RESUMO
Thymic egress is a crucial process for thymocyte maturation, strictly regulated by sphingosine-1-phosphate lyase (S1PL). Recently, cystathionine γ-lyase (CSE), one of the enzymes producing hydrogen sulfide (H2S), has emerged as a vital immune process regulator. However, the molecular connection between CSE, H2S and thymic egress remains largely unexplored. In this study, we investigated the regulatory function of CSE in the thymic egress of immune cells. We showed that genetic knockout of CSE or pharmacological inhibition by CSE enzyme inhibitor NSC4056 or D,L-propargylglycine (PAG) significantly enhanced the migration of mature lymphocytes and monocytes from the thymus to the peripheral blood, and this redistribution effect could be reversed by treatment with NaHS, an exogenous donor of H2S. In addition, the CSE-generated H2S significantly increased the levels of S1P in the peripheral blood, thymus and spleen of mice, suppressed the production of proinflammatory cytokines and rescued pathogen-induced sepsis in cells and in vivo. Notably, H2S or polysulfide inhibited S1PL activity in cells and an in vitro purified enzyme assay. We found that this inhibition relied on a newly identified C203XC205 redox motif adjacent to the enzyme's active site, shedding light on the biochemical mechanism of S1PL regulation. In conclusion, this study uncovers a new function and mechanism for CSE-derived H2S in thymic egress and provides a potential drug target for treating S1P-related immune diseases.
Assuntos
Aldeído Liases , Cistationina gama-Liase , Sulfeto de Hidrogênio , Camundongos Endogâmicos C57BL , Timo , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Sulfeto de Hidrogênio/metabolismo , Animais , Aldeído Liases/metabolismo , Aldeído Liases/antagonistas & inibidores , Timo/metabolismo , Timo/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Camundongos , Alcinos/farmacologia , Camundongos Knockout , Glicina/análogos & derivados , Glicina/farmacologia , Glicina/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Movimento Celular/efeitos dos fármacosRESUMO
The ubiquitous gasotransmitter hydrogen sulfide (H2S) has been recognized to play a crucial role in human health. Using cystathionine γ-lyase (CSE)-deficient mice, we demonstrate an unexpected role of H2S in Mycobacterium tuberculosis (Mtb) pathogenesis. We showed that Mtb-infected CSE-/- mice survive longer than WT mice, and support reduced pathology and lower bacterial burdens in the lung, spleen, and liver. Similarly, in vitro Mtb infection of macrophages resulted in reduced colony forming units in CSE-/- cells. Chemical complementation of infected WT and CSE-/- macrophages using the slow H2S releaser GYY3147 and the CSE inhibitor DL-propargylglycine demonstrated that H2S is the effector molecule regulating Mtb survival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1ß, IL-6, TNF-α, and IFN-γ levels in response to Mtb infection. Notably, Mtb infected CSE-/- macrophages show increased flux through glycolysis and the pentose phosphate pathway, thereby establishing a critical link between H2S and central metabolism. Our data suggest that excessive H2S produced by the infected WT mice reduce HIF-1α levels, thereby suppressing glycolysis and production of IL-1ß, IL-6, and IL-12, and increasing bacterial burden. Clinical relevance was demonstrated by the spatial distribution of H2S-producing enzymes in human necrotic, nonnecrotic, and cavitary pulmonary tuberculosis (TB) lesions. In summary, CSE exacerbates TB pathogenesis by altering immunometabolism in mice and inhibiting CSE or modulating glycolysis are potential targets for host-directed TB control.
Assuntos
Carbono/metabolismo , Cistationina gama-Liase/fisiologia , Sulfeto de Hidrogênio/toxicidade , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/etiologia , Alcinos/farmacologia , Animais , Cistationina gama-Liase/antagonistas & inibidores , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Glicólise , Sulfeto de Hidrogênio/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Transdução de Sinais , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologiaRESUMO
Hydrogen sulfide (H2S), synthesized by cystathionine gamma-lyase (Cth), contributes to the inflammatory response observed in sepsis. This study examines the effect of Cth-derived H2S in adhesion molecules on endothelial cells of vital organs in mice in a cecal ligation puncture (CLP)-induced model of sepsis, using two different and complementary approaches: Cth gene deletion and pharmacological inhibition. Our findings revealed a decreased level of H2S-synthesizing activity (via Cth) in both Cth-/- mice and PAG-treated wild-type (WT) mice following CLP-induced sepsis. Both treatment groups had reduced MPO activity and expression of chemokines (MCP-1 and MIP-2α), adhesion molecules (ICAM-1 and VCAM-1), ERK1/2 phosphorylation, and NF-κB in the liver and lung compared with in CLP-WT mice. Additionally, we found that PAG treatment in Cth-/- mice had no additional effect on the expression of ERK1/2 phosphorylation, NF-κB, or the production of chemokines and adhesion molecules in the liver and lung compared to Cth-/- mice following CLP-induced sepsis. The WT group with sepsis had an increased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung than the WT sham-operated control. The Cth-/-, PAG-treated WT, and Cth-/- groups of mice showed decreased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung following sepsis. Inhibition of H2S production via both approaches reduced adhesion molecule expression on endothelial cells and reduced liver and lung injury in mice with sepsis. In conclusion, this study demonstrates that H2S has an important role in the pathogenesis of sepsis and validates PAG use as a suited tool for investigating the Cth/H2S-signalling axis in sepsis.
Assuntos
Cistationina gama-Liase , Sepse , Animais , Camundongos , Moléculas de Adesão Celular , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Células Endoteliais , Deleção de Genes , NF-kappa B , Sepse/tratamento farmacológico , Sepse/genéticaRESUMO
Chronic hypoxia causes pulmonary hypertension (PH), vascular remodeling, right ventricular (RV) hypertrophy, and cardiac failure. Protein kinase G Iα (PKGIα) is susceptible to oxidation, forming an interprotein disulfide homodimer associated with kinase targeting involved in vasodilation. Here we report increased disulfide PKGIα in pulmonary arteries from mice with hypoxic PH or lungs from patients with pulmonary arterial hypertension. This oxidation is likely caused by oxidants derived from NADPH oxidase-4, superoxide dismutase 3, and cystathionine γ-lyase, enzymes that were concomitantly increased in these samples. Indeed, products that may arise from these enzymes, including hydrogen peroxide, glutathione disulfide, and protein-bound persulfides, were increased in the plasma of hypoxic mice. Furthermore, low-molecular-weight hydropersulfides, which can serve as "superreductants" were attenuated in hypoxic tissues, consistent with systemic oxidative stress and the oxidation of PKGIα observed. Inhibiting cystathionine γ-lyase resulted in decreased hypoxia-induced disulfide PKGIα and more severe PH phenotype in wild-type mice, but not in Cys42Ser PKGIα knock-in (KI) mice that are resistant to oxidation. In addition, KI mice also developed potentiated PH during hypoxia alone. Thus, oxidation of PKGIα is an adaptive mechanism that limits PH, a concept further supported by polysulfide treatment abrogating hypoxia-induced RV hypertrophy in wild-type, but not in the KI, mice. Unbiased transcriptomic analysis of hypoxic lungs before structural remodeling identified up-regulation of endothelial-to-mesenchymal transition pathways in the KI compared with wild-type mice. Thus, disulfide PKGIα is an intrinsic adaptive mechanism that attenuates PH progression not only by promoting vasodilation but also by limiting maladaptive growth and fibrosis signaling.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Artéria Pulmonar/patologia , Adulto , Animais , Linhagem Celular , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Dissulfetos/química , Feminino , Fibrose , Técnicas de Introdução de Genes , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/prevenção & controle , Hipóxia/sangue , Hipóxia/tratamento farmacológico , Pulmão/irrigação sanguínea , Pulmão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Oxidantes/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sulfetos/administração & dosagem , Sulfetos/sangue , Sulfetos/metabolismo , Regulação para Cima , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
In sleep apnea, airway obstruction causes intermittent hypoxia (IH). In animal studies, IH-dependent hypertension is associated with loss of vasodilator hydrogen sulfide (H2S), and increased H2S activation of sympathetic nervous system (SNS) activity in the carotid body. We previously reported that inhibiting cystathionine γ-lyase (CSE) to prevent H2S synthesis augments vascular resistance in control rats. The goal of this study was to evaluate the contribution of IH-induced changes in CSE signaling to increased blood pressure and vascular resistance. We hypothesized that chronic IH exposure eliminates CSE regulation of blood pressure (BP) and vascular resistance. In rats instrumented with venous catheters, arterial telemeters, and flow probes on the main mesenteric artery, the CSE inhibitor dl-propargylglycine (PAG, 50 mg/kg/day i.v. for 5 days) increased BP in Sham rats but decreased BP in IH rats [in mmHg, Sham (n = 11): 114 ± 4 to 131 ± 6; IH (n = 8): 131 ± 8 to 115 ± 7 mmHg, P < 0.05]. PAG treatment increased mesenteric vascular resistance in Sham rats but decreased it in IH rats (day 5/day 1: Sham: 1.50 ± 0.07; IH: 0.85 ± 0.19, P < 0.05). Administration of the ganglionic blocker hexamethonium (to evaluate SNS activity) decreased mesenteric resistance in PAG-treated Sham rats more than in saline-treated Sham rats or PAG-treated IH rats. CSE immunoreactivity in IH carotid bodies compared with those from Sham rats. However, CSE staining in small mesenteric arteries was less in arteries from IH than in Sham rats but not different in larger arteries (inner diameter > 200 µm). These results suggest endogenous H2S regulates blood pressure and vascular resistance, but this control is lost after IH exposure with decreased CSE expression in resistance size arteries. IH exposure concurrently increases carotid body CSE expression and relative SNS control of blood pressure, suggesting both vascular and carotid body H2S generation contribute to blood pressure regulation.NEW & NOTEWORTHY These results suggest that CSE's protective role in the vasculature is impaired by simulated sleep apnea, which also upregulates CSE in the carotid body. Thus, this enzyme system can exert both pro- and antihypertensive effects and may contribute to elevated SNS outflow in sleep apnea.
Assuntos
Circulação Sanguínea , Pressão Sanguínea , Gasotransmissores/metabolismo , Sulfeto de Hidrogênio/metabolismo , Síndromes da Apneia do Sono/metabolismo , Alcinos/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Corpo Carotídeo/efeitos dos fármacos , Corpo Carotídeo/metabolismo , Corpo Carotídeo/fisiopatologia , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Gasotransmissores/sangue , Glicina/análogos & derivados , Glicina/farmacologia , Hexametônio/farmacologia , Sulfeto de Hidrogênio/sangue , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Ratos , Ratos Sprague-Dawley , Síndromes da Apneia do Sono/fisiopatologia , Resistência VascularRESUMO
Circadian clock genes are found in almost every cell that has a nucleus; they regulate the rhythmic nature of all processes that are cyclical. Among the genes controlled by the circadian clock, there are numerous factors that regulate key processes in the functioning of the cell. Disturbances in the functioning of the circadian clock are associated with numerous disorders. A recent study has shown the key role of H2S in regulating circadian rhythm. In this study, we investigated the in vitro effect of pharmacological inhibition of cystathionine-ß-synthase (CBS) and/or cystathionine-γ-lyase (CSE) on the circadian dynamics of Per2 expression in serum-shocked NIH-3T3 cells. Alternatively, Cbs and Cse were knocked down by transfection with siRNA. The 48-h treatment of serum-shocked NIH-3T3 cells with 1 mM dl-propargylglycine (PAG), a specific CSE inhibitor, significantly decreased the amplitude and baseline expression of Per2. During exposure to an effective CBS and CSE inhibitor (aminooxyacetic acid [AOAA]), the amplitude of oscillation and baseline expression of Per2 significantly increased. Incubation of NIH-3T3 cells with both inhibitors also significantly increased the amplitude and baseline expression of Per2 messenger RNA (mRNA). siCbs or siCse knockdowan significantly reduced the baseline and amplitude of oscillation of Per2. In conclusion, we showed that CBS/CSE/H2S pathway participates in the regulation of the circadian clock system. PAG and AOAA, change the general expression and dynamics of Per2 genes, but the increase of amplitude and overall Per2 mRNA level due to exposure to AOAA is probably caused by factors other than CBS and CSE activity.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Circadianas Period/metabolismo , Soro/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/genética , Cistationina gama-Liase/deficiência , Cistationina gama-Liase/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos , Células NIH 3T3 , RNA Interferente Pequeno/genéticaRESUMO
The gaseous modulator hydrogen sulfide (H2S) is synthesized, among other routes, by the action of cystathionine-γ-lyase (CSE) and importantly participates in body fluid homeostasis. Therefore, the present study aimed to evaluate the participation of H2S in behavioral, renal and neuroendocrine homeostatic responses triggered by the acute consumption of a high Na+ diet. After habituation, adult male Wistar rats were randomly distributed and maintained for seven days on a control [CD (0.27% of Na+)] or hypersodic diet [HD (0.81% of Na+)]. CD and HD-fed animals were treated with DL-Propargylglycine (PAG, 25 mg/kg/day, ip) or vehicle (0.9% NaCl in equivalent volume) for the same period. At the end of the experiment, animals were euthanized for blood and tissue collection. We demonstrated that a short-term increase in dietary Na+ intake, in values that mimic the variations in human consumption (two times the recommended) significantly modified hydroelectrolytic homeostasis, with repercussions in the hypothalamic-neurohypophysial system and hypothalamic-pituitary-adrenal axis function. These findings were accompanied by the development of a clear inflammatory response in renal tubular cells and microvascular components. On the other hand, the inhibition of the endogenous production of H2S by CSE provided by PAG treatment prevented the inflammation induced by HD. In the kidney, PAG treatment induced the overexpression of inducible nitric oxide synthase in animals fed with HD. Taken together, these data suggest, therefore, that HD-induced H2S production plays an important proinflammatory role in the kidney, apparently counter regulating nitric oxide actions in renal tissue.
Assuntos
Alcinos/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Glicina/análogos & derivados , Sulfeto de Hidrogênio/antagonistas & inibidores , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Animais , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Aromatizantes/administração & dosagem , Glicina/farmacologia , Homeostase , Sulfeto de Hidrogênio/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Modelos Animais , Sistema Hipófise-Suprarrenal/metabolismo , Ratos , Cloreto de Sódio na Dieta/administração & dosagemRESUMO
Fifteen previously undescribed nor-clerodane diterpenoid glucosides tinosinesides C-Q (1-15), along with four known analogues (16-19), were isolated from the stems of Tinospora sinensis. The structures of the new compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation and chemical methods. All the isolates were evaluated for their inhibitory effects on cystathionine γ-lyase (CSE), a natural enzyme responsible for the synthesis of H2S. Compounds 4 and 5 represent rare examples of natural CSE inhibitors and the possible binding mode to CSE was further probed by molecular docking experiment.
Assuntos
Cistationina gama-Liase/antagonistas & inibidores , Diterpenos/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosídeos/farmacologia , Tinospora/química , Cistationina gama-Liase/metabolismo , Teoria da Densidade Funcional , Diterpenos/química , Diterpenos/isolamento & purificação , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Glucosídeos/química , Glucosídeos/isolamento & purificação , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Hydrogen sulfide (H2S) dilates isolated arteries, and knockout of the H2S-synthesizing enzyme cystathionine γ-lyase (CSE) increases blood pressure. However, the contributions of endogenously produced H2S to blood flow regulation in specific vascular beds are unknown. Published studies in isolated arteries show that CSE production of H2S influences vascular tone more in small mesenteric arteries than in renal arteries or the aorta. Therefore, the goal of this study was to evaluate H2S regulation of blood pressure, vascular resistance, and regional blood flows using chronically instrumented rats. We hypothesized that during whole animal CSE inhibition, vascular resistance would increase more in the mesenteric than the renal circulation. Under anesthesia, CSE inhibition [ß-cyanoalanine (BCA), 30 mg/kg bolus + 5 mg·kg-1·min-1 for 20 min iv) rapidly increased mean arterial pressure (MAP) more than saline administration (%Δ: saline -1.4 ± 0.75 vs. BCA 7.1 ± 1.69, P < 0.05) but did not change resistance (MAP/flow) in either the mesenteric or renal circulation. In conscious rats, BCA infusion similarly increased MAP (%Δ: saline -0.8 ± 1.18 vs. BCA 8.2 ± 2.6, P < 0.05, n = 7) and significantly increased mesenteric resistance (saline 0.9 ± 3.1 vs. BCA 15.6 ± 6.5, P < 0.05, n = 12). The H2S donor Na2S (50 mg/kg) decreased blood pressure and mesenteric resistance ,but the fall in resistance was not significant. Inhibiting CSE for multiple days with dl-proparglycine (PAG, 50 mg·kg-1·min-1 iv bolus for 5 days) significantly increased vascular resistance in both mesenteric (ratio of day 1: saline 0.86 ± 0.033 vs. PAG 1.79 ± 0.38) and renal circulations (ratio of day 1: saline 1.26 ± 0.22 vs. 1.98 ± 0.14 PAG). These results support our hypothesis that CSE-derived H2S is an important regulator of blood pressure and vascular resistance in both mesenteric and renal circulations. Furthermore, inhalation anesthesia diminishes the effect of CSE inhibition on vascular tone.NEW & NOTEWORTHY These results suggest that CSE-derived H2S has a prominent role in regulating blood pressure and blood flow under physiological conditions, which may have been underestimated in prior studies in anesthetized subjects. Therefore, enhancing substrate availability or enzyme activity or dosing with H2S donors could be a novel therapeutic approach to treat cardiovascular diseases.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Artérias Mesentéricas/metabolismo , Artéria Renal/metabolismo , Circulação Renal , Circulação Esplâncnica , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Pressão Arterial , Velocidade do Fluxo Sanguíneo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Ratos Sprague-Dawley , Artéria Renal/efeitos dos fármacos , Circulação Renal/efeitos dos fármacos , Circulação Esplâncnica/efeitos dos fármacos , Sulfetos/farmacologia , Resistência VascularRESUMO
The mechanisms underlying temporomandibular disorders following orofacial pain remain unclear. Hydrogen sulfide (H2S), a newly identified gasotransmitter, has been reported to modulate inflammation. Cystathionine γ-lyase (CSE) is responsible for the systemical production of H2S, which exerts both pro- and antinociceptive effects through inflammation. In the current study, we investigated whether the endogenous H2S production pathway contributes to arousal and maintenance of orofacial inflammatory pain, through the investigation of the effects of a CSE inhibitor, propargyglycine (PAG), in a rat CFA (Complete Freund Adjuvant)-induced temporomandibular inflammation model to mimic persistent pain in the orofacial region. For this, rats received either CFA or saline in the temporomandibular joints (TMJs), and after 3 or 14 days, they received a single injection of PAG or saline and were evaluated for nociception with the von Frey and formalin test. Also, pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were analyzed in TMJs and trigeminal ganglion (TG). In this last one, glial cells reactivity was also verified. Endogenous H2S production rate were measured in both, TMJ and TG. Our results indicated decreased allodynia and hyperalgesic responses in rats submitted to CFA after injection of PAG. Moreover, PAG inhibited leucocyte migration to temporomandibular synovial fluid after 3 and 14 days of inflammation. PAG was able to reduce levels of CBS, CSE, TNF-α, and IL-1ß in the TMJ and TG, after 13 days of CFA injection. The observed increased activation of glial cells in the trigeminal ganglia on the 14th day of inflammation can be prevented by the highest dose of PAG. Finally, CBS and CSE expression, and endogenous H2S production rate in the TMJ and TG was found higher in rats with persistent temporomandibular inflammation compared to rats injected with saline and PAG was able to prevent this elevation. Our results elucidated the molecular mechanisms by which H2S exerts its pro-inflammatory and pro-nociceptive role in the orofacial region by alterations in both local tissue and TG.
Assuntos
Alcinos/uso terapêutico , Glicina/análogos & derivados , Sulfeto de Hidrogênio/metabolismo , Hiperalgesia/tratamento farmacológico , Inflamação/metabolismo , Dor/tratamento farmacológico , Articulação Temporomandibular/metabolismo , Animais , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Glicina/uso terapêutico , Interleucina-1beta/metabolismo , Masculino , Neuroglia/efeitos dos fármacos , Ratos Wistar , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cysteine is a nonessential amino acid in poultry nutrition. Poultry diets are deficient in cysteine, but the bird's cysteine need is met through the transsulfuration pathway (TSP) where homocysteine is converted to cysteine: a process catalyzed by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). Cysteine is also a major component of keratinized protein found in feathers, but the extent to which cysteine is involved in feather and skin development in poultry is unknown. We randomly assigned chicks to control and treatment (control diet plus 100 mg/kg body weight of propargylglycine which is an inhibitor of CTH) diets. The thickness of skin layers, primary feather follicle parameters, growth, and mRNA expression of CBS and CTH were measured. Inhibition of TSP corresponded with the upregulation of liver mRNA of both CBS and CTH and reduction in growth from 35 to 40 days of age. The epidermis thickness, feather follicle length, and diameter were reduced from 10 to 40 days of age. Incorporation of cysteine into keratinized protein may be more sensitive to the level of available cysteine than into nonkeratinized proteins. Thus, disruption of the TSP could affect the thermoregulatory ability of the bird.
Assuntos
Alcinos/farmacologia , Galinhas/metabolismo , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Redes e Vias Metabólicas/efeitos dos fármacos , Animais , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Cistationina beta-Sintase/genética , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cisteína/deficiência , Dieta/veterinária , Plumas/efeitos dos fármacos , Plumas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicina/farmacologia , Fígado/efeitos dos fármacos , Distribuição Aleatória , Pele/efeitos dos fármacos , Enxofre/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Bladder pain is a prominent symptom of interstitial cystitis/painful bladder syndrome. Hydrogen sulfide (H2S) generated by cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) facilitates bladder hypersensitivity. We assessed involvement of the H2S pathway in protease-activated receptor 4 (PAR4)-induced bladder pain. A bladder pain model was induced by intravesical instillation of PAR4-activating peptide in mice. The role of H2S in this model was evaluated by intraperitoneal preadministration of d,l-propargylglycine (PAG), aminooxyacetic acid (AOAA), or S-adenosylmethionine or the preintravesical administration of NaHS. SV-HUC-1 cells were treated in similar manners. Assessments of CBS, CSE, and macrophage migration inhibitory factor (MIF) expression, bladder voiding function, bladder inflammation, H2S production, and referred bladder pain were performed. The CSE and CBS pathways existed in both mouse bladders and SV-HUC-1 cells. H2S signaling was upregulated in PAR4-induced bladder pain models, and H2S-generating enzyme activity was upregulated in human bladders, mouse bladders, and SV-HUC-1 cells. Pretreatment with AOAA or NaHS inhibited or promoted PAR4-induced mechanical hyperalgesia, respectively; however, PAG only partially inhibited PAR4-induced bladder pain. Treatment with PAG or AOAA decreased H2S production in both mouse bladders and SV-HUC-1 cells. Pretreatment with AOAA increased MIF protein levels in bladder tissues and cells, whereas pretreatment with NaHS lowered MIF protein levels. Bladder pain triggered by the H2S pathway was not accompanied by inflammation or altered micturition behavior. Thus endogenous H2S generated by CBS or CSE caused referred hyperalgesia mediated through MIF in mice with PAR4-induced bladder pain, without causing bladder injury or altering micturition behavior.
Assuntos
Cistite Intersticial/metabolismo , Sulfeto de Hidrogênio/metabolismo , Hiperalgesia/metabolismo , Limiar da Dor , Receptores de Trombina/metabolismo , Bexiga Urinária/metabolismo , Alcinos/farmacologia , Ácido Amino-Oxiacético/farmacologia , Analgésicos/farmacologia , Animais , Linhagem Celular , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Cistite Intersticial/patologia , Cistite Intersticial/fisiopatologia , Cistite Intersticial/prevenção & controle , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Hiperalgesia/prevenção & controle , Oxirredutases Intramoleculares/metabolismo , Ligantes , Liases/antagonistas & inibidores , Liases/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Limiar da Dor/efeitos dos fármacos , Transdução de Sinais , Sulfetos/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologiaRESUMO
Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H2S has recently emerged as an important gasotransmitter involved in numerous cellular signal transduction and pathophysiological responses. Interaction of nickel and H2S on fibroblast cell activation has not been studied so far. Here, we showed that a lower dose of nickel (200⯵M) induced the activation of human fibroblast cells, as evidenced by increased cell growth, migration and higher expressions of α-smooth muscle actin (αSMA) and fibronectin, while high dose of nickel (1â¯mM) inhibited cell viability. Nickel reduced intracellular thiol contents and stimulated oxidative stress. Nickel also repressed the mRNA and protein expression of cystathionine gamma-lyase (CSE, a H2S-generating gene) and blocked the endogenous production of H2S. Exogenously applied NaHS (a H2S donor) had no effect on nickel-induced cell viability but significantly attenuated nickel-stimulated cell migration and the expression of αSMA and fibronectin. In contrast, CSE deficiency worsened nickel-induced αSMA expression. Moreover, H2S incubation reversed nickel-stimulated TGFß1/SMAD1 signal and blocked TGFß1-initiated expressions of αSMA and fibronectin. Nickel inhibited the interaction of Sp1 with CSE promoter but strengthened the binding of Sp1 with TGFß1 promoter, which was reversed by exogenously applied NaHS. These data reveal that H2S protects from nickel-stimulated fibroblast activation and CSE/H2S system can be a potential target for the treatment of tissue fibrosis induced by nickel.
Assuntos
Fibroblastos/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Níquel/toxicidade , Proteína Smad1/efeitos dos fármacos , Fator de Transcrição Sp1/efeitos dos fármacos , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cistationina gama-Liase/antagonistas & inibidores , Fibronectinas/biossíntese , Fibronectinas/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo , Zinco/metabolismoRESUMO
Maintaining a certain level of hydrogen sulfide (H2S) in ischemia-reperfusion (I/R) is essential for limiting injury to the liver. Exogenous H2S exerts protective effects against this injury, but the mechanisms remain unclear. Liver injury was induced in Wistar rats undergoing hepatic I/R for 30 min, followed by a 3-h reperfusion. Administration of GYY4137 (a slow-releasing H2S donor) significantly attenuated the severity of liver injury and was reflected by reduced inflammatory cytokine production and cell apoptosis, the levels of which were elevated by I/R, while DL-propargylglycine (PAG, an inhibitor of cystathionine γ-lyase [CSE]) aggravated liver injury. Delivery of GYY4137 significantly elevated the plasma levels of H2S and upregulated the expression of microRNA-21 (miR-21), leading to the activation of the Akt pathway, in rat livers subjected to I/R. To further investigate the protective mechanisms of H2S during liver I/R injury, we established a cell model of hypoxia/reoxygenation (H/R) by incubating Buffalo rat liver (BRL) cells under hypoxia for 4 h followed by normoxia for 10 h. The regulatory effect of miR-21 on the Akt pathway by downregulating phosphatase and tensin homolog (PTEN) was validated by luciferase assays. Incubation of sodium hydrosulfide (NaHS), an H2S donor, increased the expression of miR-21, attenuated the reduced cell viability and the increased apoptosis by H/R, in BRL cells. Anti-miR-21 abolished the protective effects of NaHS by inactivating the Akt pathway. In conclusion, the present results indicate the activation of the Akt pathway regulated by miR-21 participates in the protective effects of H2S against I/R-induced liver injury.
Assuntos
Sulfeto de Hidrogênio/agonistas , Circulação Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , MicroRNAs/agonistas , Proteínas Proto-Oncogênicas c-akt/agonistas , Traumatismo por Reperfusão/prevenção & controle , Vasodilatadores/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Sulfeto de Hidrogênio/sangue , Sulfeto de Hidrogênio/metabolismo , Fígado/irrigação sanguínea , Fígado/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Compostos Organotiofosforados/farmacologia , Compostos Organotiofosforados/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Distribuição Aleatória , Ratos , Ratos Wistar , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfetos/farmacologia , Vasodilatadores/farmacologiaRESUMO
Hydrogen sulfide (H2 S) is generated from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE), and promotes nociception by targeting multiple molecules such as Cav 3.2 T-type Ca2+ channels. Bladder pain accompanying cyclophosphamide (CPA)-induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H2 S/Cav 3.2 pathway. Therefore, we investigated whether NF-κB, as an upstream signal of the CSE/H2 S system, contributes to bladder pain in mice with CPA-induced cystitis. Bladder pain-like nociceptive behaviour was observed in CPA-treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. ß-Cyano-l-alanine, a reversible selective CSE inhibitor, prevented CPA-induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF-κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor. PDTC and curcumin, which inhibits NF-κB signals, abolished CPA-induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA-induced activation of NF-κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H2 S/Cav 3.2 signaling. Therefore, NF-κB-inhibiting compounds including curcumin may be useful for the treatment of cystitis-related bladder pain.
Assuntos
Cistationina gama-Liase/metabolismo , Cistite/complicações , Sulfeto de Hidrogênio/metabolismo , NF-kappa B/metabolismo , Dor/metabolismo , Regulação para Cima , Bexiga Urinária/metabolismo , Alanina/química , Alanina/farmacologia , Animais , Canais de Cálcio Tipo T/metabolismo , Curcumina/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Camundongos , Dor/complicações , Dor/enzimologia , Dor/patologia , Prolina/análogos & derivados , Prolina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiocarbamatos/farmacologia , Regulação para Cima/efeitos dos fármacos , Bexiga Urinária/patologiaRESUMO
Sleep apnea with periodic cessation of breathing during sleep is a highly prevalent respiratory disorder affecting an estimated 10% of adults. Patients with sleep apnea exhibit several co-morbidities including hypertension, stroke, disrupted sleep, and neurocognitive and metabolic complications. Emerging evidence suggests that a hyperactive carotid body (CB) chemo reflex is an important driver of apneas in sleep apnea patients. Gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H2S) play important roles in oxygen sensing by the CB. We tested the hypothesis that an augmented CB chemo reflex stemming from disrupted CO-H2S signaling may lead to sleep apnea. This possibility was tested in mice deficient in hemeoxygenase-2 (HO-2), an enzyme involved in CO synthesis, which were shown to exhibit hyperactive CB activity due to high H2S levels. We found that HO-2-/- mice exhibit a high incidence of apneas during sleep compared to wild type mice. Blocking the CB hyperactivity with L-propargylglycine, an inhibitor of cystathionine-γ-lyase (CSE), which catalyzes H2S synthesis, prevented apneas in HO-2-/- mice. These findings suggest that targeting CB with inhibitors of CSE might be a novel therapeutic strategy for preventing sleep apnea.
Assuntos
Corpo Carotídeo/efeitos dos fármacos , Cistationina gama-Liase/antagonistas & inibidores , Síndromes da Apneia do Sono/terapia , Alcinos/farmacologia , Animais , Monóxido de Carbono/metabolismo , Gasotransmissores/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Heme Oxigenase (Desciclizante)/genética , Sulfeto de Hidrogênio/metabolismo , Camundongos , Camundongos KnockoutRESUMO
In studies on 94 male Wistar rats changes in the hydrogen sulfide content (H2S) and cystathionine γ-lyase (CSE) in the liver and skeletal muscles in hypercholesterolemia under simvastatin treatment were assessed, as well as the effect of propargylglycine (PAG) on hepato- and myotoxicity of simvastatin. It was determined, that simvastatin inhibited the CSE-mediated synthesis of H2S in the main target organs. This negatively affected their biochemical and functional status. The use of PAG significantly suppressed the H2S deficiency induced by simvastatin, and also was accompanied by a significant increase in the activity of cytolysis markers in the serum, which significantly and negatively correlated with the activity of CSE and H2S in organs. Thus, formation of H2S deficiency due to simvastatin intake is probably one of the molecular mechanisms for the realization of hepato- and myotoxicity of this drug.
Assuntos
Cistationina gama-Liase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Hipercolesterolemia/tratamento farmacológico , Fígado/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Sinvastatina/toxicidade , Alcinos/farmacologia , Animais , Cistationina gama-Liase/antagonistas & inibidores , Glicina/análogos & derivados , Glicina/farmacologia , Hipercolesterolemia/metabolismo , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Ratos WistarRESUMO
Adipocytes express the cystathionine γ lyase (CSE)-hydrogen sulfide (H2S) system. CSE-H2S promotes adipogenesis but ameliorates adipocyte insulin resistance. We investigated the mechanism of how CSE-H2S induces these paradoxical effects. First, we confirmed that an H2S donor or CSE overexpression promoted adipocyte differentiation. Second, we found that H2S donor inhibited but CSE inhibition increased phosphodiesterase (PDE) activity. H2S replacing isobutylmethylxanthine in the differentiation program induced adipocyte differentiation in part. Inhibiting PDE activity by H2S induced peroxisome proliferator activated receptor γ (PPARγ) protein and mRNA expression. Of note, H2S directly sulfhydrated PPARγ protein. Sulfhydrated PPARγ increased its nuclear accumulation, DNA binding activity and adipogenesis gene expression, thereby increasing glucose uptake and lipid storage, which were blocked by the desulfhydration reagent DTT. H2S induced PPARγ sulfhydration, which was blocked by mutation of the C139 site of PPARγ. In mice fed a high-fat diet (HFD) for 4 weeks, the CSE inhibitor decreased but H2S donor increased adipocyte numbers. In obese mice fed an HFD for 13 weeks, H2S treatment increased PPARγ sulfhydration in adipose tissues and attenuated insulin resistance but did not increase obesity. In conclusion, CSE-H2S increased PPARγ activity by direct sulfhydration at the C139 site, thereby changing glucose into triglyceride storage in adipocytes. CSE-H2S-mediated PPARγ activation might be a new therapeutic target for diabetes associated with obesity.
Assuntos
Adipócitos/enzimologia , Cistationina gama-Liase/metabolismo , Glucose/metabolismo , Sulfeto de Hidrogênio/metabolismo , Metabolismo dos Lipídeos , Obesidade/enzimologia , PPAR gama/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia , Animais , Fármacos Antiobesidade/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Cisteína , Dieta Hiperlipídica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Sulfeto de Hidrogênio/farmacologia , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Obesidade/tratamento farmacológico , Obesidade/genética , Obesidade/fisiopatologia , PPAR gama/genética , Diester Fosfórico Hidrolases/metabolismo , Fatores de Tempo , Transfecção , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Indutores da Angiogênese/farmacologia , Artérias/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Placenta/irrigação sanguínea , Trofoblastos/química , Animais , Artérias/citologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/biossíntese , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/biossíntese , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , OvinosRESUMO
OBJECTIVE: Infrarenal aortic cross-clamping (IAC) is a common procedure during infrarenal vascular operations. It often causes ischemia-reperfusion injury to lower limbs, resulting in systemic inflammation response and damage to remote organs (particularly lungs). Hydrogen sulfide (H2S) is a gaseous mediator that has been shown to have a protective effect against lung injury. METHODS: Wistar rats underwent IAC for 2 hours, followed by 4 hours of reperfusion. GYY4137 (a slow-releasing H2S donor) and dl-propargylglycine (PAG, an inhibitor of cystathionine γ-lyase) were preadministered to rats 1 hour before IAC, and their effects on severity of lung injury and related mechanisms were investigated. RESULTS: IAC induced a significant increase in plasma levels of H2S, H2S-synthesizing activity, and cystathionine γ-lyase expression in lung tissues compared with sham operation. Administration of GYY4137 significantly increased the levels of H2S but had little effect on H2S-synthesizing activity, whereas PAG reduced H2S levels and H2S-synthesizing activity. Preadministration of GYY4137 significantly attenuated acute lung injury induced by IAC, evidenced by reduced histologic scores and wet lung contents; improved blood gas parameters; reduced cell counts and protein amounts in bronchoalveolar lavage fluids; and reduced myeloperoxidase activity in lung tissues and plasma levels of tumor necrosis factor α, interleukin 6, and interleukin 1ß. However, PAG further aggravated the severity of lung injury and displayed opposite effects to GYY4137. In exploration of the mechanisms, we found that IAC increased the release of angiopoietin 2 (Ang2) and its expression in lung tissues. GYY4137 attenuated the increase of Ang2 release and expression and increased the phosphorylation of Akt and the activation of its downstream factors, glycogen synthase kinase 3ß and ribosomal protein S6 kinase; PAG showed opposite effects. CONCLUSIONS: The study indicates that H2S may play a protective role in IAC-induced acute lung injury in rats by inhibiting inflammation and Ang2 release.