Reduction Of Gap Junctional Intercellular Communication Research Articles

Effects of carbon nanotubes on intercellular communication and involvement of IL-1 genes.

An increasing amount of products containing engineered nanoparticles is emerging. Among these particles are carbon nanotubes (CNTs) which are of interest for a wide range of industrial and biomedical applications. There have been raised concerns over the effects of CNTs on human health. Some types of CNTs are classified as group 2B carcinogens by the International Agency for Research on Cancer. CNTs may also induce pulmonary inflammatory and fibrotic effects. By utilizing CNTs of different lengths, we investigated the role of the proinflammatory cytokine, interleukin-1 (IL-1) on gap junctional intercellular communication (GJIC) by using IL-1 wild-type (IL1-WT) and IL-1 knock-out (IL1-KO) cells. GJIC decreased equally in both cell types after CNT exposure. Immunofluorescence staining showed Gja1 and Gjb2 in gap junctions and hemichannels for both cell types. Gjb1 and Gjb2 expression was low in IL1-KO cells, which was confirmed by protein analysis. Gja1 was upregulated with both CNTs, whereas Gjb1 was down-regulated by CNT-2 in IL1-WT cells. Connexin mRNA expression was regulated differently by the CNTs. CNT-1 affected Gja1 and Gjb2, whereas CNT-2 had an effect on Gjb1. CNTs negatively affect GJIC through gap junctions independently of the length of CNT and IL-1 status. Furthermore, connexin gene expression was affected by IL-1 at transcriptional and translational levels. As both CNTs used in this study are cytotoxic to the cells and reduce cell survival, we suggest that CNT-induced reduction in GJIC may be important for inhibiting transfer of cell survival signals between cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s12079-016-0323-0) contains supplementary material, which is available to authorized users.

Laminin-111 Stimulates Proliferation of Mouse Embryonic Stem Cells Through a Reduction of Gap Junctional Intercellular Communication via RhoA-Mediated Cx43 Phosphorylation and Dissociation of Cx43/ZO-1/Drebrin Complex

Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18α-glycyrrhetinic acid (18α-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin β1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation.

Dexamethasone differentially regulates functional membrane properties in glioma cell lines and primary astrocytes in vitro

Similar to astrocytes, glioma cells form a well-coupled syncytium via gap junctions. This can be influenced, for example, by activated microglia, the main inflammatory cell population within the central nervous system (CNS). Under pathological conditions such as neoplastic cell growth, microglia number and activation state are enhanced. The aim of the present study is to analyze the influence of dexamethasone (DEX) on cellular and molecular properties in glial coculture models consisting of astroglia and microglia and human and rat glioma cell lines. Primary rat glial cocultures of astrocytes containing 5% (M5, representing "physiological" conditions) or 30% (M30, representing "pathological" conditions) microglia as well as rat and human glioma cell lines (F98, C6, U87) were incubated with DEX for 24 h. DEX-treated M30 cocultures showed significant increased gap junctional intercellular communication (GJIC). DEX treatment of glioma cells resulted in depolarization of the membrane resting potential (MRP) and a significant reduction of GJIC. Furthermore, DEX reduced the amount of activated microglia in M30 cocultures. DEX had no significant effects on the tested variables in the M5 coculture. DEX differentially regulates functional membrane properties of glioma cells and astrocytes in primary glial cocultures, which might resemble steroid effects in glioma cells and adjacent glial components in vivo.

Loss of gap junctional intercellular communication in rat lung epithelial cells exposed to carbon or silica-based nanoparticles

The aim of this study was to investigate whether fine and ultrafine carbon black (fC and ufC), and fine and ultrafine silica (fS, ufS) particles affect gap junctional intercellular communication (GJIC) in rat lung epithelial cells. Exposure of cells to subcytotoxic doses of ufC, fS and ufS resulted in a 63%, 59% and 77% reduction of GJIC, respectively, as determined in a dye transfer assay. In contrast to ufC, fC did not significantly alter GJIC. Changes in subcellular localization of the major gap junction protein in RLE cells, connexin-43 (Cx43), and of β-catenin were observed in cells exposed to ufC, fS or ufS. The loss of GJIC was counteracted by N-acetyl cysteine and was largely prevented by specific inhibitors of epidermal growth factor receptor-dependent signaling, pointing to the crucial role of two known major mediators of nanoparticle action, namely reactive oxygen species and membrane-receptor signaling, in particle-induced modulation of GJIC.

Cisplatin and Oxaliplatin Inhibit Gap Junctional Communication by Direct Action and by Reduction of Connexin Expression, Thereby Counteracting Cytotoxic Efficacy

Cisplatin [cis-diamminedichloroplatinum(II)]/oxaliplatin [1,2-diamminocyclohexane(trans-1)oxolatoplatinum(II)] toxicity is enhanced by functional gap junctions between treated cells, implying that inhibition of gap junctions may decrease cytotoxic activity of these platinum-based agents. This study investigates the effect of gap junction modulation by cisplatin/oxaliplatin on cytotoxicity in a transformed cell line. The effects were explored using junctional channels expressed in transfected HeLa cells and purified hemichannels. Junctional channels showed a rapid, dose-dependent decrease in dye coupling with exposure to cisplatin/oxaliplatin. With longer exposure, both compounds also decreased connexin expression. Both compounds inhibit the activity of purified connexin hemichannels, over the same concentration range that they inhibit junctional dye permeability, demonstrating that inhibition occurs by direct interaction of the drugs with connexin protein. Cisplatin/oxaliplatin reduced the clonogenic survival of HeLa cells at low density and high density in a dose-dependent manner, but to a greater degree at high density, consistent with a positive effect of gap junctional intercellular communication (GJIC) on cytotoxicity. Reduction of GJIC by genetic or pharmacological means decreased cisplatin/oxaliplatin toxicity. At low cisplatin/oxaliplatin concentrations, where effects on connexin channels are minimal, the toxicity increased with increased cell density. However, higher concentrations strongly inhibited GJIC, and this counteracted the enhancing effect of greater cell density on toxicity. The present results indicate that inhibition of GJIC by cisplatin/oxaliplatin decreases their cytotoxicity. Direct inhibition of GJIC and reduction of connexin expression by cisplatin/oxaliplatin may thereby compromise the effectiveness of these compounds and be a factor in the development of resistance to this class of chemotherapeutic agents.

Prostaglandin E2 Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Activity and Airway Surface Liquid Volume Requires Gap Junctional Communication

Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E₂ (PGE₂) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coordination of these pathways for modulating CFTR activity and mucus hydration. We used RT-PCR and Western blotting to determine connexin (Cx), CD73, and EP-R expression in a Calu-3 airway epithelial cell line grown on Transwell (Corning Costar, Cambridge, MA) inserts. We used dye coupling to evaluate gap junctional intercellular communication (GJIC). We used Ussing chamber studies and X-Z confocal microscopy to monitor Cl(-) secretion and ASL volume regulation. We found that connexin 43 (Cx43)-mediated GJIC was increased either by endogenous ADO after the hydrolysis of purine nucleotides by CD73 or by the direct activation of ADO-Rs. Inhibition of phospholipase A2 and cyclooxygenase prevented ADO-dependent increases in GJIC, suggesting the involvement of PGE₂. PGE₂ was found to increase GJIC markedly by stimulating EP4-Rs. The modulation of ADO signaling also affected the PAR-dependent activation of CFTR. The reduction of GJIC by CD73 or Cx43 inhibition prevented PAR-evoked CFTR currents in Ussing chambers. The inhibition of GJIC resulted in a failure of PGE₂ to increase ASL volume in Calu-3 cells and in primary cultures of well-differentiated human airway epithelial cells. Thus, gap junctions coordinate a signaling network comprising CFTR, ADO-Rs, PARs, and EP-Rs, and are required for ASL volume homeostasis.

Loss of gap junctional intercellular communication in rat lung epithelial cells exposed to quartz particles

Chronic inhalation of quartz particles has been implicated in lung diseases including silicosis and cancer. The aim of this study was to investigate whether quartz particles affect gap junctional intercellular communication (GJIC) in rat lung epithelial cells (RLE-6TN). Here, we demonstrate that exposure of RLE-6TN cells to subtoxic doses of DQ12 standard quartz resulted in an up to 55% reduction of GJIC, as determined in a dye transfer assay. We show that connexin-43 (Cx43) is the major connexin responsible for intercellular communication in these lung epithelial cells and that exposure to quartz particles induces a significant internalization of Cx43. Downregulation of GJIC was attenuated by N-acetyl cysteine, suggesting the involvement of reactive oxygen species and/or cellular thiol homeostasis in the regulation of GJIC. Furthermore, an inhibitor of activation of extracellular signal-regulated kinases prevented the loss of GJIC in cells exposed to DQ12 quartz, although no direct phosphorylation of Cx43 upon exposure to DQ12 was detected.

Irsogladine maleate counters the interleukin‐1β‐induced suppression in gap‐junctional intercellular communication but does not affect the interleukin‐1β‐induced zonula occludens protein‐1 levels in human gingival epithelial cells

Irsogladine maleate counters gap junctional intercellular communication reduction induced by interleukin-8 or Actinobacillus actinomycetemcomitans in cultured human gingival epithelial cells. Interleukin-1 beta is involved in periodontal disease. Little is known, however, about the effect of interleukin-1 beta on intercellular junctional complexes in human gingival epithelial cells. Furthermore, irsogladine maleate may affect the actions of interleukin-1 beta. In this study, we examined how interleukin-1 beta affected gap junctional intercellular communication, connexin 43 and zonula occludens protein-1, and how irsogladine maleate modulated the interleukin-1 beta-induced changes in the intercellular junctional complexes in human gingival epithelial cells. Human gingival epithelial cells were exposed to interleukin-1 beta, with or without irsogladine maleate. Connexin 43 and zonula occludens protein-1 were examined at mRNA and protein levels by real-time polymerase chain reaction and western blotting, respectively. Gap junctional intercellular communication was determined using the dye transfer method. The expression of zonula occludens protein-1 was also confirmed by immunofluorescence. Interleukin-1 beta decreased connexin 43 mRNA levels, but increased zonula occludens protein-1 mRNA levels. Irsogladine maleate countered the interleukin-1 beta-induced reduction in gap junctional intercellular communication and connexin 43 levels. However, irsogladine maleate did not influence the increased zonula occludens protein-1 levels. The effect of interleukin-1 beta on gap junctional intercellular communication and tight junctions of human gingival epithelial cells is different. The recovery of gap junctional intercellular communication by irsogladine maleate in the gingival epithelium may be a normal process in gingival epithelial homeostasis.

Gene Transfer of Connexin43 Mutants Attenuates Coupling in Cardiomyocytes

Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.

Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans ( A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell–cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.

Irsogladine maleate influences the response of gap junctional intercellular communication and IL-8 of human gingival epithelial cells following periodontopathogenic bacterial challenge

Gingival epithelial cells first encounter periodontopathogenic bacteria and their metabolic products to produce inflammatory cytokines. Gap junctional intercellular communication (GJIC) is thought to play a critical role in cellular coordination in tissue homeostasis. Gap junctions are structured by connexins (CXs). GJIC response of gingival epithelial cells to the bacteria may be involved in the initiation of periodontal disease. Irsogladine maleate (IM) is known to enhance GJIC through cAMP. In the present study, we examined an effect of IM on GJIC response and on interleukin-8 (IL-8) levels in human gingival epithelial cells (HGEC) exposed to a periodontopathogenic bacterium, Actinobacillus actinomycetemcomitans, and its outer membrane protein (OMP) 29 in order to test the hypothesis that IM has the ability to modulate GJIC and inflammatory responses of gingival epithelial cells to periodontopathogenic bacteria. IM countered the OMP29-induced reduction of GJIC, CX43 levels and cAMP levels in HGEC. The simultaneous addition of OMP29 and dibutyryl cAMP also abrogated the repression of GJIC by OMP29. Furthermore, IM obviated the increase in IL-8 levels in HGEC stimulated by whole live A. actinomycetemcomitans and by OMP29. These findings suggest that IM counters the OMP29-induced GJIC reduction in HGEC through cAMP. IM may eliminate initial perturbation of gingival epithelial cells by regulating responses of GJIC and IL-8 to periodontopathogenic bacterial exposure.

Nonylphenol Alters Connexin 43 Levels and Connexin 43 Phosphorylation Via an Inhibition of the p38-Mitogen-Activated Protein Kinase Pathway1

Endocrine-disrupting chemicals are exogenous compounds that mimic or inhibit the action of estrogens or other hormones. Nonylphenol, an environmental contaminant distributed along the St. Lawrence River, has been reported to act as a weak estrogen. Previous studies from our laboratory have shown that rats that were fed fish taken from nonylphenol contaminated sites have altered spermatogenesis and decreased sperm count. The mechanism responsible for this effect is unknown. Gap junctional intercellular communication (GJIC) in the testis is critical for coordinating spermatogenesis. The objectives of the study were to determine the effects of nonylphenol on GJIC and connexin 43 (Cx43) in a murine Sertoli cell line, TM4. Cells were exposed for 24 h to different concentrations (1 to 50 microM) of either nonylphenol or 17beta-estradiol. GJIC was determined using a microinjection approach in which Lucifer yellow was injected directly into a single cell, and GJIC was assessed 3 min postinjection. Nonylphenol exposure decreased GJIC between adjacent cells by almost 80% relative to controls. A significant concentration-dependent reduction in GJIC was observed at nonylphenol concentrations between 1 and 50 microM. Cx43 immunofluorescent staining was reduced at both 10 and 50 microM doses of nonylphenol. Cx43 phosphorylation, as determined by Western blot analysis, was reduced at both 10 and 50 microM concentrations, which may explain, at least in part, the inhibition of GJIC. In contrast, no effect on GJIC or Cx43 protein was observed in cells exposed to 17beta-estradiol at these concentrations. Cx43 has been reported to be phosphorylated via the p38-mitogen-activated protein kinase (MAPK) pathway. P38-MAPK activity was assessed in both control and nonylphenol-exposed cells. A dose-dependent decrease in p38-MAPK activity was observed in nonylphenol-exposed Sertoli cells. Protein kinase C activity was also measured and was not influenced by nonylphenol. These results suggest that nonylphenol inhibits GJIC between Sertoli cells and that this is modulated via nonestrogenic pathways.

Endothelin-1 Decreases Gap Junctional Intercellular Communication by Inducing Phosphorylation of Connexin 43 in Human Ovarian Carcinoma Cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Cells heterozygous for the ApcMin mutation have decreased gap junctional intercellular communication and connexin43 level, and reduced microtubule polymerization.

Mutations in the tumour suppressor gene adenomatous polyposis coli (Apc) are early and critical events in the development of colon cancer. In the absence of functional Apc, beta-catenin is not degraded in the cytoplasm and can be transported to the nucleus and turn on transcription of several genes, including the gap junction protein connexin43. Apc also stabilizes microtubules and regulates microtubule polymerization. Changes in Wnt signalling and microtubule function are reported to affect the connexin level. To study the effect of heterozygous Apc mutation we examined gap junctional intercellular communication (GJIC) in IMCE (Immorto-Min colonic epithelium) cells with one mutated Apc allele and in YAMC (Young adult mouse colon) cells with normal Apc function. IMCE cells had only half the GJIC level compared with YAMC cells. RT-PCR showed that both YAMC and IMCE cells express a common complement of seven connexin genes (Cx26, Cx31, Cx39, Cx40, Cx43, Cx45 and Cx50), with an additional Cx29 gene expression in YAMC cells. We found that the Cx43 level was correspondingly lower in IMCE cells as detected by western blotting and immunofluorescence. There were no differences in the level or localization of beta-catenin and the downstream gene E-cadherin between the cells, indicating no activation of the Wnt-signalling pathway in cells with one mutated Apc allele. We also examined the microtubule polymerization rate, and IMCE cells had markedly slower microtubule polymerization than YAMC cells. Hence, it appears that mutation in one Apc allele is sufficient to affect microtubule function, while inactivation of both wild-type Apc alleles may be necessary for activation of Wnt signalling. Reduction in GJIC and Cx43 level in IMCE cells may be caused by reduced Cx43 transport as a result of alterations in microtubule function.

Inhibitory effects of eicosapentaenoic acid (EPA) on the hypoxia/reoxygenation-induced tyrosine kinase activation in cultured human umbilical vein endothelial cells

We have previously reported that the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) inhibited the abnormal gap junctional intercellular communication (GJIC) induced by hypoxia/reoxygenation (H/R) via suppressing tyrosine kinase (TK) activation (Zhang et al., Prostaglandins Leukot Essent Fatty Acids, 1999; 61: 33–40). However, the mechanisms by which EPA-inhibited TK activation remained unidentified. In this study we investigated whether reactive oxygen species (ROS) and growth factor–receptor systems would contribute to the H/R-induced TK activation or not. The results showed that H/R-induced ROS production, which reached the peak after 30 min of reoxygenation. Pretreatment with 10 μM EPA significantly inhibited this ROS production. However, the TK inhibitor genistein (10 μM) failed to inhibit the generation of ROS, although it completely inhibited TK activation. On the other hand, the ROS inhibitor DMSO (0.5% v/v) showed little effect on TK activation while it significantly blocked ROS production. Further EPA and genistein, but not DMSO and superoxide dismutase (SOD, 300 U/ml), prevented cells from GJIC injury induced by H/R. Moreover, EPA protected against VEGF-induced reduction in GJIC and phosphorylation of connexin 43. These data suggest that growth factor, but not ROS, might be involved in the EPA-inhibited TK activation induced by H/R.

Eicosapentaenoic acid protects endothelial cell function injured by hypoxia/reoxygenation.

Eicosapentaenoic acid (EPA) may protect against atherosclerosis by improving lipid metabolism and modulating vascular cell function. Ischemia/ reperfusion injury is one risk factor for atherosclerosis. We investigated if EPA could improve hypoxia/reoxygenation (H/R)-induced endothelial cell dysfunction of gap junctional intercellular communication (GJIC). GJIC in human umbilical vascular endothelial cells (HUVECs) was measured using a photobleaching technique. Results demonstrated that H (24h)/R 2h) induced a GJIC reduction in HUVECs; however, it was inhibited by EPA pretreatment. H/R produced reactive oxygen species, but it was not affected by EPA, and it contributed little to GJIC dysfunction. By contrast, tyrosine kinase activated by H/R was inhibited by EPA pretreatment, and tyrosine kinase inhibitors also abolished H/R-induced GJIC reduction. The protective effects of EPA on the H/R-induced GJIC reduction was also observed in cells treated with tyrosine phosphatase inhibitor. These data indicate the EPA improves H/R-induced endothelial dysfunction through inhibition of tyrosine kinase activation, and it could lead to prevention of progression and/or initiation of atherosclerosis.

Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition.

The effects of hypoxia and phosphatase inhibitors on connexin43 (Cx43) phosphorylation state, gap junctional intercellular communication (GJIC) and immunolabelling with anti-Cx43 antibodies were investigated in cultured astrocytes. Astrocytes contained predominantly phosphorylated forms of Cx43 and these underwent dephosphorylation 30 min after hypoxia. This was preceded by a 77% reduction in GJIC 15 min after hypoxia, indicating that reduced GJIC occurs prior to Cx43 dephosphorylation. Hypoxia caused a reduction in punctate immunostaining (epitope masking) at cell-cell contacts with one anti-Cx43 antibody, and increased labelling with another antibody (13-8300) that detects only a dephosphorylated form of Cx43. Inhibition of protein phosphatase (PP)-1 and PP-2A with okadaic acid or calyculin A had little effect on hypoxia-induced Cx43 dephosphorylation. Inhibition of PP-2B (calcineurin) with cyclosporin A or FK506 reduced Cx43 dephosphorylation and junctional uncoupling seen after hypoxia. These results demonstrate that responses of astrocytic Cx43 to hypoxia in vitro are similar to those seen after ischaemia in vivo, and that inhibition of protein phosphatase protects astrocytes from hypoxia-induced Cx43 dephosphorylation and junctional uncoupling. In addition, calcineurin may play a direct role in the regulation of astrocytic GJIC and Cx43 phosphorylation state.

Hypoxia-reoxygenation inhibits gap junctional communication in cultured human umbilical vein endothelial cells.

We studied the change in gap junctional intercellular communication (GJIC) on human umbilical vein endothelial cells (HUVEC) under hypoxia-reoxygenation (H-R) conditions by the fluorescence redistribution after photobleaching (FRAP) method. Confluent HUVEC monolayers were exposed to hypoxia (pO2<0.1%) for 12 hours, and then were returned to normal atmospheric conditions for reoxygenation. Contrast microscopic observation showed no significant changes in the morphology of the HUVEC at any times after H-R. Reoxygenation following hypoxia caused time-dependent decrease in GJIC, that is, GJIC reduction was induced after 2 hours and reached maximum at 4-6 hours which recovered to normal levels after 18 hours. Oxidant sensitive fluorescence dye assay revealed that the generation of intracellular free radicals increased during the first 2 hours after reoxygenation. Hydroxyl radical scavengers (MCI-186, DMSO) and an iron chelator (deferoxamine) abolished the reduction of GJIC due to H-R. However, SOD, catalase and probucol were essentially inactive on this reduction. These data suggest that ischemia-reperfusion injury may be caused by a functional defect of GJIC induced by reactive oxygen radicals.

The relationship of the area under the niacin flush dose-response curve and schizophrenia status

We have previously reported that the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) inhibited the abnormal gap junctional intercellular communication (GJIC) induced by hypoxia/reoxygenation (H/R) via suppressing tyrosine kinase (TK) activation (Zhang et al., Prostaglandins Leukot Essent Fatty Acids, 1999; 61: 33–40). However, the mechanisms by which EPA-inhibited TK activation remained unidentified. In this study we investigated whether reactive oxygen species (ROS) and growth factor–receptor systems would contribute to the H/R-induced TK activation or not. The results showed that H/R-induced ROS production, which reached the peak after 30 min of reoxygenation. Pretreatment with 10 μM EPA significantly inhibited this ROS production. However, the TK inhibitor genistein (10 μM) failed to inhibit the generation of ROS, although it completely inhibited TK activation. On the other hand, the ROS inhibitor DMSO (0.5% v/v) showed little effect on TK activation while it significantly blocked ROS production. Further EPA and genistein, but not DMSO and superoxide dismutase (SOD, 300 U/ml), prevented cells from GJIC injury induced by H/R. Moreover, EPA protected against VEGF-induced reduction in GJIC and phosphorylation of connexin 43. These data suggest that growth factor, but not ROS, might be involved in the EPA-inhibited TK activation induced by H/R.

Involvement of tyrosine kinase in the hypoxia/reoxygenation-induced gap junctional intercellular communication abnormality in cultured human umbilical vein endothelial cells.

Vascular endothelial cells (EC), communicating with one another across gap junctions, are usually made dysfunctional by hypoxia and reoxygenation (H/R); however, very limited information exists regarding the effects of H/R on the endothelial gap junctions. We investigated whether H/R interferes with endothelial gap junctional intercellular communication (GJIC). After human umbilical vein EC had grown to confluence, they were exposed to hypoxia (pO2 < 0.1%) for 12-16 h and then returned to normal atmospheric conditions for reoxygenation. At 0-, 2-, 4-, 6-h reoxygenation, GJIC was detected by means of a fluorescence recovery after a photobleaching technique. The results demonstrated that a GJIC reduction (about 20% less than that under normoxia) was induced after 2 h of reoxygenation; after 4 h of reoxygenation, it began to recover (to about 10% less than that under normoxia); and after 6 h of reoxygenation, GJIC was restored to the normal level. Calphostin C (1 x 10(-7) mol/l), a specific protein kinase C inhibitor, partially inhibited the reduction in GJIC (resulting in a level about 10% less than that under normoxia), whereas the tyrosine kinase inhibitor genistein (10 micromol/L) completely blocked the reduction in GJIC. Vanadate (1.5 mmol/l), a tyrosine phosphatase inhibitor, amplified the inhibitory effect of H/R on GJIC (to about 40% less than that under normoxia). Immunofluorescence and immunoprecipitation showed that 2-h reoxygenation significantly stimulated tyrosine protein phosphorylation, and this phosphorylation event was obviously enhanced by vanadate. The results of Western blotting showed that the gap junctional protein connexin 43 (Cx43) was phosphorylated by H/R; moreover, immunoprecipitation demonstrated that 2-h reoxygenation induced a prominent increase of tyrosine phosphorylation of Cx43 compared with that under normoxia. These data indicate that H/R induces a transient endothelial GJIC dysfunction through the activation of tyrosine kinase and phosphorylation of tyrosine residues of Cx43.