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Hydrogen Peroxide Increases Gap Junctional Communication and Induces Astrocyte Toxicity: Regulation by Brain Macrophages
Abstract
Cultured astrocytes are highly coupled by gap junction channels mainly constituted by connexin 43. We have previously shown that gap junctional communication (GJC) represents a functional property of astrocytes that is a target for their interaction with other brain cell types, including neurons and brain macrophages. In pathological situations, neurons as well as brain macrophages produce superoxide ions leading to the formation of hydrogen peroxide (H2O2) that can be cytotoxic. We report here that 10-min exposure to 100 microM H2O2 increases GJC in astrocytes. Moreover, 30-min exposure to 100 microM H2O2 induces, 24 h later, an astrocyte cell death by both apoptosis and necrosis. This H2O2-induced astrocyte cell death is not affected when gap junctions are inhibited by several uncoupling agents, including 18alpha-glycyrrhetinic acid, halothane, heptanol, and endothelin-1, indicating that the proportion of cell death is not related to the level of GJC. The effect of H2O2 on gap junction channels does not result from the production of free radicals but is rather linked to modification of the redox equilibrium in astrocytes. Indeed, an oxidative agent reproduces the H2O2-evoked response while reducing agents prevent the effect of H2O2. Finally, when astrocytes are cocultured with brain macrophages, the effects of H2O2 on both GJC and toxicity are not observed, revealing a new protective role of brain macrophages during oxidative stress.
... The medium was then removed and replaced with fresh culture medium to prevent H 2 O 2 from reacting with other components of the medium and allow cells to recover. H 2 O 2 is reported to induce cell death in astrocytes by both necrotic and apoptotic pathways, 39 Ϯ 10% and 26 Ϯ 14% of the total number of cells, respectively (35). In our study, cell death was analyzed using the dye Rdex, an indicator of loss of membrane integrity, 24 h post-treatment. ...
... Moreover, whether Cx43 hemichannels and/or gap junctions are important for H 2 O 2 -mediated cell death is still under debate. The involvement of Cx43 in H 2 O 2 -mediated astrocytic death has been previously reported (19,35). Knockdown of Cx43 sensitized primary rat brain astrocytes to H 2 O 2 (19). ...
... Knockdown of Cx43 sensitized primary rat brain astrocytes to H 2 O 2 (19). However, inhibition of Cx43 GJIC by pharmacological agents did not interfere with H 2 O 2 -induced cell death in rat striatum astrocytes (35). In this study, we have demonstrated that Cx43 channel activity is crucial for Cx43-mediated ROS resistance of cortical astrocytes as astrocytes lacking Cx43 or blockage of Cx43 channels show similar elevated cell death induced by H 2 O 2 . ...
Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knockout astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggests that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.
... In endothelial cells, H 2 O 2 also causes activation of the p38 MAPK and this is followed by both activation of the MAPK-activated protein kinase-2/3 and the phosphorylation of the small heat shock protein (HSP) (Huot et al., 1998 ; et al., 2004). Oxidative stress has been shown to increase gap junction communication among astrocytes through reorganization of the actin network (Rouach et al., 2004). In this paper, we report the effects of oxidative stress induced by H 2 O 2 on plasma membrane properties, cytoskeleton arrangements and cell-cell interaction in rat astrocytes. ...
... Besides the effects on plasma membranes, oxidative stress is known to cause cytoskeletal reorganizations in cells (Qian et al., 2003; Rosado et al., 2002; Rouach et al., 2004; Zhao and Davis, 1998).Fig. 5E,G shows the effects of oxidative stress on the formation of actin-enriched protrusions (white arrowheads), similar to cytonemes described by Ramirez-Weber and Kornberg (Weber and Kornberg, 1999), and the formation of tunneling nanotube (TNT)-like connections (white arrows) similar to the TNT described by Rustom et al. (Rustom et al., 2004). ...
... It is a highly coordinated process regulated by cell signaling cascades. In astrocytes, oxidative stress is known to cause the formation of gap junction, which is another form of intercellular connection (Rouach et al., 2004). With the notion of TNTs functioning as transportation highways (Rustom et al., 2004), our results lead to the hypothesis that oxidative stress enhances communication between astrocytes. ...
Excess hydrogen peroxide (H2O2) is produced in the pathogenesis of brain injuries and neurodegenerative diseases. H2O2 may damage cells through direct oxidation of lipids, proteins and DNA or it can act as a signaling molecule to trigger intracellular pathways leading to cell death. In this study, H2O2 caused plasma membranes of primary astrocytes to become more gel-like, while artificial membranes of vesicles composed of rat brain lipid extract became more liquid crystalline-like. Besides the effects on membrane phase properties, H2O2 promoted actin polymerization, induced the formation of cell-to-cell tunneling nanotube (TNT)-like connections among astrocytes and increased the colocalization of myosin Va with F-actin. Myosin Va was also observed in the H2O2-induced F-actin-enriched TNT-like connections. Western blot analysis suggests that H2O2 triggered the phosphorylation of the p38 mitogen-activated protein kinase (MAPK), and that SB203580, a specific inhibitor of p38 MAPK, suppressed the changes in membrane phase properties and cytoskeleton resulting from H2O2 treatment. These results suggest that H2O2 alters astrocyte membranes and the cytoskeleton through activation of the p38 MAPK pathway.
... Nous avons vu que les espèces actives de l'oxygène (mEAOs) peuvent être à l'origine d'une signalisation intracellulaire lors de la détection hypothalamique du glucose, lorsqu'ils sont produits en petite quantité et rapidement éliminés. La GJC astrocytaire est également modulée par l'H2O2, qui induit une augmentation de la perméabilité in vitro (Rouach et al., 2004). ...
... Or, les Cxs sont redox-sensibles : l'H2O2 induit une augmentation du couplage astrocytaire (Rouach et al., 2004). Cet effet est relayé par la capacité des mEAOs à oxyder les groupements cystéine présents sur les boucles extracellulaires des Cxs, ce qui favorise la formation de ponts disulfures et in fine, des GJ (Sosinsky and Nicholson, 2005). ...
Numéro national (NNT) : 2012DIJOS097
... Nous avons vu que les espèces actives de l'oxygène (mEAOs) peuvent être à l'origine d'une signalisation intracellulaire lors de la détection hypothalamique du glucose, lorsqu'ils sont produits en petite quantité et rapidement éliminés. La GJC astrocytaire est également modulée par l'H2O2, qui induit une augmentation de la perméabilité in vitro (Rouach et al., 2004). ...
... Or, les Cxs sont redox-sensibles : l'H2O2 induit une augmentation du couplage astrocytaire (Rouach et al., 2004). Cet effet est relayé par la capacité des mEAOs à oxyder les groupements cystéine présents sur les boucles extracellulaires des Cxs, ce qui favorise la formation de ponts disulfures et in fine, des GJ (Sosinsky and Nicholson, 2005). ...
The hypothalamus plays a pivotal role in the nervous control of glucose homeostasis. This area contains gluco-sensitive neurons. Some of them detect increases in glucose levels and regulate glucose homeostasis by stimulating insulin secretion or inhibiting food intake. It is widely accepted that astrocytes are metabolically coupled to neurons. Lactate, resulting from the metabolism of glucose by astrocytes, is transported via the monocarboxylate transporters (MCTs). In addition, gap junctions (GJ), that form networks within astrocytes, are essential to transfer glucose from the bloodstream to the active neurons. These astroglial GJ mainly consist of connexins 43 and 30 (Cxs).The aims of my thesis are twofold: first, to show that an intracarotid lactate injection toward the brain, as for glucose, triggers insulin secretion and, second, to investigate the role of astroglial Cxs.Our results demonstrate that lactate and glucose sensing are altered in 48h hyperglycemic rats (accompanied by high blood lactate level). These alterations are not due to changes in protein expression of astroglial or neuronal MCTs in the hypothalamus. We then show that Cx43 is highly expressed in astrocytic end-feet enwraping blood vessels, in medio-basal hypothalamus (MBH) where many gluco-sensitive neurons are present. The Cx30 expression is more diffuse in this structure. We also show that the protein expression of astroglial Cxs varies very rapidly due to changes in metabolic status (fasting, refeeding and hyperglycemia). To evaluate the involvement of astroglial Cx43 (the major isoform) in the hypothalamic glucose sensing, we silenced its expression in the MBH in vivo by injecting specific siRNA. A 30% diminution in protein levels (after 72h) induced a decrease in food intake without changes in weight, blood glucose and insulin levels compared to vehicle treated animals. The central response to glucose is drastically inhibited in terms of insulin secretion in siCx43 animals. Similarly, an intracarotid injection of glucose towards the brain does not reduce refeeding in siRNA treated animals.These results demonstrate for the first time in vivo, the importance of connexins and astroglial networks in hypothalamic glucose sensing mechanism. These new data reinforce the importance of the metabolic role of astrocytes in specific neuronal functions
... Compared with neurons, the basal level of Shh in astrocytes was substantially higher (Sims et al., 2009). Recently, increasing evidence suggests that astrocytes are also vulnerable to oxidative stress (Takuma et al., 2002;Rouach et al., 2004;Takano et al., 2006;Zhu et al., 2005). Questions will naturally arise as to whether Shh expression is also upregulated in cortical astrocytes under oxidative stress and whether Shh protein is mainly secreted by astrocytes and is involved in the neuroprotective action of astrocytes under oxidative stress. ...
... The apoptosis of astrocytes exposed to H 2 O 2 is characterized by fragmentation and condensation of nuclei when stained by 4=, 6-diamidino-2-phenylindole (DAPI) (Rouach et al., 2004;Atkinson et al., 2009). In this study, we found that H 2 O 2 induced cell apoptosis and cell death, The primary cultured astrocytes were pre-treated with Shh (3 g/ml), cyclopamine (20 M, C) or/and Shh respectively, and then treated with 200 M H 2 O 2 for 24 h. ...
In our previous study, we found that the sonic hedgehog (Shh) signaling pathway is activated in neurons under oxidative stress and plays a neuro-protective role [Dai RL, et al. (2011) Neurochem Res 36:67-75]; we are led to postulate that the Shh might be released by astrocytes, thereby protecting neurons against oxidant injury. In primary cultured astrocytes of rats, we found that treatment with 100 μM H₂O₂ for 24 h induced a significant increase in the mRNA and protein levels of Shh, Patched1, and Gli-1, and the increase was substantially greater in astrocytes than in neurons. In the coculture systems of astrocytes and neurons under the H₂O₂ treatment, blocking the Shh signaling pathway with 5E1 (an antibody against the N-terminal domain of Shh) could block the neuroprotective activity of astrocytes on cocultured neurons. In this study, we found that treatment with H₂O₂ (100-800 μM) for 24 h caused cell death of astrocytes in a concentration-dependent manner. MTT reduction and Trypan Blue exclusion assay showed that exogenous Shh increased survival rate of the H₂O₂-treated astrocytes, whereas pretreatment with cyclopamine (a specific inhibitor of the Shh signaling pathway) or 5E1 decreased the survival rate of the H₂O₂-treated astrocytes. Shh also inhibited H₂O₂-induced apoptosis of astrocytes, and this effect could be partially reversed by cyclopamine. We also found that Shh promoted the phosphorylation of AKT, but had no significant effect on p38 or extracellular signal regulated kinases 1 and 2 (ERK 1/2) in H₂O₂-treated astrocytes. Blocking Shh or phosphoinositide 3-kinases (PI3-K)/AKT signaling pathway with cyclopamine or LY294002 decreased the survival rate of astrocytes, induced cell apoptosis, upregulated the expression of Bax, and downregulated the expression of Bcl-2. We are led to conclude that the oxidative stress induces astrocytes to secrete endogenous Shh and exogenous administration of Shh might protect the astrocytes from oxidative stress by activating PI3-K/AKT/Bcl-2 pathway.
... It remains controversial whether connexin-mediated GJIC are proor anti-apoptotic. While a substantial amount of data demonstrates a correlation between GJIC and apoptotic activities [129][130][131][132][133][134], others show that GJIC may hinder the occurrence of apoptosis [135][136][137][138][139]. One explanation to this apparent contradiction may be attributed to regulation of both pro-(Map4k) and anti-apoptotic (Bcl-xL) genes by Cx43, which were identified from transcriptome analysis of A: Cx43 exogenous expression has been associated with a decreased activity of the anti-apoptotic molecule bcl-2 [130] and the knockdown of Cx43 impedes apoptosis [143]. Gap junctional intercellular communication (GJIC) may promote the trafficking of molecules involved in the induction of apoptosis such as excess of Ca 2 + , inositol triphosphate (IP 3 ) or cyclic nucleotide triphosphate (cAMP/cGMP) [127,128]. ...
... Gap junctional intercellular communication (GJIC) may promote the trafficking of molecules involved in the induction of apoptosis such as excess of Ca 2 + , inositol triphosphate (IP 3 ) or cyclic nucleotide triphosphate (cAMP/cGMP) [127,128]. Hemichannels may also participate to apoptosis by their opening under oxidant stimulation (H 2 O 2 ) and by the uptake of reactive oxygen species [131]. Moreover, hemichannels may be involved in the release of intracellular molecules. ...
Despite the tremendous amount of data over the last 40years, lack of gap junctional intercellular communication (GJIC) or altered expression of gap junction proteins is still a lesser known 'hallmark' of cancer. Expression of astrocytic gap junction protein, connexin43 (Cx43), is often reduced in astrocytomas, the most common neoplasia of the central nervous system (CNS) in adults. Supported by a number of evidences, the global decrease of Cx43 expression appears to be advantageous for the growth of glioma cells. Although the mechanisms by which Cx43 regulates the expression levels of proteins involved in cell growth is unclear, there are evidences to suggest that it might be independent of their channel forming properties. In this regard, the carboxyl tail of Cx43 may have the ability to control the translocation of transcription factor regulators into the nucleus. However, this putative tumor suppressor effect of Cx43 is counterbalanced by its capacity to enhance the migration of glioma cells out of the tumor core through mechanisms that seems to implicate its carboxyl tail, possibly by interacting with the actin cytoskeleton. This ambivalence between the tumor suppressor effect and promotion of cell migration may partly be explained by the heterogeneous expression of Cx43 in the glioma core especially at the malignant glioblastoma stage; some tumor cells would be expected to migrate (Cx43 expressing cells) and others to proliferate (non-expressing Cx43 cells). Moreover, the involvement of Cx43 in glioma progression seems to be more complex since, in addition, GJIC may increase their resistance to apoptosis and Cx43 may also affect cell homeostasis in a paracrine fashion via hemichannel action. In conclusion, Cx43 appears to be involved at different levels of the glioma progression by acting on cell growth regulation, promotion of cell migration and resistance to apoptosis. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
... In agreement with a crucial role for ROS in brain aging [58], numerous studies currently highlight their relevance in neural cells and neurodegenerative diseases. It is well known that ROS can cause neuron [59] and astrocyte [60] death by both apoptosis and necrosis. Mitochondria are involved in excitotoxic nerve cell death through Ca 2C -related bursts of ROS production and opening of permeability transition pores [61]. ...
... Mitochondria are involved in excitotoxic nerve cell death through Ca 2C -related bursts of ROS production and opening of permeability transition pores [61]. Oxidative stress is also related to release of glutamate and continuous NMDA receptor activation during cerebral ischemia-reperfusion [62], production of superoxide radicals in neurons and brain macrophages [60], and glutamine-induced ROS production in astrocytes [63]. ...
Aging is characterized by decrements in maximum function and accumulation of mitochondrial DNA mutations, which are best observed in organs such as the brain that contain post-mitotic cells. Oxygen radicals are increasingly considered responsible for part of these aging changes. Comparative studies of animals with different aging rates have shown that the rate of mitochondrial oxygen radical generation is directly related to the steady-state level of oxidative damage to mitochondrial DNA and is inversely correlated with maximum longevity in higher vertebrates. The degree of unsaturation of tissue fatty acids also correlates inversely with maximum longevity. These are the two known traits connecting oxidative stress with aging. Furthermore, caloric restriction, which decreases the rate of aging, proportionately decreases mitochondrial oxygen radical generation, especially at complex I. These findings are reviewed, highlighting the results obtained in the brain.
... In liver epithelial cell WB-F344, it has also been reported that redox-active GSH peroxidase mimetics and antioxidants [39] prevent the inhibitory effect of hyper-phosphorylation of Cx43 by 12-Otetradecanoylphorbol-13-acetate on GJs-mediated coupling. However, exposure to H 2 O 2 increases gap junctional communication in astrocytes and the reducing agents dithiothreitol (DTT) and N-acetyl-cystein (NAC) reverse this phenomenon [38]. These findings suggest that protein kinase activation is involved in the modulatory effect of agents that modify the redox equilibrium (e.g., H 2 O 2 ) on the functional state of GJs. ...
Connexins (Cxs) are ubiquitous transmembrane proteins that possess both channel function (e.g., formations of gap junction and hemichannel) and non-channel properties (e.g., gene transcription and protein-protein interaction). Several factors have been identified to play a role in the regulation of Cxs, which include those acting intracellularly, as redox potential, pH, intramolecular interactions, and post-translational modifications (e.g., phosphorylation, S-nitrosylation) as well as those acting extracellularly, such as Ca2+ and Mg2+. The relationship between redox signaling and Cxs attracts considerable attention in recent years. There is ample evidence showing that redox signaling molecules (e.g., hydrogen peroxide (H2O2), nitric oxide (NO)) affect Cxs-based channel function while the opening of Cx channels also triggers the transfer of various redox-related metabolites (e.g., reactive oxygen species, glutathione, nicotinamide adenine dinucleotide, and NO). On the basis of these evidences, we propose the existence of redox-Cxs crosstalk. In this review, we briefly discuss the interaction between redox signaling and Cxs and the implications of the intersection in disease pathology and future therapeutic interventions.
... However, thin membranous TNT like structures was seen between astrocytes (Extended Fig. 2). Astrocytes have been reported to show the reorganization of the actin network and form the TNT structure under oxidative stress [18,19]. ...
Background:
Parkinson's disease (PD) is one of the neurodegeneration diseases characterized by the gradual loss of dopaminergic (DA) neurons in the substantia nigra region of the brain. Substantial evidence indicates that at the cellular level mitochondrial dysfunction is a key factor leading to pathological features such as neuronal death and accumulation of misfolded α-synuclein aggregations. Autologous transplantation of healthy purified mitochondria has shown to attenuate phenotypes in vitro and in vivo models of PD. However, there are significant technical difficulties in obtaining large amounts of purified mitochondria with normal function. In addition, the half-life of mitochondria varies between days to a few weeks. Thus, identifying a continuous source of healthy mitochondria via intercellular mitochondrial transfer is an attractive option for therapeutic purposes. In this study, we asked whether iPSCs derived astrocytes can serve as a donor to provide functional mitochondria and rescue injured DA neurons after rotenone exposure in an in vitro model of PD.
Methods:
We generated DA neurons and astrocytes from human iPSCs and hESCs. We established an astroglial-neuronal co-culture system to investigate the intercellular mitochondrial transfer, as well as the neuroprotective effect of mitochondrial transfer. We employed immunocytochemistry and FACS analysis to track mitochondria.
Results:
We showed evidence that iPSCs-derived astrocytes or astrocytic conditioned media (ACM) can rescue DA neurons degeneration via intercellular mitochondrial transfer in a rotenone induced in vitro PD model. Specifically, we showed that iPSCs-derived astrocytes from health spontaneously release functional mitochondria into the media. Mito-Tracker Green tagged astrocytic mitochondria were detected in the ACM and were shown to be internalized by the injured neurons via a phospho-p38 depended pathway. Transferred mitochondria were able to significantly reverse DA neurodegeneration and axonal pruning following exposure to rotenone. When rotenone injured neurons were cultured in presence of ACM depleted of mitochondria (by ultrafiltration), the neuroprotective effects were abolished.
Conclusions:
Our studies provide the proof of principle that iPSCs-derived astrocytes can act as mitochondria donor to the injured DA neurons and attenuate pathology. Using iPSCs derived astrocytes as a donor can provide a novel strategy that can be further developed for cellular therapy for PD.
... Les radicaux libres comme les espèces réactives de l'oxygène, peuvent aussi altérer les fonctions des connexines. Par exemple, le peroxyde d'hydrogène augmente l'activité jonction communicante entre astrocytes (Rouach et al., 2004) alors que l'oxyde nitrique la diminue (Ball et al., 2011). Mais aussi l'acide arachidonique et l'oxyde nitrique augmentent l'activité hémicanal de la Cx43 (De Vuyst et al., 2009). ...
La Neuromyélite Optique (NMO) est une maladie auto-immune démyélinisante, rare et grave, du système nerveux central (SNC). Elle est caractérisée par une démyélinisation et une perte axonale ciblant principalement le nerf optique et la moelle épinière. La découverte d'un auto-anticorps (IgG-NMO) dirigé contre l'aquaporine-4 (AQP4), un canal hydrique exprimé par l'astrocyte, a été une étape clé dans la compréhension de la physiopathologie de la NMO, actuellement définie comme une astrocytopathie. La pathogénicité de l'IgG-NMO a été démontrée : il induit une internalisation d'AQP4 et des transporteurs au glutamate, provoquant une altération de la fonction astrocytaire. Cependant les mécanismes permettant de lier la dysfonction astrocytaire aux altérations caractéristiques de la NMO, notamment la démyélinisation, restent méconnus. Les astrocytes sont des cellules gliales essentielles à l'établissement et au maintien de l'homéostasie du SNC. Ils permettent la régulation des flux hydriques et ioniques, le contrôle extracellulaire des neuromédiateurs ainsi que l'apport de métabolites énergétiques aux neurones et aux oligodendrocytes. Ils sont aussi caractérisés par une très forte expression de connexines (Cx), des molécules transmembranaires s'assemblant sous une forme hexamérique : le connexon. Les connexines forment soit des hémicanaux, permettant l'échange de petites molécules entre les milieux intra- et extra-cellulaires, soit des jonctions communicantes par la juxtaposition de connexons appartenant à deux cellules, assurant le couplage intercellulaire avec le passage de petites molécules et d'ions (ATP, glutamate, lactate, calcium). Les fonctions hemicanal et jonction communicante sont fortement régulées en condition physiologique et altérées en condition pathologique, notamment en contexte neuroinflammatoire. Nous émettons l'hypothèse que les IgG-NMO altèrent l'expression et la fonction des connexines, et conduisent ainsi à la production d'un environnement toxique pour les oligodendrocytes et la myéline, et délétère pour le fonctionnement neuronal. Mon projet de thèse avait trois objectifs : i) la caractérisation du phénotype astrocytaire induit par les IgGNMO ; ii) l'identification d'altérations des connexines et leur implication dans la pathologie ; iii) la mise en évidence d'altérations de la transmission synaptique induites par les IgG-NMO et l'implication de connexines dans cet effet. Des modèles de cultures primaires gliales traitées par des IgG-NMO issue d'une cohorte de patients m'ont permis de caractériser le phénotype acquis par les astrocytes, et de proposer le concept d'un astrocyte réactif spécifique de pathologie. Les astrocytes réactifs spécifiques de la NMO induisent un milieu inflammatoire spécifique et toxique, provoquant une démyélinisation. Grâce au développement d'une coculture gliale et neuronale produisant des neurones myélinisés, et à l'utilisation de peptides inhibiteurs des Cx, j'ai pu montrer que les NMO-IgG ont un effet démyélinisant et que celui-ci implique les Cx. La démyélinisation est en effet associée à des modifications structurales et fonctionnelles des Cx astrocytaires, observées à la fois in vitro et dans notre modèle in vivo, le rat-NMO. Enfin, la mise en place d'une étude électrophysiologique en potentiel de champs local sur des tranches d'hippocampe de rats m'a permis d'étudier l'effet des IgG-NMO sur la transmission glutamatergique basale. J'ai pu mettre en évidence un effet dépresseur des IgG-NMO, partiellement bloqué par un inhibiteur de connexines, la carbenoxolone. Comme il a déjà été démontré par des études cliniques dans des pathologies neurodégénératives, l'utilisation de modulateurs de Cx semble être une voie thérapeutique prometteuse afin de prévenir la démyélinisation et les altérations du fonctionnement neuronal de la NMO
... In conformance with a considerable function for ROS in nervous system especially in brain aging [16], various studies point out ROS level and neurodegenerative diseases of neural cells. It is specified that ROS can cause death of astrocytes [17] and neurons [18] by both necrosis and apoptosis. Also, evidences containing ROS in main degenerative disorders is consistent with their function in brain and neuronal aging. ...
... Growing evidence has shown that AgNPs action on target cells could cause the formation of excessive ROS and overwhelm cellular antioxidant defense systems, such as glutathione or superoxide dismutase in a variety of cell lines (Arora et al., 2008;Avalos et al., 2014;Foldbjerg et al., 2011;Piao et al., 2011). Earlier reports have emphasized the role played by oxidative stress in AgNP-induced cytotoxicity, and that ROS is involved in modulating GJIC activity (Kar, Riquelme, Werner, & Jiang, 2013;Le et al., 2014;Mikalsen & Sanner, 1994;Rouach, Calvo, Duquennoy, Glowinski, & Giaume, 2004). To investigate the role of ROS in GJIC enhancement by AgNPs, we further examined intracellular ROS level of AgNP-treated cells. ...
Silver nanoparticles (AgNPs) are widely used in health and consumer products that routinely contact skin. However, the biological effects and possible mechanisms of AgNPs on skin remain unclear. Gap junctional intercellular communication (GJIC) plays a critical role in multicellular organisms to maintain tissue homeostasis. The aim of this study is to examine if non-coated AgNPs affect GJIC in human keratinocytes (HaCaT cells), and to identify the possible molecular mechanisms responsible for the effects. GJIC, connexin (Cx)43 protein and mRNA expression, and the effect of siRNA-mediated knockdown of Cx43 on GJIC were assessed. HaCaT cells exposed to non-coated AgNPs at different doses after a 24 hour exposure. To explore further the underlying mechanism, reactive oxygen species and mitogen-activated protein kinase pathway were evaluated after 2, 6, 12 and 24 hours. Our results revealed that non-coated AgNP exposure at subcytotoxic doses increase GJIC partially via Cx43 upregulation. Reactive oxygen species and extracellular signal-regulated kinase and activation of c-Jun N-terminal kinase were involved in the AgNP-induced upregulation of Cx43. This study provides new insight into the potential mechanism of AgNP biological activity.
... Consistently, redoxactive glutathione peroxidase mimetics and antioxidants inhibit tumor promoterinduced downregulation of GJCs mediated coupling between WB-F344 liver epithelial cells by preventing the hyper-phosphorylation and internalization of Cx43 elicited by TPA (Hu et al., 1995). In contrast, in astrocyte this has been reported that a 10-min exposure to 100 M H 2 O 2 generate an increase in GJCs activity, which was prevented by reducing agents and reproduced by an oxidative agent (Rouach et al., 2004) suggesting that the modification in the functional state of GJCs induced by agents that modify the redox equilibrium (e.g. H 2 O 2 ) is a cell type-dependent process mostly caused by activation of protein kinases. ...
Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels.
... The enhancement of GJ communication by ammonia could facilitate the spread of stress factors, such as ROS, sodium or calcium ions, glutamate, inositol trisphosphate (IP3), and apoptotic factors, from cell to cell (Lin et al. 1998;Nodin et al. 2005;Rossi et al. 2007;Kameritsch et al. 2013;De Bock et al. 2014). Experimental evidence indicates that promoting GJ communication exacerbates glial cell death in cultures exposed to oxidative stress, whereas GJ blockers attenuate the extent of cell death (Lin et al. 1998;Frantseva et al. 2002;Rouach et al. 2004;Nodin et al. 2005;Zhang et al. 2013). Moreover, it has been suggested that calcineurin, an important phosphatase involved in signaling transduction in the brain, is activated by ammonia in astrocytes (Bodega et al. 2007). ...
Astrocytes are important brain targets of ammonia, a neurotoxin implicated in the development of hepatic encephalopathy. During hyperammonemia, the pivotal role of astrocytes in brain function and homeostasis is impaired. These cells are abundantly interconnected by gap junctions (GJ), which are intercellular channels that allow the exchange of signaling molecules and metabolites. This communication may also increase cellular vulnerability during injuries, while GJ uncoupling could limit the extension of a lesion. Therefore, the current study was performed to investigate whether astrocyte coupling through GJ contributes to ammonia-induced cytotoxicity. We found that carbenoxolone (CBX), an effective GJ blocker, prevented the following effects induced by ammonia in astrocyte primary cultures: (1) decrease in cell viability and membrane integrity; (2) increase in reactive oxygen species production; (3) decrease in GSH intracellular levels; (4) GS activity; (5) pro-inflammatory cytokine release. On the other hand, CBX had no effect on C6 astroglial cells, which are poorly coupled via GJ. To our knowledge, this study provides the first evidence that GJ play a role in ammonia-induced cytotoxicity. Although more studies in vivo are required to confirm our hypothesis, our data suggest that GJ communication between astrocytes may transmit damage signals and excitotoxic components from unhealthy to normal cells, thereby contributing to the propagation of the neurotoxicity of ammonia.
... Administration of PGF 2α resulted in a 3-fold increase in hydroxyl radicals and a 2-fold increase in malondialdehyde, an end product of membrane lipid peroxidation ). It has also been shown that H 2 O 2 is toxic to neurons (Whittemore et al., 1994, Hoyt et al., 1997, Samanta et al., 1998, Lim et al., 2002, Fatokun et al., 2007, astrocytes (Rouach et al., 2004, de Almeida et al., 2007, and oligodendrocytes (Richter-Landsberg and Vollgraf, 1998, Vollgraf et al., 1999, Mronga et al., 2004. Oxidative stressors, such as H 2 O 2 administration, also increase AA release in neurons and mesangial cells (Samanta et al., 1998, Han et al., 2003. ...
... Furthermore, gap junction channels in astrocytes seem to be modulated by different kinds of ROS. Confirming this notion, H 2 O 2 has been shown to increase GJIC in cultured astrocytes, and this response was reverted by reducing agents (Rouach et al., 2004). Therefore, although free radicals like NO, decrease GJIC, others such as H 2 O 2 or one of their sub-products (i.e., oxidized gluthathione; GSSG) may have the opposite effect. ...
... Furthermore, gap junction channels in astrocytes seem to be modulated by different kinds of ROS. Confirming this notion, H 2 O 2 has been shown to increase GJIC in cultured astrocytes, and this response was reverted by reducing agents (Rouach et al., 2004). Therefore, although free radicals like NO, decrease GJIC, others such as H 2 O 2 or one of their sub-products (i.e., oxidized gluthathione; GSSG) may have the opposite effect. ...
... The expression of IL-12 and the production of inducible nitric oxide synthase (iNOS) in activated microglia have been reported to be suppressed by astrocytes or conditioned media from astrocytes [82,111,115116117, delineating the signals from astrocytes that affect the activities of microglia. Furthermore , the communication between these two types of cells is two-way; microglia both receive and give signals, as proinflammatory cytokines released from microglia inhibit gap junctions and down-regulate connexin 43 expression in astrocytes118119120, which enhances astrocyte survival. In another study, comparative proteome analysis was performed on astrocytes that were treated with conditioned media from quiescent or activated microglia. ...
It has been nearly a century since the early description of microglia by Rio-Hortega; since then many more biological and pathological features of microglia have been recognized. Today, microglia are generally considered to be beneficial to homeostasis at the resting state through their abilities to survey the environment and phagocytose debris. However, when activated microglia assume diverse phenotypes ranging from fully inflamed, which involves the release of many pro-inflammatory cytokines, to alternatively activated, releasing anti-inflammatory cytokines or neurotrophins, the consequences to neurons can range from detrimental to supportive. Due to the different experimental sets and conditions, contradictory results have been obtained regarding the controversial question of whether microglia are "good" or "bad." While it is well understood that the dual roles of activated microglia depend on specific situations, the underlying mechanisms have remained largely unclear, and the interpretation of certain findings related to diverse microglial phenotypes continues to be problematic. In this review we discuss the functions of microglia in neuronal survival and neurogenesis, the crosstalk between microglia and surrounding cells, and the potential factors that could influence the eventual manifestation of microglia.
... However the beneficial aspects of hydrogen peroxide in regulating signal transduction in a number of cellular processes are only beginning to emerge. For example, neurons as well as brain macrophages produce superoxide in pathological situations and the hydrogen peroxide produced from superoxide is known to increase gap junctional communication in astrocytes [1]. The dismutation of superoxide produces hydrogen peroxide as well as singlet oxygen. ...
Excessive reactive oxygen species, such as hydrogen peroxide (H 2 O 2), seems to have a deleterious effect in patients with Parkinson's disease. The electrochemical behavior of this molecule in the presence of the most common biological electrolyte, sodium chloride, over a concentration range of 0.02 – 1.0 M, was investigated by cyclic voltammetry. The 'normal' and the 'activated' or 'autocatalytic' H 2 O 2 reduction were observed. The 'normal' H 2 O 2 reduction current at high cathodic potentials was nearly the same for scans 2 and 3. A slightly higher current for scan 1 was observed compared to the subsequent scans. On the other hand, the 'autocatalytic' H 2 O 2 reduction current increased with increasing number of scans and increasing sweep to higher cathodic potentials. A slight cathodic shift in the peak was also observed with increasing number of scans. Also a strong cathodic current was observed on reversing the scan from -1.0 V to less cathodic potentials. With increasing concentration of H 2 O 2 , there was a slight cathodic shift in the cyclic voltammetric peak observed for the 'autocatalytic' H 2 O 2 reduction. A new cathodic shoulder peak before the 'autocatalytic' H 2 O 2 reduction was observed for the first time. The autocatalytic process was not observed for peroxide in 0.1 M HCl. Current oscillations were observed in 0.04 M NaOH. Addition of traces of chloride enhanced the autocatalytic cathodic current in basic medium. Both the 'normal' and 'autocatalytic' H 2 O 2 reductions were observed in 0.1 M Na 2 SO 4 and 0.1 M NaClO 4 . The catalytic activity was highest in the presence of sulfate and least with perchlorate. We conclude that the 'autocatalytic' H 2 O 2 reduction process is sensitive to the range of the sweep potential, concentration of H 2 O 2 , NaCl concentration, and pH. The multiple equilibria involved in the H 2 O 2 redox process were evident in the observed cyclic voltammograms.
... On the other hand, in L2, a rat lung epithelial cell line, which expresses both Cx32 and Cx43, and Marshall cells, a fibroblastoid rat mammatory tumour cell line, which expresses Cx43, ROS (H 2 O 2 , 1 mM, 3 min after treatment) depolarized cell membranes and opened connexin hemichannels (Ramachandran et al. 2007). Moreover, a 10-min exposure to 100 lM H 2 O 2 increased Cx43 gap-junction communication in cultured rat astrocytes (Rouach et al. 2004). ...
The pair of giant reciprocally coupled neurons VD1 and RPaD2 within the CNS of the freshwater pond snail Lymnaea stagnalis was used to analyse the effect of hydrogen peroxide on gap-junction connection. Electrical activity of VD1/RPaD2 was recorded with intracellular microelectrodes in order to analyse gap-junction signalling. Hydrogen peroxide application (1 × 10⁻⁴ M) results in a rapid, 1.3-fold, increase in VD1/RPaD2 spiking frequency within 30 s after application. This was accompanied by a slight reduction in action potential amplitude. In addition, H₂O₂ induced a significant reduction in the steady-state bidirectional coupling ratio between the neurons. The maximal reduction in the coupling ratio, 1.8-1.9 fold, was measured 3 min after H₂O₂ application. However, the network input resistance did not undergo a detectable change. The voltage-gated Ca²⁺ channel blocker, nifedipine (1 × 10⁻⁴ M), abolished the effect of H₂O₂ on the coupling ratio and firing frequency. All the effects of H₂O₂ were reversible, that is, washing the preparation with standard physiological saline restored the properties of the neuronal coupling to the pre-treatment value. These data are consistent with a dynamic modulation of the gap-junction properties by H₂O₂ between these two neurons.
... The latter is in agreement with the known neuroprotective effect of resting microglia (Block et al. 2007). In fact, microglia suppress both the effect of H 2 O 2 on astroglial gap junctional communication and its toxicity (Rouach et al. 2004). In contrast, activated microglia and pro-inflammatory cytokines induce astroglial uncoupling (Faustmann et al. 2003), astroglial hemichannel opening and neuronal death (Retamal et al. 2007, Block et al. 2007. ...
J. Neurochem. (2011) 118 , 826–840.
Abstract
Inflammation contributes to neurodegeneration in post‐ischemic brain, diabetes, and Alzheimer’s disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid‐β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3 h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell‐cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ‐treated microglia (CM‐Aβ). The effects of CM‐Aβ were mimicked by TNF‐α and IL‐1β and were abrogated by neutralizing TNF‐α with soluble receptor and IL‐1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM‐Aβ made them toxic to neurons subjected to a sub‐lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM‐Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer’s disease, diabetes and ischemia.
... However, at concentrations normally found in plasma (50–100 µM), which are lower than those employed in the present experiments, AA can also oppose the inhibition of CPA-induced relaxation and smooth muscle hyperpolarization by 43Gap26 and 37,40Gap26.8 It should also be noted that in non-vascular cells H2O2 has been variously shown to enhance or inhibit intercellular coupling via gap junctions constructed from Cx43, probably by alternating the phosphorylation/oxidation status of residues present in the intracellular cytoplasmic tail of this connexin subtype.31–35 Indeed, the hyperphosphorylation of Cx43 that follows administration of H2O2 or phorbol esters (which generate ·O2−) can be prevented by a spectrum of antioxidants, including AA, with preservation of channel function.36,37 ...
Our objective was to investigate whether pro-oxidant properties of ascorbic acid (AA) and tetrahydrobiopterin (BH(4)) modulate endothelium-dependent, electrotonically mediated arterial relaxation.
In studies with rabbit iliac artery (RIA) rings, NO-independent, endothelium-derived hyperpolarizing factor (EDHF)-type relaxations evoked by the sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and the G protein-coupled agonist acetylcholine (ACh) were enhanced by AA (1 mM) and BH(4) (200 microM), which generated buffer concentrations of H(2)O(2) in the range of 40-80 microM. Exogenous H(2)O(2) potentiated cyclopiazonic acid (CPA)- and ACh-evoked relaxations with a threshold of 10-30 microM, and potentiation by AA and BH(4) was abolished by catalase, which destroyed H(2)O(2) generated by oxidation of these agents in the organ chamber. Adventitial application of H(2)O(2) also enhanced EDHF-type dilator responses evoked by CPA and ACh in RIA segments perfused intraluminally with H(2)O(2)-free buffer, albeit with reduced efficacy. In RIA rings, both control relaxations and their potentiation by H(2)O(2) were overcome by blockade of gap junctions by connexin-mimetic peptides (YDKSFPISHVR and SRPTEK) targeted to the first and second extracellular loops of the dominant vascular connexins expressed in the RIA. Superoxide dismutase attenuated the potentiation of EDHF-type relaxations by BH(4), but not AA, consistent with findings demonstrating a differential role for superoxide anions in the generation of H(2)O(2) by the two agents.
Pro-oxidant effects of AA and BH(4) can enhance the EDHF phenomenon by generating H(2)O(2), which has previously been shown to amplify electrotonic hyperpolarization-mediated relaxation by facilitating Ca(2+) release from endothelial stores.
... Administration of PGF 2α resulted in a 3-fold increase in hydroxyl radicals and a 2-fold increase in malondialdehyde, an end product of membrane lipid peroxidation [186]. It has also been shown that H 2 O 2 is toxic to neurons [203][204][205][206][207], astrocytes [208,209], and oligodendrocytes [210][211][212]. Oxidative stressors, such as H 2 O 2 administration, also increase AA release in neurons and mesanglial cells [114,204]. ...
Secretory phospholipases A(2) (sPLA(2)s) are a subfamily of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor (PAF). The hydrolysis of membrane phospholipids by PLA(2) is a rate-limiting step for generation of eicosanoids and PAF. To date, more than 10 isozymes of sPLA(2) have been found in the mammalian central nervous system (CNS). Under physiological conditions, sPLA(2)s are involved in diverse cellular responses, including host defense, phospholipid digestion and metabolism. However, under pathological situations, increased sPLA(2) activity and excessive production of free fatty acids and their metabolites may lead to inflammation, loss of membrane integrity, oxidative stress, and subsequent tissue injury. Emerging evidence suggests that sPLA(2) plays a role in the secondary injury process after traumatic or ischemic injuries in the brain and spinal cord. Importantly, sPLA(2) may act as a convergence molecule that mediates multiple key mechanisms involved in the secondary injury since it can be induced by multiple toxic factors such as inflammatory cytokines, free radicals, and excitatory amino acids, and its activation and metabolites can exacerbate the secondary injury. Blocking sPLA(2) action may represent a novel and efficient strategy to block multiple injury pathways associated with the CNS secondary injury. This review outlines the current knowledge of sPLA(2) in the CNS with emphasis placed on the possible roles of sPLA(2) in mediating CNS injuries, particularly the traumatic and ischemic injuries in the brain and spinal cord.
... There is some evidence that H2O2 can affect gap junction activity and calcium homeostasis, two factors that are integral for EDH activity. Depending on the experimental conditions, studies have shown that H2O2 can both increase 58 and decrease 59 gap junctional coupling and effect changes in intracellular calcium homeostasis, both in cultured cells and in intact arteries. [59][60][61] Although no specific evidence is currently available to support this proposal, these observations provide potential support for a mechanism to link the putative role of H2O2 as an EDH(F) with the MEGJ dependence of the EDH phenomenon. ...
1. The principal mediators of vascular tone are neural, endothelial and physical stimuli that result in the initiation of dilator and constrictor responses to facilitate the control of blood pressure. Two primary vasodilatory stimuli produced by the endothelium are nitric oxide (NO) and prostaglandins. An additional endothelium-dependent vasodilatory mechanism is characterized as the hyperpolarization-mediated relaxation that remains after the inhibition of the synthesis of NO and prostaglandins. This mechanism is due to the action of a so-called endothelium-derived hyperpolarizing factor (EDHF) and is dependent on either the release of diffusible factor(s) and/or to a direct contact-mediated mechanism.
2. Most evidence supports the concept that ‘EDHF’ activity is dependent on contact-mediated mechanisms. This involves the transfer of an endothelium-derived electrical current, as an endothelium-derived hyperpolarization (EDH), through direct heterocellular coupling of endothelial cells and smooth muscle cells via myoendothelial gap junctions (MEGJ). However, there is a lack of consensus with regard to the nature and mechanism of action of EDHF/EDH (EDH(F)), which has been shown to vary within and between vascular beds, as well as among species, strains, sex and during development, ageing and disease.
3. In addition to actual heterogeneity in EDH(F), further heterogeneity has resulted from the less-than-optimal design, analysis and interpretation of data in some key papers in the EDHF literature; with such views being perpetuated in the subsequent literature.
4. The focus of the present brief review is to examine what factors are proposed as EDH(F) and highlight the correlative structural and functional studies from our laboratory that demonstrate an integral role for MEGJ in the conduction of EDH, which account for the heterogeneity in EDH(F), while incorporating the reported diffusible mechanisms in the regulation of this activity. Furthermore, in addition to the reported heterogeneity in the nature and mechanism of action of EDH(F), the contribution of experimental design and technique to this heterogeneity will be examined.
... This inhibition may be due to closure of gap junction channels (11,12). In contrast, H 2 O 2 also is reported to increase gap junctional communication in astrocytes, and prolonged treatment with H 2 O 2 consequently caused cell death (13). Connexin proteins have numerous kinase target sites, and Ser-368 is the site of phosphorylation of Cx43 by PKC (14). ...
The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.
... Such a sequel contributes to many pathological features during ischemia and inflammation and is a putative cause for human degenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (Halliwell, 1992;Coyle and Puttfarcken, 1993;Olanow, 1993). Although the action of H 2 O 2 on astrocytes or astrocytes cultured together with neurons had been addressed (Rouach et al., 2004;Langeveld et al., 1995;Desagher et al., 1996), the direct effects of H 2 O 2 on astrocytes have not been studied. ...
Astrocytes are one of the predominant glial cell types in the adult central nervous system functioning as both supportive and metabolic cells for the brain. Our objective in this experiment is to study the direct effects of hydrogen peroxide induced oxidative stress on astrocytes in culture. These astrocytes were derived from both an aged mouse strain (P8) and a matched control strain (R1). The astrocytes for both the P8 and R1 strains were treated with increasing concentrations of hydrogen peroxide. Our results showed that the oxidative stress had a similar effect in both strains of astrocytes; decreases in 3-(4,5-dimethylthiazol-2-yl)-2,2-diphenyltetrazolium bromide (MTT) and glial fibrillary acidic protein (GFAP) levels, and increases in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, lactate dehydrogenase (LDH) staining, and superoxide dismutase (SOD), caspase-3 and B-cell lymphoma 2-associated protein X (bax) levels. At a hydrogen peroxide concentration of 400 microM , the differences of the above parameters between P8 cultures and R1 cultures were statistically significant (p<0.05). This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a hydrogen peroxide concentration of 400 microM. The inability of the P8 astrocytes to counteract the oxidative stress might lead to inadequate protection from neuronal loss possibly resulting in significantly more astrocytic death. Our results suggested that the changes of astrocytes in peroxide detoxification may play a role in aging of the central nervous system, and further aging studies should examine the oxidative status of the samples.
... However, hyperglycemia and/or oxidative stress induced by hyperglycemia have been implicated in the development of other complications of diabetes such as neuropathy and angiopathy (3). Both high glucose and oxidative stress in vitro have been shown to alter Cx expression and/or gap junctional communication in a variety of different cell types (22)(23)(24), and Cx43 and Cx26 are known to have different promoters (25,26). It should be emphasized that impaired wound healing has been demonstrated in rats as early as 1 week after induction of diabetes, before either neuropathy or angiopathy develops (16,17). ...
Dynamically regulated expression of the gap junction protein connexin (Cx)43 plays pivotal roles in wound healing. Cx43 is normally downregulated and Cx26 upregulated in keratinocytes at the edge of the wound as they adopt a migratory phenotype. We have examined the dynamics of Cx expression during wound healing in diabetic rats, which is known to be slow.
We induced diabetes with streptozotocin and examined Cx expression and communication in intact and healing skin.
We found that diabetes decreased Cx43 and Cx26 protein and communication in the intact epidermis and increased Cx43 protein and communication in the intact dermis. Diabetes also altered the dynamic changes of Cxs associated with wound healing. Within 24 h, Cx43 was upregulated in a thickened bulb of keratinocytes at the wound edge (rather than downregulated as in controls, which formed a thin process of migratory cells). Cx43 decline was delayed until 48 h, when reepithelialization began. Although Cx26 was upregulated as normal after wounding in diabetic skin, its distribution at the wound edge was abnormal, being more widespread. Application of Cx43-specific antisense gel to diabetic wounds prevented the abnormal upregulation of Cx43 and doubled the rate of reepithelialization, which exceeded control levels.
Cx expression in diabetic skin is abnormal, as is the dynamic response of Cx43 to injury, which may underlie the delayed healing of diabetic wounds. Preventing the upregulation of Cx43 in diabetic wounds significantly improves the rate of healing and clearly has potential therapeutic value.
The endothelium, a monolayer of endothelial cells, constitutes the inner cellular lining of the blood vessels (arteries, veins and capillaries) and the lymphatic system, and therefore is in direct contact with the blood/lymph and the circulating cells. The endothelium is a major player in the control of blood fluidity, platelet aggregation and vascular tone, a major actor in the regulation of immunology, inflammation and angiogenesis, and an important metabolizing and an endocrine organ. Endothelial cells controls vascular tone, and thereby blood flow, by synthesizing and releasing relaxing and contracting factors such as nitric oxide, metabolites of arachidonic acid via the cyclooxygenases, lipoxygenases and cytochrome P450 pathways, various peptides (endothelin, urotensin, CNP, adrenomedullin, etc.), adenosine, purines, reactive oxygen species and so on. Additionally, endothelial ectoenzymes are required steps in the generation of vasoactive hormones such as angiotensin II. An endothelial dysfunction linked to an imbalance in the synthesis and/or the release of these various endothelial factors may explain the initiation of cardiovascular pathologies (from hypertension to atherosclerosis) or their development and perpetuation.
Table of Contents: Introduction / Multiple Functions of the Endothelial Cells / Calcium Signaling in Vascular Cells and Cell-to-Cell Communications / Endothelium-Dependent Regulation of Vascular Tone / Conclusion / References
The endothelium controls vascular tone by releasing various vasoactive substances. Additionally, another pathway associated with the hyperpolarization of both endothelial and vascular smooth muscle cells contributes also to endothelium-dependent relaxations (EDHF-mediated responses). These responses involve an increase in the intracellular Ca₂⁺ concentration of the endothelial cells followed by the opening of Ca₂⁺-activated K⁺ channels of small and intermediate conductances (SKCa and IKCa). These channels show a distinct subcellular distribution, suggesting that their activation could be elicited by distinct stimuli. Following KCa activation, the endothelial hyperpolarization can be conducted to the underlying smooth muscle cells by electrical coupling through myo-endothelial gap junctions. In addition, the potassium efflux can lead to the accumulation of potassium ions in the intercellular space and the subsequent activation of smooth muscle Kir2.1 and/or Na⁺/K⁺-ATPase. The hyperpolarization of the smooth muscle cells produces vascular relaxation, predominantly by closing voltage-gated calcium channels, and vasodilatation. EDHFmediated responses are altered in various pathologies or, conversely, act as a compensating mechanism when other endothelial pathways are impaired. A better characterization of EDHF-mediated responses should allow determining whether or not new drugable targets can be identified within this endothelial pathway for the treatment of cardiovascular diseases.
Table of Contents: Endothelium-Dependent Hyperpolarizations: The Classical "EDHF" Pathway / Conclusion / References
Synaptic plasticity requires the careful synchronization and coordination of neurons and glial cells via various mechanisms of intercellular communication. Among them, are those mediated by i) connexin gap junction channels (GJCs), ii) connexin hemichannels and iii) pannexin channels. Whereas GJCs directly communicate the cytoplasm of contacting cells and coordinate electric and metabolic activities, connexin hemichannels and pannexin channels serve as diffusional pathways for ions and small molecules between the intra- and extracellular compartments. A growing body of evidence has revealed that intercellular communication could be critical in the spread of protective and/or deleterious signals during stroke. Here, we review the current findings on the regulation of connexin- and pannexin-based channels in ischemic stroke and how they contribute to cell damage observed in pathology. Depending on the intensity of the ischemia, brain region and connexin subtype expressed, GJCs may provide the proper diffusion of energy metabolites and dissipation of toxic substances, whereas, in other circumstances, they could increase the damage by spreading toxic molecules. Alternatively, connexin hemichannel and pannexin channel opening may favor the release of neurotoxic substances (e.g., glutamate), but in other cases, they may confer neuroprotection against an ischemic episode by the phenomenon of ischemic preconditioning. The development of new drug modulators using in silico devices for connexin and pannexin-based channels will be crucial for future therapies against stroke.
The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexins of which Cx43 is most widespread in the human body. In the brain, Cx43 GJs are mostly found in astroglia where they coordinate the propagation of Ca(2+) waves, spatial K(+) buffering, and distribution of glucose. Beyond its role in direct intercellular communication, Cx43 also forms unapposed, non-junctional hemichannels in the plasma membrane of glial cells. These allow the passage of several neuro- and gliotransmitters that may, combined with downstream paracrine signaling, complement direct GJ communication among glial cells and sustain glial-neuronal signaling. Mutations in the GJA1 gene encoding Cx43 have been identified in a rare, mostly autosomal dominant syndrome called oculodentodigital dysplasia (ODDD). ODDD patients display a pleiotropic phenotype reflected by eye, hand, teeth, and foot abnormalities, as well as craniofacial and bone malformations. Remarkably, neurological symptoms such as dysarthria, neurogenic bladder (manifested as urinary incontinence), spasticity or muscle weakness, ataxia, and epilepsy are other prominent features observed in ODDD patients. Over 10 mutations detected in patients diagnosed with neurological disorders are associated with altered functionality of Cx43 GJs/hemichannels, but the link between ODDD-related abnormal channel activities and neurologic phenotype is still elusive. Here, we present an overview on the nature of the mutants conveying structural and functional changes of Cx43 channels and discuss available evidence for aberrant Cx43 GJ and hemichannel function. In a final step, we examine the possibilities of how channel dysfunction may lead to some of the neurological manifestations of ODDD.
Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by dementia and the presence of amyloid plaques and anomalous tau aggregates. Although pathophysiological mechanisms are still unclear, neuroinflammation and glial cell dysfunction have been identified as conspicuous components of AD. Glial cell dysfunction is associated with dysregulated production of inflammation mediators and generation of both reactive oxygen species (ROS) and reactive nitrogen species (RNS), which affect synapses and induce neuronal damage. Importantly, both increased neuroinflammation and ROS/RNS production by glia dysregulate communication mediated by connexin-based channels in brain cells, which could further affect oxidative balance and neuronal viability. Recent evidence suggests that connexin-based channels could be involved in AD pathogenesis. Here we discuss how aging affects neuroinflammation, oxidative stress, and connexin-based channels and the potential relevance of these changes for AD. Understanding how they cooperate as pathogenic mechanisms of AD is promising for the discovery of new therapeutic strategies against neurodegenerative disorders.
Hydrogen peroxide (H2O2) was the main types of many peroxides produced in living mammalian cells that consumed oxygen. In the brain, the main source of H2O2 was the superoxide dismutase (SOD)-catalyzed reaction in mitochondria. However, the level of H2O2 would be elevated through administration of control drugs and alcohol by dopamine metabolism of monoamine oxidase. In this study, a H2O2 microsensor was used to investigate the level of H2O2 in the brain striatum after administration of methamphetamine (MAP), morphine (MrP) or ethanol (Eth). The placement of microsensor in the brain was done at coordinates A/P 1.1 from bregma, M/L+2.6 and D/V-1.5. A working potential of +0.05 V vs. Ag/AgCl was applied. The H2O2 concentration was measured direct from the current generated by its catalytic reaction at the electro active surface of the electrode. A significant increase of H2O2 level was observed after 7 successive injections of the controlled drugs or alcohol. The initial measurement of H2O2 is essential as excess dosage of H2O2 during treatment will contribute to the formation of neurotoxin oxygenated radicals. The H2O2 was the precursor of O2− and OH radicals. Thus, this study provided a mean to monitor H2O2 level in the brain.
In designing an amperometric l-glutamate biosensor based on the electrochemical detection of enzymatically generated H2O2, the key challenges are selectivity against electrooxidizable interferents, micromolar detection limit, and response time of seconds or less. With these issues in mind, a glutamate microbiosensor consisting of Pt black (PtBlk) electrodeposited on a 125-μm-dia. Pt wire with a permselective film of electropolymerized, overoxidized polypyrrole (OPP) and a top layer of l-glutamate oxidase (l-GluOx) crosslinked with glutaraldehyde was constructed. The OPP film permits passage of H2O2 to the electrode surface while rejecting the common interferents of brain extracellular fluid, ascorbate (AA) and dopamine (DA). The PtBlk treatment of Pt electrodes was adopted since the microscale roughness of PtBlk increases the effective surface area of the electrode and promotes electrochemical H2O2 oxidation at lower potential. This microbiosensor was highly sensitive (80 ± 10 nA μM−1 cm−2) to l-glutamate at 450 mV versus Ag/AgCl, a significantly lower potential than the ∼700 mV used with most similar l-glutamate sensors prepared on polished Pt electrodes. Notably, microbiosensor response to both AA and DA was undetectable at concentrations exceeding physiological extracellular concentrations of these compounds. The described l-glutamate biosensor also showed excellent response time (∼1–2 s) and temporal stability (∼3 week half-life). In addition, this microbiosensor was robust enough for insertion into live brain tissue where it responded rapidly to l-glutamate, but was insensitive to dopamine.
Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP(3), and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.
It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca(2+)-activated K(+) channels channels by InsP(3)-induced Ca(2+) release from the endoplasmic reticulum and/or store-operated Ca(2+) entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.
Research on oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with their macromolecular machinery, particularly lipids, proteins, and DNA. However, many chronic diseases are not always a consequence of tissue necrosis, DNA, or protein damage, but rather to altered gene expression. Gene expression is highly regulated by the coordination of cell signaling systems that maintain tissue homeostasis. Therefore, much research has shifted to the understanding of how ROS reversibly control gene expression through cell signaling mechanisms. However, most research has focused on redox regulation of signal transduction within a cell, but we introduce a more comprehensive-systems biology approach to understanding oxidative signaling that includes gap junctional intercellular communication, which plays a role in coordinating gene expression between cells of a tissue needed to maintain tissue homeostasis. We propose a hypothesis that gap junctions are critical in modulating the levels of second messengers, such as low molecular weight reactive oxygen, needed in the transduction of an external signal to the nucleus in the expression of genes. Thus, any comprehensive-systems biology approach to understanding oxidative signaling must also include gap junctions, in which aberrant gap junctions have been clearly implicated in many human diseases.
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.
To investigate whether ascorbic acid (AA) can influence endothelium-dependent relaxation by modulating the spread of endothelial hyperpolarization through the arterial wall via gap junctions.
Force development and membrane potential were monitored by myography and sharp electrode techniques in isolated rabbit iliac arteries.
AA prevented the ability of the gap junction blocker 2-aminoethoxydiphenyl borate to inhibit endothelium-dependent relaxations and subintimal smooth muscle hyperpolarizations evoked by cyclopiazonic acid in the presence of nitric oxide (NO) synthase and cyclooxygenase blockade. AA also prevented the ability of a connexin-mimetic peptide targeted against Cx37 and Cx40 (37,40Gap 26) to attenuate the transmission of endothelial hyperpolarization to subintimal smooth muscle, and a peptide targeted against Cx43 (43Gap 26) to attenuate the spread of subintimal hyperpolarization to subadventitial smooth muscle and the associated mechanical relaxation. Parallel studies with endothelium-denuded preparations demonstrated that AA and cyclopiazonic acid both depressed relaxation evoked by the NO donor MAHMA NONOate.
The data suggest that AA can modulate arterial function through a previously unrecognized ability to preserve electrotonic signalling via myoendothelial and homocellular smooth muscle gap junctions under conditions where cell coupling is depressed. Underlying mechanisms do not involve amplification of 'residual' NO activity by AA.
The properties of astroglial gap junction channels and the protein that constitutes the channels were characterized by immunocytochemical, molecular biological, and physiological techniques. Comparative immunocytochemical labeling utilizing different antibodies specific for liver connexin 32 and connexin 26 and antibodies to peptides corresponding to carboxy-terminal sequences of the heart gap junction protein (connexin 43) indicates that the predominant gap junction protein in astrocytes is connexin 43. The expression of this connexin in cultured astrocytes was also established by Western and Northern blot analyses. Cultured astrocytes expressed connexin 43 mRNA and did not contain detectable levels of the mRNAs encoding connexin 32 or connexin 26. Further, the cells contained the same primary connexin 43 translation product and the same phosphorylated forms as heart. Finally, electrophysiological recordings under voltage-clamp conditions revealed that astrocyte cell pairs were moderately well coupled, with an average junctional conductance of about 13 nS. Single-channel recordings indicated a unitary junctional conductance of about 50–60 pS, which is of the same order as that found in cultured rat cardiac myocytes, where the channel properties of connexin 43 were first described. Thus, physiological properties of gap junction channels appear to be determined by the connexin expressed, independent of the tissue type.
A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication.
Gap junctions are highly conductive channels that allow the direct transfer of intracellular messengers such as Ca2+ and inositol triphosphate (IP3) between interconnected cells. In brain, astrocytes are coupled extensively by gap junctions. We found here that gap junctions among astrocytes in acutely prepared brain slices as well as in culture remained open during ischemic conditions. Uncoupling first occurred after the terminal loss of plasma membrane integrity. Gap junctions therefore may link ischemic astrocytes in an evolving infarct with the surroundings. The free exchange of intracellular messengers between dying and potentially viable astrocytes might contribute to secondary expansion of ischemic lesions.
The properties of astroglial gap junction channels and the protein that constitutes the channels were characterized by immunocytochemical, molecular biological, and physiological techniques. Comparative immunocytochemical labeling utilizing different antibodies specific for liver connexin 32 and connexin 26 and antibodies to peptides corresponding to carboxy-terminal sequences of the heart gap junction protein (connexin 43) indicates that the predominant gap junction protein in astrocytes is connexin 43. The expression of this connexin in cultured astrocytes was also established by Western and Northern blot analyses. Cultured astrocytes expressed connexin 43 mRNA and did not contain detectable levels of the mRNAs encoding connexin 32 or connexin 26. Further, the cells contained the same primary connexin 43 translation product and the same phosphorylated forms as heart. Finally, electrophysiological recordings under voltage-clamp conditions revealed that astrocyte cell pairs were moderately well coupled, with an average junctional conductance of about 13 nS. Single-channel recordings indicated a unitary junctional conductance of about 50-60 pS, which is of the same order as that found in cultured rat cardiac myocytes, where the channel properties of connexin 43 were first described. Thus, physiological properties of gap junction channels appear to be determined by the connexin expressed, independent of the tissue type.
H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate, or H2O2 alone. Employing Western blotting with anti-P-Tyr antibodies, we have identified in Fao cells at least four proteins (pp180, 150, 114, and 100) whose P-Tyr content is rapidly increased upon treatment of the cells with 3 mM H2O2. Tyrosine phosphorylation of these and additional proteins was markedly potentiated (6-10-fold) when 100 microM sodium orthovanadate was added together with H2O2. The effects of H2O2 and vanadate on protein tyrosine phosphorylation were rapid and specific. The enhanced tyrosine phosphorylation was accompanied by a concomitant inhibition of a cytosolic protein tyrosine phosphatase activity. The latter was inhibited by 50% in 3 mM H2O2-treated cells. The inhibitory effect was augmented in the combined presence of H2O2 and vanadate. Half- and maximal effects of vanadate were obtained at 15 microM and 1 mM, respectively. Vanadate (1 mM) alone, added to the cells, had only a trivial effect on protein tyrosine phosphatase activity. A 45-s challenge with insulin (10(-7) M) of cells pretreated with H2O2 largely mimicked the potentiating effects of vanadate on protein tyrosine phosphorylation but not on protein tyrosine phosphatase activity. Our results suggest the involvement of multiple tyrosine-phosphorylation proteins in mediating the biological effects of H2O2/vanadate. Their enhanced phosphorylation can be attributed at least in part, to the inhibitory effects exerted by H2O2 alone, or in combination with vanadate, on protein tyrosine phosphatase activity. The similarity between proteins phosphorylated in Fao cells in response to H2O2/vanadate or H2O2/insulin, suggests that either treatment stimulates protein tyrosine kinases having similar substrate specificities. The insulin receptor kinase is a likely candidate as its activity is markedly enhanced either by insulin (plus H2O2) or by H2O2/vanadate.
Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system are thought to be key events in the development of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be interdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cortical astrocytic cultures to either xanthine + xanthine oxidase (X/XO), a free radical-generating system, or hydrogen peroxide (H2O2) results in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximately 60% of its control value. In parallel no detectable release of lactate dehydrogenase occurs. X/XO effect is abolished in the presence of a mixture of scavenger enzymes (superoxide dismutase+catalase) or by the disulfide-reducing agents glutathione and dithiothreitol (DTT), but not by lipophilic antioxidants or ascorbate. The time course of inhibition shows an almost linear decline of glutamate transport during cell exposure to free radicals, while upon their inactivation the decline stops but established inhibition persists for at least 1 hr. In this situation, application of DTT significantly restores transport function. These data suggest that free radicals inhibit glutamate uptake primarily by long-lasting oxidation of protein sulfhydryl (SH) groups. Chemical modifiers of free SH groups, such as p-chloromercuribenzoate and N-ethylmaleimide, also induce uptake inhibition. Na+/K+ ATPase is a known target of oxygen radicals and may be involved in glutamate uptake inhibition. Indeed, ouabain, a blocker of the pump, reduces uptake in astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of K2CrO4, H2O2, benzoyl peroxide, menadione, KBrO3 and UV365nm on gap junctional intercellular communication (GJIC) have been studied in the 12-O-tetra-decanoylphorbol-13-acetate (TPA)-sensitive Syrian hamster embryo (SHE) cell line BPNi. All agents were found to increase
the level of GJIC by 50–100%. Also, in early passage SHE cells, a tendency for increased GJIC was found for the oxidative
agents studied. Hydrogen peroxide was used as a model compound in the subsequent studies. The increase in GJIC was reversible,
and it was not due to an increased non-junctional permeability. Hydrogen peroxide counteracted the TPA-induced decrease in
GJIC, regardless of whether the cells were exposed to the compounds simultaneously or the cells were pre-exposed to TPA before
addition of H2O2. The GJIC enhancement by H2O2 was slightly reduced by the addition of the hydroxyl radical scavenger dimethylsulphoxide or by the inhibition of catalase
by amitrole. The cAMP/ protein kinase A system is the only characterized signal transduction system that is known to increase
GJIC in most cell types. Hydrogen peroxide did not increase the amount of cAMP (or cGMP) in BPNi cells, while forskolin and
a phosphodiesterase inhibitor had to increase the cAMP level several-fold to affect GJIC to the same degree as the oxidative
agents. Some inhibitors of protein kinase A were assayed for their ability to inhibit the increases in GJIC caused by H2O2 and forskolin. Staurosporine inhibited the forskolin-induced increase in GJIC, with much less effect on the H2O2-induced increase. H8, H88 and H89 had less effect than staurosporine on the forskolin-induced increase in GJIC. The results
suggest that the cAMP/protein kinase A system may not be involved in the increase in GJIC caused by H2O2, although this cannot be completely ruled out.
Recent reports indicate that neurons are particularly sensitive to hydrogen peroxide (H2O2). The present study was undertaken to investigate the putative role of astrocytes in the modulation of the neurotoxic effect of H2O2. The exposure to H2O2 of cultured striatal neurons from mouse embryos induced a concentration-dependent (10-1000 microM) cell death as estimated 24 hr later. Two methods were used to estimate neuronal survival: the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay or an enzyme-linked immunosorbent assay with antibodies directed against an antigen located in neurons (microtubule-associated protein-2). The neurotoxic effect of H2O2 on neurons cocultured with astrocytes was strongly attenuated compared with that observed on a pure population of neurons seeded at the same density. Moreover, the protective effect of astrocytes depended on the astrocytes/neurons ratio, a significant neuroprotection being detectable for 1 astrocyte to 20 neurons. Catalase seems to be the main hydrogen peroxidase activity involved in the neuroprotective effect of astrocytes. Indeed, in the culture conditions used, this enzymatic activity was enriched in this cell type compared with neurons; its inhibition, and not that of glutathione peroxidase, reduced the disappearance rate of the oxidant. On the contrary, glutathione peroxidase appeared to be the main enzymatic activity involved in the neuronal defense against H2O2 toxicity. Therefore, astrocytes could delay neuronal death in pathological situations in which H2O2 has been, at least partially, demonstrated to be involved.
Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 microM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox, did not prevent the inhibition of GJIC by H2O2, which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione-sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH.
Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of approximately 34-37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel beta sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed.
Brain injury induces reactive gliosis, characterized by increased expression of glial fibrillary acidic protein (GFAP), astrocytes hypertrophy, and hyperplasia of astrocytes and microglia. One hypothesis tested in this study was whether ganglioside GD3+ glial precursor cells would contribute to macroglial proliferation following injury. Adult rats received a cortical stab wound. Proliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA) and by [³H]-thymidine autoradiography, and cell phenotypes by immunocytochemical staining for GD3, GFAP, ED1 (for reactive microglia) and for Bandeiraea Simplicifolia isolectin-B4 binding (all microglia). Animals were labeled with thymidine at 1, 2, 3, and 4 days postlesion (dpl) and sacrificed at various times thereafter. Proliferating cells of each phenotype were quantified. A dramatic upregulation of GD3 on ramified microglia was seen in the ipsilateral hemisphere by 2 dpl. Proliferating cells consisted of microglia and fewer astrocytes. Microglia proliferated maximally at 2–3 dpl and one third to one half were GD3+. Astrocytes proliferated maximally at 3–4 dpl, and some were also GD3+. Both ramified and ameboid forms of microglia proliferated and by 4 dpl all GD3+ microglia were ED1+ and vice versa. In the contralateral cortex microglia expressed neither GD3 nor ED1. Thus they acquired these antigens when activated. Neither microglia nor astrocytes that were thymidine-labeled at 2, 3, or 4 dpl changed in number in subsequent days. Most thymidine+ astrocytes were large GFAP+ reactive cells that clearly arose from pre-existing astrocytes, not from GD3+ glial precursors. In this model of injury microglia proliferate earlier and to a much greater extent than astrocytes, they can divide when in ramified form, and GD3 is up-regulated in most reactive microglia and in a subset of reactive astrocytes. We also conclude that microglial proliferation precedes proliferation of invading blood-borne macrophages. © 1996 Wiley-Liss, Inc.
Gap junctional communication is likely one means by which neurons can endure glutamate cytotoxicity associated with CNS insults (i.e. ischemia). To examine this neuroprotective role of gap junctions, we employed gap junctional blockers to neuronal and astrocytic co-cultures during exposure to a high concentration of extracellular glutamate. Co-cultures were treated with the blocking agents carbenoxolone (CBX; 25 microM), 18alpha-glycyrrhetinic acid (AGA; 10 microM), vehicle or the inactive blocking analogue glycyrrhizic acid (GZA; 25 microM). Twenty-four hours following the insult, cell mortality was analyzed and quantified by the release of lactate dehydrogenase (LDH) into the media, the cells' inability to exclude propidium iodide, and terminal dUTP nick end labeling (TUNEL). Measurement of LDH release revealed that the glutamate insult was detrimental to the co-cultures when gap junctions were blocked with CBX and AGA. Based on propidium iodide and TUNEL labeling, the glutamate insult caused significant cell death compared to sham vehicle and mortality was amplified in the presence of CBX and AGA. Since blockers were not themselves toxic and did not affect astrocytic uptake of glutamate, it is likely that blocked gap junctions lead to the increased glutamate cytotoxicity. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.
Several lines of evidence indicate that the extent of ischemic injury is not defined immediately following arterial occlusion; rather that infarction expands over time. Episodes of spreading depression have been linked to this secondary increase in infarct volume. Tissue bordering the infarct fails to repolarize following spreading depression and is incorporated into the infarction. The result is that ischemic infarcts expand stepwise following each episode of spreading depression. Another line of evidence has demonstrated that gap junction blockers effectively inhibit spreading depression.
These observations suggest that the efflux of potentially harmful cytosolic messengers from ischemic cells into surrounding nonischemic cells might cause amplification of injury in focal stroke. It is therefore conceivable that minimizing gap junction permeability might reduce final infarct volume. To test this hypothesis, the authors pretreated rats with the gap junction blocker, octanol, before occluding the middle cerebral artery and compared the sizes of the ischemic lesions to those in rats that received vehicle dimethyl sulfoxide prior to arterial occlusion. Histopathological analysis was performed 24 hours later. The 12 octanol-treated animals showed a significantly decreased mean infarction volume (80 ± 16 mm ³ ) compared with the nine control rats (148 ± 9 mm ³ ). In a separate set of experiments, the frequency of experimentally induced waves of spreading depression was evaluated following octanol treatment. Octanol pretreatment resulted in complete inhibition in two of nine animals, transient inhibition in five of nine, and no inhibition in two of nine.
The results indicate that gap junction inhibitors, when not limited by toxicity, have significant therapeutic potential in the treatment of acute stroke.
Conditioned media from embryonic mixed cells from the rat brain were used in a chemotaxis assay to look for potential chemotactic activity which could account for the infiltration of the developing central nervous system (CNS) by macrophage precursors. The most potent chemotactic activity was found in the conditioned medium from E17 mixed brain cells (E17-CM). Based upon checkerboard analysis, this activity was shown to be chemotactic rather than chemokinetic. This chemoattraction was not restricted to brain macrophages (BM) because it was as pronounced on bone marrow-derived macrophages. The implication of a peptide compound in this activity was suggested by its resistance to heat as well as acid treatments, and by its sensitivity to aminopeptidase M digestion. In agreement with the opioid nature of the peptide, not only naloxone, but also the delta opioid receptor antagonist ICI-174 reduced the migration of BM in response to E17-CM by 60%. This migratory activity was no longer effective when pertussis toxin-treated BM were used. When the chemotactic effects of selective opioid agonists were compared to that of E17-CM, DPDPE, the delta agonist, was the most efficient in attracting BM. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that delta as well as other known opioid receptors were expressed in both BM and E17 mixed brain cells. Finally, a Met-enkephalin-like reactivity was found by RIA in the E17-CM. Altogether, these observations suggest that a delta-like opioid peptide released from embryonic mixed brain cells could be responsible for the infiltration of the developing CNS by macrophages precursors.
Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.
Acute (10-30 min) treatment of intact rat hepatoma (Fao) cells with H2O2, inhibits in vivo protein tyrosine phosphatase activity. Vanadate markedly potentiates this effect although it has only trivial effects of its own. Here we show that H2O2 inhibits a protein tyrosine phosphatase activity, but not a p-nitro phenyl phosphate hydrolysing activity, in cytosolic extracts of these cells. This effect is completely reversed by 10 mM dithiothreitol. Other oxidants have similar inhibitory effects. Vanadate inhibits the protein tyrosine phosphatase activity in vitro, and its effects are additive with those of H2O2. These findings suggest that H2O2 and vanadate interact with the protein tyrosine phosphatases at two independent sites. They also suggest that in intact cells H2O2 has a direct inhibitory effect on protein tyrosine phosphatase activity and an indirect effect of facilitating the entry of vanadate.
A free radical is any species capable of independent existence that contains one or more unpaired electrons. Free radicals and other reactive oxygen species are frequently proposed to be involved in the pathology of several neurological disorders. Criteria for establishing such involvement are presented. Development of new methods for measuring oxidative damage should enable elucidation of the precise role of reactive oxygen species in neurological disorders.
Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.
This review addresses current understanding of oxygen radical mechanisms as they relate to the brain during ischemia and reperfusion. The mechanism for radical production remains speculative in large part because of the difficulty of measuring radical species in vivo. Breakdown of lipid membranes during ischemia leads to accumulation of free fatty acids. Decreased energy stores during ischemia result in the accumulation of adenine nucleotides. During reperfusion, metabolism of free fatty acids via the cyclooxygenase pathway and metabolism of adenine nucleotides via the xanthine oxidase pathway are the most likely sources of oxygen radicals. Although leukocytes have been found to accumulate in some models of ischemia and reperfusion, their mechanistic role remains in question. Therapeutic strategies aimed at decreasing brain injury have included administration of radical scavengers at the time of reperfusion. Efficacy of traditional oxygen radical scavengers such as superoxide dismutase and catalase may be limited by their inability to cross the blood-brain barrier. Lipid-soluble antioxidants appear more efficacious because of their ability to cross the blood-brain barrier and because of their presence in membrane structures where peroxidative reactions can be halted.
SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100 mM) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.
H2O2 compromises a multitude of cellular functions the combination of which leads to cell death. DNA is an important target for oxidant-induced injury. The formation of DNA strand breaks leads to activation of poly-ADP-ribose polymerase (24) which in turn causes depletion of NAD and ATP, followed by Ca++ influx and eventually by cell lysis. Inhibitors of poly-ADP-ribose polymerase prevented cell lysis, but not DNA damage. A similar sequence of events has been described for cell injury following DNA damage induced by gamma-irradiation and alkylating agents, and was proposed to be a suicide mechanism for cells with irreversibly damaged DNA. Sublethal doses of H2O2 will delay cell division, but not necessarily prevent it.
Human lung carcinoma cells (A549) were oxidatively stressed with mildly-toxic or non-toxic amounts of hydrogen peroxide (H2O2, 0.1 mM to 120 mM) for 5 min. Hydrogen peroxide exposure resulted in a dose dependent inhibition of binding (pH 7) of the thiol reagent iodoacetic acid (IAA) to a 38 kDa cell protein. Incubation of cells in saline for 60 min following H2O2 removal restored the ability of IAA to bind to the protein. Treatment with 20 mM dithiothreitol or 2 M urea also restored IAA binding, but 10% Triton X102 or 1 mM Brij 58 had no effect. Increasing to pH 11 during the IAA binding also increased thiol availability. Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) has been identified as the protein undergoing thiol/disulfide redox status and enzymic activity changes.
Treatment of WB-F344 liver epithelial cells with buthionine sulfoximine (BSO, 100 microM) for 24 h caused a greater than 95% depletion in cellular glutathione (GSH) and potentiated the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA) to inhibit gap junctional intercellular communication (GJIC) between the cells (IC50 shifted from 5 microM to 2 microM). Similarly, acute depletion of GSH by up to 30%, either with the thiol oxidant diamide or with BSO, also potentiated the inhibitory effect of the phorbol ester on GJIC. The treatment of the control cells with TPA caused a concomitant increase in the accumulation of oxidation products of 2',7'-dichlorofluorescein (DCF), indicating elevated production of oxidants in the cells during the blockade of GJIC. The depletion of GSH over a 24 h period with BSO itself increased the flux of oxidants in the cells but did not inhibit GJIC. Treatment of these GSH-depleted cells with TPA caused an additive elevation in the accumulation of oxidised DCF metabolites. Direct application of H2O2 (25-200 microM) or benzoyl peroxide (25-150 microM) to the control cells for 60 min caused weak, dose-dependent inhibitions of gap junctional communication in these cells but these responses were accompanied by the induction of acute, sub-lethal cytotoxicity. The depletion of GSH from the cells did not potentiate these responses to the peroxides but did facilitate synergistic inhibition of gap junctional communication in response to both TPA and sub-toxic doses of either peroxide. The results of the above studies indicate that oxidants are produced in WB-F344 cells in response to TPA and that these function in a co-operative manner with other cellular responses to the phorbol ester in the inhibition of gap junctional communication. This may explain why priming the cells for the induction of oxidative stress by the depletion of GSH potentiates the inhibitory activity of TPA on gap junctional communication.
Toxic reactive oxygen species have been implicated as important mediators of tissue injury after reperfusion of ischemic organs. When rats are subject to 30 min global forebrain ischemia, 24 h following this insult, there is substantial loss of medium-sized neurones as revealed by histological sectioning of the striatal region of the forebrain. The goal of this study was to utilize microdialysis to directly measure one of the more stable intermediates of reduced molecular oxygen, H2O2 in the rat striatum following 4-vessel occlusion and reperfusion, and to correlate these levels with H2O2 toxicity to neurones grown in culture. A significant rise in striatal H2O2 levels was observed for about 1 h during reperfusion, amounting to an increase of approximately 100 microM at the peak. In control experiments where the dialysis probe was embedded in cortical regions surrounding the striatum (where there is no neuronal loss due to the ischemic episode), there was no measurable increase in tissue H2O2 levels. H2O2 has been previously shown to be neurotoxic to PC12 cells as well as rat primary hippocampal neurones at comparable concentrations striatal neurones experience during reperfusion. We demonstrate that H2O2 is also neurotoxic to the human cortical neuronal cell line, HCN-1A. These experiments establish an important link between oxidant generation and neuronal loss in this tissue following global forebrain ischemia.
Astroglia are extensively coupled by gap junctions and form a functional syncytium. Astroglial gap junctions are thought to be involved in the spatial buffering of K+ in vivo and in the Ca2+ waves seen on glutamate receptor activation. The conductivity of gap junctions is regulated by several second messengers, with up-regulation by cyclic AMP and down-regulation through activation of protein kinase C, decreases in intracellular pH, or increases in the free cytosolic Ca2+ concentration. The results presented here indicate that dye coupling of astroglia is significantly up-regulated by membrane depolarization, both by increases in the extracellular K+ concentration and directly by ionophores. Furthermore, glutamate, kainate, and quisqualate, which depolarize astroglial cells through activation of ionotropic receptors, also increase dye coupling in astroglia. The effect of kainate and quisqualate was reversed by 6-cyano-7-nitroquinoxaline-2,3-dione, an inhibitor of the ionotropic glutamate receptor. A dose-dependent decrease in dye coupling was seen when the cells were injected with increasing concentrations of Ca2+. However, if the cells were simultaneously depolarized, the inhibitory effect of Ca2+ on gap junctional conductance was reversed. Significant increases over basal coupling was attained when the cells were injected with Ca2+ if they were treated with kainate or K+. These data suggest that ligands that depolarize astroglia enhance gap junction communication between astroglia and that this enhancement may be important in maintaining communication between astroglia in the face of elevated Ca2+ levels.
The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.
Intracerebral administration of kainic acid (KA) in rats was previously shown to abolish immunohistochemical labelling for the astrocytic gap junction protein connexin43 (Cx43) at sites depleted of neurons (Vukelic et al: Neurosci Lett 130:120-124, 1991). This response of Cx43 has now been further investigated with a number of different sequence-specific anti-Cx43 antibodies. At lesion sites in the thalamus, striatum, and hippocampus examined immunohistochemically with an antibody against amino acids (aa's) 346-363 in the Cx43 sequence, the antibody used in the earlier study, Cx43-immunoreactivity was increased 5 h after KA injections, absent by 24 h and for up to 2 weeks post-injection, and began to return to less than normal levels by 2 to 3 weeks post-injection. Analyses of KA lesion sites with antibodies against other sequences of Cx43 (amino acids 283-298, 253-270, 241-260, 113-123, and 49-61) revealed not only the presence but in some cases an increased density of Cx43 immunoreactivity after a survival time of 1 week. Immunolabelling patterns at these sites consisted of relatively large, coarse profiles rather the fine punctate labelling typically seen in sections of normal brain. In homogenates of KA-injected striatum analyzed by Western blots, Cx43 was detected at near normal or slightly increased levels at various survival times examined. The 43 kDa phosphorylated form of Cx43 and its faster migrating 41 kDa dephosphorylated form which is generated post-mortem by a brain phosphatase were both present after standard methods of tissue preparation for Western blot analysis, while only the 43 kDa form was present in normal and KA-injected striatum after inactivation of brain metabolism by focused cranial microwave irradiation. Ultrastructural investigations of lesions sites within the thalamus revealed a virtual absence of astrocytic gap junctions. These results demonstrate that Cx43 levels initially increase after intracerebral KA treatment, that its molecular organization in resident astrocytes is altered such that epitopes that are normally accessible to antibody are hidden while those that may be hidden or relatively inaccessible are exposed, and that this molecular alteration in Cx43 is associated with loss of astrocytic gap junctions.
After peripheral transection of the facial nerve, immunostaining of astrocytic gap junction protein changed in the corresponding brainstem nucleus of the rat. Enhanced connexin-43 immunoreactivity was restricted to the ipsilateral facial nucleus and to astrocytes surrounding lesioned motoneurons. This reaction is focally distinct, and marks only a part of the astrocytic network indicating a local plasticity of intercellular coupling. These results suggest that astrocytes work as sensors of signals which either depend on the integrity of neighboring neurons or inform about neuronal disorders.
Brain injury induces reactive gliosis, characterized by increased expression of glial fibrillary acidic protein (GFAP), astrocyte hypertrophy, and hyperplasia of astrocytes and microglia. One hypothesis tested in this study was whether ganglioside GD3+ glial precursor cells would contribute to macroglial proliferation following injury. Adult rats received a cortical stab wound. Proliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA) and by [3H]-thymidine autoradiography, and cell phenotypes by immunocytochemical staining for GD3, GFAP, ED1 (for reactive microglia) and for Bandeiraea Simplicifolia isolectin-B4 binding (all microglia). Animals were labeled with thymidine at 1,2,3, and 4 days postlesion (dpl) and sacrificed at various times thereafter. Proliferating cells of each phenotype were quantified. A dramatic upregulation of GD3 on ramified microglia was seen in the ipsilateral hemisphere by 2 dpl. Proliferating cells consisted of microglia and fewer astrocytes. Microglia proliferated maximally at 2-3 dpl and one third to one half were GD3+. Astrocytes proliferated maximally at 3-4 dpl, and some were also GD3+. Both ramified and ameboid forms of microglia proliferated and by 4 dpl all GD3+ microglia were ED1+ and vice versa. In the contralateral cortex microglia expressed neither GD3 nor ED1. Thus they acquired these antigens when activated. Neither microglia nor astrocytes that were thymidine-labeled at 2, 3, or 4 dpl changed in number in subsequent days. Most thymidine+ astrocytes were large GFAP+ reactive cells that clearly arose from pre-existing astrocytes, not from GD3+ glial precursors. In this model of injury microglia proliferate earlier and to a much greater extent than astrocytes, they can divide when in ramified form, and GD3 is up-regulated in most reactive microglia and in a subset of reactive astrocytes. We also conclude that microglial proliferation precedes proliferation of invading blood-borne macrophages.
Oxidative stress refers to the cytopathologic consequences of a mismatch between the production of free radicals and the ability of the cell to defend against them. Growing data from experimental models and human brain studies suggest oxidative stress may play an important role in neuronal degeneration in diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Mitochondrial oxidative metabolism, nitric oxide, phospholipid metabolism, and proteolytic pathways are potential sources of intracellular free radicals. Alterations in free radical defense systems may also contribute to oxidative stress. A net increase in reactive oxygen species can produce damage to lipids, proteins, and DNA and induce necrosis or apoptosis. Elucidating the pathways important in the production of and defense from free radicals may be important in devising new pharmacologic strategies to slow or halt neuronal degeneration.
Gap junctions are formed by a multigene family of polytopic membrane channel proteins, connexins, that have four hydrophobic transmembrane domains and their N and C termini located on the cytoplasmic membrane face. The C-terminal tail plays important roles in channel regulation by pH and phosphorylation. Conserved cysteine residues stabilize the conformation of the extracellular loops that mediate the 'docking' between connexons in the intercellular channel. Over the past year, electron cryocrystallography of two-dimensional crystals of a truncated recombinant alpha 1 (Cx43) has revealed that the transmembrane boundary of the intercellular channel is lined with alpha helices. Furthermore, a ring of alpha helices resides at the interface with the membrane lipids. A three-dimensional analysis based on images recorded from tilted crystals should reveal the location and secondary structure of additional transmembrane domains, as well as provide important structural details about the interactions between connexins within a hemi-channel and connexon-connexon interactions in the extracellular gap.
Astrocytes, which constitute the most abundant cell type in mammalian brain, are extensively coupled to one another through gap junctions composed mainly of connexin43. In regions exhibiting high levels of connexin43 expression, tens of astrocytes are labeled following single-cell intracellular injection. Importantly, both the expression and the permeability of gap junctions are tightly regulated. Such long- and short-term regulations indicate that astrocytic networks might be subject to remodeling and to some plasticity. Since evidence for neuro-glial interaction exists, the degree of coupling between astrocytes could participate to set the tone of neuronal activity and to determine the sphere of influenced neurons. Research in this area is still at its early stages and significant progress requires a transition from the understanding of basic properties to the study of function.
We present evidence to support the premise that many of the pathological correlates of Alzheimer's disease are precipitated by free radical- and oxidative stress-induced mechanisms. We propose that amyloid-beta deposition in senile plaques, intracellular accumulation of protein in neurofibrillary tangles, and the degeneration of specific neuronal populations can be attributed to specific oxidative stress-type mechanisms. Free radicals in disease pathogenesis, generated in part as a result of Fenton-type reactions, suggest that lowering the level of available iron, intervention with antioxidants, or the administration of free radical scavengers could provide a therapeutic inroad in the fight against Alzheimer's disease.
Cultured brain cells are capable of generating many molecules associated with inflammatory and immune functions. They constitute the endogenous immune response system of brain. They include complement proteins and their regulators, inflammatory cytokines, acute phase reactants and many proteases and protease inhibitors. Most of the proteins are made by microglia and astrocytes, but even neurons are producers. Many appear in association with Alzheimer disease lesions, indicating a state of chronic inflammation in Alzheimer disease brain. Such a state can apparently exist without stimulation by peripheral inflammatory mediators or the peripheral immune system. A strong inflammatory response may be autotoxic to neurons, exacerbating the fundamental pathology in Alzheimer disease and perhaps other neurological disorders. Autotoxic processes may contribute to cellular death in chronic inflammatory diseases affecting other parts of the body, suggesting the general therapeutic value of anti-inflammatory agents. With respect to Alzheimer disease, multiple epidemiological studies indicate that patients taking anti-inflammatory drugs or suffering from conditions in which such drugs are routinely used, have a decreased risk of developing Alzheimer disease. In one very preliminary clinical trial, the anti-inflammatory drug indomethacin arrested progress of the disease. New agents directed against the inflammatory processes revealed in studies of Alzheimer disease lesions may have broad therapeutic applications.
The protein-tyrosine phosphatase inhibitors pervanadate, permolybdate, H2O2, and to a much lesser extent vanadate, increased the amount of cellular phosphotyrosine and induced tyrosine phosphorylation of connexin43 (Cx43) in early passage hamster embryo fibroblasts. The presence of phosphotyrosine in Cx43 immunoprecipitates from pervanadate-treated cells was shown by a phosphotyrosine-specific antibody and a phosphotyrosine-specific phosphatase. Pervanadate-induced Cx43 tyrosine phosphorylation was further verified by phosphoamino acid analysis, while no phosphotyrosine was present in control cells. This is the first observation of tyrosine phosphorylation of connexins in normal cells.
Studies of ischemic brain injury in cell culture, animal models, and humans have revealed inter- and intra-cellular signaling pathways that increase resistance to cell degeneration and death. Brain injury induces expression of many different growth factors and cytokines which can protect neurons against insults relevant to the pathogenesis of ischemic brain injury including excitotoxicity, hypoxia, hypoglycemia, acidosis, and pro-oxidants. Neuroprotective signal transduction pathways elicit changes that promote the maintenance of cellular ion homeostasis and/or suppress the accumulation of free radicals. For example: basic fibroblast growth factor suppresses expression of a glutamate receptor protein and induces antioxidant enzymes; tumor necrosis factor induces expression of a Ca(2+)-binding protein and Mn-superoxide dismutase; and secreted forms of beta-amyloid precursor protein hyperpolarize neurons by activating K+ channels. Transcriptional regulation involves activation of tyrosine phosphorylation cascades and NFkB. Interestingly, similar neuroprotective pathways can be activated by moderate levels of cell "stress" such as that induced by glutamate in cell culture or a brief period of cerebral ischemia in vivo. Novel rapid and delayed intracellular neuroprotective signaling mechanisms are being revealed, such as the regulation of Ca2+ influx by actin filaments and the induction of genes by Ca2+ and radicals. New therapeutic approaches arising from this research include low molecular weight lipophilic compounds that activate neurotrophic factor signaling pathways and agents that selectively depolymerize actin.
Halothane is a strong inhibitor of potassium evoked spreading depression (SD) in cats. In the current study, we investigate halothane effects on induction of perifocal SD-like depolarizations, CBF, and infarct evolution in focal ischemia. Calomel and platinum electrodes measured cortical direct current potential and CBF in ectosylvian, suprasylvian, and marginal gyri. Left middle cerebral artery occlusion (MCAO) induced permanent focal ischemia for 16 hours in artificially ventilated cats (30% oxygen, 70% nitrous oxide) under halothane (0.75%, n = 8) or alpha-chloralose anesthesia (60 mg/kg intravenously, n = 7). Under alpha-chloralose, MCAO induced severe ischemia in ectosylvian and suprasylvian gyri(mean CBF < 10 mL/100 g/min), and direct current potentials turned immediately into terminal depolarization. In marginal gyri, CBF reduction was mild (more than 20 mL/100 g/min), and in six of seven animals, frequent SD-like depolarizations turned into terminal depolarization at a later stage of the experiments. Under halothane, MCAO induced severe ischemia (less than 10 mL/100 g/min) and immediate terminal depolarization only in ectosylvian gyrus. In suprasylvian gyrus, residual CBF remained significantly higher (more than 10 mL/100 g/min) than under alpha-chloralose, whereas in marginal gyri, CBF did not differ between groups. Compared with chloralose, the number of transient depolarizations was significantly reduced in marginal gyrus, and in suprasylvian gyrus transient but significantly longer depolarizations than in marginal gyrus were recorded. Except for one animal, transient depolarizations did not turn into terminal depolarization under halothane, and infarct volume reduction was particularly seen in suprasylvian gyrus. We conclude that halothane, the most commonly used anesthetic in studies of experimental brain ischemia, has protective properties, which may depend on both cerebrovascular and electrophysiologic influences.
We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18-alpha-glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7-dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4-hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte-astrocyte coupling but no astrocyte-neuron or neuron-neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA-treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L-type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate- and FeSO4-induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.
In this study, we investigated the involvement of reactive oxygen species (ROS) in the motorcycle exhaust particle (MEP)-induced genotoxic and non-genotoxic activity in mammalian cell systems. Initially, the capability of MEP to induce ROS was evaluated by using 2',7'-dichlorofluorescin diacetate (DCFH-DA) to detect hydrogen peroxide (H2O2). A five-fold increase in H2O2 was observed in Chinese hamster lung V79 and human lung carcinoma Calu-1 cells treated with 100 microg/ml MEP for 2 h. Under the same experimental conditions, only a two-fold elevation in H2O2 was detected in hepatic cell systems such as BNL.Cl.2, HepG2, and Hep3B. Treatment of the V79 cells with varying concentrations of MEP caused a dose-dependent increase in sister chromatid exchanges (SCEs), which are effectively inhibited by addition of antioxidants, N-acetyl-l-cysteine (NAC) and ascorbic acid. Furthermore, we determined the oxidized bases in the V79 cells after exposure to MEP. The result showed that 500 microg/ml MEP induced a 3.7-fold increase in thymine glycol (TG) and a seven-fold increase in 8-hydroxy-guanosine (8-OHGua) as compared to untreated cells. We subsequently examined whether MEP would affect gap junctional intercellular communication (GJIC), a tumor promotion process, in V79 cells. We found that MEP inhibited GJIC in a dose-response fashion. Maximal inhibition occurred at 500 microg/ml. The concentration that inhibited at 0.5 of the fraction of the control was 200 microg/ml. Interestingly, when cells were pretreated with NAC or ascorbic acid, they could abolish the MEP-mediated inhibition of GJIC. In addition, a moderate decrease of glutathione was observed in the V79 cells during exposure to MEP. Taken together, our findings suggest that MEP can induce oxidative stress in a broad range of cell lines, especially in lung cell systems. The MEP-induced oxidative stress was critically involved in both genotoxic and non-genotoxic activity.
Indo‐1 and fluo‐3 imaging techniques were used to investigate the role of gap junctions in the changes in cytosolic calcium concentrations ([Ca ²⁺ ] i ) induced by several receptor agonists. Subpopulations of confluent cultured astrocytes from the rat striatum were superfused with submaximal concentrations of endothelin‐1 (Et1) and the α1‐adrenergic and muscarinic receptor agonists, methoxamine and carbachol, respectively.
Combined binding and autoradiographic studies indicated that all striatal astrocytes possess binding sites for Et1. In contrast, α1‐adrenergic and muscarinic binding sites were found to be heterogeneously distributed. In agreement with these findings, Et1 induced fast calcium responses in all cells while only subsets of striatal astrocytes responded to the application of either methoxamine or carbachol.
Halothane, heptanol and octanol, which are commonly used as gap junction inhibitors, drastically reduced the amplitude of Et1‐induced calcium responses. In contrast, 18‐α‐glycyrrhetinic acid (αGA) used at a concentration known to block gap junction permeability in astrocytes had no significant effect on the amplitude of these calcium responses.
As demonstrated by quantitative and topological analysis, Et1 application similarly increased [Ca ²⁺ ] i levels in all astrocytes in both the absence and presence of αGA.
In control conditions, subpopulations of cells responding to methoxamine or carbachol exhibited two main types of calcium responses which differed in their shape and kinetic characteristics. In the presence of αGA the number of cells responding to these receptor agonists was significantly reduced. Indeed, responses characterized by their long latency, slow rise time and weak amplitude disappeared in the presence of αGA while responses with short latency and fast rise time were preserved.
These results indicate that permeable gap junction channels tend to attenuate the pharmacological and functional heterogeneity of populations of astrocytes, while their inhibition restricts calcium responses in astrocytes expressing high densities of transmitter receptors coupled to phospholipase C.
It is now generally accepted that massive neuronal death due to oxidative stress is a regular feature of brains in neurodegenerative diseases. However, much less attention has been given to the death of glial cells. In this study, we examined p53-sensitive apoptosis of cells by using human glioblastoma A172 cells and p53-deficient mouse astrocytes. In human A172 cells, hydrogen peroxide (H2O2) caused cell death in a time- and concentration-dependent manner, accompanied by nucleosomal DNA fragmentation and chromatin condensation. After treatment with H2O2, p53 protein was highly expressed and protein levels of Bak, p21WAF1/CIP1 and GADD45 were also enhanced. However, the protein levels of Bcl-2 and Bax did not change. On the other hand, primary cultured astrocytes from p53-deficient mouse brain grew faster than wild-type and heterozygous astrocytes. In addition, p53-deficient astrocytes were more resistant to H2O2-induced apoptosis than wild-type and heterozygous astrocytes. These results suggest that glial proliferation and the repair of damaged DNA may be regulated by p53-induced p21WAF1/CIP1 and GADD45, and that glial apoptosis caused by oxidative stress may be mediated by p53-induced Bak.
Cytokines and chemokines have been implicated in contributing to the initiation, propagation and regulation of immune and inflammatory responses. Also, these soluble mediators have important roles in contributing to a wide array of neurological diseases such as multiple sclerosis, AIDS Dementia Complex, stroke and Alzheimer's disease. Cytokines and chemokines are synthesized within the central nervous system by glial cells and neurons, and have modulatory functions on these same cells via interactions with specific cell-surface receptors. In this article, I will discuss the ability of glial cells and neurons to both respond to, and synthesize, a variety of cytokines. The emphasize will be on three select cytokines; interferon-gamma (IFN-gamma), a cytokine with predominantly proinflammatory effects; interleukin-6 (IL-6), a cytokine with both pro- and anti-inflammatory properties; and transforming growth factor-beta (TGF-beta), a cytokine with predominantly immunosuppressive actions. The significance of these cytokines to neurological diseases with an immunological component will be discussed.
Gap junctions are conductive channels that connect the interiors of coupled cells. We determined whether gap junctions propagate transcellular signals during metabolic stress and whether such signaling exacerbates cell injury. Although overexpression of the human proto-oncogene bcl2 in C6 glioma cells normally increased their resistance to injury, the relative resistance of bcl2+ cells to calcium overload, oxidative stress and metabolic inhibition was compromised when they formed gap junctions with more vulnerable cells. The likelihood of death was in direct proportion to the number and density of gap junctions with their less resistant neighbors. Thus, dying glia killed neighboring cells that would otherwise have escaped injury. This process of glial 'fratricide' may provide a basis for the secondary propagation of brain injury in cerebral ischemia.
We previously reported that incubation of cultured astrocytes in Ca2 + -containing medium after exposure to Ca2 + -free medium caused Ca2 + influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca2 + -mediated injury of cultured astrocytes. Our results show that Ca2 + reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase-3 inhibitors. Paradoxical Ca2 + challenge stimulated rapidly reactive oxygen species (ROS) production. Ca2 + reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H2O2) for 30 min and then incubated without H2O2 for 1-5 days, cell toxicity including apoptosis was observed. Ca2 + reperfusion injury induced by Ca2 + depletion or H2O2 exposure was blocked by the iron chelator 1, 10-phenanthroline, the NF-kappaB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O2 exposure rapidly increased the level of nuclear NF-kappaB p65 subunit, and the effect was blocked by 1,10-phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca2 + reperfusion-induced apoptosis is mediated at least partly by ROS production and ROS cause NF-kappaB activation in cultured astrocytes.
The anticarcinogenic effects of epicatechin (EC) and ginsenoside Rb(2) (Rb(2)), which are major components of green tea and Korea ginseng, respectively, were investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydrogen peroxide, known as cancer promoters, inhibited GJIC in the epithelial cells as determined by the scrape loading/dye transfer assay, fluorescence redistribution assay after photobleaching, and immunofluorescent staining of connexin 43 using a laser confocal microscope. The inhibition of GJIC by TPA and H(2)O(2) was prevented with treatment of Rb(2) or EC. The effect of EC on GJIC was stronger in TPA-treated cells than in H(2)O(2)-treated cells, while the effect of Rb(2) was opposite to that of EC. EC, at the concentration of 27.8 microg/ml, prevented the TPA-induced GJIC inhibition by about 60%. Rb(2,) at the concentration of 277 microg/ml, recovered the H(2)O(2)-induced GJIC inhibition by about 60%. These results suggest that Rb(2) and EC may prevent human cancers by preventing the down-regulation of GJIC during the cancer promotion phase and that the anticancer effect of green tea and Korea ginseng may come from the major respective components, EC and Rb(2).