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Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.
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The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.
Assuntos
Movimento Celular/fisiologia , Conexinas/fisiologia , Células Dendríticas/fisiologia , Leucócitos/fisiologia , Fagócitos/fisiologia , Nucleotídeos de Adenina/fisiologia , Envelhecimento/imunologia , Envelhecimento/fisiologia , Animais , Astrócitos/fisiologia , Polaridade Celular , Quimiotaxia de Leucócito/fisiologia , Citoesqueleto/fisiologia , Fibroblastos/fisiologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Queratinócitos/fisiologia , Mecanotransdução Celular/fisiologia , Neoplasias/imunologia , Degeneração Neural/imunologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Purinérgicos/fisiologiaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive T and B cells, autoantibody production, and immune complex deposition in various organs. Previous evidence showed abnormal accumulation of B cells in the thymus of lupus-prone mice, but the role of this population in the progression of the disease remains mostly undefined. Here we analyzed the spatial distribution, function, and properties of this thymic B cell population in the BWF1 murine model of SLE. We found that in diseased animals, thymic B cells proliferate, and cluster in structures that resemble ectopic germinal centers. Moreover, we detected antibody-secreting cells in the thymus of diseased-BWF1 mice that produce anti-dsDNA IgG autoantibodies. We also found that thymic B cells from diseased-BWF1 mice induced the differentiation of thymocytes to follicular helper T cells (TFH). These data suggest that the accumulation of B cells in the thymus of BWF1 mice results in the formation of germinal center-like structures and the expansion of a TFH population, which may, in turn, activate and differentiate B cells into autoreactive plasma cells. Therefore, the thymus emerges as an important niche that supports the maintenance of the pathogenic humoral response in the development of murine SLE.
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Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Humoral , Lúpus Eritematoso Sistêmico/imunologia , Células T Auxiliares Foliculares/imunologia , Timo/imunologia , Animais , Autoanticorpos/imunologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , DNA/imunologia , Modelos Animais de Doenças , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Plasmócitos/imunologiaRESUMO
Mast cells (MCs) release pro-inflammatory mediators through a process called degranulation response. The latter may be induced by several conditions, including antigen recognition through immunoglobulin E (IgE) or "cross-linking," classically associated with Type I hypersensitivity reactions. Early in this reaction, Ca2+ influx and subsequent increase of intracellular free Ca2+ concentration are essential for MC degranulation. Several membrane channels that mediate Ca2+ influx have been proposed, but their role remains elusive. Here, we evaluated the possible contribution of pannexin-1 channels (Panx1 Chs), well-known as ATP-releasing channels, in the increase of intracellular Ca2+ triggered during cross-linking reaction of MCs. The contribution of Panx1 Chs in the degranulation response was evaluated in MCs from wild type (WT) and Panx1 knock out (Panx1-/-) mice after anti-ovalbumin (OVA) IgE sensitization. Notably, the degranulation response (toluidine blue and histamine release) was absent in Panx1-/- MCs. Moreover, WT MCs showed a rapid and transient increase in Ca2+ signal followed by a sustained increase after antigen stimulation. However, the sustained increase in Ca2+ signal triggered by OVA was absent in Panx1-/- MCs. Furthermore, OVA stimulation increased the membrane permeability assessed by dye uptake, a prevented response by Panx1 Ch but not by connexin hemichannel blockers and without effect on Panx1-/- MCs. Interestingly, the increase in membrane permeability of WT MCs was also prevented by suramin, a P2 purinergic inhibitor, suggesting that Panx1 Chs act as ATP-releasing channels impermeable to Ca2+. Accordingly, stimulation with exogenous ATP restored the degranulation response and sustained increase in Ca2+ signal of OVA stimulated Panx1-/- MCs. Moreover, opening of Panx1 Chs in Panx1 transfected HeLa cells increased dye uptake and ATP release but did not promote Ca2+ influx, confirming that Panx1 Chs permeable to ATP are not permeable to Ca2+. These data strongly suggest that during antigen recognition, Panx1 Chs contribute to the sustained Ca2+ signal increase via release of ATP that activates P2 receptors, playing a critical role in the sequential events that leads to degranulation response during Type I hypersensitivity reactions.
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Degranulação Celular/fisiologia , Conexinas/imunologia , Hipersensibilidade Imediata/imunologia , Mastócitos/imunologia , Proteínas do Tecido Nervoso/imunologia , Animais , Conexinas/metabolismo , Células HeLa , Humanos , Hipersensibilidade Imediata/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismoRESUMO
OBJECTIVE: Pregestational maternal obesity (PGMO) associates with foetoplacental vascular endothelial dysfunction and higher risk for insulin resistance in the neonate. We characterised the PGMO consequences on the insulin response of the human foetoplacental vasculature. METHODS: Umbilical veins were from pregnancies where the mother was with PGMO (body mass index 30-42.3â¯kg/m2, nâ¯=â¯33) or normal pregestational weight (PGMN) (body mass index 19.5-24.4â¯kg/m2, nâ¯=â¯21) with total gestational weight gain within the physiological range. Umbilical vein ring segments were mounted in a myograph for isometric force measurements. Primary cultures of human umbilical vein endothelial cells were used in passage 3. Vessel rings and cells were exposed to 1â¯nmol/L insulin (20â¯min) in the absence or presence of 100⯵mol/L NG-nitro-l-arginine methyl ester (inhibitor of nitric oxide synthase, NOS). RESULTS: Vessel rings from PGMO showed reduced nitric oxide synthase-activity dependent dilation to insulin or calcitonin-gene related peptide compared with PGMN. PGMO associated with higher inhibitor phosphorylation of the insulin receptor substrate 1 (IRS-1) and lower activator phosphorylation of protein kinase B/Akt (Akt). Cells from PGMO also showed lower nitric oxide level and reduced activator serine1177 but increased inhibitor threonine495 phosphorylation of endothelial nitric oxide synthase (eNOS) and saturable transport of l-arginine. HUVECs from PGMO were not responsive to insulin. CONCLUSION: The lack of response to insulin by the foetoplacental endothelium may result from reduced IRS-1/Akt/eNOS signalling in PGMO. These findings may result in higher risk of insulin resistance in neonates to PGMO pregnancies.
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Endotélio Vascular/fisiopatologia , Insulina , Obesidade/fisiopatologia , Complicações na Gravidez/fisiopatologia , Veias Umbilicais/fisiopatologia , Adulto , Arginina/metabolismo , Estudos de Casos e Controles , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Recém-Nascido , Proteínas Substratos do Receptor de Insulina/metabolismo , Miografia , Gravidez , Cultura Primária de Células , Adulto JovemRESUMO
Obesity associates with the endoplasmic reticulum (ER) stress-induced endothelial dysfunction. Pregnant women with pre-pregnancy maternal obesity (PGMO) may transfer this potential risk to their offspring; however, whether ER stress occurs and associates with foetoplacental endothelial dysfunction in PGMO is unknown. We studied the l-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs) from women with PGMO or with a normal pre-pregnancy weight. We analysed the expression and activation of the ER stress sensors protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). PGMO associated with lower endothelial NO synthase activity due to increased Thr495-inhibitor and decreased Ser1177-stimulator phosphorylation. However, higher expression and activity of the human cationic amino acid transporter 1 was found. PGMO caused activation of PERK and its downstream targets eukaryotic initiation factor 2 (eIF2α), C/EBP homologous protein 10 (CHOP), and tribbles-like protein 3 (TRB3). Increased IRE1α protein abundance (but not its phosphorylation or X-box binding protein 1-mRNA splicing) and increased c-Jun N-terminal kinase 1 phosphorylation was seen in PGMO. A preferential nuclear location of the activating transcription factor 6 (ATF6) was found in HUVECs from PGMO. All the changes seen in PGMO were blocked by TUDCA but unaltered by tunicamycin. Thus, PGMO may determine a state of ER stress via upregulation of the PERK-eIF2α-CHOP-TRB3 axis signalling in HUVECs. This phenomenon results in foetoplacental vascular endothelial dysfunction at birth.
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Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Fator 6 Ativador da Transcrição/metabolismo , Adulto , Arginina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endorribonucleases/metabolismo , Feminino , Humanos , Óxido Nítrico/metabolismo , Gravidez , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição CHOP/metabolismo , Adulto Jovem , eIF-2 Quinase/metabolismoRESUMO
The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal â¼1 h after scratch. Two connexin hemichannel blockers, La(3+) and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at â¼17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K(+) . The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.
Assuntos
Astrócitos/metabolismo , Conexina 43/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Astrócitos/efeitos dos fármacos , Lesões Encefálicas , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Camundongos Knockout , Peptídeos/administração & dosagem , Potássio/metabolismo , Antagonistas do Receptor Purinérgico P2/farmacologia , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismoRESUMO
Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.
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Células Apresentadoras de Antígenos/metabolismo , Conexinas/metabolismo , Citocinas/metabolismo , Animais , Junções Comunicantes/metabolismo , HumanosRESUMO
Adverse microenvironmental stimuli can trigger the endoplasmic reticulum (ER) stress pathway, which initiates the unfolded protein response (UPR), to restore protein-folding homeostasis. Several studies show induction of ER stress during obesity. Chronic UPR has been linked to different mechanisms of disease in obese and diabetic individuals, including insulin resistance (IR) and impaired angiogenesis. Endothelial cell (EC) migration is an initial step for angiogenesis, which is associated with remodeling of existing blood vessels. EC migration occurs according to the leader-follower model, involving coordinated processes of chemotaxis, haptotaxis, and mechanotaxis. Thus, a fine-tuning of EC migration is necessary to provide the right timing to form the required vessels during angiogenesis. ER stress modulates EC migration at different levels, usually impairing migration and angiogenesis, although different effects may be observed depending on the tissue and/or microenvironment. In the context of pregnancy, maternal obesity (MO) induces IR in the offspring. Interestingly, several proteins associated with obesity-induced IR are also involved in EC migration, providing a potential link with the ER stress-dependent alterations observed in obese individuals. Different signaling cascades that converge on cytoskeleton regulation directly impact EC migration, including the Akt and/or RhoA pathways. In addition, ER is the main intracellular reservoir for Ca(2+), which plays a pivotal role during EC migration. Therefore, ER stress-related alterations in Ca(2+) signaling or Ca(2+) levels might also produce distorted EC migration. However, the above findings have been studied in the context of adult obesity, and no information has been reported regarding the effect of MO on fetal EC migration. Here we summarize the state of knowledge about the possible mechanisms by which ER stress and IR might impact EC migration and angiogenesis in fetal endothelium exposed to MO during pregnancy.
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Satellite glial cells (SGCs) are the main glia in sensory ganglia. They surround neuronal bodies and form a cap that prevents the formation of chemical or electrical synapses between neighboring neurons. SGCs have been suggested to establish bidirectional paracrine communication with sensory neurons. However, the molecular mechanism involved in this cellular communication is unknown. In the central nervous system (CNS), astrocytes present connexin43 (Cx43) hemichannels and pannexin1 (Panx1) channels, and the opening of these channels allows the release of signal molecules, such as ATP and glutamate. We propose that these channels could play a role in glia-neuron communication in sensory ganglia. Therefore, we studied the expression and function of Cx43 and Panx1 in rat and mouse nodose-petrosal-jugular complexes (NPJcs) using confocal immunofluorescence, molecular and electrophysiological techniques. Cx43 and Panx1 were detected in SGCs and in sensory neurons, respectively. In the rat and mouse, the electrical activity of vagal nerve increased significantly after nodose neurons were exposed to a Ca(2+)/Mg(2+)-free solution, a condition that increases the open probability of Cx hemichannels. This response was partially mimicked by a cell-permeable peptide corresponding to the last 10 amino acids of Cx43 (TAT-Cx43CT). Enhanced neuronal activity was reduced by Cx hemichannel, Panx1 channel and P2X7 receptor blockers. Moreover, the role of Panx1 was confirmed in NPJc, because in those from Panx1 knockout mice showed a reduced increase of neuronal activity induced by Ca(2+)/Mg(2+)-free extracellular conditions. The data suggest that Cx hemichannels and Panx channels serve as paracrine communication pathways between SGCs and neurons by modulating the excitability of sensory neurons.
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Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X 7 receptors (P2X 7Rs). However, a link between P2X 7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1-/- mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1-/- mice, in which levels of P2X 7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X 7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.
Assuntos
Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Linfócitos T/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Calcimicina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Conexinas/antagonistas & inibidores , Conexinas/genética , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Imagem com Lapso de TempoRESUMO
Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1â»/â») showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1â»/â» hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1â»/â» astrocytes also showed more intracellular Ca²âº signal oscillations mediated by functional connexin 43 hemichannels and P2Y1 receptors. Therefore, Npc1â»/â» astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas/genética , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Sinalização do Cálcio , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Junções Comunicantes/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/patologia , Proteínas/metabolismo , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Técnicas de Cultura de TecidosRESUMO
Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-α/IFN-γ-induced dye coupling, probably through the induction of IL-1ß release. Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.
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Citocinas/farmacologia , Junções Comunicantes/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Trifosfato de Adenosina , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Camundongos , Microglia/citologia , Ratos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Therapeutic approaches for CKD (chronic kidney disease) have been able to reduce proteinuria, but not diminish the disease progression. We have demonstrated beneficial effects by injection of BM (bone marrow)-derived MSCs (mesenchymal stem cells) from healthy donors in a rat model with CKD. However, it has recently been reported that BM-MSCs derived from uraemic patients failed to confer functional protection in a similar model. This suggests that autologous BM-MSCs are not suitable for the treatment of CKD. In the present study, we have explored the potential of MSCs derived from adipose tissue (AD-MSCs) as an alternative source of MSCs for the treatment of CKD. We have isolated AD-MSCs and evaluated their effect on the progression of CKD. Adult male SD (Sprague-Dawley) rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×10(6) AD-MSCs or MSC culture medium alone. The therapeutic effect was evaluated by plasma creatinine measurement, structural analysis and angiogenic/epitheliogenic protein expression. AD-MSCs were detected in kidney tissues from NPX animals. This group had a significant reduction in plasma creatinine levels and a lower expression of damage markers ED-1 and α-SMA (α-smooth muscle actin) (P<0.05). In addition, treated rats exhibited a higher level of epitheliogenic [Pax-2 and BMP-7 (bone morphogenetic protein 7)] and angiogenic [VEGF (vascular endothelial growth factor)] proteins. The expression of these biomarkers of regeneration was significantly related to the improvement in renal function. Although many aspects of the cell therapy for CKD remain to be investigated, we provide evidence that AD-MSCs, a less invasive and highly available source of MSCs, exert an important therapeutic effect in this pathology.
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Tecido Adiposo/citologia , Falência Renal Crônica/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Falência Renal Crônica/patologia , Falência Renal Crônica/fisiopatologia , Masculino , Neovascularização Fisiológica , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição PAX2/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Connexin hemichannel (Cx HC) opening is involved in physiological and pathological processes, allowing the cellular release of autocrine/paracrine signaling molecules. Linoleic acid (LA) is known to modulate the functional state of connexin46 (Cx46) HCs. However, the molecular mechanism involved in this effect, or whether LA affects HCs constituted of other connexins, remains unknown. Here, we report the effects of LA on HCs in HeLa cells that express Cx26, one of the main Cxs in the cochlear sensory epithelium. Cx26 HC activity (dye uptake) was increased in a concentration-dependent manner by bath application of LA and inhibited by HC blockers. Moreover, intracellular BAPTA, a Ca(2+) chelator, and PI3K/AKT inhibitors were found to reduce the LA-induced Cx26 HC opening, suggesting that the LA effect is mediated by an increase of free intracellular Ca(2+) concentration and activation of the PI3K/Akt-dependent pathway. The LA-induced increase in free intracellular Ca(2+) concentration was mainly due to Ca(2+) influx through Cx26 HCs. In addition, the involvement of SH groups was ruled out, because dithiothreitol (DTT) did not block the LA-induced dye uptake. LA also increased the membrane current mediated by Cx26 HCs expressed in Xenopus oocytes and the dye uptake in HeLa cells expressing Cxs 32, 43 or 45. Since LA is an essential polyunsaturated fatty acid, its effect on HCs might be relevant to cell growth as well as to cellular functions of differentiated cells such as audition.
Assuntos
Cálcio/química , Conexinas/química , Ácido Linoleico/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Biotinilação , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio , Conexina 26 , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Ácidos Graxos Insaturados/metabolismo , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Oócitos/metabolismo , Estrutura Terciária de Proteína , Fatores de Tempo , XenopusRESUMO
The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose transporter 2, glucokinase, and ATP-sensitive K(+) (K(ATP) ) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose-induced rise of intracellular free Ca(2+) concentration ([Ca(2+) ](i) ) in cultured ß-tanycytes was examined. Fura-2AM time-lapse fluorescence images revealed that glucose increases the intracellular Ca(2+) signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K(ATP) channel-dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y(1) receptors followed by inositol trisphosphate receptor activation and Ca(2+) release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca(2+) ](i) in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Conexina 43/metabolismo , Glucose/farmacologia , Líquido Intracelular/metabolismo , Neuroglia/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Cátions/metabolismo , Células Cultivadas , Conexina 43/antagonistas & inibidores , Citocalasina B/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glucoquinase/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Hipotálamo/citologia , Antígeno Ki-67/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia Confocal , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Técnicas de Patch-Clamp , Probenecid/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de von Willebrand/metabolismoRESUMO
CKD (chronic kidney disease) has become a public health problem. The therapeutic approaches have been able to reduce proteinuria, but have not been successful in limiting disease progression. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of MSC (mesenchymal stem cells) on the progression of CKD and the expression of molecular biomarkers associated with regenerative effects. Adult male Sprague-Dawley rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×106 MSC or culture medium. A sham group subjected to the same injection was used as the control. Rats were killed 5 weeks after MSC infusion. Dye tracking of MSC was followed by immunofluorescence analysis. Kidney function was evaluated using plasma creatinine. Structural damage was evaluated by H&E (haematoxylin and eosin) staining, ED-1 abundance (macrophages) and interstitial α-SMA (α-smooth muscle actin). Repairing processes were evaluated by functional and structural analyses and angiogenic/epitheliogenic protein expression. MSC could be detected in kidney tissues from NPX animals treated with intravenous cell infusion. This group presented a marked reduction in plasma creatinine levels and damage markers ED-1 and α-SMA (P<0.05). In addition, treated rats exhibited a significant induction in epitheliogenic [Pax-2, bFGF (basic fibroblast growth factor) and BMP-7 (bone morphogenetic protein-7)] and angiogenic [VEGF (vascular endothelial growth factor) and Tie-2] proteins. The expression of these biomarkers of regeneration was significantly related to the increase in renal function. Many aspects of the cell therapy in CKD remain to be investigated in more detail: for example, its safety, low cost and the possible need for repeated cell injections over time. Beyond the undeniable importance of these issues, what still needs to be clarified is whether MSC administration has a real effect on the treatment of this pathology. It is precisely to this point that the present study aims to contribute.
Assuntos
Falência Renal Crônica/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Proteína Morfogenética Óssea 7/metabolismo , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/metabolismo , Rim/metabolismo , Rim/fisiologia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Masculino , Células-Tronco Mesenquimais/fisiologia , Fator de Transcrição PAX2/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor TIE-2/metabolismo , Regeneração/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The mechanisms involved in Alzheimer's disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-ß peptide (Aß) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aß. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aß(25-35) increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aß. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aß(25-35)-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aß(25-35)-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aß leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aß.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Morte Celular/fisiologia , Neuroglia/fisiologia , Neurônios/patologia , Fragmentos de Peptídeos/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Conexina 43/antagonistas & inibidores , Conexina 43/deficiência , Conexina 43/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/fisiologiaRESUMO
In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process.
Assuntos
Junções Comunicantes/metabolismo , Junções Comunicantes/fisiologia , Degeneração Neural/metabolismo , Animais , Humanos , Degeneração Neural/patologia , Transdução de Sinais/fisiologiaRESUMO
Astrocytes have a role in maintaining normal neuronal functions, some of which depend on connexins, protein subunits of gap junction channels and hemichannels. Under inflammatory conditions, microglia release cytokines, including interleukin-1beta and tumor necrosis factor-alpha, that reduce intercellular communication via gap junctions. Now, we demonstrate that either conditioned medium harvested from activated microglia or a mixture of these two cytokines enhances the cellular exchange with the extracellular milieu via Cx43 hemichannels. These changes in membrane permeability were not detected in astrocytes cultured from Cx43 knock-out mice and were abrogated by connexin hemichannel blockers, including La3+, mimetic peptides, and niflumic acid. Both the reduction in gap junctional communication and the increase in membrane permeability were mediated by a p38 mitogen-activated protein kinase-dependent pathway. However, the increase in membrane permeability, but not the gap junction inhibition, was rapidly reversed by the sulfhydryl reducing agent dithiothreitol, indicating that final regulatory mechanisms are different. Treatment with proinflammatory cytokines reduced the total and cell surface Cx43 levels, suggesting that the increase in membrane permeability was attributable to an increase in hemichannels activity. Indeed, unitary events of approximately 220 pS corresponding to Cx43 hemichannels were much more frequent in astrocytes treated with microglia conditioned medium than under control conditions. Finally, the effect of cytokines enhanced the uptake and reduced the intercellular diffusion of glucose, which might explain changes in the metabolic status of astrocytes under inflammatory conditions. Accordingly, this opposite regulation may affect glucose trafficking and certainly will modify the metabolic status of astrocytes involved in brain inflammation.