RESUMO
BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a complicated disease with low survival rate partially due to frequent recurrence and no efficient therapy. Promoter hypermethylation of tumor suppressor genes has been demonstrated as one of the molecular mechanisms contributing to tumorigenesis and progression in HCC. This study aims to investigate regulation of NKAPL expression by promoter methylation and its clinical relevance as a biomarker for HCC. METHODS: We measured mRNA expression of NKAPL in 5 HCC cell lines and a cohort of 62 pairs of primary HCC tumor and their adjacent non-cancer liver tissues. NKAPL protein expression on HCC cell lines and clinical samples was assessed by Western blot and immunohistochemistry, respectively. Association analyses between NKAPL expression and clinicopathologic characteristics in the cohort were conducted. Methylation statuses of NKAPL promoter in 18 pairs of tumor and adjacent non-tumor HCC samples were studied using methylation-specific PCR. Biological functions of NKAPL in HCC were investigated by ectopic expression of NKAPL in HCC cells, and cell viability and cell cycle analyses were performed. RESULTS: Our present study showed suppressed expression and promoter hypermethylation are common events in HCC. Demethylation experiment in HCC cells demonstrated that the NKAPL expression was regulated by promoter methylation. In addition, high methylation level of NKAPL and its low expression predict poor outcome. Furthermore, ectopic expression of NKAPL in the HCC cells inhibited cell growth. CONCLUSIONS: Our findings suggest that methylation of NKAPL is a frequent event and is a potential prognosis biomarker in HCC.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Proteínas Correpressoras/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Proteínas Correpressoras/genética , Feminino , Humanos , Masculino , Metilação , Proteínas Nucleares/genética , Prognóstico , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
Four and a Half LIM protein 2 (FHL2) is a LIM domain only protein that is able to form various protein complexes and regulate gene transcription. Recent findings showed that FHL2 is a potential tumor suppressor gene that was down-regulated in hepatocellular carcinoma (HCC). Moreover, FHL2 can bind to and activate the TP53 promoter in hepatic cells. In this study, the activity of the two promoters of FHL2, 1a and 1b, were determined in the human embryonic kidney cell line HEK293 and the activation of these two promoters by p53 was investigated. Our results showed that the 1b promoter has a higher activity than the 1a promoter in HEK 293 cells but the 1a promoter is more responsive to the activation by p53 when compared with the 1b promoter. The regulation of FHL2 by p53 was further confirmed in liver cells by the overexpression of p53 in Hep3B cells and the knockdown of p53 in HepG2 cells. Combining promoter activity results of truncated mutants and predictions by bioinformatics tools, a putative p53 binding site was found in the exon 1a of FHL2 from +213 to +232. The binding between the p53 protein and the putative p53 binding site was then validated by the ChIP assay. Furthermore, the expression of FHL2 and TP53 were down-regulated in majority of HCC tumour samples (nâ=â41) and significantly correlated (Pâ=â0.026). Finally, we found that the somatic mutation 747 (GâT), a hot spot mutation of the TP53 gene, is potentially associated with a higher expression of FHL2 in HCC tumour samples. Taken together, this is the first in-depth study about the transcriptional regulation of FHL2 by p53.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Rim/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Biologia Computacional/métodos , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Mutagênese Sítio-Dirigida , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Deleção de Sequência , Ativação Transcricional , Proteína Supressora de Tumor p53/genéticaRESUMO
Outdoor air pollution has been recently classified as a class I human carcinogen by the World Health Organization (WHO). Cumulative evidence from across the globe shows that polluted air is associated with increased risk of lung, head and neck, and nasopharyngeal cancers--all of which affect the upper aerodigestive tract. Importantly, these cancers have been previously linked to smoking. In this article, we review epidemiologic and experimental evidence of the genotoxic and mutagenic effects of air pollution on DNA, purportedly a key mechanism for cancer development. The alarming increase in cancers of the upper aerodigestive tract in Asia suggests a need to focus government efforts and research on reducing air pollution, promoting clean energy, and investigating the carcinogenic effects of air pollution on humans.
Assuntos
Poluição do Ar/efeitos adversos , Dano ao DNA , Mutagênese , Ásia , Carcinógenos , Neoplasias de Cabeça e Pescoço , Humanos , Neoplasias Pulmonares , Neoplasias Nasofaríngeas , FumarRESUMO
The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Receptores ErbB/antagonistas & inibidores , Melanoma/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/enzimologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Receptores ErbB/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Indóis/farmacologia , Concentração Inibidora 50 , Ligantes , Melanoma/genética , Melanoma/patologia , Terapia de Alvo Molecular , Morfolinas/farmacologia , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , VemurafenibRESUMO
The putative tumor suppressor Mst1, when cleaved to its 36kDa cleaved form, amplifies apoptotic signals. We found that Mst1 was predominantly expressed in its full-length form in 76% (17/25 cases) of hepatocellular carcinoma (HCC) tumors. Mst1 cleavage was basically absent in HCC cells. Ectopic full-length Mst1 expression increased the growth of HCC cells by 55-80% within 3days after transfection. Expression of exogenous NORE1B, a tumor suppressor commonly lost in HCC tumors (~56% of our cohort), was sufficient to suppress the growth promotion of full-length Mst1. Hence, Mst1 exhibits a growth promoting activity in HCC cells upon NORE1B downregulation.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Fator de Crescimento de Hepatócito/fisiologia , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas Reguladoras de Apoptose , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Análise Mutacional de DNA , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Peso Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Regulação para CimaRESUMO
Recent studies revealed an unexpected role of the neurotrophin receptor pathway, BDNF/TrkB signaling, in cancer metastasis and anoikis (i.e. detachment-induced cell death). Survival of cancer cells in detached state (known as anoikis-resistance) is known to be pre-requisite for metastasis. Nasopharyngeal carcinoma (NPC), an endemic head and neck cancer in Southeast Asia, is highly invasive, metastatic, and etiologically associated with Epstein-Barr virus (EBV, an oncovirus) infection. Mechanistic studies on the invasive/metastatic nature of NPC can facilitate the development of anti-metastatic therapy in NPC. Thus far, the role of BDNF/TrkB signaling in virus-associated human cancer is unclear. Here, using multiple cell line models of NPC with EBV-association (HONE-1-EBV, HK1-LMP1 and C666-1), we investigated the potential involvement of BDNF/TrkB signaling in cellular migration and anoikis-resistant characteristics of NPC. We found that all three EBV-associated NPC cell lines tested were intrinsically anoikis-resistant (i.e. survived in detached state) and expressed both BDNF and TrkB. BDNF stimulation induced cellular migration, but not proliferation of these cells. Further, we examined if pharmacologic targeting of anoikis-resistance of NPC cells can be achievable by a proof-of-concept Trk inhibitor, K252a, in these EBV-associated NPC models. Our results demonstrated that K252a, was able to attenuate BDNF-induced migration and proliferation of NPC cells. More importantly, we demonstrated for the first time that K252a harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against EBV-associated human cancer cells, namely NPC cells. This proof-of-concept study demonstrated that K252a, a Trk inhibitor, can potentially be used as an anoikis-sensitizing agent in NPC.
Assuntos
Anoikis/efeitos dos fármacos , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Movimento Celular/efeitos dos fármacos , Herpesvirus Humano 4/patogenicidade , Alcaloides Indólicos/farmacologia , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/virologia , Inibidores de Proteínas Quinases/farmacologia , Receptor trkB/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carcinoma , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica , Receptor trkB/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The four-and-a-half LIM protein 2 (FHL2) was originally identified to be expressed abundantly in the heart, as well as in a wide range of tissues demonstrated in various studies. The human FHL2 gene expresses different transcripts which are known to differ only in the 5'UTR region. However, little is known about the functional role of the different variants and the mechanism of gene regulation. In the present study, we characterized the different alternative spliced transcripts of FHL2 by in silico analysis and RT-PCR analysis. A novel transcript variant was identified. The FHL2 gene produces transcripts by different 5' exons, which may be responsible for tissue-specific regulation. To study the mechanism of FHL2 gene regulation, the potential promoter region was investigated. We have identified a functional promoter region upstream of the transcription start site. Deletion mutation analysis of 5' flanking region showed that the fragment from -138 to +292 bp have positive regulatory effect. We identified the binding sites of Pax-5/ZF5 in this region and found that Pax-5 and ZF5 expression in HCC samples had a significant positive correlation with FHL2 expression, suggesting a possible role for these transcription factors in the regulation of FHL2 expression.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Homeodomínio/genética , Neoplasias Hepáticas/genética , Proteínas Musculares/genética , Isoformas de Proteínas , Fatores de Transcrição/genética , Região 5'-Flanqueadora , Sítios de Ligação , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Homeodomínio LIM , Proteínas Musculares/metabolismo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
FHL2 displays tumor promoting or tumor suppressing activities depending on the types of tumor cells. In this study, we demonstrated that FHL2 overexpression inhibits the proliferation of human HCC cells Hep3B through cell cycle regulation by decreasing cyclin D1 expression while increasing the expressions of p21 and p27. FHL2 overexpression also inhibits migration and invasion of Hep3B cells through the regulation of epithelial-mesenchymal transition. Surprisingly, we also demonstrated an antiapoptotic function for FHL2 overexpression with increased resistance to doxorubicin-induced apoptosis, which indicates the separation of anti-proliferative and anti-apoptotic role of FHL2. Taken together, our results indicate FHL2 could exert anti-apoptotic effect independent of tumor growth suppression.
Assuntos
Apoptose/genética , Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Separação Celular , Transição Epitelial-Mesenquimal/genética , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Musculares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , TransfecçãoRESUMO
PURPOSE: Patients with nocturnal enuresis may have small functional bladder capacity or altered bladder fullness sensation. We determined whether reducing bladder volume would affect the central inhibition of voiding that is normally present between birth and 2 weeks of life in neonatal rats. MATERIALS AND METHODS: One and 3-week-old Sprague-Dawley rats underwent 50% bladder volume reduction by suture closure of the bladder dome. T8-T10 spinal cord injury was done in select animals. Latency of the perigenital-bladder reflex, spontaneous voiding onset and body weight were measured. Cystometry using urethane anesthesia, and measurements of in vitro spontaneous and KCl evoked contractions were done. RESULTS: Bladder reduction surgery led to the immediate appearance of spontaneous voiding in 1-week-old rats. Cystometry at 2 weeks showed voiding contractions in rats with bladder reduction, which was abolished by acute T8-T10 spinalization. Voiding contractions were not seen in animals with sham surgery or concurrent T8-T10 spinalization and bladder reduction. The perigenital-bladder reflex, somatic growth, spontaneous bladder contractions and bladder contractility were not affected by bladder reduction. Bladder capacity at 9 weeks was significantly larger in animals that underwent bladder reduction at 1 week than in sham treated animals (540 vs 256 microl, p = 0.04) but not in animals that underwent bladder reduction at 3 weeks. CONCLUSIONS: Bladder reduction removes the central inhibition of spontaneous voiding in neonatal rats. This suggests that decreased neonatal bladder capacity may alter how the brain regulates the bladder.
Assuntos
Bexiga Urinária/fisiologia , Bexiga Urinária/cirurgia , Micção/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Nasopharyngeal carcinoma (NPC) is an Asian-prevalent head and neck cancer with high invasiveness. Although several important risk factors for NPC development have been identified, there is currently no preventive strategy for NPC, even in endemic regions. Signal transducer and activator of transcription 3 (STAT3) has been implicated in NPC carcinogenesis, which may serve as a potential target for cancer prevention. Here, we examined the chemopreventive potential of Cucurbitacin I, a natural-occurring selective inhibitor of JAK/STAT3, in NPC models. We hypothesized that Cucurbitacin I would prevent NPC invasion and tumor formation. Our data demonstrated that brief exposure of NPC cells to Cucurbitacin I was sufficient to significantly reduce the in vitro clonogenicity and in vivo tumorigenicity of NPC cells. The chemopreventive potential of Cucurbitacin I was further demonstrated by pre-dosing of the animals with Cucurbitacin I prior to tumor inoculation, which was found to be able to suppress tumor growth up to 7 days post-inoculation. The anti-proliferation activity of Cucurbitacin I was accompanied by downregulation of phospho-STAT3 and STAT3 target gene expression (e.g. cyclin D1 and Mcl-1). Cucurbitacin I also reduced the invasiveness of invasive NPC cell lines with elevated STAT3 activation. Furthermore, our data demonstrated for the first time that Cucurbitacin I harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against human cancer. Taken together, our results suggested that Cucurbitacin I may be a potent chemopreventive agent for NPC with anti-invasion and anoikis-sensitizing activities.
Assuntos
Anoikis , Carcinoma/metabolismo , Carcinoma/patologia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Triterpenos/metabolismo , Animais , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular , Colágeno/química , Combinação de Medicamentos , Feminino , Humanos , Laminina/química , Camundongos , Camundongos Nus , Neoplasias Nasofaríngeas/tratamento farmacológico , Invasividade Neoplásica , Transplante de Neoplasias , Proteoglicanas/química , Fator de Transcrição STAT3/metabolismo , Triterpenos/farmacologiaRESUMO
PURPOSE: Congenital bladder anomalies are a major challenge to pediatric urologists. Understanding the mechanism of bladder development is crucial for advancing patient treatment. Current evidence suggests that Shh (R&D Systems) is an epithelial signal regulating bladder development, although the mechanism of the regulation is still unclear. We examined the regulation of bladder mesenchymal development. MATERIALS AND METHODS: Mutation analysis, immunohistochemistry, immunoblot, in situ hybridization, and primary cell culture and transfection were performed. The mesenchyma proximal to the epithelium was defined as the inner zone and that distal to the epithelium was defined as the outer zone. RESULTS: We found that the Shh transcriptional factor Gli2 and the Shh target gene Bmp4 (R&D Systems) were expressed in the inner mesenchymal zone of the bladder, where active cell proliferation was observed. In Gli2(-/-) bladder primary mesenchymal cell cultures transfection with adenoviruses expressing DeltaNGli2, a constitutionally active form of Gli2, up-regulated Bmp4 expression and promoted cell proliferation. In the outer mesenchymal zone, where Gli2 and Bmp4 expression was not detectable, smooth muscle alpha-actin was expressed. In Gli2(-/-) embryo bladders Bmp4 expression in the inner zone was lost and ectopic smooth muscle was detected in the inner mesenchymal zone. Exogenous Bmp4 (10 ng/ml) in primary smooth muscle cell culture repressed smooth muscle differentiation and repression was partially rescued by the Bmp4 antagonist Noggin (R&D Systems) (300 ng/ml). CONCLUSIONS: Our data suggests that the Shh transcriptional factor Gli2 regulates radial patterning of the bladder mesenchyma.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Fatores de Transcrição Kruppel-Like/genética , Mesoderma/patologia , Bexiga Urinária/embriologia , Bexiga Urinária/patologia , Análise de Variância , Animais , Proliferação de Células , Modelos Animais de Doenças , Feminino , Proteínas Hedgehog/metabolismo , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Fatores de Transcrição Kruppel-Like/metabolismo , Mesoderma/embriologia , Camundongos , Reação em Cadeia da Polimerase , Gravidez , Prenhez , Probabilidade , RNA/análise , Ratos , Sensibilidade e Especificidade , Transfecção , Proteína Gli2 com Dedos de ZincoRESUMO
Spontaneous bladder contractions (SBCs) in the neonatal rat urinary bladder change from a high-amplitude, low-frequency pattern to a low-amplitude, high-frequency pattern during the first 6 wk of life. Understanding the mechanism of this developmental change may provide insights into the causes of bladder overactivity in adults. In vitro whole bladder preparations from Sprague-Dawley rats were used to study the modulation of SBCs by calcium-activated potassium channels (K(Ca)) and electrical field stimulation from 3 days to 6 wk of life. SBCs in 3-day-old bladders were unmasked by treatment with iberiotoxin (100 nM), an inhibitor of large conductance K(Ca) (BK) channels, or apamin (100 nM), an inhibitor of small conductance K(Ca) (SK) channels. Iberiotoxin significantly increased the magnitude of SBCs at 2-3 wk, whereas apamin was only effective at 6 wk. In 1-2 wk bladders, exposure to room temperature Krebs solution decreased SBCs. This decrease was reversed by activating intramural nerves with electrical field stimulation. The effect of electrical field stimulation was inhibited by atropine (1 microM), suramin (10 microM), or pretreatment with tetrodotoxin (1 microM) but was not reversed by tetrodotoxin applied after electrical field stimulation. BK-alpha mRNA increased threefold, and BK-alpha protein increased fivefold from 3 days to 6 wk. These data suggest that BK channels play an important role in the regulation of SBCs in the neonatal bladder and that both increased BK channel activity, as well as changes in smooth muscle sensitivity to locally released neurotransmitters contribute to the downregulation of SBCs during early postnatal development.
Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Neurônios/fisiologia , Bexiga Urinária/crescimento & desenvolvimento , Bexiga Urinária/inervação , Envelhecimento , Animais , Atropina/farmacologia , Regulação para Baixo , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Suramina/farmacologia , Tetrodotoxina/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiologiaRESUMO
In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in bladders from 1- to 2-wk-old Sprague-Dawley rats were not affected by M2 or M3 receptor antagonists. However, administration of 0.5 microM physostigmine, an anticholinesterase agent that increases the levels of endogenous acetylcholine, or 50-100 nM carbachol, a cholinergic agonist at low concentrations, which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M2 receptors with 0.1 microM AF-DX 116 or 1 microM methoctramine or blockade of M3 receptors with 50 nM 4-diphenylacetoxy-N-methylpiperidine methiodide or 0.1 microM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP mustard) reversed the physostigmine and carbachol responses. M2 and M3 receptor blockade did not alter the facilitation of spontaneous contractions induced by 10 nM BAY K 8644, an L-type Ca2+ channel opener, or 0.1 microM iberiotoxin, a large-conductance Ca2+-activated K+ channel blocker. NS-1619 (30 microM), a large-conductance Ca2+-activated K+ channel opener, decreased carbachol-augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of M2 and M3 receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.
Assuntos
Contração Muscular/fisiologia , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/fisiologia , Bexiga Urinária/inervação , Bexiga Urinária/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Animais Recém-Nascidos , Agonistas dos Canais de Cálcio/farmacologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Inibidores da Colinesterase/farmacologia , Diaminas/farmacologia , Estimulação Elétrica , Contração Muscular/efeitos dos fármacos , Hipertonia Muscular/fisiopatologia , Músculo Liso/inervação , Músculo Liso/fisiologia , Parassimpatolíticos/farmacologia , Peptídeos/farmacologia , Fisostigmina/farmacologia , Piperidinas/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M3/antagonistas & inibidoresRESUMO
Most stored neuropeptide cannot be released from nerve terminals suggesting the existence of a refractory pool of dense core vesicles (DCVs). Past fluorescence photobleaching recovery, single particle tracking and release experiments suggested that the refractory neuropeptide pool corresponds to a distinct immobile fraction of cytoplasmic DCVs. However, tracking of hundreds of individual green fluorescent protein-labeled neuropeptidergic vesicles by wide-field or evanescent-wave microscopy shows that a separate immobile fraction is not evident. Instead, the DCV diffusion coefficient (D) distribution is unusually broad and asymmetric. Furthermore, the distribution shifts with a release facilitator. This unexpected variation, which could reflect heterogeneity among vesicles or in their medium, is shown to generate the appearance of a regulated refractory neuropeptide pool.
Assuntos
Movimento Celular/fisiologia , Microscopia de Fluorescência/métodos , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Neuropeptídeos/metabolismo , Transporte Proteico/fisiologia , Vesículas Secretórias/fisiologia , Animais , Simulação por Computador , Difusão , Células PC12 , Ratos , Reprodutibilidade dos Testes , Vesículas Secretórias/ultraestrutura , Sensibilidade e EspecificidadeRESUMO
It has been speculated that neurosecretion can be enhanced by increasing the motion, and hence, the availability of cytoplasmic secretory vesicles. However, facilitator-induced physical mobilization of secretory vesicles has not been observed directly in living cells, and recent experimental results call this hypothesis into question. Here, high resolution green fluorescent protein (GFP)-based measurements in nerve growth factor-differentiated PC12 cells are used to test whether altering dense core vesicle (DCV) motion affects neuropeptide release. Experiments with mycalolide B and jasplakinolide demonstrate that neuropeptidergic DCV motion at the ends of processes is proportional to F-actin. Furthermore, Ba2+ increases DCV mobility without detectably modifying F-actin. Finally, we show that altering DCV motion by changing F-actin or stimulating with Ba2+ proportionally changes sustained neuropeptide release. Therefore, increasing DCV mobility facilitates prolonged neuropeptide release.
Assuntos
Actinas/fisiologia , Diferenciação Celular/efeitos dos fármacos , Depsipeptídeos , Fator de Crescimento Neural/farmacologia , Neuropeptídeos/metabolismo , Vesículas Secretórias/fisiologia , Actinas/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Toxinas Marinhas , Movimento , Oxazóis/farmacologia , Células PC12 , Peptídeos Cíclicos/farmacologia , Feocromocitoma , Ratos , Proteínas Recombinantes/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/ultraestruturaRESUMO
PC12 cells, like endocrine chromaffin cells, undergo neuronal-like differentiation in response to nerve growth factor (NGF). Here we report that this phenotype conversion produces major changes in release of a green fluorescent protein-tagged neuropeptide-hormone. First, the spatial distribution of the releasable pool is altered; peptide release from untreated cells is supported predominantly by membrane-proximal vesicles, whereas a diffuse pool at the ends of processes is used by NGF-treated cells. Second, the time course of release evoked by photolysis of caged Ca(2+) is faster after differentiation. High-resolution measurements suggest that a slow step before membrane fusion dominates the kinetics of release in untreated cells. Finally, the effect of actin microfilament depolymerization on total release is altered by NGF treatment. This implies that the mechanism that limits the size of the releasable pool is altered by phenotype conversion. Therefore, the cellular organization of peptide release is plastic and changes in response to NGF. This flexibility may be used to generate cell-specific release properties.