RESUMEN

Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3 methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.

Asunto(s)

Acetilcisteína , Epigénesis Genética , Retardo del Crecimiento Fetal , Sulfuro de Hidrógeno , Hipoxia , Animales , Sulfuro de Hidrógeno/metabolismo , Acetilcisteína/farmacología , Embrión de Pollo , Humanos , Femenino , Embarazo , Retardo del Crecimiento Fetal/metabolismo , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/fisiopatología , Hipoxia/metabolismo , Hipoxia/fisiopatología , Metilación de ADN , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Vasodilatación/efectos de los fármacos , Placenta/metabolismo , Placenta/irrigación sanguínea , Arterias Umbilicales/metabolismo

RESUMEN

Hydrogen sulfide (H2S) is a gasotransmitter implied in metabolic diseases, insulin resistance, obesity, and type 2 Diabetes Mellitus. This study aimed to determine the effect of chronic administration of sodium hydrosulfide (NaHS; inorganic H2S donor), L-Cysteine (L-Cys; substrate of H2S producing enzymes) and DL-Propargylglycine (DL-PAG; cystathionine-gamma-lyase inhibitor) on the vascular dysfunction induced by insulin resistance in rat thoracic aorta. For this purpose, 72 animals were divided into two main sets that received: 1) tap water (control group; n = 12); and 2) fructose 15% w/v in drinking water [insulin resistance group (IR); n = 60] for 20 weeks. After 16 weeks, the group 2 was divided into five subgroups (n = 12 each), which received daily i. p. injections during 4 weeks of: 1) non-treatment (control); 2) vehicle (phosphate buffer saline; PBS, 1 ml/kg); 3) NaHS (5.6 mg/kg); 4) L-Cys (300 mg/kg); and (5) DL-PAG (10 mg/kg). Hemodynamic variables, metabolic variables, vascular function, ROS levels and the expression of p-eNOS and eNOS were determined. IR induced: 1) hyperinsulinemia; 2) increased HOMA-index; 3) decreased Matsuda index; 4) hypertension, vascular dysfunction, increased ROS levels; 5) increased iNOS, and 6) decreased CSE, p-eNOS and eNOS expression. Furthermore, IR did not affect contractile responses to norepinephrine. Interestingly, NaHS and L-Cys treatment, reversed IR-induced impairments and DL-PAG treatment decreased and increased the HOMA and Matsuda index, respectively. Taken together, these results suggest that NaHS and L-Cys decrease the metabolic and vascular alterations induced by insulin resistance by reducing oxidative stress and activating eNOS. Thus, hydrogen sulfide may have a therapeutic application.

Asunto(s)

Diabetes Mellitus Tipo 2 , Sulfuro de Hidrógeno , Hipertensión , Resistencia a la Insulina , Animales , Ratas , Cistationina gamma-Liasa/antagonistas & inhibidores , Cistationina gamma-Liasa/metabolismo , Cisteína/farmacología , Cisteína/uso terapéutico , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/uso terapéutico , Sulfuro de Hidrógeno/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Resistencia a la Insulina/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno

RESUMEN

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.

Asunto(s)

Quistes , Trypanosoma cruzi , Humanos , Animales , Antioxidantes/metabolismo , Cisteína/metabolismo , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Mamíferos

RESUMEN

Deodorized garlic (DG) may favor the activity of the antioxidant enzymes and promote the synthesis of hydrogen sulfide (H2S). The objective was to test if DG favors an increase in H2S and if it decreases the oxidative stress caused by lipopolysaccharide (LPS) in rat hearts. A total of 24 rats were divided into 4 groups: Group 1 control (C), Group 2 LPS, Group 3 DG, and Group 4 LPS plus DG. The cardiac mechanical performance (CMP), coronary vascular resistance (CVR), and oxidative stress markers, such as total antioxidant capacity (TAC), glutathione (GSH), selenium (Se), lipid peroxidation (LPO), thiols, hydrogen sulfide (H2S), and the activities and expressions of thioredoxin reductase (TrxR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST), cystathionine synthetase (CBS), cystathionine γ-lyase (CTH), iNOS, and eNOS-p, were analyzed in the heart. Infarct zones in the cardiac tissue were present (p = 0.01). The CMP and CVR decreased and increased (p ≤ 0.05), TAC, GSH, H2S, NO, thiols, and GST activity (p ≤ 0.01) decreased, and LPO and iNOS increased (p ≤ 0.05). The activities and expressions of TrxR, GPx, eNOS-p, CTH, and CBS (p ≤ 0.05) decreased with the LPS treatment; however, DG normalized this effect. DG treatment decreases heart damage caused by LPS through the cross-talk between the H2S and NO systems.

Asunto(s)

Ajo , Sulfuro de Hidrógeno , Selenio , Animales , Ratas , Antioxidantes/farmacología , Antioxidantes/metabolismo , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Ajo/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/metabolismo , Lipopolisacáridos/farmacología , Estrés Oxidativo , Selenio/farmacología , Compuestos de Sulfhidrilo/farmacología , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Transferasas/metabolismo

RESUMEN

PURPOSE: This study aimed to assess the possible healing effect of combination treatment with a hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS) plus tadalafil on partial bladder outlet obstruction (PBOO)-induced bladder dysfunction. MATERIALS AND METHODS: A total of 75 male Sprague-Dawley rats aged 10-wk and 300-350g were divided into five groups; control; PBOO; PBOO+NaHS (5.6mg/kg/day, i.p., 6-wk); PBOO+tadalafil (2mg/kg/day, oral, 6-wk) and PBOO+NaHS+tadalafil. PBOO was created by partial urethral ligation. 6 weeks after obstruction, the in vitro contractile responses of the detrusor muscle and Western blotting, H2S and malondialdehyde assay were performed in bladder tissues. RESULTS: There was an increase in bladder weight(p<0.001) and a decrease in contractile responses to KCL(p<0.001), carbachol(p<0.01), electrical field stimulation(p<0.05) and ATP (p<0.001) in the detrusor smooth muscle of obstructed rats which was normalized after the combination treatment. Cystathionine γ-lyase and cystathionine ß-synthase, and nuclear factor kappa B protein levels did not significantly differ among groups. The obstruction induced decrement in 3-mercaptopyruvate sulfur transferase protein expression(p<0.001) and H2S levels(p<0.01) as well as increment in protein expressions of neuronal nitric oxide synthase (NO, p<0.001), endothelial NOS (p<0.05), inducible NOS(p<0.001), hypoxia-inducible factor 1-alpha (p<0.01), and malondialdehyde levels (p<0.01), when combined treatment entirely normalized. CONCLUSIONS: Combination therapy has beneficial effects on bladder dysfunction via regulating both H2S and nitric oxide pathways as well as downregulation of oxidative stress and hypoxia. The synergistic effect of H2S and nitric oxide is likely to modulate bladder function, which supports the combined therapy for enhancing clinical outcomes in men with BPH/LUTS.

Asunto(s)

Sulfuro de Hidrógeno , Obstrucción del Cuello de la Vejiga Urinaria , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/uso terapéutico , Animales , Carbacol/metabolismo , Carbacol/farmacología , Carbacol/uso terapéutico , Cistationina betasintasa/metabolismo , Cistationina betasintasa/farmacología , Cistationina betasintasa/uso terapéutico , Cistationina gamma-Liasa/metabolismo , Cistationina gamma-Liasa/farmacología , Cistationina gamma-Liasa/uso terapéutico , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/uso terapéutico , Hipoxia/tratamiento farmacológico , Hipoxia/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Factor 1 Inducible por Hipoxia/farmacología , Factor 1 Inducible por Hipoxia/uso terapéutico , Masculino , Malondialdehído , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Sulfuros , Azufre/metabolismo , Azufre/farmacología , Azufre/uso terapéutico , Tadalafilo/farmacología , Tadalafilo/uso terapéutico , Transferasas/metabolismo , Transferasas/farmacología , Transferasas/uso terapéutico , Vejiga Urinaria , Obstrucción del Cuello de la Vejiga Urinaria/tratamiento farmacológico

RESUMEN

Hydrogen sulfide (H2S) has been known for its toxicity. However, recent studies have focused on the mechanisms involved in endogenous production and function. To date, the H2S role in insulin signaling and glucose homeostasis is unclear. This uncertainty is even more evident in skeletal muscle, a physiological niche highly relevant for regulating glycemia in response to insulin. This study aimed to investigate the role of H2S on insulin signaling and glucose uptake in the L6 skeletal muscle cell line. We evaluated the endogenous synthesis with the fluorescent dye, 7-azido-4-methyl coumarin (7-AzMC). Glucose restriction-induced an increase in the endogenous levels of H2S, likely through stimulation of cystathionine γ-lyase activity, as its specific inhibitor, PAG (5 mM) prevented this increase, and mRNA levels of CSE decreased with glucose and amino acid restriction. Exogenous H2S reduced insulin-induced glucose uptake at 0.5 up to 24 h, an effect dissociated from the level of Akt phosphorylation. Our results show that glucose restriction induces endogenous production of H2S via CSE. In addition, H2S disrupts insulin-induced glucose uptake independent of the Akt pathway. These results suggest that H2S antagonism over insulin-induced glucose uptake could help maintain the plasmatic glucose levels in conditions that provoke hypoglycemia, which could serve as an H2S-regulated mechanism for maintaining glucose plasmatic levels through the inhibition of the skeletal muscle insulin-depended glucose uptake.

Asunto(s)

Sulfuro de Hidrógeno , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Glucosa/metabolismo , Sulfuro de Hidrógeno/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética

RESUMEN

PURPOSE: To explore the role and molecular mechanisms of neuroprotective effects of octreotide in alcohol-induced neuropathic pain. METHODS: Male Wistar rats were employed and were administered a chronic ethanol diet containing 5% v/v alcohol for 28 days. The development of neuropathic pain was assessed using von Frey hair (mechanical allodynia), pinprick (mechanical hyperalgesia) and cold acetone drop tests (cold allodynia). The antinociceptive effects of octreotide (20 and 40 µg·kg-1) were assessed by its administration for 28 days in ethanol-treated rats. ANA-12 (0.25 and 0.50 mg·kg-1), brain-derived neurotrophic factor (BDNF) receptor blocker, was coadministered with octreotide. The sciatic nerve was isolated to assess the biochemical changes including hydrogen sulfide (H2S), cystathionine ß synthase (CBS), cystathionine γ lyase (CSE), tumor necrosis factor-α (TNF-α), BDNF and nuclear factor erythroid 2-related factor 2 (Nrf2). RESULTS: Octreotide significantly attenuated chronic ethanol-induced neuropathic pain and it also restored the levels of H2S, CBS, CSE, BDNF, Nrf2 and decreased TNF-α levels. ANA-12 abolished the effects of octreotide on pain, TNF-α, BDNF, Nrf2 without any significant effects on H2S, CBS, CSE. CONCLUSIONS: Octreotide may attenuate the behavioral manifestations of alcoholic neuropathic pain, which may be due to an increase in H2S, CBS, CSE, BDNF, Nrf2 and a decrease in neuroinflammation.

Asunto(s)

Analgésicos/farmacología , Neuralgia , Octreótido/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo , Cistationina betasintasa , Cistationina gamma-Liasa , Etanol , Sulfuro de Hidrógeno , Hiperalgesia , Masculino , Factor 2 Relacionado con NF-E2 , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa

RESUMEN

BACKGROUND: H2S is proved to be functioning as a signaling molecule in an array of physiological processes in the plant and animal kingdom. However, the H2S synthesis pathway and the responses to cold conditions remain unclear in postharvest mushroom. RESULTS: The biosynthesis of H2S in the Agaricus bisporus mushroom tissues exhibited an increasing tendency during postharvest storage and was significantly triggered by cold treatment. The cystathionine clyase (AbCSE) and cystathionine b-synthase (AbCBS) genes were cloned and proved responsible for H2S biosynthesis. Furthermore, transcriptional and posttranscriptional regulation of AbCSE and AbCBS were crucial for the enzyme activities and subsequent H2S levels. However, the AbMST was not involved in this process. Moreover, the AbCSE and AbCBS genes displayed low identity to the characterized genes, but typical catalytic domains, activity sites, subunit interface sites, and cofactor binding sites were conserved in the respective protein sequences, as revealed by molecular modeling and docking study. The potential transcription factors responsible for the H2S biosynthesis in cold conditions were also provided. CONCLUSIONS: The H2S biosynthetic pathway in postharvest mushroom was unique and distinct to that of other horticultural products.

Asunto(s)

Agaricus/química , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Sulfuro de Hidrógeno/síntesis química , Producción de Cultivos , Agaricus campestris , Frío , Almacenamiento de Alimentos

RESUMEN

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.

Asunto(s)

Alquinos/farmacología , Cistationina gamma-Liasa/antagonistas & inhibidores , Glicina/análogos & derivados , Sulfuro de Hidrógeno/antagonistas & inhibidores , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Animales , Cistationina gamma-Liasa/metabolismo , Inhibidores Enzimáticos/farmacología , Aromatizantes/administración & dosificación , Glicina/farmacología , Homeostasis , Sulfuro de Hidrógeno/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Modelos Animales , Sistema Hipófiso-Suprarrenal/metabolismo , Ratas , Cloruro de Sodio Dietético/administración & dosificación

RESUMEN

ABSTRACT Purpose To explore the role and molecular mechanisms of neuroprotective effects of octreotide in alcohol-induced neuropathic pain. Methods Male Wistar rats were employed and were administered a chronic ethanol diet containing 5% v/v alcohol for 28 days. The development of neuropathic pain was assessed using von Frey hair (mechanical allodynia), pinprick (mechanical hyperalgesia) and cold acetone drop tests (cold allodynia). The antinociceptive effects of octreotide (20 and 40 µg·kg-1) were assessed by its administration for 28 days in ethanol-treated rats. ANA-12 (0.25 and 0.50 mg·kg-1), brain-derived neurotrophic factor (BDNF) receptor blocker, was coadministered with octreotide. The sciatic nerve was isolated to assess the biochemical changes including hydrogen sulfide (H2S), cystathionine β synthase (CBS), cystathionine γ lyase (CSE), tumor necrosis factor-α (TNF-α), BDNF and nuclear factor erythroid 2-related factor 2 (Nrf2). Results Octreotide significantly attenuated chronic ethanol-induced neuropathic pain and it also restored the levels of H2S, CBS, CSE, BDNF, Nrf2 and decreased TNF-α levels. ANA-12 abolished the effects of octreotide on pain, TNF-α, BDNF, Nrf2 without any significant effects on H2S, CBS, CSE. Conclusions Octreotide may attenuate the behavioral manifestations of alcoholic neuropathic pain, which may be due to an increase in H2S, CBS, CSE, BDNF, Nrf2 and a decrease in neuroinflammation.

Asunto(s)

Animales , Masculino , Ratas , Octreótido/farmacología , Analgésicos/farmacología , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Factor de Necrosis Tumoral alfa , Ratas Wistar , Factor Neurotrófico Derivado del Encéfalo , Cistationina betasintasa , Cistationina gamma-Liasa , Etanol , Factor 2 Relacionado con NF-E2 , Sulfuro de Hidrógeno , Hiperalgesia

RESUMEN

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.

Asunto(s)

Alquinos/uso terapéutico , Glicina/análogos & derivados , Sulfuro de Hidrógeno/metabolismo , Hiperalgesia/tratamiento farmacológico , Inflamación/metabolismo , Dolor/tratamiento farmacológico , Articulación Temporomandibular/metabolismo , Animales , Cistationina gamma-Liasa/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Glicina/uso terapéutico , Interleucina-1beta/metabolismo , Masculino , Neuroglía/efectos de los fármacos , Ratas Wistar , Ganglio del Trigémino/citología , Ganglio del Trigémino/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Hydrogen sulfide (H2S) is an endogenous neuromodulator produced mainly by the enzyme cystathionine gamma-lyase (CSE) in peripheral tissues. A pronociceptive role of endogenously produced H2S has been previously reported by our group in a model of orofacial inflammatory pain. Using the established persistent orofacial pain rat model induced by complete Freund's adjuvant (CFA) injection into temporomandibular joint (TMJ), we have now investigated the putative role of endogenous H2S modulating hypernociceptive responses. Additionally, plasmatic extravasation on TMJ was measured following different treatments by Evans blue dye quantification. Thus, rats were submitted to Von Frey and Formalin tests in orofacial region before and after pharmacological inhibition of the CSE-H2S system combined or not with CFA-induced TMJ inflammation. Pretreatment with CSE inhibitor, propargylglycine (PAG; 88.4 µmol/kg) reduced temporomandibular inflammatory pain when injected locally as well as systemically. In particular, local PAG injection seems to be more effective for hypernociceptive responses in orofacial persistent inflammation since its action is evidenced in the majority analyzed periods of the inflammatory process compared to its systemic use. Moreover, local injection seems to act on temporomandibular vascular permeability, evidenced by decreased plasmatic extravasation induced by local PAG administration. Our data are consistent with the notion that the endogenous synthetized gas H2S modulates persistent orofacial pain responses revealing the pharmacological importance of the CSE inhibitor as a possible therapeutic target for their control.

Asunto(s)

Cistationina gamma-Liasa/metabolismo , Dolor Facial/enzimología , Dolor Facial/etiología , Inflamación/complicaciones , Inflamación/patología , Articulación Temporomandibular/patología , Alquinos/uso terapéutico , Análisis de Varianza , Animales , Inhibidores Enzimáticos/uso terapéutico , Dolor Facial/complicaciones , Dolor Facial/tratamiento farmacológico , Adyuvante de Freund/toxicidad , Glicina/análogos & derivados , Glicina/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inflamación/inducido químicamente , Masculino , Dimensión del Dolor , Ratas , Ratas Wistar , Factores de Tiempo , Resultado del Tratamiento

RESUMEN

ABSTRACT Background and aim: It is well established that the rate of gastric lesions increases in diabetic rats. Recently, the protective effect of hydrogen sulfide (H2S) in gastric mucosa has been proven. This study aimed to determine the release of H2S and mRNA expression of cystathionine gamma lyase (CSE) in gastric mucosa in alloxan-diabetic rats in response to distention-induced gastric acid secretion. Twenty-four rats were randomly assigned to 4 groups (6 in each). They were the normal-control, distention-control, diabetic-control, and distention-diabetic groups. Under anesthesia, animals underwent a tracheotomy and midline laparotomy. To washout the gastric contents, a catheter was inserted in the stomach through the duodenum. To determine the effect of distention-induced gastric acid secretion on H2S release and mRNA expression of CSE, the stomachs were distended by normal saline. At the end of experiments, animals were sacrificed and the gastric mucosa was collected to determine H2S concentration and to quantify mRNA expression of CSE by quantitative real-time PCR. Mucosal release of H2S and mRNA expression of CSE significantly increased in response to stimulated gastric acid secretion in normal rats (P<0.01), while the increases in diabetic rats were not significant. Basal release of H2S and mRNA expression of CSE in gastric mucosa were significantly in diabetic rats lower than normal rats. On the basis of the results, we conclude that the decreased release of H2S in response to basal and stimulated gastric acid output in alloxan-diabetic rats compared to normal rats is largely due to downregulation of mRNA expression of CSE.

Asunto(s)

Animales , Ratas , Cistationina gamma-Liasa , Ácido Gástrico , Sulfuro de Hidrógeno , Aloxano

RESUMEN

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.

Asunto(s)

Regulación de la Temperatura Corporal/fisiología , Fiebre/fisiopatología , Sulfuro de Hidrógeno/metabolismo , Condicionamiento Físico Animal/fisiología , Área Preóptica/metabolismo , Animales , Corticosterona/sangre , Corticosterona/metabolismo , Cistationina betasintasa/biosíntesis , Cistationina betasintasa/genética , Cistationina gamma-Liasa/biosíntesis , Cistationina gamma-Liasa/genética , Citocinas/sangre , Citocinas/metabolismo , Endotoxemia/inducido químicamente , Endotoxemia/complicaciones , Endotoxemia/fisiopatología , Endotoxinas/toxicidad , Inducción Enzimática , Fiebre/etiología , Inflamación , Masculino , Área Preóptica/fisiopatología , Ratas , Ratas Wistar , Carrera , Conducta Sedentaria , Sulfurtransferasas/biosíntesis , Sulfurtransferasas/genética

RESUMEN

AIM: The objective was to evaluate the effects of nitric oxide (NO) and hydrogen sulfide (H2S) donors and possible interactions between these two systems in modulating gastric function. METHODS: Mice received saline, sodium nitroprusside (SNP), or sodium hydrosulfite (NaHS), and after 1 h, the animals were killed for immunofluorescence analysis of CSE or eNOS expressions, respectively. Other groups received saline, SNP, NaHS, Lawesson's reagent (H2S donor), PAG + SNP, L-NAME, L-NAME + NaHS, or L-NAME + Lawesson's reagent. Then, the gastric secretions (mucous and acid), gastric blood flow, gastric defense against ethanol, and gastric motility (gastric emptying and gastric contractility) were evaluated. RESULTS: SNP and NaHS increased the expression of CSE or eNOS, respectively. SNP or Lawesson's reagent did not alter gastric acid secretion but increased mucus production, and these effects reverted with PAG and L-NAME treatment, respectively. SNP or NaHS increased gastric blood flow and protected the gastric mucosa against ethanol injury, and these effects reverted with PAG and L-NAME treatments, respectively. SNP delayed gastric emptying when compared with saline, and PAG partially reversed this effect. NaHS accelerate gastric emptying, and L-NAME partially reversed this effect. SNP and NaHS alone induced gastric fundus and pylorus relaxation. However, pretreatment with PAG or L-NAME reversed these relaxant effects only in the pylorus but not in the gastric fundus. CONCLUSION: NO and H2S interact in gastric physiological functions, and this "cross-talk" is important in the control of mucus secretion, gastric blood flow, gastric mucosal defense, and gastric motility, but not in the control of basal gastric acid secretion.

Asunto(s)

Cistationina gamma-Liasa/efectos de los fármacos , Vaciamiento Gástrico/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Nitroprusiato/farmacología , Estómago/efectos de los fármacos , Sulfuros/farmacología , Alquinos/farmacología , Animales , Depresores del Sistema Nervioso Central/farmacología , Cistationina gamma-Liasa/antagonistas & inhibidores , Cistationina gamma-Liasa/metabolismo , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Etanol/farmacología , Técnica del Anticuerpo Fluorescente , Ácido Gástrico/metabolismo , Fundus Gástrico/efectos de los fármacos , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Flujometría por Láser-Doppler , Masculino , Malondialdehído/metabolismo , Ratones , Moco/efectos de los fármacos , Moco/metabolismo , Contracción Muscular/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Píloro/efectos de los fármacos , Ratas , Ratas Wistar , Flujo Sanguíneo Regional , Estómago/irrigación sanguínea

RESUMEN

The present research aimed to isolate the non-polar secondary metabolites that produce the vasodilator effects induced by the dichloromethane extract of Prunus serotina (P. serotina) fruits and to determine whether the NO/cGMP and the H2S/KATP channel pathways are involved in their mechanism of action. A bioactivity-directed fractionation of the dichloromethane extract of P. serotina fruits led to the isolation of ursolic acid and uvaol as the main non-polar vasodilator compounds. These compounds showed significant relaxant effect on rat aortic rings in an endothelium- and concentration-dependent manner, which was inhibited by NG-nitro-L-arginine methyl ester (L-NAME), DL-propargylglycine (PAG) and glibenclamide (Gli). Additionally, both triterpenes increased NO and H2S production in aortic tissue. Molecular docking studies showed that ursolic acid and uvaol are able to bind to endothelial NOS and CSE with high affinity for residues that form the oligomeric interface of both enzymes. These results suggest that the vasodilator effect produced by ursolic acid and uvaol contained in P. serotina fruits, involves activation of the NO/cGMP and H2S/KATP channel pathways, possibly through direct activation of NOS and CSE.

Asunto(s)

Sulfuro de Hidrógeno/agonistas , Óxido Nítrico/agonistas , Prunus avium/química , Triterpenos/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Alquinos/antagonistas & inhibidores , Alquinos/farmacología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , GMP Cíclico/metabolismo , Cistationina gamma-Liasa/química , Cistationina gamma-Liasa/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Frutas/química , Gliburida/antagonistas & inhibidores , Gliburida/farmacología , Glicina/análogos & derivados , Glicina/antagonistas & inhibidores , Glicina/farmacología , Sulfuro de Hidrógeno/metabolismo , Canales KATP/agonistas , Canales KATP/metabolismo , Masculino , Simulación del Acoplamiento Molecular , NG-Nitroarginina Metil Éster/antagonistas & inhibidores , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Extractos Vegetales/química , Unión Proteica , Ratas , Triterpenos/aislamiento & purificación , Vasodilatadores/aislamiento & purificación , Ácido Ursólico

RESUMEN

Posthemorrhagic shock mesenteric lymph (PHSML) is a key factor in multiple organ injury following hemorrhagic shock. We investigated the role of hydrogen sulfide (H2S) in PHSML drainage in alleviating acute kidney injury (AKI) by administering D,L-propargylglycine (PPG) and sodium hydrosulfide hydrate (NaHS) to 12 specific pathogen-free male Wistar rats with PHSML drainage. A hemorrhagic shock model was established in 4 experimental groups: shock, shock+drainage, shock+drainage+PPG (45 mg/kg, 0.5 h prehemorrhage), and shock+drainage+NaHS (28 µmol/kg, 0.5 h prehemorrhage). Fluid resuscitation was performed after 1 h of hypotension, and PHMSL was drained in the last three groups for 3 h after resuscitation. Renal function and histomorphology were assessed along with levels of H2S, cystathionine-γ-lyase (CSE), Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)-α in renal tissue. Hemorrhagic shock induced AKI with increased urea and creatinine levels in plasma and higher H2S, CSE, TLR4, IL-10, IL-12, and TNF-α levels in renal tissue. PHSML drainage significantly reduced urea, creatinine, H2S, CSE, and TNF-α but not TLR4, IL-10, or IL-12. PPG decreased creatinine, H2S, IL-10, and TNF-α levels, but this effect was reversed by NaHS administration. In conclusion, PHSML drainage alleviated AKI following hemorrhagic shock by preventing increases in H2S and H2S-mediated inflammation.

Asunto(s)

Lesión Renal Aguda/prevención & control , Drenaje/métodos , Gasotransmisores/uso terapéutico , Sulfuro de Hidrógeno/uso terapéutico , Linfa/fisiología , Choque Hemorrágico/terapia , Lesión Renal Aguda/fisiopatología , Alquinos/uso terapéutico , Animales , Creatinina/sangre , Cistationina gamma-Liasa/análisis , Citocinas/análisis , Inhibidores Enzimáticos/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Gasotransmisores/análisis , Glicina/análogos & derivados , Glicina/uso terapéutico , Sulfuro de Hidrógeno/análisis , Masculino , Mesenterio , Ratas Wistar , Reproducibilidad de los Resultados , Choque Hemorrágico/complicaciones , Sulfitos/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Urea/sangre

RESUMEN

AIM: Hydrogen sulphide (H2S) is endogenously produced and plays an important role as a modulator of neuronal functions; however, its modulatory role in the central CO2 chemoreception is unknown. The aim of the present study was to assess the role of endogenously produced H2S in the ventilatory response to hypercapnia in adult conscious rats. METHODS: Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) inhibitors (aminooxyacetate: AOA and propargylglycine: PAG respectively) and a H2S donor (sodium sulphide: Na2S) were microinjected into the fourth ventricle (4V). Ventilation (V̇(E)), oxygen consumption (V̇O2) and body temperature were recorded before (room air) and during a 30-min CO2 exposure (hypercapnia, 7% CO2). Endogenous H2S levels were measured in the nucleus tractus solitarius (NTS). RESULTS: Microinjection of Na2S (H2S donor), AOA (CBS inhibitor) or PAG (CSE inhibitor) did not affect baseline of the measured variables compared to control group (vehicle). In all experimental groups, hypercapnia elicited an increase in V̇(E). However, AOA microinjection, but not PAG, attenuated the ventilatory response to hypercapnia (P < 0.05), whereas Na2S elicited a slight, not significant, enhancement. Moreover, endogenous H2S levels were found higher in the NTS after hypercapnia (P < 0.05) compared to room air (normoxia) condition. CONCLUSION: There are a few reports on the role of gaseous transmitters in the control of breathing. Importantly, the present data suggest that endogenous H2S via the CBS-H2S pathway mediates the ventilatory response to hypercapnia playing an excitatory role.

Asunto(s)

Sulfuro de Hidrógeno/farmacología , Hipercapnia/tratamiento farmacológico , Envejecimiento/metabolismo , Ácido Aminooxiacético/metabolismo , Animales , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Modelos Animales de Enfermedad , Sulfuro de Hidrógeno/metabolismo , Hipercapnia/metabolismo , Masculino , Ratas Wistar , Sulfuros/farmacología

RESUMEN

Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine ß-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ-lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild-type phenotype of a Saccharomyces cerevisiae CGL-null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL-propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC50 of approximately 65 µM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.

Asunto(s)

Vías Biosintéticas , Cistationina gamma-Liasa/metabolismo , Cisteína/biosíntesis , Leishmania major/enzimología , Azufre/metabolismo , Alquinos/metabolismo , Inhibidores Enzimáticos/metabolismo , Prueba de Complementación Genética , Glicina/análogos & derivados , Glicina/metabolismo , Concentración 50 Inhibidora , Leishmania major/genética , Saccharomyces cerevisiae/genética

RESUMEN

CONTEXT: Hydrogen sulphide (H2S) has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H2S synthesis is essential to gastric protection against ethanol. OBJECTIVE: To better understanding the role of H2S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE) and cystathionine-ß-synthetase (CBS) isoforms in gastric mucosa of mice treated with saline or 50% ethanol. METHODS: Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g). After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS. RESULTS: We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol. CONCLUSION: Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol.

Asunto(s)

Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Mucosa Gástrica/enzimología , Sulfuro de Hidrógeno/metabolismo , Animales , Modelos Animales de Enfermedad , Etanol/farmacología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/patología , Inmunohistoquímica , Ratones