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1.
Cell Biol Int ; 45(5): 1030-1037, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33404163

RESUMO

Antimetastatic effect of Metformin has been documented in epithelial ovarian cancer (EOC). Presently, we investigated the regulatory mechanism of Metformin in EOC metastasis. First, Girdin was significantly enhanced in EOC tumorous tissues and cell lines. Seconded, knockdown of Girdin significantly suppressed EOC cell viability, migration, and invasion, while upregulation of Girdin produced the opposite effects in vitro and facilitated lung metastasis in EOC cell xenograft in vivo. In addition, we confirmed that the inhibitory effect of Metformin on Girdin expression. Mechanistically, the oncogenic effects of Girdin could be reversed by LY294002 (an AKT pathway inhibitor) and Metformin. These results suggested that Metformin attenuated EOC metastasis through Girdin and targeting Girdin may be a promising therapeutic strategy for EOC in the future.


Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Proteínas dos Microfilamentos/genética , Metástase Neoplásica/tratamento farmacológico , Proteínas de Transporte Vesicular/genética , Adulto , Animais , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metformina/metabolismo , Metformina/farmacologia , Camundongos Nus , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Transcriptoma/genética , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo
2.
Toxicology ; 442: 152537, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663520

RESUMO

Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.


Assuntos
Anestésicos Inalatórios/toxicidade , Flavonoides/farmacologia , Isoflurano/antagonistas & inibidores , Isoflurano/toxicidade , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/prevenção & controle , Polifenóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte Vesicular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Camundongos , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo
3.
J Cell Sci ; 130(14): 2251-2265, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28536105

RESUMO

Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.


Assuntos
Antígenos de Neoplasias/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células A549 , Fator 1 de Ribosilação do ADP/metabolismo , Antibacterianos/farmacologia , Antígenos de Neoplasias/efeitos dos fármacos , Autofagia/fisiologia , Brefeldina A/farmacologia , Disenteria Bacilar/tratamento farmacológico , Disenteria Bacilar/metabolismo , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HT29 , Células HeLa , Humanos , Shigella flexneri/efeitos dos fármacos , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/efeitos dos fármacos
4.
Arthritis Rheumatol ; 67(9): 2447-56, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26016600

RESUMO

OBJECTIVE: Giant cell arteritis (GCA) is pathologically characterized by dysfunctional angiogenesis and inflammatory cell infiltration. Acute-phase serum amyloid A (A-SAA) is an acute-phase reactant, but is also produced at sites of inflammation and may contribute to vascular inflammation in atherosclerosis. This study was undertaken to examine the effect of A-SAA on proinflammatory pathways and angiogenesis in GCA, using a novel ex vivo temporal artery tissue explant model. METHODS: Serum A-SAA levels were measured by enzyme-linked immunosorbent assay (ELISA). Temporal artery explants and peripheral blood mononuclear cell (PBMC) cultures were established from patients with GCA. Temporal artery explant morphology, viability, and spontaneous release of proinflammatory mediators following 24-hour culture were assessed by hematoxylin and eosin, calcein viability staining, and ELISA. Temporal artery explants and PBMC cultures were stimulated with A-SAA (10 µg/ml), and interleukin-6 (IL-6), IL-8, vascular endothelial growth factor, Ang2, and matrix metalloproteinase 2 (MMP-2)/MMP-9 were quantified by ELISA and gelatin zymography. The effect of conditioned medium from temporal artery explants on angiogenesis was assessed using endothelial cell Matrigel tube-formation assays. Temporal artery explants were also embedded in Matrigel, and myofibroblast outgrowth was assessed. RESULTS: Serum A-SAA levels were significantly higher in GCA patients versus healthy controls (P < 0.0001). Intact tissue morphology, cell viability, and spontaneous cytokine secretion were demonstrated in temporal artery explants. A-SAA treatment induced a significant increase in the levels of IL-6 and IL-8 from temporal artery explants (P < 0.05) and IL-8 from PBMCs (P < 0.05) compared to basal conditions. Conditioned medium from A-SAA-treated explants significantly induced angiogenic tube formation (P < 0.05 versus basal controls). Finally, A-SAA induced myofibroblast outgrowth and MMP-9 activation. CONCLUSION: Our findings demonstrate a functional role for A-SAA in regulating temporal artery inflammation, angiogenesis, and invasion, all key processes in the pathogenesis of GCA.


Assuntos
Arterite de Células Gigantes/imunologia , Miofibroblastos/efeitos dos fármacos , Neovascularização Patológica/imunologia , Proteína Amiloide A Sérica/farmacologia , Artérias Temporais/efeitos dos fármacos , Proteínas de Fase Aguda/imunologia , Proteínas de Fase Aguda/farmacologia , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Arterite de Células Gigantes/metabolismo , Humanos , Técnicas In Vitro , Inflamação/imunologia , Interleucina-6/imunologia , Interleucina-8/efeitos dos fármacos , Interleucina-8/imunologia , Leucócitos Mononucleares , Masculino , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/imunologia , Pessoa de Meia-Idade , Proteína Amiloide A Sérica/imunologia , Artérias Temporais/imunologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/imunologia , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/imunologia
5.
Salud colect ; 11(1): 99-114, ene.-mar. 2015. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-746687

RESUMO

El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.


In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.


Assuntos
Animais , Ratos , Apoptose/genética , Regulação Enzimológica da Expressão Gênica/genética , Heme/deficiência , Degeneração Neural/genética , Neurônios/metabolismo , Porfirias/complicações , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colágeno Tipo XI/efeitos dos fármacos , Colágeno Tipo XI/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inibidores Enzimáticos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme/biossíntese , Heptanoatos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/efeitos dos fármacos , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Poli(ADP-Ribose) Polimerases , Porfirias/metabolismo , Porfirias/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
6.
An. bras. dermatol ; 89(6): 891-897, Nov-Dec/2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-727638

RESUMO

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results. .


Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anticorpos Monoclonais Murinos/análise , Vasos Linfáticos/patologia , Psoríase/tratamento farmacológico , Fatores de Necrose Tumoral/antagonistas & inibidores , Fatores de Crescimento do Endotélio Vascular/análise , Proteínas de Transporte Vesicular/análise , Anticorpos Monoclonais Murinos/efeitos dos fármacos , Biópsia , Biomarcadores/análise , Imuno-Histoquímica , Imunoglobulina G/uso terapêutico , Fatores Imunológicos/uso terapêutico , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Psoríase/metabolismo , Psoríase/patologia , Valores de Referência , Receptores do Fator de Necrose Tumoral/uso terapêutico , Estatísticas não Paramétricas , Pele/efeitos dos fármacos , Pele/patologia , Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Proteínas de Transporte Vesicular/efeitos dos fármacos
7.
An Bras Dermatol ; 89(6): 891-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25387493

RESUMO

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results.


Assuntos
Anticorpos Monoclonais Murinos/análise , Vasos Linfáticos/patologia , Psoríase/tratamento farmacológico , Inibidores do Fator de Necrose Tumoral , Fatores de Crescimento do Endotélio Vascular/análise , Proteínas de Transporte Vesicular/análise , Adulto , Anticorpos Monoclonais Murinos/efeitos dos fármacos , Biomarcadores/análise , Biópsia , Etanercepte , Feminino , Humanos , Imunoglobulina G/uso terapêutico , Imuno-Histoquímica , Fatores Imunológicos/uso terapêutico , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Psoríase/metabolismo , Psoríase/patologia , Receptores do Fator de Necrose Tumoral/uso terapêutico , Valores de Referência , Pele/efeitos dos fármacos , Pele/patologia , Estatísticas não Paramétricas , Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Proteínas de Transporte Vesicular/efeitos dos fármacos
8.
Clin Cancer Res ; 12(23): 6952-9, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17145813

RESUMO

PURPOSE: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development. EXPERIMENTAL DESIGN: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done. RESULTS: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log(2) ratio less than -1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis. CONCLUSIONS: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.


Assuntos
Neoplasias do Colo/genética , Regulação para Baixo/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosforilação , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Nexinas de Classificação , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/efeitos dos fármacos
9.
Cell Metab ; 4(2): 143-54, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16890542

RESUMO

Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/farmacologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Palmitatos/farmacologia , Palmitatos/toxicidade , Potássio/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas de Transporte Vesicular/efeitos dos fármacos
10.
Semin Nephrol ; 25(5): 322-7, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16139687

RESUMO

In the current report we review the results that lay grounds for the model of intracellular sodium-mediated dopamine-induced endocytosis of Na,K-ATPase. Under conditions of a high salt diet, dopamine activates PKCzeta, which phosphorylates NKA alpha1 Ser-18. The phosphorylation produces a conformational change of alpha1 NH2-terminus, which through interaction with other domains of alpha1 exposes PI3K- and AP-2-binding domains. PI3K bound to the NKA alpha1 induces the recruitment and activation of other proteins involved in endocytosis, and PI3K-generated 3-phosphoinositides affect the local cytoskeleton and modify the biophysical conditions of the membrane for development of clathrin-coated pits. Plasma membrane phosphorylated NKA is internalized to specialized intracellular compartments where the NKA will be dephosphorylated. The NKA internalization results in a reduced Na+ transport by proximal tubule epithelial cells.


Assuntos
Cardiotônicos/farmacologia , Dopamina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Túbulos Renais Proximais/citologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo
11.
Brain Res Mol Brain Res ; 137(1-2): 23-30, 2005 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-15950757

RESUMO

Defective heme synthesis may cause acute porphyrias, which are associated with a wide array of neurological disturbances involving both the central and peripheral nervous systems. Thus, the understanding of the roles of heme in neuronal cell function may provide insights into the molecular events underlying the pathogenesis of neuropathies associated with defective heme synthesis. In this report, we use rat pheochromocytoma (PC12) clonal cells as a model system for studying the role of heme in neuronal cell survival. We examined the effects of inhibition of heme synthesis on signaling pathways and gene expression in nerve growth factor (NGF)-induced PC12 cells. We found that succinyl acetone-induced heme deficiency selectively caused apoptosis in NGF-induced PC12 cells. Further, we found that in succinyl acetone-treated, NGF-induced cells, the pro-survival Ras-ERK1/2 signaling pathway was inactivated and the pro-apoptotic JNK signaling pathway was activated. In these cells, the activation of caspase and the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) were also evident. Importantly, microarray gene expression analysis showed that more than 20 key neuronal genes that were induced by NGF were suppressed by succinyl acetone. These genes include those encoding survival motor neuron protein, synaptic vesicle protein SVOP, and neural cell adhesion molecule NCAM. These results indicate that heme is important for neuronal cell signaling and the proper functioning of neuronal cells.


Assuntos
Apoptose/genética , Regulação Enzimológica da Expressão Gênica/genética , Heme/deficiência , Degeneração Neural/genética , Neurônios/metabolismo , Porfirias/complicações , Animais , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colágeno Tipo XI/efeitos dos fármacos , Colágeno Tipo XI/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inibidores Enzimáticos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme/biossíntese , Heptanoatos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/efeitos dos fármacos , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Células PC12 , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Porfirias/metabolismo , Porfirias/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Proteínas do Complexo SMN , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
12.
Am J Physiol Cell Physiol ; 287(5): C1366-74, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15240346

RESUMO

Exocytic insertion of H(+)-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H(+)-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H(+)-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 +/- 7% and increased the interaction between GFP-syntaxin 1A and H(+)-ATPase by 170 +/- 23%. Apical membrane Munc-18-2 decreased by 27.5 +/- 4.6% and H(+)-ATPase increased by 246 +/- 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H(+)-ATPase. In a pull-down assay of H(+)-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H(+)-ATPase pulled down by 64 +/- 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H(+)-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H(+)-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H(+)-ATPase vesicles.


Assuntos
Adenosina Trifosfatases/metabolismo , Exocitose/fisiologia , Túbulos Renais Coletores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Animais , Antígenos de Superfície/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Medula Renal/efeitos dos fármacos , Medula Renal/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Proteínas Munc18 , Proteínas do Tecido Nervoso/efeitos dos fármacos , Testes de Precipitina , Proteína Quinase C/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sintaxina 1 , Proteínas de Transporte Vesicular/efeitos dos fármacos
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