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1.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540675

RESUMO

Restraint stress causes various maternal diseases during pregnancy. ß2-Adrenergic receptor (ß2-AR) and Forkhead transcription factor class O 1 (FOXO1) are critical factors not only in stress, but also in reproduction. However, the role of FOXO1 in restraint stress, causing changes in the ß2-AR pathway in pregnant mice, has been unclear. The aim of this research was to investigate the ß2-AR pathway of restraint stress and its impact on the oxidative stress of the maternal uterus. In the study, maternal mice were treated with restraint stress by being restrained in a transparent and ventilated device before sacrifice on Pregnancy Day 5 (P5), Pregnancy Day 10 (P10), Pregnancy Day 15 (P15), and Pregnancy Day 20 (P20) as well as on Non-Pregnancy Day 5 (NP5). Restraint stress augmented blood corticosterone (CORT), norepinephrine (NE), and blood glucose levels, while oestradiol (E2) levels decreased. Moreover, restraint stress increased the mRNA levels of the FOXO family, ß2-AR, and even the protein levels of FOXO1 and ß2-AR in the uterus and ovaries. Furthermore, restraint stress increased uterine oxidative stress level. In vitro, the protein levels of FOXO1 were also obviously increased when ß2-AR was activated in endometrial stromal cells (ESCs). In addition, phosphorylated-nuclear factor kappa-B p65 (p-NF-κB p65) and its target genes decreased significantly when FOXO1 was inhibited. Overall, it can be said that the ß2-AR/FOXO1/p-NF-κB p65 pathway was activated when pregnant mice were under restraint stress. This study provides a scientific basis for the origin of psychological stress in pregnant women.


Assuntos
Endométrio/metabolismo , Proteína Forkhead Box O1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Animais , Corticosterona/sangue , Endométrio/citologia , Estradiol/sangue , Feminino , Camundongos , Norepinefrina/sangue , Gravidez , Restrição Física/efeitos adversos , Células Estromais/metabolismo
2.
Am J Physiol Endocrinol Metab ; 320(3): E619-E628, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33522400

RESUMO

Prolonged supplementation with the ß2-agonist clenbuterol improves glucose homeostasis in diabetic rodents, likely via ß2-adrenoceptor (ß2-AR)-mediated effects in the skeletal muscle and liver. However, since rodents have, in contrast to-especially diabetic-humans, substantial quantities of brown adipose tissue (BAT) and clenbuterol has affinity to ß1- and ß3-ARs, the contribution of BAT to these improvements is unclear. Therefore, we investigated clenbuterol-mediated improvements in glucose homeostasis in uncoupling protein 1-deficient (UCP1-/-) mice, lacking thermogenic BAT, versus wild-type (WT) mice. Anesthetized WT and UCP1-/- C57Bl/6 mice were injected with saline or clenbuterol and whole body oxygen consumption was measured. Furthermore, male WT and UCP1-/- C57Bl/6 mice were subjected to 17-wk of chow feeding, high-fat feeding, or high-fat feeding with clenbuterol treatment between weeks 13 and 17. Body composition was measured weekly with MRI. Oral glucose tolerance and insulin tolerance tests were performed in week 15 and 17, respectively. Clenbuterol increased oxygen consumption approximately twofold in WT mice. This increase was blunted in UCP1-/- mice, indicating clenbuterol-mediated activation of BAT thermogenesis. High-fat feeding induced diabetogenic phenotypes in both genotypes. However, low-dose clenbuterol treatment for 2 wk significantly reduced fasting blood glucose by 12.9% in WT and 14.8% in UCP1-/- mice. Clenbuterol treatment improved glucose and insulin tolerance in both genotypes compared with HFD controls and normalized to chow-fed control mice independent of body mass and composition alterations. Clenbuterol improved whole body glucose homeostasis independent of UCP1. Given the low human abundancy of BAT, ß2-AR agonist treatment provides a potential novel route for glucose disposal in diabetic humans.NEW & NOTEWORTHY Improvements in whole body glucose homeostasis of rodents upon prolonged ß2-adrenergic agonist supplementation could potentially be attributed to UCP1-mediated BAT thermogenesis. Indeed, we show that acute injection with the ß2-AR agonist clenbuterol induces BAT activation in mice. However, we also demonstrate that prolonged clenbuterol supplementation robustly improves whole body glucose and insulin tolerance in a similar way in both DIO WT and UCP1-/- mice, indicating that ß2-AR agonist supplementation improves whole body glucose homeostasis independent of UCP1-mediated BAT thermogenesis.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Glucose/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Proteína Desacopladora 1/genética , Tecido Adiposo Marrom/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Clembuterol/administração & dosagem , Clembuterol/farmacologia , Dieta Hiperlipídica , Esquema de Medicação , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Homeostase/genética , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/etiologia , Obesidade/patologia , Receptores Adrenérgicos beta 2/metabolismo , Termogênese/efeitos dos fármacos , Termogênese/genética , Fatores de Tempo , Proteína Desacopladora 1/deficiência
3.
Mol Carcinog ; 60(3): 172-178, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33482042

RESUMO

Although beta 2 adrenergic receptors (ß2 ADR) are present in the keratinocytes, their role in cutaneous squamous cell tumorigenesis needs to be ascertained. For the first time, we report here that selective ß2 ADR antagonists by inhibiting ß2 ADR actions significantly retarded the progression of ultraviolet B (UVB) induced premalignant cutaneous squamous cell lesions. These antagonists acted by inhibiting vascular endothelial growth factor-A (VEGF) mediated angiogenesis to prevent UVB radiation-induced squamous cell carcinoma of the skin.


Assuntos
Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Neoplasias de Células Escamosas/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Raios Ultravioleta/efeitos adversos , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Butoxamina/farmacologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Masculino , Camundongos Endogâmicos , Neoplasias Induzidas por Radiação/irrigação sanguínea , Neoplasias Induzidas por Radiação/tratamento farmacológico , Neoplasias Induzidas por Radiação/etiologia , Neoplasias de Células Escamosas/irrigação sanguínea , Neoplasias de Células Escamosas/etiologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/etiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Xamoterol/farmacologia
4.
Anal Chem ; 93(4): 2010-2017, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33400862

RESUMO

G protein-coupled receptor (GPCR) is activated by extracellular signals. After their function at plasma membrane, GPCRs are internalized to be desensitized, while emerging evidence suggests that some GPCRs maintain their activity even after internalization. The endosomal trafficking pathway of a prototypic GPCR, ß adrenergic receptor 2 (B2AR), is in the range of several hours, however, spatiotemporal B2AR activity during this long-term endosomal trafficking pathway has not been characterized yet. Here, we analyze an agonist-induced real-time B2AR activity and its downstream function at the level of individual vesicles, utilizing a fluorescence resonance energy transfer (FRET)-based B2AR biosensor and cAMP reporters tethered at different trafficking stages of endosomes. Our results report that the internalized B2ARs sustain the activity and maintain the production of cAMP for several hours during the endosomal trafficking pathway. Temporal kinetics of B2AR activity is mathematically well explained by our active-vesicle population model modified from the Ricker model. Therefore, our GPCR monitoring system and a new kinetics model can be applied to understand the spatiotemporal GPCR activity and its downstream function during the endosomal trafficking pathway.


Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais , AMP Cíclico , Endossomos , Células HEK293 , Humanos , Isoproterenol/farmacologia , Plasmídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão , Análise Espaço-Temporal
5.
Am J Physiol Heart Circ Physiol ; 320(2): H867-H880, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33356961

RESUMO

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.


Assuntos
Lesões das Artérias Carótidas/prevenção & controle , Proliferação de Células , Células Progenitoras Endoteliais/metabolismo , Hipertensão/metabolismo , Reepitelização , Receptores Adrenérgicos beta 2/metabolismo , Animais , Apoptose , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Estudos de Casos e Controles , Caspase 3/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Células Progenitoras Endoteliais/patologia , Células Progenitoras Endoteliais/transplante , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipertensão/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
J Chromatogr A ; 1637: 461835, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33383241

RESUMO

High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (ß2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that ß2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to ß2-AR were calculated to be 2.02 × 104 M-1, 1.15 × 104 M-1, 1.75 × 104 M-1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.


Assuntos
Albuterol/metabolismo , Cromatografia em Papel/métodos , Receptores Adrenérgicos beta 2/metabolismo , Terbutalina/análogos & derivados , Terbutalina/metabolismo , Adsorção , Interações Medicamentosas , Proteínas de Ligação ao GTP/metabolismo , Ligantes
7.
Nat Commun ; 11(1): 4857, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978402

RESUMO

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.


Assuntos
Arrestina/química , Arrestina/genética , Arrestina/metabolismo , Ligantes , Espectroscopia de Prótons por Ressonância Magnética/métodos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fenilalanina , Ligação Proteica , Conformação Proteica , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , beta-Arrestina 1/química , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(37): 23096-23105, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868434

RESUMO

The ß2-adrenergic receptor (ß2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the ß2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the ß2AR-Gs complex revealed the interaction interface of ß2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549-555 (2011)], yet, the dynamic process of the ß2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the ß2AR in response to the full agonist BI-167107 and Gs and Gi1 These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the ß2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the ß2AR-Gs and the ß2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs These differences between the ß2AR-Gs and ß2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Benzoxazinas/farmacologia , Sítios de Ligação/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
9.
Nat Commun ; 11(1): 3160, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572026

RESUMO

Heterotrimeric G proteins are categorized into four main families based on their function and sequence, Gs, Gi/o, Gq/11, and G12/13. One receptor can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. However, the precise mechanism underlying different coupling efficiencies is unknown. Here, we study the structural mechanism underlying primary and secondary Gi/o coupling, using the muscarinic acetylcholine receptor type 2 (M2R) as the primary Gi/o-coupling receptor and the ß2-adrenergic receptor (ß2AR, which primarily couples to Gs) as the secondary Gi/o-coupling receptor. Hydrogen/deuterium exchange mass spectrometry and mutagenesis studies reveal that the engagement of the distal C-terminus of Gαi/o with the receptor differentiates primary and secondary Gi/o couplings. This study suggests that the conserved hydrophobic residue within the intracellular loop 2 of the receptor (residue 34.51) is not critical for primary Gi/o-coupling; however, it might be important for secondary Gi/o-coupling.


Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas-G , Animais , Sítios de Ligação , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Estrutura Molecular , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais/fisiologia
10.
Cardiovasc Ther ; 2020: 3480276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32565909

RESUMO

Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an "ingredient-gene-path" network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P < 0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P < 0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P < 0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Potenciais de Ação/genética , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Frequência Cardíaca/genética , Masculino , Medicina Tradicional Chinesa , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Mapas de Interação de Proteínas , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Suínos , Porco Miniatura , Fatores de Tempo
11.
Nat Chem Biol ; 16(7): 749-755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483378

RESUMO

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Antagonistas Adrenérgicos beta/química , Alprenolol/química , Norepinefrina/química , Receptores Adrenérgicos beta 2/química , Xinafoato de Salmeterol/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Regulação Alostérica , Sítio Alostérico , Alprenolol/farmacologia , Células HEK293 , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Norepinefrina/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores Adrenérgicos beta 2/metabolismo , Xinafoato de Salmeterol/farmacologia , Termodinâmica , Água/química
12.
J Chromatogr A ; 1622: 461091, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376022

RESUMO

Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis and in vivo biological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a ß2-adrenergic receptor (ß2-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized ß2-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized ß2-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/isolamento & purificação , Agonistas Adrenérgicos beta/metabolismo , Sítios de Ligação , Interações Medicamentosas , Ligantes , Receptores Adrenérgicos beta 2/metabolismo
13.
Sci Rep ; 10(1): 7101, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345990

RESUMO

Alveolar ß2-receptor blockade worsens lung diffusion in heart failure (HF). This effect could be mitigated by stimulating alveolar ß2-receptors. We investigated the safety and the effects of indacaterol on lung diffusion, lung mechanics, sleep respiratory behavior, cardiac rhythm, welfare, and exercise performance in HF patients treated with a selective (bisoprolol) or a non-selective (carvedilol) ß-blocker. Study procedures were performed before and after indacaterol and placebo treatments according to a cross-over, randomized, double-blind protocol in forty-four patients (27 on bisoprolol and 17 on carvedilol). No differences between indacaterol and placebo were observed in the whole population except for a significantly higher VE/VCO2 slope and lower maximal PETCO2 during exercise with indacaterol, entirely due to the difference in the bisoprolol group (VE/VCO2 31.8 ± 5.9 vs. 28.5 ± 5.6, p < 0.0001 and maximal PETCO2 36.7 ± 5.5 vs. 37.7 ± 5.8 mmHg, p < 0.02 with indacaterol and placebo, respectively). In carvedilol, indacaterol was associated with a higher peak heart rate (119 ± 34 vs. 113 ± 30 bpm, with indacaterol and placebo) and a lower prevalence of hypopnea during sleep (3.8 [0.0;6.3] vs. 5.8 [2.9;10.5] events/hour, with indacaterol and placebo). Inhaled indacaterol is well tolerated in HF patients, it does not influence lung diffusion, and, in bisoprolol, it increases ventilation response to exercise.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Antagonistas Adrenérgicos beta/administração & dosagem , Bisoprolol/administração & dosagem , Carvedilol/administração & dosagem , Insuficiência Cardíaca/tratamento farmacológico , Indanos/administração & dosagem , Quinolonas/administração & dosagem , Agonistas de Receptores Adrenérgicos beta 2/efeitos adversos , Antagonistas Adrenérgicos beta/efeitos adversos , Idoso , Bisoprolol/efeitos adversos , Carvedilol/efeitos adversos , Estudos Cross-Over , Método Duplo-Cego , Feminino , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Indanos/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Quinolonas/efeitos adversos , Receptores Adrenérgicos beta 2/metabolismo
14.
Nat Commun ; 11(1): 1821, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286326

RESUMO

The abscopal effect following ionizing radiation therapy (RT) is considered to be a rare event. This effect does occur more frequently when combined with other therapies, including immunotherapy. Here we demonstrate that the frequency of abscopal events following RT alone is highly dependent upon the degree of adrenergic stress in the tumor-bearing host. Using a combination of physiologic, pharmacologic and genetic strategies, we observe improvements in the control of both irradiated and non-irradiated distant tumors, including metastatic tumors, when adrenergic stress or signaling through ß-adrenergic receptor is reduced. Further, we observe cellular and molecular evidence of improved, antigen-specific, anti-tumor immune responses which also depend upon T cell egress from draining lymph nodes. These data suggest that blockade of ß2 adrenergic stress signaling could be a useful, safe, and feasible strategy to improve efficacy in cancer patients undergoing radiation therapy.


Assuntos
Adrenérgicos/farmacologia , Imunidade , Neoplasias/imunologia , Neoplasias/radioterapia , Radiação Ionizante , Estresse Fisiológico , Antagonistas Adrenérgicos beta/farmacologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfonodos/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
PLoS One ; 15(4): e0228121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236094

RESUMO

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and ß-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.


Assuntos
Transdução de Sinal Luminoso/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Opsinas de Bastonetes/metabolismo , beta-Arrestina 1/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosforilação/fisiologia , Ligação Proteica , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes de Fusão/genética , Opsinas de Bastonetes/química , Opsinas de Bastonetes/genética , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , beta-Arrestina 1/química
16.
Biol Pharm Bull ; 43(4): 731-735, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32238715

RESUMO

Alzheimer's disease (AD) is characterized by the formation of extracellular amyloid plaques containing the amyloid ß-protein (Aß) within the parenchyma of the brain. Aß is considered to be the key pathogenic factor of AD. Recently, we showed that Angiotensin II type 1 receptor (AT1R), which regulates blood pressure, is involved in Aß production, and that telmisartan (Telm), which is an angiotensin II receptor blocker (ARB), increased Aß production via AT1R. However, the precise mechanism underlying how AT1R is involved in Aß production is unknown. Interestingly, AT1R, a G protein-coupled receptor, was strongly suggested to be involved in signal transduction by heterodimerization with ß2-adrenergic receptor (ß2-AR), which is also shown to be involved in Aß generation. Therefore, in this study, we aimed to clarify whether the interaction between AT1R and ß2-AR is involved in the regulation of Aß production. To address this, we analyzed whether the increase in Aß production by Telm treatment is affected by ß-AR antagonist using fibroblasts overexpressing amyloid precursor protein (APP). We found that the increase in Aß production by Telm treatment was decreased by the treatment of ß2-AR selective antagonist ICI-118551 more strongly than the treatment of ß1-AR selective antagonists. Furthermore, deficiency of AT1R abolished the effect of ß2-AR antagonist on the stimulation of Aß production caused by Telm. Taken together, the interaction between AT1R and ß2-AR is likely to be involved in Aß production.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Atenolol/farmacologia , Bisoprolol/farmacologia , Células Cultivadas , Camundongos Endogâmicos C57BL , Propanolaminas/farmacologia , Propranolol/farmacologia , Telmisartan/farmacologia
17.
Arch Oral Biol ; 113: 104712, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234582

RESUMO

OBJECTIVE: Chronic stress hormone norepinephrine (NE) has been previously reported to play a role in the development of cancer, but the correlation between NE and oral squamous cell carcinoma (OSCC) progression is not well understood. METHOD: To address this, the expression of adrenergic receptors (ARs) in human OSCC cell lines and clinic OSCC samples was detected, and the role of NEin vivo and in vitro was further investigated. RESULTS: It was found that ß2-AR was the main AR of NE in OSCC. Stimulation of OSCC cells with NE significantly increased the OSCC proliferation and invasion, which was, however, blocked by ß2-AR inhibitor. NE could induce the phosphorylation of extracellular regulated protein kinases (ERK) and cAMP-response element binding protein (CREB). Inhibition of ERK and CREB pathway abrogated NE-induced OSCC invasion and proliferation. NE could enhance cancer stem cells (CSCs)-like phenotype and up-regulate the expression of stemness marker. In tumor-bearing nude mice, it was found that consecutive administration of NE significantly promoted the tumor growth, while daily injection of ß2-AR inhibitor blocked this phenomenon. CONCLUSIONS: Those findings indicated a critical role of the chronic stress hormone NE in OSCC progression. Inhibition of ß2-AR may serve as a potential therapeutic strategy for protecting OSCC patients from chronic stress related deleterious effect.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Norepinefrina/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2 , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , MAP Quinases Reguladas por Sinal Extracelular , Humanos , Camundongos , Camundongos Nus , Transdução de Sinais
18.
Biol Pharm Bull ; 43(3): 493-502, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115508

RESUMO

The ß-adrenoceptor (ß-AR)-mediated pharmacological effects of catecholamine (CA) metabolites are not well known. We examined the effects of seven CA metabolites on smooth muscle relaxation in mouse and guinea pig (GP) tracheas and rat thoracic aorta. Among them, metadrenaline (MA) significantly relaxed GP trachea (ß2-AR dominant), even in the presence of clorgiline, a monoamine oxidase-A inhibitor. In mouse trachea (ß1-AR dominant), normetadrenaline (NMA) and MA (10-4 M each) apparently did not affect isoprenaline (ISO)-induced relaxation, but significantly inhibited it in the presence of clorgiline. ISO-induced relaxation was also unaffected by 3,4-dihydroxyphenylglycol (DHPG) (10-4 M), but significant suppression was observed with the addition of 3,5-dinitrocatechol, a catechol-O-methyltransferase inhibitor. In GP trachea, NMA, MA, 3,4-dihydroxymandelic acid (DOMA), and DHPG (10-4 M each) significantly augmented ISO-induced relaxation. However, in the presence of clorgiline plus 3,5-dinitrocatechol, both NMA and MA (10-4 M) significantly suppressed ISO-induced relaxation. DHPG (10-4 M) also significantly suppressed ISO-induced relaxation in the presence of 3,5-dinitrocatechol. In rat thoracic aorta, DHPG (10-4 M) significantly suppressed relaxation induced by CGP-12177 A (a ß3-AR partial agonist) in the presence of 3,5-dinitrocatechol plus propranolol. Our findings indicate that 1) MA may possess ß2-AR agonistic action; 2) NMA and MA augment ß2-AR-mediated tracheal relaxation in the absence of CA metabolic inhibitors, though themselves possessing ß1-, ß2-AR antagonistic action (ß2 > ß1); 3) DHPG exhibits ß1-, ß2-, ß3-AR antagonistic action, and this is particularly marked for ß3-AR. Our observations may help explain some of the pathologies associated with pheochromocytoma, which is characterized by increased CA metabolite levels.


Assuntos
Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Aorta/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Animais , Carbacol/farmacologia , Cobaias , Isoproterenol/farmacologia , Masculino , Camundongos , Propranolol/farmacologia , Ratos , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 2/metabolismo
19.
Invest Ophthalmol Vis Sci ; 61(3): 26, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32182332

RESUMO

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway. Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock. Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. ß2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm. Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.


Assuntos
Ritmo Circadiano/fisiologia , Corticosterona/farmacologia , Pressão Intraocular/fisiologia , Norepinefrina/farmacologia , Sistema Nervoso Simpático/fisiologia , Fatores de Transcrição ARNTL/metabolismo , Administração Oftálmica , Adrenalectomia , Animais , Células Cultivadas , Corpo Ciliar/efeitos dos fármacos , Corpo Ciliar/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ganglionectomia , Imuno-Histoquímica , Iris/efeitos dos fármacos , Iris/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Circadianas Period/metabolismo , Fotoperíodo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glucocorticoides/metabolismo , Gânglio Cervical Superior/cirurgia , Tonometria Ocular
20.
Int J Clin Oncol ; 25(6): 1137-1144, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32185627

RESUMO

PURPOSE: The ß2-adrenergic receptor (ß2AR) is highly expressed in various human cancers. The prognostic significance of its expression in patients with colorectal cancer (CRC) remains unclear. The aim of this study was to assess the prognostic role of ß2AR expression in patients with surgically resected CRC. METHODS: One hundred and forty-seven patients with surgically resected CRC were examined using immunohistochemistry. The expression of ß2AR was assessed in the specimens of resected primary tumors. RESULTS: ß2AR was expressed in 52.3% of the patients' tumors. ß2AR expression was significantly associated with T factor, N factor, and tumor cell proliferation (Ki-67 labeling index). Univariate analysis demonstrated that T factor, N factor, tumor stage, lymphatic permeation, vascular invasion, perineural invasion, ß2AR expression, and Ki-67 labeling index were significant prognostic factors for worse disease-free survival (DFS); all but T factor were also significant predictors for worse overall survival (OS). Multivariate analysis confirmed that expression of ß2AR was a significant prognostic marker for predicting worse DFS and OS. CONCLUSION: ß2AR expression was identified as a significant independent prognostic factor in patients with surgically resected CRC.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Receptores Adrenérgicos beta 2/metabolismo , Idoso , Proliferação de Células , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico
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