Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.198
Filtrar
1.
Chem Biol Interact ; 319: 108979, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32045570

RESUMO

Heart rhythm disturbances have been widely recognized as major triggers of cardiovascular (CV) mortality in chronic kidney disease (CKD) patients. Connexin 43 (Cx43)-composed gap junctions are essential in cardiomyocyte synchronization and may be involved in the pathological response to uremic toxins. Indoxyl sulfate (IS) is one of the most dominant uremic toxins that contribute to CKD-related cardiovascular diseases. In primary cultures of rat neonatal cardiomyocytes, we demonstrated that IS treatment decreased spontaneous contraction without impairing viability. In addition, there was disruption of gap junction intercellular communication (GJIC) between cardiomyocytes after 30 min of IS stimulation. IS caused time- and dose-dependent Cx43 redistribution, and the patterns of Cx43 immunostaining returned to baseline while IS stimulation was removed. Furthermore, IS exposure downregulated Cx43 protein and mRNA levels. Elevated JNK1 and JNK2 phosphorylation was further identified after IS exposure in both rat cardiomyocytes and H9c2 cells. The above changes as well as GJIC and Cx43 suppression were reversed by pretreatment with a JNK inhibitor (SP600125). Inhibition of p-JNK attenuated IS-mediated downward trends in Cx43 transcription and translation. In cardiac muscle from nephrectomy-induced CKD mice, an alteration in Cx43 level was identified at intercalated discs. Our findings disclosed that JNK activation might participate in the remodeling of gap junction and Cx43 expression by uremic toxin-IS both in vitro and in vivo.


Assuntos
Conexina 43/metabolismo , Indicã/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antracenos/farmacologia , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
2.
Cell Physiol Biochem ; 53(4): 606-622, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550088

RESUMO

BACKGROUND/AIMS: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model. METHODS: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity. RESULTS: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate. CONCLUSION: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway.


Assuntos
Conexina 26/metabolismo , Receptor A2B de Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Carbenoxolona/farmacologia , Linhagem Celular , Conexina 26/antagonistas & inibidores , Conexina 26/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espectroscopia Dielétrica , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor A2B de Adenosina/química , Receptor A2B de Adenosina/genética , Transdução de Sinais/efeitos dos fármacos
3.
Nat Commun ; 10(1): 4095, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506439

RESUMO

Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion and pharyngeal pumping, phenotypes requiring FLP-11 neuropeptides and GABA. RIS photoactivation allows the animal to maintain its body posture by sustaining muscle tone, yet inactivating motor neuron oscillatory activity. During locomotion, RIS axonal Ca2+ signals revealed functional compartmentalization: Activity in the nerve ring process correlated with locomotion stop, while activity in a branch correlated with induced reversals. GABA was required to induce, and FLP-11 neuropeptides were required to sustain locomotion stop. RIS attenuates neuronal activity and inhibits movement, possibly enabling sensory integration and decision making, and exemplifies dual use of one cell across development in a compact nervous system.


Assuntos
Caenorhabditis elegans/fisiologia , Cálcio/metabolismo , Neurônios GABAérgicos/metabolismo , Locomoção/fisiologia , Neuropeptídeos/metabolismo , Sono/fisiologia , Animais , Axônios/metabolismo , Caenorhabditis elegans/citologia , Neurônios Colinérgicos/fisiologia , Junções Comunicantes/metabolismo , Luz , Modelos Biológicos , Neurônios Motores/fisiologia , Músculos/citologia , Fenótipo , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismo
4.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370156

RESUMO

Diabetes mellitus is one of the major risk factors for cardiovascular disease and is an important health issue worldwide. Long-term diabetes causes endothelial dysfunction, which in turn leads to diabetic vascular complications. Endothelium-derived nitric oxide is a major vasodilator in large-size vessels, and the hyperpolarization of vascular smooth muscle cells mediated by the endothelium plays a central role in agonist-mediated and flow-mediated vasodilation in resistance-size vessels. Although the mechanisms underlying diabetic vascular complications are multifactorial and complex, impairment of endothelium-dependent hyperpolarization (EDH) of vascular smooth muscle cells would contribute at least partly to the initiation and progression of microvascular complications of diabetes. In this review, we present the current knowledge about the pathophysiology and underlying mechanisms of impaired EDH in diabetes in animals and humans. We also discuss potential therapeutic approaches aimed at the prevention and restoration of EDH in diabetes.


Assuntos
Fatores Biológicos/genética , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio Cálcio-Ativados/genética , Animais , Fatores Biológicos/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Regulação da Expressão Gênica , Humanos , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Canais de Potássio Cálcio-Ativados/metabolismo , Fatores de Risco , Transdução de Sinais , Vasodilatação/efeitos dos fármacos
5.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269652

RESUMO

Growing evidence suggests dietary antioxidants reduce the risk of several cancers. Grape seeds extracts (GSE) are a rich source of polyphenols known to have antioxidant, chemopreventive and anticancer properties. Herein, we investigated the in vitro effects and putative action mechanisms of a grape seed extract (GSE) on human breast cancer cells (MCF-7). The effects of GSE were evaluated on cell proliferation, apoptosis and gap-junction-mediated cell-cell communications (GJIC), as basal mechanism involved in the promotion stage of carcinogenesis. GSE (0.05-100 µg/mL) caused a significant dose- and time-dependent inhibition of MCF-7 viability and induced apoptotic cell death, as detected by Annexin-V/Propidium Iodide. Concurrently, GSE induced transient but significant enhancement of GJIC in non-communicating MCF-7 cells, as demonstrated by the scrape-loading/dye-transfer (SL/DT) assay and an early and dose-dependent re-localization of the connexin-43 (Cx43) proteins on plasma membranes, as assayed by immunocytochemistry. Finally, real-time-PCR has evidenced a significant increase in cx43 mRNA expression. The results support the hypothesis that the proliferation inhibition and pro-apoptotic effect of GSE against this breast cancer cell model are mediated by the GJIC improvement via re-localization of Cx43 proteins and up-regulation of cx43 gene, and provide further insight into the action mechanisms underlying the health-promoting action of dietary components.


Assuntos
Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/prevenção & controle , Extrato de Sementes de Uva/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Comunicação Celular/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Humanos , Células MCF-7 , Regulação para Cima/efeitos dos fármacos
6.
Chin Med J (Engl) ; 132(14): 1700-1705, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31283648

RESUMO

BACKGROUND: More than ten genome-wide association studies have identified the significant association between the gap junction delta-2 (GJD2) gene and myopia. However, no functional studies have been performed to confirm that this gene is correlated with myopia. This study aimed to observe how this gene changed in mRNA and protein level in the form-deprivation myopia (FDM) animal model. METHODS: Four-week-old guinea pigs were randomly divided into two groups: control and FDM groups (n = 12 for each group). The right eyes of the FDM group were covered with opaque hemispherical plastic lenses for 3 weeks. For all the animals, refractive status, axial length (AL), and corneal radius of curvature were measured at baseline and 3 weeks later by streak retinoscope, A-scan ultrasonography, and keratometer, respectively. Retinal GJD2 mRNA expression and connexin 36 (Cx36) levels in FDM and control groups were measured by quantitative real-time PCR and Western blot analyses, respectively. Those results were compared using independent t test, Mann-Whitney U test, or paired t test. A significance level of P < 0.05 was used. RESULTS: Three weeks later, the FDM group (form-deprived eyes) showed about a myopic shift of approximately -6.75 (-7.94 to -6.31) D, while the control group remained hyperopic with only a shift of -0.50 (-0.75 to 0.25) D (Z = -3.38, P < 0.01). The AL increased by 0.74 (0.61-0.76) and 0.10 (0.05-0.21) mm in FDM and control groups, respectively (Z = -3.37, P < 0.01). The relative mRNA expression of GJD2 in the FDM group decreased 31.58% more than the control group (t = 11.44, P < 0.01). The relative protein expression of CX36 on the retina was lowered by 37.72% in form-deprivation eyes as compared to the controls (t = 17.74, P < 0.01). CONCLUSION: Both the mRNA expression of GJD2 and Cx36 protein amount were significantly decreased in the retina of FDM guinea pigs. This indicates that Cx36 is involved in FDM development, providing compensating evidence for the results obtained from genome-wide association studies.


Assuntos
Conexinas/metabolismo , Miopia/metabolismo , RNA Mensageiro/metabolismo , Animais , Western Blotting , Conexinas/genética , Córnea/metabolismo , Modelos Animais de Doenças , Junções Comunicantes/metabolismo , Estudo de Associação Genômica Ampla , Cobaias , Miopia/genética , RNA Mensageiro/genética
7.
Cells ; 8(7)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31247948

RESUMO

Myopia is a substantial public health problem worldwide. Although it is known that defocused images alter eye growth and refraction, their effects on retinal ganglion cell (RGC) signaling that lead to either emmetropization or refractive errors have remained elusive. This study aimed to determine if defocused images had an effect on signaling of RGCs in the mouse retina. ON and OFF alpha RGCs and ON-OFF RGCs were recorded from adult C57BL/6J wild-type mice. A mono green organic light-emitting display presented images generated by PsychoPy. The defocused images were projected on the retina under a microscope. Dark-adapted mouse RGCs were recorded under different powers of projected defocused images on the retina. Compared with focused images, defocused images showed a significantly decreased probability of spikes. More than half of OFF transient RGCs and ON sustained RGCs showed disparity in responses to the magnitude of plus and minus optical defocus (although remained RGCs we tested exhibited similar response to both types of defocus). ON and OFF units of ON-OFF RGCs also responded differently in the probability of spikes to defocused images and spatial frequency images. After application of a gap junction blocker, the probability of spikes of RGCs decreased with the presence of optical defocused image. At the same time, the RGCs also showed increased background noise. Therefore, defocused images changed the signaling of some ON and OFF alpha RGCs and ON-OFF RGCs in the mouse retina. The process may be the first step in the induction of myopia development. It appears that gap junctions also play a key role in this process.


Assuntos
Junções Comunicantes/metabolismo , Miopia/fisiopatologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/fisiologia , Visão Ocular/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Estimulação Luminosa , Células Ganglionares da Retina/citologia
8.
Int J Mol Sci ; 20(11)2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185593

RESUMO

Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive-a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions.


Assuntos
Encéfalo/fisiologia , Modelos Neurológicos , Neuroglia/fisiologia , Animais , Biotina/análogos & derivados , Biotina/farmacocinética , Encéfalo/citologia , Feminino , Junções Comunicantes/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/citologia
9.
Invest Ophthalmol Vis Sci ; 60(7): 2525-2531, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31195409

RESUMO

Purpose: We reported previously that aquaporin 0 (AQP0) modulates lens fiber cell gap junction (GJ) channel function. The present study was conducted to find out whether the C-terminal end of AQP0 is involved in this regulation. Methods: A mouse model, AQP0ΔC/ΔC, was genetically engineered to express AQP0 with 1-246 amino acids, without the normal intact AQP0 (1-263 amino acids) in the lens. Transparency and focusing of the lens were assessed. Intracellular impedance was measured to determine GJ coupling resistance. Intracellular hydrostatic pressure (HP) was also determined. Western blotting was performed to determine connexin (Cx46 and Cx50) expression levels. Results: At postnatal day 10, AQP0ΔC/ΔC mouse lenses relative to age-matched wild-type lenses showed loss of transparency and abnormal optical distortion; GJ coupling resistance increased in the differentiating (1.6-fold) and mature (8-fold) fiber cells; lens HP increased approximately 1.5-fold at the junction between the differentiating and mature fiber cells and approximately 2.0-fold in the center; there was no significant change (P > 0.05) in expression levels of Cx46 or Cx50. Conclusions: The increase in GJ coupling resistance was not associated with reduced connexin expression, suggesting either a reduction in the open probability or some physical change in plaque location. The increase in resistance was significantly greater than the increase in HP, suggesting less pressure-driven water flow through each open GJ channel. These changes may lead to a loss of transparency and abnormal optical distortion. Overall, our data demonstrate the C-terminal end of AQP0 is involved in modulating GJ coupling to maintain lens transparency and homeostasis.


Assuntos
Aquaporinas/fisiologia , Proteínas do Olho/fisiologia , Junções Comunicantes/metabolismo , Cristalino/fisiologia , Animais , Aquaporinas/química , Western Blotting , Catarata/patologia , Conexinas/metabolismo , Proteínas do Olho/química , Técnicas de Introdução de Genes , Homeostase , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína
10.
Brain Struct Funct ; 224(6): 2183-2197, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31172263

RESUMO

Connexin-36 (Cx36) is the major constituent of mammalian retinal gap junctions positioned in key signal pathways. Here, we examined the laminar and large-scale topographical distribution of Cx36 punctate immunolabels in the retina of the cat, a classical model of the mammalian visual system. Calretinin-immunoreactive (CaR-IR) cell populations served to outline the nuclear and plexiform layers and to stain specific neuronal populations. CaR-IR cells included horizontal cells in the outer retina, numerous amacrine cells, and scattered cells in the ganglion cell layer. Cx36-IR plaques were found among horizontal cell dendrites albeit without systematic colocalization of the two labels. Diffuse Cx36 immunoreactivity was found in the cytoplasm of AII amacrine cells, but no colocalization of Cx36 plaques was observed with either the perikarya or the long varicose dendrites of the CaR-IR non-AII amacrine cells. Cx36 puncta were seen throughout the entire inner plexiform layer showing their highest density in the ON sublamina. The densities of AII amacrine cell bodies and Cx36 plaques in the ON sublamina were strongly correlated across a wide range of eccentricities suggesting their anatomical association. However, the high number of plaques per AII cell suggests that a considerable fraction of Cx36 gap junctions in the ON sublamina is formed by other cell types than AII amacrine cells drawing attention to extensive but less studied electrically coupled networks.


Assuntos
Conexinas/metabolismo , Dendritos/metabolismo , Retina/metabolismo , Vias Visuais/fisiologia , Células Amácrinas/metabolismo , Animais , Calbindina 2/metabolismo , Gatos , Junções Comunicantes/metabolismo , Imuno-Histoquímica/métodos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo
11.
Int J Mol Sci ; 20(12)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216630

RESUMO

An imbalance of excitatory and inhibitory neurotransmission leading to over excitation plays a crucial role in generating seizures, while enhancing GABAergic mechanisms are critical in terminating seizures. In recent years, it has been reported in many studies that astrocytes are deeply involved in synaptic transmission. Astrocytes form a critical component of the "tripartite" synapses by wrapping around the pre- and post-synaptic elements. From this location, astrocytes are known to greatly influence the dynamics of ions and transmitters in the synaptic cleft. Despite recent extensive research on excitatory tripartite synapses, inhibitory tripartite synapses have received less attention, even though they influence inhibitory synaptic transmission by affecting chloride and GABA concentration dynamics. In this review, we will discuss the diverse actions of astrocytic chloride and GABA homeostasis at GABAergic tripartite synapses. We will then consider the pathophysiological impacts of disturbed GABA homeostasis at the tripartite synapse.


Assuntos
Astrócitos/metabolismo , Neurônios GABAérgicos/metabolismo , Receptores de GABA/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Transporte Biológico , Junções Comunicantes/metabolismo , Humanos , Transmissão Sináptica , Canais de Ânion Dependentes de Voltagem/metabolismo
12.
Int J Mol Sci ; 20(10)2019 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-31109150

RESUMO

Gap junction (GJ) channels in invertebrates have been used to understand cell-to-cell communication in vertebrates. GJs are a common form of intercellular communication channels which connect the cytoplasm of adjacent cells. Dysregulation and structural alteration of the gap junction-mediated communication have been proven to be associated with a myriad of symptoms and tissue-specific pathologies. Animal models relying on the invertebrate nervous system have exposed a relationship between GJs and the formation of electrical synapses during embryogenesis and adulthood. The modulation of GJs as a therapeutic and clinical tool may eventually provide an alternative for treating tissue formation-related diseases and cell propagation. This review concerns the similarities between Hirudo medicinalis innexins and human connexins from nucleotide and protein sequence level perspectives. It also sets forth evidence of computational techniques applied to the study of proteins, sequences, and molecular dynamics. Furthermore, we propose machine learning techniques as a method that could be used to study protein structure, gap junction inhibition, metabolism, and drug development.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Animais , Simulação por Computador , Conexinas/análise , Conexinas/antagonistas & inibidores , Junções Comunicantes/química , Humanos , Aprendizado de Máquina , Modelos Biológicos , Sistema Nervoso/química , Sistema Nervoso/metabolismo , Conformação Proteica
13.
Invest Ophthalmol Vis Sci ; 60(6): 2336-2346, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31117126

RESUMO

Purpose: Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation. Methods: Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca2+]i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images. Results: In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca2+]i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts. Conclusions: Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.


Assuntos
Cálcio/metabolismo , Catarata/metabolismo , Conexinas/fisiologia , Cristalino/metabolismo , Animais , Conexinas/metabolismo , Modelos Animais de Doenças , Junções Comunicantes/metabolismo , Camundongos
14.
Mol Med Rep ; 19(6): 5177-5184, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059036

RESUMO

Gap junctions (GJs) and tight junctions (TJs) are essential to maintain the function of hepatocytes. Changes in biliary tract pressure and the effect of lipopolysaccharide (LPS) may lead to acute obstructive cholangitis (AOC) and cause liver injury via GJ and TJ dysfunction. Hydrogen has been confirmed to have a protective role in various organs during pathological conditions and inflammation. The present study investigated the function of junction proteins and the potential application of H2 in AOC­induced liver injury. An AOC rat model was established by LPS injection through a bile duct catheter, while the distal bile duct was closed. The catheter sealing caps were removed and bile was allowed to flow out from the catheters at 12 h after LPS infusion. The potential application of H2 was studied in the AOC rat model with biliary drainage. It was observed that AOC induced the disruption of junction proteins of both GJs and TJs. H2 administration reversed AOC­induced disruption of GJs and TJs after biliary drainage. The mechanism of this phenomenon suggests that H2 may have effectively attenuated AOC­induced inflammatory and oxidative damage, and decreased matrix metalloproteinase activity. H2 may accelerate the reversal of AOC­induced liver dysfunction, and this phenomenon may depend on reversing the inhibition of GJs and TJs.


Assuntos
Colangite/patologia , Junções Comunicantes/metabolismo , Hidrogênio/farmacologia , Hepatopatias/etiologia , Junções Íntimas/metabolismo , Doença Aguda , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Colangite/complicações , Colangite/tratamento farmacológico , Conexinas/metabolismo , Modelos Animais de Doenças , Hidrogênio/química , Hidrogênio/uso terapêutico , Lipopolissacarídeos/toxicidade , Fígado/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Solução Salina/química , Proteína da Zônula de Oclusão-1/metabolismo
15.
Environ Pollut ; 251: 328-337, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31091496

RESUMO

Microcystin-leucine-arginine (MC-LR) can cause male reproductive disorder. However, the underlying mechanism are not yet entirely elucidated. In this study, we aimed to investigated the effects of MC-LR on the integrity of blood-testis barrier (BTB) and the related molecular mechanisms. Both in vivo and in vitro experiments revealed that MC-LR caused disruption of BTB and gap junctions between Sertoli cells respectively, which was paralleled by the alteration of connexin43 (Cx43). Our data demonstrated that MC-LR decreased gap junction intercellular communication (GJIC) and impaired Cx43 expression by activating the phosphatidylinositol 3-kinase/Akt cascades. In addition, a possible protective effect of Icariin (ICA), a flavonoid isolated from Chinese medicinal herb, against MC-LR toxicity was investigated. The ICA prevented the degradation of GJIC and impairment of Cx43 induced by MC-LR via suppressing the Akt pathway. Together, our results confirmed that the expression of Cx43 induced by MC-LR was regulated in vivo and in vitro, which was involved in the destruction of BTB. Additionally, ICA seems to be able to mitigate the MC-LR toxic effects.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Flavonoides/farmacologia , Junções Comunicantes/efeitos dos fármacos , Microcistinas/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Sertoli/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Masculino , Camundongos , Células de Sertoli/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
PLoS Genet ; 15(5): e1008153, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31071084

RESUMO

Electrical synapses between neurons, also known as gap junctions, are direct cell membrane channels between adjacent neurons. Gap junctions play a role in the synchronization of neuronal network activity; however, their involvement in cognition has not been well characterized. Three-hour olfactory associative memory in Drosophila has two components: consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). Here, we show that knockdown of the gap junction gene innexin5 (inx5) in mushroom body (MB) neurons disrupted ARM, while leaving ASM intact. Whole-mount brain immunohistochemistry indicated that INX5 protein was preferentially expressed in the somas, calyxes, and lobes regions of the MB neurons. Adult-stage-specific knockdown of inx5 in αß neurons disrupted ARM, suggesting a specific requirement of INX5 in αß neurons for ARM formation. Hyperpolarization of αß neurons during memory retrieval by expressing an engineered halorhodopsin (eNpHR) also disrupted ARM. Administration of the gap junction blocker carbenoxolone (CBX) reduced the proportion of odor responsive αß neurons to the training odor 3 hours after training. Finally, the α-branch-specific 3-hour ARM-specific memory trace was also diminished with CBX treatment and in inx5 knockdown flies. Altogether, our results suggest INX5 gap junction channels in αß neurons for ARM retrieval and also provide a more detailed neuronal mechanism for consolidated memory in Drosophila.


Assuntos
Conexinas/genética , Sinapses Elétricas/fisiologia , Corpos Pedunculados/metabolismo , Animais , Encéfalo/metabolismo , Carbenoxolona/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Sinapses Elétricas/metabolismo , Junções Comunicantes/metabolismo , Junções Comunicantes/fisiologia , Memória/fisiologia , Corpos Pedunculados/fisiologia , Neurônios/metabolismo , Odorantes , Olfato/genética , Transmissão Sináptica/fisiologia
17.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 555-561, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31056639

RESUMO

Neuropathic pain is caused by the damage or dysfunction of the nervous system. In many neuropathic pain models, there is an increase in the number of gap junction (GJ) channels, especially the upregulation of the expression of connexin43 (Cx43), leading to the secretion of various types of cytokines and involvement in the formation of neuropathic pain. GJs are widely distributed in mammalian organs and tissues, and Cx43 is the most abundant connexin (Cx) in mammals. Astrocytes are the most abundant glial cell type in the central nervous system (CNS), which mainly express Cx43. More importantly, GJs play an important role in regulating cell metabolism, signaling, and function. Many existing literatures showed that Cx43 plays an important role in the nervous system, especially in the CNS under normal and pathological conditions. However, many internal mechanisms have not yet been thoroughly explored. In this review, we summarized the current understanding of the role and association of Cx and pannexin channels in neuropathic pain, especially after spinal cord injury, as well as some of our own insights and thoughts which suggest that Cx43 may become an emerging therapeutic target for future neuropathic pain, bringing new hope to patients.


Assuntos
Astrócitos/efeitos dos fármacos , Conexina 43/metabolismo , Neuralgia/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Astrócitos/metabolismo , Carbenoxolona/uso terapêutico , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Conexina 43/antagonistas & inibidores , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Humanos , Terapia de Alvo Molecular/métodos , Neuralgia/fisiopatologia , Neuralgia/prevenção & controle , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia
18.
Cancer Sci ; 110(6): 1947-1958, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012516

RESUMO

MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18-27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA-34a. Additionally, these effects of microRNA-34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.


Assuntos
Comunicação Celular/genética , Proliferação de Células/genética , Junções Comunicantes/metabolismo , MicroRNAs/genética , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/genética , Técnicas de Cocultura , Conexina 43/genética , Conexinas/genética , Glioma/genética , Glioma/patologia , Células HeLa , Humanos , Interferência de RNA
19.
BMC Evol Biol ; 19(Suppl 1): 46, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30813901

RESUMO

BACKGROUND: Gap junctions (GJ) are one of the most common forms of intercellular communication. GJs are assembled from proteins that form channels connecting the cytoplasm of adjacent cells. They are considered to be the main or the only type of intercellular channels and the universal feature of all multicellular animals. Two unrelated protein families are currently considered to be involved in this function, namely, connexins and pannexins (pannexins/innexins). Pannexins were hypothesized to be the universal GJ proteins of multicellular animals, distinct from connexins that are characteristic of chordates only. Here we have revised this supposition by applying growing high throughput sequencing data from diverse metazoan species. RESULTS: Pannexins were found in Chordates, Ctenophores, Cnidarians, and in the most major groups of bilateral protostomes. Yet some metazoans appear to have neither connexins nor pannexins in their genomes. We detected no connexins or pannexins/innexins homologues in representatives of all five classes of echinoderms and their closest relatives hemichordates with available genomic sequences. Despite this, our intracellular recordings demonstrate direct electrical coupling between blastomeres at the 2-cell embryo of the echinoderm (starfish Asterias rubens). In these experiments, carboxyfluorescein fluorescent dye did not diffuse between electrically coupled cells. This excludes the possibility that the observed electrical coupling is mediated by incomplete cytoplasm separation during cleavage. CONCLUSION: Functional GJs are present in representatives of the clade that lack currently recognized GJ protein families. New undiscovered protein families utilized for intercellular channels are predicted. It is possible that the new type(s) of intercellular channels are present in parallel to pannexin and connexin gap junctions in animal groups, other than Echinodermata.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Animais , Comunicação Celular , Equinodermos/citologia , Equinodermos/genética , Equinodermos/metabolismo , Genoma
20.
Oncogene ; 38(23): 4429-4451, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30814684

RESUMO

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Neoplasias/metabolismo , Animais , Carcinogênese , Comunicação Celular , Diferenciação Celular , Membrana Celular/metabolismo , Proliferação de Células , Citosol/metabolismo , Regulação da Expressão Gênica , Homeostase , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/citologia , Prognóstico , Domínios Proteicos , Isoformas de Proteínas , Pesquisa Médica Translacional , Resultado do Tratamento , Microambiente Tumoral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA