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1.
J Pharmacol Exp Ther ; 370(3): 437-446, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31248979

RESUMO

Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice. Chymase-specific enzymatic activity was also measured in the lung, brain, and mast cell extracts in vitro. Finally, the impact of EAE immunization was studied on the pulmonary and brain mRNA expression of different genes of the endothelin pathway, interleukin-33 (IL-33), and monitoring of immunoreactive tumor necrosis factor-α (TNF-α). Systemically or intrathecally administered big-ET-1 triggered increases in blood pressure in conscious mice. One week post-EAE, the pressor responses to big-ET-1 were potentiated in wild-type (WT) mice but not in mMCP-4 knockout (KO) mice. EAE triggered mMCP-4-specific activity in cerebral homogenates and peritoneal mast cells. Enhanced pulmonary, but not cerebral preproendothelin-1 and IL-33 mRNA were found in KO mice and further increased 1 week post-EAE immunization, but not in WT animals. Finally, TNF-α levels were also increased in serum from mMCP-4 KO mice, but not WT, 1 week post-EAE. Our study suggests that mMCP-4 activity is enhanced both centrally and systemically in a mouse model of EAE.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Endotelina-1/administração & dosagem , Endotelina-1/farmacologia , Serina Endopeptidases/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Técnicas de Inativação de Genes , Hemodinâmica/efeitos dos fármacos , Injeções Espinhais , Interleucina-33/deficiência , Interleucina-33/genética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Regulação para Cima/efeitos dos fármacos
2.
Eur J Pharmacol ; 855: 124-136, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31063771

RESUMO

In the pulmonary vasculature there is clearance of endothelin-1 from the circulation mediated by endothelin ETB receptors. This study explored the haemodynamic effects of endothelin-1 and its clearance in the pulmonary and hindquarter vasculature in anaesthetised rats. Carotid and pulmonary artery pressures and pulmonary and hindquarter blood flows were measured. In each rat, a single endothelin-1 or sarafotoxin S6C cumulative dose-response curve was generated with or without antagonist pretreatment (i.v.). Endothelin-1 caused an acute fall in MAP and rise in hindquarter vascular conductance (HVC) followed by a marked increase in MAP at 5 min with falls in HVC and pulmonary vascular conductance (PVC). Bosentan (10, 20 & 30 mg/kg) pretreatment caused dose-dependent inhibition of the MAP increase as well as PVC and HVC decreases to endothelin-1. Similarly, macitentan (30 mg/kg) or ambrisentan (10 mg/kg) caused significant block of responses to endothelin-1. Sarafotoxin S6C caused acute falls in MAP and increases in HVC and then small falls in PVC and HVC, all prevented by pretreatment with ETB antagonist BQ788 (1 mg/kg). Pretreatment with BQ788 enhanced endothelin-1 potency by 2.5-fold in PVC and 2.4-fold in HVC. With BQ788 and bosentan, the fall in HVC response was completely blocked, but there were residual MAP rises and PVC falls at the highest endothelin-1 dose. Our work confirms the role of ETB receptors in the pulmonary vasculature that decrease the circulating levels of endothelin-1. This has important consequences in selecting an appropriate ETA and ETB dual receptor antagonist to effectively block endothelin-1-mediated pulmonary vasoconstriction.


Assuntos
Endotelina-1/farmacologia , Hemodinâmica/efeitos dos fármacos , Pulmão/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Anestesia , Animais , Bosentana/farmacologia , Interações de Medicamentos , Feminino , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Venenos de Víboras/farmacologia
3.
J Pharmacol Sci ; 140(1): 102-105, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31103330

RESUMO

Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization.


Assuntos
Sinalização do Cálcio/fisiologia , Endotelina-1/farmacologia , Receptor de Endotelina A/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Feminino , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Transdução de Sinais/efeitos dos fármacos
4.
PLoS One ; 14(3): e0214336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30893362

RESUMO

Evidence suggests that ethanol-induced hypertension is associated with increased cardiovascular responsiveness to vasopressors in vivo and enhanced reactivity of isolated arteries to vasopressors ex vivo. The underlying mechanisms are not well understood and the contribution of ethanol metabolites to vascular effects induced by ethanol consumption are unclear. Mesenteric resistance arteries were harvested from Sprague-Dawley rats. Pressure myography was utilized to test effects of ethanol, acetaldehyde and phosphatidylethanol on myogenic tone and on vasoconstriction induced by phenylephrine, arginine vasopressin (aVP), endothelin-1 and KCl. Ethanol, acetaldehyde and phosphatidylethanol concentrations were monitored during the experiments. Ethanol concentrations in the vessel bath decreased with a half-life of 25min; acetaldehyde and phosphatidylethanol concentrations remained constant. Pretreatment with ethanol dose-dependently increased the potency of phenylephrine to induce vasoconstriction 4-fold (p<0.01). These effects were comparable when arteries were pre-treated with a single dose of ethanol for 30min and when ethanol concentrations were kept constant during 30min and 60min of pretreatment. While ethanol also dose-dependently increased the potency of aVP to induce vasoconstriction 1.7-fold (p<0.05), it did not affect vasoconstriction induced by endothelin-1 or KCl. Acetaldehyde pre-treatment (30 min) dose-dependently increased the potency of phenylephrine to induce vasoconstriction 2.7-fold (p<0.01) but did not affect other vasoconstrictor responses. Phosphatidylethanol did not affect any vasoconstrictor responses. Ethanol and its metabolites did not affect myogenic tone. These data suggest that ethanol and acetaldehyde selectively sensitize intrinsic constrictor responses upon activation of vascular α1-adrenergic and/or vasopressin receptors at clinically relevant concentrations. Our findings support the concept that enhanced vasoreactivity to vasoactive hormones contributes to the development of hypertension induced by ethanol consumption. Ex vivo exposure of resistance arteries to ethanol and acetaldehyde resembles effects of chronic ethanol consumption on intrinsic vascular function, and thus could serve as test platform to evaluate interventions aimed to mitigate vascular effects associated with ethanol consumption.


Assuntos
Etanol/farmacologia , Artérias Mesentéricas/fisiologia , Resistência Vascular/efeitos dos fármacos , Vasoconstritores/farmacologia , Acetaldeído/farmacologia , Animais , Arginina Vasopressina/farmacologia , Endotelina-1/farmacologia , Etanol/química , Glicerofosfolipídeos/farmacologia , Masculino , Artérias Mesentéricas/química , Artérias Mesentéricas/efeitos dos fármacos , Miografia , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Vasoconstrição
5.
J Biol Chem ; 294(11): 3920-3933, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30670587

RESUMO

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ciclina D1/metabolismo , Endotelina-1/farmacologia , Proteínas Quinases Associadas a Fase S/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Astrócitos/citologia , Astrócitos/enzimologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/genética , Relação Dose-Resposta a Droga , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Proteínas Quinases Associadas a Fase S/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Relação Estrutura-Atividade
6.
Eur J Pharmacol ; 846: 109-118, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30653947

RESUMO

Subarachnoid hemorrhage (SAH) is associated with increased cerebral artery sensitivity to vasoconstrictors and release of the perivascular sensory vasodilator CGRP. In the current study the constrictive phenotype and the vasodilatory effects of exogenous and endogenous perivascular CGRP were characterized in detail applying myograph technology to cerebral artery segments isolated from experimental SAH and sham-operated rats. Following experimental SAH, cerebral arteries exhibited increased vasoconstriction to endothelin-1, 5-hydroxytryptamine and U46419. In addition, depolarization-induced vasoconstriction (60 mM potassium) was significantly increased, supporting a general SAH-associated vasoconstrictive phenotype. Using exogenous CGRP, we demonstrated that sensitivity of the arteries to CGRP-induced vasodilation was unchanged after SAH. However, vasodilation in response to capsaicin (100 nM), a sensory nerve activator used to release perivascular CGRP, was significantly reduced by SAH (P = 0.0079). Because CGRP-mediated dilation is an important counterbalance to increased arterial contractility, a reduction in CGRP release after SAH would exacerbate the vasospasms that occur after SAH. A similar finding was obtained with artery culture (24 h), an in vitro model of SAH-induced vascular dysfunction. The arterial segments maintained sensitivity to exogenous CGRP but showed reduced capsaicin-induced vasodilation. To test whether a metabolically stable CGRP analogue could be used to supplement the loss of perivascular CGRP release in SAH, SAX was systemically administered in our in vivo SAH model. SAX treatment, however, induced CGRP-desensitization and did not prevent the development of vasoconstriction in cerebral arteries after SAH.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Artérias Cerebrais/patologia , Hemorragia Subaracnóidea/patologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Capsaicina/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Endotelina-1/farmacologia , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologia , Vasoconstritores/farmacologia
7.
Biol Sex Differ ; 10(1): 1, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606254

RESUMO

BACKGROUND: The pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1. METHODS: Male and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10-12 to 10-8 M) and sarafotoxin 6c (S6c, ETB agonist, 10-12 to 10-8 M) using the blood-perfused juxtamedullary nephron preparation. RESULTS: Control afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 µm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10-8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10-8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10-8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10-12 to 10-9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation. CONCLUSION: These data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.


Assuntos
Arteríolas/efeitos dos fármacos , Endotelina-1/farmacologia , Receptor de Endotelina A/agonistas , Receptor de Endotelina B/agonistas , Animais , Arteríolas/fisiologia , Castração , Feminino , Masculino , Microcirculação/efeitos dos fármacos , Ratos Sprague-Dawley , Receptor de Endotelina A/fisiologia , Receptor de Endotelina B/fisiologia , Caracteres Sexuais , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Venenos de Víboras/farmacologia
8.
Behav Brain Res ; 362: 1-6, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30597250

RESUMO

In the olfactory epithelium, olfactory sensitive neurons and their axons are surrounded by glia-like cells called sustentacular cells, which maintain both the structural and ionic integrity of the olfactory mucosa. We have previously found that endothelin-1 (ET-1) can uncouple sustentacular cell gap junctions in vitro similarly as carbenoxolone, a known gap junction uncoupling agent. The role of gap junctions in odorant transduction remains controversial and we explored here if ET-1 naturally produced by the olfactory mucosa could impact odorant detection. Using calcium imaging on olfactory mucosa explant, we first confirmed that ET-1 uncouples gap junctions in an olfactory mucosa preparation preserving the tissue integrity. We next measured the olfactory epithelium responses to odorant stimulation using electro-olfactogram recordings. While the amplitude of the response was not modified by application of ET-1 and carbenoxolone, its repolarizing phase was slower after both treatments. We finally examined the behavioral performances of rat pups in an orientation test based on maternal odor recognition after intranasal instillations of ET-1 or carbenoxolone. While rat pups performances were decreased after ET-1 treatment, it was unchanged after carbenoxolone treatment. Overall, our results indicate that ET-1 modulates olfactory responses at least partly through gap junction uncoupling.


Assuntos
Comportamento Animal/efeitos dos fármacos , Endotelina-1/farmacologia , Mucosa Olfatória/efeitos dos fármacos , Olfato/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Masculino , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Odorantes , Mucosa Olfatória/fisiologia , Ratos Wistar , Olfato/fisiologia
9.
Diab Vasc Dis Res ; 16(1): 57-68, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30482051

RESUMO

AIM: This study aims to investigate the altered expression signature of long non-coding RNAs, mRNAs and deregulated pathways related to diabetic cardiomyopathy disease pathogenesis. METHOD: We utilize the previously established in vitro diabetic cardiomyopathy model of human induced pluripotent stem cell-derived human cardiomyocytes to perform long non-coding RNA and mRNA expression analysis on glucose (11 mM), endothelin-1 (10 nM) and cortisol (1 µM) stimulated human induced pluripotent stem cell-derived human cardiomyocytes to interrogate diabetic cardiomyopathy associated RNA expression profile. RESULT: Out of 20,730 mRNAs and 40,173 long non-coding RNAs being screened, 2046 long non-coding RNAs and 1582 mRNAs were differentially regulated (fold change > 2, p < 0.05) between diabetic cardiomyopathy and control group, of which more than half were intergenic and antisense long non-coding RNAs. Most of the coding transcripts were associated with processes like inflammation, structural reorganization, metabolism, smooth muscle contraction, focal adhesion and repair contributing towards the development of diabetic cardiomyopathy. The subgroup analysis further revealed 411 long non-coding RNAs being co-expressed with neighbouring genes. However, our coding-non-coding co-expression analysis showed an overall 48,155 co-expression network connections. In addition to that, the long non-coding RNAs with highest network connections were profoundly enriched for focal adhesion, cell-matrix adhesion and muscle contraction. CONCLUSION: These results provide comprehensive data about the pathways and regulatory mechanisms associated with diabetic cardiomyopathy and indicate that long non-coding RNAs may play a crucial role in diabetic cardiomyopathy.


Assuntos
Cardiomiopatias Diabéticas/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Transcriptoma , Diferenciação Celular , Células Cultivadas , Cardiomiopatias Diabéticas/metabolismo , Endotelina-1/farmacologia , Redes Reguladoras de Genes , Glucose/farmacologia , Humanos , Hidrocortisona/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transcriptoma/efeitos dos fármacos
10.
Am J Physiol Heart Circ Physiol ; 316(1): H245-H254, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30444664

RESUMO

Inadequate perfusion of solid cancer tissue results in low local nutrient and oxygen levels and accumulation of acidic waste products. Previous investigations have focused primarily on tumor blood vessel architecture, and we lack information concerning functional differences between arteries that deliver blood to solid cancer tissue versus normal tissue. Here, we use isometric myography to study resistance-sized arteries from human primary colon adenocarcinomas and matched normal colon tissue. Vasocontraction of colon cancer feed arteries in response to endothelin-1 and thromboxane stimulation is attenuated compared with normal colon arteries despite similar wall dimensions and comparable contractions to arginine vasopressin and K+-induced depolarization. Acetylcholine-induced vasorelaxation and endothelial NO synthase expression are increased in colon cancer feed arteries compared with normal colon arteries, whereas vasorelaxation to exogenous NO donors is unaffected. In congruence, the differences in vasorelaxant and vasocontractile function between colon cancer feed arteries and normal colon arteries decrease after NO synthase inhibition. Rhythmic oscillations in vascular tone, known as vasomotion, are of lower amplitude but similar frequency in colon cancer feed arteries compared with normal colon arteries. In conclusion, higher NO synthase expression and elevated NO signaling amplify vasorelaxation and attenuate vasocontraction of human colon cancer feed arteries. We propose that enhanced endothelial function augments tumor perfusion and represents a potential therapeutic target. NEW & NOTEWORTHY Local vascular resistance influences tumor perfusion. Arteries supplying human colonic adenocarcinomas show enhanced vasorelaxation and reduced vasocontraction mainly due to elevated nitric oxide-mediated signaling. Rhythmic oscillations in tone, known as vasomotion, are attenuated in colon cancer feed arteries.


Assuntos
Adenocarcinoma/patologia , Artérias/metabolismo , Neoplasias do Colo/patologia , Neovascularização Patológica/metabolismo , Óxido Nítrico/metabolismo , Vasodilatação , Acetilcolina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artérias/efeitos dos fármacos , Artérias/fisiopatologia , Endotelina-1/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais , Tromboxanos/farmacologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia
11.
Mol Med Rep ; 18(6): 5229-5236, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30272323

RESUMO

Cerebral vasospasm (CVS) is a severe complication of subarachnoid hemorrhage (SAH), and endothelin­1 (ET­1) may be involved in its pathogenesis. The present study aimed to investigate the expression of ET­1 in cerebrospinal fluid (CSF) in patients with SAH and to analyze rat arterial contractility and the expression levels of ET­1 receptors in vitro. CSF samples were collected from 28 patients and the expression levels of ET­1 were measured. Rat cerebral basilar arteries were isolated and incubated with hemorrhagic or clear CSF. Contractility, as well as ETA and ETB mRNA expression were measured. ET­1 levels in CSF increased and reached a peak within the initial 5 days after SAH onset and then gradually subsided. After 12 or 24 h, the contraction of arteries incubated in hemorrhagic CSF was substantially stronger than those in clear CSF. The mRNA expression levels of endothelin receptor type A and B in arteries incubated in hemorrhagic CSF were significantly higher than those in clear CSF. ET­1 and its receptors may be involved in the pathogenic mechanism of CVS following SAH. ET­1 expression in CSF may be used as a marker in CVS and its receptors may provide novel therapeutic targets in CVS.


Assuntos
Endotelina-1/metabolismo , Receptor de Endotelina A/metabolismo , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/fisiopatologia , Adulto , Idoso , Animais , Angiografia por Tomografia Computadorizada , Modelos Animais de Doenças , Endotelina-1/genética , Endotelina-1/farmacologia , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor de Endotelina A/genética , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Hemorragia Subaracnóidea/diagnóstico , Fatores de Tempo , Tomografia Computadorizada por Raios X
12.
Invest Ophthalmol Vis Sci ; 59(12): 4886-4895, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30347083

RESUMO

Purpose: To correlate outflow function and outflow tract vessel diameter changes induced by nitric oxide (NO). Methods: In a porcine anterior segment perfusion model, the effects of a nitric oxide donor (100 µM DETA-NO) on outflow facility were compared with controls (n = 8 per group) with trabecular meshwork (TM) and after circumferential ab interno trabeculectomy (AIT). Outflow structures were assessed with spectral-domain optical coherence tomography (SD-OCT) before and after NO, or an NO synthase inhibitor (100 µM L-NAME) and the vasoconstrictor, endothelin-1 (100 pg/mL ET-1). Scans were processed with a custom macroscript and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA). Results: The facility increased after DETA-NO (Δ of 0.189 ± 0.081 µL/min·mm Hg, P = 0.034) and AIT (Δ of 0.251 ± 0.094 µL/min·mm Hg, P = 0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (Δ of 0.190 ± 0.074 µL/min·mm Hg, P = 0.023) and CSA by 13.9% (P < 0.001). L-NAME + ET-1 decreased CSA by -8.6% (P < 0.001). NO increased the diameter of focal constrictions 5.0 ± 3.8-fold. Conclusions: NO can dilate vessels of the distal outflow tract and increase outflow facility in a TM-independent fashion. There are short, focally constricting vessel sections that display large diameter changes and may have a substantial impact on outflow.


Assuntos
Humor Aquoso/fisiologia , Fatores Relaxantes Dependentes do Endotélio/farmacologia , Óxido Nítrico/farmacologia , Malha Trabecular/efeitos dos fármacos , Animais , Endotelina-1/farmacologia , Inibidores Enzimáticos/farmacologia , Pressão Intraocular/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Suínos , Tomografia de Coerência Óptica , Malha Trabecular/diagnóstico por imagem , Malha Trabecular/fisiopatologia , Trabeculectomia
13.
Invest Ophthalmol Vis Sci ; 59(12): 5167-5175, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30372743

RESUMO

Purpose: Endothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in retinal venous pathologies such as diabetic retinopathy and retinal vein occlusion. However, underlying mechanisms contributing to venular constriction remain unknown. Thus, we examined the roles of ET-1 receptors, extracellular calcium (Ca2+), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) in ET-1-induced constriction of retinal venules. Methods: Porcine retinal venules were isolated and pressurized for vasoreactivity study using videomicroscopic techniques. Protein and mRNA were analyzed using molecular tools. Results: Retinal venules developed basal tone and constricted concentration-dependently to ET-1. The ETA receptor (ETAR) antagonist BQ123 abolished venular constriction to ET-1, but ETB receptor (ETBR) antagonist BQ788 had no effect on vasoconstriction. The ETBR agonist sarafotoxin S6c did not elicit vasomotor activity. In the absence of extracellular Ca2+, venules lost basal tone and ET-1-induced constriction was nearly abolished. Although L-VOCC inhibitor nifedipine also reduced basal tone and blocked vasoconstriction to L-VOCC activator Bay K8644, constriction of venules to ET-1 remained. The ROCK inhibitor H-1152 but not PKC inhibitor Gö 6983 prevented ET-1-induced vasoconstriction. Protein and mRNA expressions of ETARs and ETBRs, along with ROCK1 and ROCK2 isoforms, were detected in retinal venules. Conclusions: Extracellular Ca2+ entry via L-VOCCs is essential for developing and maintaining basal tone of porcine retinal venules. ET-1 causes significant constriction of retinal venules by activating ETARs and extracellular Ca2+ entry independent of L-VOCCs. Activation of ROCK signaling, without involvement of PKC, appears to mediate venular constriction to ET-1 in the porcine retina.


Assuntos
Cálcio/metabolismo , Endotelina-1/farmacologia , Receptor de Endotelina A/metabolismo , Veia Retiniana/fisiologia , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Animais , Western Blotting , Canais de Cálcio Tipo L/metabolismo , Antagonistas do Receptor de Endotelina B/farmacologia , Antagonistas dos Receptores de Endotelina/farmacologia , Feminino , Masculino , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperidinas/farmacologia , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Endotelina A/genética , Sus scrofa , Vênulas/fisiologia
14.
Neuroscience ; 393: 110-122, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30300704

RESUMO

Cerebral palsy is an irreversible movement disorder resulting from cerebral damage sustained during prenatal or neonatal brain development. As survival outcomes for preterm injury improve, there is increasing need to model ischemic injury at earlier neonatal time-points to better understand the subsequent pathological consequences. Here we demonstrate a novel neonatal ischemic model using focal administration of the potent vasoconstrictor peptide, endothelin-1 (ET-1), in newborn rats. The functional and histopathological outcomes compare favourably to those reported following the widely used hypoxic ischemia (HI) model. These include a robust motor deficit sustained into adulthood and recapitulation of hallmark features of preterm human brain injury, including atrophy of subcortical white matter and periventricular fiber bundles. Compared to procedures involving carotid artery manipulation and periods of hypoxia, the ET-1 ischemia model represents a rapid and technically simplified model more amenable to larger cohorts and with the potential to direct the locus of ischemic damage to specific brain areas.


Assuntos
Lesões Encefálicas/patologia , Encéfalo/patologia , Endotelina-1/farmacologia , Hipóxia-Isquemia Encefálica/patologia , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Lesões Encefálicas/tratamento farmacológico , Modelos Animais de Doenças , Endotelina-1/administração & dosagem , Feminino , Hipóxia/patologia , Hipóxia-Isquemia Encefálica/induzido quimicamente , Gravidez , Ratos
15.
Am J Physiol Heart Circ Physiol ; 315(6): H1759-H1764, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30265150

RESUMO

H2S is an endogenous gasotransmitter that increases cerebral blood flow. In the cerebral vascular endothelium, H2S is produced by cystathionine δ-lyase (CSE). Endothelin-1 (ET-1) has constrictor and dilator influences on the cerebral circulation. The mechanism of the vasodilation caused by ET-1 may involve endothelium-derived factors. We hypothesize that ET-1-elicited dilation of pial arterioles requires an elevation of H2S production in the cerebral vascular endothelium. We investigated the effects of ET-1 on CSE-catalyzed brain H2S production and pial arteriolar diameter using cranial windows in newborn pigs in vivo. H2S was measured in periarachnoid cerebrospinal fluid. ET-1 (10-12-10-8 M) caused an elevation of H2S that was reduced by the CSE inhibitors propargylglycine (PPG) and ß-cyano-l-alanine (BCA). Low doses of ET-1 (10-12-10-11 M) produced vasodilation of pial arterioles that was blocked PPG and BCA, suggesting the importance of H2S influences. The vasodilator effects of H2S may require activation of smooth muscle cell membrane ATP-sensitive K+ (KATP) channels and large-conductance Ca2+-activated K+ (BK) channels. The KATP inhibitor glibenclamide and the BK inhibitor paxilline blocked CSE/H2S-dependent dilation of pial arterioles to ET-1. In contrast, the vasoconstrictor response of pial arterioles to 10-8 M ET-1 was not modulated by PPG, BCA, glibenclamide, or paxilline and, therefore, was independent of CSE/H2S influences. Pial arteriolar constriction response to higher levels of ET-1 was independent of CSE/H2S and KATP/BKCa channel activation. These data suggest that H2S is an endothelium-derived factor that mediates the vasodilator effects of ET-1 in the cerebral circulation via a mechanism that involves activation of KATP and BK channels in vascular smooth muscle. NEW & NOTEWORTHY Disorders of the cerebral circulation in newborn infants may lead to lifelong neurological disabilities. We report that vasoactive peptide endothelin-1 exhibits vasodilator properties in the neonatal cerebral circulation by stimulating production of H2S, an endothelium-derived messenger with vasodilator properties. The ability of endothelin-1 to stimulate brain production of H2S may counteract the reduction in cerebral blood flow and prevent the cerebral vascular dysfunction caused by stroke, asphyxia, cerebral hypoxia, ischemia, and vasospasm.


Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular , Endotelina-1/farmacologia , Sulfitos/líquido cefalorraquidiano , Vasodilatação , Alanina/análogos & derivados , Alanina/farmacologia , Alquinos/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Arteríolas/fisiologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Canais KATP/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Suínos
16.
Arch Dermatol Res ; 310(8): 625-637, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30046896

RESUMO

We have already reported that glucosamine (GlcN) distinctly abrogates the pigmentation of human epidermal equivalents stimulated by stem cell factor + endothelin-1 (SE). In this study, we characterized the molecular mechanism involved in the anti-melanogenic effects of GlcN using normal human melanocytes (NHMs) in culture. The SE-stimulated gene (12 h) and protein (24 h) expression levels of melanocyte-specific proteins (at the indicated times post-stimulation) were significantly abrogated by pretreatment with GlcN for 72 h. Western blotting analysis of the phosphorylation of intracellular signaling molecules in the MAPK pathway revealed that despite the significantly decreased level of total CREB protein at all times post-stimulation, the SE-stimulated phosphorylation of ERK, CREB and MITF is not attenuated at 15 min post-stimulation in GlcN-treated NHMs. However, the SE-stimulated protein expression level of total MITF at 2 and 6 h post-stimulation was significantly abrogated by 72 h pretreatment with GlcN. Consistently, pretreatment with GlcN for 72 h abrogated the stimulated gene and protein expression levels of MITF at 1 h and 2 h post-stimulation, respectively. Analysis of gene and protein expression levels also demonstrated that pretreatment with GlcN for 72 h significantly reduced the protein levels of CREB and MITF without affecting their gene expression levels prior to the SE stimulation. Silencing with a CREB siRNA distinctly abrogated the SE-stimulated expression of MITF (at 2 h post-stimulation) and melanocyte-specific proteins (at 24 h post-stimulation). Similarly, transfection of MITF siRNA markedly abrogated the SE-stimulated expression of MITF protein and melanocyte-specific proteins at 2 and 24 h post-stimulation, respectively. Finally, the decreased levels of CREB and MITF proteins induced by 72 h pretreatment with GlcN were abrogated by the co-addition of the proteosomal degradation inhibitor MG132. These findings suggest that the anti-melanogenic effect elicited by GlcN is mediated via the decreased expression of MITF which results from the attenuated transcriptional activity of CREB due to proteolytic degradation.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotelina-1/farmacologia , Glucosamina/farmacologia , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Células-Tronco/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação para Baixo , Humanos , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Transdução de Sinais/efeitos dos fármacos
17.
Kidney Blood Press Res ; 43(3): 860-871, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29870994

RESUMO

BACKGROUND/AIMS: Canonical Wnt signaling is involved in oxidative stress, vasculopathy and diabetes mellitus but its role in diabetic renal microvascular dysfunction is unclear. We tested the hypothesis that enhanced canonical Wnt signaling in renal afferent arterioles from diabetic mice increases reactive oxygen species (ROS) and contractions to endothelin-1 (ET-1). METHODS: Streptozotocin-induced diabetes or control C57Bl/6 mice received vehicle or sulindac (40 mg·kg-1·day-1) to block Wnt signaling for 4 weeks. ET-1 contractions were measured by changes of afferent arteriolar diameter. Arteriolar H2O2, O2 -, protein expression and enzymatic activity were assessed using sensitive fluorescence probes, immunoblotting and colorimetric assay separately. RESULTS: Compared to control, diabetic mouse afferent arteriole had increased O2- (+ 84%) and H2O2 (+ 91%) and enhanced responses to ET-1 at 10-8 mol·l-1 (-72±4% of versus -43±4%, P< 0.05) accompanied by reduced protein expressions and activities for catalase and superoxide dismutase 2 (SOD2). Arteriolar O2 - was increased further by ET-1 and contractions to ET-1 reduced by PEG-SOD in both groups whereas H2O2 unchanged by ET-1 and contractions were reduced by PEG-catalase selectively in diabetic mice. The Wnt signaling protein ß-catenin was upregulated (3.3-fold decrease in p-ß-catenin/ß-catenin) while the glycogen synthase kinase-3ß (GSK-3ß) was downregulated (2.6-fold increase in p-GSK-3ß/ GSK-3ß) in preglomerular vessels of diabetic mice. Sulindac normalized the Wnt signaling proteins, arteriolar O2 -, H2O2 and ET-1 contractions while doubling microvascular catalase and SOD2 expression in diabetic mice. CONCLUSION: Increased ROS, notably H2O2 contributes to enhanced afferent arteriolar responses to ET-1 in diabetes, which is closely associated with Wnt signaling. Antioxidant pharmacological strategies targeting Wnt signaling may improve vascular function in diabetic nephropathy.


Assuntos
Arteríolas/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Endotelina-1/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Vasoconstrição/efeitos dos fármacos , Via de Sinalização Wnt , Animais , Arteríolas/metabolismo , Peróxido de Hidrogênio , Rim/irrigação sanguínea , Camundongos
18.
Spine J ; 18(9): 1669-1677, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29886166

RESUMO

BACKGROUND CONTEXT: Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/ß-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown. PURPOSE: The purpose of the study was to evaluate the influence of the endothelin-A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous end plate cells (CECs) associated with CEP degeneration via the Wnt/ß-catenin signaling pathway. STUDY DESIGN/SETTING: The influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs and the effect of BQ-123 in this context were investigated. METHODS: To establish a model for CEP degeneration, three lumbar discs (L3-L4, L4-L5, and L5-L6 levels) in New Zealand white rabbits were punctured close to the vertebral end plate using a 14G needle. Intervertebral disc degeneration was evaluated by magnetic resonance imaging 4 weeks after vertebral end plate injury. CECs were then isolated from the degenerated CEPs to allow evaluation of the role of ET-1 and BQ-123 and to investigate their effects on the Wnt/ß-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction and by Western blotting. The Wnt/ß-catenin signaling pathway was also investigated by Western blotting. RESULTS: After 4 weeks, IVDs with vertebral end plate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. Endothelin-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, whereas BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of ß-catenin, cyclin D1, and Dvl1 in the Wnt/ß-catenin signaling pathway of CECs from degenerated discs and reduced the expression of GSK-3ß, whereas BQ-123 had the opposite effect. CONCLUSIONS: Endothelin-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/ß-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration.


Assuntos
Antagonistas dos Receptores de Endotelina/farmacologia , Endotelina-1/farmacologia , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Via de Sinalização Wnt , Agrecanas/metabolismo , Animais , Colágeno/metabolismo , Disco Intervertebral/metabolismo , Coelhos , beta Catenina/metabolismo
19.
Stroke ; 49(6): 1496-1503, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29752347

RESUMO

BACKGROUND AND PURPOSE: Reach training in concert with environmental enrichment provides functional benefits after experimental stroke in rats. The present study extended these findings by assessing whether intensive task-specific reach training or enrichment initiated alone would provide similar functional benefit. Additionally, we investigated whether the 70% recovery rule, or a combined model of initial poststroke impairment, cortical infarct volume, and rehabilitation intensity, could predict recovery in the single-pellet task, as previously found for the Montoya staircase. METHODS: Rats were trained on single-pellet reaching before middle cerebral artery occlusion via intracerebral injection of ET-1 (endothelin-1). There were 4 experimental groups: stroke+enrichment, stroke+reaching, stroke+enrichment+reaching, and sham+enrichment+reaching. Reaching rehabilitation utilized a modified Whishaw box that encouraged impaired forelimb reaching for 6 hours per day, 5 days per week, for 4 weeks. All treatment paradigms began 7 days after ischemia with weekly assessment on the single-pellet task during rehabilitation and again 4 weeks after rehabilitation concluded. RESULTS: Rats exposed to the combination of enrichment and reaching showed the greatest improvement in pellet retrieval and comparable performance to shams after 3 weeks of treatment, whereas those groups that received a monotherapy remained significantly impaired at all time points. Initial impairment alone did not significantly predict recovery in single-pellet as the 70% rule would suggest; however, a combined model of cortical infarct volume and rehabilitation intensity predicted change in pellet retrieval on the single-pellet task with the same accuracy as previously shown with the staircase, demonstrating the generalizability of this model across reaching tasks. CONCLUSIONS: Task-specific reach training and environmental enrichment have synergistic effects in rats that persist long after rehabilitation ends, and this recovery is predicted by infarct volume and rehabilitation intensity.


Assuntos
Endotelina-1/farmacologia , Destreza Motora/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Reabilitação do Acidente Vascular Cerebral , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Endotelina-1/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Destreza Motora/fisiologia , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/fisiopatologia , Reabilitação do Acidente Vascular Cerebral/métodos
20.
Microvasc Res ; 119: 84-90, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29738719

RESUMO

Endothelin-1 (ET-1) is one of the key factors regulating tension of smooth muscles in blood vessels. It is believed that ET-1 plays an important role in pathogenesis of hypertension, and cardiovascular diseases; therefore, research in order to limit ET-1-mediated action is still in progress. The main objective of this paper was to evaluate the role of Rho-kinase in the ET-1-induced constriction of arteries. The analysis also included significance of intra- and extracellular pool of calcium ions in constriction triggered by ET-1. The studies were performed on perfused Wistar rat tail arteries. Concentration response curve (CRC) was determined for ET-1 in the presence of increased concentrations of Rho-kinase inhibitor (Y-27632) and IP3-receptor antagonist (2APB), both in reference to constriction triggered by solely ET-1. Afterwards, the influence of calcium ions present in the perfusion fluid was evaluated in terms of the effect triggered by 2APB and occurring in arteries constricted by ET-1. ET-1, in concentration dependent manner, leads to increase in perfusion pressure. Y-27632 and 2APB lead to shift of the concentration response curve for ET-1 to the right with simultaneously lowered maximum effect. There was no difference in reaction of the artery constricted by ET-1 and treated with 2APB in solution containing calcium and in calcium-free solution. Vasoconstrictive action of endothelin is not significantly dependent on the inflow of extracellular calcium, but it is proportional to inflow of Ca2+ related to activation of IP3 receptors and to Rho-kinase activity.


Assuntos
Artérias/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Endotelina-1/farmacologia , Cauda/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Quinases Associadas a rho/metabolismo , Animais , Artérias/enzimologia , Relação Dose-Resposta a Droga , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratos Wistar
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