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1.
J Cell Biol ; 223(5)2024 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-38451221

RESUMO

Polycomb repressive complexes regulate developmental gene programs, promote DNA damage repair, and mediate pericentromeric satellite repeat repression. Expression of pericentromeric satellite repeats has been implicated in several cancers and diseases, including facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4-mediated transcription of HSATII regions causes nuclear foci formation of KDM2A/B-PRC1 complexes, resulting in a global loss of PRC1-mediated monoubiquitination of histone H2A. Loss of PRC1-ubiquitin signaling severely impacts DNA damage response. Our data implicate DUX4-activation of HSATII and sequestration of KDM2A/B-PRC1 complexes as a mechanism of regulating epigenetic and DNA repair pathways.


Assuntos
Reparo do DNA , Proteínas de Homeodomínio , Complexos Multiproteicos , Núcleo Celular/genética , Epigenômica , Histonas/genética , Humanos , Proteínas F-Box/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Complexos Multiproteicos/metabolismo
2.
Nat Commun ; 15(1): 866, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38286805

RESUMO

Homologous recombination (HR) plays critical roles in repairing lesions that arise during DNA replication and is thus essential for viability. RAD51 plays important roles during replication and HR, however, how RAD51 is regulated downstream of nucleofilament formation and how the varied RAD51 functions are regulated is not clear. We have investigated the protein c1orf112/FLIP that previously scored in genome-wide screens for mediators of DNA inter-strand crosslink (ICL) repair. Upon ICL agent exposure, FLIP loss leads to marked cell death, elevated chromosomal instability, increased micronuclei formation, altered cell cycle progression and increased DNA damage signaling. FLIP is recruited to damage foci and forms a complex with FIGNL1. Both proteins have epistatic roles in ICL repair, forming a stable complex. Mechanistically, FLIP loss leads to increased RAD51 amounts and foci on chromatin both with or without exogenous DNA damage, defective replication fork progression and reduced HR competency. We posit that FLIP is essential for limiting RAD51 levels on chromatin in the absence of damage and for RAD51 dissociation from nucleofilaments to properly complete HR. Failure to do so leads to replication slowing and inability to complete repair.


Assuntos
Cromatina , Replicação do DNA , Dano ao DNA , Reparo do DNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Humanos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo
3.
bioRxiv ; 2023 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-37808755

RESUMO

Homologous recombination (HR) plays critical roles in repairing lesions that arise during DNA replication and is thus essential for viability. RAD51 plays important roles during replication and HR, however, how RAD51 is regulated downstream of nucleofilament formation and how the varied RAD51 functions are regulated is not clear. We have investigated the poorly characterized protein c1orf112/RADIF that previously scored in genome-wide screens for mediators of DNA inter-strand crosslink (ICL) repair. Upon ICL agent exposure, RADIF loss leads to marked cell death, elevated chromosomal instability, increased micronuclei formation, altered cell cycle progression and increased DNA damage signaling. RADIF is recruited to damage foci and forms a complex with FIGNL1. Both proteins have epistatic roles in ICL repair, forming a co-stable complex. Mechanistically, RADIF loss leads to increased RAD51 amounts and foci on chromatin both with or without exogenous DNA damage, defective replication fork progression and reduced HR competency. We posit that RADIF is essential for limiting RAD51 levels on chromatin in the absence of damage and for RAD51 dissociation from nucleofilaments to properly complete HR. Failure to do so leads to replication slowing and inability to complete repair.

5.
Cell Stem Cell ; 30(4): 396-414.e9, 2023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-37028405

RESUMO

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.


Assuntos
Edição de Genes , Miócitos Cardíacos , Humanos , Animais , Suínos , Miócitos Cardíacos/metabolismo , Linhagem Celular , Arritmias Cardíacas/genética , Arritmias Cardíacas/terapia , Arritmias Cardíacas/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Diferenciação Celular/genética
6.
Stem Cell Reports ; 16(10): 2473-2487, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34506727

RESUMO

Heart failure remains a significant cause of morbidity and mortality following myocardial infarction. Cardiac remuscularization with transplantation of human pluripotent stem cell-derived cardiomyocytes is a promising preclinical therapy to restore function. Recent large animal data, however, have revealed a significant risk of engraftment arrhythmia (EA). Although transient, the risk posed by EA presents a barrier to clinical translation. We hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. This study uses a porcine model to provide proof-of-concept evidence that a combination of amiodarone and ivabradine can effectively suppress EA. None of the nine treated subjects experienced the primary endpoint of cardiac death, unstable EA, or heart failure compared with five out of eight (62.5%) in the control cohort (hazard ratio = 0.00; 95% confidence interval: 0-0.297; p = 0.002). Pharmacologic treatment of EA may be a viable strategy to improve safety and allow further clinical development of cardiac remuscularization therapy.


Assuntos
Amiodarona/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Ivabradina/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/transplante , Transplante de Células-Tronco/efeitos adversos , Taquicardia/tratamento farmacológico , Animais , Antiarrítmicos/uso terapêutico , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Modelos Animais de Doenças , Combinação de Medicamentos , Humanos , Masculino , Células-Tronco Pluripotentes/transplante , Suínos
7.
J Imaging ; 7(7)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-39080895

RESUMO

(1) Background: The accuracy of the left ventricular volume (LVV) and contractility measurements with cardiac magnetic resonance imaging (CMRI) is decreased if the papillary muscles are abnormally enlarged, such as in hypertrophic cardiomyopathy in human patients or in pig models of human diseases. The purpose of this work was to establish the best method of LVV quantification with CMRI in pigs. (2) Methods: The LVV in 29 Yucatan minipig hearts was measured using two different techniques: the "standard method", which uses smooth contouring along the endocardial surface and adds the papillary volume to the ventricular cavity volume, and the "detailed method", which traces the papillary muscles and trabeculations and adds them to the ventricular mass. (3) Results: Papillary muscles add 21% to the LV mass in normal and infarcted hearts of Yucatan minipigs. The inclusion or exclusion of these from the CMRI analysis significantly affected the study results. In the normal pig hearts, the biggest differences were found in measurements of the LVV, ejection fraction (EF), LV mass and indices derived from the LV mass (p < 0.001). The EF measurement in the normal pig heart was 11% higher with the detailed method, and 19% higher in the infarcted pig hearts (p < 0.0001). The detailed method of endocardium tracing with CMRI closely represented the LV mass measured ex vivo. (4) Conclusions: The detailed method, which accounts for the large volume of the papillary muscles in the pig heart, provides better accuracy and interobserver consistency in the assessment of LV mass and ejection fraction, and might therefore be preferable for these analyses.

8.
Nucleosides Nucleotides Nucleic Acids ; 39(1-3): 292-309, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31509065

RESUMO

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.


Assuntos
DNA/química , Oligonucleotídeos/química , RNA de Cadeia Dupla/química , Linhagem Celular Tumoral , Técnicas de Química Sintética , DNA/síntese química , Inativação Gênica , Humanos , Interferon-alfa/biossíntese , Leucócitos Mononucleares/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Clivagem do RNA , Interferência de RNA , RNA de Cadeia Dupla/síntese química , RNA Mensageiro/genética , Relação Estrutura-Atividade , Transfecção
9.
Pulm Pharmacol Ther ; 54: 31-38, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30448291

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease, and consequently, effective antifibrotic drugs are strongly desired. Although we have previously reported a validated Col1a1-Luc Tg rat model for fibrosis, there are only a few mouse models that enable the evaluation of fibrosis in a short time period and with high sensitivity. Therefore, we generated a Col1a1-internal ribosome entry site (IRES)-Luc knock-in (KI) mouse in which the IRES-luciferase gene construct was inserted into the 3'-UTR of the type I collagen alpha 1 gene (Col1a1). There was a high correlation between luciferase activity and hydroxyproline content in the KI mice, which is similar to the result that we have previously reported for the Col1a1-Luc Tg rat model. In a bleomycin (BLM)-induced lung fibrosis model, luciferase activity in the lung showed a significant increase 3 days after BLM treatment, while only a slight increase was observed in the hydroxyproline content. An ALK-5 inhibitor-R-268712-was effective in inhibiting the luciferase activity in both the in vivo BLM-induced lung fibrosis model and in vitro primary mouse lung fibroblasts. This suggests that fibroblasts are the major collagen-producing cells in lung fibrosis. In human lung fibroblasts, TGF-ß stimulation induced α-smooth muscle actin as observed by immunostaining, suggesting that myofibroblast transdifferentiation (MTD) plays an important role in lung fibrosis. Together, these results indicated that ALK-5 inhibitors might affect lung fibrosis mainly via the inhibition of MTD. Thus, the Col1a1-IRES-Luc KI mouse might be useful for the evaluation of antifibrotic effects and their underlying mechanisms.


Assuntos
Colágeno Tipo I/genética , Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Animais , Bleomicina/administração & dosagem , Transdiferenciação Celular , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Fibroblastos/metabolismo , Técnicas de Introdução de Genes , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Miofibroblastos/citologia , Fibrose Pulmonar/fisiopatologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo
11.
Nat Biotechnol ; 36(7): 597-605, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29969440

RESUMO

Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias Humanas/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Animais , Criopreservação , Modelos Animais de Doenças , Humanos , Macaca , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/transplante , Primatas
12.
Bioorg Med Chem Lett ; 24(3): 893-9, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24412069

RESUMO

A PDE4B subtype selective inhibitor is expected to have a wider therapeutic window than non-selective PDE4 inhibitors. In this Letter, two series of 7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives and 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives were evaluated for their PDE4B subtype selectivity using human PDE4B2 and PDE4D2 full length enzymes. To improve their PDE4B selectivity over PDE4D, we optimized the substituents on the pyrimidine ring and the side chain phenyl ring, resulting in several derivatives with more than 100-fold selectivity for PDE4B. Consequently, we identified 2-(3-chloro-4-methoxy-phenyl)-5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative 54 as a highly selective PDE4B inhibitor, which had potent hPDE4B inhibitory activity with an IC50 value of 3.0 nM and 433-fold PDE4B selectivity over PDE4D.


Assuntos
Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacologia , Fenilacetatos/química , Fenilacetatos/farmacologia , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/farmacologia , Sítios de Ligação , Óxidos S-Cíclicos/isolamento & purificação , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Fenilacetatos/isolamento & purificação , Inibidores da Fosfodiesterase 4/isolamento & purificação
13.
J Pharmacol Sci ; 123(3): 219-26, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24152964

RESUMO

Phosphodiesterase 4 (PDE4) inhibitors have been developed for the treatment of pulmonary inflammatory diseases, but their clinical use was dose-limited by mainly gastric adverse effects. Recent studies suggested PDE4B-selective inhibitors over PDE4D are supposed to display a wider therapeutic index than subtype non-selective PDE4 inhibitors such as roflumilast. Compound A was identified as an orally active PDE4B-selective inhibitor over PDE4D both in humans (80-fold selective) and mice (29-fold selective). In this study, the therapeutic effects of compound A and roflumilast were evaluated on lipopolysaccaride (LPS) injection-induced plasma TNF-α elevation and on LPS inhalation-induced pulmonary neutrophilia in mice. The inhibitory effect on gastric emptying in mice was evaluated as a gastric adverse effect. The therapeutic index for TNF-α production (TI(TNF) = ID50 in gastric emptying / ID50 in LPS injection-induced plasma TNF-α elevation) of compound A was larger than roflumilast (9.0 and 0.2, respectively), whereas the therapeutic index for pulmonary neutrophilia (TI(Neu) = ID50 in gastric emptying / ID50 in LPS inhalation-induced pulmonary neutrophilia) of compound A was comparable to roflumilast (1.0 and 0.5, respectively). In conclusion, the TI(Neu) of compound A was not superior compared to that of roflumilast in spite of its high selectivity for PDE4B over PDE4D in mice.


Assuntos
Aminopiridinas/uso terapêutico , Benzamidas/uso terapêutico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Compostos Heterocíclicos com 2 Anéis/uso terapêutico , Transtornos Leucocíticos/tratamento farmacológico , Neutrófilos , Fenilacetatos/uso terapêutico , Inibidores da Fosfodiesterase 4/uso terapêutico , Pneumonia/tratamento farmacológico , Administração Oftálmica , Aminopiridinas/administração & dosagem , Aminopiridinas/efeitos adversos , Animais , Benzamidas/administração & dosagem , Benzamidas/efeitos adversos , Ciclopropanos/administração & dosagem , Ciclopropanos/efeitos adversos , Ciclopropanos/uso terapêutico , Esvaziamento Gástrico/efeitos dos fármacos , Compostos Heterocíclicos com 2 Anéis/efeitos adversos , Humanos , Transtornos Leucocíticos/induzido quimicamente , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos , Fenilacetatos/efeitos adversos , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/efeitos adversos , Pneumonia/induzido quimicamente , Fator de Necrose Tumoral alfa/sangue
14.
Am J Physiol Renal Physiol ; 299(4): F792-801, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20660018

RESUMO

Fibrosis is the final common pathway for various tissue lesions that lead to chronic progressive organ failure, and consequently effective antifibrotic drugs are strongly desired. However, there are few animal models in which it is possible to evaluate fibrosis sensitively in a short period of time. We therefore generated two transgenic rats harboring a firefly luciferase reporter gene under the control of the 5'-flanking region of rat α(1)(I) collagen (Col1a1-Luc Tg rats) and α(2)(I) collagen (Col1a2-Luc Tg rats). The luciferase activities of these transgenic rats were highly correlated with the hydroxyproline content in various organs. In unilateral ureteral obstruction (UUO), a well-characterized model of renal fibrosis, the luciferase activity in obstructed kidneys showed a significant increase after even 3 days of UUO, while the hydroxyproline content showed little increase. In addition, the renal hydroxyproline content had a higher correlation with the luciferase activity than α(1)(I) collagen mRNA level for over 2 wk after UUO. Although both an ANG II type 1 receptor blocker (ARB), olmesartan, and a transforming growth factor-ß (TGF-ß) type I receptor kinase (ALK5) inhibitor, SB-431542, inhibited renal luciferase activities in UUO, only SB-431542 inhibited luciferase activity induced by TGF-ß1 in isolated glomeruli. Double immunostaining for luciferase and α-smooth muscle actin (α-SMA) revealed that some α-SMA-positive tubular epithelial cells and tubular interstitial cells produced type I collagen, which would lead to renal fibrosis. Thus collagen reporter transgenic rats would be very useful for the evaluation of antifibrotic effects and analysis of their mechanisms.


Assuntos
Colágeno Tipo I/metabolismo , Colágeno/metabolismo , Genes Reporter , Rim/metabolismo , Rim/patologia , Luciferases/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Benzamidas/farmacologia , Colágeno/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Dioxóis/farmacologia , Modelos Animais de Doenças , Fibrose , Hidroxiprolina/metabolismo , Imidazóis/farmacologia , Rim/efeitos dos fármacos , Luciferases/genética , Masculino , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Transgênicos , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Sensibilidade e Especificidade , Tetrazóis/farmacologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
15.
Eur J Pharmacol ; 596(1-3): 153-9, 2008 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-18706408

RESUMO

Neurokinins are known to induce neurogenic inflammation related to respiratory diseases. The effects of CS-003 ([1-{2-[(2R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl}spiro[benzo[c]thiophene-1(3H),4'-piperidine]-(2S)-oxide hydrochloride]), a novel triple neurokinin receptor antagonist, on several respiratory disease models were evaluated in guinea pigs. As we have already shown that CS-003 is intravenously effective, we first determined if CS-003 was orally effective. CS-003 dose-dependently inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID(50) values of 3.6, 1.3 and 0.89 mg/kg (p.o.), respectively. CS-003 (10 mg/kg, p.o.) inhibited the number of coughs induced by capsaicin aerosol (P<0.01) and the antitussive effect was comparable to that of codeine. CS-003 (10 mg/kg, p.o.) also inhibited airway hyperresponsiveness to methacholine chloride in ovalbumin-induced asthma models (P<0.01), a milder one and a severer one. On the other hand, montelukast (10 mg/kg, p.o.), a leukotriene receptor antagonist, significantly inhibited the hyperresponsiveness only in the milder model (P<0.05). In an ovalbumin-induced rhinitis model, oral administration of CS-003 inhibited nasal blockade in a dose-dependent manner and the inhibitory effect was comparable to that of dexamethasone (10 mg/kg, p.o.). CS-003 (i.v.) also dose-dependently inhibited cigarette smoke-induced bronchoconstriction, tracheal vascular hyperpermeability and mucus secretion. These data show that CS-003, a potent orally active triple neurokinin receptor antagonist, may be useful for the treatment of respiratory diseases associated with neurokinins, such as allergic asthma, allergic rhinitis, chronic obstructive pulmonary disease and cough.


Assuntos
Óxidos S-Cíclicos/farmacologia , Morfolinas/farmacologia , Receptores de Taquicininas/antagonistas & inibidores , Doenças Respiratórias/tratamento farmacológico , Administração Oral , Animais , Asma/tratamento farmacológico , Asma/imunologia , Broncoconstrição/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Capsaicina , Tosse/induzido quimicamente , Tosse/tratamento farmacológico , Óxidos S-Cíclicos/administração & dosagem , Óxidos S-Cíclicos/uso terapêutico , Modelos Animais de Doenças , Cobaias , Masculino , Morfolinas/administração & dosagem , Morfolinas/uso terapêutico , Muco/metabolismo , Ovalbumina , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Rinite/tratamento farmacológico , Rinite/imunologia , Fumaça , Nicotiana , Traqueia/irrigação sanguínea , Traqueia/metabolismo
16.
Eur J Pharmacol ; 586(1-3): 306-12, 2008 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-18353309

RESUMO

Neurokinins are known to induce neurogenic inflammation related to respiratory diseases, though there is little information on triple neurokinin receptor antagonists. The pharmacological properties of the novel triple neurokinin 1, 2 and 3 receptor antagonist [1-(2-[(2R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl)spiro[benzo[c]thiophene-1(3H),4'-piperidine]-(2S)-oxide hydrochloride] (CS-003) were evaluated in this study. The binding affinities of CS-003 were evaluated with human and guinea pig neurokinin receptors. As well, the in vivo antagonism of CS-003 against exogenous neurokinins and effects on capsaicin-induced and citric acid-induced responses were investigated in guinea pigs. CS-003 exhibited high affinities for human neurokinin 1, neurokinin 2 and neurokinin 3 receptors with Ki values (mean+/-S.E.M.) of 2.3+/-0.52, 0.54+/-0.11 and 0.74+/-0.17 nM, respectively, and for the guinea pig receptors with Ki values of 5.2+/-1.4, 0.47+/-0.075 and 0.71+/-0.27 nM, respectively. Competitive antagonism was indicated in a Schild analysis of substance P-, neurokinin A- and neurokinin B-induced inositol phosphate formation with pA2 values of 8.7, 9.4 and 9.5, respectively. CS-003 inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID50 values of 0.13, 0.040 and 0.063 mg/kg (i.v.), respectively. CS-003 also inhibited capsaicin-induced bronchoconstriction (ID50: 0.27 mg/kg, i.v.), which is caused by endogenous neurokinins. CS-003 significantly inhibited citric acid-induced coughs and the effect was greater than those of other selective neurokinin receptor antagonists. CS-003 is a potent antagonist of triple neurokinin receptors and may achieve the best therapeutic efficacy on respiratory diseases associated with neurokinins compared to selective neurokinin receptor antagonists.


Assuntos
Óxidos S-Cíclicos/farmacologia , Morfolinas/farmacologia , Neurocinina A/antagonistas & inibidores , Neurocinina A/farmacologia , Neurocinina B/antagonistas & inibidores , Neurocinina B/farmacologia , Antagonistas dos Receptores de Neurocinina-1 , Receptores da Neurocinina-2/antagonistas & inibidores , Receptores da Neurocinina-3/antagonistas & inibidores , Animais , Brônquios/efeitos dos fármacos , Células COS , Permeabilidade Capilar/efeitos dos fármacos , Capsaicina/antagonistas & inibidores , Capsaicina/farmacologia , Chlorocebus aethiops , Ácido Cítrico , Tosse/induzido quimicamente , Tosse/prevenção & controle , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Cobaias , Humanos , Técnicas In Vitro , Indóis/farmacologia , Fosfatos de Inositol/biossíntese , Masculino , Dados de Sequência Molecular , Receptores da Neurocinina-1/metabolismo , Receptores da Neurocinina-2/metabolismo , Receptores da Neurocinina-3/metabolismo , Substância P/farmacologia , Traqueia/irrigação sanguínea , Traqueia/efeitos dos fármacos
17.
J Neurosci ; 26(14): 3626-33, 2006 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-16597715

RESUMO

The glutamate receptor delta2 subunit (GluRdelta2) is selectively expressed in cerebellar Purkinje neurons (PNs) and is involved in the long-term depression (LTD). However, little is known about the mechanism of its action. Acute expression of the wild-type GluRdelta2 in the GluRdelta2-deficient PN rescued the induction of LTD, suggesting the direct role of GluRdelta2 in LTD. To identify the critical region of GluRdelta2 necessary for LTD, we constructed and expressed various mutant GluRdelta2 proteins in the GluRdelta2-deficient PNs. The mutant GluRdelta2 possessing the membrane-proximal 21 aa residues in the C-terminal cytoplasmic region rescued the induction of LTD, whereas the mutant with membrane-proximal 13 aa failed. In addition, overexpression of 865 approximately 871 aa of GluRdelta2 (corresponding to membrane-proximal 14-20 aa) fused to EGFP (enhanced green fluorescent protein) suppressed LTD in a wild-type PN. These results suggest that 865 approximately 871 aa of GluRdelta2 play an essential role in LTD. We next identified protein interacting with C kinase 1 (PICK1) as a molecule interacting with the membrane-proximal C-terminal region of GluRdelta2 by yeast two-hybrid screening. PICK1 plays an essential role in LTD. It colocalized with GluRdelta2 at spines of PNs, and immunoprecipitation assays showed that GluRdelta2 bound to PICK1 mainly through 865-871 aa. These results indicate that 865-871 aa of GluRdelta2 are essential for both LTD and interaction with PICK1, and suggest that interaction between GluRdelta2 and PICK1 might be critical for the induction of LTD.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Proteínas Nucleares/metabolismo , Células de Purkinje/fisiologia , Receptores de Glutamato/metabolismo , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia
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