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1.
Toxicol Appl Pharmacol ; 403: 115154, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32710959

RESUMO

Exposure to air pollution is associated with the incidence of respiratory diseases. The present study evaluated the pulmonary vascular system injury by chronic real-time particulate matter (PM10) exposure and investigated the underlying mechanisms. Rats were exposed to PM10 or filtered air for 2 to 4 months using a whole body exposure system, and intraperitoneally injected with the MEK1/2 inhibitor U0126. Right heart catheterization and myography were performed to detect lung function and pulmonary vascular reactivity, respectively. Western blotting, qRT-PCR, enzyme-linked immunosorbent assay and histological analyses were used to detect the effects and mechanisms by which PM10 exposure-induced pulmonary vascular dysfunction. Functional experiment results showed that PM10 exposure increased the pulmonary artery pressure of rats and caused endothelin B receptor (ETBR)-mediated pulmonary arteriole hyperreactivity. U0126 significantly rescued these pathological changes. PM10 exposure upregulated the contractile ETBR of pulmonary arteriolar smooth muscle, and damaged pulmonary artery endothelial cells to induce the release of more endothelin 1 (ET-1). The upregulated ETBR bound to increased ET-1 induced pulmonary arteriolar hyperresponsiveness and remodeling. U0126 inhibited the PM10 exposure-induced upregulation of ETBR in pulmonary arteriole, ETBR-mediated pulmonary arterial hyperresponsiveness and vascular remodeling. In conclusion, chronic real-time particulate matter exposure can activate the ERK1/2 signaling, thereby inducing the upregulation of contractile ETBR in pulmonary arteriole, which may be involved in pulmonary arteriole hyperresponsiveness and remodeling in rats. These findings provide new mechanistic evidence of PM10 exposure-induced respiratory diseases, and a new possible target for treatment.


Assuntos
Arteríolas/efeitos dos fármacos , Butadienos/farmacologia , Pulmão/irrigação sanguínea , Nitrilos/farmacologia , Material Particulado/toxicidade , Receptor de Endotelina B/metabolismo , Remodelação Vascular/efeitos dos fármacos , Animais , Esquema de Medicação , Endotelina-1/genética , Endotelina-1/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina B/genética
2.
Am J Physiol Heart Circ Physiol ; 319(1): H242-H247, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32559137

RESUMO

The endothelin system plays an important role in mediating vascular function. The endothelin-B receptor (ETBR) on endothelial cells mediates vasodilation via nitric oxide production. The vasodilatory effect of the ETBR is lost following menopause and may contribute to impaired vascular endothelial function in postmenopausal women (PMW). However, it is unclear if these functional changes are due to changes in ETBR expression on the endothelium. Therefore, the purpose of this study was to test the hypothesis that endothelial cell ETBR expression is lower in PMW compared with young women (YW). Primary endothelial cells were harvested from the antecubital vein of healthy PMW (n = 15, 60 ± 6 yr) and YW (n = 15, 22 ± 2 yr). Cells were identified as endothelial cells by staining for vascular endothelial cadherin, and nuclear integrity was assessed using 4',6-diamidino-2-phenylindole (DAPI). Within those cells, ETBR was quantified using immunocytochemistry; fluorescence intensity was measured in 30 cells and averaged for each participant. Endothelial function was assessed using brachial artery flow-mediated dilation (FMD). Endothelial cell ETBR expression was lower in PMW [0.46 ± 0.11 arbitrary units (AU)] compared with YW (0.58 ± 0.14 AU; P = 0.02). Furthermore, significant correlations between ETBR expression and FMD (r = 0.47, P < 0.01), total cholesterol (r = -0.38, P = 0.04), and LDL cholesterol (r = -0.39, P = 0.03) were observed. These data demonstrate that endothelial cell ETBR expression is attenuated in PMW. These novel findings provide additional insight into the mechanisms underlying vascular endothelial dysfunction in PMW.NEW & NOTEWORTHY Our study provides novel data demonstrating attenuated endothelial ETBR expression in postmenopausal women. Furthermore, our data extend current knowledge by demonstrating a positive relation between ETBR expression and brachial artery flow-mediated dilation. These findings provide additional mechanistic insight into vascular endothelial dysfunction in postmenopausal women.


Assuntos
Endotélio Vascular/metabolismo , Pós-Menopausa/metabolismo , Receptor de Endotelina B/genética , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Receptor de Endotelina B/metabolismo , Veias/metabolismo , Adulto Jovem
3.
Exp Neurol ; 328: 113255, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32084451

RESUMO

We have demonstrated previously that activation of either the ETA or ETB receptor can induce acute electrographic seizures following the intrahippocampal infusion of endothelin-1 (ET-1) in immature (P12) rats. We also demonstrated that activation of the ETA receptor is associated with marked focal ischemia, while activation of the ETB receptor is not. Exploring the mechanisms underlying seizures induced by these two ET-1 receptor interactions can potentially provide insight into how focal ischemia in immature animals produces seizures and whether ischemiarelated seizures differ from seizures not associated with ischemia. To explore these seizure mechanisms we used microdialysis to determine biomarkers associated with seizures in P12 rats following the intrahippocampal infusion of two different agents: (1) ET-1, which activates both the ETA and ETB receptors and causes focal ischemia and (2) Ala-ET-1, which selectively activates only the ETB receptor and does not cause ischemia. Our results show that seizures associated with combined ETA and ETB receptor activation (and ischemia) have a different temporal distribution and microdialysis profile from seizures associated with ETB activation alone (and without ischemia). Seizures with combined activation peak within the first hour after infusion and the microdialysis profile is characterized by a significant increase in the ratio of glutamic acid to GABA. By contrast, seizures with activation of only the ETB receptor peak in the second hour after infusion and microdialysis shows a significant increase in the ratio of leukotriene B4 to prostaglandin E2. These findings suggest that ischemia-related seizures in immature animals involve an imbalance of excitation and inhibition, while non-ischemiarelated seizures involve an inflammatory process resulting from an excess of leukotrienes.


Assuntos
Endotelina-1/toxicidade , Hipocampo/efeitos dos fármacos , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Convulsões/metabolismo , Animais , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Masculino , Ratos , Ratos Wistar , Convulsões/induzido quimicamente
4.
Mol Biol Rep ; 47(3): 2137-2147, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32080807

RESUMO

The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.


Assuntos
Afinidade de Anticorpos , Antineoplásicos Imunológicos/farmacologia , Antagonistas do Receptor de Endotelina B/farmacologia , Receptor de Endotelina B/metabolismo , Anticorpos de Domínio Único/farmacologia , Afinidade de Anticorpos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Melanoma/tratamento farmacológico , Melanoma/imunologia , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Receptor de Endotelina B/imunologia , Análise de Sequência de DNA , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação
5.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023824

RESUMO

Chronic renal insufficiency (CRI) is characterized by increased endothelin 1 (ET-1) synthesis. We studied rat kidney endothelin receptor A (ETA) and receptor B (ETB) expressions after 12 and 27 weeks of 5/6 nephrectomy, and after 12 weeks of 0.3% adenine diet, representing proteinuric and interstitial inflammation models of CRI, respectively. Uric acid and calcium-phosphate metabolism were modulated after 5/6 nephrectomy, while ETA blocker and calcimimetic were given with adenine. Endothelin receptor mRNA levels were measured using RT-qPCR and protein levels using autoradiography (5/6 nephrectomy) or ELISA (adenine model). Both 12 and 27 weeks after 5/6 nephrectomy, kidney cortex ETA protein was increased by ~60% without changes in ETB protein, and the ETB:ETA ratio was reduced. However, the ETB:ETA mRNA ratio did not change. In the adenine model, kidney ETA protein was reduced by ~70%, while ETB protein was suppressed by ~95%, and the ETB:ETA ratio was reduced by ~85%, both at the protein and mRNA levels. The additional interventions did not influence the observed reductions in the ETB:ETA ratio. To conclude, unfavorable reduction in the ETB:ETA protein ratio was observed in two different models of CRI. Therefore, ETA blockade may be beneficial in a range of diseases that cause impaired kidney function.


Assuntos
Adenina/efeitos adversos , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Insuficiência Renal Crônica/genética , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Córtex Renal/metabolismo , Masculino , Nefrectomia/efeitos adversos , Fosfatos/metabolismo , Ratos , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/metabolismo , Ácido Úrico/metabolismo
6.
Life Sci ; 244: 117306, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31953159

RESUMO

AIMS: Accumulated evidence indicates that the dysregulation of circular RNAs (circRNAs) plays pivotal roles in many human diseases including preeclampsia (PE). Circ_0063517 has been verified to be down-regulated in PE. But the role of circ_0063517 in PE is still unclear. This research aims to probe into the effect of circ_0063517 on angiogenesis in PE development. MAIN METHODS: The expression of circ_0063517, endothelin receptor type B (ETBR) and miR-31-5p was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MTT assay, colony formation assay, scratch assay, transwell assay, and tube formation assay were performed to detect proliferation, migration, and angiogenesis, respectively. Dual luciferase reporter system and RNA immunoprecipitation (RIP) assay were carried out to determine the interaction between miR-31-5p and circ_0063517(or ETBR). ETBR, VEGFRA, and VEGFR2 levels were detected by western blot analysis. The effect of circ_0063517 and ETBR on angiogenesis was evaluated in N-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced PE in vivo. KEY FINDINGS: The levels of circ_0063517 and ETBR were down-regulated in the placenta tissue of PE patients. Conversely, the level of miR-31-5p was up-regulated in PE. Overexpression of circ_0063517 or knockdown of miR-31-5p facilitated growth, migration, and angiogenesis of vascular endothelial cells. Circ_0063517 knockdown-induced repression of the expression of ETBR, VEGFA, and VEGFR2 was partly counteracted by ETBR overexpression. Mechanistically, circ_0063517 sponged miR-31-5p to regulate ETBR expression. Finally, circ_0063517 promoted angiogenesis via enhancing ETBR expression in PE in vivo. SIGNIFICANCE: Our findings suggest that circ_0063517-miR-31-5p-ETBR axis regulates angiogenesis during the pathological process of PE.


Assuntos
MicroRNAs/metabolismo , Pré-Eclâmpsia/metabolismo , RNA Circular/metabolismo , Receptor de Endotelina B/metabolismo , Animais , Linhagem Celular , Proliferação de Células/fisiologia , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/genética , Neovascularização Patológica/genética , Pré-Eclâmpsia/genética , Gravidez , RNA Circular/genética , Ratos , Ratos Sprague-Dawley
7.
Am J Physiol Renal Physiol ; 318(3): F710-F719, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31904281

RESUMO

Kidney function follows a 24-h rhythm subject to regulation by circadian genes including the transcription factor Bmal1. A high-salt diet induces a phase shift in Bmal1 expression in the renal inner medulla that is dependent on endothelin type B (ETB) receptors. Furthermore, ETB receptor-mediated natriuresis is sex dependent. Therefore, experiments tested the hypothesis that collecting duct Bmal1 regulates blood pressure in a sex-dependent manner. We generated a mouse model that lacks Bmal1 expression in the collecting duct, where ETB receptor abundance is highest. Male, but not female, collecting duct Bmal1 knockout (CDBmal1KO) mice had significantly lower 24-h mean arterial pressure (MAP) than flox controls (105 ± 2 vs. 112 ± 3 mmHg for male mice and 106 ± 1 vs. 108 ± 1 mmHg for female mice, by telemetry). After 6 days on a high-salt (4% NaCl) diet, MAP remained significantly lower in male CDBmal1KO mice than in male flox control mice (107 ± 2 vs. 113 ± 1 mmHg), with no significant differences between genotypes in female mice (108 ± 2 vs. 109 ± 1 mmHg). ETB receptor blockade for another 6 days increased MAP similarly in both male and female CDBmal1KO and flox control mice. However, MAP remained lower in male CDBmal1KO mice than in male flox control mice (124 ± 2 vs. 130 ± 2 mmHg). No significant differences were observed between female CDBmal1KO and flox mice during ETB blockade (130 ± 2 vs. 127 ± 2 mmHg). There were no significant genotype differences in amplitude or phase of MAP in either sex. These data suggest that collecting duct Bmal1 has no role in circadian MAP but plays an important role in overall blood pressure in male, but not female, mice.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Pressão Sanguínea/fisiologia , Regulação da Expressão Gênica/fisiologia , Túbulos Renais Coletores/metabolismo , Fatores de Transcrição ARNTL/genética , Aldosterona/metabolismo , Aldosterona/urina , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Potássio/urina , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Fatores Sexuais , Sódio/metabolismo , Sódio/urina , Cloreto de Sódio na Dieta/administração & dosagem
8.
Life Sci ; 239: 117062, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31734261

RESUMO

AIMS: Endothelin has been implicated in various processes in the brain, including the modulation of sympathetic responses. The present study examined the pathophysiologic role of brain endothelin-receptors in the setting of acute myocardial infarction, characterized by high incidence of ventricular tachyarrhythmias. MAIN METHODS: We investigated the effects of intracerebroventricular administration of antagonists of endothelin-receptors ETA, ETB, or both, during a 24 h-observation period post-coronary ligation in (n = 70) rats. Continuous recording was performed via implanted telemetry transmitters, followed by arrhythmia-analysis and calculation of autonomic indices derived from heart rate variability. The regional myocardial electrophysiologic properties were assessed by monophasic action potentials and multi-electrode recordings. KEY FINDINGS: Sympathetic-activity was decreased and vagal-activity was enhanced after intracerebroventricular ETA-receptor blockade, thus attenuating regional myocardial repolarization inhomogeneity. As a result, the incidence of ventricular tachyarrhythmias was markedly lower in this group. Such effects were also observed after intracerebroventricular blockade of ETB-, or both, ETA- and ETB-receptors, although to a lesser extent. SIGNIFICANCE: ETA-receptors in the brain modulate sympathetic and vagal responses and alter arrhythmogenesis during evolving myocardial necrosis in rats. These findings provide insights into arrhythmogenic mechanisms during acute myocardial infarction and call for further investigation on the role of endothelin in the central autonomic network.


Assuntos
Endotelinas/farmacologia , Infarto do Miocárdio/fisiopatologia , Receptores de Endotelina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/metabolismo , Sistema Nervoso Autônomo/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiologia , Antagonistas dos Receptores de Endotelina/metabolismo , Antagonistas dos Receptores de Endotelina/farmacologia , Endotelina-1/farmacologia , Endotelinas/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Masculino , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Wistar , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Receptores de Endotelina/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia
9.
Elife ; 82019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31657718

RESUMO

Experience and changes in neuronal activity can alter CNS myelination, but the signalling pathways responsible remain poorly understood. Here we define a pathway in which endothelin, signalling through the G protein-coupled receptor endothelin receptor B and PKC epsilon, regulates the number of myelin sheaths formed by individual oligodendrocytes in mouse and zebrafish. We show that this phenotype is also observed in the prefrontal cortex of mice following social isolation, and is associated with reduced expression of vascular endothelin. Additionally, we show that increasing endothelin signalling rescues this myelination defect caused by social isolation. Together, these results indicate that the vasculature responds to changes in neuronal activity associated with experience by regulating endothelin levels, which in turn affect the myelinating capacity of oligodendrocytes. This pathway may be employed to couple the metabolic support function of myelin to activity-dependent demand and also represents a novel mechanism for adaptive myelination.


Assuntos
Endotelinas/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteína Quinase C-épsilon/metabolismo , Receptor de Endotelina B/metabolismo , Transdução de Sinais , Animais , Camundongos , Córtex Pré-Frontal/fisiologia , Peixe-Zebra
10.
Pediatr Surg Int ; 35(12): 1339-1343, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31555862

RESUMO

BACKGROUND: Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. Hypoxia is known as one of the major risk factors for the development of NEC. Endothelin, known to regulate vasoconstriction, has two receptors (A and B). However, the role of endothelin receptor B (EDNRB) in neonatal intestinal injury remains unclear. We aimed to investigate whether EDNRB is involved in NEC pathophysiology. METHODS: Following ethical approval (#44032), EDNRB hetero knockout mice pups (EDNRB±) and their wild-type (WT) littermates were studied. NEC was induced from postnatal day 5-9 (P5-P9) by hypoxia, gavage feeding of formula and administration of lipopolysaccharide. On P9, the ileum was harvested. RESULTS: NEC induction in WT mice was associated with mucosal injury. However, EDNRB± NEC mice had reduced mucosal injury. Similarly, EDNRB± mice had significantly lower expression of IL-6 mRNA compared to WT NEC mice. Pimonidazole immunostaining was also significantly lower in EDNRB± compared to WT NEC, suggesting reduced tissue hypoxia. CONCLUSIONS: Partial knockout of EDNRB results in reduced NEC severity and reduced tissue hypoxia. Intestinal perfusion and hypoxia are important elements of NEC pathogenesis. These findings are relevant to the understanding of NEC pathophysiology and to the development of novel preventive strategies for NEC.


Assuntos
Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Receptor de Endotelina B/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , Recém-Nascido , Intestinos/patologia , Camundongos , Camundongos Knockout
11.
Mol Syst Biol ; 15(8): e8828, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31464372

RESUMO

Endothelins (EDN) are peptide hormones that activate a GPCR signalling system and contribute to several diseases, including hypertension and cancer. Current knowledge about EDN signalling is fragmentary, and no systems level understanding is available. We investigated phosphoproteomic changes caused by endothelin B receptor (ENDRB) activation in the melanoma cell lines UACC257 and A2058 and built an integrated model of EDNRB signalling from the phosphoproteomics data. More than 5,000 unique phosphopeptides were quantified. EDN induced quantitative changes in more than 800 phosphopeptides, which were all strictly dependent on EDNRB. Activated kinases were identified based on high confidence EDN target sites and validated by Western blot. The data were combined with prior knowledge to construct the first comprehensive logic model of EDN signalling. Among the kinases predicted by the signalling model, AKT, JNK, PKC and AMP could be functionally linked to EDN-induced cell migration. The model contributes to the system-level understanding of the mechanisms underlying the pleiotropic effects of EDN signalling and supports the rational selection of kinase inhibitors for combination treatments with EDN receptor antagonists.


Assuntos
Endotelinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Melanócitos/metabolismo , Fosfoproteínas/genética , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Endotelinas/genética , Endotelinas/metabolismo , Redes Reguladoras de Genes , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo
12.
Drug Deliv ; 26(1): 680-688, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31274009

RESUMO

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.


Assuntos
Butadienos/administração & dosagem , Glicerol/análogos & derivados , Nitrilos/administração & dosagem , Receptor de Endotelina B/metabolismo , Animais , Butadienos/líquido cefalorraquidiano , Butadienos/farmacologia , Butadienos/toxicidade , Portadores de Fármacos , Sinergismo Farmacológico , Feminino , Glicerol/administração & dosagem , Glicerol/farmacologia , Glicerol/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Modelos Biológicos , Nitrilos/líquido cefalorraquidiano , Nitrilos/farmacologia , Nitrilos/toxicidade , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina B/agonistas , Regulação para Cima
13.
J Biol Chem ; 294(33): 12495-12506, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31248984

RESUMO

Endothelin-1 (ET-1) is a neuroactive peptide produced by neurons, reactive astrocytes, and endothelial cells in the brain. Elevated levels of ET-1 have been detected in the post-mortem brains of individuals with Alzheimer's disease (AD). We have previously demonstrated that overexpression of astrocytic ET-1 exacerbates memory deficits in aged mice or in APPK670/M671 mutant mice. However, the effects of ET-1 on neuronal dysfunction remain elusive. ET-1 has been reported to mediate superoxide formation in the vascular system via NADPH oxidase (NOX) and to regulate the actin cytoskeleton of cancer cell lines via the cofilin pathway. Interestingly, oxidative stress and cofilin activation were both reported to mediate one of the AD histopathologies, cofilin rod formation in neurons. This raises the possibility that ET-1 mediates neurodegeneration via oxidative stress- or cofilin activation-driven cofilin rod formation. Here, we demonstrate that exposure to 100 nm ET-1 or to a selective ET type B receptor (ETB) agonist (IRL1620) induces cofilin rod formation in dendrites of primary hippocampal neurons, accompanied by a loss of distal dendrites and a reduction in dendritic length. The 100 nm IRL1620 exposure induced superoxide formation and cofilin activation, which were abolished by pretreatment with a NOX inhibitor (5 µm VAS2870). Moreover, IRL1620-induced cofilin rod formation was partially abolished by pretreatment with a calcineurin inhibitor (100 nm FK506), which suppressed cofilin activation. In conclusion, our findings suggest a role for ETB in neurodegeneration by promoting cofilin rod formation and dendritic loss via NOX-driven superoxide formation and cofilin activation.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Dendritos/metabolismo , Estresse Oxidativo , Receptor de Endotelina B/metabolismo , Fatores de Despolimerização de Actina/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Dendritos/patologia , Antagonistas do Receptor de Endotelina B/farmacologia , Endotelina-1/genética , Endotelina-1/metabolismo , Endotelinas/farmacologia , Camundongos , Fragmentos de Peptídeos/farmacologia , Receptor de Endotelina B/genética
14.
Neurotox Res ; 36(4): 688-699, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31228092

RESUMO

The aim of this study was to evaluate the participation of the endothelin ETA and ETB receptors and the effects of bosentan in oxaliplatin-induced peripheral sensory neuropathy (OIN) in mice. Adult male Swiss mice received 1 mg/kg of oxaliplatin intravenously, twice a week for 5 weeks. Dorsal root ganglia (DRG) and spinal cords were removed for evaluation of the endothelin ETA and ETB receptor expression. Afterwards, selective (BQ-123 and BQ-788; 10 nmol in 30 µL, intraplantarly) and non-selective (bosentan, 100 mg/kg, orally) antagonists were administered in order to evaluate the involvement of the endothelin receptors in OIN. Mechanical and thermal nociception tests were performed once a week for 56 days. Oxaliplatin induced mechanical and thermal hypersensitivity and increased the endothelin ETA receptor expression in both the DRG and spinal cord (P < 0.05). Endothelin ETB receptor expression was increased in the DRG (P < 0.05) but not in the spinal cord. Both endothelin ETA and ETB receptor selective antagonists partially prevented mechanical hyperalgesia in mice with OIN (P < 0.05). Moreover, bosentan prevented mechanical and thermal hypersensitivity in oxaliplatin-treated mice (P < 0.05). In conclusion, both endothelin ETA and ETB receptors seem to be involved in the OIN in mice and they should be considered possible targets for the management of this clinical feature.


Assuntos
Oxaliplatina/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Animais , Bosentana/administração & dosagem , Antagonistas dos Receptores de Endotelina , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Doenças do Sistema Nervoso Periférico/metabolismo , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo
15.
Placenta ; 77: 8-15, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30827357

RESUMO

INTRODUCTION: It is well established that upregulation of endothelin type B (ETB) receptors in vascular smooth muscle cells plays a role in pathophysiological artery remodeling as response to ischemia and atherosclerosis. This study aimed to investigate the ETB receptors function and localization under normal physiological remodeling. Specifically, in the guinea pig uterine arteries during pregnancy. METHODS: Uterine artery contractility was assessed with sarafotoxin 6c and endothelin-1 in wire-myography in uterine arteries from non-pregnant and pregnant guinea pigs at gestational day 37 ±â€¯5. Localization of ETB receptors, proliferation marker Ki-67, and SMC differentiation marker SM22α in uterine arteries were investigated with immunohistochemistry. RESULTS: Uterine arteries from pregnant guinea pigs showed significantly increased ETB receptor-mediated vasoconstriction compared to uterine arteries from non-pregnant and to coronary arteries from pregnant guinea pigs (p < 0.001), suggesting that ETB-receptor upregulation in uterine artery SMCs is a normal physiological mechanism taking place during remodeling. Furthermore, uterine arteries from pregnant guinea pigs showed enhanced expression of ETB receptors, high density of Ki-67 positive SMCs and sparse SM22α staining in SMCs localized in the outer layer of the vessel wall. DISCUSSION: Our results suggest that ETB receptors are expressed in dedifferentiated proliferating SMCs of uterine arteries in pregnant guinea pigs. This study provides novel insight into the function and expression of ETB receptors in uterine vascular remodeling during pregnancy.


Assuntos
Miócitos de Músculo Liso/metabolismo , Prenhez/metabolismo , Receptor de Endotelina B/metabolismo , Artéria Uterina/metabolismo , Animais , Desdiferenciação Celular , Proliferação de Células , Endotelina-1/farmacologia , Feminino , Cobaias , Miócitos de Músculo Liso/citologia , Técnicas de Cultura de Órgãos , Gravidez , Receptor de Endotelina B/agonistas , Artéria Uterina/citologia , Artéria Uterina/efeitos dos fármacos , Remodelação Vascular/fisiologia , Vasoconstritores/farmacologia , Venenos de Víboras/farmacologia
16.
Ann N Y Acad Sci ; 1448(1): 5-18, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30900271

RESUMO

Endothelin receptor B (EDNRB) is one of the receptors in the endothelin axis and its upregulated expression is associated with tumorigenesis and metastasis of several types of solid tumors. However, the expression profile of EDNRB in breast cancer and its role in the progression of breast cancer are unclear. Here, we show that EDNRB expression is higher in metastatic tumors than in primary breast cancer, and is associated significantly with lymph node metastasis and poor survival in Chinese patients with breast cancer. EDNRB expression was particularly upregulated in triple-negative breast cancer (TNBC) cells. Moreover, EDNRB silencing by a specific shRNA significantly attenuated the proliferation, migration, and invasiveness of MDA-MB-231 and BT549 cells and increased their apoptosis, as well as retarded the growth of implanted tumors in mice. Tandem mass spectrometry analysis indicated that 248 proteins were differentially expressed in EDNRB-silenced cells and their cellular organelles, and these proteins participate in many processes. EDNRB silencing decreased protein kinase B and extracellular regulated protein kinase phosphorylation and promoted the mesenchymal-to-epithelial transition process in MDA-MB-231 cells. Therefore, our findings provide strong evidence for the first time that knockdown of EDNRB expression inhibits the progression of TNBC and that EDNRB can serve as a prognostic biomarker and therapeutic target for the treatment of TNBC.


Assuntos
Transformação Celular Neoplásica/genética , Invasividade Neoplásica/genética , Receptor de Endotelina B/genética , Neoplasias de Mama Triplo Negativas/genética , Adolescente , Adulto , Idoso , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor de Endotelina B/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Adulto Jovem
17.
J Cardiovasc Pharmacol ; 73(3): 178-185, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30839511

RESUMO

INTRODUCTION: Hepatopulmonary syndrome and portopulmonary hypertension are common complications of liver disorders. This study aimed to determine roles of ET-B receptors and endothelial-derived NO synthase in the regulation of pulmonary hemodynamic in cirrhotic rats. METHODS: Male Sprague-Dawley rats were divided into the Sham and common bile duct ligation (CBDL) groups. After 28 days, animals were anesthetized, and the right ventricle, femoral artery, and vein cannulated. Then, intravenous injection of BQ-788 (a selective ET-B receptor antagonist) and L-NAME (eNOS inhibitor) were performed sequentially. RESULTS: After the first injection of BQ-788, the right ventricular systolic pressure (RVSP) and mean arterial systemic pressure increased only in the Sham group. L-NAME increased RVSP in the Sham and CBDL groups, whereas mean arterial systemic pressure elevated only in the Sham group significantly. Reinjection of BQ-788 increased RVSP in the Sham group, whereas it decreased RVSP in the CBDL group. Both plasma NO metabolites and lung endothelin-1 increased in the CBDL group. CONCLUSION: ET-B receptors on the endothelial cells play roles in the regulation of pulmonary and systemic vascular tone in normal condition through the NO-mediated pathway, whereas ET-B receptors on the smooth muscle cells have a role in the pulmonary vascular tone in liver cirrhosis.


Assuntos
Hemodinâmica , Síndrome Hepatopulmonar/enzimologia , Cirrose Hepática Experimental/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Artéria Pulmonar/enzimologia , Circulação Pulmonar , Receptor de Endotelina B/metabolismo , Animais , Pressão Arterial/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotelina-1/metabolismo , Síndrome Hepatopulmonar/fisiopatologia , Cirrose Hepática Experimental/fisiopatologia , Masculino , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Transdução de Sinais , Função Ventricular Direita/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacos
18.
Acta Derm Venereol ; 99(6): 579-586, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30809683

RESUMO

Prurigo nodularis is a highly pruritic and hyperplastic chronic dermatosis with unknown pathogenesis. Many pruritogenic mediators, including nerve growth factor, interleukin (IL)-31, thymic stromal lymphopoietin, and endothelin-1, are implicated in chronic itch and inflammation. This study investigated the mRNA levels and immunoreactivity of the nerve growth factor, IL-31, thymic stromal lymphopoietin, and endothelin axes in both lesional and perilesional skin in prurigo nodularis by using quantitative real-time PCR and immunohistochemistry studies. The nerve growth factor high-affinity receptor tyrosine kinase receptor A was upregulated while the low affinity receptor p75 neurotrophin receptor was downregulated in prurigo nodularis lesions. Downregulated expression of IL-31/IL-31 receptor A and endothelin-3/endothelin receptor B and upregulation of thymic stromal lymphopoietin receptor were found in prurigo nodularis lesions. Aberrant expression of nerve growth factor, IL-31, thymic stromal lymphopoietin and endothelin axes was found in prurigo nodularis lesions, especially in the epidermis, indicating the importance of keratinocytes in prurigo nodularis pathogenesis.


Assuntos
Epiderme/metabolismo , Queratinócitos/metabolismo , Prurigo/genética , Prurigo/metabolismo , Adulto , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo , Endotelina-3/genética , Endotelina-3/metabolismo , Feminino , Expressão Gênica , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/genética , Hipersensibilidade/metabolismo , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Prurigo/complicações , RNA Mensageiro/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Regulação para Cima
19.
J Pharm Pharmacol ; 71(6): 988-995, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30809816

RESUMO

OBJECTIVES: TGF-ß through hyperelongation of glycosaminoglycan (GAG) chains leads to binding of low-density lipoproteins to the proteoglycans. The vasoactive peptide, endothelin-1 (ET-1), plays a key role in the development of atherosclerosis. This study addressed the question whether ET-1 by activating the Rho kinase and cytoskeletal rearrangement can transactivate the TGF-ß receptor leading to phosphorylation of the transcription factor Smad2 and increased expression of the GAG chain synthesizing enzyme such as chondroitin synthase-1 (CHSY-1) in bovine aortic endothelial cells (BAECs). METHODS: In this study, intermediates in ET-1-induced Smad2C phosphorylation and the protein level of CHSY-1 were identified and quantified by Western blotting. KEY FINDINGS: Endothelin-1 caused time-dependent phosphorylation of Smad2C which was inhibited in the presence of the endothelin B receptor antagonist, BQ788. The response to ET-1 was inhibited by the Rho/ROCK kinase antagonist, Y27632 and by cytochalasin D, an inhibitor of actin polymerization but the ET-1-mediated pSmad2C was not inhibited by the matrix metalloproteinase (MMP) inhibitor, GM6001. ET-1 increased CHSY-1 protein level, which was inhibited in the presence of BQ788, cytochalasin D and Y27632. CONCLUSIONS: Endothelin-1 signalling via the ETB receptor utilizes cytoskeletal rearrangement and Rho kinase but not MMPs leading to TßRI transactivation signalling and phosphorylation of Smad2C and through this pathway increased the level of CHSY-1.


Assuntos
Células Endoteliais/metabolismo , Endotelina-1/metabolismo , N-Acetilgalactosaminiltransferases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Amidas/farmacologia , Animais , Aorta/citologia , Western Blotting , Bovinos , Células Cultivadas , Citocalasina D/farmacologia , Oligopeptídeos/farmacologia , Fosforilação/fisiologia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Tempo , Ativação Transcricional/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Quinases Associadas a rho/metabolismo
20.
FEBS Lett ; 593(6): 644-651, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30801683

RESUMO

Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ETA R) and ET type B receptor (ETB R). ETA R and ETB R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ETA R- or ETB R-specific binding proteins to understand the differences in ETA R- and ETB R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ETB R than towards ETA R. Moreover, GRP78 overexpression promoted ETB R-mediated ERK activation and GRP78 silencing suppressed ETB R-mediated ERK activation. Furthermore, ETB R can localize GRP78 to the cell periphery. These results suggest that the interaction of ETB R with GRP78 affects ERK activation and GRP78 localization.


Assuntos
Endotelina-1/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Endotelina-1/genética , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
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