Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Am J Physiol Endocrinol Metab ; 307(12): E1085-96, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25336523

RESUMO

Despite the presence of vitamin D receptor (VDR) in endothelial cells, the effect of vitamin D on endothelial function is unknown. An unbalanced production of vasoactive endothelial factors such as nitric oxide (NO) or endothelin-1 (ET-1) results in endothelial dysfunction, which can alter the normal cardiovascular function. Present experiments were devoted to assess the effect of active vitamin D (calcitriol) on the synthesis of endothelial vasoactive factors. The results were that, in cells, calcitriol increased ET-1 and NO productions, which were measured by ELISA and fluorimetric assay, respectively. Calcitriol also increased endothelin-converting enzyme-1 (ECE-1) and endothelial-nitric oxide synthase (eNOS) activities, their mRNA (qPCR), their protein expressions (Western-blot), and their promoter activities (transfection assays). Calcitriol did not change prepro-ET-1 mRNA. The effect was specific to VDR activation because when VDR was silenced by siRNA, the observed effects disappeared. Mechanisms involved in each upregulation differed. ECE-1 upregulation depended on AP-1 activation, whereas eNOS upregulation depended directly on VDR activation. To evaluate the in vivo consequences of acute calcitriol treatment, normal Wistar rats were treated with a single ip injection of 400 ng/kg calcitriol and euthanized 24 h later. Results confirmed those observed in cells, that production and expression of both factors were increased by calcitriol. Besides, calcitriol-treated rats showed a slight rise in mean blood pressure, which decreased when pretreated with FR-901533, an ECE-1 antagonist. We conclude that calcitriol increases the synthesis of both ET-1 and NO in endothelial cells. However, the ET-1 upregulation seems to be biologically more relevant, as animals acutely treated with calcitriol show slight increases in blood pressure.


Assuntos
Calcitriol/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotelinas/metabolismo , Óxido Nítrico/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Enzimas Conversoras de Endotelina , Humanos , Masculino , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Wistar , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
2.
Pharmacol Res ; 76: 106-18, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23911580

RESUMO

Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and Western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP.


Assuntos
Compostos de Anilina/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Cálcio/agonistas , Metaloendopeptidases/metabolismo , Animais , Ácido Aspártico Endopeptidases/análise , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotelina-1/sangue , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina , Humanos , Masculino , Metaloendopeptidases/análise , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo III/metabolismo , Fenetilaminas , Propilaminas , Ratos , Ratos Wistar
3.
Arterioscler Thromb Vasc Biol ; 31(11): 2577-85, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21852564

RESUMO

OBJECTIVE: Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells. METHODS AND RESULTS: Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3'-untranslated region (3'-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3'-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content. CONCLUSION: Our results support that NO regulates ECE-1 expression through a cGMP/PKG-dependent regulatory mechanism at the post-transcriptional level via the 3'-UTR of the ECE-1 gene.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Ácido Aspártico Endopeptidases/genética , Bovinos , Linhagem Celular , Técnicas de Cocultura , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação para Baixo/fisiologia , Enzimas Conversoras de Endotelina , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Rim/embriologia , Rim/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Modelos Animais , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , RNA Mensageiro/efeitos dos fármacos , Ratos Wistar
4.
Cardiovasc Res ; 113(2): 207-221, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28025386

RESUMO

AIM: To analyse the ability of TWEAK to modify the endothelin system, particularly endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1), studying the intracellular mechanisms implied. TNF-like weak inducer of apoptosis (TWEAK) is a member of TNF superfamily; it has different biological functions such as inflammation, angiogenesis, proliferation, and apoptosis. TWEAK and fibroblast growth-factor-inducible 14 are expressed in different cell types, including endothelial and smooth muscle cells. Despite their presence in endothelial cells, the effect of TWEAK on endothelial function is incompletely defined. METHODS AND RESULTS: In cells, TWEAK induced protein (Western blot) and mRNA (quantitative polymerase chain reaction) expression of ECE-1. Results were related to transcriptional changes, as ECE-1 promoter activity (transfection assays) was also increased. Transfections with serial deletions of ECE-1 promoter suggest a potential role for AP-1 and NFkB, which were confirmed by electrophoretic mobility shift assays. When AP-1 or NFkB activations were inhibited by specific inhibitors of AP-1, PD-98059 (Erk1/2 inhibitor), or SP-600125 (JNK inhibitor), and also with an inhibitor of NFKB and PDTC, TWEAK effect was partially blocked in both cases, suggesting that both transcription factors are implied in ECE-1 regulation. Moreover, the endothelial changes induced by TWEAK were also tested in vivo, using 3-month-old male CD-1 mice treated with TWEAK 10 µg/kg body weight for 24 h, finding similar effects, a rise in ET-1 production (enzyme-linked immunosorbent assay), and ECE-1 expression in aorta and lung tissues. Mice showed slight hypertension after 4 h of treatment, which disappeared at 24 h. CONCLUSIONS: In pathological situations such as chronic inflammation, TWEAK could be more harmful through this effect at endothelial level. Pharmacological blockade of this cytokine could prevent the haemodynamic and structural changes related to an increased ET-1 synthesis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Fatores de Necrose Tumoral/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/enzimologia , Endotelina-1/genética , Enzimas Conversoras de Endotelina/genética , Humanos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Camundongos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Transfecção , Fatores de Necrose Tumoral/toxicidade , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA