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1.
Clin Sci (Lond) ; 138(17): 1071-1087, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39136472

RESUMO

Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. The present study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg·kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3ß and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.


Assuntos
Tecido Adiposo , Regulação para Baixo , Endotelina-1 , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Obesidade , Espécies Reativas de Oxigênio , Animais , Endotelina-1/metabolismo , Obesidade/metabolismo , Obesidade/fisiopatologia , Masculino , Tecido Adiposo/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Bosentana/farmacologia , Dieta Hiperlipídica , Camundongos , Estresse Oxidativo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina A/genética , Enzimas Conversoras de Endotelina/metabolismo , Aorta Torácica/metabolismo , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiopatologia
2.
Cells ; 12(3)2023 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-36766848

RESUMO

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.


Assuntos
Glioblastoma , Humanos , Glioblastoma/metabolismo , Enzimas Conversoras de Endotelina/genética , Enzimas Conversoras de Endotelina/metabolismo , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/patologia , Fenótipo
3.
Dis Markers ; 2022: 6412238, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35178130

RESUMO

Lung transplantation requires optimization of donor's organ use through ex vivo lung perfusion (EVLP) to avoid primary graft dysfunction. Biomarkers can aid in organ selection by providing early evidence of suboptimal lungs during EVLP and thus avoid high-risk transplantations. However, predictive biomarkers of pulmonary graft function such as endothelin-converting enzyme (ECE-1) and vascular endothelial growth factor (VEGF) have not been described under EVLP with standard prolonged hypothermic preservation, which are relevant in situations where lung procurement is difficult or far from the transplantation site. Therefore, this study is aimed at quantifying ECE-1 and VEGF, as well as determining their association with hemodynamic, gasometric, and mechanical ventilatory parameters in a swine model of EVLP with standard prolonged hypothermic preservation. Using a protocol with either immediate (I-) or delayed (D-) initiation of EVLP, ECE-1 levels over time were found to remain constant in both study groups (p > 0.05 RM-ANOVA), while the VEGF protein was higher after prolonged preservation, but it decreased throughout EVLP (p > 0.05 RM-ANOVA). Likewise, hemodynamic, gasometric, mechanical ventilatory, and histological parameters had a tendency to better results after 12 hours of hypothermic preservation in the delayed infusion group.


Assuntos
Enzimas Conversoras de Endotelina/análise , Circulação Extracorpórea/métodos , Hipotermia Induzida , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Biomarcadores/análise , Hipotermia Induzida/métodos , Pulmão/fisiologia , Pulmão/cirurgia , Transplante de Pulmão , Preservação de Órgãos/métodos , Suínos , Fatores de Tempo
4.
Mol Oncol ; 14(2): 347-362, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31788944

RESUMO

Endothelin-1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin-1 rely on its activation by endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N-terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R ) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self-renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2-dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC-like traits. Therefore, phospho-ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.


Assuntos
Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Carcinogênese/genética , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Enzimas Conversoras de Endotelina/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Naftiridinas/farmacologia , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Fenazinas/farmacologia , Fosforilação , Prognóstico , Estabilidade Proteica , Proteínas Recombinantes , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Oncotarget ; 6(40): 42749-60, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26543229

RESUMO

Endothelin-converting enzyme-1c (ECE-1c) is a membrane metalloprotease involved in endothelin-1 synthesis, which has been shown in vitro to have a role in breast, ovary and prostate cancer cell invasion. N-terminal end of ECE-1c displays three putative phosphorylation sites for the protein kinase CK2. We studied whether CK2 phosphorylates N-terminal end of ECE-1c as well as whether this has a role in migration and invasion of colon cancer cells. CK2 phosphorylated the N-terminal end of ECE-1c and this was precluded upon inhibition of CK2. Inhibition also led to diminished protein levels of both endogen ECE-1 or GFP-fused N-terminal end of ECE-1c in 293T embryonic and DLD-1 colon cancer cells, which highlighted the importance of this motif on UPS-dependent ECE-1c degradation. Full-length ECE-1c mutants designed either to mimic or abrogate CK2-phosphorylation displayed increased or decreased migration/invasion of colon cancer cells, respectively. Moreover, ECE-1c overexpression or its silencing with a siRNA led to increased or diminished cell migration/invasion, respectively. Altogether, these data show that CK2-increased ECE-1c protein stability is related to augmented migration and invasion of colon cancer cells, shedding light on a novel mechanism by which CK2 may promote malignant progression of this disease.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Neoplasias do Colo/patologia , Metaloendopeptidases/metabolismo , Invasividade Neoplásica/patologia , Western Blotting , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Enzimas Conversoras de Endotelina , Humanos , Microscopia Confocal , Estabilidade Proteica , RNA Interferente Pequeno , Transfecção
6.
Alcohol Alcohol ; 48(6): 657-66, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23797280

RESUMO

AIMS: We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. METHODS: Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. RESULTS: Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. CONCLUSION: The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Endotelina-1/biossíntese , Etanol/farmacologia , Músculo Liso/metabolismo , Pênis/metabolismo , Animais , Ácido Aspártico Endopeptidases/biossíntese , Western Blotting , Peso Corporal/efeitos dos fármacos , Depressores do Sistema Nervoso Central/sangue , Endotelina-1/sangue , Enzimas Conversoras de Endotelina , Etanol/sangue , Luminescência , Masculino , Metaloendopeptidases/biossíntese , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Endotelina A/biossíntese , Receptor de Endotelina B/biossíntese , Superóxidos/metabolismo
7.
Br J Pharmacol ; 157(4): 568-80, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19371338

RESUMO

BACKGROUND AND PURPOSE: There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. EXPERIMENTAL APPROACH: Vascular reactivity to ET-1 and ET(A) and ET(B) receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ET(A) and ET(B) receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [(125)I]-ET-1. KEY RESULTS: HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ET(A) or ET(B) receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ET(A) but not of ET(B) receptors abolished enhancement in HHcy tissues. ET(A) and ET(B) receptor gene expressions were not up-regulated. ET(A) receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A(2) receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. CONCLUSIONS AND IMPLICATIONS: Increased ET(A) receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ET(A) receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility.


Assuntos
Artérias Carótidas/fisiopatologia , Endotelina-1/fisiologia , Endotélio Vascular/fisiopatologia , Hiper-Homocisteinemia/metabolismo , Hiper-Homocisteinemia/fisiopatologia , Animais , Ácido Aspártico Endopeptidases/metabolismo , Cálcio/farmacologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Relação Dose-Resposta a Droga , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Endotelina-1/biossíntese , Enzimas Conversoras de Endotelina , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Técnicas In Vitro , Masculino , Metaloendopeptidases/metabolismo , Óxidos de Nitrogênio/metabolismo , Óxidos de Nitrogênio/farmacologia , Ratos , Ratos Wistar , Receptor de Endotelina A/biossíntese , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/biossíntese , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia
8.
Alcohol ; 41(2): 77-85, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17466482

RESUMO

The purpose of the present work was to investigate whether conversion of exogenous applied big-endothelin-1 (Big-ET-1) as well as the basal release and mRNA levels of endothelin-1 (ET-1) is altered by ethanol consumption in the rat carotid. The measurement of the contraction induced by Big-ET-1 served as an indicative of functional endothelin (ET)-converting enzyme (ECE) activity. Cumulative application of exogenous Big-ET-1 elicited a concentration-related contraction with the concentration-response curve shifted to the right when compared to ET-1. In endothelium-intact rings, phosphoramidon (1 mmol/l), a nonselective ECE/neutral endopeptidase (NEP) inhibitor, produced a rightward displacement of the concentration-response curves and reduced the maximal contractile response to Big-ET-1. However, in endothelium-denuded rings phosphoramidon reduced the maximum contraction for Big-ET-1 but did not alter the potency when compared to the curves obtained in the absence of the inhibitor. Ethanol consumption for 2, 6, or 10 weeks reduced the contractile effect elicited by Big-ET-1 in carotid rings with intact endothelium when compared to control or isocaloric rings. However, no differences on Big-ET-1-induced contraction were observed after endothelial denudation. On the other hand, ethanol consumption increased ET-1-induced contraction. Finally, chronic ethanol consumption did not alter either the mRNA levels for pre-pro-ET-1 nor the basal release of ET-1. The present findings show that chronic ethanol consumption does not alter the mRNA levels for ET-1 or its basal release in the rat carotid. Moreover, ethanol intake reduces the contraction induced by exogenously applied Big-ET-1 in carotid rings with intact endothelium, a fact that might be the result of a reduced conversion of this peptide by ECE on its mature active peptide ET-1.


Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Ácido Aspártico Endopeptidases/metabolismo , Artérias Carótidas/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Endotelina-1/metabolismo , Etanol/farmacologia , Metaloendopeptidases/metabolismo , Vasoconstrição/efeitos dos fármacos , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Artérias Carótidas/metabolismo , Artérias Carótidas/fisiopatologia , Depressores do Sistema Nervoso Central/administração & dosagem , Relação Dose-Resposta a Droga , Endotelina-1/genética , Endotelina-1/farmacologia , Enzimas Conversoras de Endotelina , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Etanol/administração & dosagem , Glicopeptídeos/farmacologia , Masculino , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo
9.
Inflammation ; 30(1-2): 28-37, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17221318

RESUMO

Neutrophils isolated from human peripheral blood added to a monolayer of human endothelial cells (ECV-304 cell line) stimulated with LPS (100 ng ml(-1)) resulted in: (a) neutrophil activation, measured by spreading and release of leukotriene B(4) (LTB(4)); (b) neutrophil degranulation, measured by release of matrix pro-metalloproteinase-9 (proMMP-9) and (c) loss of the monolayer integrity due to detachment of the endothelial cells. Stimulation of endothelial cells with tumor necrosis factor-alpha (TNF-alpha 10 ng ml(-1)) or interleukin-1 (IL-1; 10 ng ml(-1)) induced a similar dose-dependent increase in the neutrophil activation and endothelial cell detachment. Pre-treatment of LPS-activated ECV-304 cells with [Phe22]BigET-1(19-37) (10(-9) M; an inhibitor of endothelin converting enzyme (ECE)) or addition of BQ-123 (10(-6) M; a selective endothelin A (ET(A)) receptor antagonist) to the co-cultures, significantly reduced neutrophil spreading (50-70% inhibition) as well as the levels of LTB(4) (70-100% inhibition) and proMMP-9 (40-50% inhibition) in the co-culture supernatants. In addition, the detachment of endothelial cells was also reduced (60-75% inhibition). Moreover, the exogenous addition of ET-1 (10(-9) M) to neutrophil suspensions induced neutrophil spreading and release of LTB(4) and proMMP-9. Taken together, these findings indicate that neutrophils added to stimulated endothelial cells in the co-culture system employed in this study, get activated by products of these cells and degranulate. In parallel, the detachment of endothelial cell monolayer from the culture plates, possibly by the action of neutrophil granule-derived gelatinases, is observed. Endothelins (ETs) produced by the endothelial cells are suggested to play an essential role in these phenomena.


Assuntos
Adesão Celular , Células Endoteliais/metabolismo , Endotelina-1/metabolismo , Precursores Enzimáticos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Comunicação Parácrina , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Adesão Celular/efeitos dos fármacos , Degranulação Celular , Linhagem Celular , Forma Celular , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Antagonistas do Receptor de Endotelina A , Enzimas Conversoras de Endotelina , Endotelinas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Interleucina-1/metabolismo , Leucotrieno B4/metabolismo , Lipopolissacarídeos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Receptor de Endotelina A/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Subcell Biochem ; 38: 129-45, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15709476

RESUMO

Amyloid beta (Abeta) accumulates in the neuropil and within the walls of cerebral vessels in association with normal aging, dementia or stroke. Abeta is released from its precursor protein as soluble monomeric species yet, under pathological conditions, it self-aggregates to form soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Abeta levels could be lowered by inhibiting its generation or by promoting its clearance by transport or degradation. Here we will summarize recent findings on brain proteases capable of degrading Abeta, with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence supporting a role in the proteolysis of Abeta in vivo.


Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/enzimologia , Peptídeo Hidrolases/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Enzimas Conversoras de Endotelina , Humanos , Insulisina/metabolismo , Metaloendopeptidases/metabolismo , Neprilisina/metabolismo , Especificidade por Substrato
11.
Eur J Pharmacol ; 276(1-2): 113-21, 1995 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-7781681

RESUMO

The endothelin receptors controlling sympathetic neurotransmission and the presence of endothelin-converting enzyme were investigated in the mouse vas deferens. Endothelin-1 or endothelin-3 (0.01-100 nM) enhanced contractions evoked by field stimulation, yielding EC50 (geometric mean and 95% confidence limits) of 0.7 nM (0.4-1.6) and 13.7 nM (10.2-14.1) and Emax (mean +/- S.E.M. increase in twitch tension, in mg/10 mg wet tissue) of 473 +/- 35 and 520 +/- 51, respectively. The selective endothelin ETB receptor agonists IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1) and sarafotoxin S6c were inactive up to 100 nM. Responses to endothelin-3 were progressively inhibited by the selective endothelin ETA receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]) (10, 30 and 100 nM). At 100 nM, BQ-123 almost abolished the response to endothelin-3 (100 nM). In contrast, at 100, 300 nM and 1 microM, BQ-123 shifted the curve to endothelin-1 to the right only 2-, 5- and 6-fold, respectively. The selective endothelin ETB receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-++ +methoxycarbonyltryptophanyl-D-norleucine) (100 nM) did not modify responses to endothelin-1 or endothelin-3 (0.01-100 nM). Big-endothelin-1 (0.3-30 nM) was 10-fold less potent than endothelin-1 in increasing neurogenic responses (EC50 6.8 nM, 4.7-9.6; Emax 457 +/- 37 mg/10 mg wet tissue). Preincubation with phosphoramidon (100 microM) reduced responses to big-endothelin-1, but not endothelin-1.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Receptores de Endotelina/fisiologia , Ducto Deferente/fisiologia , Animais , Estimulação Elétrica , Enzimas Conversoras de Endotelina , Endotelinas/farmacologia , Masculino , Metaloendopeptidases , Camundongos , Contração Muscular , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperidinas/farmacologia , Receptores de Endotelina/efeitos dos fármacos , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/enzimologia
12.
Eur J Pharmacol ; 240(2-3): 113-9, 1993 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-8243532

RESUMO

The presence of a phosphoramidon-sensitive endothelin-1-converting enzyme was investigated in the rat isolated uterus. Endothelin-1 and its precursor, big-endothelin-1, increased the rate of spontaneous contractions and caused tonic contractions. Responses to big-endothelin-1 had a slower start than those to endothelin-1. The tonic contraction induced by big-endothelin-1 (10 nM) was nearly abolished by phosphoramidon (100 microM), but the response to an equieffective concentration of endothelin-1 (3 nM) was not affected. Big-endothelin-1 (EC50 6.7 nM) was only 7-fold less potent than endothelin-1 (EC50 0.9 nM), whereas endothelin-3 was much less potent (EC50 > 100 nM). The endothelin ETA receptor antagonist, BQ-123 (40, 150 and 600 nM), induced graded rightward shifts of the concentration-response curve for endothelin-1. Schild analysis yielded a straight line with a slope not different from unity, and a pA2 value of 7.76. At 100 nM, BQ-123 specifically blocked responses to both endothelin-1 (3 nM) and big-endothelin-1 (10 nM), without modifying those to oxytocin (5 nM), acetylcholine (3 microM) or bradykinin (0.5 nM). Our results suggest the presence of phosphoramidon-sensitive endothelin-converting enzyme and demonstrate the occurrence of functional endothelin ETA receptors in the rat uterus.


Assuntos
Endotelinas/farmacologia , Glicopeptídeos/farmacologia , Músculo Liso/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Receptores de Endotelina/metabolismo , Útero/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Antagonistas dos Receptores de Endotelina , Endotelina-1 , Enzimas Conversoras de Endotelina , Endotelinas/metabolismo , Feminino , Técnicas In Vitro , Metaloendopeptidases , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Precursores de Proteínas/metabolismo , Ratos , Ratos Wistar , Útero/metabolismo
13.
J Cardiovasc Pharmacol ; 22 Suppl 8: S192-5, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7509942

RESUMO

Like endothelin-1 (ET-1), its immediate human precursor big ET-1 (1-100 nM) increased the rate of spontaneous phasic contractions and caused graded tonic contractions of isolated rat uterus strips. The tonic contraction to big ET-1 (10 nM) was markedly blocked by phosphoramidon (100 microM), which did not modify the response to an equipotent concentration of ET-1 (3 nM). Responses to big-ET-1 (30 nM) were abolished in calcium-free medium, but those to ET-1 (10 nM) were only reduced by this condition. The EC50 of big ET-1 for inducing tonic contraction was only sevenfold greater than that of ET-1, and both peptides produced a maximal response similar to that evoked by KCl 80 mM. ET-3 was much less potent. The selective ETA receptor antagonist BQ-123 (40-600 nM) caused graded rightward shifts of the ET-1 curve without affecting the maximal response, yielding a Schild plot with a slope not different from unity and a pA2 value of 7.76. BQ-123 (100 nM) did not affect contractions induced by oxytocin (5 nM), acetylcholine (3 microM), or bradykinin (0.3 nM), but inhibited responses to both big ET-1 and ET-1. Therefore, the rat uterus contains a phosphoramidon-sensitive, calcium-dependent endothelin-converting enzyme that readily converts big ET-1 into ET-1, which then contracts the myometrium via activation of ETA receptors.


Assuntos
Endotelinas/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Endotelina/fisiologia , Contração Uterina/fisiologia , Útero/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Antagonistas dos Receptores de Endotelina , Endotelina-1 , Enzimas Conversoras de Endotelina , Feminino , Glicopeptídeos/farmacologia , Técnicas In Vitro , Metaloendopeptidases , Dados de Sequência Molecular , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Wistar , Receptores de Endotelina/efeitos dos fármacos , Útero/enzimologia
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