Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a prototype reporter-based assay for the evaluation of gap junctional intercellular communication

A new reporter-based assay for the evaluation of gap junctional intercellular communication (GJIC) is presented. This assay was applied to the study of endogenous GJIC as well as to the evaluation of cell-to-cell communication exogenously induced in non-coupling cells by transfection with connexin 32. The results obtained with 18--glycyrrhetinic acid indicate that this assay system can be used to monitor the GJIC induced by transport of cAMP induced by activation of the dopamine 1 receptor cascade.     

v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap junctional communication (GJC) is not clear. In this study, we determined that Tyr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We established an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in the absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y265F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. Most importantly, GJC established by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Src. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.     

Measurement of gap junctional communication by fluorescence activated cell sorting

Summary Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.     

Repeated simulated ischemia and protection against gap junctional uncoupling

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The aim of this study was to investigate the effect of early and delayed preconditioning on gap junction communication, connexin abundance, and phosphorylation in cultured neonatal rat cardiac myocytes. Prolonged ischemia followed 5 minutes after preconditioning in the early protocol, whereas 20 hours separated preconditioning and prolonged ischemia in the delayed preconditioning protocol. Gap junctional intercellular communication (GJIC) was assessed by Lucifer yellow dye transfer. An initial reduction in communication in response to sublethal ischemia was observed. This may be one mechanism whereby neighboring cells are protected from damaging substances produced during the first phase of subsequent regional ischemia in early preconditioning protocols. With respect to delayed preconditioning, the transient decrease in GJIC disappeared prior to prolonged ischemia, indicating that other mechanisms are responsible for delayed protection. Both early and delayed preconditioning preserved intercellular coupling after prolonged ischemia and this correlated with presence of less connexin43 dephosphorylation assessed by immunoblot.     

Selective astrocytic gap junctional trafficking of molecules involved in the glycolytic pathway: impact on cellular brain imaging

To assess the specificity of metabolite trafficking among gap junction-coupled astrocytes, we developed novel, real-time, single-cell enzymatic fluorescence assays to assay cell-to-cell transfer of unlabeled glycolytic intermediates and report (i) highly restricted transfer of glucose-6-phosphate (P) and two analogs, deoxyglucose (DG)-6-P, and 2-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG-6-P, compared with DG and 2- and 6-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG, (ii) extensive junctional diffusion of glyceraldehyde-3-P, NADH, and NADPH plus three anionic fluorescent dyes used as internal standards for transfer assays, and (iii) stimulation of gap junctional communication by increased intracellular Na⁺ that also evokes metabolic responses in nearby coupled astrocytes. Thus, dye transfer does not predict gap junctional permeability of endogenous metabolites. Intracellular retention of flux-regulating compounds (e.g. glucose-6-P) may be necessary for local metabolic control, whereas 'syncytial sharing' may dissipate the work load on peri-synaptic astrocytes. Imaging of brain functional activity depends on local accumulation of exogenous or endogenous signals, and DG-6-P is trapped in the cell where it is phosphorylated, whereas rapid dispersal of cytoplasmic NAD(P)H and labeled glucose metabolites throughout the astrocytic syncytium can interfere with cellular assessment of neuron–astrocyte relationships in autoradiographic, fluorescence microscopic, and magnetic resonance spectroscopic studies.     

Connexin expression and gap junctional coupling in human cumulus cells: contribution to embryo quality

Gap junctional coupling among cumulus cells is important for oogenesis since its deficiency in mice leads to impaired folliculogenesis. Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles. The aim of this study was to determine which connexins contribute to gap junctions in human cumulus cells and to explore the possible relationship between connexin expression and pregnancy outcome from in vitro fertilization (IVF). Cumulus cells were obtained from IVF patients undergoing intra-cytoplasmic sperm injection (ICSI). Connexin expression was examined by RT-PCR and confocal microscopy. Cx43 was quantified by immunoblotting and gap junctional coupling was measured by patch-clamp electrophysiology. All but 5 of 20 connexin mRNAs were detected. Of the connexin proteins detected, Cx43 forms numerous gap junction-like plaques but Cx26, Cx30, Cx30.3, Cx32 and Cx40 appeared to be restricted to the cytoplasm. The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age. Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not. Thus, despite the presence of multiple connexins, Cx43 is a major contributor to gap junctions in human cumulus cells and its expression level may influence pregnancy outcome after ICSI.     

Microinjected anti-actin antibodies decrease gap junctional intercellular commmunication in cultured astrocytes

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.     

Regulation of osteoclastogenesis by gap junction communication

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.     

Glial connexins and gap junctions in CNS inflammation and disease

Gap junctions facilitate direct cytoplasmic communication between neighboring cells, facilitating the transfer of small molecular weight molecules involved in cell signaling and metabolism. Gap junction channels are formed by the joining of two hemichannels from adjacent cells, each composed of six oligomeric protein subunits called connexins. Of paramount importance to CNS homeostasis are astrocyte networks formed by gap junctions, which play a critical role in maintaining the homeostatic regulation of extracellular pH, K⁺, and glutamate levels. Inflammation is a hallmark of several diseases afflicting the CNS. Within the past several years, the number of publications reporting effects of cytokines and pathogenic stimuli on glial gap junction communication has increased dramatically. The purpose of this review is to discuss recent observations characterizing the consequences of inflammatory stimuli on homocellular gap junction coupling in astrocytes and microglia as well as changes in connexin expression during various CNS inflammatory conditions.     

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 μmol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a “hyperphosphorylated” connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.     

The effect of acrylonitrile on gap junctional intercellular communication in rat astrocytes

Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.     

Pharmacological evidence for system-dependent involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication

Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.     

Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communication

It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells.     

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.     

Expression of a dominant negative mutant innexin in identified neurons and glial cells reveals selective interactions among gap junctional proteins

Neurons and glia of the medicinal leech CNS express different subsets of the 21 innexin genes encoded in its genome. We report here that the punctal distributions of fluorescently tagged innexin transgenes varies in a stereotypical pattern depending on the innexin expressed. Furthermore, whereas certain innexins colocalize extensively (INX1 and INX14), others do not (e.g., INX1 and INX2 or INX6). We then demonstrate that the mutation of a highly conserved proline residue in the second transmembrane domain of innexins creates a gap junction protein with dominant negative properties. Coexpressing the mutated INX1 gene with its wild type blocks the formation of fluorescent puncta and decouples the expressing neuron from its normal gap junction‐coupled network of cells. Similarly, expression of an INX2 mutant transgene (a glial cell innexin), blocks endogenous INX2 puncta and wild‐type transgene puncta, and decouples the glial cell from the other glial cells in the ganglion. We show in cell culture with dye‐uptake and plasma membrane labeling experiments that the mutant innexin transgene is not expressed on the cell membrane but instead appears to accumulate in the cell's perinuclear region. Lastly, we use these mutant innexin transgenes to show that the INX1 mutant transgene blocks not only INX1 puncta formation, but also puncta of INX14, with which INX1 usually colocalizes. By contrast, the formation of INX6 puncta was unaffected by the INX1 mutant. Together, these experiments suggest that leech innexins can selectively interact with one another to form gap junction plaques, which are heterogeneously located in cellular arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 571–586, 2013     

Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication

Mutations in the zebrafish connexin43 (cx43) gene cause the short fin phenotype, indicating that direct cell-cell communication contributes to bone length. Three independently generated cx43 alleles exhibit short segments of variable sizes, suggesting that gap junctional intercellular communication may regulate bone growth. Dye coupling assays showed that all alleles are capable of forming gap junction channels. However, ionic coupling assays revealed allele-specific differences in coupling efficiency and gating. For instance, oocyte pairs expressing the weakest allele exhibited much higher levels of coupling than either of the strong alleles. Therefore, measurable differences in Cx43 function may be correlated with the severity of defects in bone length.