, inflammation, and cellular signaling. The generation of H2S is catalyzed by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). The expression of CBS and CSE is tissue specific, with CBS being expressed predominantly in the brain, and CSE in peripheral tissues, including lungs. CSE expression and activity are developmentally regulated, and recent studies suggest that CSE plays an important role in lung alveolarization during fetal development. In the respiratory tract, endogenous H2S has been shown to participate in the regulation of important functions such as airway tone, pulmonary circulation, cell proliferation or apoptosis, fibrosis, oxidative stress, and inflammation. In the past few years, changes in the generation of H2S have been linked to the pathogenesis of a variety of acute and chronic inflammatory lung diseases, including asthma and chronic obstructive pulmonary disease. Recently, our laboratory made the critical discovery that cellular H2S exerts broad-spectrum antiviral activity both in vitro and in vivo, in addition to independent antiinflammatory activity. These findings have important implications for the development of novel therapeutic strategies for viral respiratory infections, as well as other inflammatory lung diseases, especially in light of recent significant efforts to generate controlled-release H2S donors for clinical therapeutic applications.

Effect of Physical Exercise on the Febrigenic Signaling is Modulated by Preoptic Hydrogen Sulfide Production.

We tested the hypothesis that the neuromodulator hydrogen sulfide (H2S) in the preoptic area (POA) of the hypothalamus modulates the febrigenic signaling differently in sedentary and trained rats. Besides H2S production rate and protein expressions of H2S-related synthases cystathionine ß-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MPST) and cystathionine γ-lyase (CSE) in the POA, we also measured deep body temperature (Tb), circulating plasma levels of cytokines and corticosterone in an animal model of systemic inflammation. Rats run on a treadmill before receiving an intraperitoneal injection of lipopolysaccharide (LPS, 100 µg/kg) or saline. The magnitude of changes of Tb during the LPS-induced fever was found to be similar between sedentary and trained rats. In sedentary rats, H2S production was not affected by LPS. Conversely, in trained rats LPS caused a sharp increase in H2S production rate that was accompanied by an increased CBS expression profile, whereas 3-MPST and CSE expressions were kept relatively constant. Sedentary rats showed a significant LPS-induced release of cytokines (IL-1ß, IL-6, and TNF-α) which was virtually abolished in the trained animals. Correlation between POA H2S and IL-6 as well as TNF-α was observed. Corticosterone levels were augmented after LPS injection in both groups. We found correlations between H2S and corticosterone, and corticosterone and IL-1ß. These data are consistent with the notion that the responses to systemic inflammation are tightly regulated through adjustments in POA H2S production which may play an anti-inflammatory role downmodulating plasma cytokines levels and upregulating corticosterone release.

Heme-dependent Metabolite Switching Regulates H2S Synthesis in Response to Endoplasmic Reticulum (ER) Stress.

Substrate ambiguity and relaxed reaction specificity underlie the diversity of reactions catalyzed by the transsulfuration pathway enzymes, cystathionine ß-synthase (CBS) and γ-cystathionase (CSE). These enzymes either commit sulfur metabolism to cysteine synthesis from homocysteine or utilize cysteine and/or homocysteine for synthesis of H2S, a signaling molecule. We demonstrate that a kinetically controlled heme-dependent metabolite switch in CBS regulates these competing reactions where by cystathionine, the product of CBS, inhibits H2S synthesis by the second enzyme, CSE. Under endoplasmic reticulum stress conditions, induction of CSE and up-regulation of the CBS inhibitor, CO, a product of heme oxygenase-1, flip the operating preference of CSE from cystathionine to cysteine, transiently stimulating H2S production. In contrast, genetic deficiency of CBS leads to chronic stimulation of H2S production. This metabolite switch from cystathionine to cysteine and/or homocysteine renders H2S synthesis by CSE responsive to the known modulators of CBS: S-adenosylmethionine, NO, and CO. Used acutely, it regulates H2S synthesis; used chronically, it might contribute to disease pathology.

Endogenous CBS-H2S Pathway Contributes to the Development of CCI-Induced Neuropathic Pain.

Studies showed a complex relationship between hydrogen sulfide (H2S) and neuropathic pain. In this study, the relationship between endogenous CBS-H2S pathway in L4-6 spinal cord and neuropathic pain was explored. A total of 163 adult Kunming mice were used in this study. CBS expression and H2S formation in L4-6 spinal cord were detected in the development of neuropathic pain firstly. Then, effect of AOAA, an CBS inhibitor, on treatment of neuropathic pain by chronic construction injury surgery (CCI) was detected. Pain thresholds and activation of NF-κB(p65), ERK1/2 and CREB were measured as biomarks of neuropathic pain. Results showed that CCI surgery significantly upregulated protein expression of CBS and H2S formation. Correlation analysis showed pain thresholds had negative relationships with protein expression of CBS and H2S formation. Treatment with AOAA, a CBS inhibitor, inhibited CCI-induced upregulation of CBS expression and H2S formation (P < 0.05). Further, AOAA significantly decreased activation of NF-κB(p65), ERK1/2 and CREB pathway, and reversed CCI-induced allodynia (P < 0.05). This indicated that CBS-H2S pathway promoted the development of neuropathic pain. CBS-H2S pathway could be a promising target for treatment of neuropathic pain.

Expression profile of hydrogen sulfide and its synthases correlates with tumor stage and grade in urothelial cell carcinoma of bladder.

BACKGROUND: Hydrogen sulfide (H2S) is a newly discovered gas transmitter. It is synthesized by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MPST). Endogenous hydrogen sulfide has never been studied in bladder cancer. PURPOSE: We evaluated H2S production and its synthases expression levels in transitional cell carcinoma (urothelial cell carcinoma of bladder [UCB]) of human bladder tissue and cell lines. MATERIALS AND METHODS: Immunostaining was performed in urothelial cell lines and bladder specimens from 94 patients with UCB of different stages/grades. The expression levels/activities of CBS, CSE, and MPST of specimens and cell lines were analyzed by image semiquantity assay, western blot, and a sulfur-sensitive electrode. We tried to find the correlation between hydrogen sulfide and its synthases with tumor stage in UCB. All experiments were repeated at least 3 times. RESULTS: Immunoreactivity for CBS, CSE, and MPST was detected in malignant uroepithelium and muscular layer of all tissues examined and cultured cells. The expression levels of CBS, CSE, and MPST were associated with UCB stage/grade. Muscle-invasive bladder cancer samples showed the highest production of H2S (52.6±2.91 nmol/[mg·min]) among all tested samples and EJ cells (transitional cell carcinoma, grade IIIshowed the highest production of H2S among all tested cell lines (53.3±7.02nmol/[mg·min]). CONCLUSIONS: Protein levels and catalytic activities of CBS, CSE, and MPST increased with the increase of malignant degrees in human bladder tissues and human UCB cell lines. Our findings may promote the application of these novel enzymes to UCB diagnosis or treatment.

Calcitriol prevents peripheral RSC96 Schwann neural cells from high glucose & methylglyoxal-induced injury through restoration of CBS/H2S expression.

A meta-analysis has suggested that vitamin D deficiency is involved in diabetic peripheral neuropathy (DPN) and the levels of hydrogen sulfide (H2S) are also decreased in type 2 diabetes. The injection of vitamin D induces cystathionine-ß-synthase (CBS) expression and H2S generation. However, it remains unclear whether the supplementation of vitamin D prevents DPN through improvement of CBS/H2S expression. In the present study, RSC96 cells, a rat Schwann cell line, were exposed to high glucose and methylglyoxal (HG&MG) to simulate diabetic peripheral nerve injury in vivo. Before the exposure to HG&MG, the cells were preconditioned with calcitriol (CCT), an active form of vitamin D, and then CCT-mediated neuroprotection was investigated in respect of cellular viability, superoxide anion (O2(-)) generation, inducible nitric oxide (NO) synthase (iNOS)/NO expression, mitochondrial membrane potential (MMP), as well as CBS expression and activity. It was found that both high glucose and MGO decreased cell viability and co-treatment with the two induced a more serious injury in RSC96 cells. Therefore, the exposure to HG&MG was used in the present study. The exposure to HG&MG markedly induced iNOS expression, NO and O2(-) generation, as well as MMP loss. In addition, the exposure to HG&MG depressed CBS expression and activity in RSC96 cells. However, the preconditioning with CCT significantly antagonized HG&MG-induced cell injury including the decreased viability, iNOS overexpression, NO and O2(-) accumulation, as well as MMP loss. CCT also partially restored the decreased CBS expression and activity triggered by HG&MG, while the inhibition of CBS with hydroxylamine attenuated CCT-mediated neuroprotection. Moreover, the exogenous donation of H2S produced similar cellular protective effects to CCT. The data indicate that the supplementation of vitamin D prevents HG&MG-induced peripheral nerve injury involving the restoration of endogenous H2S system, which may provide a basal support for the treatment of DPN with vitamin D clinically.

The H2S-generating enzymes cystathionine ß-synthase and cystathionine γ-lyase play a role in vascular development during normal lung alveolarization.

The gasotransmitter hydrogen sulfide (H2S) is emerging as a mediator of lung physiology and disease. Recent studies revealed that H2S administration limited perturbations to lung structure in experimental animal models of bronchopulmonary dysplasia (BPD), partially restoring alveolarization, limiting pulmonary hypertension, limiting inflammation, and promoting epithelial repair. No studies have addressed roles for endogenous H2S in lung development. H2S is endogenously generated by cystathionine ß-synthase (Cbs) and cystathionine γ-lyase (Cth). We demonstrate here that the expression of Cbs and Cth in mouse lungs is dynamically regulated during lung alveolarization and that alveolarization is blunted in Cbs(-/-) and Cth(-/-) mouse pups, where a 50% reduction in the total number of alveoli was observed, without any impact on septal thickness. Laser-capture microdissection and immunofluorescence staining indicated that Cbs and Cth were expressed in the airway epithelium and lung vessels. Loss of Cbs and Cth led to a 100-500% increase in the muscularization of small- and medium-sized lung vessels, which was accompanied by increased vessel wall thickness, and an apparent decrease in lung vascular supply. Ablation of Cbs expression using small interfering RNA or pharmacological inhibition of Cth using propargylglycine in lung endothelial cells limited angiogenic capacity, causing a 30-40% decrease in tube length and a 50% decrease in number of tubes formed. In contrast, exogenous administration of H2S with GYY4137 promoted endothelial tube formation. These data confirm a key role for the H2S-generating enzymes Cbs and Cth in pulmonary vascular development and homeostasis and in lung alveolarization.

Cystathionine-ß-Synthase Gene Transfer Into Rostral Ventrolateral Medulla Exacerbates Hypertension via Nitric Oxide in Spontaneously Hypertensive Rats.

BACKGROUND: Rostral ventrolateral medulla (RVLM) plays a crucial role in the central regulation of cardiovascular functions. Cystathionine-ß-synthase (CBS) is a major hydrogen sulfide (H2S)-generating enzyme that has been identified mainly in the brain. The present study was designed to examine CBS expression and determine its roles and mechanisms of regulating sympathetic outflow and blood pressure (BP) in the RVLM in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: CBS expression was decreased in the RVLM in SHR compared to Wistar-Kyoto (WKY) rats. Accumulating evidences suggest that H2S interacts with nitric oxide (NO) to regulate cardiovascular function. Therefore, we hypothesize that the decrease in CBS expression in the RVLM may be involved in the disorder of l-arginine/NO pathway, which subsequently affects BP in SHR. Overexpression of CBS in the RVLM caused significant increases in BP, heart rate, and urinary norepinephrine excretion in SHR but not in WKY. Acute experiments were carried out at day 7 after gene transfer. NO metabolite levels, neuronal NO synthase, and γ-amino butyric acid were decreased in SHR after CBS gene transfer. Furthermore, pressor responses to microinjection of NG-monomethyl-l-arginine into RVLM were blunt in SHR transfected with AdCBS compared to SHR transfected with AdEGFP. CONCLUSIONS: Overexpression of CBS in the RVLM elicits enhanced pressor responses in SHR, but not in WKY, and the NO system is involved in these effects. The results suggest that alterations of H2S signaling in the brain may be associated with the development of hypertension.

Gene transfer of cystathionine ß-synthase into RVLM increases hydrogen sulfide-mediated suppression of sympathetic outflow via KATP channel in normotensive rats.

Hydrogen sulfide has been shown to have a sympathoinhibitory effect in the rostral ventrolateral medulla (RVLM). The present study examined the function of cystathionine ß-synthase (CBS)/hydrogen sulfide system in the RVLM, which plays a crucial role in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding CBS (AdCBS) or enhanced green fluorescent protein (AdEGFP) were transfected into the RVLM in normotensive rats. Identical microinjection of AdCBS into the RVLM had no effect on systolic blood pressure and heart rate (HR) in conscious rats. Acute experiments were performed at day 7 after gene transfer in anesthetized rats. Microinjection of the CBS inhibitors hydroxylamine (HA) or amino-oxyacetate into the RVLM produced an increase in the renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and HR. There was a potentiation of the increases in RSNA, MAP, and HR because of the CBS inhibitors in AdCBS-injected rats compared with AdEGFP-injected rats. Pretreatment with pinacidil, a ATP-sensitive potassium (KATP) channel activator, abolished the effects of HA in two groups. Microinjection of glibenclamide, a KATP channel blocker, produced increases in RSNA, MAP, and HR in AdCBS-injected rats. No changes in behavior were observed in AdEGFP-injected rats. Furthermore, Western blot analysis indicated an increase in the expression of sulfonylurea receptor 2 and inward rectifier K(+) 6.1 in AdCBS-injected rats. These results suggest that the increase in KATP channels in the RVLM may be responsible for the greater sympathetic outflow and pressor effect of HA in AdCBS-injected rats compared with AdEGFP-injected rats.

Hypoxia-inducible factors regulate human and rat cystathionine ß-synthase gene expression.

Increased catalytic activity of CBS (cystathionine ß-synthase) was recently shown to mediate vasodilation of the cerebral microcirculation, which is initiated within minutes of the onset of acute hypoxia. To test whether chronic hypoxia was a stimulus for increased CBS expression, U87-MG human glioblastoma and PC12 rat phaeochromocytoma cells were exposed to 1% or 20% O2 for 24-72 h. CBS mRNA and protein expression were increased in hypoxic cells. Hypoxic induction of CBS expression was abrogated in cells transfected with vector encoding shRNA targeting HIF (hypoxia-inducible factor) 1α or 2α. Exposure of rats to hypobaric hypoxia (0.35 atm; 1 atm=101.325 kPa) for 3 days induced increased CBS mRNA, protein and catalytic activity in the cerebral cortex and cerebellum, which was blocked by administration of the HIF inhibitor digoxin. HIF-binding sites, located 0.8 and 1.2 kb 5' to the transcription start site of the human CBS and rat Cbs genes respectively, were identified by ChIP assays. A 49-bp human sequence, which encompassed an inverted repeat of the core HIF-binding site, functioned as a hypoxia-response element in luciferase reporter transcription assays. Thus HIFs mediate tissue-specific CBS expression, which may augment cerebral vasodilation as an adaptive response to chronic hypoxia.

High-fat diet stimulates hepatic cystathionine ß-synthase and cystathionine γ-lyase expression.

Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) catalyze homocysteine (Hcy) metabolism via the trans-sulfuration pathway. They are also responsible for hydrogen sulfide (H2S) production via desulfuration reactions. The liver contributes significantly to the regulation of Hcy and H2S homeostasis, which might participate in many physiological and pathological processes. The aim of this study was to investigate the effect of a high-fat diet (HFD) on hepatic CBS and CSE expression and its impact on Hcy and H2S metabolism. Mice (C57BL/6) fed a HFD (60% kcal fat) for 5 weeks developed fatty liver. The mRNA and protein levels of CBS and CSE in the liver were significantly elevated in mice fed a HFD. Subsequently the metabolism of Hcy by CBS and CSE was increased in the liver, and its level decreased in the circulation. Increased CBS and CSE expression also caused a significant elevation in H2S production in the liver. The level of lipid peroxides was elevated, indicating oxidative stress, while the level of total glutathione remained unchanged in the liver of HFD-fed mice. Upregulation of the trans-sulfuration pathway might play an adaptive role against oxidative stress by maintaining total glutathione levels in the liver.

Further evidence of endogenous hydrogen sulphide as a mediator of relaxation in human and rat bladder.

We investigated the expression of hydrogen sulphide (H2S) in human and rat lower urinary tract (including bladder, prostate and urethra) tissues, and we sought to determine whether H2S induces relaxation of human and Sprague-Dawley (SD) rat bladder strips. Human normal lower urinary tract tissue was obtained for the evaluation of endogenous H2S productivity using a sulphide-sensitive electrode and for the analysis of the expression levels of all three synthases of endogenous H2S, cystathionine ß-synthase (CBS), cystathionine γ lyase (CSE) and 3-mercaptopyruvate sulphur transferase (MPST, as known as 3-MST) by Western blot assay. CBS, CSE and MPST were located in human sample slides by immunohistochemistry. Human and male adult SD rat bladder strips were tested for H2S function with a transducer and recorded. All experiments were repeated six times. The endogenous H2S productivity and the H2S synthases had various distributions in the human and rat lower urinary tract tissues and were located in both epithelial and stromal sections. L-cysteine (L-Cys, a substrate of CBS, CSE and MPST) elicited relaxation in a dose-dependent manner on human bladder strips pre-contracted by acetylcholine chloride. This effect could be diminished by the ATP-sensitive potassium ion (KATP) channel blocker glibenclamide (GLB), the CSE inhibitor DL-propargylglycine (PPG) and the CBS inhibitor hydroxylamine (HA). H2S and its three synthases were present in the human and rat lower urinary tract tissues and relaxed human and rat bladder strips, which implied that endogenous H2S might play a role in physiological function and pathological disorders of the lower urinary tract symptoms (LUTS) or overactive bladder (OAB).

Neonatal colonic inflammation sensitizes voltage-gated Na(+) channels via upregulation of cystathionine ß-synthetase expression in rat primary sensory neurons.

The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood, and treatment remains difficult. We have previously reported that colon-specific dorsal root ganglion (DRG) neurons were hyperactive in a rat model of IBS induced by neonatal colonic inflammation (NCI). This study was designed to examine plasticity of voltage-gated Na(+) channel activities and roles for the endogenous hydrogen sulfide-producing enzyme cystathionine ß-synthetase (CBS) in chronic visceral hyperalgesia. Abdominal withdrawal reflex (AWR) scores were recorded in response to graded colorectal distention in adult male rats as a measure of visceral hypersensitivity. Colon-specific DRG neurons were labeled with 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate and acutely dissociated for measuring Na(+) channel currents. Western blot analysis was employed to detect changes in expressions of voltage-gated Na(+) (Na(V)) channel subtype 1.7, Na(V)1.8, and CBS. NCI significantly increased AWR scores when compared with age-matched controls. NCI also led to an ~2.5-fold increase in Na(+) current density in colon-specific DRG neurons. Furthermore, NCI dramatically enhanced expression of Na(V)1.7, Na(V)1.8, and CBS in colon-related DRGs. CBS was colocalized with Na(V)1.7 or -1.8 in colon-specific DRG neurons. Administration of O-(carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, remarkably suppressed Na(+) current density and reduced expression of Na(V)1.7 and Na(V)1.8. More importantly, intraperitoneal or intrathecal application of AOAA attenuated AWR scores in NCI rats in a dose-dependent manner. These data suggest that NCI enhances Na(+) channel activity of colon DRG neurons, which is most likely mediated by upregulation of CBS expression, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Down-regulation of hydrogen sulfide biosynthesis accompanies murine interstitial cells of Cajal dysfunction in partial ileal obstruction.

PURPOSE: To investigate the role of endogenous hydrogen sulfide (H(2)S) in partial obstruction-induced dysfunction of the interstitial cells of Cajal (ICC) in mice ileum. MATERIALS AND METHODS: Partial intestinal obstruction was induced surgically in male imprinting control region (ICR) mice. ICC networks were studied by Immunohistochemistry. Electrical activity was recorded by intracellular recording techniques. The expression of ICC phenotype marker c-kit receptor tyrosine kinase (c-kit), membrane binding stem cell factor (mSCF), the endogenous H(2)S biosynthesis enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) was studied by Western blotting. The expression of tumor necrosis factor-α (TNF-α) mRNA was observed by using real-time polymerase chain reaction. RESULTS: Partial intestinal obstruction resulted in ICC networks were disrupted above obstruction 14 days after the operation. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed and their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized. The expression of c-kit and mSCF was significantly decreased, also suggesting the disruption of the ICC network. The expression of TNF-α was significantly increased in the tunica muscularis of the obstructed intestine. Treatment of cultured intestinal smooth muscle cells with TNF-α caused dramatic down regulation of mSCF. The expression of CBS and CSE was significantly decreased in the tunica muscularis of the obstructed intestine. Intraperitoneal injection (i.p) of DL-propargylglycine, an irreversible inhibitor of CSE, and aminooxyacetic acid, an inhibitor of CBS, elevated the expression of TNF-α mRNA in the tunica muscularis of the ileum. Obstruction-induced over expression of TNF-α was significantly improved by supplementation of NaHS, but not the expressions of mSCF and c-kit. CONCLUSIONS: The down regulation of endogenous H(2)S biosynthesis is related to over expression of TNF-α in obstructed small intestine. TNF-α-mediated mSCF down-regulation is not the only reason of partial intestinal obstruction-induced loss of ICC.