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1.
Cell Commun Signal ; 21(1): 8, 2023 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-36639804

RESUMO

Gap junctions (GJs), which are composed of connexins (Cxs), provide channels for direct information exchange between cells. Cx expression has a strong spatial specificity; however, its influence on cell behavior and information exchange between cells cannot be ignored. A variety of factors in organisms can modulate Cxs and subsequently trigger a series of responses that have important effects on cellular behavior. The expression and function of Cxs and the number and function of GJs are in dynamic change. Cxs have been characterized as tumor suppressors in the past, but recent studies have highlighted the critical roles of Cxs and GJs in cancer pathogenesis. The complex mechanism underlying Cx and GJ involvement in cancer development is a major obstacle to the evolution of therapy targeting Cxs. In this paper, we review the post-translational modifications of Cxs, the interactions of Cxs with several chaperone proteins, and the effects of Cxs and GJs on cancer. Video Abstract.


Assuntos
Conexinas , Neoplasias , Humanos , Conexinas/metabolismo , Conexinas/farmacologia , Junções Comunicantes/metabolismo , Neoplasias/metabolismo
2.
Int J Mol Sci ; 24(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36675184

RESUMO

Previously, we reported a mechanosensitive ion channel, TRPV4, along with functional connexin hemichannels on the basolateral surface of the ocular nonpigmented ciliary epithelium (NPE). In the lens, TRPV4-mediated hemichannel opening is part of a feedback loop that senses and respond to swelling. The present study was undertaken to test the hypothesis that TRPV4 and hemichannels in the NPE respond to a mechanical stimulus. Porcine NPE cells were cultured on flexible membranes to study effects of cyclic stretch and ATP release was determined by a luciferase assay. The uptake of propidium iodide (PI) was measured as an indicator of hemichannel opening. NPE cells subjected to cyclic stretch for 1-10 min (10%, 0.5 Hz) displayed a significant increase in ATP release into the bathing medium. In studies where PI was added to the bathing medium, the same stretch stimulus increased cell PI uptake. The ATP release and PI uptake responses to stretch both were prevented by a TRPV4 antagonist, HC067047 (10 µM), and a connexin mimetic peptide, Gap 27 (200µm). In the absence of a stretch stimulus, qualitatively similar ATP release and PI uptake responses were observed in cells exposed to the TRPV4 agonist GSK1016790A (10 nM), and Gap 27 prevented the responses. Cells subjected to an osmotic swelling stimulus (hypoosmotic medium: 200 mOsm) also displayed a significant increase in ATP release and PI uptake and the responses were abolished by TRPV4 inhibition. The findings point to TRPV4-dependent connexin hemichannel opening in response to mechanical stimulus. The TRPV4-hemichannel mechanism may act as a mechanosensor that facilitates the release of ATP and possibly other autocrine or paracrine signaling molecules that influence fluid (aqueous humor) secretion by the NPE.


Assuntos
Cílios , Conexinas , Epitélio , Canais de Cátion TRPV , Animais , Trifosfato de Adenosina , Conexinas/metabolismo , Epitélio/metabolismo , Suínos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Cílios/genética , Cílios/metabolismo
3.
Int J Oral Sci ; 15(1): 7, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36646698

RESUMO

Severe muscle injury is hard to heal and always results in a poor prognosis. Recent studies found that extracellular vesicle-based therapy has promising prospects for regeneration medicine, however, whether extracellular vesicles have therapeutic effects on severe muscle injury is still unknown. Herein, we extracted apoptotic extracellular vesicles derived from mesenchymal stem cells (MSCs-ApoEVs) to treat cardiotoxin induced tibialis anterior (TA) injury and found that MSCs-ApoEVs promoted muscles regeneration and increased the proportion of multinucleated cells. Besides that, we also found that apoptosis was synchronized during myoblasts fusion and MSCs-ApoEVs promoted the apoptosis ratio as well as the fusion index of myoblasts. Furthermore, we revealed that MSCs-ApoEVs increased the relative level of creatine during myoblasts fusion, which was released via activated Pannexin 1 channel. Moreover, we also found that activated Pannexin 1 channel was highly expressed on the membrane of myoblasts-derived ApoEVs (Myo-ApoEVs) instead of apoptotic myoblasts, and creatine was the pivotal metabolite involved in myoblasts fusion. Collectively, our findings firstly revealed that MSCs-ApoEVs can promote muscle regeneration and elucidated that the new function of ApoEVs as passing inter-cell messages through releasing metabolites from activated Pannexin 1 channel, which will provide new evidence for extracellular vesicles-based therapy as well as improving the understanding of new functions of extracellular vesicles.


Assuntos
Conexinas , Creatina , Vesículas Extracelulares , Mioblastos , Creatina/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Regeneração , Conexinas/metabolismo
4.
Int J Mol Sci ; 24(2)2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36674690

RESUMO

Atherosclerotic lesions preferentially develop at bifurcations, characterized by non-uniform shear stress (SS). The aim of this study was to investigate SS-induced endothelial activation, focusing on stress-regulated mitogen-activated protein kinases (MAPK) and downstream signaling, and its relation to gap junction proteins, Connexins (Cxs). Human umbilical vein endothelial cells were exposed to flow ("mechanical stimulation") and stimulated with TNF-α ("inflammatory stimulation"). Phosphorylated levels of MAPKs (c-Jun N-terminal kinase (JNK1/2), extracellular signal-regulated kinase (ERK), and p38 kinase (p38K)) were quantified by flow cytometry, showing the activation of JNK1/2 and ERK. THP-1 cell adhesion under non-uniform SS was suppressed by the inhibition of JNK1/2, not of ERK. Immunofluorescence staining and quantitative real-time PCR demonstrated an induction of c-Jun and c-Fos and of Cx43 in endothelial cells by non-uniform SS, and the latter was abolished by JNK1/2 inhibition. Furthermore, plaque inflammation was analyzed in human carotid plaques (n = 40) using immunohistochemistry and quanti-gene RNA-assays, revealing elevated Cx43+ cell counts in vulnerable compared to stable plaques. Cx43+ cell burden in the plaque shoulder correlated with intraplaque neovascularization and lipid core size, while an inverse correlation was observed with fibrous cap thickness. Our results constitute the first report that JNK1/2 mediates Cx43 mechanoinduction in endothelial cells by atheroprone shear stress and that Cx43 is expressed in human carotid plaques. The correlation of Cx43+ cell counts with markers of plaque vulnerability implies its contribution to plaque progression.


Assuntos
Conexina 43 , Placa Aterosclerótica , Humanos , Conexina 43/genética , Conexina 43/metabolismo , Mecanotransdução Celular , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Placa Aterosclerótica/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Conexinas/metabolismo
5.
Subcell Biochem ; 102: 113-137, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36600132

RESUMO

Gap junctions, comprising connexin proteins, create conduits directly coupling the cytoplasms of adjacent cells. Expressed in essentially all tissues, dynamic gap junction structures enable the exchange of small molecules including ions and second messengers, and are central to maintenance of homeostasis and synchronized excitability. With such diverse and critical roles throughout the body, it is unsurprising that alterations to gap junction and/or connexin expression and function underlie a broad array of age-related pathologies. From neurological dysfunction to cardiac arrhythmia and bone loss, it is hard to identify a human disease state that does not involve reduced, or in some cases inappropriate, intercellular communication to affect organ function. With a complex life cycle encompassing several key regulatory steps, pathological gap junction remodeling during ageing can arise from alterations in gene expression, translation, intracellular trafficking, and posttranslational modification of connexins. Connexin proteins are now known to "moonlight" and perform a variety of non-junctional functions in the cell, independent of gap junctions. Furthermore, connexin "hemichannels" on the cell surface can communicate with the extracellular space without ever coupling to an adjacent cell to form a gap junction channel. This chapter will focus primarily on gap junctions in ageing, but such non-junctional connexin functions will be referred to where appropriate and the full spectrum of connexin biology should be noted as potentially causative/contributing to some findings in connexin knockout animals, for example.


Assuntos
Conexinas , Junções Comunicantes , Animais , Humanos , Junções Comunicantes/metabolismo , Conexinas/genética , Conexinas/metabolismo , Comunicação Celular , Processamento de Proteína Pós-Traducional , Envelhecimento/genética
6.
PLoS Biol ; 20(12): e3001891, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36477165

RESUMO

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.


Assuntos
Astrócitos , Conexinas , Modelos Animais de Doenças , Animais , Camundongos , Conexinas/metabolismo , Astrócitos/metabolismo
7.
Sci Signal ; 15(765): eabl6781, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36538593

RESUMO

Pyroptosis is a mechanism of programmed, necrotic cell death mediated by gasdermins, a family of pore-forming proteins. Caspase-1 activates gasdermin D (GSDMD) under inflammatory conditions, whereas caspase-3 activates GSDME under apoptotic conditions, such as those induced by chemotherapy. These pathways are thought to be separate. However, we found that they are part of an integrated network of gatekeepers that enables pyroptotic cell death. We observed that GSDMD was the primary pyroptotic mediator in cultured blood cells in response to doxorubicin and etoposide, two common chemotherapies for hematopoietic malignancies. Upon treatment, the channel protein pannexin-1 (PANX1), which is stimulated by the initiation of apoptosis, increased membrane permeability to induce K+ efflux-driven activation of the NLRP3 inflammasome and GSDMD. However, either PANX1 or GSDME could also be the primary mediator of chemotherapy-induced pyroptosis when present at higher amounts. The most abundant pore-forming protein in acute myeloid leukemias from patients predicted the cell death pathway in response to chemotherapy. This interconnected network, a multistep switch that converts apoptosis to pyroptosis, could be clinically titratated to modulate cell death with regard to antitumor immunity or tumor lysis syndrome in patients.


Assuntos
Antineoplásicos , Neoplasias Hematológicas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Apoptose , Necrose , Inflamassomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Conexinas/genética , Conexinas/metabolismo
8.
Int J Mol Sci ; 23(24)2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36555237

RESUMO

Gap junctions (GJs) are specialized transmembrane channels assembled by two hemi-channels of six connexin (Cx) proteins that facilitate neuroglial crosstalk in the central nervous system (CNS). Previous studies confirmed the crucial role of glial GJs in neurodegenerative disorders with dementia or motor dysfunction including Alzheimer's disease (AD). The aim of this study was to examine the alterations in astrocyte and related oligodendrocyte GJs in association with Aß plaques in the spinal cord of the 5xFAD mouse model of AD. Our analysis revealed abundant Aß plaque deposition, activated microglia, and astrogliosis in 12-month-old (12M) 5xFAD mice, with significant impairment of motor performance starting from 3-months (3M) of age. Additionally, 12M 5xFAD mice displayed increased immunoreactivity of astroglial Cx43 and Cx30 surrounding Aß plaques and higher protein levels, indicating upregulated astrocyte-to-astrocyte GJ connectivity. In addition, they demonstrated increased numbers of mature CC1-positive and precursor oligodendrocytes (OPCs) with higher immunoreactivity of Cx47-positive GJs in individual cells. Moreover, total Cx47 protein levels were significantly elevated in 12M 5xFAD, reflecting increased oligodendrocyte-to-oligodendrocyte Cx47-Cx47 GJ connectivity. In contrast, we observed a marked reduction in Cx32 protein levels in 12M 5xFAD spinal cords compared with controls, while qRT-PCR analysis revealed a significant upregulation in Cx32 mRNA levels. Finally, myelin deficits were found focally in the areas occupied by Aß plaques, whereas axons themselves remained preserved. Overall, our data provide novel insights into the altered glial GJ expression in the spinal cord of the 5xFAD model of AD and the implicated role of GJ pathology in neurodegeneration. Further investigation to understand the functional consequences of these extensive alterations in oligodendrocyte-astrocyte (O/A) GJ connectivity is warranted.


Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/metabolismo , Junções Comunicantes/metabolismo , Conexinas/metabolismo , Neuroglia/metabolismo , Medula Espinal/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças
9.
Elife ; 112022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36378164

RESUMO

Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in Caenorhabditis elegans, in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sinapses/metabolismo , Neurônios Motores/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
10.
Biochem Biophys Res Commun ; 636(Pt 1): 33-40, 2022 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-36332480

RESUMO

BACKGROUND: Inflammation can contribute to the initiation and progression of atrial fibrillation (AF), and pinocembrin can suppress downstream inflammatory cytokine production by inhibiting the inflammation pathway. In our previous studies, pinocembrin was also beneficial in ameliorating cardiac arrhythmia in different models of rats, such as depression, myocardial infarction, and heart failure. This study aims to investigate the effect of pinocembrin on the susceptibility to AF in isoproterenol-induced rats. METHODS: Rats were randomly divided into four groups. Pinocembrin was injected through the tail vein. Isoproterenol was treated by intraperitoneal injection for one week (5 mg/kg/day). We evaluated the susceptibility to AF by atrial electrophysiological experiments. Masson staining was used to evaluate the fibrosis area. The protein levels of connexin (Cx) 40, Cav1.2, Kv4.2, collagen I, collagen III, α-SMA, transforming growth factor (TGF)-ß, NLRP3, caspase 1, and interleukin (IL)-1ß were detected by western blot. RESULTS: Our data demonstrated that pinocembrin could prolong the atrial effective refractory period (ERP) and action potential duration (APD), and decrease AF inducibility. Isoproterenol increased the expression of Cav1.2 and Kv4.2 ion channels whereas pinocembrin could alleviate this change. Pinocembrin could reduce the fibrosis area, fibrosis-related protein collagen I, collagen III, α-SMA, and TGF-ß and upregulate gap junction protein Cx40. In addition, pinocembrin reduced the expression of NLRP3, caspase 1, and IL-1ß. CONCLUSIONS: Our study indicated that pinocembrin was beneficial to alleviate atrial electrical remodeling and fibrosis. Accompanied the downregulation of ion channels and upregulation of gap junction protein Cx40. Pinocembrin may produce these effects by inhibiting the NLRP3 pathway.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Ratos , Animais , Fibrilação Atrial/induzido quimicamente , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/metabolismo , Isoproterenol/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspase 1/metabolismo , Ratos Sprague-Dawley , Átrios do Coração , Fibrose , Conexinas/metabolismo , Inflamação/patologia , Colágeno Tipo I/metabolismo , Canais Iônicos/metabolismo , Modelos Animais de Doenças
11.
Proc Natl Acad Sci U S A ; 119(45): e2213162119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322757

RESUMO

Temporal lobe epilepsy (TLE) is one of the most common types of epilepsy, yet approximately one-third of patients are refractory to current anticonvulsive drugs, which target neurons and synapses. Astrocytic and microglial dysfunction is commonly found in epileptic foci and has been shown to contribute to neuroinflammation and hyperexcitability in chronic epilepsy. Accumulating evidence points to a key role for glial hemichannels in epilepsy, but inhibiting both connexin (Cx) gap junctions and hemichannels can lead to undesirable side effects because the former coordinate physiological functions of cell assemblies. It would be a great benefit to use an orally available small molecule to block hemichannels to alleviate epileptic symptoms. Here, we explored the effect of D4, a newly developed compound that inhibits the Cx hemichannels but not Cx gap junctions using the pilocarpine mouse model of TLE. In vitro application of D4 caused a near-complete reduction in the pilocarpine-induced cell membrane permeability associated with increased Cx hemichannel activity. Moreover, preadministration of D4 in vivo effectively reduced neuroinflammation and altered synaptic inhibition, which then enhanced the animal survival rate. Posttreatment with a single dose of D4 in vivo has prolonged effects on suppressing the activation of astrocytes and microglia and rescued the changes in neuroinflammatory and synaptic gene expression induced by pilocarpine. Collectively, these results indicate that targeting Cx hemichannels by D4 is an effective and promising strategy for treating epilepsy in which neuroinflammation plays a critical role.


Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Animais , Camundongos , Conexinas/metabolismo , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/metabolismo , Pilocarpina , Doenças Neuroinflamatórias
12.
Cells ; 11(22)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36429074

RESUMO

Enhanced activity and overexpression of Pannexin 1 (Panx1) channels contribute to neuronal pathologies such as epilepsy and Alzheimer's disease (AD). The Panx1 channel ablation alters the hippocampus's glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, Panx1-knockout (Panx1-KO) mice still retain the ability to learn, suggesting that compensatory mechanisms stabilize their neuronal activity. Here, we show that the absence of Panx1 in the adult brain promotes a series of structural and functional modifications in the Panx1-KO hippocampal synapses, preserving spontaneous activity. Compared to the wild-type (WT) condition, the adult hippocampal neurons of Panx1-KO mice exhibit enhanced excitability, a more complex dendritic branching, enhanced spine maturation, and an increased proportion of multiple synaptic contacts. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in the actin polymerization and an imbalance between the Rac1 and the RhoA GTPase activities were observed in Panx1-KO brain tissues. Our findings highlight a novel interaction between Panx1 channels, actin, and Rho GTPases, which appear to be relevant for synapse stability.


Assuntos
Actinas , Conexinas , Animais , Camundongos , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo
13.
Int J Mol Sci ; 23(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36361888

RESUMO

Renal disease is a major public health challenge since its prevalence has continuously increased over the last decades. At the end stage, extrarenal replacement therapy and transplantation remain the only treatments currently available. To understand how the disease progresses, further knowledge of its pathophysiology is needed. For this purpose, experimental models, using mainly rodents, have been developed to unravel the mechanisms involved in the initiation and progression of renal disease, as well as to identify potential targets for therapy. The gap junction protein connexin 43 has recently been identified as a novel player in the development of kidney disease. Its expression has been found to be altered in many types of human renal pathologies, as well as in different animal models, contributing to the activation of inflammatory and fibrotic processes that lead to renal damage. Furthermore, Cx43 genetic, pharmacogenetic, or pharmacological inhibition preserved renal function and structure. This review summarizes the existing advances on the role of this protein in renal diseases, based mainly on different in vivo animal models of acute and chronic renal diseases.


Assuntos
Conexina 43 , Insuficiência Renal Crônica , Animais , Humanos , Conexina 43/metabolismo , Rim/metabolismo , Conexinas/metabolismo , Insuficiência Renal Crônica/metabolismo , Fibrose
14.
Int J Mol Sci ; 23(21)2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-36362016

RESUMO

Studies suggest that astrocytic connexins (Cx) have an important role in the regulation of high brain functions through their ability to establish fine-tuned communication with neurons within the tripartite synapse. In light of these properties, growing evidence suggests a role of Cx in psychiatric disorders such as major depression but also in the therapeutic activity of antidepressant drugs. However, the real impact of Cx on treatment response and the underlying neurobiological mechanisms remain yet to be clarified. On this ground, the present study was designed to evaluate the functional activity of Cx in a mouse model of depression based on chronic corticosterone exposure and to determine to which extent their pharmacological inactivation influences the antidepressant-like activity of venlafaxine (VENLA). On the one hand, our results indicate that depressed mice have impaired Cx-based gap-junction and hemichannel activities. On the other hand, while VENLA exerts robust antidepressant-like activity in depressed mice; this effect is abolished by the pharmacological inhibition of Cx with carbenoxolone (CBX). Interestingly, the combination of VENLA and CBX is also associated with a higher rate of relapse after treatment withdrawal. To our knowledge, this study is one of the first to develop a model of relapse, and our results reveal that Cx-mediated dynamic neuroglial interactions play a critical role in the efficacy of monoaminergic antidepressant drugs, thus providing new targets for the treatment of depression.


Assuntos
Astrócitos , Conexinas , Transtorno Depressivo , Animais , Camundongos , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Carbenoxolona/farmacologia , Conexinas/efeitos dos fármacos , Conexinas/metabolismo , Fenótipo , Recidiva , Depressão/tratamento farmacológico , Depressão/metabolismo , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/metabolismo
15.
J Neuroinflammation ; 19(1): 244, 2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36195881

RESUMO

BACKGROUND: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. METHODS: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. RESULTS: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. CONCLUSION: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.


Assuntos
Carbenoxolona , Neuralgia , Trifosfato de Adenosina/farmacologia , Animais , Carbenoxolona/farmacologia , Carbenoxolona/uso terapêutico , Conexinas/genética , Conexinas/metabolismo , Citocinas/metabolismo , Etídio/metabolismo , Etídio/farmacologia , Etídio/uso terapêutico , Hiperalgesia/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/metabolismo , Probenecid/metabolismo , Probenecid/farmacologia , Probenecid/uso terapêutico , RNA Interferente Pequeno/metabolismo , Células de Schwann
16.
Life Sci ; 310: 121080, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36252698

RESUMO

AIMS: Disorganization of the subcutaneous tissue due to inflammation and fibrosis is a common feature in patients with myofascial pain. Dermal accumulation of adenosine favours collagen production by human subcutaneous fibroblasts (HSCF) via A2A receptors (A2AR) activation. Adenosine mimics the fibrogenic effect of inflammatory mediators (e.g. histamine, bradykinin), which promote ATP release from HSCF via plasma-membrane-bound pannexin-1 (Panx1) and/or connexin-43 (Cx43) channels, but this mechanism has never been implicated in A2AR actions. MATERIALS AND METHODS: A2AR-mediated effects on Panx1 and Cx43 protein amounts were evaluated in primary cultures of HSCF by confocal microscopy and Western blot analysis. Functional repercussions in collagen production, intracellular [Ca2+]i oscillations and ATP release were also evaluated. KEY FINDINGS: NECA and CGS21680, two enzymatically-stable A2AR agonists, increased Panx1, but reduced Cx43, protein density in HSCF. This effect was accompanied by increases in ATP release and collagen III production by HSCF. The involvement of the A2AR was confirmed by blockage with the selective A2AR antagonist, SCH442416. Inhibition of Panx1 channels by probenecid and the Panx1 mimetic inhibitory peptide, 10Panx, also decreased ATP release and collagen production by HSCF under similar conditions. Superfluous ATP release by HSCF exposed to A2AR agonists overexpressing Panx1 channels contributes to keeping high [Ca2+]i levels when the cells were exposed to histamine. SIGNIFICANCE: Adenosine A2AR-induced Panx1 overexpression was shown here for the first time in HSCF; this feature indirectly implicates ATP release in the fibrogenic vicious cycle operated by adenosine accumulating in subcutaneous tissue fibrosis and myofascial pain associated to dermal inflammation.


Assuntos
Conexina 43 , Conexinas , Proteínas do Tecido Nervoso , Receptor A2A de Adenosina , Humanos , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Colágeno/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Fibroblastos/metabolismo , Fibrose , Histamina/metabolismo , Inflamação/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Dor/metabolismo , Receptor A2A de Adenosina/metabolismo , Tela Subcutânea/metabolismo
17.
Mol Vis ; 28: 245-256, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36284672

RESUMO

Purpose: Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release. Methods: Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca2+, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space. Results: The incubation of the rat lenses in the AAH that lacked Ca2+ induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid. Conclusions: Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.


Assuntos
Carbenoxolona , Probenecid , Ratos , Animais , Probenecid/farmacologia , Carbenoxolona/farmacologia , Ácido Flufenâmico , Dextranos , Conexinas/metabolismo , Trifosfato de Adenosina/metabolismo
18.
Int J Mol Sci ; 23(19)2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36232905

RESUMO

Lens gap junctions (GJs) formed by Cx46 and Cx50 are important to keep lens transparency. Functional studies on Cx46 and Cx50 GJs showed that the Vj-gating, single channel conductance (γj), gating polarity, and/or channel open stability could be modified by the charged residues in the amino terminal (NT) domain. The role of hydrophobic residues in the NT on GJ properties is not clear. Crystal and cryo-EM GJ structures have been resolved, but the NT domain structure has either not been resolved or has showed very different orientations depending on the component connexins and possibly other experimental conditions, making it difficult to understand the structural basis of the NT in Vj-gating and γj. Here, we generated missense variants in Cx46 and Cx50 NT domains and studied their properties by recombinant expression and dual whole-cell patch clamp experiments on connexin-deficient N2A cells. The NT variants (Cx46 L10I, N13E, A14V, Q15N, and Cx50 I10L, E13N, V14A, N15Q) were all able to form functional GJs with similar coupling%, except Cx46 N13E, which showed a significantly reduced coupling%. The GJs of Cx46 N13E, A14V and Cx50 E13N, N15Q showed a reduced coupling conductance. Vj-gating of all the variant GJs were similar to the corresponding wild-type GJs except Cx46 L10I. The γj of Cx46 N13E, A14V, Cx50 E13N, and N15Q GJs was reduced to 51%, 82%, 87%, and 74%, respectively, as compared to their wild-type γjs. Structural models of Cx46 L10I and A14V predicted steric clashes between these residues and the TM2 residues, which might be partially responsible for our observed changes in GJ properties. To verify the importance of hydrophobic interactions, we generated a variant, Cx50 S89T, which also shows a steric clash and failed to form a functional GJ. Our experimental results and structure models indicate that hydrophobic interactions between the NT and TM2 domain are important for their Vj-gating, γj, and channel open stability in these and possibly other GJs.


Assuntos
Junções Comunicantes , Ativação do Canal Iônico , Conexinas/metabolismo , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/metabolismo
19.
Cells ; 11(20)2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36291086

RESUMO

Pannexin1 (Panx1) is expressed in both neurons and glia where it forms ATP-permeable channels that are activated under pathological conditions such as epilepsy, migraine, inflammation, and ischemia. Membrane lipid composition affects proper distribution and function of receptors and ion channels, and defects in cholesterol metabolism are associated with neurological diseases. In order to understand the impact of membrane cholesterol on the distribution and function of Panx1 in neural cells, we used fluorescence recovery after photobleaching (FRAP) to evaluate its mobility and electrophysiology and dye uptake to assess channel function. We observed that cholesterol extraction (using methyl-ß-cyclodextrin) and inhibition of its synthesis (lovastatin) decreased the lateral diffusion of Panx1 in the plasma membrane. Panx1 channel activity (dye uptake, ATP release and ionic current) was enhanced in cholesterol-depleted Panx1 transfected cells and in wild-type astrocytes compared to non-depleted or Panx1 null cells. Manipulation of cholesterol levels may, therefore, offer a novel strategy by which Panx1 channel activation might modulate various pathological conditions.


Assuntos
Astrócitos , Colesterol , Conexinas , Proteínas do Tecido Nervoso , Neuroblastoma , Humanos , Trifosfato de Adenosina/metabolismo , Anticolesterolemiantes/farmacologia , Astrócitos/metabolismo , Colesterol/metabolismo , Conexinas/metabolismo , Canais Iônicos/metabolismo , Lovastatina/farmacologia , Lipídeos de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Estabilidade Proteica
20.
Elife ; 112022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36107487

RESUMO

Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in 'metabolic genes' will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial respiration (NDUFS1) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as 'essential' do not influence the growth of human cancers.


Assuntos
Conexinas , Junções Comunicantes , Conexina 26/genética , Conexina 26/metabolismo , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Humanos , Mutação , Fenótipo , Isoformas de Proteínas/metabolismo
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