RESUMO
Doxorubicin (Doxo)-associated cardio-and vasotoxicity has been recognised as a serious complication of cancer chemotherapy. The purpose of this novel paper was to determine the effect of Doxo on G-protein coupled receptor (GPCR)-mediated vasocontraction located on vascular smooth muscle cells. Rat left anterior descending artery segments were incubated for 24 h with 0.5 µM Doxo. The vasocontractile responses by activation of endothelin receptor type A (ETA) and type B (ETB), serotonin receptor 1B (5-HT1B) and thromboxane A2 prostanoid receptor (TP) were investigated by a sensitive myography using specific agonists, while the specificity of the GPCR agonists was verified by applying selective antagonists (i.e. ETA and ETB agonist = 10- 14-10- 7.5 M endothelin-1 (ET-1); ETA antagonist = 10 µM BQ123; ETB agonists = 10- 14-10- 7.5 M sarafotoxin 6c (S6c) and ET-1; ETB antagonist = 0.1 µM BQ788; 5-HT1B agonist = 10- 12-10- 5.5 M 5-carboxamidotryptamine (5-CT); 5-HT1B antagonist = 1 µM GR55562; TP agonist = 10- 12-10- 6.5 M U46619; TP antagonist = 1 µM Seratrodast). Our results show that 0.5 µM Doxo incubation of LAD segments leads to an increased VSMC vasocontraction through the ETB, 5-HT1B and TP GPCRs, with a 2.2-fold increase in ETB-mediated vasocontraction at 10- 10.5 M S6c, a 2.0-fold increase in 5-HT1B-mediated vasocontraction at 10- 5.5 M 5-CT, and a 1.3-fold increase in TP-mediated vasocontraction at 10- 6.5 M U46619. Further studies unravelling the involvement of intracellular GPCR signalling pathways will broaden our understanding of the Doxo-induced vasotoxicity, and thus pave the way to mitigate the adverse effects by potential implementation of adjunct therapy options.
Assuntos
Vasos Coronários , Doxorrubicina , Vasoconstrição , Animais , Masculino , Doxorrubicina/farmacologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasoconstrição/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/toxicidade , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Ratos Wistar , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/agonistas , Receptor 5-HT1B de Serotonina/metabolismo , Ratos , Receptor de Endotelina B/metabolismo , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/efeitos dos fármacos , Técnicas In Vitro , Receptor de Endotelina A/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Chemical hepatotoxicity, especially alcoholic liver injury (ALI), commonly occurs in young and middle-aged people who drink heavily. ALI is extremely harmful and can induce severe disease states, such as hepatitis, liver fibrosis, cirrhosis, or liver cancer, which are similar to CCl4-induced liver disease states in animals. In recent studies, the pathological changes of hepatocytes and the hepatic stellate cell have shown a significant connection between endoplasmic reticulum (ER) stress and the development of liver pathology in patients. However, the detailed pathological mechanism needs to be further studied. Schisandra chinensis, (S. chinensis), a fruit-bearing vine used in Traditional Chinese Medicine (TCM), has been used to treat chronic or acute diseases, including liver disease. S. chinensis-derived lignans (SCDLs) in particular have been shown to alleviate liver pathological changes. AIM OF THE STUDY: This study sought to elucidate the mechanisms underlying SCDL-mediated hepatoprotection. MATERIALS AND METHODS: We first used in silico target prediction and computational simulation methods to identify putative lignan-binding targets relative to the hepatoprotective effect. A gene microarray analysis was performed to identify differently expressed genes that might have significance in the disease pathological process. We then used histological analyses in a mice hepatotoxicity model to test the effectiveness of SCDLs in vivo, and a hepatocellular toxicity model to analyze the candidate-compound-mediated hepatoprotection and expression states of the key targets in vitro. RESULTS: The in silico analysis results indicated that endothelin receptor B (ETBR/EDNRB) is likely a significant node during the liver pathological change process and a promising key target for the SCDL compound schisantherin D on the hepatoprotective effect; experimental studies showed that schisantherin D alleviated the EtOH- and ET-1-induced HL-7702â¯cell (belongs to liver parenchymal cell lines) injury ratio, decreased the expression of ETBR, and inhibited ECMs and ET-1 secretion in LX-2â¯cells (one form of hepatic stellate cells). SCDLs ameliorated EtOH- and CCl4-induced fibrosis formation in mice liver tissue. Liver tissue western blots of SCDL-treated mice showed downregulated α-SMA, ETBR, PLCß, CHOP, Bax, and the apoptotic factors of cleaved-caspase 12, cleaved-caspase 9, and cleaved-caspase 3 hinted at an anti-apoptosis and hepatoprotective effect. The SCDL treatment also elevated serum glutathione (GSH) and reduced the serum-transforming growth factor-ß1 (TGF-ß1) level. CONCLUSION: The findings indicated that SCDLs prevent hepatotoxicity via their anti-fibrotic, anti-oxidant, and anti-apoptosis properties. ETBR may be the key factor in promoting chemical hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Lignanas/farmacologia , Hepatopatias Alcoólicas/prevenção & controle , Schisandra/química , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/patologia , Simulação por Computador , Modelos Animais de Doenças , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Lignanas/isolamento & purificação , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologia , Receptor de Endotelina B/efeitos dos fármacosRESUMO
AIMS: Although effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria. METHODS AND RESULTS: Male Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of â¼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan. CONCLUSIONS: Our results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury.
Assuntos
Albuminúria/prevenção & controle , Anti-Hipertensivos/farmacologia , Artérias/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Antagonistas do Receptor de Endotelina A/farmacologia , Antagonistas do Receptor de Endotelina B/farmacologia , Hipertensão/prevenção & controle , Albuminúria/induzido quimicamente , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Artérias/metabolismo , Artérias/fisiopatologia , Modelos Animais de Doenças , Epoprostenol/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Isoxazóis/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/fisiopatologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Pirimidinas/farmacologia , Ratos Endogâmicos WKY , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/metabolismo , Transdução de Sinais , Sulfonamidas/farmacologia , Sunitinibe , Tiofenos/farmacologiaRESUMO
INTRODUCTION: Vascular calcification (VC) is a complex, regulated process involved in many disease entities. So far, there are no treatments to reverse it. Exploring novel strategies to prevent VC is important and necessary for VC-related disease intervention. OBJECTIVE: In this study, we evaluated whether MOTS-c, a novel mitochondria-related 16-aa peptide, can reduce vitamin D3 and nicotine-induced VC in rats. METHODS: Vitamin D3 plus nicotine-treated rats were injected with MOTS-c at a dose of 5 mg/kg once a day for 4 weeks. Blood pressure, heart rate, and body weight were measured, and echocardiography was performed. The expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and the angiotensin II type 1 (AT-1) and endothelin B (ET-B) receptors was determined by Western blot analysis. RESULTS: Our results showed that MOTS-c treatment significantly attenuated VC. Furthermore, we found that the level of phosphorylated AMPK was increased and the expression levels of the AT-1 and ET-B receptors were decreased after MOTS-c treatment. CONCLUSIONS: Our findings provide evidence that MOTS-c may act as an inhibitor of VC by activating the AMPK signaling pathway and suppressing the expression of the AT-1 and ET-B receptors.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Mitocondriais/metabolismo , Calcificação Vascular/metabolismo , Animais , Colecalciferol/administração & dosagem , Colecalciferol/efeitos adversos , Colecalciferol/metabolismo , Masculino , Proteínas Mitocondriais/administração & dosagem , Proteínas Mitocondriais/efeitos adversos , Proteínas Mitocondriais/farmacologia , Modelos Animais , Nicotina/administração & dosagem , Nicotina/efeitos adversos , Nicotina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Calcificação Vascular/induzido quimicamente , Remodelação Ventricular/efeitos dos fármacosRESUMO
OBJECTIVE: We recently demonstrated ETBR mediate vasodilation in young but not postmenopausal women; it is unclear if this is related to age or a decline in ovarian hormones. The purpose of this study was to test the hypothesis that ETBR responses are modulated by ovarian hormones. METHODS: We measured cutaneous vasodilatory responses in 12 young women (22 ± 1 years, 23 ± 1 kg/m2 ) during the ML (days 20-25) and EF (days 2-5) phases of the menstrual cycle. Cutaneous microdialysis perfusions of lactated Ringer (control), ETBR antagonist (BQ-788, 300 nmol/L), and ETAR antagonist (BQ-123, 500 nmol/L) were performed, followed by local heating to 42°C. RESULTS: Serum estradiol (ML: 118 ± 16 vs EF: 44 ± 9 pg/mL, P < 0.05) and progesterone (ML: 8.3 ± 1.0 vs EF: 0.7 ± 0.2 ng/mL, P < 0.05) were higher during ML vs EF phase. ETBR blockade decreased vasodilation during ML (control: 91 ± 2 vs BQ-788: 83 ± 2%CVCmax, P < 0.05) but not EF (control: 89 ± 2 vs BQ-788: 89 ± 1%CVCmax). ETAR blockade also decreased vasodilation during ML (control: 91 ± 2 vs BQ-123: 87 ± 2%CVCmax, P < 0.05) but not EF (control: 89 ± 2 vs BQ-123: 92 ± 2%CVCmax). CONCLUSIONS: These data suggest that fluctuations in ovarian hormones modulate ETBR and ETAR responses in young women.
Assuntos
Hormônios/farmacologia , Ovário/metabolismo , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Estradiol/sangue , Estradiol/farmacologia , Feminino , Hormônios/sangue , Humanos , Progesterona/sangue , Progesterona/farmacologia , Pele/irrigação sanguínea , Vasodilatação , Adulto JovemRESUMO
INTRODUCTION: Endothelin-1 receptor (ET-1R)/ß-arrestin1 (ß-arr1) signaling is dysregulated in ovarian cancer. This signaling circuit enables cancer cells to engage several signaling and transcriptional networks that are pervasively intertwined, and represent a potential therapeutic target for developing novel agents for ovarian cancer treatment. Areas covered: In this article, we discuss the role of the signaling network between ET-1R and key pathways mediated by the scaffold protein ß-arr1, as part of signaling complex, or as a transcription co-activator, promoting precise control of transcription of different genes, including ET-1. Therefore ET-1R/ß-arr1 is an actionable node involved in the activation of a persistent feedback loop that contributes to bypass signaling. Targeting ET-1R empowering this circuit can represent a necessary measure to reach clinical efficacy. Preclinical studies demonstrate that blocking ET-1R by FDA approved dual ETAR/ETBR antagonist prevents ß-arr1 network formation, offering a novel therapeutic strategy in ovarian cancer patients. Expert opinion: The information provided in this review about the ET-1R/ß-arr1 hub represents an invaluable tool for both identifying the interconnected pathways involved in ovarian cancer and targeting them more effectively. The new perspective arising from ET-1R therapeutics will likely prompt a valuable frame for the design of new promising combinatorial therapy, blocking compensatory networks.
Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Neoplasias Ovarianas/tratamento farmacológico , Desenho de Fármacos , Feminino , Humanos , Neoplasias Ovarianas/patologia , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/metabolismo , Transdução de Sinais , beta-Arrestina 1/metabolismoRESUMO
Brain-blood barrier (BBB) disruption results in vasogenic edema, which is involved in the pathogenesis of epilepsy. Following status epilepticus (SE), up-regulated transient receptor potential canonical channel-3 (TRPC3), a Ca2+-permeable cation channels in endothelial cells, is relevant to vasogenic edema formation in the rat piriform cortex. In addition, pyrazole-3 (Pyr-3, a TRPC3 inhibitor) attenuated SE-induced vasogenic edema. However, the upstream regulators of TRPC3 expression in vasogenic edema formation have been unclear. In the present study, soluble tumor necrosis factor p55 receptor (sTNFp55R, a TNF-α inhibitor), SN50 (a nuclear factor-κB (NFκB) inhibitor), BQ-788 (an endothelin B (ETB) receptor inhibitor) and Pyr-3 effectively prevented vasogenic edema following SE. sTNFp55R and SN50 (but not BQ-788) attenuated SE-induced up-regulation of endothelial TRPC3 expression. Pyr-3 ameliorated SE-induced NFκB p65-Thr435 phosphorylation and ETB receptor expression. In addition, Pyr-3 mitigated NFκB p65-Thr435 phosphorylation induced by recombinant TNF-α. These findings indicate that TNF-α-mediated NFκB p65-Thr435 phosphorylation may up-regulate TRPC3 expression, which participates in vasogenic edema formation via increasing endothelial nitric oxide synthase expression following SE, independent of ETB receptor activation. Therefore, we suggest that TRPC3 may be involved in a positive feedback loop of NFκB/ETB receptor signaling pathway.
Assuntos
Edema Encefálico/metabolismo , Células Endoteliais/metabolismo , Estado Epiléptico/metabolismo , Canais de Cátion TRPC/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Edema Encefálico/patologia , Células Endoteliais/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Ratos Sprague-Dawley , Receptor de Endotelina B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológicoRESUMO
Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.
Assuntos
Butadienos/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilas/farmacologia , Receptor de Endotelina A/biossíntese , Receptor de Endotelina B/biossíntese , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Cotinina/urina , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Regulação para Cima , Vasoconstrição/efeitos dos fármacosRESUMO
AIMS: Vascular remodeling results from aberrations in the balance between cell proliferation and death, which is seen in the obstructive vasculature of pulmonary arterial hypertension (PAH). Endothelin (ET)-1 has a potent proliferative activity on vascular smooth muscle cells, and ET receptor inhibitors are used to treat PAH; however, it remains unclear whether ET receptor inhibition contributes to the apoptosis of pulmonary arterial smooth muscle cells (PASMCs), another cause of pulmonary vascular remodeling. MAIN METHODS: Cultured human PASMCs were treated with the ETA receptor antagonist BQ-123 (100µM), or the ETB antagonist A-192621 (1-100µM) or BQ-788 (1-100µM) for 48h. The cells were then incubated for another 24h with or without doxorubicin (DOX, 1µM), an anthracyclin antitumor antibiotic that promotes p53-mediated apoptosis. Cell viability and apoptosis were evaluated by MTT assays, caspase-3/7 activity assays, and Western blots for cleaved caspase-3 expression. KEY FINDINGS: The viability of PASMCs was significantly decreased by A-192621 and BQ-788, in a dose-dependent manner. A-192621 and BQ-788 significantly increased the caspase-3/7 activity and cleaved caspase-3 expression in PASMCs. The PASMCs' susceptibility to DOX-induced apoptosis was significantly higher in the presence of A-192621 and BQ-788 than with vehicle. However, BQ-123 did not affect these parameters. SIGNIFICANCE: Blockade of the ETB receptor increases the extent of apoptosis and susceptibility to DOX-induced apoptosis in PASMCs. Apoptosis caused by ETB receptor blockade in PASMCs may be one of the mechanisms by which vascular remodeling is reduced in ET receptor inhibitor-based PAH treatments.
Assuntos
Apoptose/efeitos dos fármacos , Antagonistas dos Receptores de Endotelina/farmacologia , Músculo Liso Vascular/citologia , Artéria Pulmonar/citologia , Receptor de Endotelina B/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Humanos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/enzimologia , Artéria Pulmonar/metabolismoRESUMO
In human cancers, the autocrine and paracrine loop mediated by the aberrantly activation of endothelin-1 (ET-1) receptor (ET-1R) elicits pleiotropic effects, preferentially mediated by the scaffold protein ß-arrestin 1 (ß-arr1), on tumor cells and on the host microenvironment, providing a strong rationale for targeting ET-1 receptors. This review describes the most up-to-date preclinical and clinical results obtained by using ET-1 therapeutics. The previous negative clinical results of ET-1 therapeutics should not prevent us from setting the standard of this class of drugs for future well-designed clinical trials. The preclinical data obtained with the dual ETAR and ETBR antagonist macitentan indicate that this molecule, which targets cancer cells and tumor-associated microenvironmental elements, could be a cancer therapeutic option. The field of ET-1 therapeutics will be improved in the next decade, facilitated by the new knowledge on the genomic landscape of the human stroma and tumor, and by the low invasive approaches based on liquid biopsies for the discovery of predictive biomarkers. The information obtained from preclinical studies in patient-derived models and from the Cancer Genome Atlas will set the scene of precision medicine for cancer. Results from these studies are expected to open the possibility that ET-1R antagonists might be more efficacious as molecular cancer therapeutics, able to hamper the functional ß-arr1-dependent signaling complexes, either alone or coupled with new targeted approaches.
Assuntos
Antineoplásicos/uso terapêutico , Endotelina-1/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Animais , Biomarcadores Tumorais , Humanos , Neoplasias/diagnósticoRESUMO
AKI is associated with high mortality rates and the development of CKD. Ischemia/reperfusion (IR) is an important cause of AKI. Unfortunately, there is no available pharmacologic approach to prevent or limit renal IR injury in common clinical practice. Renal IR is characterized by diminished nitric oxide bioavailability and reduced renal blood flow; however, the mechanisms leading to these alterations are poorly understood. In a rat model of renal IR, we investigated whether the administration of the novel nonsteroidal mineralocorticoid receptor (MR) antagonist BR-4628 can prevent or treat the renal dysfunction and tubular injury induced by IR. Renal injury induced by ischemia was associated with increased oxidant damage, which led to a cysteine sulfenic acid modification in endothelin B receptor and consequently decreased endothelial nitric oxide synthase activation. These modifications were efficiently prevented by nonsteroidal MR antagonism. Furthermore, we demonstrated that the protective effect of BR-4628 against IR was lost when a selective endothelin B receptor antagonist was coadministered. These data describe a new mechanism for reduced endothelial nitric oxide synthase activation during renal IR that can be blocked by MR antagonism with BR-4628.
Assuntos
Rim/irrigação sanguínea , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Receptor de Endotelina B/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Ácidos Sulfênicos/farmacologia , Animais , Cromonas/uso terapêutico , Di-Hidropiridinas/uso terapêutico , Masculino , Ratos , Ratos WistarRESUMO
Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential of ERA and warrant further investigation of their utility in chronic inflammatory airway diseases.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , RNA Mensageiro/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Anti-Hipertensivos/farmacologia , Bosentana , Células Cultivadas , Quimiocina CXCL2/biossíntese , Humanos , Metaloproteinase 12 da Matriz/biossíntese , Músculo Liso/patologia , Oligopeptídeos/farmacologia , Fenilpropionatos/farmacologia , Piperidinas/farmacologia , Piridazinas/farmacologia , Receptor de Endotelina A/efeitos dos fármacos , Sulfonamidas/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The clinical efficiency of the highly potent antitumor agent doxorubicin is limited by cardiotoxic effects. In a murine doxorubicin cardiotoxicity model, increased endothelin-1 (ET-1) expression and cardioprotective effects of the dual ET-1 blocker bosentan were demonstrated. To date it is unclear if combined blocking of endothelin A/B receptors is necessary or whether selective inhibition of one of the ET-1 receptors is sufficient for the observed cardioprotection. Therefore, we investigated the impact of dual (bosentan) and single endothelin receptor antagonism through sitaxentan (receptor A blocker) or BQ788 (receptor B blocker) in a murine doxorubicin cardiotoxicity model (C57BL/6N). Simultaneous administration of each endothelin receptor antagonist (ERA) with doxorubicin resulted in a significantly improved hemodynamic performance in comparison to the impaired cardiac function in control mice with bosentan being most effective but closely followed by sitaxentan and also BQ788. This cardioprotection was not caused by diminished doxorubicin levels in heart since the doxorubicin content in cardiac tissue was not altered by ERAs significantly. However, whole transcript expression profiling showed partly different effects of the ERAs on doxorubicin-modulated cardiac gene expression of genes involved in signal transduction (e.g. Stat3, Pim1, Akt1, Plcb2), fibrosis (e.g. Myl4), energy production (e.g. Ant1) or oxidative stress (e.g. Aox1). Furthermore, doxorubicin-mediated gene regulations were verified in the murine cardiomyocyte model HL-1 showing partly reversed expression patterns after co-administration of the ERAs. In summary, our results demonstrate strong cardioprotective effects of blocking ET-1 receptors against the doxorubicin-related cardiomyopathy and provide evidence to potential underlying signaling pathways.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/prevenção & controle , Cardiotônicos/farmacologia , Doxorrubicina/toxicidade , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Animais , Cardiomiopatias/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Expressions of VEGF-R1 and -R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF-R3 and Tie-2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF-R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF-E, a selective VEGF-R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF165 were reduced by selective antagonists for VEGF-R1 and -R2. Treatment with ET-1 increased VEGF-R1 mRNA and protein levels. The effects of ET-1 on VEGF-R1 mRNA were mimicked by Ala(1,3,11,15) -ET-1, a selective agonist for ETB receptors, and inhibited by BQ788, an ETB antagonist. ET-1 did not affect the mRNA levels of VEGF-R2, -R3, and Tie-2. Pre-treatment with ET-1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues. To clarify the regulatory mechanisms of vascular endothelial growth factor (VEGF) receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Effects of selective VEGF-R1 and R2 agonist showed that these receptors were linked to focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Treatment with ET-1 increased expression of VEGF-R1, which was mediated by ETB receptors. Pre-treatment with ET-1 potentiated the VEGF-R1-mediated activations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes.
Assuntos
Astrócitos/efeitos dos fármacos , Endotelina-1/farmacologia , Receptor de Endotelina B/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Western Blotting , Células Cultivadas , Quinase 1 de Adesão Focal/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Therapies targeting the mitogen-activated protein (MAP) kinase pathway in melanoma have produced significant clinical responses; however, duration of response is limited by acquisition of drug resistance. Rational drug combinations may improve outcomes in this setting. We assessed the therapeutic combination of an antibody-drug conjugate (ADC) targeting the endothelin B receptor (EDNRB) with small-molecule inhibitors of the MAP kinase signaling pathway in melanoma. Cell lines and tumor models containing either mutant BRAF or NRAS, or wild-type for both, were exposed to small-molecule inhibitors of BRAF and MEK. Expression of EDNRB was analyzed and the therapeutic impact of combining the anti-EDNRB ADC with the BRAF and MEK inhibitors was assessed. Increased expression of EDNRB in response to inhibition of BRAF and/or MEK was observed and augmented the antitumor activity of the ADC. Enhanced target expression and ADC antitumor activity were realized irrespective of the response of the tumor model to the BRAF or MEK inhibitors alone and could be achieved in melanoma with mutant NRAS, BRAF, or neither mutation. Cells that acquired resistance to BRAF inhibition through long-term culture retained drug-induced elevated levels of EDNRB expression. Expression of EDNRB was not enhanced in normal human melanocytes by inhibition of BRAF and the combination of the ADC with MAPK inhibitors was well-tolerated in mice. The anti-EDNRB ADC combines well with BRAF and MEK inhibitors and could have therapeutic use in the majority of human melanoma cases.
Assuntos
Antagonistas do Receptor de Endotelina B/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/genética , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Receptor de Endotelina B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/regulation of endothelin-1 (ET-1) to its receptor subtypes A (ETAR) and B (ETBR) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [(125)I]-ET-1 was employed to estimate ET-1 binding affinity (τ = 1/K-n) to ETAR and ETBR at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of ETAR and ETBR were detected using Western blot. No significance differences were detected in the τ of ETAR and ETBR between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the τ value for ETAR and ETBR on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, ETAR and ETBR densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated ETAR and down-regulated ETBR band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus.
Assuntos
Amidas/farmacologia , Vasos Coronários/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Endotelina-1/metabolismo , Fumaratos/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Peptídeos/farmacologia , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Peçonhas/farmacologia , Animais , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/prevenção & controle , Vasos Coronários/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Exenatida , Masculino , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Estreptozocina , Fatores de TempoRESUMO
In coronary artery diseases, cigarette smoking is a risk factor and the endothelin system plays a key role in the pathogenesis. This study was to examine if dimethylsulfoxide-soluble smoke particles (DSP) upregulate endothelin type-B (ETB) receptors in the coronary artery and investigate the mechanism. The isolated rat coronary arteries were organ-cultured for 24 h. The contractile response of the coronary artery was recorded by myograph. The mRNA and protein expression of the ETB receptors was studied using quantitative real-time PCR and immunohistochemistry. Results showed that the ETB receptor agonist, sarafotoxin 6c, induced a weak contraction in the fresh coronary artery. After culture, the contraction curve mediated by ETB receptor was shifted towards the left with an increased Emax of 152 ± 12%. DSP of 0.2 and 0.4 µl/ml shifted the concentration-contractile curves towards the left with further increased Emax of 270 ± 26 and 280 ± 29%, respectively. The culture increased ETB receptor mRNA and protein levels from fresh arteries, which was further enhanced by DSP. PD98059 (ERK1/2 inhibitor), wedelolactone (NF-κB inhibitor), actinomycin D or cycloheximide significantly inhibited the DSP-enhanced contraction and expression of mRNA and protein of the ETB receptor. However, SB203580 (p38 inhibitor) further increased DSP-enhanced contraction and protein expression of the ETB receptor. The results indicate that DSP upregulates ETB receptors in rat coronary artery via ERK1/2 and the NF-κB pathway.
Assuntos
Vasos Coronários/metabolismo , Dimetil Sulfóxido , Nicotiana , Receptor de Endotelina B/genética , Fumaça/análise , Animais , Vasos Coronários/química , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Feminino , Flavonoides/farmacologia , Expressão Gênica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Técnicas de Cultura de Órgãos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina B/análise , Receptor de Endotelina B/efeitos dos fármacos , Fumaça/efeitos adversos , Solubilidade , Vasoconstritores/farmacologia , Venenos de Víboras/farmacologiaRESUMO
AIMS: The involvement of endothelin receptors in the contraction of the lower urinary tract smooth muscle is well established. There is scarce information, however, about endothelin receptors mediating relaxation of the bladder outlet region. The current study investigates the possible existence of endothelin ET(B) receptors involved in the relaxation of pig bladder neck. METHODS: ET(B) receptor expression was determined by immunohistochemistry and urothelium-denuded bladder neck strips were mounted in organ baths for isometric force recording. RESULTS: ET(B) -immunoreactivity (ET(B) -IR) was observed within nerve fibers among smooth muscle bundles and urothelium. BQ3020 (0.01-300 nM), an ET(B) receptor agonist, produced concentration-dependent relaxations which were reduced by BQ788, an ET(B) receptor antagonist, and by inhibitors of protein kinase A (PKA) and large (BK(Ca) )- or small (SK(Ca) )-conductance Ca(2+) -activated K(+) channels. Pretreatment with BK(Ca) or SK(Ca) channel inhibitors plus PKA blocking did not cause further inhibition compared with that exerted by inhibiting BK(Ca) or SK(Ca) channels only. BQ3020-induced relaxation was not modified by blockade of either nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX) or of intermediate-conductance Ca(2+) -activated-(IK(Ca) ), ATP-dependent-(K(ATP) ), or voltage-gated-(K(v) ) K(+) channels. Under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation (0.5-16 Hz) evoked frequency-dependent relaxations, which were reduced by BQ788 and potentiated by threshold concentrations of BQ3020. CONCLUSIONS: These results suggest that BQ3020 produces relaxation of the pig bladder neck via activation of muscle endothelin ET(B) receptors, NO/cGMP- and COX-independent-, cAMP-PKA pathway-dependent-mechanisms, and involving BK(Ca) and SK(Ca) channel activation. ET(B) receptors are also involved in the NANC inhibitory neurotransmission.
Assuntos
Relaxamento Muscular , Músculo Liso/metabolismo , Receptor de Endotelina B/metabolismo , Bexiga Urinária/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Endotelinas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Fibras Nervosas/metabolismo , Neurotransmissores/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Piperidinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Guanilil Ciclase Solúvel , Suínos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/inervação , Urotélio/metabolismoRESUMO
We have previously reported that endothelin-1 and -3 modulate different steps of noradrenergic transmission in the hypothalamus. We showed that endothelins modify neuronal norepinephrine transport activity through the regulation of the kinetic constant and internalization. In the present work we sought to define the endothelin receptors and intracellular mechanisms involved in the down-regulation of neuronal norepinephrine uptake induced by endothelin-1 and -3 in the rat posterior hypothalamic region. Results showed that endothelin-1 reduced norepinephrine uptake through ET(B) receptors, whereas endothelin-3 through a non-conventional or atypical endothelin receptor. In both cases, the effect on norepinephrine uptake was coupled to protein kinase A and C as well as nitric oxide pathways. However, neither protein kinase G nor intracellular or extracellular calcium and calcium/calmodulin-dependent protein kinase II were involved. In addition, the same intracellular mechanisms participated in the reduction of nisoxetine binding (norepinephrine transporter internalization index) induced by both endothelins. Present findings reveal the underlying mechanisms involved in the regulation of the neuronal norepinephrine transporter by endothelins and further support the role of these peptides in the modulation of noradrenergic transmission at the presynaptic nerve endings in the posterior hypothalamus.
Assuntos
Endotelina-1/farmacologia , Endotelina-3/farmacologia , Hipotálamo Posterior/metabolismo , Neurônios/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fluoxetina/análogos & derivados , Fluoxetina/metabolismo , Hipotálamo Posterior/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Norepinefrina/metabolismo , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/metabolismoRESUMO
BACKGROUND: We investigated the contractile function in responses to endothelin-1 (ET-1) in the human coronary microvasculature as well as the roles of endothelin receptors and protein kinase C-alpha (PKC-alpha) in these responses. METHODS: Human atrial tissue was harvested from patients who underwent cardiac surgery pre- and post-cardioplegia (CP)/cardiopulmanory bypass (CPB). Microvascular constriction was assessed in pre- and post-CP/CPB samples in responses to ET-1, in the presence and absence of an endothelin-A (ET-A) receptor antagonist, an endothelin-B (ET-B) receptor antagonist, or a PKC-alpha inhibitor, respectively. The expression and localization of the ET-A and ET-B receptors were also examined using immunoblot and immunofluorescence photomicroscopy. RESULTS: The post-CP/CPB contractile response of coronary arterioles to ET-1 was significantly decreased compared with the pre-CP/CPB responses. The response to ET-1 was significantly inhibited in the presence of the ET-A antagonist BQ123 (10(-7)mol/L), but these values remained unchanged with the ET-B receptor antagonist BQ788 (10(-7)mol/L). Pretreatment with the PKC-alpha inhibitor safingol (2.5 x 10(-5) mol/L) reversed the ET-1 responses from contraction into relaxation. The total polypeptide levels of ET-A and ET-B receptors were not altered post-CP/CPB. Immunoblot and immunofluorescent staining displayed strong signals for ET-A receptors and relatively weak signals for ET-B receptors localized on coronary microvasculature. CONCLUSION: CP/CPB decreases the contractile function of human coronary microvessels in responses to ET-1. ET-A receptors are predominantly localized in the human coronary microcirculation, whereas ET-B receptors seem to be less abundant. The contractile response to ET-1 is in part through the activation of ET-A receptors and PKC-alpha. These results suggest a role of ET-1-induced contraction in the vasomotor dysfunction after cardiac surgery.