Cystathionine Β-synthase Research Articles

Hydrogen sulfide production in the medullary respiratory center modulates the neural circuit for respiratory pattern and rhythm generations

Hydrogen sulfide (H2S), which is synthesized in the brain, modulates the neural network. Recently, the importance of H2S in respiratory central pattern generation has been recognized, yet the function of H2S in the medullary respiratory network remains poorly understood. Here, to evaluate the functional roles of H2S in the medullary respiratory network, the Bötzinger complex (BötC), the pre-Bötzinger complex (preBötC), and the rostral ventral respiratory group (rVRG), we observed the effects of inhibition of H2S synthesis at each region on the respiratory pattern by using an in situ arterially perfused preparation of decerebrated male rats. After microinjection of an H2S synthase inhibitor, cystathionine β-synthase, into the BötC or preBötC, the amplitude of the inspiratory burst decreased and the respiratory frequency increased according to shorter expiration and inspiration, respectively. These alterations were abolished or attenuated in the presence of a blocker of excitatory synaptic transmission. On the other hand, after microinjection of the H2S synthase inhibitor into the rVRG, the amplitude of the inspiratory burst was attenuated, and the respiratory frequency decreased, which was the opposite effect to those obtained by blockade of inhibitory synaptic transmission at the rVRG. These results suggest that H2S synthesized in the BötC and preBötC functions to limit respiratory frequency by sustaining the respiratory phase and to maintain the power of inspiration. In contrast, H2S synthesized in the rVRG functions to promote respiratory frequency by modulating the interval of inspiration and to maintain the power of inspiration. The underlying mechanism might facilitate excitatory synaptic transmission and/or attenuate inhibitory synaptic transmission.

Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants.

Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.

Acute pancreatitis as a key to diagnosis of hyperhomocysteinemia in a 12-year-old boy caused by CBS gene mutation

Acute pancreatitis as a key to diagnosis of hyperhomocysteinemia in a 12-year-old boy caused by CBS gene mutation

Exploration of cystathionine β-synthase and cystathionine γ-lyase gene expression poor prognostic predictive value with the application of bioinformatics data mining in liver cancer

Objective: To study the relationship between cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) genes-related signaling pathways in liver cancer cells. Methods: We conducted a correlation analysis between the clinical features of CBS and CTH gene expression by mining the GEO (Gene Expression Omnibus) and TCGA (The Cancer Genome Atlas) databases of liver cancer. Additionally, liver cancer cell lines were verified by immunoblotting. Results: CBS and CTH expressions were significantly lower in tumors than in non-tumors (P < 0.05). COX regression result showed that CBS was an independent risk factor for the poor prognosis of liver cancer cells (HR=0.65, P = 0.02). A univariate logistic regression analysis was performed on the different tumor stages focusing on the CBS gene, which showed that TNM stage II verses I (P = 0.01, OR=0.50), stage III verses I (P = 0.03, OR=0.56), T stage T2 verses T1 (P < 0.01, OR=0.43), and T3 stage verses T1 (P = 0.02, OR=0.54) were significantly lower in liver cancer. TNM stage III verses I (P = 0.01, OR=0.50), Edmondson stage II verses I (P = 0.03, OR=0.48), stage III verses stage I (P < 0.01, OR=0.30), stage IV verses I (P = 0.03, OR=0.22), and T stage T3 verses T1(P = 0.03, OR=0.22) of the CTH gene expressions were significantly lower in liver cancer. GSEA enrichment analysis result revealed that the signaling pathway most correlated with the expression of CTH and CBS genes in liver cancer cells was cytochrome P450 (CYP450) (FDR Q < 0.01, FWER P < 0.01). Western blot results showed that the expression of the CTH downstream protein CSE was reduced in HCC cell lines such as HLE and Hep3B cells compared with the human immortalized liver cell line HL-7702. Conclusion: CBS and CTH gene expressions are lower in tumor tissue than in normal tissue groups. The CBS gene is an independent risk factor for poor prognosis in stem cell carcinoma. The cytochrome P450 is the signaling pathway most closely related to the CBS and CTH genes.

Disease-causing cystathionine β-synthase linker mutations impair allosteric regulation

Cystathionine β-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (∼8-fold) decrease in basal activity, which is either unresponsive to, or inhibited by, AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects, and that the differences between them are expressed in the second half reaction, i.e. homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.

Inborn errors of amino acid metabolism - from underlying pathophysiology to therapeutic advances.

Amino acids are organic molecules that serve as basic substrates for protein synthesis and have additional key roles in a diverse array of cellular functions, including cell signaling, gene expression, energy production and molecular biosynthesis. Genetic defects in the synthesis, catabolism or transport of amino acids underlie a diverse class of diseases known as inborn errors of amino acid metabolism. Individually, these disorders are rare, but collectively, they represent an important group of potentially treatable disorders. In this Clinical Puzzle, we discuss the pathophysiology, clinical features and management of three disorders that showcase the diverse clinical presentations of disorders of amino acid metabolism: phenylketonuria, lysinuric protein intolerance and homocystinuria due to cystathionine β-synthase (CBS) deficiency. Understanding the biochemical perturbations caused by defects in amino acid metabolism will contribute to ongoing development of diagnostic and management strategies aimed at improving the morbidity and mortality associated with this diverse group of disorders.

The Role of Hydrogen Sulfide in the Localization and Expression of p53 and Cell Death in the Nervous Tissue in Traumatic Brain Injury and Axotomy.

Traumatic brain injury (TBI) is one of the leading causes of disability and death worldwide. It is characterized by various molecular-cellular events, with the main ones being apoptosis and damage to axons. To date, there are no clinically effective neuroprotective drugs. In this study, we examined the role of hydrogen sulfide (H2S) in the localization and expression of the key pro-apoptotic protein p53, as well as cell death in the nervous tissue in TBI and axotomy. We used a fast donor (sodium sulphide, Na2S) H2S and a classic inhibitor (aminooxyacetic acid, AOAA) of cystathionine β-synthase (CBS), which is a key enzyme in H2S synthesis. These studies were carried out on three models of neurotrauma in vertebrates and invertebrates. As a result, it was found that Na2S exhibits a pronounced neuroprotective effect that reduces the number of TUNEL-positive neurons and glial cells in TBI and apoptotic glia in axotomy. This effect could be realized through the Na2S-dependent decrease in the level of p53 in the cells of the nervous tissue of vertebrates and invertebrates, which we observed in our study. We also observed the opposite effect when using AOAA, which indicates the important role of CBS in the regulation of p53 expression and death of neurons and glial cells in TBI and axotomy.

Metabolic control analysis of the transsulfuration pathway and the compensatory role of the cysteine transport in Trypanosoma cruzi

Trypanosoma cruzi is the causal agent of American Trypanosomiasis or Chagas Disease in humans. The current drugs for its treatment benznidazole and nifurtimox have inconveniences of toxicity and efficacy; therefore, the search for new therapies continues. Validation through genetic strategies of new drug targets against the parasite metabolism have identified numerous essential genes. Target validation can be further narrowed by applying Metabolic Control Analysis (MCA) to determine the flux control coefficients of the pathway enzymes. That coefficient is a quantitative value that represents the degree in which an enzyme/transporter determines the flux of a metabolic pathway; those with the highest coefficients can be promising drug targets. Previous studies have demonstrated that cysteine (Cys) is a key precursor for the synthesis of trypanothione, the main antioxidant metabolite in the parasite. In this research, MCA was applied in an ex vivo system to the enzymes of the reverse transsulfuration pathway (RTP) for Cys synthesis composed by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL). The results indicated that CGL has 90% of the control of the pathway flux. Inhibition of CGL with propargylglycine (PAG) decreased the levels of Cys and trypanothione and depleted those of glutathione in epimastigotes (proliferative stage in the insect vector); these metabolite changes were prevented by supplementing with Cys, suggesting a compensatory role of the Cys transport (CysT). Indeed, Cys supplementation (but not PAG treatment) increased the activity of the CysT in epimastigotes whereas in trypomastigotes (infective stage in mammals) CysT was increased when they were incubated with PAG. Our results suggested that CGL could be a potential drug target given its high control on the RTP flux and its effects on the parasite antioxidant defense. However, the redundant Cys supply pathways in the parasite may require inhibition of the CysT as well. Our findings also suggest differential responses of the Cys supply pathways in different parasite stages.

Hypoxia-Induced Changes in L-Cysteine Metabolism and Antioxidative Processes in Melanoma Cells.

This study was performed on human primary (WM115) and metastatic (WM266-4) melanoma cell lines developed from the same individual. The expression of proteins involved in L-cysteine metabolism (sulfurtransferases, and cystathionine β-synthase) and antioxidative processes (thioredoxin, thioredoxin reductase-1, glutathione peroxidase, superoxide dismutase 1) as well as the level of sufane sulfur, and cell proliferation under hypoxic conditions were investigated. Hypoxia in WM115 and WM266-4 cells was confirmed by induced expression of carbonic anhydrase IX and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 by the RT-PCR and Western blot methods. It was shown that, under hypoxic conditions the inhibition of WM115 and WM266-4 melanoma cell proliferation was associated with decreased expression of thioredoxin reductase-1 and cystathionine β-synthase. These two enzymes may be important therapeutic targets in the treatment of melanoma. Interestingly, it was also found that in normoxia the expression and activity of 3-mercaptopyruvate sulfurtransferase in metastatic WM266-4 melanoma cells was significantly higher than in primary melanoma WM115 cells.

Clinical and Genetic Description of Hereditary Chronic Pancreatitis in Pakistani Children.

The purpose of this study was to identify the spectrum and frequency of pathogenic variants as well as the clinical and genetic insight of hereditary chronic pancreatitis in Pakistani children. The deoxyribonucleic acid of affected probands of 44 unrelated Pakistani families, having hereditary chronic pancreatitis-affected children, were subjected to massive parallel sequencing for candidate reported genes (SPINK1, PRSS1, CFTR, CPA1, CTRC, CBS, AGL, PHKB, and LPL). Data were analyzed using different bioinformatics tools for the variants and in-silico analysis. All the identified variants were validated by direct sequencing of the targeted exons in the probands and their parents. There were 50 patients included in this study with confirmed hereditary chronic pancreatitis. Nine known mutations in SPINK1, PRSS1, CFTR, CTRC, CBS, and AGL genes, and 10 novel variants in LPL, CFTR, CTR, and PHKB genes were identified. The identified variants were found in heterozygous, compound heterozygous, and trans-heterozygous forms, with rare allele frequency in the normal population. The novel variants were [c.378C>T(p.Lys126Asn) and c.719G>A(p.Arg240Gln) in CTRC, c.586-3C>A and c.763A>G(p.Arg255Gly) in CPA1, c.1160_1161insT(p.Lys387Asnfs*26), c.784C>T(p.Gln262*), c.1139+1G>A, c.175G>A(p.Gly59Arg) in LPL, c.388C>G(p.leu130val) in CFTR, and c.2327G>A(p.Arg776His in PHKB)]. The phenotypic characteristics were variable and correlated with the relevant variant. The genetic composition plays a significant role in the predisposition of hereditary chronic pancreatitis. The clinical presentation varies with the genetic determinant involved. This information would help in building up a diagnostic algorithm for our population that can be used for genetic screening services in affected cohorts.

H2S-Reactivating Antitumor Immune Response after Microwave Thermal Therapy for Long-Term Tumor Suppression.

Microwave thermal therapy (MWTT) is one of the most potent ablative treatments known, with advantages like deep penetration, minimal invasion, repeatable operation, and low interference from bone and gas. However, microwave (MW) is not selective against tumors, and residual tumors after incomplete ablation will generate immunosuppression, ultimately making tumors prone to recurrence and metastasis. Herein, a nano-immunomodulator (Bi-MOF-l-Cys@PEG@HA, BMCPH) is proposed to reverse the immunosuppression and reactivate the antitumor immune effect through responsively releasing H2S in tumor cells for improving MWTT. Under MW irradiation, BMCPH will mediate MWTT to ablate tumors and release l-cysteine (l-Cys) to react with the highly expressed cystathionine β-synthase in tumor to generate H2S. The generated H2S can inhibit the accumulation of myeloid-derived suppressor cells (MDSCs) and promote the expression of cytotoxic T lymphocytes (CTLs). Moreover, Bi-MOF can also scavenge reactive oxygen species (ROS), a major means of MDSCs-mediated immunosuppression, to further weaken the immunosuppressive effect. Simultaneously, the surface-covered HA will gather CTLs around the tumor to enhance the immune response. This nano gas immunomodulator provides an idea for the sensitive and tunable release of unstable gas molecules at tumor sites. The strategy of H2S gas to reverse immunosuppression and reactivate antitumor immune response introduces a direction to reduce the risk of tumor recurrence and metastasis after thermal ablation.

Dual targeting of H2S synthesizing enzymes; cystathionine β-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer

IntroductionHydrogen sulfide (H2S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H2S synthesizing enzymes; Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. ObjectivesFirstly, this study aimed to demonstrate a comparative expression profile of H2S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H2S synthesizing enzymes that could cease H2S production and reduce TNBC pathogenicity. MethodsPairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H2S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H2S levels were detected using a AzMc fluorescent probe. ResultsBC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H2S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H2S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. ConclusionThis study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H2S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.

Classical homocystinuria presenting with transient basal ganglia pathology and dystonia.

Classical homocystinuria is caused by pathogenic variants in the CBS gene leading to a deficiency of the vitamin B6-dependent enzyme cystathionine beta synthase. The disease is typically associated with high blood homocysteine concentrations. Clinical features include developmental delay/intellectual disability, psychiatric problems, thromboembolism, lens dislocation, and marfanoid habitus. We report on a child with classical homocystinuria presenting with acute episodes of dystonia and symmetrical basal ganglia abnormalities mimicking a mitochondrial disease. After starting treatment with vitamin B6, homocysteine levels rapidly normalized and dystonic episodes did not re-occur. Moreover, brain-imaging findings almost completely disappeared. The case illustrates that homocystinuria should be considered as a treatable differential diagnosis of dystonia.

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5′-phosphate (PLP) via Schiff base formation

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5′-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 μM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

B-265 Prevalence of 844ins68 Polymorphism in the Cystathionine β-Synthase Gene in Brazilian Individuals

Abstract Background Deficiency of Cystathionine β-Synthase (CBS) is the most common cause of the homocysteine increase in blood and urine. This is an important autosomal recessive disorder with pleiotropic clinical manifestations in the vascular system, the skeletal structure, the ocular lens, and the central nervous system. Several polymorphisms in CBS gene have been associated with homocystinuria, with consequent premature arterial and/or venous occlusive diseases. The thymine-to-cytosine substitution at nucleotide 833 of the CBS gene (T833C) is the majority predominant mutation in individuals with homocystinuria. Several studies of T833C have been performed on patients where venous or arterial occlusive diseases are an important risk factor. The T833C causes a substitution of hydrophobic isoleucine by a more hydrophilic threonine at codon 278 (I278T) and affects the CBS conformation or the interaction of the CBS subunits. Most occurrences of T833C are known to segregate in cis with 844ins68 insertion polymorphism in the exon 8, duplicating the intron 7 splice acceptor. At the mRNA level, polymorphisms T833C and 844ins68 are skipped next to the use of this alternative splicing site. Therefore, previous studies about 844ins68 produced conflicting results that show a neutral or even effect protective of the insertion against occlusive venous and arterial occlusive disease. Thus, this study aims to describe the genotypic profile of the 844ins68 mutation in CBS gene in Brazilian individuals affect with vascular diseases or patients at risk for genetic variants segregating in the family. Methods The study group consisted of 1301 individuals submitted to the identification of the CBS 844ins68 in Grupo Pardini Laboratory. Genomic DNA was isolated from EDTA blood using QIAcard FTA Classic (Qiagen). To detect the insertion, the genomic fragments comprising the intron 7 and exon 8 in the coding region and splicing junctions of the CBS gene were amplified by Polymerase Chain Reaction (PCR). Results Our sampling included 214 men’s blood samples (16.4%) and 1087 women’s blood samples (83.5%). We found a prevalence of 21.6% individuals with the insertion. Males without the 844ins68 mutation (wild type) predominated 161/214, (75.2%), carriers of 844ins68 heterozygotes were 45/214 (21%), and homozygotes were 8/214 (3.7%). In female individuals, was found 859/1087 (79%) allele without 844ins68 (wild type), 214/1087 (19.7%) carriers of 844ins68 heterozygotes and 14/1087 (1.3%) homozygotes. The number of solicitations for the exam in women is greater compared to men. Conclusions Our findings show that the prevalence of 844ins68 in Brazil is compatible with others studies involving Brazilian women with cardiovascular defects. According to the literature, the insertion is most frequent in females than in males. Considering the possible restoration of non-mutated RNA through preferential splicing of the 844ins68, our study suggest that mutated individuals have the insertion as a protective factor for the development of venous or arterial occlusive diseases. The search of 844ins68 should be evaluated in association with the clinical aspects.

Nanomotor-based H2S donor with mitochondrial targeting function for treatment of Parkinson's disease

Reduction of endogenous hydrogen sulfide (H2S) is considered to have an important impact on the progress of Parkinson's disease (PD), thus exogenous H2S supplementation is expected to become one of the key means to treat PD. However, at present, it is difficult for H2S donors to effectively penetrate the blood brain barrier (BBB), selectively release H2S in brain, and effectively target the mitochondria of neuron cells. Herein, we report a kind of nanomotor-based H2S donor, which is obtained by free radical polymerization reaction between l-cysteine derivative modified-polyethylene glycol (PEG-Cys) and 2-methacryloyloxyethyl phosphorylcholine (MPC). This kind of H2S donor can not only effectively break through BBB, but also be specifically catalyzed by cystathionine β-synthase (CBS) in neurons of PD site in brain and 3-mercaptopyruvate sulfurtransferase (3-MST) in mitochondria to produce H2S, endowing it with chemotaxis/motion ability. Moreover, the unique chemotaxis effect of nanomotor can realize the purpose of precisely targeting brain and the mitochondria of damaged neuron cytopathic diseases. This kind of nanomotor-based H2S donor is expected to enrich the current types of H2S donors and provide new ideas for the treatment of PD.

Biphasic inflammatory response induced by intra-plantar injection of L-cysteine: Role of CBS-derived H2S and S1P/NO signaling

This study investigates the inflammatory response to intra-plantar injection of L-cysteine in a murine model. L-cysteine induces a two-phase response: an early phase lasting 6 h and a late phase peaking at 24 h and declining by 192 h. The early phase shows increased neutrophil accumulation at 2 h up to 24 h, followed by a reduction at 48 h. On the other hand, the late phase exhibits increased macrophage infiltration peaking at 96 h. Inhibition of cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway, significantly reduces L-cysteine-induced edema, suggesting its dependence on CBS-derived hydrogen sulfide (H2S). Sequential formation of sphingosine-1-phosphate (S1P) preceding nitric oxide (NO) generation suggests the involvement of a CBS/S1P/NO axis in the inflammatory response. Inhibition of de novo sphingolipid biosynthesis, S1P1 receptor, and endothelial NO synthase (eNOS) attenuates L-cysteine-induced paw edema. These findings indicate a critical role of the CBS/H2S/S1P/NO signaling pathway in the development and maintenance of L-cysteine-induced inflammation. The co-presence of H2S and NO is necessary for inducing and sustaining the inflammatory response, as NaHS or L-arginine alone do not replicate the marked and prolonged inflammatory effect observed with L-cysteine. This study enhances our understanding of the complex molecular mechanisms of the interplay between NO and H2S pathways in inflammation and identifies potential therapeutic targets for inflammatory disorders.

ICI 182,780 Attenuates Selective Upregulation of Uterine Artery Cystathionine β-Synthase Expression in Rat Pregnancy.

Endogenous hydrogen sulfide (H2S) produced by cystathionine β-synthase (CBS) and cystathionine-γ lyase (CSE) has emerged as a novel uterine vasodilator contributing to pregnancy-associated increases in uterine blood flow, which safeguard pregnancy health. Uterine artery (UA) H2S production is stimulated via exogenous estrogen replacement and is associated with elevated endogenous estrogens during pregnancy through the selective upregulation of CBS without altering CSE. However, how endogenous estrogens regulate uterine artery CBS expression in pregnancy is unknown. This study was conducted to test a hypothesis that endogenous estrogens selectively stimulate UA CBS expression via specific estrogen receptors (ER). Treatment with E2β (0.01 to 100 nM) stimulated CBS but not CSE mRNA in organ cultures of fresh UA rings from both NP and P (gestational day 20, GD20) rats, with greater responses to all doses of E2β tested in P vs. NP UA. ER antagonist ICI 182,780 (ICI, 1 µM) completely attenuated E2β-stimulated CBS mRNA in both NP and P rat UA. Subcutaneous injection with ICI 182,780 (0.3 mg/rat) of GD19 P rats for 24 h significantly inhibited UA CBS but not mRNA expression, consistent with reduced endothelial and smooth muscle cell CBS (but not CSE) protein. ICI did not alter mesenteric and renal artery CBS and CSE mRNA. In addition, ICI decreased endothelial nitric oxide synthase mRNA in UA but not in mesenteric or renal arteries. Thus, pregnancy-augmented UA CBS/H2S production is mediated by the actions of endogenous estrogens via specific ER in pregnant rats.

Hydrogen sulfide promotes migration of trophoblast cells by a Rho GTPase mediated actin cytoskeleton reorganization

IntroductionPreeclampsia (PE) arises due to defective spiral artery remodelling which may be due to deficient migration of trophoblast cells. Migration of human endothelial cells has been shown to be promoted via Hydrogen sulphide(H2S)/Rho GTPase Rac1 axis. This novel role of H2S and its downstream processes have not yet been studied in the development and function of the placental trophoblast cells. MethodsPlacental tissues were obtained post-delivery from consented preeclamptic and normotensive mothers (n = 60). The protein expression levels of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS) along with its downstream migratory molecules were compared in both the arms. The pro-migratory role of H2S was investigated in a first trimester placental cell line. ResultsH2S promoted the migration of trophoblast cells in a Rho GTPase dependent manner mediated by actin cytoskeleton reorganization. The reduced levels of H2S producing enzymes in the PE placentae along with decreased levels of Rho GTPases (Rac1 and Rho A) corroborate the results of PAG and AOAA treatment in down regulating the Rho GTPases in the in vitro grown placental cultures. Reduction of the migratory potential of trophoblastic cells caused due to hypoxia/reoxygenation was rescued by upregulating the H2S expression with the use of NaHS as a H2S donor. DiscussionExogenous H2S increases the migratory potential of the placental cells in culture conditions and also post hypoxia/reoxygenation injury. H2S as a gaso-transmitter holds a great potential as a therapeutic agent. Its long-term effects need to be investigated using model systems (rat/mouse) of PE following it up with clinical regulatory trials.

Treatment with aripiprazole and N-acetylcysteine affects anaerobic cysteine metabolism in the hippocampus and reverses schizophrenia-like behavior in the neurodevelopmental rat model of schizophrenia.

Preclinical and clinical studies have shown that the antipsychotic drug aripiprazole and the antioxidant N-acetylcysteine have unique biological properties. The aim of the study was to investigate, in a rat model of schizophrenia, the effects of chronic administration of these drugs on schizophrenia-like behaviors and anaerobic cysteine metabolism in the hippocampus (HIP). The schizophrenia-type changes were induced in Sprague-Dawley rats by repeated administration of the glutathione synthesis inhibitor l-butionine-(S,R)-sulfoximine in combination with the dopamine reuptake inhibitor GBR 12909 in the early postnatal period. Adult model rats were chronically treated with aripiprazole (0.3 mg·kg-1 , i.p.) or N-acetylcysteine (30 mg·kg-1 , orally), and their effects on schizophrenia-like behaviors were assessed using the social interaction test and novel object recognition test. In the HIP, the level of anaerobic cysteine metabolites, H2 S, and bound sulfane sulfur were determined by a fluorescence method, while the expression of H2 S-synthetizing enzymes: cystathionine β-synthase (CBS) and mercaptopyruvate sulfurtransferase (MST) by western blot. Long-term treatment with aripiprazole or N-acetylcysteine reversed social and cognitive deficits and reduced the exploratory behaviors. In the HIP of 16-day-old model pups, H2 S levels and MST protein expression were significantly decreased. In adult model rats, H2 S levels remained unchanged, bound sulfane sulfur significantly increased, and the expression of CBS and MST slightly decreased. The studied drugs significantly reduced the level of bound sulfane sulfur and the expression of tested enzymes. The reduction in bound sulfane sulfur level coincided with the attenuation of exploratory behavior, suggesting that modulation of anaerobic cysteine metabolism in the HIP may have therapeutic potential in schizophrenia.