Connexin 43 Prevents Radiation-Induced Intestinal Damage via the Ca2+-Dependent PI3K/Akt Signaling Pathway.

Radiation-induced intestinal damage (RIID) is a common side effect of radiotherapy in patients with abdominopelvic malignancies. Gap junctions are special structures consisting of connexins (Cxs). This study aimed to investigate the expression and role of connexins in RIID and underlying mechanism. In this study, a calcein-AM fluorescence probe was used to detect changes in gap junctional intercellular communication in intestinal epithelial IEC-6 cells. Our results show that gap junctional intercellular communication of IEC-6 cells was reduced at 6, 12, 24, and 48 h after irradiation, with the most pronounced effect at 24 h. Western blotting and immunofluorescence results showed that the expression of Cx43, but not other connexins, was reduced in irradiated intestinal epithelial cells. Silencing of Cx43 reduced gap junctional intercellular communication between irradiated intestinal epithelial cells with increased ROS and intracellular Ca2+ levels. Furthermore, knockdown of Cx43 reduced the number of clonal clusters, decreased cell proliferation with increased cytotoxicity and apoptosis. Western blotting results showed that silencing of Cx43 resulted in changed γ-H2AX and PI3K/AKT pathway proteins in irradiated intestinal epithelial cells. Administration of the PI3K/AKT pathway inhibitor LY294002 inhibited the radioprotective effects in Cx43-overexpressing intestinal epithelial cells. Our study demonstrated that Cx43 expression is decreased by ionizing radiation, which facilitates the radioprotection of intestinal epithelial cells.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

Panx1 knockout promotes preneoplastic aberrant crypt foci development in a chemically induced model of mouse colon carcinogenesis.

Colorectal cancer, which is the third leading cause of cancer-related deaths worldwide, is a multistep disease, featuring preneoplastic aberrant crypt foci (ACF) as the early morphological manifestation. The roles of hemichannel-forming transmembrane Pannexin 1 (Panx1) protein have not been investigated in the context of colon carcinogenesis yet, although it has contrasting roles in other cancer types. Thus, this study was conducted to examine the effects of Panx1 knockout (Panx1-/- ) on the early events of chemically induced colon carcinogenesis in mouse. Wild type (WT) and Panx1-/- female C57BL6J mice were submitted to a chemically induced model of colon carcinogenesis by receiving six intraperitoneal administrations of 1,2-dimethylhydrazine (DMH) carcinogen. Animals were euthanized 8 h (week 7) or 30 weeks (week 37) after the last DMH administration in order to evaluate sub-acute colon toxicity outcomes or the burden of ACF, respectively. At week 7, Panx1 genetic ablation increased DMH-induced genotoxicity in peripheral blood cells, malondialdehyde levels in the colon, and apoptosis (cleaved caspase-3) in colonic crypts. Of note, at week 37, Panx1-/- animals showed an increase in aberrant crypts (AC), ACF mean number, and ACF multiplicity (AC per ACF) by 56%, 57% and 20%, respectively. In essence, our findings indicate that Panx1 genetic ablation promotes preneoplastic ACF development during chemically induced mouse colon carcinogenesis, and a protective role of Panx1 is postulated.

Cadmium Transforms Astrocytes into the A1 Subtype via Inducing Gap Junction Protein Connexin 43 into the Nucleus.

Cadmium is highly toxic and present in the environment and can be accumulated among various levels of the food chain. Both humans and animals are at risk from toxicity associated with cadmium. However, the neurological endpoint caused by cadmium has not been revealed. The aim of our research is to explore the potential target of cadmium attack when causing neurotoxicity. 80 male chickens (one day old, weighing 36.49 ± 2.88 g) were randomly divided into four groups and independently treated with 0, 35, 70, or 140 mg/kg CdCl2 in diet for 90 days. The result showed that the striatum was damaged due to a high dose of cadmium in the brain, which was characterized by degeneration of neurons and astrocyte dysfunction. Transcriptome analysis demonstrated that striatal astrocytes were transformed into the A1 state under cadmium exposure. Deeper investigation revealed that the internalization of gap junction protein connexin 43 was responsible for this transformation. Eventually, we can conclude that the internalized gap junction protein connexin 43 of astrocytes is the target of cadmium anchoring, and this process was accompanied by the transformation of astrocytes into the A1 subtype. This study provides a new direction for exploring the effects of cadmium on the nervous system and the treatment of subsequent nervous system diseases.

CX43 down-regulation promotes cell aggressiveness and 5-fluorouracil-resistance by attenuating cell stiffness in colorectal carcinoma.

Chemotherapy is one of the most commonly treatments of advanced colorectal cancer (CRC). However, the drug resistant following chemotherapeutic treatment is a significant challenge in the clinical management of CRC. Therefore, understanding the resistance mechanisms and developing new strategies for enhancing the sensitivity are urgently needed to improve CRC outcome. Connexins contribute to the formation of gap junctions among neighboring cells and then advance gap junctional intercellular communication (GJIC) for transportation of ions and small molecules. Although the drug resistance resulted from GJIC dysfunctional by aberrant expression of connexins is relatively well understood, the underlying mechanisms of mechanical stiffness mediated by connexin responsible for chemoresistance are largely unknown in CRC. Here, we demonstrated that connexin 43 (CX43) expression was downregulated in CRC and that loss of CX43 expression was positively correlated with metastasis and poor prognosis of CRC patients. The CX43 overexpressing suppressed CRC progression and increased the sensitivity to 5-fluorouracil (5-FU) via enhanced GJIC in vitro and in vivo. Moreover, we also highlight that the downregulation of CX43 in CRC increases the stemness of cells via reducing the cell stiffness, thus promoting the drug resistance. Our results further suggest that both effects, that is changes in the mechanical stiffness of the cell and GJIC mediated by CX43 deregulated, are closely related to drug resistance in CRC, which indicating CX43 as a target against cancer growth and chemoresistance in CRC.

Analgesics can affect the sensitivity of temozolomide to glioma chemotherapy through gap junction.

This study investigated the effect of frequently used analgesics in cancer pain management (flurbiprofen (FLU), tramadol (TRA), and morphine (MOR)) and a novel α2-adrenergic agonist (dexmedetomidine, DEX) on temozolomide (TMZ) sensitivity in glioma cells. Cell counting kit-8 and colony-formation assays were performed to analyze the viability of U87 and SHG-44 cell lines. A high and low cell density of colony method, pharmacological methods, and connexin43 mimetic peptide GAP27 were used to manipulate the function of gap junctions; "Parachute" dye coupling and western blot were employed to determine junctional channel transfer ability and connexin expression. The results showed that DEX (in the concentration range of 0.1 to 5.0 ng/ml) and TRA (in the concentration range of 1.0 to 10.0 µg/ml) reduced the TMZ cytotoxicity in a concentration-dependent manner but was only observed with high cell density (having formed gap junction). The cell viability percentage was 71.3 to 86.8% when DEX was applied at 5.0 ng/ml, while tramadol showed 69.6 to 83.7% viability at 5.0 µg/ml in U87 cells. Similarly, 5.0 ng/ml of DEX resulted in 62.6 to 80.5%, and 5.0 µg/ml TRA showed 63.5 to 77.3% viability in SHG-44 cells. Further investigating the impact of analgesics on gap junctions, only DEX and TRA were found to decrease channel dye transfer through connexin phosphorylation and ERK pathway, while no such effect was observed for FLU and MOR. Analgesics that can affect junctional communication may compromise the effectiveness of TMZ when used simultaneously.

The roles of connexins and gap junctions in the progression of cancer.

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

Cx32 promotes autophagy and produces resistance to SN­induced apoptosis via activation of AMPK signalling in cervical cancer.

The roles of gap junctions (GJs) and its components, connexins, in the autophagy of cervical cancer cells have been rarely investigated. Our previous study demonstrated that connexin 32 (Cx32) exerted an anti­apoptotic effect on cervical cancer. However, as an important regulator of apoptosis, whether the autophagy is involved in the function of Cx32 on cervical cancer cells is not well defined. The present study aimed to investigate the role of Cx32 on autophagy and apoptosis inhibition in cervical cancer cells. The expression levels of Cx32 and the autophagy­associated protein LC3­â…¡ in paracancerous cervical tissues (n=30) and cervical cancer (n=50) tissues were determined via western blotting. In total, 45 cervical cancer specimens were used to evaluate the clinical relevance of Cx32 and LC3­â…¡. It was found that both Cx32 and LC3­â…¡ were upregulated in cervical cancer tissues compared with those in paracancerous cervical tissues. The effect of Cx32 on autophagy was examined by detecting the change of LC3­â…¡ using western blotting, transfection with enhanced green fluorescent protein­LC3 plasmid and transmission electron microscopy analysis. Overexpression of Cx32 significantly enhanced autophagy in HeLa­Cx32 cells, whereas knockdown of Cx32 suppressed autophagy in C­33A cells. The flow cytometry results demonstrated that Cx32 inhibited the apoptosis of cervical cancer cells by promoting autophagy. Moreover, Cx32 triggered autophagy via the activation of the AMP­activated protein kinase (AMPK) signalling, regardless of the presence or absence of GJs. Collectively, it was identified that Cx32 exerted its anti­apoptotic effect by activating autophagy via the AMPK pathway in cervical cancer, which demonstrates a novel mechanism for Cx32 in human cervical cancer progression.

Dysregulation of Connexin Expression Plays a Pivotal Role in Psoriasis.

BACKGROUND: Psoriasis, a chronic inflammatory disease affecting 2-3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. METHODS: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. RESULTS: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a "psoriatic phenotype" in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. CONCLUSION: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.

Targeting connexins with Gap27 during cold storage of the human donor uterus protects against cell death.

Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to in situ warm ischemia with or without reperfusion. Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4ᵒC) or warm (37ᵒC) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority.

Adenosine Triphosphate Release is Required for Toll-Like Receptor-Induced Monocyte/Macrophage Activation, Inflammasome Signaling, Interleukin-1ß Production, and the Host Immune Response to Infection.

OBJECTIVES: Monocytes and macrophages produce interleukin-1ß by inflammasome activation which involves adenosine triphosphate release, pannexin-1 channels, and P2X7 receptors. However, interleukin-1ß can also be produced in an inflammasome-independent fashion. Here we studied if this mechanism also involves adenosine triphosphate signaling and how it contributes to inflammasome activation. DESIGN: In vitro studies with human cells and randomized animal experiments. SETTING: Preclinical academic research laboratory. SUBJECTS: Wild-type C57BL/6 and pannexin-1 knockout mice, healthy human subjects for cell isolation. INTERVENTIONS: Human monocytes and U937 macrophages were treated with different inhibitors to study how purinergic signaling contributes to toll-like receptor-induced cell activation and interleukin-1ß production. Wild-type and pannexin-1 knockout mice were subjected to cecal ligation and puncture to study the role of purinergic signaling in interleukin-1ß production and host immune defense. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor agonists triggered mitochondrial adenosine triphosphate production and adenosine triphosphate release within seconds. Inhibition of mitochondria, adenosine triphosphate release, or P2 receptors blocked p38 mitogen-activated protein kinase and caspase-1 activation and interleukin-1ß secretion. Mice lacking pannexin-1 failed to activate monocytes, to produce interleukin-1ß, and to effectively clear bacteria following cecal ligation and puncture. CONCLUSIONS: Purinergic signaling has two separate roles in monocyte/macrophage activation, namely to facilitate the initial detection of danger signals via toll-like receptors and subsequently to regulate nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 inflammasome activation. Further dissection of these mechanisms may reveal novel therapeutic targets for immunomodulation in critical care patients.

Connexin43 intercellular communication drives the early differentiation of human bone marrow stromal cells into osteoblasts.

Although it has been demonstrated that human bone marrow stromal cells (hBMSCs) express the ubiquitous connexin43 (Cx43) and form functional gap junctions, their role in the early differentiation of hBMSCs into osteoblasts remains poorly documented. Using in vitro assays, we show that Cx43 expression and gap junctional intercellular communication (GJIC) are increased during the differentiation of hBMSCs into osteoblasts, both at the protein and mRNA levels. Two independent procedures to reduce GJIC, a pharmacological approach with GJIC inhibitors (18α-glycyrrhetinic acid and Gap27 peptide) and a molecular approach using small interfering RNA against Cx43, demonstrated that the presence of Cx43 and functional junctional channels are essential to the ability of hBMSCs to differentiate into osteoblasts in vitro. In addition, a reduced GJIC decreases the expression of Runx2, the major transcription factor implicated in the control of osteoblast commitment and early differentiation of hBMSCs into osteoblasts, suggesting that GJIC mediated by Cx43 is implicated in this process. Together our results demonstrate that GJIC mediated by the Cx43 channels plays a central role throughout the differentiation of hBMSC into osteoblasts, from the early stages to the process of mineralization.

Inhibition of connexin43 improves functional recovery after ischemic brain injury in neonatal rats.

Connexin43 (Cx43) is one of the most abundant gap junction proteins in the central nervous system. Abnormal opening of Cx43 hemichannels after ischemic insults causes apoptotic cell death. In this study, we found persistently increased expression of Cx43 8 h to 7 d after hypoxia/ischemia (HI) injury in neonatal rats. Pre-treatment with Gap26 and Gap27, two Cx43 mimetic peptides, significantly reduced cerebral infarct volume. Gap26 treatment at 24 h after ischemia improved functional recovery on muscle strength, motor coordination, and spatial memory abilities. Further, Gap26 inhibited Cx43 expression and reduced active astrogliosis. Gap26 interacted and co-localized with Cx43 together in brain tissues and cultured astrocytes. After oxygen glucose deprivation, Gap26 treatment reduced the total Cx43 level in cultured astrocytes; but Cx43 level in the plasma membrane was increased. Degradation of Cx43 in the cytoplasm was mainly via the ubiquitin proteasome pathway. Concurrently, phosphorylated Akt, which phosphorylates Cx43 on Serine(373) and facilitates the forward transport of Cx43 to the plasma membrane, was increased by Gap26 treatment. Microdialysis showed that increased membranous Cx43 causes glutamate release by opening Cx43 hemichannels. Extracellular glutamate concentration was significantly decreased by Gap26 treatment in vivo. Finally, we found that cleaved caspase-3, an apoptosis marker, was attenuated after HI injury by Gap26 treatment. Effects of Gap27 were analogous to those of Gap26. In summary, our findings demonstrate that modulation of Cx43 expression and astroglial function is a potential therapeutic strategy for ischemic brain injury.

Activated microglia impairs neuroglial interaction by opening Cx43 hemichannels in hippocampal astrocytes.

Glia plays an active role in neuronal functions and dysfunctions, some of which depend on the expression of astrocyte connexins, the gap junction channel and hemichannel proteins. Under neuroinflammation triggered by the endotoxin lipopolysacharide (LPS), microglia is primary stimulated and releases proinflammatory agents affecting astrocytes and neurons. Here, we investigate the effects of such microglial activation on astrocyte connexin-based channel functions and their consequences on synaptic activity in an ex vivo model. We found that LPS induces astroglial hemichannel opening in acute hippocampal slices while no change is observed in gap junctional communication. Based on pharmacological and genetic approaches we found that the LPS-induced hemichannel opening is mainly due to Cx43 hemichannel activity. This process primarily requires a microglial stimulation resulting in the release of at least two proinflammatory cytokines, IL-1ß and TNF-α. Consequences of the hemichannel-mediated increase in membrane permeability are a calcium rise in astrocytes and an enhanced glutamate release associated to a reduction in excitatory synaptic activity of pyramidal neurons in response to Schaffer's collateral stimulation. As a whole our findings point out astroglial hemichannels as key determinants of the impairment of synaptic transmission during neuroinflammation.

Regulation of activity of P2X7 receptor by its splice variants in cultured mouse astrocytes.

Of purinergic receptors, P2X7 receptor (P2X7R, defined as a full-length receptor) has unique characteristics, and its activation leads to ion channel activity and pore formation, causing cell death. Previously, we demonstrated that P2X7R expressed by nonstimulated astrocyte cultures obtained from SJL-strain mice exhibits constitutive activation, implying its role in maintenance of cellular homeostasis. To obtain novel insights into its physiological roles, we examined whether constitutive activation of P2X7R is regulated by expression of its splice variants in such resting astrocytes, and whether their distinct expression profiles in different mouse strains affect activation levels of astrocytic P2X7Rs. In SJL- and ddY-mouse astrocytes, spontaneous YO-PRO-1 uptake, an indicator of pore activity of P2X7R, was detected, but the uptake by the formers was significantly greater than that by the latter. Between the two mouse strains, there was a difference in their sensitivity of YO-PRO-1 uptake to antagonists, but not in the expression levels and sequences of P2X7R and pannexin-1. Regarding expression of splice variants of P2X7R, expression of P2X7R variant-3 (P2X7R-v3) and -4 (P2X7R-v4), but not variant-2 and -k, was lower in SJL-mouse astrocytes than in ddY-mouse ones. On transfection of P2X7R-v3 and -v4 into SJL-mouse astrocytes, the pore activity was attenuated as in the case of the HEK293T cell-expression system. These findings demonstrate that basal activity of P2X7R expressed by resting astrocytes is negatively regulated by P2X7R-v3 and -v4, and that their distinct expression profiles result in the different activation levels of astrocytic P2X7Rs in different mouse strains.

Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

OBJECTIVES: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels.

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-ß peptide (Aß) 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 Aß-treated microglia (CM-Aß). The effects of CM-Aß were mimicked by TNF-α and IL-1ß and were abrogated by neutralizing TNF-α with soluble receptor and IL-1ß with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aß made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aß 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.

Quantification of gap junctional intercellular communication based on digital image analysis.

Intercellular communication via gap junction channels can be quantified by several methods based on diffusion of fluorescent dyes or metabolites. Given the variation in intercellular coupling of cells, even under untreated control conditions, it is of essence to quantify the coupling between numerous cells to obtain reliable estimates of metabolic coupling. Quantification is often based on manual counting of fluorescent cells, which is time consuming and may include some degree of subjectivity. In this report, we introduce a technique based on digital image analysis, and the software for the analysis is presented together with a detailed protocol in the online supplemental material (http://bmi.ku.dk/matlab_program/). Fluorescent dye was introduced in connexin 43-expressing C6 glioma cells by in situ electroporation, and fluorescence intensity was measured in the electroporated cells and in cells receiving dye by intercellular diffusion. The analysis performed is semiautomatic, and comparison with traditional cell counting shows that this method reliably determines the effect of uncoupling by several interventions. This new method of analysis yields a rapid and objective quantification process with a high degree of reproducibility.

Connexin mimetic peptides improve cell migration rates of human epidermal keratinocytes and dermal fibroblasts in vitro.

Nonhealing cutaneous wounds, a major cause of morbidity and mortality, are difficult to treat. Recent studies suggest that significant increases in skin wound-healing rates occur by altering gap junction intercellular communication (GJIC). As migration of keratinocytes and fibroblasts is an important feature of wound healing, this study investigated whether migration rates in cultured normal human epidermal keratinocytes and dermal fibroblasts could be altered by modulating GJIC via connexin mimetic peptides. First, HeLa cells stably transfected with connexin43 (Cx43), Cx40, or Cx26 were used as a model to determine connexin specificity and the doses of connexin mimetic peptides required to attenuate GJIC. Gap26 and Gap26M inhibited GJIC dose dependently and were nonconnexin specific, whereas Gap27 was Cx43-selective. Skin keratinocytes and fibroblasts expressed a variety of connexins, with Cx43 predominating. Cx43 protein expression was reduced at leading edges 3 hours after scraping confluent monolayers, resolving at 24 hours. Gap26M and Gap27 significantly increased migration rates across scrapes in keratinocytes and fibroblasts by blocking gap junction functionality. GJIC inhibition can thus directly influence keratinocyte and fibroblast migration. Furthermore, our results support the therapeutic potential of connexin mimetic peptides to aid wound closure, and provide a simple approach to screening new agents.

Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles.

Vascular conducted responses are believed to play a central role in controlling the microcirculatory blood flow. The responses most likely spread through gap junctions in the vascular wall. At present, four different connexins (Cx) have been detected in the renal vasculature, but their role in transmission of conducted vasoconstrictor signals in the preglomerular arterioles is unknown. Connexin mimetic peptides were previously reported to target and inhibit specific connexins. We, therefore, investigated whether conducted vasoconstriction in isolated renal arterioles could be blocked by the use of mimetic peptides directed against one or more connexins. Preglomerular resistance vessels were microdissected from kidneys of Sprague-Dawley rats and loaded with fura 2. The vessels were stimulated locally by applying electrical current through a micropipette, and the conducted calcium response was measured 500 mum from the site of stimulation. Application of connexin mimetic peptides directed against Cx40, 37/43, 45, or a cocktail with equimolar amounts of each, did not inhibit the propagated response, whereas the nonselective gap junction uncoupler carbenoxolone completely abolished the propagated response. However, the connexin mimetic peptides were able to reduce dye coupling between rat aorta endothelial cells shown to express primarily Cx40. In conclusion, we did not observe any attenuating effects on conducted calcium responses in isolated rat interlobular arteries when exposed to connexin mimetic peptides directed against Cx40, 37/43, or 45. Further studies are needed to determine whether conducted vasoconstriction is mediated via previously undescribed pathways.