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Sample records for connexins cx36 cx43

  1. Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice.

    Science.gov (United States)

    Oguro, K; Jover, T; Tanaka, H; Lin, Y; Kojima, T; Oguro, N; Grooms, S Y; Bennett, M V; Zukin, R S

    2001-10-01

    Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

  2. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells

    OpenAIRE

    Pollok, Simone; Pfeiffer, Ann-Catherine; Lobmann, Ralf; Wright, Catherine S; Moll, Ingrid; Martin, Patricia E M; Johanna M Brandner

    2010-01-01

    Abstract During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its t...

  3. The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration.

    Science.gov (United States)

    Gerhart, Sarah V; Eble, Diane M; Burger, R Michael; Oline, Stefan N; Vacaru, Ana; Sadler, Kirsten C; Jefferis, Rebecca; Iovine, M Kathryn

    2012-01-01

    Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.

  4. The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration.

    Directory of Open Access Journals (Sweden)

    Sarah V Gerhart

    Full Text Available Connexins (Cx are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.

  5. Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

    Science.gov (United States)

    Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

    2015-11-01

    The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.

  6. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo.

    Science.gov (United States)

    Wrenzycki, C; Herrmann, D; Carnwath, J W; Niemann, H

    1996-09-01

    In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity

  7. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells.

    Science.gov (United States)

    Pollok, Simone; Pfeiffer, Ann-Catherine; Lobmann, Ralf; Wright, Catherine S; Moll, Ingrid; Martin, Patricia E M; Brandner, Johanna M

    2011-04-01

    During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non-diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non-diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non-diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non-diabetic cells. The cause of the discrepancy between diabetic and non-diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368-phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

  8. Correlations of differentially expressed gap junction connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with breast cancer progression and prognosis.

    Directory of Open Access Journals (Sweden)

    Ivett Teleki

    Full Text Available Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other's prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.Differential expression of Cx43 and Cx

  9. [Relationship between Helicobacter pylori infection and expression of connexin (Cx) 32 and Cx43 genes in gastric cancer and gastric precancerous lesions].

    Science.gov (United States)

    Xu, Can-Xia; Jia, Yan; Yang, Wen-Bin; Wang, Fen; Shen, Shou-Rong

    2008-06-10

    To investigate the expression of connexin (Cx)32 and Cx43 genes in gastric cancer and precancerous lesion, and to investigate the relation between the changes of expression of Cx32 and Cx43 genes and Helicobacter pylori (Hp) infection. Gastroscopy and biopsy of gastric mucosa were conducted on 33 patients with chronic superficial gastritis (CSG), 88 with precancerous lesion, and 70 with gastric cancer. Hp was detected by rapid urease test, basic fuchsin staining, and 14C-urea breath test. The CagA gene of Hp was determined by PCR. SABC immunohistochemical method was used to detect the expression of Cx32 and Cx43 genes in gastric mucosa biopsy specimens. The positive expression rates of Cx32 and Cx43 genes were 15.7% and 32.9% respectively in the gastric cancer patients, 51.1% and 54.5% in the patients with precancerous lesion, and 100.0% and 93.9% in the CSG patients. The positive Cx32 and Cx43 expression rates of the gastric cancer and precancerous lesion patients were significantly lower than those of the CSG patients (all P infection was 16.7%, not significantly different from that of the gastric cancer patients without Hp infection (13.6%). The positive Cx43 expression rate of the gastric cancer patients with Hp infection was 25%, significantly lower than that of the gastric cancer patients without Hp infection (50%, P = 0.039). The positive Cx32 and Cx43 expression rates and expression intensity of the precancerous lesion patients with Hp infection were all significantly lower than those of the precancerous lesion patients without Hp infection (all P infection was 17.9%, significantly lower than that of the CagA- Hp group (55.6%, P = 0.027), however, the positive Cx32 expression rate of the gastric cancer patients with CagA+ Hp infection was 12.8%, not significantly different from that of the gastric cancer patients with CagA- Hp infection (33.3%, P = 0.159). The positive Cx32 and Cx43 expression rates of the CSG patients with CagA+ Hp and CagA- Hp infection

  10. Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用%Construction of a lentiviral vector for RNA interference targeting rat cardiac myocytes connexin 43 gene and its effect

    Institute of Scientific and Technical Information of China (English)

    陈立; 钟国强; 涂荣会; 黎庆捷; 何艳

    2013-01-01

    目的 构建缝隙连接蛋白(Cx)43基因shRNA慢病毒载体,并检测其对大鼠心肌细胞Cx43基因的作用.方法 针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,挑选阳性克隆行PCR鉴定及测序.用pHelper1.0和pHelper2.0质粒转染293T细胞,包装产生具备感染能力的慢病毒.以293T细胞中绿色荧光蛋白的细胞数量计算病毒滴度;以最适感染复数感染大鼠心肌细胞,通过荧光显微镜观察感染效率;应用real-time PCR和Western blot法检测大鼠心肌细胞.Cx43 mRNA及Cx43蛋白表达,并评价其抑制效果.结果 经PCR鉴定和测序证实,Cx43慢病毒载体构建正确,其病毒滴度为8×108 TU/mL、转染效率为82.59%.荧光定量real-time PCR法检测Cx43 mRNA的抑制率为96.10%,Western blot法检测Cx43蛋白的抑制率为77.16%.结论 成功构建了Cx43基因shRNA慢病毒载体,其能显著抑制大鼠心肌细胞Cx43基因的表达.%Objective To construct a lentiviral RNAi vector and to detect its effect on connexin 43 (Cx43) gene in rat cardiac myocytes.Methods Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43 gene was designed.After synthesis and annealing,the double-stranded oligonucleotides (ds oligo) were connecting to pGC-LV vectors.The positive clones were selected and conducted by PCR identification and sequencing.Then,The viral particles were gen erated by cotransfection of 293T cells with the pGC-lv-Cx43 and two packaging vector (pHelper1.0,pHelper2.0),and the virus titer was determined by counting the number of GFP positive cells.After transfection of lentiviral vector into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43 mRNA in rat cardiac myocytes was determined by realtime PCR and the level of Cx43 protein was determined by Western blot assay.The inhibitory effect was evaluated.Results The construction of Cx43 lentiviral vector was confirmed by PCR identification

  11. Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice.

    Science.gov (United States)

    Munger, Stephanie J; Geng, Xin; Srinivasan, R Sathish; Witte, Marlys H; Paul, David L; Simon, Alexander M

    2016-04-15

    Venous valves (VVs) are critical for unidirectional blood flow from superficial and deep veins towards the heart. Congenital valve aplasia or agenesis may, in some cases, be a direct cause of vascular disease, motivating an understanding of the molecular mechanisms underlying the development and maintenance of VVs. Three gap junction proteins (Connexins), Cx37, Cx43, and Cx47, are specifically expressed at VVs in a highly polarized fashion. VVs are absent from adult mice lacking Cx37; however it is not known if Cx37 is required for the initial formation of valves. In addition, the requirement of Cx43 and Cx47 for VV development has not been studied. Here, we provide a detailed description of Cx37, Cx43, and Cx47 expression during mouse vein development and show by gene knockout that each Cx is necessary for normal valve development. The valve phenotypes in the knockout lines exhibit Cx-specific differences, however, including whether peripheral or central VVs are affected by gene inactivation. In addition, we show that a Cx47 null mutation impairs peripheral VV development but does not affect lymphatic valve formation, a finding of significance for understanding how some CX47 mutations cause inherited lymphedema in humans. Finally, we demonstrate a striking segregation of Foxc2 and NFATc1 transcription factor expression between the downstream and upstream faces, respectively, of developing VV leaflets and show that this segregation is closely associated with the highly polarized expression of Cx37, Cx43, and Cx47. The partition of Foxc2 and NFATc1 expression at VV leaflets makes it unlikely that these factors directly cooperate during the leaflet elongation stage of VV development.

  12. 缝隙连接蛋白Cx43在SD大鼠电点燃癫痫模型中的表达%Expression of gap junction connexin43 in the SD rats electrical kindling of epilepsy model

    Institute of Scientific and Technical Information of China (English)

    宋建华; 谢锡驹

    2012-01-01

    Objective To investigate the role of connexin (Cx) in pathogenesis of epilepsy. Methods Thirty - two male SD rats were randomly divided into 4 groups and 4 models were set up. The behaviors of all SD rats were observed meanwhile the EEGs were recorded. Before the end Cx43 in hippocampus was detected with Western blot. The relationship between gap junction connexin and the formation of epilepsy was observed through the intervention of medicine to the kindling - model. Results Rats displayed typical epileptic behaviors,and epileptic discharges were observed in the epilepsy model group. The expression of connexin 43 in the hippocampal tissue was significantly enhanced in the epilepsy model group,but reduced by carbenoxolone and phenytoin sodium. Carbenoxolone and phenytoin sodium reduced frequency and amplitude of epileptic waves. The increasing expression of Cx43 was inhibited. Conclusion Gap junction Cx43 is the material base for the formation of epilepsy. The inhibition of excessive expression of Cx43 can reduce the formation of abnormal gap junction. Consequently the formation of epilepsy is inhibited.%目的 探讨缝隙连接蛋白(Cx)在癫痫形成中的作用.方法 将32只雄性SD大鼠随机分为4组,分别造模.观察SD大鼠行为,记录脑电图.通过免疫蛋白印迹的方法检测Cx43在大鼠海马区的表达变化.通过药物对癫痫电点燃模型成模过程的干预来观察癫痫形成与缝隙连接蛋白间的关系.结果 模型组大鼠出现癫痫发作.蛋白印迹显示Cx43的表达异常增高.药物干预的2组SD大鼠模型痫性发作滞后,发作频率降低,程度减轻,痫性脑电波波幅降低,蛋白印迹显示Cx43的表达增高受到抑制.结论 缝隙连接蛋白是癫痫形成的物质基础.抑制Cx43的过度表达,减少异常缝隙连接的形成,癫痫形成也受到抑制,从而预防癫痫产生.

  13. 胃癌和癌前病变患者Cx32,Cx43表达与幽门螺杆菌感染的相关性%Relationship between Helicobacter pylori infection and expression of connexin(Cx)32 and Cx43 genes in gastric cancer and gastric precancerous lesions

    Institute of Scientific and Technical Information of China (English)

    徐灿霞; 贾燕; 杨文斌; 王芬; 沈守荣

    2008-01-01

    Objective To investigate the expression of connexin (Cx) 32 and Cx43 genes in gastric cancer and precancerous lesion,and to investigate the relation between the changes of expression of Cx32 and Cx43 genes and Helicobacter pylori (Hp) infection.Methods Gastroscopy and biopsy of gastric mucosa were conducted on 33 patients with chronic superficial gastritis(CSG),88 with precancerous lesion,and 70 with gastric cancer.Hp was detected by rapid urease test,basic fuchsin staining,and 14C-urea breath test.The CagA gene of Hp Was determined by PCR.SABC immunohistochemical method was used to detect the expression of Cx32 and Cx43 genes in gastric mucosa biopsy specimens.Results The positive expression rates of Cx32 and Cx43 genes were 15.7%and 32.9%respectively in the gastric cancer patients,51.1% and 54.5%in the patients with precancerous lesion,and 100.0%and 93.9%in the CSG patients.The positive Cx32 and Cx43 expression rates of the gastric cancer and precancerous lesion patients were significantly lower than those of the CSG patients(all P<0.05).The positive Cx32 expression rate of the gastric cancer patients with Hp infection was 16.7%,not significantly different from that of the gastric cancer patients without Hp infection(13.6%).The positive Cx43 expression rate of the gastric cancer patients with Hp infection was 25%,significantly lower than that of the gastric cancer patients without Hp infection(50%,P=0.039).The positive Cx32 and Cx43 expression rates and expression intensity of the precancerous lesion patients with Hp infection were all significantly lower than those of the precancerous lesion patients without Hp infection(all P<0.05).The positive Cx43 expression rate of the gastric cancer patients with CasA+ Hp infection was 17.9%,significantly lower than that of the CasA- Hp group (55.6%,P=0.027),however,the positive Cx32 expression rate of the gastric cancer patients with CasA+Hp infection was 12.8%,not significantly different from that of the gastric cancer

  14. Up-Regulation Effect of 8-Br-cAMP on Expression of Connexin Gene Cx43 in Tongue Carcinoma Cell Line%8-Br-cAMP上调舌癌细胞株间隙连接蛋白基因Cx43表达的研究

    Institute of Scientific and Technical Information of China (English)

    王安训; 黄洪章; 孔庆瑜

    2001-01-01

    目的:探讨8-Br-cAMP(8-Bromo3':5'-cyclic adenosine monophosphate)对舌癌细胞株(Tca8113)细胞间隙连接蛋白基因Cx43表达的调节作用。方法:应用MTT法检测8-Br-cAMP对舌癌细胞株生长的抑制作用;应用免疫细胞化学、流式细胞仪等技术,对8-Br-cAMP诱导舌癌细胞株细胞间隙连接蛋白基因Cx43的表达进行分析。结果:舌癌细胞经10-5mol/L、10-4mol/L、10-3mol/L8-Br-cAMP处理后,细胞生长受抑制,其抑制率随浓度升高而升高;免疫细胞化学检测可见Cx43蛋白的表达明显提高;流式细胞仪分析,Cx43蛋白荧光强度增强,阳性细胞计数率由4.8%上升至26.5%,经t检验两组间存在显著性差异(P<0.01)。结论:8-Br-cAMP对舌癌细胞的抑制作用可能通过上调Cx43蛋白的表达而实现。?%Objectives:This study was designed to investigate the regulation effect of 8-Br-cAMP ( 8-Bromo 3′ :5′ -cyclic adenosine monophosphate) on the expression of connexin gene (Cx43) in human tongue carcinoma cell line (Tca8113) . Methods:The inhibitory effect of 8-Br-cAMP on growth of Tca8113 cell line was examined by methyl thiazolyl tetrazolium (MTT) assay. Cx43 gene expression in Tca8113 cells after treated with 8-Br-cAMP was analyzed by immunocytochemistry and flow cytometry . Results:After treated with 8-Br-cAMP(10-5 mol/L, 10-4 mol/L, 10-3 mol/L), Tca8113 cells was inhibited and the inhibition rate was increased with the concentration of 8-Br-cAMP; Immunocytochemistry showed Cx43 protein detectability and up-regulation in 8-Br-cAMP treated cells; The positive rate of Cx43 protein in Tca8113 ce1ls increased from 4.8% to 26.5% . There was significant difference between 8-Br-cAMP treated cells and untreated cells ( P<0.01) . Conclusion: The anti-tumor effect of 8-Br-cAMP on Tca8113 cells might be due to up-regu1ation on Cx43 gene.

  15. Construction of Mouse Melanoma Cell Models of Wild-type Overexpression Connexin 43 and Site-specific Mutant Connexin 43%过表达及定点突变缝隙连接蛋白Cx43小鼠黑色素瘤细胞模型的构建

    Institute of Scientific and Technical Information of China (English)

    李玢; 张广献; 刘娟; 赵青; 谭宇蕙; 吴映雅; 易华; 杜标炎

    2014-01-01

    目的:构建小鼠黑色素瘤细胞(B16)过表达野生型及点突变型缝隙连接蛋白43(Connexin43, Cx43)细胞模型,为以缝隙连接(Gap Junction, GJ)为靶点的中药复方、中药单药及药物单体的研究提供可靠阳性对照和阴性对照。方法构建野生型Cx43、突变型Cx43G21R、突变型Cx43G138R重组荧光蛋白融合慢病毒表达质粒,用定点突变技术获得Cx43G21R和Cx43G138R突变体,对上述3种质粒进行双酶切和测序鉴定,并分别包装病毒感染B16细胞,使B16细胞过表达野生型Cx43、突变型Cx43G21R、突变型Cx43G138R。 Western blot检测Cx43蛋白表达水平变化,荧光示踪法观察缝隙连接通讯(Gap Junction Intercellular Communication, GJIC)功能变化。结果①酶切及测序证明,成功构建 pLVCx43-mCherry、 pLVCx43-mCherryG21R、 pLVCx43-mCherryG138R重组荧光蛋白融合慢病毒表达质粒。②成功感染B16细胞并筛选稳定过表达Cx43Cx43G21R、Cx43G138R细胞株, Western blot检测显示上述细胞株Cx43蛋白表达均高于对照组。③过表达野生型Cx43后B16细胞GJIC功能较对照组增强;过表达突变型Cx43后B16细胞GJIC功能较对照组减弱。结论过表达野生型Cx43可增强B16细胞GJIC功能,过表达突变型Cx43可抑制B16细胞GJIC功能。%Objective To construct mouse B16 melanoma cell models of wild-type overexpression Cx43 and site-specific mutant Cx43, thus to provide reliable positive and negative control for the study of gap junction targeted Chinese herbal formula, Chinese medicine ingredient, and drug monomer. Methods We constructed the recombined fluorescent protein(mCherry) infused with Lentivirus expression plasmid of wild-type Cx43(pLVCx43-mCherry), mutant R (pLVCx43G21R-mCherry) and mutant Cx43G138R(pLVCx43 G138R-mCherry). The Cx43G21R and Cx43G138R mutants were obtained by site-specific mutagenesis. The three kinds of plasmid were performed double restriction

  16. Researches on Regulation Mechanism of Connexin 43 Expression in Reproductive System by Sexual Hormones%性激素对生殖系统Cx43表达调控机制的研究进展

    Institute of Scientific and Technical Information of China (English)

    孙馥箐; 段华; 汪沙

    2015-01-01

    由连接蛋白43 (connexin 43,Cx43)构成的细胞间间隙连接(gap junction,GJ)是介导细胞间直接的物质、能量交换及电、化学信号耦合的重要通路,其可保证细胞间功能活动的协调一致,在生殖系统细胞发育、分化及成熟等生理过程及功能活动中发挥非常重要的调控作用.性激素可在转录及翻译等层面调节生殖系统细胞Cx43的结构及表达,一方面通过核受体基因组机制调节Cx43基因的转录,另一方面通过非基因组快速信号传导通路机制调节Cx43的磷酸化水平,共同影响细胞间隙连接通讯(gapjunction intracellular communication,GJIC),进一步干预生殖系统细胞的病理、生理过程及功能活动.近年来,性激素在心血管系统病生理状态下对连接蛋白调节的变化及机制研究较为成熟,而在子宫、卵巢等生殖器官中,对性激素通过调节连接蛋白及间隙连接进而影响细胞间信息流通的研究较少,其作用机制并不清晰.故本文结合近年来文献,综述性激素对生殖系统细胞Cx43表达及GJIC的调控机制的研究进展,旨在为今后深入地研究提供可行的思路.%Gap junction (G J) composed of connexin 43 (Cx43) is a kind of cell-cell junction which significantly mediates the direct interchange of metabolites,energy and electrochemical signals from one cell to another.GJ ensures the coordination of intercellular activities and plays a vital role in the regulation of cell development,differentiation,maturation and functional activities in the reproductive system.Sexual hormones can influence the pathophysiological processes and functional activities of the reproductive cells by regulating gap junction intracellular communication (GJIC),which depends on the expression of Cx43.On one hand,sexual hormones regulate Cx43 gene transcription via the nuclear receptor mechanism,on the other hand,sexual hormones regulate Cx43 phosphorylation level through the signal

  17. 异氟烷对心肌动作电位及缝隙连接蛋白Cx43的作用%Effects of Isoflurane on Action Potential of Heart and Expression of Myocardial Connexin 43

    Institute of Scientific and Technical Information of China (English)

    谷宇; 高鸿; 杨烨

    2012-01-01

    目的:观察异氟烷对心肌动作电位及缝隙连接蛋白Cx43表达的影响.方法:健康家兔40只,采用Langendorff离体心脏平衡灌注15min后,随机分为5组:I0.65组,用0.65MAC异氟烷的K-H液灌注;I0,65H用0.65MAC异氟烷加0.5mmol/L庚醇的K-H液灌注;I1.3组,用1.3MAC异氟烷的K-H液灌注;H0.5组,用庚醇0.5mmol/L的K-H液灌注;S组,用K-H液灌注.观察记录灌流15min时(基础值)和给药15min时的心率(HR)及心内膜心肌(Endo)、中层心肌(M)、心外膜心肌(Epi)的动作电位时程(APD)和振幅(APA),灌注完毕取左心室心肌组织,用免疫组织化学检测心肌Cx43蛋白表达.结果:I0.65组心肌动作电位无明显影响,I1.3组APD显著延长(P<0.05),H0.5组HR显著减慢(P<0.05),APD显著延长(P<0.05),k0.65H组给药后有发生心律失常甚至心电活动停止现象;Cx43蛋白表达I0.65H组较H0.5组表达少(P<0.05),H0.5组较I0.65、I1.3组表达少(P<0.05),I1.3组较I0.65组、S组表达少(P<0.05).结论:异氟烷与庚醇对心脏动作电位和缝隙连接蛋白Cx43可能有着相似的作用,延长动作单位时程,减少Cx43蛋白表达,改变Cx43蛋白分布;异氟烷可能通过阻滞缝隙连接,对心脏动作电位产生作用.%Objective: To observe the effects of isoflurane on action potential ( AP) of heart and the expression of myocardial connexin43 (Cx43). Methods: Forty isolated rabbit hearts were perfused for 15 minutes with modified Krebs-Hensleit buffer using Langendorff device. Then, all the hearts were randomly divided into five groups (re =8 in each) : group I0.65, group I0.65H , group 1.3, group t0. 5 and group S, in which hearts were perfused with Kreb-Hensleit buffer containing 0. 65 MAC isoflurane, 0.65 MAC isoflurane plus 0.5 mmol/L heptanol, 1.3 MAC isoflurane, 0.5 mmol/L heptanol and Kreb-Hensleit buffer only respectively. Heart rates ( HR) , AP course ( APD) and AP amplitude ( APA) of endocardium ( Endo) , mid-layer myocardium ( M ) and

  18. Connexin-36 contributes to control function of insulin-producing cells.

    Science.gov (United States)

    Le Gurun, Sabine; Martin, David; Formenton, Andrea; Maechler, Pierre; Caille, Dorothee; Waeber, Gérard; Meda, Paolo; Haefliger, Jacques-Antoine

    2003-09-26

    Connexin-36 (Cx36) is a gap junction protein expressed by the insulin-producing beta-cells. We investigated the contribution of this protein in normal beta-cell function by using a viral gene transfer approach to alter Cx36 content in the insulin-producing line of INS-1E cells and rat pancreatic islets. Transcripts for Cx43, Cx45, and Cx36 were detected by reverse transcriptase-PCR in freshly isolated pancreatic islets, whereas only a transcript for Cx36 was detected in INS-1E cells. After infection with a sense viral vector, which induced de novo Cx36 expression in the Cx-defective HeLa cells we used to control the transgene expression, Western blot, immunofluorescence, and freeze-fracture analysis showed a large increase of Cx36 within INS-1E cell membranes. In contrast, after infection with an antisense vector, Cx36 content was decreased by 80%. Glucose-induced insulin release and insulin content were decreased, whether infected INS-1E cells expressed Cx36 levels that were largely higher or lower than those observed in wild-type control cells. In both cases, basal insulin secretion was unaffected. Comparable observations on basal secretion and insulin content were made in freshly isolated rat pancreatic islets. The data indicate that large changes in Cx36 alter insulin content and, at least in INS-1E cells, also affect glucose-induced insulin release.

  19. Acetylation mediates Cx43 reduction caused by electrical stimulation

    Science.gov (United States)

    Meraviglia, Viviana; Azzimato, Valerio; Colussi, Claudia; Florio, Maria Cristina; Binda, Anna; Panariti, Alice; Qanud, Khaled; Suffredini, Silvia; Gennaccaro, Laura; Miragoli, Michele; Barbuti, Andrea; Lampe, Paul D.; Gaetano, Carlo; Pramstaller, Peter P.; Capogrossi, Maurizio C.; Recchia, Fabio A.; Pompilio, Giulio; Rivolta, Ilaria; Rossini, Alessandra

    2015-01-01

    Communication between cardiomyocytes depends upon Gap Junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylases (HAT) and deacetylases (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 hours significantly reduced Connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43. PMID:26264759

  20. Cardioprotective effect of connexin 43 expression regulated by PI3K/Akt on hydrogen sulfide postconditioning in isolated ischemic and reperfused rat hearts%PI3K/Akt调节线粒体Cx43蛋白表达在H2S后处理离体大鼠心肌中的保护作用

    Institute of Scientific and Technical Information of China (English)

    张可璇; 毛洪雅; 孟雪; 惠夏; 张邓新; 季永

    2013-01-01

    Aim To investigate whether the PBK/Akt signaling pathway regulates connexin 43 expression to protect isolated rat hearts against ischemia/reperfusion ( I/R ) injury in hydrogen sulfide postconditioning. Methods Hearts of 56 male SD rats were isolated and linked to the Langendorff apparatus. They were randomly divided into 4 groups( n = 14): ischemia reper-fusion group ( I/R ), PBK/Akt signaling pathway inhibitor LY294002 group ( LY ), hydrogen sulfide post-conditioning group ( NP ), and hydrogen sulfide with to all the nuclei counted was used as apoptotic index ( AI ). The expression of total Cx43 and phosphoryla-tion Cx43 in mitochondria were determined with Western blot analysis at the end of reperfusion. Results No differences in baseline hemodynamics were observed a-mong the experimental groups ( P > 0. 05 ). After reperfusion, compared with I/R group, NP group had better hemodynamics; the myocardial infarct size and cardiocyte apoptotic index were much lower ( P < 0. 05 ); the expression of tCx43 ( total connexin 43 , tCx43 ) and pCx43 ( phosphorylated connexin 43, pCx43 ) in mitochondria increased significantly. How- LY294002 group( N + L ). The heart rate ( HR ) , the left ventricular diastolic pressure ( LVEDP ), the left ventricular developed pressure ( LVDP ), the maximum rate of increase or decrease of left ventricular pressure ( ± dp/dtmax ) were recorded at 20 min of equilibrium at 30 min of reperfusion and at the end of reperfusion, respectively. Myocardial infarct size was measured by triphenyltet tetrazolium chloride ( TTC ) staining. Myocardial TUNEL staining was determined by in situ cell death detection kit. The ratio of TUNEL positive nuclei ever , LY294002 abolished the cardioprotection offered by hydrogen sulfide postconditioning and the increase in tCx43 and pCx43 expression in mitochondria. Conclusion The PI3K/Akt pathway upregulates connexin 43 expression to protect isolated rat hearts against is-chemia/reperfusion ( I/R ) injury in

  1. Effects of microtubule stabilizer on connexin 43 of isolated cardiac myocytes under hypoxia injury%微管稳定剂Taxol对缺氧心肌细胞Cx43蛋白的影响

    Institute of Scientific and Technical Information of China (English)

    王德国; 王新; 刘玉; 王星; 邢文; 李祥东; 杨胜; 王安才; 孙贤林

    2012-01-01

    Aim To observe the effects of mierotu-bule stabilizer ( Taxol) on eonnexin43 ( Cx43 ) expression and distribution of eardiae myoeyles and investi-gate its new value as an anti-arrhylhmie agent. Methods Cardiae myoevles isolated from aduk rats were exposed to hypoxia for 120 min with or without taxol ( a kind of mierotubule stabilizer ) in different. eoneentra-tions. Cellular survival was assayed by trypan blue dye-exelusion. Cx43 expression was determined by western blots. Cx43 distribution was deteeted by immu-nofluoreseenee staining and observed under laser sean-ning eonfoeal mieroseope. Results The cell viability under hypoxie eondition was redueed signifieantly whieh showed only few numbers of rod-shaped eells. Taxol promoted cell survival in a dose-dependent man- ner at the range of0. 1~10 nmol · L-1 . At the range of 100 nmol o L-1~ 10 μmol · L~1 , taxol lead toredueed cell survival. Taxol in a dose-dependent manner improved Cx43 expression even although exeessive taxol also suppressed Cx43. Cx43 was loeat ed in both ends of intaet eardiae myoeyles ( interealated disk ) and redistributed in both sides of the cell under hypoxie eonditions. Taxol dose-dependently inhibited lateraliza-tion of Cx43 in lower eoneentration and further de-ereased Cx43 in the interealated disk. Conclusions Cx43 redistribution indueed by hypoxie is proteeted by low dosage of taxol. Mierotubule stabilizer may be ae-ted as a new anti-arrhythmie agent.%目的 观察微管稳定剂Taxol对缺氧心肌细胞Cx43表达和分布的影响,探讨其作为新的抗心律失常药物的潜在价值.方法 酶解法分离的大鼠心肌细胞缺氧120 min,并以不同浓度的Taxol干预;台盼蓝排斥实验测定细胞存活率;免疫印迹检测Cx43的蛋白表达,免疫荧光染色后激光共聚焦显微镜观察分析Cx43的分布.结果 Taxol在0.1~10 nmol·L-1浓度下成剂量依赖性地提高杆状细胞数,促进细胞存活,而100 nmol·L-1~10 μmol·L-1浓度下杆状细胞数

  2. Mutations of Cx43 that affect B cell spreading in response to BCR signaling

    Directory of Open Access Journals (Sweden)

    Letitia Falk

    2014-07-01

    Full Text Available The gap junction (GJ protein connexin 43 (Cx43 is both necessary and sufficient for B cell receptor (BCR-mediated cell spreading. To address how Cx43 mediates this effect, we blocked its function genetically, by expressing mutants of Cx43, and pharmacologically, by using chemical inhibitors. While various point mutations of Cx43 inhibited B cell spreading, treatment with channel blocking drugs did not, suggesting that this response was independent of channel function. The critical region of Cx43 appears to be the cytoplasmic carboxyl-terminal (CT domain, which has previously been shown to be important for B cell spreading. Consistent with this, mutations of either tyrosine 247 or 265 found in the CT were sufficient to inhibit spreading. Thus Cx43 may influence B cell spreading by mechanisms requiring protein binding to, or modification of, these sites in the CT tail.

  3. 维甲酸对HeLa细胞间隙连接蛋白基因cx43表达的调节作用%The regulation effect of all-trans-retinoic acid on expression of connexin gene cx43 in HeLa

    Institute of Scientific and Technical Information of China (English)

    陈必良; 马向东; 王德堂; 辛晓燕

    1999-01-01

    目的:探讨分化诱导剂维甲酸对肿瘤抑制基因--细胞间隙连接蛋白基因cx43在人子宫颈癌细胞系HeLa中表达的调节作用.方法:应用核酸原位杂交、流式细胞仪、Westen blot及Lucifer Yellow划痕标记荧光传输技术,研究维甲酸作用对HeLa细胞cx43 mRNA及其蛋白表达,以及对HeLa细胞生长和通讯功能的调节作用.结果:HeLa细胞经维甲酸处理后,原位杂交显示,HeLa细胞cx43 mRNA水平上调;流式细胞仪分析,Cx43蛋白荧光强度增强,阳性细胞计数率由1.9%上升至26.3%;Wester blot检测到分子量约43KDa的Cx43蛋白表达;HeLa细胞生长明显受抑;细胞间隙连接通讯功能有所恢复.结论:维甲酸可通过上调肿瘤抑制基因cx43及其蛋白在HeLa细胞中的表达,实现对宫颈癌细胞恶性表型的逆转作用,这可能是维甲酸肿瘤抑制作用的重要机制之一.

  4. The role of the Cx43 C-terminus in GJ plaque formation and internalization.

    Science.gov (United States)

    Wayakanon, Praween; Bhattacharjee, Rajib; Nakahama, Ken-ichi; Morita, Ikuo

    2012-04-06

    Connexin 43 (Cx43) is a major gap junction (GJ) protein found in many mammalian cell types. The C-terminal (CT) domain of Cx43 has unique characteristics in terms of amino acid (aa) sequence and its length differs from other connexins. This CT domain can be associated with protein partners to regulate GJ assembly and degradation, which results in the direct control of gap junction intercellular communication (GJIC). However, the essential roles of the CT regions involved in these mechanisms have not been fully elucidated. In this study, we aimed to investigate the specific regions of Cx43CT involved in GJ formation and internalization. Wild type Cx43((382aa)) and 10 CT truncated mutants were stably expressed in HeLa cells as GFP or DsRed tagged proteins. First, we found that the deletion of 235-382aa from Cx43 resulted in failure to make GJ and establish GJIC. Second, the Cx43 with 242-382aa CT deletion could form functional GJs and be internalized as annular gap junctions (AGJs). However, the plaques consisting of Cx43 with CT deletions (Δ242-382aa to Δ271-382aa) were longer than the plaques consisting of Cx43 with CT deletions (Δ302-382aa). Third, co-culture experiments of cells expressing wild type Cx43((382)) with cells expressing Cx43CT mutants revealed that the directions of GJ internalization were dependent on the length of the respective CT. Moreover, a specific region, 325-342aa residues of Cx43, played an important role in the direction of GJ internalization. These results showed the important roles of the Cx43 C-terminus in GJ expression and its turnover.

  5. Pathological implications of Cx43 down-regulation in human colon cancer.

    Science.gov (United States)

    Ismail, Rehana; Rashid, Rabiya; Andrabi, Khurshid; Parray, Fazl Q; Besina, Syed; Shah, Mohd Amin; Ul Hussain, Mahboob

    2014-01-01

    Connexin 43 is an important gap junction protein in vertebrates and is known for its tumor suppressive properties. Cx43 is abundantly expressed in the human intestinal epithelial cells and muscularis mucosae. To explore the role of Cx43 in the genesis of human colon cancer, we performed the expression analysis of Cx43 in 80 cases of histopathologically confirmed and clinically diagnosed human colon cancer samples and adjacent control tissue and assessed correlations with clinicopathological variables. Western blotting using anti-Cx43 antibody indicated that the expression of Cx43 was significantly down regulated (75%) in the cancer samples as compared to the adjacent control samples. Moreover, immunohistochemical analysis of the tissue samples confirmed the down regulation of the Cx43 in the intestinal epithelial cells. Cx43 down regulation showed significant association (pcancer. Our data demonstrated that loss of Cx43 may be an important event in colon carcinogenesis and tumor progression, providing significant insights about the tumor suppressive properties of the Cx43 and its potential as a diagnostic marker for colon cancer.

  6. 温胆汤对地卓西平马来酸盐诱发精神分裂症模型大鼠脑组织中Cx43 mRNA表达的影响%Effect of Wendan Decoction on Connexin 43 mRNA Expression of in Rat Brain Tissue with Schizophrenia Induced by Dizocilpine Maleate

    Institute of Scientific and Technical Information of China (English)

    付艳丽; 万红娇; 朱金华; 马广强

    2012-01-01

    目的:探讨温胆汤对精神分裂症模型大鼠脑组织中神经胶质细胞连接蛋白(Cx43) mRNA的影响.方法:将健康的SD大鼠50只随机分为5组:正常组(A)、模型组(B)、温胆汤高、中、低剂量组(C,D,E组,剂量为生药40,20,10 g·kg-1).A,B组生理盐水ig,C,D,E组温胆汤ig,1次/d,21 d后,按照0.6 mg· kg -1一次性ip地卓西平马来酸盐(MK-801)建立拟精神分裂症模型,然后行为学观察3d,处死大鼠.应用逆转录聚合酶链式反应(RT-PCR)法,检测大鼠Cx43 mRNA的表达量.结果:模型组Cx43 mRNA的表达量低于正常组,有显著性意义(P<0.05);温胆汤3个剂量组Cx43 mRNA的表达量明显高于模型组,有显著性意义(P<0.05).结论:温胆汤能够增强精神分裂症模型大鼠脑组织中Cx43 mRNA的表达.%Objective; To study the effect of Wendan decoction on connexin 43 ( Cx43 ) mRNA expression in brain tissue of rats with schizophrenia. Method; Fifty SD rats were divided five groups by random, including normal group ( A ) , model group ( B ) , Wendan decoction high-dosage group ( C ) , medium-dosage group (D) and low-dosage group (E) , respectively. The A and B groups were given normal saline and the C, D, E groups were given Wendan decoction (40, 20, 10 g - kg-1 ) every day, which lasted for 21 days. Dizocilpine maleate ( MK-801 ) 0. 6 mg - kg was ip given to establishthe rat schizophrenic model. Then rat behaviors were observed for 3 days all rats were sacrificed. Then RT-PCR method was used to detect Cx43 mRNA expression in the nervecells of the every group rats. Result; The Cx43 mRNA expression of group B was lower than that of group A ( P < 0. 05) . And the Cx43 mRNA expression of group C , D and E was rose compared with that of group B ( P < 0. 05 ) . Conclusion; Wendan decoction can reinforce the expression of Cx43 mRNA in the brain tissue of rats with schizophrenia.

  7. Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 expression and reduced sodium current that accounts for arrhythmia vulnerability in conditional Cx43 knockout mice.

    NARCIS (Netherlands)

    Jansen, J.; Noorman, M.; Musa, H.; Stein, M.; Jong, S. de; Nagel, R. van der; Hund, T.J.; Mohler, P.J.; Vos, M.E. de; Veen, T.A. van; Bakker, J.M. de; Delmar, M.; Rijen, H.V. van

    2012-01-01

    BACKGROUND: Reduced expression of connexin43 (Cx43) and sodium channel (Nav1.5) and increased expression of collagen (fibrosis) are important determinants of impulse conduction in the heart. OBJECTIVE: To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx

  8. Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication

    Science.gov (United States)

    Zhang, Yiyao; Isayev, Orkhan; Heilmann, Katharina; Schoensiegel, Frank; Liu, Li; Nessling, Michelle; Richter, Karsten; Labsch, Sabrina; Nwaeburu, Clifford C.; Mattern, Juergen; Gladkich, Jury; Giese, Nathalia; Werner, Jens; Schemmer, Peter; Gross, Wolfgang; Gebhard, Martha M.; Gerhauser, Clarissa; Schaefer, Michael; Herr, Ingrid

    2014-01-01

    The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA. PMID:24742583

  9. HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

    Directory of Open Access Journals (Sweden)

    Peng Sun

    2015-10-01

    Full Text Available Human papillomavirus type 16 (HPV16 causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large. Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43, a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

  10. The role of the Cx43 C-terminus in GJ plaque formation and internalization

    Energy Technology Data Exchange (ETDEWEB)

    Wayakanon, Praween; Bhattacharjee, Rajib [Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Cx43-GFP or -DsRed fusion proteins were expressed in HeLa cells. Black-Right-Pointing-Pointer Roles of C-terminus were examined using various mutants. Black-Right-Pointing-Pointer Gap junction plaque size was dependent on the length of C-terminus. Black-Right-Pointing-Pointer C-terminus dependent gap junction plaque internalization was observed. -- Abstract: Connexin 43 (Cx43) is a major gap junction (GJ) protein found in many mammalian cell types. The C-terminal (CT) domain of Cx43 has unique characteristics in terms of amino acid (aa) sequence and its length differs from other connexins. This CT domain can be associated with protein partners to regulate GJ assembly and degradation, which results in the direct control of gap junction intercellular communication (GJIC). However, the essential roles of the CT regions involved in these mechanisms have not been fully elucidated. In this study, we aimed to investigate the specific regions of Cx43CT involved in GJ formation and internalization. Wild type Cx43{sub (382aa)} and 10 CT truncated mutants were stably expressed in HeLa cells as GFP or DsRed tagged proteins. First, we found that the deletion of 235-382aa from Cx43 resulted in failure to make GJ and establish GJIC. Second, the Cx43 with 242-382aa CT deletion could form functional GJs and be internalized as annular gap junctions (AGJs). However, the plaques consisting of Cx43 with CT deletions ({Delta}242-382aa to {Delta}271-382aa) were longer than the plaques consisting of Cx43 with CT deletions ({Delta}302-382aa). Third, co-culture experiments of cells expressing wild type Cx43{sub (382)} with cells expressing Cx43CT mutants revealed that the directions of GJ internalization were dependent on the length of the respective CT. Moreover, a specific region, 325-342aa residues of Cx43, played an important role in the direction of GJ internalization. These results showed the important roles of the Cx43 C-terminus in GJ

  11. Expression of Cx43-related microRNAs in patients with tetralogy of Fallot

    Institute of Scientific and Technical Information of China (English)

    Yao Wu; Xiao-Jing Ma; Hui-Jun Wang; Wen-Can Li; Long Chen; Duan Ma; Guo-Ying Huang

    2014-01-01

    Background: Abnormal expression of connexin 43 (Cx43) has been reported to play an important role in the development of conotrunccal anomalies. However, less is known about the underlying reason for its abnormal expression. MicroRNAs (miRNAs), as an important part of gene expression regulation, have been implicated in some cardiac diseases. This study aimed to investigate the expression of Cx43 and its related miRNAs in patients with tetralogy of Fallot (TOF), and illustrate the potential role of abnormal miRNAs regulation to Cx43 expression in the pathology of TOF. Methods: Real-time polymerase chain reaction was used to detect the expression of Cx43 and 10 Cx43-related miRNAs in the myocardium from 30 TOF patients and 10 normal controls. Immunohistochemistry was used to detect Cx43 protein expression. Putative miRNA binding sites in the 3'UTR of Cx43 were examined in 200 TOF patients and 200 healthy individuals, using Sanger sequencing, to exclude sequence variations resulting in binding diffi culties of miRNAs. Results: Cx43 mRNA and protein expression in the myocardium tissue was significantly increased in TOF patients. The expression of MiR-1 and 206 was significantly decreased in the TOF patients as compared with the controls (P0.05). No meaningful sequence variation was detected in the putative miR1/206 binding sites in the 3'UTR of Cx43. Conclusions: This study indicated that miR-1 and 206 is down-regulated in TOF patients, which may cause an up-regulation of Cx43 protein's synthesis. It provided a clue that miR-1 and 206 might be involved in the pathogenesis of TOF, additional experiments are needed to determine if in fact, miR-1 and 206 contribute substantially to TOF.

  12. Functional expression of gap junction gene Cx43 and the myogenic differentiation of rhabdomyosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    林仲翔; 张志谦; 韩亚玲; C.C.G.Naus; K.R.Yu; H.Holtzer

    1995-01-01

    Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably

  13. Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression

    NARCIS (Netherlands)

    Serre-Beinier, Veronique; Bosco, Domenico; Zulianello, Laurence; Charollais, Anne; Caille, Dorothee; Charpantier, Eric; Gauthier, Benoit R.; Diaferia, Giuseppe R.; Giepmans, Ben N.; Lupi, Roberto; Marchetti, Piero; Deng, Shaoping; Buhler, Leo; Berney, Thierry; Cirulli, Vincenzo; Meda, Paolo

    2009-01-01

    Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observ

  14. Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression

    NARCIS (Netherlands)

    Serre-Beinier, Veronique; Bosco, Domenico; Zulianello, Laurence; Charollais, Anne; Caille, Dorothee; Charpantier, Eric; Gauthier, Benoit R.; Diaferia, Giuseppe R.; Giepmans, Ben N.; Lupi, Roberto; Marchetti, Piero; Deng, Shaoping; Buhler, Leo; Berney, Thierry; Cirulli, Vincenzo; Meda, Paolo

    2009-01-01

    Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observ

  15. Neurological manifestations of oculodentodigital dysplasia: a Cx43 channelopathy of the central nervous system?

    Directory of Open Access Journals (Sweden)

    Marijke eDe Bock

    2013-09-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexins of which Cx43 is most widespread in the human body. In the brain, Cx43 GJs are mostly found in astroglia where they coordinate the propagation of Ca2+ waves, spatial K+ buffering and distribution of glucose. Beyond its role in direct intercellular communication, Cx43 also forms unapposed, non-junctional hemichannels in the plasma membrane of glial cells. These allow the passage of several neuro- and gliotransmitters that may, combined with downstream paracrine signaling, complement direct GJ communication among glial cells and sustain glial-neuronal signaling. Mutations in the GJA1 gene encoding Cx43 have been identified in a rare, mostly autosomal dominant syndrome called oculodentodigital dysplasia (ODDD. ODDD patients display a pleiotropic phenotype reflected by eye, hand, teeth and foot abnormalities, as well as craniofacial and bone malformations. Remarkably, neurological symptoms such as dysarthria, neurogenic bladder (manifested as urinary incontinence, spasticity or muscle weakness, ataxia, and epilepsy are other prominent features observed in ODDD patients. Over 10 mutations detected in patients diagnosed with neurological disorders are associated with altered functionality of Cx43 GJs/hemichannels, but the link between ODDD-related abnormal channel activities and neurologic phenotype is still elusive. Here, we present an overview on the nature of the mutants conveying structural and functional changes of Cx43 channels and discuss available evidence for aberrant Cx43 GJ and hemichannel function. In a final step, we examine the possibilities of how channel dysfunction may lead to some of the neurological manifestations of ODDD.

  16. The relation of Cx43 and NMDA to visceral sensitization in rats with irritable bowel syndrome

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    Jing-yu ZHANG

    2016-01-01

    Full Text Available Objective  To study the relationship between connexin 43 (Cx43 and N-methyl-D-aspartate (NMDA receptors and visceral sensitization in the rats with irritable bowel syndrome (IBS. Methods  Thirty rats were gavaged with Triehinella spiralis to reproduce the IBS model. These rats were randomly divided into IBS group, IBS+colon distension group, and IBS+STI-571+colon distension group, and other groups of normal rats were randomized into normal group and normal+colon distension group, with 10 rats in each group. Immunofluorescent double staining were used to observe the expressions of intestine Cx43 and sacral NMDA re ceptors of rats in all the groups. Results  The Cx43 and sacral NMDA expressions in the normal group, normal+colon distension group and IBS group showed no significant changes (P>0.05, however, Cx43 and sacral NMDA expressions were significantly higher in IBS rats with colon distension as compared with those in normal group, normal+colon distension group, and IBS group (P<0.05, while they were significantly lower in the IBS+STI-571+colon distension group after STI-571 intervention (P<0.05. Conclusion  Cx43 and sacral NMDA may be the most important factor of visceral sensitization in IBS rats. DOI: 10.11855/j.issn.0577-7402.2015.12.02

  17. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

  18. Infection by Cx43 adenovirus increased chemotherapy sensitivity in human gastric cancer BGC-823 cells: not involving in induction of cell apoptosis.

    Science.gov (United States)

    Liu, Dan; Zhou, Hongfeng; Wu, Jin; Liu, Wentao; Li, Yongqing; Shi, Guangyue; Yue, Xiaolong; Sun, Xiwen; Zhao, Yanbin; Hu, Xiaowei; Wang, Tianjiao; Zhang, Xufeng

    2015-12-15

    There is a lower basal expression of Connexin43 (Cx43) in human gastric cancer BGC-823 cells. In the present study, BGC-823 cells were transfected with recombinant Cx43 adenovirus plasmid vector, and we explored the influences of Cx43 expression on cell proliferation, chemo-sensitivity, colony forming ability, invasion ability and apoptosis. Moreover, we also determined the expression of Pgp, Cx43, as well as apoptosis-related proteins (bcl-2, bax, caspase3 and caspase 9). MTT assay was performed to determine the proliferation of BGC-823 cells before and after Cx43 transfection. The influences of Cx43 infection on sensitivity of chemotherapy (including Doxorubicin, fluorouracil, oxaliplatin) were detected by MTT assay. Expression levels of Pgp, Cx43, as well as apoptosis-related proteins (bcl-2, bax, caspase-3 and caspase-9) in BGC-823 cells were determined by Western blotting analysis before and after the infection with Cx43 adenovirus. MDR expression was determined by RT-PCR before and after Cx43 infection. Invasive ability was detected by invasion chamber. Influence of Cx43 adenovirus infection on apoptosis of BGC-823 cells was determined by flow cytometry. After infection by Cx43 adenovirus, colony forming rate and invasive ability of BGC-823 cells were decreased. Flow cytometry results revealed that cell apoptosis were insignificantly increased. The data of MTT assay revealed that infection with Cx43 adenovirus, cell proliferation ability decreased and sensitivity to chemotherapy drugs (including doxorubicin, fluorouracil, oxaliplatin) increased. Results of Western blotting analysis revealed that increasing expression levels of Cx43, decreasing expression levels of Pgp, and insignificant changes of bcl-2, bax, caspase3 and caspase 9 were detected. RT-PCR revealed the expression of MDR1 gene, the gene encoding Pgp, decreased significantly (pinfected with Cx43-IRES2-EGFP recombinant adenovirus vector. Colony formation, invasive ability and cell proliferation all

  19. Connexin43 hemichannel-mediated regulation of connexin43.

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    Kai Li

    Full Text Available BACKGROUND: Many signaling molecules and pathways that regulate gap junctions (GJs protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43 hemichannels were activated by cadmium (Cd(2+, we tested this hypothesis. PRINCIPAL FINDINGS: Incubation of Cx43-transfected LLC-PK1 cells with Cd(2+ led to an increased expression of Cx43. This effect of Cd(2+ was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd(2+ on JNK activation and Cx43 expression. Indeed, Cd(2+ induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd(2+. CONCLUSIONS: Collectively, our results thus indicate that Cd(2+-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.

  20. Keratitis-ichthyosis-deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43.

    Science.gov (United States)

    García, Isaac E; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshmi; Olivero, Pablo; Perez-Acle, Tomas; González, Carlos; Sáez, Juan C; Contreras, Jorge E; Martínez, Agustín D

    2015-05-01

    Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like the Keratitis-Ichthyosis-Deafness syndrome (KID). Because in the human skin connexin 26 (Cx26) is co-expressed with other connexins, like Cx43 and Cx30, and as the KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild-type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channel (GJC) functions remain unknown. In this study, we demonstrate that syndromic mutations, at the N terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) shows exacerbated hemichannel activity but nonfunctional GJCs; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca(2+) overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin.

  1. Cx43和Pax3在人胚胎食管肌层组织中的表达及意义%Expression and Significance of Cx43 and Pax3 in the Esophagus of a Human Embryo

    Institute of Scientific and Technical Information of China (English)

    张剑; 刘学红

    2009-01-01

    应用免疫组织化学SABC法检测第2,3,4三个月胎龄段,Cx43和Pax3蛋白在人胚胎食管组织中的表达.结果表明:第2个月胚龄时,Cx43和Pax3在食管肌层肌细胞中呈阳性表达,在肌间神经丛处神经细胞呈阴性表达;第3个月胎龄段,Cx43和Pax3在食管肌层肌细胞阳性表达与第2个月胚龄相似,在肌间神经丛处大部分神经细胞呈阳性表达;第4个月胎龄段,Cx43和Pax3在食管肌层神经细胞和肌细胞均呈弱阳性表达.因此,Cx43和Pax3蛋白与人胚胎早期食管肌层组织细胞的生长发育关系密切.%The objective of the paper is to explore the patterns of Connexin43 (Cx43) and Pairedbox3 (Pax3) protein expressions in the esophageal muscular layers of a human embryo.The SABC method in Immunohistochemistry has been employed to examine the expressions of Cx43 and Pax3 proteins in the muscular layers of the esophagus in human embryos from the second to fourth months of gestation.The following are the results:In the second month of gestation,their muscle cells of the esophageal muscular layers are positive while the nerve cells in the myenteric plexus are negative;In the third month,the Cx43 and Pax3 expressions in the muscle cells in the esophageal muscular layers are similar to those of the second month,with most nerve cells positive in the myenteric plexus;In the fourth month,the Cx43 and Pax3 protein expressions are weakly positive both in the muscle cells and nerve cells of the esophageal muscular layers.A conclusion is reached:Their Cx43 and Pax3 proteins are closely related to the growth and development of the cells and tissues in the esophageal muscular layers in human embryos.

  2. Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43

    Science.gov (United States)

    García, Isaac E.; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshimi; Olivero, Pablo; Pérez-Acle, Tomás; González, Carlos; Sáez, Juan C.; Contreras, Jorge E.; Martínez, Agustín D.

    2015-01-01

    Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin. PMID:25625422

  3. Effects of Taxol on the expression and distribution of connexin 43 in cultured cardiac myocytes under hypoxia%Taxol对缺氧培养乳鼠心肌细胞Cx43蛋白表达及分布的影响

    Institute of Scientific and Technical Information of China (English)

    王德国; 王新; 王安才; 朱红军; 孙贤林

    2013-01-01

    目的:观察微管稳定剂Taxol对缺氧导致的培养乳鼠心肌细胞Cx43表达和分布的影响.方法:差速贴壁梯度离心法分离培养乳大鼠心肌细胞,取培养4d细胞缺氧120 min,分别以不同浓度的Taxol干预;免疫印迹法检测聚合态微管蛋白含量、心肌细胞Cx43的蛋白表达,免疫荧光染色后激光共聚焦显微镜观察Cx43的分布.结果:正常培养心肌细胞Cx43分布在核膜和细胞的闰盘处,缺氧120 min可导致心肌细胞微管解聚,Cx43蛋白表达降低,在心肌细胞间连接处分布规律散失,核膜Cx43分布减弱或消失,而均匀分布在细胞膜上;在低剂量的Taxol作用下,心肌1细胞微管解聚状态缓解,Cx43蛋白表达和分布异常明显改善;随着Taxol剂量增加,这种改善作用更明显,呈现剂量依赖性.结论:缺氧引起乳鼠心肌细胞微管解聚,Cx43蛋白表达降低分布紊乱,微管稳定剂Taxol可以显著地保护缺氧导致的心肌Cx43异常,具有潜在的抗缺血性心律失常价值.

  4. Dephosphorylation of cardiomyocyte Cx43 is associated with myocardial ischemia and reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Zhijuan Cao; Xuan Xu; Linli Que; Qi Chen; Yuehua Li

    2009-01-01

    Objective:Myocardial ischemia/reperfusion(I/R) injury is the leading cause of death in the world. However, the details of the mechanism of its pathophysioiogy are still unknown. The present study was designed to investigate the role of connexin 43(Cx63) in acute models of myocardial I/R injury. Methods: Male C57BL/6 mice were subjected to myocardial ischemia(45 rain) followed by reperfusion(4 hrs) in vivo. The whole operation was monitored using a two-lead ECG. Hearts were harvested and the level of protein was assessed by western blot analysis. Haematoxylin and Eosin(HE) staining was used to detect the extent of neutrophil infiltration. The expression level of IL-6 was detected by ELISA. Results: A murine myocardial I/R injury model was constructed successfully. Phosphorylated Cx43 decreased 83.45% while non-phosphorylated Cx43 increased 1.62- fold in the myocardium after I/R injury. Neutrophil infiltration and the expression of the inflammatory cytokine IL-6 increased in the myocardium following I/R. Conclusion: During myocardial I/R injury, cardiomyocyte Cx43 is dephosphorylated, and this may be associated with an inflammatory response.

  5. Syndromic and non-syndromic disease-linked Cx43 mutations.

    Science.gov (United States)

    Laird, Dale W

    2014-04-17

    There are now at least 14 distinct diseases linked to germ line mutations in the 21 genes that encode the connexin (Cx) family of gap junction proteins. This review focuses on the links between germ-line mutations in the gene encoding Cx43 (GJA1) and the human disease termed oculodentodigital dysplasia (ODDD). This disease is clinically characterized by soft tissue fusion of the digits, abnormal craniofacial bone development, small eyes and loss of tooth enamel. However, the disease is considerably more complex and somewhat degenerative as patients often suffer from other syndromic effects that include incontinence, glaucoma, skin diseases and neuropathies that become more pronounced during aging. The challenge continues to be understanding how distinct Cx43 gene mutations cause such a diverse range of tissue phenotypes and pathophysiological changes while other Cx43-rich organs are relatively unaffected. This review will provide an overview of many of these studies and distill some themes and outstanding questions that need to be addressed in the coming years. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Engineered Cx26 variants established functional heterotypic Cx26/Cx43 and Cx26/Cx40 gap junction channels.

    Science.gov (United States)

    Karademir, Levent B; Aoyama, Hiroshi; Yue, Benny; Chen, Honghong; Bai, Donglin

    2016-05-15

    Gap junction (GJ) channels mediate direct intercellular communication and are composed of two docked hemichannels (connexin oligomers). It is well documented that the docking and formation of GJs are possible only between compatible hemichannels (or connexins). The mechanisms of heterotypic docking compatibility are not fully clear. We aligned the protein sequences of docking-compatible and -incompatible connexins with that of connexin26 (Cx26). We found that two docking hydrogen bond (HB)-forming residues on the second extracellular domain (E2) of Cx26 and their equivalent residues are well conserved within docking-compatible connexins, but different between docking-incompatible connexins. Replacing one or both of these residues of Cx26 into the corresponding residues in the docking incompatible connexins (K168V, N176H or K168V-N176H) increased the formation of morphological and functional heterotypic GJs with connexin43 (Cx43) or connexin40 (Cx40), indicating that these two residues are important for docking incompatibility between Cx26 and these connexins. Our homology structure models predict that both HBs and hydrophobic interactions at the E2 docking interface are important docking mechanisms in heterotypic Cx26 K168V-N176H/Cx43 GJs and probably other docking compatible connexins. Revealing the key residues and mechanisms of heterotypic docking compatibility will assist us in understanding why these putative docking residues are hotspots of disease-linked mutants. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  7. Lentivirus-mediated RNAi knockdown of the gap junction protein, Cx43, attenuates the development of vascular restenosis following balloon injury.

    Science.gov (United States)

    Han, Xiao-Jian; Chen, Min; Hong, Tao; Zhu, Ling-Yu; He, Dan; Feng, Jiu-Geng; Jiang, Li-Ping

    2015-04-01

    Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular restenosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.

  8. Involvement of connexin 43 in acupuncture analgesia

    Institute of Scientific and Technical Information of China (English)

    HUANG Guang-ying; ZHENG Cui-hong; YU Wei-chang; TIAN Dai-shi; WANG Wei

    2009-01-01

    Background Connexin 43 (Cx43) is one of the major components of human keratinocyte gap junctions. To study whether gap junctional intercellular communication participates in the transfer of acupoint signals and acupuncture analgesia, the expression of Cx43 was studied in Zusanli (ST36) acupoints compared with control non-acupoint regions in rats after acupuncture. In addition, Cx43 heterozygous gene knockout mice were used to further explore the relationship between Cx43 and acupuncture analgesia. Methods The expression of Cx43 was detected by immunohistochemistry, immunoblotting, and RT-PCR for the Cx43 protein and mRNA. The influence of the Cx43 gene knockout on acupuncture analgesia was measured by a hot plate and observing the writhing response on Cx43 heterozygous gene knockout mice. Results Immunohistochemistry showed abundant Cx43 expression in some cells in the skin and subcutaneous tissue of rat ST36 acupoints. The mRNA and protein levels of Cx43 in acupoints were significantly higher than those in the control points in the non-acupuncture group, and even more so after acupuncture. The hot plate and writhing response experiments showed that partial knockout of the Cx43 gene decreased acupuncture analgesia. Conclusion Cx43 expression and acupuncture analgesia showed a positive correlation.

  9. Peroxisome proliferator-activated receptor gamma promotes exogenous mesenchymal stem cells to express connexin 43:its role and mechanism%过氧化物酶体增殖物激活受体γ促进外源性骨髓间充质干细胞表达Cx43的作用及机制

    Institute of Scientific and Technical Information of China (English)

    晏平; 侯婧瑛; 郑韶欣; 龙会宝; 周长青; 郭天柱; 伍权华; 钟婷婷; 王彤

    2016-01-01

    大鼠在干预开始时心功能参数无明显差异性。干预2周后,骨髓间充质干细胞+吡格列酮联合治疗组左室舒张末径、左室收缩末径明显减小,左室射血分数明显增高;②骨髓间充质干细胞+吡格列酮组和吡格列酮组左心室心肌组织不同区域过氧化物酶体增殖物激活受体γ表达量显著增加;③骨髓间充质干细胞+吡格列酮组在梗死区和梗死边缘区Cx43表达较吡格列酮组和磷酸缓冲液组显著增高,TGF-β1、SMAD2、SMAD3表达明显下降,吡格列酮组与磷酸缓冲液组在上述指标方面表达差异无显著性意义;④结果表明,过氧化物酶体增殖物激活受体γ激动剂吡格列酮并不能刺激内源性骨髓间充质干细胞的增殖分化并改进心功能,吡格列酮联合外源性骨髓间充质干细胞能改善心功能,其机制可能与过氧化物酶体增殖物激活受体γ抑制TGF-β1/SMAD通路进而促进外源性骨髓间充质干细胞表达Cx43有关。

  10. 产妇子宫平滑肌组织中MAPK信号转导通路及CX43表达与产后出血关系的研究%Expression and Significance of Mitogen-Activated Protein Kinase Pathway and Connexin43 in Myometrium of Parturient Women with Postpartum Hemorrhage

    Institute of Scientific and Technical Information of China (English)

    颜建英; 黄科华; 刘青闽; 黄晓燕; 徐榕莉; 余爱丽; 邓婷

    2012-01-01

    目的:分析宫缩乏力性产后出血产妇离体子宫平滑肌自发性收缩功能和收缩潜能的变化,并探讨子宫平滑肌组织中丝裂原激活蛋白激酶(MAPK)信号转导通路活化水平和连接蛋白( CX43)表达变化与宫缩乏力性产后出血的关系.方法:选择2009年11月至2010年12月在福建省妇幼保健院产科择期剖宫产分娩的宫缩乏力性产后出血产妇30例为病例组,另选同期无产后出血产妇30例为对照组.采用等长张力测定方法检测产妇离体子宫下段平滑肌收缩功能和缩宫素诱导的收缩潜能;采用蛋白免疫印迹技术检测产妇子宫下段平滑肌组织中MAPK信号转导通路中的细胞外信号调节激酶( ERK1/2)、c-Jun N端蛋白激酶(JNK1/2)、p38磷酸化蛋白和CX43蛋白水平;采用RT-PCR技术检测产妇子宫下段平滑肌组织中CX43 mRNA表达.结果:①病例组离体子宫平滑肌自发性收缩的收缩频率、收缩活动力均低于对照组(P<0.05);缩宫素诱导后病例组的离体子宫平滑肌收缩频率、收缩幅度、收缩活动力和收缩潜能均低于对照组(P <0.05,P<0.01).②以子宫平滑肌自发性收缩活动力72.66g·次/h作为诊断点,评估宫缩乏力性产后出血的ROC曲线下面积(AUC)为0.802.③病例组产妇子宫平滑肌组织中p-ERK1/2、p-JNK1/2、p-P38、CX43蛋白的表达量和CX43 mRNA的表达水平均分别低于对照组(P<0.05).④两组产妇子宫平滑肌组织中p-ERK1/2、p-JNK1/2、p-P38、CX43蛋白表达量以及CX43 mRNA表达水平分别与其离体子宫的自发性收缩活动力呈正相关关系(P<0.05).⑤两组产妇子宫平滑肌组织中p-ERK1/2、p-JNK1/2、p-P38、CX43蛋白和CX43 mRNA表达量相互两两之间均呈正相关关系(P<0.05).结论:宫缩乏力性产后出血产妇离体子宫平滑肌收缩功能和收缩潜能明显降低,其子宫平滑肌组织中MAPK信号通路活化水平及CX43表达水平明显降低,且两者

  11. Study on connexin gene and protein expression and cellular distribution in relation to real-time proliferation of porcine granulosa cells.

    Science.gov (United States)

    Kempisty, B; Ziółkowska, A; Ciesiółka, S; Piotrowska, H; Antosik, P; Bukowska, D; Nowicki, M; Brüssow, K P; Zabel, M

    2014-01-01

    Granulosa cells (GCs) play an important role during follicle growth and development in preovulatory stage. Moreover, the proteins such as connexins are responsible for formation of protein channel between follicular-cumulus cells and oocyte. This study was aimed to investigate the role of connexin expression in porcine GCs in relation to their cellular distribution and real-time cell proliferation. In the present study, porcine GCs were isolated from the follicles of puberal gilts and then cultured in a real-time cellular analyzer (RTCA) system for 168 h. The expression levels of connexins (Cxs) Cx36, Cx37, Cx40 and Cx43 mRNA were measured by RQ-PCR analysis, and differences in the expression and distribution of Cx30, Cx31, Cx37, Cx43 and Cx45 proteins were analyzed by confocal microscopic visualization. We found higher level of Cx36, Cx37, and Cx43 mRNA expression in GCs at recovery (at 0 h of in vitro culture, IVC) compared to all analyzed time periods of IVC (24, 48, 72, 96, 120, 144 and 168 h; Pproteins were higher before (0 h) compared to after 168 h of IVC. The expression of Cx30 and Cx43, however, did not vary between the groups. In all, the proteins were distributed throughout the cell membrane rather than in the cytoplasm both before and after IVC. After 24 h of IVC, we observed a significant increase in the proliferation of GCs (log phase). We found differences in the proliferation index between 72-96 and 96- 140 h within the same population of GCs. In conclusion, the decrease in the expression of Cx mRNAs and proteins following IVC could be associated with a breakdown in gap-junction connections (GJCs), and leads to the decreased of their activity, which may be a reason of non-functional existence of connexon in follicular granulosa cells. These data indicated that the differentiation and proliferation of GCs and lutein cells are regulated by distinct mechanisms in pigs.

  12. Targeting Cx43 and N-cadherin, which are abnormally upregulated in venous leg ulcers, influences migration, adhesion and activation of Rho GTPases.

    Directory of Open Access Journals (Sweden)

    Ariadna Mendoza-Naranjo

    Full Text Available BACKGROUND: Venous leg ulcers can be very hard to heal and represent a significant medical need with no effective therapeutic treatment currently available. PRINCIPAL FINDINGS: In wound edge biopsies from human venous leg ulcers we found a striking upregulation of dermal N-cadherin, Zonula Occludens-1 and the gap junction protein Connexin43 (Cx43 compared to intact skin, and in stark contrast to the down-regulation of Cx43 expression seen in acute, healing wounds. We targeted the expression of these proteins in 3T3 fibroblasts to evaluate their role in venous leg ulcers healing. Knockdown of Cx43 and N-cadherin, but not Zonula Occludens-1, accelerated cell migration in a scratch wound-healing assay. Reducing Cx43 increased Golgi reorientation, whilst decreasing cell adhesion and proliferation. Furthermore, Connexin43 and N-cadherin knockdown led to profound effects on fibroblast cytoskeletal dynamics after scratch-wounding. The cells exhibited longer lamelipodial protrusions lacking the F-actin belt seen at the leading edge in wounded control cells. This phenotype was accompanied by augmented activation of Rac-1 and RhoA GTPases, as revealed by Förster Resonance Energy Transfer and pull down experiments. CONCLUSIONS: Cx43 and N-cadherin are potential therapeutic targets in the promotion of healing of venous leg ulcers, by acting at least in part through distinct contributions of cell adhesion, migration, proliferation and cytoskeletal dynamics.

  13. Presence of Cx43 in extracellular vesicles reduces the cardiotoxicity of the anti-tumour therapeutic approach with doxorubicin

    Directory of Open Access Journals (Sweden)

    Tania Martins-Marques

    2016-09-01

    Full Text Available Extracellular vesicles (EVs are major conveyors of biological information, mediating local and systemic cell-to-cell communication under physiological and pathological conditions. These endogenous vesicles have been recognized as prominent drug delivery vehicles of several therapeutic cargoes, including doxorubicin (dox, presenting major advantages over the classical approaches. Although dox is one of the most effective anti-tumour agents in the clinical practice, its use is very often hindered by its consequent dramatic cardiotoxicity. Despite significant advances witnessed in the past few years, more comprehensive studies, supporting the therapeutic efficacy of EVs, with decreased side effects, are still scarce. The main objective of this study was to evaluate the role of the gap junction protein connexin43 (Cx43 in mediating the release of EV content into tumour cells. Moreover, we investigated whether Cx43 improves the efficiency of dox-based anti-tumour treatment, with a concomitant decrease of cardiotoxicity. In the present report, we demonstrate that the presence of Cx43 in EVs increases the release of luciferin from EVs into tumour cells in vitro and in vivo. In addition, using cell-based approaches and a subcutaneous mouse tumour model, we show that the anti-tumour effect of dox incorporated into EVs is similar to the administration of the free drug, regardless the presence of Cx43. Strikingly, we demonstrate that the presence of Cx43 in dox-loaded EVs reduces the cardiotoxicity of the drug. Altogether, these results bring new insights into the concrete potential of EVs as therapeutic vehicles and open new avenues toward the development of strategies that help to reduce unwanted side effects.

  14. Connexin43 is dispensable for phagocytosis.

    Science.gov (United States)

    Glass, Aaron M; Wolf, Benjamin J; Schneider, Karin M; Princiotta, Michael F; Taffet, Steven M

    2013-05-01

    Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

  15. Diabetes Increases Cryoinjury Size with Associated Effects on Cx43 Gap Junction Function and Phosphorylation in the Mouse Heart

    Directory of Open Access Journals (Sweden)

    Joseph A. Palatinus

    2016-01-01

    Full Text Available Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43 in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.

  16. Association of connexin43 with a receptor protein tyrosine phosphatase

    NARCIS (Netherlands)

    Giepmans, Ben N G; Feiken, Elles; Gebbink, Martijn F B G; Moolenaar, Wouter H

    2003-01-01

    Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap juncti

  17. Changes of transmural heterogeneity of Cx43 expression in acute myocardial ischemia reperfusion

    Institute of Scientific and Technical Information of China (English)

    Jiang-Hua Zhong; Shi-Juan Lu; Xiao-Pan Chen; Qi Zuo; Zheng-Wang Liu; Ding-Jun Sun; Miao Wu

    2016-01-01

    Objective:To observe the change of transmural heterogeneity of Cx43 protein among three myocardial layers and explore physiological mechanisms of malignant ventricular arrhythmia (MVA) in myocardial ischemia reperfusion (MIR).Methods: Twenty rabbits were randomly divided into MIR group (n=10) which rabbits with MIR were made in and sham group (n=10). 90% monophasic action potential repolarization duration (APD90), transmural dispersion of repolarization (TDR) and Cx43 protein (Cx43-pro) and mRNA (Cx43-Cq) expression among three myocardial layers were measured in both groups.Results:Compared with APD90 and TDR among three myocardial layers in the sham group, those in the MIR group were significant increased (P<0.01), which showed that transmural dispersion of repolarization increased significantly in MIR. Compared to those in the sham group, all three myocardial Cx43-pro significantly decreased (P<0.05) and△Cx43-pro obviously increased (P<0.05) in the MIR group which indicated transmural expression heterogeneity of Cx43 protein enlarged in MIR. 3. Cx43-Cq in all myocardium were significantly reduced (P<0.05) and△Cx43-Cq was significantly increased (P<0.05) in the MIR group compared to those in the sham group, which proved that transmural heterogeneity of Cx43 mRNA raised in MIR.Conclusions:Transmural heterogeneity of Cx43 expression among three myocardial layers in MIR significantly increased, which may enlarge dispersion of repolarization and prone to MVA.

  18. The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein

    NARCIS (Netherlands)

    Giepmans, B N; Moolenaar, W H

    1998-01-01

    Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated

  19. Connexin 43 impacts on mitochondrial potassium uptake

    Directory of Open Access Journals (Sweden)

    Kerstin eBoengler

    2013-06-01

    Full Text Available In cardiomyocytes, connexin 43 (Cx43 forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria. Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake.Subsarcolemmal mitochondria were isolated from rat left ventricular (LV myocardium and loaded with the potassium-sensitive dye PBFI. Intramitochondrial potassium was replaced by TEA (tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 µM Gap19, a peptide that specifically inhibits Cx43-dependent hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100±11.6 % in control mitochondria to 65.5±10.7 % (n=6, p<0.05. In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (Cx43Cre-ER(T/fl + 4-OHT. The mitochondria of the Cx43Cre-ER(T/fl + 4-OHT mice contained 3±1% Cx43 (n=6 of that in control mitochondria (100±11%, n=8, p<0.05. The ablation of Cx43 (n=5 reduced the velocity of the potassium influx from 100±11.2 % in control mitochondria (n=9 to 66.6±5.5 % (p<0.05.Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  20. Role of Cx43 gene in the process of myocardialization of proximal outflow tract septum in the mouse heart%Cx43基因对近端流出道隔心肌化过程的调控机制

    Institute of Scientific and Technical Information of China (English)

    赵晓晴; 黄国英; 谢利剑; 彭涛; 陈萍; 周国民

    2006-01-01

    目的:探讨connexin43(Cx43)基因在小鼠近端流出道隔心肌化的作用及其机制.方法:选用胚胎(ED)11.5 d至出生后1 d的Cx43基因剔除(KO)纯合型(Cx43-/-)、杂合型(Cx43+/-)及野生型(Cx43+/+)C57/BL6小鼠作为研究对象;采用PCR方法鉴定基因型;HE染色观察心脏结构,免疫组化法测定横纹肌肌动蛋白α-SCA、凋亡相关分子active caspase-3及神经嵴细胞的标志物AP-2的表达.结果:①Cx43-/-小鼠大多出生后24 h内即死亡.大体解剖见明显的右室流出道圆锥部异常膨隆,右心房扩张;组织切片HE染色示右室流出道壁大量异常小梁状组织增生突起,形成多个囊状结构,右室流出道腔明显狭窄,右室腔扩张.Cx43+/-和Cx43+/+心脏无明显异常.②Cx43-/-小鼠近端流出道隔中央区域α-SCA的表达明显滞后.③Active caspase-3组化显示Cx43+/+凋亡细胞主要出现在近端流出道隔,ED12.5至ED15.5均可见到;Cx43+/-凋亡减少,Cx43-/-则仅见很少凋亡细胞.④Cx43-/-流出道AP-2的表达增多,且表达位置异常.结论:Cx43 KO小鼠以右室流出道异常增生引起的梗阻性畸形为主要特征,该病变过程可能与胚胎期近端流出道隔心肌化迟滞有关,其近端流出道隔细胞凋亡减少和神经嵴细胞表达异常很可能参与了流出道心肌化异常的形成,表明Cx43在近端流出道隔心肌化过程中具有重要作用.

  1. Connexin Hemichannels in Astrocytes

    DEFF Research Database (Denmark)

    Nielsen, Brian Skriver; Hansen, Daniel Bloch; Ransom, Bruce R.

    2017-01-01

    are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate......Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli...

  2. Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts.

    Science.gov (United States)

    Zhang, Yan; Kanter, Evelyn M; Laing, James G; Aprhys, Colette; Johns, David C; Kardami, Elissavet; Yamada, Kathryn A

    2008-09-01

    Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

  3. Targeting connexin 43 protects against the progression of experimental chronic kidney disease in mice.

    Science.gov (United States)

    Abed, Ahmed; Toubas, Julie; Kavvadas, Panagiotis; Authier, Florence; Cathelin, Dominique; Alfieri, Carlo; Boffa, Jean-Jacques; Dussaule, Jean-Claude; Chatziantoniou, Christos; Chadjichristos, Christos E

    2014-10-01

    Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43+/- mice. The renal cortex of 5-month-old RenTgCx43+/- mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43+/- mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43+/- or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.

  4. Gap junction protein connexin-43 interacts directly with microtubules

    NARCIS (Netherlands)

    Giepmans, B N; Verlaan, I; Hengeveld, T; Janssen, H; Calafat, J; Falk, M M; Moolenaar, W H

    2001-01-01

    Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated

  5. Hindered Diffusion through an Aqueous Pore Describes Invariant Dye Selectivity of Cx43 Junctions☆

    OpenAIRE

    Heyman, Nathanael S.; Burt, Janis M.

    2008-01-01

    The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junct...

  6. Cx43 Mediates Resistance against MPP+-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway

    Directory of Open Access Journals (Sweden)

    In-Su Kim

    2016-11-01

    Full Text Available Neuronal apoptosis in the substantia nigra par compacta (SNpc appears to play an essential role in the pathogenesis of Parkinson’s disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+, to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC mediated by gap junctions (GJs formed by transmembrane proteins called connexins (Cx. Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP+-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP+-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP+-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway.

  7. The effects of FuFangTengLiGen preparation regulating the expressions of Cx43 and E-cad of transplantation tumor of PG cell%复方藤梨根制剂对PG荷瘤裸鼠Cx43基因表达水平及E-钙粘附素影响的研究

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To study the anticancer effect of Chinese compound recipe FuFangTengLiGen (FFTLG) against transplantation tumor of PG cell and preliminarily explore the connection with connexin43 (Cx43) and epithelial cadherin (E-cad).Methods: Model rats of transplantation tumor of human large cell cancer PG were established. The low, medium and high dose groups of FFTLG, the blank control group were set up. After 21 days medication of FFTLG through gastrogavage, the antitumor effect of each group was compared. The gene expressions of Cx43 and E-cad in each group were quantitatively detected and compared by the application of immunohistochemistry technique. Results: FFTLG could significantly inhibit the growth of transplantation tumor. FFTLG could obviously up-regulate the expressions of Cx43 and E-cad, which in the low/medium and high dose groups were separately higher than that in the blank group. Conclusion: FFTLG has definite antitumor effect against human large cell cancer and can obviously up-regulate the expressions of Cx43 and E-cad, which may be correlated with its effect of anti-tumor.

  8. Effects of different types of Helicobacter pylori on expressions of Cx32 and Cx43 in GES-1 cells%不同类型幽门螺杆菌对GES-1细胞Cx32、Cx43表达的影响

    Institute of Scientific and Technical Information of China (English)

    徐灿霞; 陈玉林; 邹惠芳; 陈雄

    2011-01-01

    目的 观察不同类型幽门螺杆菌(Helicobacter pylori,H.pylori)对人胃黏膜上皮细胞系GES-1细胞间隙连接蛋白(Connexin,Cx)32和43表达的影响,探讨与Cx32、Cx43表达异常有关的H.pylori菌株类型.方法 将临床培养分离的不同H.pylori菌株类型包括东亚型CagA+ H.pylori、西方型CagA+H.pylori及CagA-H.pylori与GES-1细胞共培养24 h及48 h,对照组不加H.pylori培养24 h及48 h.采用间接免疫荧光方法(IIF)及计算机图像分析技术检测GES-1细胞Cx32、Cx43表达.结果 对照组24 h和48 h及加H.pylori各组24 h GES-1细胞Cx32、Cx43表达阳性率均为100%,东亚型CagA+H.pylori组48 h Cx32、Cx43表达阳性率均低于对照组、CagA-H.pylori组和西方型CagA+H.Pylori组(P<0.05);对照组24 h和48 h Cx32、Cx43绿色荧光位于细胞膜,西方型CagA+H.pylori组和东亚型CagA+H,pylori组24 h和48 h Cx32绿色荧光大部分位于细胞膜,少部分位于细胞浆,Cx43绿色荧光大部分位于细胞浆,少部分位于细胞膜;东亚型CagA+H.pylori组和西方型Cag+H,pylori组24 h及48 hCx32、Cx43表达强度低于对照组和CagA-H.pylori组(P<0.05),且东亚型CagA+H.pylori组较西方型CagA+H.pylori组减弱更明显(P<0.05).结论 H.pylori下调GES-1细胞Cx32、Cx43表达,以CagA+H.pylori菌株特别是东亚型CagA+ H.pylori菌株作用更明显.%Objective To study the effects of different types of H. pylori on the expressions of Connexin(Cx) 32 and Cx43 in human gastric epithelial cell line GES-1 cells, and investigate the types of H. pylorirelated to the expressions of Cx32 and Cx43. Method Different types of clinical H.pylori strains were isolated and cultured, including the EasternAsian type CagA + H. pylori, the Western type CagA + H.pylori, and the CngA- H.pylori. These H.pylori strains were cocultured with GES-1 cells for 24 hours and 48 hours, respectively. The control groups were cultured without any H. pylori for 24 hours and 48 hours. The indirect immunofluorescence

  9. A role for connexin43 in macrophage phagocytosis and host survival after bacterial peritoneal infection.

    Science.gov (United States)

    Anand, Rahul J; Dai, Shipan; Gribar, Steven C; Richardson, Ward; Kohler, Jeff W; Hoffman, Rosemary A; Branca, Maria F; Li, Jun; Shi, Xiao-Hua; Sodhi, Chhinder P; Hackam, David J

    2008-12-15

    The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.

  10. THE EFFECT OF TRANSFECTED CX43 GENE ON THE GJIC AND PROLIFERATION OF GLIOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    浦佩玉; 夏之柏; 黄强; 王春艳; 王广秀

    2002-01-01

    Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofectamine. The expression of Cx43 was identified by Northern blot analyses, in situ hybridization and immunohistochemistry. MTT assay and average number of AgNORs (Argyrophlic nuclear organizer regions) were used to determine the cell proliferation. TUNEL method was used for detection of cell apoptosis, and scrape loading and dye tranfer method for examination of GJIC. Results: The Cx43 expression was greatly upregulated when Cx43 gene was transfected into TJ905 glioma cells. The cell proliferation was inhibited while the cell apoptosis was not increased and GJIC was significantly restored in the glioma cells tranfected with Cx43 gene. Conclusion: Cx43 gene has an inhibitory effect on the glioma cell proliferation, but no effect on induction of cell apoptosis. The restoration of GJIC may be the major mechanism involved in its effect. Cx43 gene can be the candidate for gene therapy of gliomas.

  11. The study of the expression of the Cx43 in the oral squamous cell carcinoma and Tca8113%间隙连接蛋白43在口腔颌面部鳞癌组织及舌癌细胞株(Tca8113)中表达的研究

    Institute of Scientific and Technical Information of China (English)

    宋娟; 张汉东

    2012-01-01

    目的:探讨间隙连接蛋白43(Cx43)在口腔鳞癌组织(oscc)和舌鳞癌细胞株(Tca8113)中的表达及其生物学行为的关系:细胞分化诱导剂全反式维甲酸(ATRA)对人舌鳞癌细胞株(Tca8113)中Cx43表达的影响.方法:应用免疫组织、细胞化学、免疫荧光及结合免疫印迹技术,观察OSCC组织中以及ATRA对Tca8113细胞株进行培养及分化诱导后Cx43在蛋白水平的变化.结果:Cx43在正常口腔鳞状上皮主要表达于相邻细胞间相互接触的细胞膜上,而在OSCC组织中,Cx43细胞膜表达与组织的分化程度、转移之间有显著性差异(Cx43:x2=7.42、12.43,P<0.05、P<0.01).免疫细胞化学检测可见舌癌细胞中Cx43异常定位于细胞浆中和/或细胞膜不与邻近细胞接触的游离缘上.Tca8113经ATRA诱导后,Cx43蛋白表达增加.结论:Cx43表达的改变与OSCC的发生和发展有关.ATRA可以通过上调Cx43表达,实现对肿瘤生长的抑制%Objective: To investigate the expression of connexin 43 and its relationship with the biological behavior in oral squamous cell carcinoma (OSCC) and Tca8113;to investigate the effects of connexin 43 in tongue carcinoma cell line (Tca8113) by all-trans-retinoic acid (ATRA). Method: Immunohistochemical and immunofluorescence was used to detect the expressions of Cx43 in oral squamous carcinoma. Western blot was adopted to examine the expressions of Cx43 in tongue carcinoma cell line (Tca8113) by all-trans-retinoic acid (ATRA). Result: In normal oral squamous epithelium the expression of Cx43 was typically shown in the membrane at intercellular contact,but in OSCC they were aberrantly expressed in cytoplasm.The significant difference was observed between low Cx43 expression and differentiation,metastasis (Cx43:X2= 7.42,12.43,P <0.05,P <0.01).In Tca8113 cell line Cx43 was detected mainly either intracytoplas-mically or in plasma membrane free from contact with other cells by immunocytochemistry and immunofluorescence

  12. Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate

    NARCIS (Netherlands)

    van Zeijl, Leonie; Ponsioen, Bas; Giepmans, Ben N G; Ariaens, Aafke; Postma, Friso R; Várnai, Péter; Balla, Tamas; Divecha, Nullin; Jalink, Kees; Moolenaar, Wouter H

    2007-01-01

    Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosp

  13. No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning.

    Science.gov (United States)

    Totzeck, Andreas; Boengler, Kerstin; van de Sand, Anita; Konietzka, Ina; Gres, Petra; Garcia-Dorado, David; Heusch, Gerd; Schulz, Rainer

    2008-11-01

    Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1alpha, PP2Aalpha, and PP2Balpha and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Göttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 +/- 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 +/- 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2Aalpha and PP1alpha, but not PP2Balpha, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2Aalpha but not with PP1alpha. Although the total PP2Aalpha immunoreactivity (confocal laser scan) was increased to 154 +/- 24% and 194 +/- 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2Aalpha coimmunoprecipitated with Cx43 remained unchanged. Only PP2Aalpha coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2Aalpha is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.

  14. Prenatal nicotine exposure enhances Cx43 and Panx1 unopposed channel activity in brain cells of adult offspring mice fed a high-fat/cholesterol diet

    Directory of Open Access Journals (Sweden)

    Juan Andrés Orellana

    2014-12-01

    Full Text Available Nicotine, the most important neuroteratogen of tobacco smoke, can reproduce brain and cognitive disturbances per se when administered prenatally. However, it is still unknown if paracrine signaling among brain cells participates in prenatal nicotine-induced brain impairment of adult offspring. Paracrine signaling is partly mediated by unopposed channels formed by connexins (hemichannels and pannexins serving as aqueous pores permeable to ions and small signaling molecules, allowing exchange between the intra- and extracellular milieus. Our aim was to address whether prenatal nicotine exposure changes the activity of those channels in adult mice offspring under control conditions or subjected to a second challenge during young ages: high-fat/cholesterol (HFC diet. To induce prenatal exposure to nicotine, osmotic minipumps were implanted in CF1 pregnant mice at gestational day 5 to deliver nicotine bitartrate or saline (control solutions. After weaning, offspring of nicotine-treated or untreated pregnant mice were fed ad libitum with chow or HFC diets for 8 weeks. The functional state of Cx43 and Panx1 unopposed channels was evaluated by dye uptake experiments in hippocampal slices from 11-week-old mice. We found that prenatal nicotine increased the opening of Cx43 hemichannels in astrocytes, and Panx1 channels in microglia and neurons only if offspring mice were fed with HFC diet. Blockade of iNOS, COX2 and EP1, P2X7 and NMDA receptors, showed differential inhibition of prenatal nicotine-induced channel opening in glial cells and neurons. Importantly, inhibition of the above mentioned enzymes and receptors, or blockade of Cx43 and Panx1 unopposed channels greatly reduced ATP and glutamate release from hippocampal slices of prenatally nicotine-exposed offspring. We propose that unregulated gliotransmitter release through Cx43 and Panx1 unopposed channels may participate in brain alterations observed in offspring of mothers exposed to tobacco smoke

  15. Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

    Science.gov (United States)

    Longchamp, Alban; Allagnat, Florent; Alonso, Florian; Kuppler, Christopher; Dubuis, Céline; Ozaki, Charles-Keith; Mitchell, James R.; Berceli, Scott; Corpataux, Jean-Marc

    2015-01-01

    Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment. PMID:26398895

  16. The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress.

    Science.gov (United States)

    Hutnik, Cindy M L; Pocrnich, Cady E; Liu, Hong; Laird, Dale W; Shao, Qing

    2008-02-01

    To determine the role of connexin43 (Cx43) and gap junctional intercellular communication (GJIC) in the response of the human retinal pigment epithelial cell line ARPE-19 to oxidative stress. ARPE-19 cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH), and cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays, and Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of ARPE-19 cells with shRNA vectors targeting Cx43 or vectors encoding Cx43, Cx26, and a disease-linked dominant negative Cx43 mutant (G21R) were used, and the effect on cell viability was assessed. t-BOOH-induced ARPE-19 cell death was correlated with reductions in GJIC and in the total level of Cx43 protein expression. Overexpression of Cx26 and Cx43 increased the viability of oxidant-treated ARPE-19 cells. Conversely, shRNA knockdown of Cx43, expression of a disease-linked dominant negative Cx43 mutant, and blocking GJIC with 18beta-glycyrrhetinic acid and flufenamic acid all increased t-BOOH-induced ARPE-19 cell death. Cx43-mediated protection of ARPE-19 cells from oxidative stress-induced death is dependent on functional Cx43 channels.

  17. Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication

    NARCIS (Netherlands)

    Giepmans, B N; Hengeveld, T; Postma, F R; Moolenaar, W H

    2001-01-01

    Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junct

  18. Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

    Science.gov (United States)

    Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

    2013-01-01

    The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

  19. Expression of Connexin43 in Rat Epithelial Cells and Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy . And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EAgroup, n=10). EA ( 0.5-1.5 V, 4-16 Hz , 30 min) was applied to"Zusanli"acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected.The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91 % and 29.53 % respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

  20. Connexin26 Mutations Causing Palmoplantar Keratoderma and Deafness Interact with Connexin43, Modifying Gap Junction and Hemichannel Properties.

    Science.gov (United States)

    Shuja, Zunaira; Li, Leping; Gupta, Shashank; Meşe, Gülistan; White, Thomas W

    2016-01-01

    Mutations in GJB2 (connexin [Cx]26) cause either deafness or deafness associated with skin diseases. That different disorders can be caused by distinct mutations within the same gene suggests that unique channel activities are influenced by each class of mutation. We have examined the functional characteristics of two human mutations, Cx26-H73R and Cx26-S183F, causing palmoplantar keratoderma (PPK) and deafness. Both failed to form gap junction channels or hemichannels when expressed alone. Coexpression of the mutants with wild-type Cx43 showed a transdominant inhibition of Cx43 gap junction channels, without reductions in Cx43 protein synthesis. In addition, the presence of mutant Cx26 shifted Cx43 channel gating and kinetics toward a more Cx26-like behavior. Coimmunoprecipitation showed Cx43 being pulled down more efficiently with mutant Cx26 than wild-type, confirming the enhanced formation of heteromeric connexons. Finally, the formation of heteromeric connexons resulted in significantly increased Cx43 hemichannel activity in the presence of Cx26 mutants. These findings suggest a common mechanism whereby Cx26 mutations causing PPK and deafness transdominantly influence multiple functions of wild-type Cx43. They also implicate a role for aberrant hemichannel activity in the pathogenesis of PPK and further highlight an emerging role for Cx43 in genetic skin diseases.

  1. Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.

    Science.gov (United States)

    Basu, Rahul; Bose, Abhishek; Thomas, Deepthi; Das Sarma, Jayasri

    2017-09-08

    Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-β-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo, was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

    Science.gov (United States)

    Le, Hoa T; Sin, Wun Chey; Lozinsky, Shannon; Bechberger, John; Vega, José Luis; Guo, Xu Qiu; Sáez, Juan C; Naus, Christian C

    2014-01-17

    Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

  3. Gap junction protein connexin43 exacerbates lung vascular permeability.

    Directory of Open Access Journals (Sweden)

    James J O'Donnell

    Full Text Available Increased vascular permeability causes pulmonary edema that impairs arterial oxygenation and thus contributes to morbidity and mortality associated with Acute Respiratory Distress Syndrome and sepsis. Although components of intercellular adhesive and tight junctions are critical for maintaining the endothelial barrier, there has been limited study of the roles of gap junctions and their component proteins (connexins. Since connexins can modulate inflammatory signaling in other systems, we hypothesized that connexins may also regulate pulmonary endothelial permeability. The relationships between connexins and the permeability response to inflammatory stimuli were studied in cultured human pulmonary endothelial cells. Prolonged treatment with thrombin, lipopolysaccharide, or pathological cyclic stretch increased levels of mRNA and protein for the major connexin, connexin43 (Cx43. Thrombin and lipopolysaccharide both increased intercellular communication assayed by transfer of microinjected Lucifer yellow. Although thrombin decreased transendothelial resistance in these cells, the response was attenuated by pretreatment with the connexin inhibitor carbenoxolone. Additionally, the decreases of transendothelial resistance produced by either thrombin or lipopolysaccharide were attenuated by reducing Cx43 expression by siRNA knockdown. Both carbenoxolone and Cx43 knockdown also abrogated thrombin-induced phosphorylation of myosin light chain. Taken together, these data suggest that increased lung vascular permeability induced by inflammatory conditions may be amplified via increased expression of Cx43 and intercellular communication among pulmonary endothelial cells.

  4. Prenatal nicotine exposure enhances Cx43 and Panx1 unopposed channel activity in brain cells of adult offspring mice fed a high-fat/cholesterol diet.

    Science.gov (United States)

    Orellana, Juan A; Busso, Dolores; Ramírez, Gigliola; Campos, Marlys; Rigotti, Attilio; Eugenín, Jaime; von Bernhardi, Rommy

    2014-01-01

    Nicotine, the most important neuroteratogen of tobacco smoke, can reproduce brain and cognitive disturbances per se when administered prenatally. However, it is still unknown if paracrine signaling among brain cells participates in prenatal nicotine-induced brain impairment of adult offspring. Paracrine signaling is partly mediated by unopposed channels formed by connexins hemichannels (HCs) and pannexins serving as aqueous pores permeable to ions and small signaling molecules, allowing exchange between the intra- and extracellular milieus. Our aim was to address whether prenatal nicotine exposure changes the activity of those channels in adult mice offspring under control conditions or subjected to a second challenge during young ages: high-fat/cholesterol (HFC) diet. To induce prenatal exposure to nicotine, osmotic minipumps were implanted in CF1 pregnant mice at gestational day 5 to deliver nicotine bitartrate or saline (control) solutions. After weaning, offspring of nicotine-treated or untreated pregnant mice were fed ad libitum with chow or HFC diets for 8 weeks. The functional state of connexin 43 (Cx43) and pannexin 1 (Panx1) unopposed channels was evaluated by dye uptake experiments in hippocampal slices from 11-week-old mice. We found that prenatal nicotine increased the opening of Cx43 HCs in astrocytes, and Panx1 channels in microglia and neurons only if offspring mice were fed with HFC diet. Blockade of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2) and prostaglandin E receptor 1 (EP1), ionotropic ATP receptor type 7 (P2X7) and NMDA receptors, showed differential inhibition of prenatal nicotine-induced channel opening in glial cells and neurons. Importantly, inhibition of the above mentioned enzymes and receptors, or blockade of Cx43 and Panx1 unopposed channels greatly reduced adenosine triphosphate (ATP) and glutamate release from hippocampal slices of prenatally nicotine-exposed offspring. We propose that unregulated gliotransmitter

  5. Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

    Indian Academy of Sciences (India)

    Anshul I Arora; Kaid Johar; Devarshi U Gajjar; Darshini A Ganatra; Forum B Kayastha; Anuradha K Pal; Alpesh R Patel; Rajkumar S; Abhay R Vasavada

    2012-12-01

    Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (=52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (=11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s -test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.

  6. Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish.

    Science.gov (United States)

    Misu, Akihiro; Yamanaka, Hiroaki; Aramaki, Toshihiro; Kondo, Shigeru; Skerrett, I Martha; Iovine, M Kathryn; Watanabe, Masakatsu

    2016-06-10

    Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stp(tl28d)). Vertebrae of stp(tl28d) (/+) fish look normal in larvae (∼30 days) but are distinctly shorter (59-81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43(stp) or Cx43(sof) was reduced compared with Cx43-WT. On the other hand, only Cx43(stp) induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.

  7. Depression of Intraocular Pressure Following Inactivation of Connexin43 in the Nonpigmented Epithelium of the Ciliary Body

    Science.gov (United States)

    Calera, Mónica R.; Wang, Zhao; Sanchez-Olea, Roberto; Paul, David L.; Civan, Mortimer M.; Goodenough, Daniel A.

    2010-01-01

    Purpose Conditional inactivation of connexin43 (Cx43) in the pigmented epithelium of the mouse eye results in a reduction in aqueous humor production and complete loss of the vitreous chamber. It was proposed that gap junctions between pigmented and nonpigmented epithelia of the ciliary body are critical for the production of the aqueous humor. To form such junctions, Cx43 in the pigmented epithelium must interact with connexin(s) present in the adjacent cells of the nonpigmented epithelium. The importance of Cx43 expression in the nonpigmented epithelium for the establishment of gap junctions and the regulation of intraocular pressure was tested. Methods To inactivate Cx43 in the nonpigmented epithelium of the mouse eye, a mouse line was crossed with a floxed Cx43 locus (Cx43flox/flox) and a transgenic mouse line expressing cre recombinase under the control of the Pax6α promoter. General eye structure was evaluated by light microscopy, gap junctions were analyzed by electron microscopy, and intraocular pressure was directly assessed with micropipettes. Results In Pax6α-cre/Cx43flox/flox mice, Cx43 was partially inactivated in the nonpigmented epithelium of the ciliary body and iris. Animals developed dilatations between the pigmented and nonpigmented epithelia and displayed a significant reduction in intraocular pressure. However, gap junctions between the ciliary epithelial layers were decreased but not eliminated. Conclusions Cx43 expression in the nonpigmented epithelium of the ciliary body contributes to the formation of gap junctions with the cells of the pigmented epithelium. These gap junctions play a critical role in maintaining the physical integrity of the ciliary body epithelium. Although the partial loss of Cx43 from the nonpigmented epithelium was correlated with a measurable drop in intraocular pressure, possible changes in Cx43 in the aqueous outflow pathway may provide an additional contribution to the observed phenotype. PMID:19168903

  8. TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

    Science.gov (United States)

    Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

    2009-08-14

    Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

  9. Mutations of connexin43 in fetuses with congenital heart malformations

    Institute of Scientific and Technical Information of China (English)

    CHEN Ping; XIE Li-jian; HUANG Guo-ying; ZHAO Xiao-qing; CHANG Cai

    2005-01-01

    Background Gap junction channels formed by connexin43 (Cx43) protein are important in cardiac morphogenesis, and Cx43 gene is thought to be associated with congenital heart malformation (CHM). This study was undertaken to detect the mutations of Cx43 in fetuses with CHM.Methods Cx43 extron DNA was amplified by PCR from 16 fetuses with a variety of CHM. The PCR products were analyzed by SSCP and DNA sequencing. Thirty children who had no CHM were selected as controls. Results Eight homozygous mutations of Cx43 were observed in a fetus with double outlet right ventricule (DORV), five of the 8 mutations were missense mutations including Arg239Trp, Ser251Thr, Ala253Pro, Pro283Leu and Thr290Asn, and the remaining 3 were silent polymorphisms including Gly252Gly, Pro256Pro and Thr275Thr. No mutations were found in other fetuses and the control group.Conclusions Mutations of Cx43 may be associated with congenital conotruncal anomalies. PCR-SSCP is an effective method for screening the mutations of Cx43.

  10. The modulatory effects of connexin 43 on cell death/survival beyond cell coupling.

    Science.gov (United States)

    Rodríguez-Sinovas, Antonio; Cabestrero, Alberto; López, Diego; Torre, Iratxe; Morente, Miriam; Abellán, Arancha; Miró, Elisabet; Ruiz-Meana, Marisol; García-Dorado, David

    2007-01-01

    Connexins form a diverse and ubiquitous family of integral membrane proteins. Characteristically, connexins are assembled into intercellular channels that aggregate into discrete cell-cell contact areas termed gap junctions (GJ), allowing intercellular chemical communication, and are essential for propagation of electrical impulses in excitable tissues, including, prominently, myocardium, where connexin 43 (Cx43) is the most important isoform. Previous studies have shown that GJ-mediated communication has an important role in the cellular response to stress or ischemia. However, recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. Connexin hemichannels, located at the plasma membrane, may be important in paracrine signaling that could influence intracellular calcium and cell survival by releasing intracellular mediators as ATP, NAD(+), or glutamate. In addition, recent studies have shown the presence of connexins in cell structures other than the plasma membrane, including the cell nucleus, where it has been suggested that Cx43 influences cell growth and differentiation. In addition, translocation of Cx43 to mitochondria appears to be important for certain forms of cardioprotection. These findings open a new field of research of previously unsuspected roles of Cx43 intracellular signaling.

  11. Hindered Diffusion through an Aqueous Pore Describes Invariant Dye Selectivity of Cx43 Junctions☆

    Science.gov (United States)

    Heyman, Nathanael S.; Burt, Janis M.

    2008-01-01

    Abstract The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA ∼ Alexa 350 > Lucifer yellow > Alexa 488 ≫ Alexa 594 (Stokes radii of 4.3 Å, 4.4 Å, 4.9 Å, 5.8 Å, and 7.4 Å, respectively) and the conductance sequence of KCl > TEACl ∼ Kglutamate are well described by hindered diffusion through an aqueous pore with radius ∼10 Å and length 160 Å. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable. PMID:17921206

  12. Distinct permeation profiles of the connexin 30 and 43 hemichannels

    DEFF Research Database (Denmark)

    Hansen, Daniel Bloch; Braunstein, Thomas Hartig; Nielsen, Morten Schak;

    2014-01-01

    , lactate, and glucose, did not permeate the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was observed in oocytes expressing Cx30. Exposure to divalent cation free solutions induced a robust membrane conductance in Cx30-expressing oocytes but none in Cx43-expressing oocytes......Connexin 43 (Cx43) hemichannels may form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules and presumably a range of smaller physiologically relevant molecules. However......, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of biological molecules smaller than ethidium, such as glutamate...

  13. Expression of connexin36 in the adult and developing rat brain.

    Science.gov (United States)

    Belluardo, N; Mudò, G; Trovato-Salinaro, A; Le Gurun, S; Charollais, A; Serre-Beinier, V; Amato, G; Haefliger, J A; Meda, P; Condorelli, D F

    2000-05-19

    The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.

  14. Effects of Angiotensin Ⅱ on Expression of the Gap Junction Channel Protein Connexin 43 in Neonatal Rat Ventricular Myocytes

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Wei Wu

    2007-01-01

    To study the effects of angiotensin Ⅱ,as a mediator of cardiac hypertrophy,on expression of connexin 43 (Cx43) in cultured neonatal rat ventricular myocytes and correlation of expression of Cx43 and cardiomyocyte hypertrophy.Methods Cardiomyocytes were isolated from newborn SD rats.Angiotensin Ⅱ was added into the media to induce myocyte hypertrophy.Cultures were exposed to 10 ~6 mol/L angiotensin Ⅱ for 72 h,Cx43 expression was characterized by RT-PCR and Immunofluorescence methods.Results Immunofluorescence analysis revealed decreased Cx43 immunoreactivity in cells treated for 72 h with angiotensin Ⅱ.RT-PCR analysis demonstrated there was an obvious decrease of Cx43 mRNA level in cells exposed to angiotensin Ⅱ for 72 h.The changes of expression of connexin 43 were related to its entrance into S phase of the cell cycle.Cultured neonatal rat cardiomyocytes were exposed for 72 h to increase concentrations of angiotensin Ⅱ ( 1.0 × 10-9 ~ 1.0 × 10-6mol/L),resulting in significantly decreased Cx43 expression.Conclusions Angiotensin Ⅱ leads to a concentration-dependent decrease in Cx43 protein in cultured neonatal rat ventricular myocytes by decreasing Cx43 mRNA synthesis.Signal transduction pathways activated by angiotensin Ⅱ under pathophysiologic conditions of cardiac hypertrophy could initiate remodeling of gap junctions.

  15. BRMS1 and Cx43 expression in fine needle aspiration thyroid cancer tissue and their correlation with tumor malignancy

    Institute of Scientific and Technical Information of China (English)

    Jian-Guo Sheng; Bin Wang; Zong-Ping Diao; Kun-Kun Cao; Sai Zhang; Zheng-Guo Pu

    2016-01-01

    Objective:To study the BRMS1 and Cx43 expression in fine needle aspiration thyroid cancer tissue and their correlation with tumor malignancy.Methods:Patients undergoing thyroid fine needle aspiration biopsy in our hospital from April 2012 to October 2015 were selected for study, 60 patients with thyroid cancer and 60 patients with benign thyroid tumor were screened after pathological diagnosis, biopsy tissue was collected to determine the expression of BRMS1 and Cx43, and serum specimens were collected to determin Gal-3, CEACAM1, MMP2 and MMP9 content.Results: mRNA levels and positive expression rate of BRMS1 andCx43in thyroid cancer tissue were significantly lower than those in benign thyroid tumor tissue; mRNA levels ofBRMS1andCx43in thyroid cancer tissue with different pathological types and tumor diameters were not different, mRNA level ofCx43in thyroid cancer tissue with TNM III-IV stage was significantly lower than that in thyroid cancer tissue with TNM I-II stage, mRNA levels ofBRMS1 in thyroid cancer tissue with different TNM stages were not different, and mRNA levels ofBRMS1andCx43in thyroid carcinoma tissue with lymph node metastasis were significantly lower than those in thyroid carcinoma tissue without lymph node metastasis; serum Gal-3, CEACAM1, MMP2 and MMP9 levels in patients with positive BRMS1 and Cx43 expression in thyroid cancer tissue were significantly lower than those in patients with negative BRMS1 and Cx43 expression in thyroid cancer tissue.Conclusions:Lower expression of BRMS1 and Cx43 in fine needle aspiration thyroid cancer tissue is associated with the distant metastasis and malignant degree of tumor, and lower expression of Cx43 is also associated with the growth of tumor and cancer cell proliferation.

  16. Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo.

    Science.gov (United States)

    Schulz, Rainer; Gres, Petra; Skyschally, Andreas; Duschin, Alexej; Belosjorow, Sergej; Konietzka, Ina; Heusch, Gerd

    2003-07-01

    During myocardial ischemia, connexin 43 (Cx43) is dephosphorylated in vitro, and the subsequent opening of gap junctions formed by two opposing Cx43 hexamers was suggested to propagate ischemia/reperfusion injury. Reduction of infarct size (IS) by ischemic preconditioning (IP) involves activation of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK), both of which can phosphorylate Cx43. We now studied in anesthetized pigs whether IP impacts on Cx43 phosphorylation by measuring the density of non-phosphorylated and total Cx43 (confocal laser) during normoperfusion and 90-min ischemia in non-preconditioned and preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 and the activity of p38MAPK were assessed. IP by 10 min ischemia and 15 min reperfusion reduced IS. Non-phosphorylated Cx43 remained unchanged during ischemia in preconditioned hearts, while it increased from 35+/-3 to 75+/-8 AU (P<0.05) in non-preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 during ischemia increased only in preconditioned hearts. While the ischemia-induced increase in p38MAPKalpha activity was comparable in preconditioned and non-preconditioned hearts, p38MAPKbeta activity was increased only in preconditioned hearts. Blockade of p38MAPK by SB203580 attenuated the IS-reduction and the increased p38MAPK-Cx43 co-localization by IP. We conclude that IP increases co-localization of protein kinases with Cx43 and preserves phosphorylation of Cx43 during ischemia.

  17. Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes.

    Science.gov (United States)

    Martins-Marques, Tania; Catarino, Steve; Zuzarte, Monica; Marques, Carla; Matafome, Paulo; Pereira, Paulo; Girão, Henrique

    2015-04-15

    GJIC (gap junction intercellular communication) between cardiomyocytes is essential for synchronous heart contraction and relies on Cx (connexin)-containing channels. Increased breakdown of Cx43 has been often associated with various cardiac diseases. However, the mechanisms whereby Cx43 is degraded in ischaemic heart remain unknown. The results obtained in the present study, using both HL-1 cells and organotypic heart cultures, show that simulated ischaemia induces degradation of Cx43 that can be prevented by chemical or genetic inhibitors of autophagy. Additionally, ischaemia-induced degradation of Cx43 results in GJIC impairment in HL-1 cells, which can be restored by autophagy inhibition. In cardiomyocytes, ubiquitin signals Cx43 for autophagic degradation, through the recruitment of the ubiquitin-binding proteins Eps15 (epidermal growth factor receptor substrate 15) and p62, that assist in Cx43 internalization and targeting to autophagic vesicles, via LC3 (light chain 3). Moreover, we establish that degradation of Cx43 in ischaemia or I/R (ischaemia/reperfusion) relies upon different molecular players. Indeed, degradation of Cx43 during early periods of ischaemia depends on AMPK (AMP-activated protein kinase), whereas in late periods of ischaemia and I/R Beclin 1 is required. In the Langendorff-perfused heart, Cx43 is dephosphorylated in ischaemia and degraded during I/R, where Cx43 degradation correlates with autophagy activation. In summary, the results of the present study provide new evidence regarding the molecular mechanisms whereby Cx43 is degraded in ischaemia, which may contribute to the development of new strategies that aim to preserve GJIC and cardiac function in ischaemic heart.

  18. Atorvastatin prevents connexin43 remodeling in hypertrophied left ventricular myocardium of spontaneously hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-juan; YAO Lei; CHEN Tu-gang; YU Min; WANG Li-hong; CHEN Jun-zhu

    2007-01-01

    Background Connexin43 (Cx43) is the predominant gap junction protein in heart and is involved in the control of cell-to-cell communication to modulate the contractility and the electrical coupling of cardiac myocytes. Left ventricular(LV) hypertrophy is accompanied by changes of Cx43 expression. Recent studies have demonstrated that statins reduced cardiac hypertrophy. However, it is unknown whether statins can affect Cx43 expression in hypertrophied left ventricular myocardium. This study was designed to assess the effects of atorvastatin on LV hypertrophy and Cx43 expression in spontaneously hypertensive rats (SHR).Methods Nine-week old SHRs were randomly divided into two groups. Some received atorvastatin at 30 mg/kg by oral gavage once daily for 8 weeks (SHR-A); others received vehicle. Age-matched Wistar-Kyoto rats (WKY) received atorvastatin or vehicle for 8 weeks were used as controls. At the end of the experiment, we investigated LV hypertrophy and the expression of Cx43 in LV myocardium in four groups. Cx43 expression was investigated by the methods of Western blotting, immunohistochemistry, and transmission electron microscope. LV hypertrophy was accessed by pathological analysis and plasma brain natriuretic peptide (BNP) level.Results LV hypertrophy was prominent in untreated SHR. In SHR, LV myocardium Cx43 level was upregulated, and the distribution of Cx43 was displaced from their usual locations to other sites at various distances away from the intercalated disks. After atorvastatin treatment, myocardium Cx43 level was reduced in SHR-A, and the distribution of Cx43 gap junction became much regular and confined to intercalated disk. Statins also prevented LV hypertrophy in SHR.Conclusions These results provide novel in vivo evidence for the key role of Cx43 gap junctions in LV hypertrophy and the possible mechanism in anti-hypertrophic effect of statins. Atorvastatin treatment may have beneficial effects on LV hypertrophy in spontaneously hypertensive

  19. Cardiac Connexin 43 and Ischemic Cardioprotection

    Institute of Scientific and Technical Information of China (English)

    Jin-yi LI; Guo-qiang ZHONG; Yan HE; Yun LING

    2009-01-01

    The connexin 43 (Cx43) proteins, which is the predominant protein that can form gap junctions and non-junctional hemichannels in ventricular myocardium, are central to the cardioprotection afforded by ischemic precondi-tioning (IP) and maybe ischemic postconditioning (PC) too. Recent studies showed that recruitment of Cx43 to the mitochondria in IP might play a role in the production of reactive oxygen species (ROS) that mediates IP. The localiza-tion of Cx43 at mitochondria appears to be important for the achieved cardioprotection and opens a new door for us to reveal the exact mechanisms of ischemia/reperfusion (I/R) injury and cardioprotection, and it might be new targets of pharmacological modulator to achieved cardioprotection.

  20. Role of connexin 43 in cardiovascular diseases.

    Science.gov (United States)

    Michela, Pecoraro; Velia, Verrilli; Aldo, Pinto; Ada, Popolo

    2015-12-05

    Gap junctions (GJs) channels provide the basis for intercellular communication in the cardiovascular system for maintenance of the normal cardiac rhythm, regulation of vascular tone and endothelial function as well as metabolic interchange between the cells. They allow the transfer of small molecules and may enable slow calcium wave spreading, transfer of "death" or of "survival" signals. In the cardiomyocytes the most abundant isoform is Connexin 43 (Cx43). Alterations in Cx43 expression and distribution were observed in myocardium disease; i.e. in hypertrophic cardiomyopathy, heart failure and ischemia. Recent reports suggest the presence of Cx43 in the mitochondria as well, at least in the inner mitochondrial membrane, where it plays a central role in ischemic preconditioning. In this review, the current knowledge on the relationship between the remodeling of cardiac gap junctions and cardiac diseases are summarized. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. 戊四氮点燃癫痫大鼠海马Cx32和Cx43表达及甘珀酸对其表达的影响%The expression of Cx32 and Cx43 in the hippocampus of PTZ-kindled epileptic rats

    Institute of Scientific and Technical Information of China (English)

    兰莉; 兰怡; 段丽; 曹荣

    2013-01-01

    目的 探讨点燃癫痫大鼠神经元、星形胶质细胞及其缝隙蛋白的变化,甘珀酸(CBX)对缝隙连接的影响.方法 30只SD雄性大鼠随机分为3组:对照组(NS组)、点燃组(K组)、点燃后干预组(KCBX组),每组10只大鼠.NS组每日腹腔注射生理盐水.K组和KCBX组大鼠腹腔注射戊四氮(PTZ)[35mg/(kg·d)]至点燃,再分别腹腔注射生理盐水、CBX(10mg/kg)干预3d.观察3组大鼠惊厥行为变化,并观察Cx32、Cx43在海马内的表达变化.结果 ①PTZ点燃癫痫大鼠出现自发性抽搐,KCBX组自发性抽搐次数明显减少(P<0.05).②点燃癫痫大鼠海马Cx32、Cx43的表达增加;CBX干预后,Cx32、Cx43的表达减少(P<0.05).结论 CBX抑制点燃大鼠Cx32、Cx43的表达和癫痫活动,提示缝隙连接在癫癎的发生发展过程中具有重要的作用.%Objective To investigate the changes of neurons, astrocytes and their connexin in PTZ-kindled rats as well as the effect of carbenoxolone (CBX) on gap junction. Methods Totally 30 male SD rats were randomly divided into normal saline (NS) group, kindled (K) group and KCBX group, with 10 rats in each. NS group rats were intraperitoneally injected (i.p.) with saline daily. Rats in KCBX group and K group received PTZ [i.p. 35 mg/kg · d)] to establish epilepsy model of kindled rats. After kindling, KCBX group rats were injected with CBX (i.p. 10 mg/kg) and K group rats NS for three days. Seizure changes were analyzed for all the groups. Immunohistochemistry method was used to observe the expressions of Cx32 and Cx43 in the hippocampus of the rats. Results Spontaneous seizures appeared in PTZ-kindled epileptic rats. Accumulative times of seizures were significantly fewer in KCBX group rats than in K group rats (P<0. 05). The expressions of Cx32 and Cx43 increased significantly in the hippocampus of K group rats, but decreased in KCBX group rats (P<0. 05). Conclusion Gap junction blocker CBX inhibits not only the expressions of Cx32 and Cx43

  2. Connexin 43 Upregulation in Mouse Lungs during Ovalbumin-Induced Asthma.

    Directory of Open Access Journals (Sweden)

    Yin Yao

    Full Text Available Connexin (Cx-based gap junction channels play important roles in the inflammatory response. Cx43 is involved in the pathogenesis of some lung diseases such as acute lung injury. However, the Cx43 expression in asthma is unclear. In the present study, we used a murine model of ovalbumin (OVA-induced allergic airway disease to examine the levels of Cx43 and analyze the relationship between Cx43 and airway inflammation in allergic airway disease.Asthma was induced in mice via sensitization and challenge with OVA. Cx43 mRNA and protein expression levels were investigated via QT-PCR, western blot, and immunohistochemistry 0 h, 8 h, 1 d, 2 d and 4 d after the first challenge. The relationship between Cx43 protein levels and inflammatory cell infiltration, cytokine levels was analyzed.The OVA-induced mice exhibited typical pathological features of asthma, including airway hyper-responsiveness; strong inflammatory cell infiltration surrounding the bronchia and vessels; many inflammatory cells in the bronchoalveolar lavage fluid (BALF; higher IL-4, IL-5 and IL-13 levels; and high OVA specific IgE levels. Low Cx43 expression was detected in the lungs of control (PBS mice. A dramatic increase in the Cx43 mRNA and protein levels was found in the asthmatic mice. Cx43 mRNA and protein expression levels increased in a time-dependent manner in asthma mice, and Cx43 was mostly localized in the alveolar and bronchial epithelial layers. Moreover, lung Cx43 protein levels showed a significant positive correlation with inflammatory cell infiltration in the airway and IL-4 and IL-5 levels in the BALF at different time points after challenge. Interestingly, the increase in Cx43 mRNA and protein levels occurred prior to the appearance of the inflammatory cell infiltration.Our data suggest that there is a strong upregulation of Cx43 mRNA and protein levels in the lungs in asthma. Cx43 levels also exhibited a positive correlation with allergic airway inflammation. Cx43

  3. Differential Connexin Function Enhances Self-Renewal in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Masahiro Hitomi

    2015-05-01

    Full Text Available The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43, but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.

  4. [Impacts of early metoprolol intervention on connexin 43 and phosphorylated connexin 43 expression in rabbits with experimental myocardial infarction].

    Science.gov (United States)

    Zhou, M; Lu, Q; Jiang, J Q; Chen, Z N; Gong, Z G; Li, Z G; Fu, W W; Ding, S F

    2017-04-24

    Objective: To investigate the early intervention effects of metoprolol on connexin 43(Cx43) and phosphorylated Cx43 (p-Cx43) expression in rabbits with post myocardial infarction. Methods: A total of 24 adult male New Zealand white rabbits were divided into sham group (n=6), early treatment group(n=6), routine treatment group(n=6), and myocardial infarction group(n=6) with a randomized block design blocked by weight. Myocardial infarction was induced by left anterior descending coronary artery (LAD) ligation. Rabbits in sham group received similar surgical procedure without LAD ligation. Metoprolol (12.5 mg/kg dissolved in 2 ml distilled water) was applied to rabbits in early treatment group and routine treatment group per gavage immediately after recovery from anesthesia and at 24 hours after myocardial infarction, respectively, then treated daily for 40 days. Rabbits in sham group and myocardial infarction group received 2 ml distilled water per gavage daily for 40 days. Plasma lactate dehydrogenase (LDH) and creatine kinase (CK) level were detected by automatic biochemistry analyzer after 6 hours in all rabbits. Ventricular fibrillation threshold (VFT) was measured in vivo by bipolar pacing electrodes at 40 days. Cx43 and p-Cx43 distribution in ventricular tissue was detected by immunofluorescence analyses. Cx43 and p-Cx43 protein level in ventricular tissue was determined by Western blot. Results: (1) Plasma LDH ((851.7±85.9)U/L vs. (332.3±39.6)U/L, Pmyocardial infarction group than in sham group (both Pmyocardial infarction group than that in sham group ((470.0±91.0) beats per minute vs. (683.3±60.9) beats per minute, Pmyocardial infarction group (both Pmyocardial infarction group. The expression of p-Cx43 in myocardial infarction group was less than in sham group, which was significantly upregulated in in early treatment group and routine treatment group when compared with myocardial infarction group, and expression of p-Cx43 was significantly higher in

  5. Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.

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    Oloris, Silvia Catarina Salgado; Mesnil, Marc; Reis, Viviane Neri de Souza; Sakai, Mônica; Matsuzaki, Patrícia; Fonseca, Evelise de Souza Monteiro; da Silva, Tereza Cristina; Avanzo, José Luís; Sinhorini, Idércio Luiz; Guerra, José Luiz; Costa-Pinto, Frederico Azevedo; Maiorka, Paulo Cesar; Dagli, Maria Lúcia Zaidan

    2007-03-06

    Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.

  6. Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry

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    Guoqiang Zhong

    2017-05-01

    Full Text Available In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2 is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge of the crossing molecules.

  7. Transforming growth factor-β stimulates human ovarian cancer cell migration by up-regulating connexin43 expression via Smad2/3 signaling.

    Science.gov (United States)

    Qiu, Xin; Cheng, Jung-Chien; Zhao, Jianfang; Chang, Hsun-Ming; Leung, Peter C K

    2015-10-01

    Reduced connexin43 (Cx43) expression is frequently detected in different types of human cancer. Cx43 has been shown to regulate cancer cell migration in a cell-type dependent manner. In both primary and recurrent human ovarian cancer, overexpression of TGF-β ligand and its receptors have been detected. TGF-β can regulate Cx43 expression in other cell types and stimulate human ovarian cancer cell migration. However, whether Cx43 can be regulated by TGF-β and is involved in TGF-β-stimulated cell migration in human ovarian cancer cells remain unknown. In this study, we demonstrate that TGF-β up-regulates Cx43 in two human ovarian cancer cell lines, SKOV3 and OVCAR4. The stimulatory effect of TGF-β on Cx43 expression is blocked by inhibition of TGF-β receptor. Treatment with TGF-β activates Smad2 and Smad3 signaling pathways in both ovarian cancer cell lines. In addition, siRNA-mediated knockdown of Smad2 or Smd3 abolishes TGF-β-induced up-regulation of Cx43 expression. Moreover, knockdown of Cx43 attenuates TGF-β-stimulated cell migration. This study demonstrates an important role for Cx43 in mediating the effects of TGF-β on human ovarian cancer cell migration.

  8. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

    Science.gov (United States)

    Rodriguez-Sinovas, Antonio; Boengler, Kerstin; Cabestrero, Alberto; Gres, Petra; Morente, Miriam; Ruiz-Meana, Marisol; Konietzka, Ina; Miró, Elisabet; Totzeck, Andreas; Heusch, Gerd; Schulz, Rainer; Garcia-Dorado, David

    2006-07-07

    We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

  9. Upregulation of Connexin 43 Expression Via C-Jun N-Terminal Kinase Signaling in Prion Disease.

    Science.gov (United States)

    Lee, Geon-Hwi; Jang, Byungki; Choi, Hong-Seok; Kim, Hee-Jun; Park, Jeong-Ho; Jeon, Yong-Chul; Carp, Richard I; Kim, Yong-Sun; Choi, Eun-Kyoung

    2016-01-01

    Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

  10. Ischemia Alters the Expression of Connexins in the Aged Human Brain

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    Taizen Nakase

    2009-01-01

    Full Text Available Although the function of astrocytic gap junctions under ischemia is still under debate, increased expression of connexin 43 (Cx43 has been observed in ischemic brain lesions, suggesting that astrocytic gap junctions could provide neuronal protection against ischemic insult. Moreover, different connexin subtypes may play different roles in pathological conditions. We used immunohistochemical analysis to investigate alterations in the expression of connexin subtypes in human stroke brains. Seven samples, sectioned after brain embolic stroke, were used for the analysis. Data, evaluated semiquantitatively by computer-assisted densitometry, was compared between the intact hemisphere and ischemic lesions. The results showed that the coexpression of Cx32 and Cx45 with neuronal markers was significantly increased in ischemic lesions. Cx43 expression was significantly increased in the colocalization with astrocytes and relatively increased in the colocalization with neuronal marker in ischemic lesions. Therefore, Cx32, Cx43, and Cx45 may respond differently to ischemic insult in terms of neuroprotection.

  11. Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation.

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    Su, Guo-Ying; Wang, Jing; Xu, Zhen-Xing; Qiao, Xiao-Jun; Zhong, Jing-Quan; Zhang, Yun

    2015-10-01

    The present study investigated the effects of rotigaptide (ZP123) on the expression, distribution and phosphorylation of connexin43 (Cx43) in myocardial cell membranes in cardioversion of ventricular fibrillation (VF). A model of prolonged VF (8, 12 and 30 min) was established in mongrel dogs (n=8/group), following treatment with ZP123 or normal saline (NS control). A sham control was included. Cardiopulmonary resuscitation was begun at the start of VF followed by defibrillation. Animals received a maximum of three defibrillations of increasing energy (70, 100 and 150 J biphasic shock) as required. The average defibrillation energy, defibrillation success rate, return of spontaneous circulation and survival rate were recorded. Cx43 and phosphorylated (p-)Cx43 expression in cardiomyocyte membranes was detected by western blot and immunofluorescence analyses. Compared with the NS-treated control groups, the success defibrillation rate in the 8-min and 12-min ZP123 groups was significantly higher (P<0.05), while the average defibrillation energy was significantly lower (P<0.05). Cx43 expression in the VF groups was significantly lower than that in the sham control group (P<0.05). Cx43 expression was higher in the 12-min and 30-min ZP123 groups than that in the NS control group (P<0.05), while p-Cx43 expression decreased, although the levels were significantly higher than those in the control groups (P<0.05). Cx43 expression was positively correlated with the defibrillation success rate (r=0.91; P<0.01) and negatively with the mean defibrillation energy (r=-0.854; P<0.01), while p-Cx43 expression was positively correlated with the success rate of the previous three defibrillations (r=0.926; P<0.01).In conclusion, ZP123 reduced Cx43 remodeling through regulating the expression, distribution and phosphorylation of Cx43, thereby reducing the defibrillation energy required for successful cardioversion.

  12. Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria.

    Science.gov (United States)

    Boengler, Kerstin; Stahlhofen, Sabine; van de Sand, Anita; Gres, Petra; Ruiz-Meana, Marisol; Garcia-Dorado, David; Heusch, Gerd; Schulz, Rainer

    2009-03-01

    Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase alpha (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 microg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 microg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 +/- 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 +/- 4%, n = 7), whereas the immunoreactivities of ATP synthase alpha (49.1 +/- 6.4%, n = 7) and MnSOD (79.9 +/- 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of

  13. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.

    Science.gov (United States)

    Boengler, Kerstin; Dodoni, Giuliano; Rodriguez-Sinovas, Antonio; Cabestrero, Alberto; Ruiz-Meana, Marisol; Gres, Petra; Konietzka, Ina; Lopez-Iglesias, Carmen; Garcia-Dorado, David; Di Lisa, Fabio; Heusch, Gerd; Schulz, Rainer

    2005-08-01

    Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline. These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

  14. Dynamic alterations of connexin43, matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 during ventricular fibrillation in canine.

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    Wang, Jing; Li, Jing-sha; Liu, Hong-zhen; Yi, Shao-lei; Su, Guo-ying; Zhang, Yun; Zhong, Jing-quan

    2014-06-01

    The aim of this study is to investigate the dynamic alterations of cardiac connexin 43 (Cx43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF) duration. In this study, thirty-two dogs were randomly divided into sham control group, 8-min VF group, 12-min VF group, and 30-min VF group. Cx43 and phosphorylated Cx43 (p-Cx43) in tissues were detected by western blot and immunofluorescence analysis. MMP-2 and TIMP-2 were detected by western blot and immunohistochemistry analysis. The results showed that Cx43 levels in three VF groups were significantly decreased compared with sham control group. p-Cx43 levels in 12-min and 30-min VF groups were significantly reduced compared with sham control group. The ratio of p-Cx43/Cx43 was also decreased in VF groups. Compared with sham controls, no significant difference was observed between the sham control group and 8-min VF group in MMP-2 level, but MMP-2 level increased in 12-min and 30-min VF groups. The ratios of MMP-2/TIMP-2 were higher in VF groups, and were correlated with the duration of VF. A remarkable correlation was observed between the ratio of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate prognosis of VF.

  15. Omega-3 fatty acids and atorvastatin affect connexin 43 expression in the aorta of hereditary hypertriglyceridemic rats.

    Science.gov (United States)

    Dlugosová, Katarína; Weismann, Peter; Bernátová, Iveta; Sotníková, Ruzena; Slezák, Ján; Okruhlicová, Ludmila

    2009-12-01

    Statins and omega-3 polyunsaturated fatty acids (n-3 PUFA) reduce cardiovascular disease incidence during hypertriglyceridemia (HTG). To elucidate possible cardioprotective mechanisms, we focused on gap junction protein connexin 43 (Cx43). Its expression is disturbed during atherogenesis, but little information is available on its expression during HTG. Experiments were performed on adult male hereditary HTG (hHTG) rats treated with n-3 PUFA (30 mg/day) and atorvastatin (0.5 mg/100 g body weight per day) for 2 months. Cx43 expression and distribution in the aorta were investigated by using Western blotting and immunolabeling, followed by quantitative analysis. Transmission electronmicroscopy was used to study ultrastructure of endothelial contact sites. In contrast to age-matched Wistar, Cx43 expression in aorta of hHTG rats was significantly higher (p < 0.05), and prominent Cx43 immunospots were seen in tunica media and less in endothelium of hHTG rats. Changes in Cx43 expression were accompanied by local qualitative subcellular alterations of interendothelial connections. Treatment of hHTG rats with n-3 PUFA and atorvastatin markedly lowered Cx43 expression in aorta and modified connexin distribution in endothelium and media (p < 0.05 vs. untreated hHTG). The protective effect of treatment of HTG was observed on the structural integrity of the endothelium and was readily visible at the molecular level. Results indicate the involvement of altered Cx43 expression in vascular pathophysiology during HTG and during HTG treatment.

  16. Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice.

    Science.gov (United States)

    Morioka, Norimitsu; Zhang, Fang Fang; Nakamura, Yoki; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-10-01

    Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca(2+) channel subunit α2δ1 blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been

  17. RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

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    Xie, Xi [Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Department of Pharmaceutical Engineering, Ocean College, Hainan University, Haikou 570228 (China); Chen, Cheng; Huang, Kaipeng; Wang, Shaogui; Hao, Jie; Huang, Junying [Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Huang, Heqing, E-mail: huangheq@mail.sysu.edu.cn [Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China)

    2014-10-01

    RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs. - Highlights: • RhoA/ROCK signaling induces Cx43 degradation in GMCs. • F-actin and ZO-1 have functions in the regulation of Cx43 by RhoA/ROCK signaling. • We reveal the relationship between RhoA/ROCK and Cx43 in the activation of NF-κB.

  18. The Expressions of Survivin, PTEN and Cx43 Gene in Skin Basal Cell Carcinoma%Survivin、PTEN和Cx43基因在皮肤基底细胞癌中的表达

    Institute of Scientific and Technical Information of China (English)

    杜金锋; 郭瑞珍

    2009-01-01

    背景与目的:探讨Survivin、PTEN和Cx43基因与皮肤基底细胞癌(basal cell carcinoma,BCC)的相关性.材料与方法:采用免疫组织化学S-P法分别检测35例皮肤BCC、10例正常皮肤组织中Survivin、PTEN和Cx43蛋白的表达;运用核酸分子原位杂交技术检测Cx43 mRNA的表达.应用图像分析系统采集分析图像,分别检测BCC和正常皮肤组织中各项检测指标的表达水平(阳性面积)和表达强度(光密度).结果:Survivin蛋白在正常皮肤中呈阴性表达,在BCC中呈弱阳性表达,其差异有统计学意义(P<0.05):PTEN蛋白在正常皮肤组织中呈强阳性表达,在BCC中呈阳性或弱阳性表达,其差异具有统计学意义(P<0.05).而Cx43蛋白和Cx43 mRNA在正常皮肤组织中的表达均呈强阳性,而在BCC组织中均呈阳性或弱阳性,差异均具有统计学意义(P均<0.01).相关性分析显示,BCC组织中,PTEN蛋白的表达与Cx43蛋白呈正相关(r=0.519,P<0.01),Cx43蛋白的表达与Cx43 mRNA也呈正相关(r=0.732,P<0.01). 结论:PTEN蛋白、Cx43蛋白和Cx43 mRNA在BCC中的低表达;Survivin蛋白在BCC中的高表达,可能在BCC发生发展的过程中发挥重要作用.

  19. Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Yi XIA; Kai-zheng GONG; Ming XU; You-yi ZHANG; Ji-hong GUO; Yao SONG; Ping ZHANG

    2009-01-01

    Aim:β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties.Connexin 43 (Cx43),the predominant gap-junction protein in the heart,has an indispensable role in modulating cardiac electric activities by affecting gap-junction function.The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function.Methods:The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay.The GJIC function was evaluated by scrape loading/dye transfer assay.Results:Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level,but not total Cx43.Selective β2-AR inhibitor ICI 118551,but not β-AR inhibitor CGP20712,could fully abolish the effect.Moreover,pretreatment with both protein kinase A inhibitor H89 and G,protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression.Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.Conclusion:Taken together,β2-AR stimulation increases the expression of nonphosphorylated Cx43,thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A-and G1-dependent manner.

  20. Mechanism of Mitochondrial Connexin43′s Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Shuai Hou

    2016-05-01

    Full Text Available We observed mitochondrial connexin43 (mtCx43 expression under cerebral ischemia-reperfusion (I/R injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO. Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD activity and malondialdehyde (MDA content. MtCx43, p-mtCx43, protein kinase C (PKC, and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX and diazoxide (DZX groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists.

  1. Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

    Energy Technology Data Exchange (ETDEWEB)

    Talhouk, Rabih S., E-mail: rtalhouk@aub.edu.lb [Department of Biology, Faculty of Arts and Sciences, American University of Beirut, P.O. Box 11-0236, Beirut (Lebanon); Fares, Mohamed-Bilal; Rahme, Gilbert J.; Hariri, Hanaa H.; Rayess, Tina; Dbouk, Hashem A.; Bazzoun, Dana; Al-Labban, Dania [Department of Biology, Faculty of Arts and Sciences, American University of Beirut, P.O. Box 11-0236, Beirut (Lebanon); El-Sabban, Marwan E., E-mail: me00@aub.edu.lb [Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut, P.O. Box 11-0236, Beirut (Lebanon)

    2013-12-10

    Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus. - Highlights: • Cx43 over-expressing MCF-7 and MDA-MB-231 were grown in 2D and 3D cultures. • Proliferation and growth morphology were affected in a context dependent manner. • Extravasation ability of both MCF

  2. Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells.

    Science.gov (United States)

    Uzu, Miaki; Sato, Hiromi; Shimizu, Ayaka; Shibata, Yukihiro; Ueno, Koichi; Hisaka, Akihiro

    2017-06-01

    The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  3. Downregulation of connexin43 by microRNA-130a in cardiomyocytes results in cardiac arrhythmias.

    Science.gov (United States)

    Osbourne, Appledene; Calway, Tyler; Broman, Michael; McSharry, Saoirse; Earley, Judy; Kim, Gene H

    2014-09-01

    MicroRNAs (miRNAs) are now recognized as critical regulators of diverse physiological and pathological processes; however, studies of miRNAs and arrhythmogenesis remain sparse. Connexin43 (Cx43), a major cardiac gap junction protein, has elicited great interest in its role in arrhythmias. Additionally, Cx43 was a potential target for miR-130a as predicted by several computational algorithms. This study investigates the effect of miR-130a overexpression in the adult heart and its effect on cardiac rhythm. Using a cardiac-specific inducible system, transgenic mice demonstrated both atrial and ventricular arrhythmias. We performed ventricular-programmed electrical stimulation and found that the αMHC-miR130a mice developed sustained ventricular tachycardia beginning 6weeks after overexpression. Western blot analysis demonstrated a steady decline in Cx43 after 2weeks of overexpression with over a 90% reduction in Cx43 levels by 10weeks. Immunofluorescent staining confirmed a near complete loss of Cx43 throughout the heart. To validate Cx43 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts and HL-1 cardiomyocytes, both known to endogenously express miR-130a. Using a luciferase reporter fused to the 3'UTR of Cx43, we found a 52.9% reduction in luciferase activity in 3T3 cells (parrhythmias.

  4. Connexin hemichannels in the lens

    Directory of Open Access Journals (Sweden)

    Eric C Beyer

    2014-02-01

    Full Text Available The normal function and survival of cells in the avascular lens is facilitated by intercellular communication through an extensive network of gap junctions formed predominantly by three connexins (Cx43, Cx46, and Cx50. In expression systems, these connexins can all induce hemi-channel currents, but other lens proteins (e.g., pannexin1 can also induce similar currents. Hemichannel currents have been detected in isolated lens fiber cells. These hemichannels may make significant contributions to normal lens physiology and pathophysiology. Studies of some connexin mutants linked to congenital cataracts have implicated hemichannels with aberrant voltage-dependent gating or modulation by divalent cations in disease pathogenesis. Hemichannels may also contribute to age- and disease-related cataracts.

  5. Connexin43与心血管疾病关系的研究进展%Progress on correlation between connexin 43 and cardiovascular disease

    Institute of Scientific and Technical Information of China (English)

    吴瑶

    2011-01-01

    Connexin( Cx43)作为最重要的间隙连接蛋白,是哺乳动物心脏中最主要的连接蛋白。Cx43对心脏发育、心脏电生理活动、心肌的缺血后修复起重要作用,Cx43异常是很多心血管疾病包括先天性心脏病、缺血性心脏病、心律失常、高血压的重要病因。Cx43表达受多种因素影响,包括甲基化修饰、组蛋白修饰和非编码RNA调控等,这些调控机制的异常可能是其影响疾病发生、发展的重要因素。%As the most important gap junction protein, connexin 43 ( Cx43 ) is the most important connexin in the mammalian heart. Recent studies suggest that it plays an important role in heart development, cardiac electrophysiological activity and cell repair after myocardial ischemia. Cx43 abnormity is an important cause of many cardiovascular diseases including congenital heart disease, ischemic heart disease, arrhythmias and hypertension. The expression of Cx43 is regulated by various factors, including methylation, histone modification and non-coding RNA regulation, etc. These abnormal regulations may be important for the development of diseases.

  6. Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

    Science.gov (United States)

    Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

    2001-01-01

    A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

  7. Dexamethasone-induced muscular atrophy is mediated by functional expression of connexin-based hemichannels.

    Science.gov (United States)

    Cea, Luis A; Balboa, Elisa; Puebla, Carlos; Vargas, Aníbal A; Cisterna, Bruno A; Escamilla, Rosalba; Regueira, Tomás; Sáez, Juan C

    2016-10-01

    Long-term treatment with high glucocorticoid doses induces skeletal muscle atrophy. However, the molecular mechanism of such atrophy remains unclear. We evaluated the possible involvement of connexin-based hemichannels (Cx HCs) in muscle atrophy induced by dexamethasone (DEX), a synthetic glucocorticoid, on control (Cx43(fl/fl)Cx45(fl/fl)) and Cx43/Cx45 expression-deficient (Cx43(fl/fl)Cx45(fl/fl):Myo-Cre) skeletal myofibers. Myofibers of Cx43(fl/fl)Cx45(fl/fl) mice treated with DEX (5h) expressed several proteins that form non-selective membrane channels (Cx39, Cx43, Cx45, Panx1, P2X7 receptor and TRPV2). After 5h DEX treatment in vivo, myofibers of Cx43(fl/fl)Cx45(fl/fl) mice showed Evans blue uptake, which was absent in myofibers of Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice. Similar results were obtained in vitro using ethidium as an HC permeability probe, and DEX-induced dye uptake in control myofibers was blocked by P2X7 receptor inhibitors. DEX also induced a significant increase in basal intracellular Ca(2+) signal and a reduction in resting membrane potential in Cx43(fl/fl)Cx45(fl/fl) myofibers, changes that were not elicited by myofibers deficient in Cx43/Cx45 expression. Moreover, treatment with DEX induced NFκB activation and increased mRNA levels of TNF-α in control but not in Cx43/Cx45 expression-deficient myofibers. Finally, a prolonged DEX treatment (7days) increased atrogin-1 and Murf-1 and reduced the cross sectional area of Cx43(fl/fl)Cx45(fl/fl) myofibers, but these parameters remained unaffected in Cx43(fl/fl)Cx45(fl/fl):Myo-Cre myofibers. Therefore, DEX-induced expression of Cx43 and Cx45 plays a critical role in early sarcolemma changes that lead to atrophy. Consequently, this side effect of chronic glucocorticoid treatment might be avoided by co-administration with a Cx HC blocker.

  8. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

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    Zahra Moinfar

    Full Text Available Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43 and estrogen receptors (ERs. Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2 on Cx43 expression in two glioma cell lines with variable native expression of Cx43.F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

  9. Losartan reduced connexin43 expression in left ventricular myocardium of spontaneously hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    Li-li ZHAO; Hong-juan CHEN; Jun-zhu CHEN; Min YU; Yun-lan NI; Wei-fang ZHANG

    2008-01-01

    Objective: To assess the effect of angiotensin Ⅱ type 1 (AT1) receptor antagonist losartan on myocardium connexin43 (Cx43) gap junction (GJ) expression in spontaneously hypertensive rats (SHRs) and investigate possible mechanisms. Methods: Sixteen 9-week-old male SHRs and 8 age-matched male Wistar-Kyoto (WKY) rats were included in this study. SHRs were randomly divided into two groups to receive losartan at 30mg/(kg·d) by oral gavage once daily for 8 weeks (SHR-L) or vehicle (0.9% saline) to act as controls (SHR-V); WKY rats receiving vehicle for 8 weeks served as normotensive controls. At the end of the experiment, rats were sacrificed and the hearts were removed. Expressions of Cx43 and nuclear factor-kappaB p65 (NF-κB p65) proteins in all three groups were observed and further investigations on the effect of angiotensin Ⅱ type 1 receptor antagonist losartan (30mg/(kg·d), 8 weeks) on Cx43 expression were conducted with Western blot and immunohistochemistry. NF-κB p65 protein in nuclear extracts was determined by Western blot. Results: Left ventricular (LV) hypertrophy was prominent in SHRs, Cx43 and NF-κB p65 protein expressions were obviously upregulated and Cx43 distribution was dispersed over the cell surface. Treatment with losarton reduced the over-expressions of Cx43 and NF-κB p65 in LV myocardium. The distribution of Cx43 gap junction also became much regular and confined to intercalated disk after losartan treatment. Conclusion: Cx43 level was upregulated in LV myocardium of SHR during early stage of hypertrophy. Angiotensin Ⅱ type l receptor antagonist losartan prevented Cx43 gap junction remodeling in hypertrophied left ventricles, possibly through the NF-κB pathway.

  10. Connexin 43 expression in human and mouse testes with impaired spermatogenesis

    Directory of Open Access Journals (Sweden)

    M Kotula-Balak

    2009-08-01

    Full Text Available Connexin 43 (Cx43 belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter’s syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter’s patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter’s patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.

  11. Maturational changes in connexin 43 expression in the seminiferous tubules may depend on thyroid hormone action

    Science.gov (United States)

    Marchlewska, Katarzyna; Kula, Krzysztof; Walczak-Jedrzejowska, Renata; Kula, Wojciech; Oszukowska, Elzbieta; Filipiak, Eliza; Moszura, Tomasz

    2013-01-01

    Introduction Connexin 43 (Cx43) mediates the effect of thyroid hormone on Sertoli cell maturation in vitro. We investigated the influence of triiodothyronine (T3) administration on Cx43 expression in relation to the progress in seminiferous tubule maturation. Material and methods Male rats were daily injected with 100 µg T3/kg body weight from birth until postnatal day (pnd) 5 (transient treatment – tT3) or until pnd 15 (continuous treatment – cT3) or solvent – control (C). On pnd 16 serum hormone levels, body and testes weight, seminiferous tubule morphometry, Cx43 immunostaining and germ cell degeneration were investigated. Cx43 expression was also assessed in six 50-day-old adult untreated rats. Result tT3 increased 2.6-fold serum level of T3, testes weight, and seminiferous tubule diameter, and induced maturation-like dislocation of Cx43 expression from the apical to the peripheral region of Sertoli cell cytoplasm. In addition, incidence of Cx43-positive tubules declined from 86% in C to 46% after tT3, being similar to the adult value (30% of tubules Cx43-positive). In turn, cT3 increased serum T3 level 12-fold, and decreased body weight. Seminiferous tubules became shortened and distended, Sertoli cell cytoplasm vacuolated, Cx43 expression had minimal intensity and germ cell degeneration increased. Conclusions Cx43 might intermediate a short and transient stimulatory effect of T3 on seminiferous tubule maturation that disappeared together with exposure to the toxic effect of a continuously high level of the hormone. PMID:23515877

  12. Knockdown of connexin 43 attenuates balloon injury-induced vascular restenosis through the inhibition of the proliferation and migration of vascular smooth muscle cells.

    Science.gov (United States)

    Han, Xiao-Jian; He, Dan; Xu, Liang-Jing; Chen, Min; Wang, Yi-Qi; Feng, Jiu-Geng; Wei, Min-Jun; Hong, Tao; Jiang, Li-Ping

    2015-11-01

    Coronary artery disease (CAD) or atherosclerotic heart disease is one of the most common types of cardiovascular disease. Although percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] is a mature, well-established technique used to treat atherosclerotic heart disease, its long‑term therapeutic effects are compromised by a high incidence of vascular restenosis (RS) following angioplasty. In our previous study, we found that the principal gap junction protein, connexin 43 (Cx43), in vascular smooth muscle cells (VSMCs) was involved in the development of vascular RS following angioplasty-induced balloon injury. However, the exact role action of Cx43 in vascular RS remains unclear. In the present study, we aimed to further examine whether the knockdown of Cx43 attenuates the development of vascular RS through the inhibition of the proliferation and migration of VSMCs. We found that the use of a lentiviral vector expressing shRNA targeting Cx43 (Cx43‑RNAi-LV) efficiently silenced the mRNA and protein expression of Cx43 in cultured VSMCs. In addition, MTT and Transwell assays were used to examined the proliferation and migration of the VSMCs, respectively. The results revealed that the knockdown of Cx43 by Cx43-RNAi-LV at a multiplicity of infection (MOI) of 100 significantly inhibited the proliferation and migration of the VSMCs in vitro. Notably, the knockdown of Cx43 also effectively attenuated the development of vascular RS and intimal hyperplasia following balloon injury in vivo. Taken together, our data suggest that Cx43 is involved in the development of vascular RS and intimal hyperplasia through the regulation of the proliferation and migration of VSMCs. Thus, the present study provides new insight into the pathogenesis of vascular RS, and suggests that further comfirms that Cx43 may well be a novel potential pharmacological target for preventing vascular RS following PCI.

  13. 地西他滨对白血病细胞系中连接蛋白43的作用和机制%Effect and mechanism of decitabine on expression of connexin 43 protein in leukemic cells

    Institute of Scientific and Technical Information of China (English)

    韦炜; 徐娅蓓; 孙云; 莫蓓蕾; 阎骅

    2012-01-01

    目的:探讨地西他滨对白血病细胞中连接蛋门43(Cx43)的作用和机制.方法:蛋白印迹法(Western blot)检测去甲基化药物地西他滨对NB4-MR2、U937细胞中Cx43水平表达的影响以及加入蛋白酶体抑制剂MG132对Cx43表达的影响.实时定量PCR检测地西他滨对NB4-MR2、U937细胞中Cx43 RNA水平的影响.结果:地西他滨明显下调MR2细胞中Cx43表达,对其RNA水平没有明显影响;地西他滨明显缩短Cx43蛋白的半衰期.加入蛋白酶体抑制剂MG132可明显抑制地西他滨引起的Cx43蛋白的减少.结论:地西他滨通过蛋白酶体途径减少Cx43的水平,可能会影响其治疗效果.%Objective To study the effect and mechanism of decitabine on expression of connexin 43(Cx43) protein in leukemic cells. Methods Western blot was used to assess the Cx43 protein level in leukemic NB4-MR2 and U937 ceil lines for determining the effect of decitabine on expression of Cx43 protein and the effect of adding proteasome inhibitor MG132 on the expression. Level of Cx43 RNA expression was determined by real-time PCR. Results Decitabine significantly down-regulated the expression of Cx43 protein in both leukemic cell lines, while no significant change in Cx43 RNA level was found. Moreover, decitabine significantly reduced the half-life of Cx43 protein. The addition of proteasome inhibitor MG132 could block the inhibiting effect of decitabine on Cx43 protein expression. Conclusions Decitabine down-regulated the expression of Cx43 protein in leukemic cells through proteasome pathway.

  14. Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells

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    Ralph Brehm

    2006-06-01

    Full Text Available Carcinoma in situ (CIS represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap functional proteins, connexin 43 (Cx43 represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semi quantitative reverse transcription-polymerase chain reaction (RT-PCR from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

  15. Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells1

    Science.gov (United States)

    Brehm, Ralph; Rüttinger, Christina; Fischer, Petra; Gashaw, Isabella; Winterhager, Elke; Kliesch, Sabine; Bohle, Rainer M; Steger, Klaus; Bergmann, Martin

    2006-01-01

    Abstract Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development. PMID:16820096

  16. Late cardioprotection of exercise preconditioning against exhaustive exercise-induced myocardial injury by up-regulatation of connexin 43 expression in rat hearts

    Institute of Scientific and Technical Information of China (English)

    Kai Wang; Bai-Chao Xu; Hai-Yun Duan; Hua Zhang; Fu-Song Hu

    2015-01-01

    Objective:To investigate the expression of myocardium connexin 43 (Cx43) in late exercise preconditioning (LEP) cardioprotection.Methods: Eight-week-old adult male Sprague Dawley rats were randomly assigned into four groups (n=8). Myocardial injury was judged in accordance with serum levels of cTnⅠ and NT-proBNP as well as hematoxylin basicfuchsin picric acid staining of myocardium.Cx43mRNA was detected byin situhybridization and qualified by real-time fluorescence quantitative PCR. Cx43 protein was localized by immunohistochemistry and its expression level was determined by western blotting.Results:The LEP obviously attenuated the myocardial ischemia/hypoxia injury caused by exhaustive exercise. There was no significant difference of Cx43mRNA level between the four groups. Cx43 protein level was decreased significantly in group EE (P<0.05). However, LEP produced a significant increase in Cx43 protein level (P<0.05), and the decreased Cx43 protein level in exhaustive exercise was significantly up-regulated by LEP (P<0.05).Conclusions:LEP protects rat heart against exhaustive exercise-induced myocardial injury by up-regulating the expression of myocardial Cx43.

  17. C-kit、SCF、Cx43在先天性巨结肠中的表达及作用%Expression and correlation study of C-kit, SCF and Cx43 in hirschsprung's disease

    Institute of Scientific and Technical Information of China (English)

    谢丽微; 夏丽娜; 赵志光

    2013-01-01

    Objective:To explore the action of C-kit,SCF and Cx43 in the pathogenesis of HD by examining the expression and distribution of C-kit,SCF and Cx43.Methods:The colon specimens of 68 cases of HD and 8 cases of accidental death without digestive tract malformations (control) were collected.The case group composed of a expansion group in which the ganglion cells were normal in the intestinal wall and a narrow group in which the ganglion cells were absent in the intestinal wall according to diagnosis by HE.Immunohistochemical staining was used to detect the proteins of C-Kit,SCF and Cx43,and hybridization in situ to detect the mRNA of C-Kit in all specimens.Results:Expression of C-kit,SCF and Cx43 in the expansion group was not significantly different from the control group (P > 0.05),while expression of C-kit,SCF and Cx43 in narrow group decreased significantly compared with the control group (P < 0.05).Conclusion:The development of Hirschsprung's disease is associated with the decreased expression of C-kit,SCF and Cx43.%目的:探讨酪氨酸激酶膜受体基因(C-kit)、干细胞因子(SCF)与缝隙连接蛋白(Cx43)在先天性巨结肠发病机制中的作用.方法:收集68例先天性巨结肠(HD)患儿手术切除标本的存档蜡块,根据HE染色结果,将神经节细胞正常的扩张段标本设为扩张组,无神经节细胞的狭窄段标本设为狭窄组.并收集8例无消化道畸形新生儿尸检结肠组织作为对照.用免疫组织化学EliVision法检测C-kit、SCF及Cx43蛋白的表达,原位杂交法检测 C-kit mRNA的表达.结果:与对照组比较,HD扩张组中C-kit、SCF、Cx43的表达差异无统计学意义(P>0.05),狭窄组中C-kit、SCF、Cx43的表达差异有统计学意义(P<0.05).结论:先天性巨结肠的发生与C-kit、SCF及Cx43表达减少有关.

  18. Connexin 43 is Involved in Aldosterone-Induced Podocyte Injury

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    Min Yang

    2014-11-01

    Full Text Available Background/Aims: Connexin43 (Cx43 belongs to a family of transmembrane proteins that build cell-to-cell channels in gap junctions, which play an essential role in intercellular communication, cell proliferation, differentiation and apoptosis. The aim of this study is to clarify the effect of Cx43 in aldosterone (Aldo-induced podocyte injury and explore the possible molecular mechanism behind this effect. Methods: Uninephrectomized Sprague-Dawley rats were given 1% NaCl (salt in their water and an Aldo infusion (0.75 μg/h for 28 days to induce podocyte injury. Podocytes were incubated in media containing either buffer or increasing concentrations of Aldo (10-9 - 10-7 M for variable time periods. The podocytes were then examined and the mechanism of injury investigated using TUNEL assay, ELISA, immunofluorescence staining, western blot, RNA interference, and DCFDA fluorescence. Results: Here, we report that in vivo administration of Aldo caused greater numbers of TUNEL-positive podocytes, accompanied by increased Cx43 expression, but a reduction in WT1-positive podocytes. In vitro studies indicated that Aldo induces podocyte apoptosis in a dose- and time-dependent manner and is accompanied by increases in Cx43 expression, gap junction intercellular communication (GJIC, ROS production, and the Bax/Bcl-2 ratio. Silencing of Cx43 expression attenuates the increase of ROS production and the subsequent up-regulation of the Bax/Bcl-2 ratio, which partially inhibited the podocytes apoptosis. Conclusions: Our study provides preliminary evidence that upregulation of Cx43 expression is involved in Aldo-induced podocyte injury, and that Cx43 is a potentially relevant therapeutic target for the treatment of chronic kidney diseases (CKD.

  19. Levonorgestrel Inhibits Human Endometrial Cell Proliferation through the Upregulation of Gap Junctional Intercellular Communication via the Nuclear Translocation of Ser255 Phosphorylated Cx43

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    Xiaomiao Zhao

    2015-01-01

    Full Text Available Objects. To assess whether LNG exerts antiproliferation effects on human endometrial cells through changes of GJIC function and the phosphorylated Cx43. Methods. Cell proliferation and apoptosis of human endometrial stromal cells (HESCs and glandular cells (HEGCs treated with LNG in a dose- and time-dependent manner. GJIC change and further total Cx43 and serine 368 and 255 phosphorylated Cx43 were measured. Results. 5 × 10−5 mol/L LNG revealed a time-dependent inhibition of cell proliferation and an increase of apoptosis in both HESCs and HEGCs. Furthermore, these cells demonstrated a significant GJIC enhancement upon treatment with 5 × 10−5 mol/L for 48 hours. The effects of LNG were most noticeable in HESCs rather than in HEGCs. Associated with these changes, LNG induced a relative increase in total Cx43 in a time-dependent manner but not Ser368 phosphorylated Cx43. Moreover, laser scanning confocal microscope confirmed the increased expression of total Cx43 in the cytoplasm and, interestingly, the nuclear translocation of Ser255 phosphorylated Cx43. Conclusions. LNG likely inhibits the proliferation and promotes apoptosis in HESCs and HEGCs though an increase in gap junction permeability in vitro, which is achieved through the upregulation of Cx43 expression and the translocation of serine 255 phosphorylated Cx43 from the plasma to the nuclear compartment.

  20. Specificity of gap junction communication among human mammary cells and connexin transfectants in culture.

    Science.gov (United States)

    Tomasetto, C; Neveu, M J; Daley, J; Horan, P K; Sager, R

    1993-07-01

    In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC

  1. HIV-Associated Cardiovascular Disease: Role of Connexin 43.

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    Prevedel, Lisa; Morocho, Camilla; Bennett, Michael V L; Eugenin, Eliseo A

    2017-09-01

    Chronic HIV infection due to effective antiretroviral treatment has resulted in a broad range of clinical complications, including accelerated heart disease. Individuals with HIV infection have a 1.5 to 2 times higher incidence of cardiovascular diseases than their uninfected counterparts; however, the underlying mechanisms are poorly understood. To explore the link between HIV infection and cardiovascular diseases, we used postmortem human heart tissues obtained from HIV-infected and control uninfected individuals to examine connexin 43 (Cx43) expression and distribution and HIV-associated inflammation. Here, we demonstrate that Cx43 is dysregulated in the hearts of HIV-infected individuals. In all HIV heart samples analyzed, there were areas where Cx43 was overexpressed and found along the lateral membrane of the cardiomyocyte and in the intercalated disks. Areas of HIV tissue with anomalous Cx43 expression and localization also showed calcium overload, sarcofilamental atrophy, and accumulation of collagen. All these changes were independent of viral replication, CD4 counts, inflammation, and type of antiretroviral treatment. Overall, we propose that HIV infection increases Cx43 expression in heart, resulting in tissue damage that likely contributes to the high rates of cardiovascular disease in HIV-infected individuals. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Hyperthermia Differently Affects Connexin43 Expression and Gap Junction Permeability in Skeletal Myoblasts and HeLa Cells

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    Ieva Antanavičiūtė

    2014-01-01

    Full Text Available Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK in hyperthermia-induced changes of connexin43 (Cx43 expression and permeability of Cx43 gap junctions (GJs in the rabbit skeletal myoblasts (SkMs and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC. In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  3. Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and HeLa cells.

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    Antanavičiūtė, Ieva; Mildažienė, Vida; Stankevičius, Edgaras; Herdegen, Thomas; Skeberdis, Vytenis Arvydas

    2014-01-01

    Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK) in hyperthermia-induced changes of connexin43 (Cx43) expression and permeability of Cx43 gap junctions (GJs) in the rabbit skeletal myoblasts (SkMs) and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h) enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC). In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  4. Connexins and the kidney

    DEFF Research Database (Denmark)

    Hanner, Fiona; Sørensen, Charlotte Mehlin; Holstein-Rathlou, Niels-Henrik

    2010-01-01

    Connexins (Cxs) are widely-expressed proteins that form gap junctions in most organs, including the kidney. In the renal vasculature, Cx37, Cx40, Cx43, and Cx45 are expressed, with predominant expression of Cx40 in the endothelial cells and Cx45 in the vascular smooth muscle cells. In the tubules......, the major function of Cxs in the kidney appears to be intercellular communication, although they may also form hemichannels that allow cellular secretion of large signaling molecules. Renal Cxs facilitate vascular conduction, juxtaglomerular apparatus calcium signaling, and tubular purinergic signaling....... Accordingly, current evidence points to roles for these Cxs in several important regulatory mechanisms in the kidney, including the renin angiotensin system, tubuloglomerular feedback, and salt and water reabsorption. At the systemic level, renal Cxs may help regulate blood pressure and may be involved...

  5. Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study.

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    Maria Sobaniec-Lotowska

    2005-02-01

    Full Text Available Gap junctional intercellular communication (GJIC is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs, which consist of proteins called connexins (Cxs. GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.

  6. Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study.

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    Kanczuga-Koda, Luiza; Sulkowski, Stanislaw; Koda, Mariusz; Sobaniec-Lotowska, Maria; Sulkowska, Mariola

    2004-01-01

    Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases) were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.

  7. High glucose induces dysfunction of airway epithelial barrier through down-regulation of connexin 43.

    Science.gov (United States)

    Yu, Hongmei; Yang, Juan; Zhou, Xiangdong; Xiao, Qian; Lü, Yang; Xia, Li

    2016-03-01

    The airway epithelium is a barrier to the inhaled antigens and pathogens. Connexin 43 (Cx43) has been found to play critical role in maintaining the function of airway epithelial barrier and be involved in the pathogenesis of the diabetic retinal vasculature, diabetes nephropathy and diabetes skin. Hyperglycemia has been shown to be an independent risk factor for respiratory infections. We hypothesize that the down-regulation of Cx43 induced by HG alters the expression of tight junctions (zonula occludens-1 (ZO-1) and occludin) and contributes to dysfunction of airway epithelial barrier, and Cx43 plays a critical role in the process in human airway epithelial cells (16 HBE). We show that high glucose (HG) decreased the expression of ZO-1 and occludin, disassociated interaction between Cx43 and tight junctions, and then increased airway epithelial transepithelial electrical resistance (TER) and permeability by down-regulation of Cx43 in human airway epithelial cells. These observations demonstrate an important role for Cx43 in regulating HG-induced dysfunction of airway epithelial barrier. These findings may bring new insights into the molecular pathogenesis of pulmonary infection related to diabetes mellitus and lead to novel therapeutic intervention for the dysfunction of airway epithelial barrier in chronic inflammatory airway diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

    Science.gov (United States)

    Mahoney, Vanessa M.; Mezzano, Valeria; Mirams, Gary R.; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E.

    2016-05-01

    Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.

  9. Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex.

    Science.gov (United States)

    Liu, Xiuxin; Sun, Lin; Torii, Masaaki; Rakic, Pasko

    2012-05-22

    The prospective pyramidal neurons, migrating from the proliferative ventricular zone to the overlaying cortical plate, assume multipolar morphology while passing through the transient subventricular zone. Here, we show that this morphogenetic transformation, from the bipolar to the mutipolar and then back to bipolar again, is associated with expression of connexin 43 (Cx43) and, that knockdown of Cx43 retards, whereas its overexpression enhances, this morphogenetic process. In addition, we have observed that knockdown of Cx43 reduces expression of p27, whereas overexpression of p27 rescues the effect of Cx43 knockdown in the multipolar neurons. Furthermore, functional gap junction/hemichannel domain, and the C-terminal domain of Cx43, independently enhance the expression of p27 and promote the morphological transformation and migration of the multipolar neurons in the SVZ/IZ. Collectively, these results indicate that Cx43 regulates the passage of migrating neurons through their multipolar stage via p27 signaling and that interference with this process, by either genetic and/or environmental factors, may cause cortical malformations.

  10. Common mechanisms linking connexin43 to neural progenitor cell migration and glioma invasion.

    Science.gov (United States)

    Naus, Christian C; Aftab, Qurratulain; Sin, Wun Chey

    2016-02-01

    Cell migration is critical for cell differentiation, tissue formation and organ development. Several mechanisms come to play in the process of cell migration, orchestrating changes in cell polarity, adhesion, process extension and motility. Recent findings have shown that gap junctions, and specifically connexin43 (Cx43), can play a significant role in these processes, impacting adhesion and cytoskeletal rearrangements. Thus Cx43 within a cell regulates its motility and migration via intracellular signaling. Furthermore, Cx43 in the host cells can impact the degree of cellular migration through that tissue. Similarities in these connexin-based processes account for both neural progenitor migration in the developing brain, and for glioma cell invasion in the mature brain. In both cases, Cx43 in the tissue ("soil") in which cells ("seeds") exist facilitates their migration and, for glioma cells, tissue invasion. Cx43 mediates these effects through channel- and non-channel-dependent mechanisms which have similarities in both paradigms of cell migration. This provides insight into developmental processes and pathological situations, as well as possible therapeutic approaches regarding specific functional domains of gap junction proteins.

  11. Inhibition of Connexin43 Hemichannels Impairs Spatial Short-Term Memory without Affecting Spatial Working Memory.

    Science.gov (United States)

    Walrave, Laura; Vinken, Mathieu; Albertini, Giulia; De Bundel, Dimitri; Leybaert, Luc; Smolders, Ilse J

    2016-01-01

    Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory.

  12. Inhibition of Connexin43 Hemichannels Impairs Spatial Short-Term Memory without Affecting Spatial Working Memory

    Science.gov (United States)

    Walrave, Laura; Vinken, Mathieu; Albertini, Giulia; De Bundel, Dimitri; Leybaert, Luc; Smolders, Ilse J.

    2016-01-01

    Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory. PMID:28066184

  13. Early disruption of glial communication via connexin gap junction in multiple sclerosis, Baló's disease and neuromyelitis optica.

    Science.gov (United States)

    Masaki, Katsuhisa

    2015-10-01

    Multiple sclerosis (MS), neuromyelitis optica (NMO), and Baló's disease (BD) are inflammatory demyelinating diseases of the CNS. We previously reported anti-aquaporin-4 (anti-AQP4) antibody-dependent AQP4 loss occurs in some NMO patients, while antibody-independent AQP4 astrocytopathy can occur in heterogeneous demyelinating conditions, including MS, NMO and BD. To investigate the relationship between astrocytopathy and demyelination, we focused on connexins (Cxs), which form gap junctions (GJs) between astrocytes and oligodendrocytes and maintain homeostasis in the CNS. We evaluated expression of astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 in autopsied materials from MS, NMO and BD patients. Astrocytic Cx43 and oligodendrocytic Cx32/Cx47 expressions were significantly diminished in both demyelinated and preserved myelin layers in all BD samples. In the leading edge of BD lesions, Cx43 and AQP4 loss preceded Cx32/Cx47 loss. Half of the NMO and MS samples showed preferential loss of astrocytic Cx43 expression in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte-oligodendrocyte GJs were lost. Cases with Cx43 loss were significantly associated with rapid disease progression, regardless of the disease phenotype. Pathologically, Cx43 loss was frequently accompanied by distal oligodendrogliopathy. Our findings suggest that Cx43 astrocytopathy can occur in MS, BD and NMO. Moreover, astrocytic Cx43 loss may be associated with disease aggressiveness and distal oligodendrogliopathy in demyelinating conditions. Early disruption of glial communications via GJs may cause loss of glia syncytium, thereby inducing oligodendroglial damage and myelin loss. Inhibition of Cx hemichannels and restoration of GJs may be a possible therapeutic target for demyelinating disorders. © 2015 Japanese Society of Neuropathology.

  14. Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression.

    Science.gov (United States)

    Moffitt, Julia A; Henry, Matthew K; Welliver, Kathryn C; Jepson, Amanda J; Garnett, Emily R

    2013-03-01

    Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43.

  15. Connexin 43 is not essential for the control of renin synthesis and secretion.

    Science.gov (United States)

    Gerl, Melanie; Kurt, Birgül; Kurtz, Armin; Wagner, Charlotte

    2014-05-01

    The juxtaglomerular areas of mammalian kidneys express the gap junction proteins connexin 37, 40, 43, and 45. Among these, Cx40 plays a major role for the function of juxtaglomerular renin-expressing cells, while Cx37 and Cx45 appear to be less relevant in this context. Since the role of the remaining Cx43 for the function of renin expression is not well understood, this study aimed to systematically characterize the direct role of Cx43 for renin expression and secretion. For this aim, we generated mice with endothelium and with renin cell-specific deletions of Cx43, and we characterized the regulation of renin expression and renin secretion in the kidneys of these mice on normal salt diet and during chronic challenge of the renin system by pretreatment of mice with a low-salt diet in combination with an angiotensin I-converting enzyme inhibitor. We found that renal renin mRNA abundance, plasma renin concentration, and systolic blood pressure did not differ between wild-type, Cx43(fl/fl) Ren1d(+/Cre) mice as well as Cx43(fl/fl) Tie-2(+/Cre) mice under basal conditions nor under chronic stimulation by salt depletion. The localization of renin-expressing cells was also regular in kidneys of all genotypes, and moreover, regulation of renin secretion by beta-adrenergic stimulation and renal perfusion pressure measured in isolated perfused kidneys of Cx43(fl/fl) Ren1d(+/Cre) and Cx43(fl/fl) Tie-2(+/Cre) mice was not different from control. We infer from these results that Cx43 plays if at all only a minor role for the functional control of renin-producing cells in the kidney.

  16. Involvement of connexin43 hemichannel in ATP release after γ-irradiation

    Science.gov (United States)

    Ohshima, Yasuhiro; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Kojima, Shuji

    2012-01-01

    Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that γ-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X7 receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X7 receptor-dependent ATP release after 0.5 Gy γ-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked γ-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy γ-irradiation tyrosine phosphorylation was significantly blocked by P2X7 receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X7 receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X7 receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X7 receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43. PMID:22843620

  17. Control of Vascular Smooth Muscle Cell Growth by Connexin 43

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    Chintamani eJoshi

    2012-06-01

    Full Text Available Connexin 43 (Cx43, the principal gap junction protein in vascular smooth muscle cells (VSMCs, regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC stimulator BAY 41-2272 (BAY, or the Cx inducer diallyl disulfide (DADS significantly reduced proliferation after 72 h compared to vehicle controls. Bromodeoxyuridine uptake revealed reduction (p<.001 in DNA synthesis after 6 h and flow cytometry showed reduced (40% S phase cell numbers after 16 h in DADS-treated cells compared to controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at the MAPK-sensitive serine (Ser255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and the PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared to controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays an important role in regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSMCs.

  18. Bone morphogenetic protein-2 (BMP-2 and transforming growth factor-β1 (TGF-β1 alter connexin 43 phosphorylation in MC3T3-E1 Cells

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    Rudkin George H

    2001-07-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs and transforming growth factor-βs (TGF-βs are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC in MC3T3-E1 cells. Connexin 43 (Cx43 has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC. Results Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-β1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. Conclusions BMP-2 and TGF-β1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-β1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

  19. Gap junction signalling mediated through connexin-43 is required for chick limb development.

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    Makarenkova, H; Patel, K

    1999-03-15

    During chick limb development the gap junction protein Connexin-43 (Cx43) is expressed in discrete spatially restricted domains in the apical ectodermal ridge (AER) and mesenchyme of the zone of polarising activity. Antisense oligonucleotides (ODNs) were used to investigate the role of Connexin-43 (Cx43) in the development of the chick limb bud. We have used unmodified ODNs in Pluronic F-127 gel, which is liquid at low temperature but sets at room temperature and so remains situated at the point of application. As a mild surfactant, the gel increases antisense ODN penetration and supplies ODNs to the embryo continually for 12-18 h. We have shown a strong decrease in Cx43 protein expression after application of specific antisense oligonucleotides but the abundance of a closely related protein, Connexin-32 (Cx32), was not affected. Application of antisense Cx43 ODNs at stages 8-15 HH before limb outgrowth resulted in dramatic limb phenotypes. About 40% of treated embryos exhibited defects such as truncation of the limb bud, fragmentation into two or more domains, or complete splitting of the limb bud into two or three branches. Molecular analysis of antisense treated embryos failed to detect Shh or Bmp-2 in anterior structures and suggested that extra lobes seen in nicked and split limbs were not a result of establishment of new signalling centres as found after the application of FGF to the flank. However, examination of markers for the AER showed a number of abnormalities. In severely truncated specimens we were unable to detect the expression of either Fgf-4 or Fgf-8. In both nicked and split limbs the expression of these genes was discontinuous. Down-regulation of Cx43 after the antisense application could be comparable to AER removal and results in distal truncation of the limb bud. Taken together these data suggest the existence of a feedback loop between the FGFs and signalling mediated by Cx43.

  20. Connexin 43 astrocytopathy linked to rapidly progressive multiple sclerosis and neuromyelitis optica.

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    Katsuhisa Masaki

    Full Text Available BACKGROUND: Multiple sclerosis (MS and neuromyelitis optica (NMO occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx pathology and disease aggressiveness in Asian patients with MS and NMO. METHODS/PRINCIPAL FINDINGS: Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD, six with MS, and 20 with other neurological diseases (OND. Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59, chronic active (n=58 and chronic inactive (n=23. Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, P=0.0090. Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, P=0.0296. Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients. CONCLUSIONS

  1. Effects of sevoflurance preconditioning on myocardial celluar CX43 protein during ischemia/reperfusion%七氟醚预处理对缺血再灌注心肌细胞CX43蛋白表达的影响

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    章征兵; 明腾; 杨文萍; 谢学良

    2012-01-01

    目的 观察七氟醚预处理对缺血再灌注心肌细胞缝隙连接蛋白43(CX43)表达的影响,探讨七氟醚预处理对心肌保护作用的机制.方法 选取需接受手术的先天性心脏病患儿38例,随机分为七氟醚预处理组(S组)和对照组(C组),每组19例.患儿于麻醉诱导气管插管后,S组吸入七氟醚1.0 MAC 30 min,随后洗脱10 min;C组则只吸入空氧混合气体.两组麻醉诱导及维持用药相同,均按常规行体外循环(CPB).监测两组患儿年龄、体质量、主动脉阻断时间、CPB时间、机械通气时间、重症监护对间和手术后住院天数及麻醉诱导后和主动脉开放6h后血浆CK-MB、cTnI的浓度;免疫组织化学方法显示心肌CX43的分布特征及CX43含量的变化.结果 两组患儿年龄、体质量、主动脉阻断时间、CPB时间、机械通气时间、重症监护时间和手术后住院天数、术前CK-MB、cTnI浓度及转流前CX43含量和分布无统计学差异(P>0.05);S组术后6 h CK.MB、cTnI浓度低于C组(P<0.05);转流后S组CX43阳性染色的平均灰度值低于C组,阳性染色的平均面积高于C组(P<0.05),且S组cX43蛋白主要分布在闰盘上,而C组主要分布在侧连接胞膜上.结论 七氟醚预处理能减少CPB后心肌细胞CX43蛋白的降解,稳定CX43的分布,发挥心肌保护作用.%Objective To observe the effects of sevoflurance preconditioning on myocaidial celluar conexin 43 (CX43) protein during ischemia/reperfusion and explore the mechanism of myocardial protection. Methods Thirty-eight pediatric patients with congenital heart disease who need surgery were randomly allocated into sevofiurance preconditioning group (group S) and control group (group C). After induction of anesthesia, sevoflurance were inhaled for 30 minutes and followed by 10 minutes air-oxygen mixture washout in group S, group C inhale air-oxygen mixture only. Establishing car-diopulmonary bypass (CPB) as a matter of routine in two

  2. 17β estradiol regulation of connexin 43-based gap junction and mechanosensitivity through classical estrogen receptor pathway in osteocyte-like MLO-Y4 cells.

    KAUST Repository

    Ren, Jian

    2013-04-01

    Connexin 43 (Cx43) plays an essential role in osteocyte mechanotransduction. Although estrogen involves in the adaptive responses of bone cells to mechanical loadings, its effects on osteocytic Cx43-based gap junction intercellular communication (GJIC) remain obscure. We found that 17β estradiol (E2) up-regulated Cx43, and enhanced GJIC in osteocyte-like MLO-Y4 cells in fluorescence recovery after photobleaching (FRAP) assay. Combination of E2 pre-treatment and oscillating fluid flow (OFF) further enhanced Cx43 expression and mitogen-activated protein kinase (MAPK) phosphorylation, comparing to E2 or OFF treatment alone. Both blocking of classical estrogen receptors (ERα/β) by fulvestrant and ERα knockdown by small interfering RNA inhibited E2-mediated Cx43 increase, while a GPR30-specific agonist G-1 failed to promote Cx43 expression. Our results suggest that the presence of E2 enhanced Cx43-based GJIC mainly via ERα/β pathway, and sensitized osteocytes to mechanical loading. © 2012 Elsevier Inc. All rights reserved.

  3. Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis.

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    Khan, Zahidul; Yaiw, Koon-Chu; Wilhelmi, Vanessa; Lam, Hoyin; Rahbar, Afsar; Stragliotto, Giuseppe; Söderberg-Nauclér, Cecilia

    2014-01-01

    A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

  4. A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43-Ezrin Interaction Disruptors Using IncuCyte ZOOM.

    Science.gov (United States)

    Dukic, Aleksandra R; McClymont, David W; Taskén, Kjetil

    2017-01-01

    Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin-protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.

  5. Effects of all-trans-retinoic acid on the expression and tyrosine phosphorylation of gap junction connexin 43 in HeLa cell line and its significance

    Institute of Scientific and Technical Information of China (English)

    CHEN Bi-liang; MA Xiang-dong; XIN Xiao-yan; WANG De-tang; WANG Chun-mei

    2001-01-01

    Objective: To investigate the signal transduction mechanism of gap junctional genes connexin43 in human cervical carcinogenesis. Methods: Human cervical carcinoma cell line HeLa was cultured and treated by all-trans-retinoic acid (ATRA). Flow cytometer (FCM) was employed to detect expression of Cx43 protein in HeLa cells. Fluo-3 AM loading and laser scanning confocal microscope (LSCM) were used to measure the concentrations of intracellular calcium ([Ca2+]i) in HeLa cells. Phosphorylation on tyrosine of connexin43 protein was examined by immunoblot. Results: The positive rate of Cx43 protein increased from 1.9% in untreated HeLa cells to 26.3% in RA-treated HeLa cells as shown by FCM. [Ca2+]i was 35.73 nmol/L in untreated HeLa cells which was increased to 58.16 nmol/L in ATRA-treated cells.Immunoblot showed that ATRA-treated HeLa cells had phosphorylation on tyrosine in Cx43 protein whereas untreated cells had not. Conclusions: Carcinogenesis of human cervical carcinoma is related with the abnormal expression of cx43gene and disorder of signal transduction manifested as the decrease of [Ca2+]i and post-translation phosphorylation on tyrosine of Cx43 protein. The anti-tumor effect of ATRA in HeLa cells might be due to the up-regulation of cx43 gene and its signal transduction pathway.

  6. Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo.

    Science.gov (United States)

    Basu, Rahul; Banerjee, Kaveri; Bose, Abhishek; Das Sarma, Jayasri

    2015-12-16

    Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition to gap junction

  7. Adenoviral delivery of human connexin37 induces endothelial cell death through apoptosis.

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    Seul, Kyung H; Kang, Keum Y; Lee, Kyung S; Kim, Suhn H; Beyer, Eric C

    2004-07-09

    Gap junction channels formed of connexins directly link the cytoplasm of adjacent cells and have been implicated in intercellular signaling that may regulate the functions of vascular cells. To facilitate connexin manipulation and analysis of their roles in adult endothelial cells, we developed adenoviruses containing the vascular connexins (Cx37, Cx40, and Cx43). We infected cultured human umbilical vein endothelial cells with control or connexin adenoviruses. Connexin expression was verified by immunoblotting and immunofluorescence. Infection with the Cx37 adenovirus (but not control or other connexin adenoviruses) led to a dose-dependent death of the endothelial cells that was partially antagonized by the gap junction blocker alpha-glycyrrhetinic acid and altered the intercellular transfer of Lucifer yellow and neurobiotin. Cell morphology, Annexin V and TUNEL staining, and caspase 3 assays all implicated apoptosis in the cell death. These data suggest that connexin-specific alterations of intercellular communication may modulate endothelial cell growth and death.

  8. On the role of the gap junction protein Cx43 (GJA1 in human cardiac malformations with Fallot-pathology. a study on paediatric cardiac specimen.

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    Aida Salameh

    Full Text Available INTRODUCTION: Gap junction channels are involved in growth and differentiation. Therefore, we wanted to elucidate if the main cardiac gap junction protein connexin43 (GJA1 is altered in patients with Tetralogy of Fallot or double-outlet right ventricle of Fallot-type (62 patients referred to as Fallot compared to other cardiac anomalies (21 patients referred to as non-Fallot. Patients were divided into three age groups: 0-2years, 2-12years and >12years. Myocardial tissue samples were collected during corrective surgery and analysis of cell morphology, GJA1- and N-cadherin (CDH2-distribution, as well as GJA1 protein- and mRNA-expression was carried out. Moreover, GJA1-gene analysis of 16 patients and 20 healthy subjects was performed. RESULTS: Myocardial cell length and width were significantly increased in the oldest age group compared to the younger ones. GJA1 distribution changed significantly during maturation with the ratio of polar/lateral GJA1 increasing from 2.93±0.68 to 8.52±1.41. While in 0-2years old patients ∼6% of the lateral GJA1 was co-localised with CDH2 this decreased with age. Furthermore, the changes in cell morphology and GJA1-distribution were not due to the heart defect itself but were significantly dependent on age. Total GJA1 protein expression decreased during growing-up, whereas GJA1-mRNA remained unchanged. Sequencing of the GJA1-gene revealed only few heterozygous single nucleotide polymorphisms within the Fallot and the healthy control group. CONCLUSION: During maturation significant changes in gap junction remodelling occur which might be necessary for the growing and developing heart. In our study point mutations within the Cx43-gene could not be identified as a cause of the development of TOF.

  9. Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

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    González-Sánchez, Ana; Jaraíz-Rodríguez, Myriam; Domínguez-Prieto, Marta; Herrero-González, Sandra; Medina, José M.; Tabernero, Arantxa

    2016-01-01

    Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such as phosphatase and tensin homolog (PTEN). Our results showed that the antiproliferative effect of Cx43 is reduced when Csk and PTEN are silenced in glioma cells, suggesting the involvement of both enzymes. Confocal microscopy and immunoprecipitation assays confirmed that Cx43, in addition to c-Src, binds to PTEN and Csk in glioma cells transfected with Cx43 and in astrocytes. Pull-down assays showed that region 266–283 in Cx43 is sufficient to recruit c-Src, PTEN and Csk and to inhibit the oncogenic activity of c-Src. As a result of c-Src inhibition, PTEN was increased with subsequent inactivation of Akt and reduction of proliferation of human glioblastoma stem cells. We conclude that the recruitment of Csk and PTEN to the region between residues 266 and 283 within the C-terminus of Cx43 leads to c-Src inhibition. PMID:27391443

  10. Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    XING Yifei; XIAO Yajun; ZENG FuQing; ZHAO Jun; XIAO Chuanguo; XIONG Ping; FENG Wei

    2007-01-01

    Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis.mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.

  11. Mechanism of Regulatory Effect of MicroRNA-206 on Connexin 43 in Distant Metastasis of Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Zi-Jing Lin; Jia Ming; Lu Yang; Jun-Ze Du; Ning Wang; Hao-Jun Luo

    2016-01-01

    Background: MicroRNA-206 (miR-206) and connexin 43 (Cx43) are related with the distant metastasis of breast cancer.It remains unclear whether the regulatory effect of miR-206 on Cx43 is involved in metastasis of breast cancer.Methods: Using quantitative real-time polymerase chain reaction and Western blot, the expressions of miR-206 and Cx43 were determined in breast cancer tissues, hepatic and pulmonary metastasis (PM), and cell lines (MCF-10A, MCF-7, and MDA-MB-231).MCF-7/MDA-M-231 cells were transfected with lentivirus-shRNA vectors to enhance/inhibit miR-206, and then Cx43 expression was observed.Cell counting kit-8 assay and Transwell method were used to detect their changes in proliferation, migration, and invasion activity.The mutant plasmids of Cx43-3' untranslated region (3'UTR) at position 478-484 and position 1609-1615 were constructed.Luciferase reporter assay was performed to observe the effects of miR-206 on luciferase expression of different mutant plasmids and to confirm the potential binding sites of Cx43.Results: Cx43 protein expression in hepatic and PM was significantly higher than that in the primary tumor, while no significant difference was showed in messenger RNA (mRNA) expression.MiR-206 mRNA expression in hepatic and PM was significantly lower than that in the primary tumor.Cx43 mRNA and protein levels, as well as cell proliferation, migration, and invasion capabilities, were all significantly improved in MDA-MB-231 cells after reducing miR-206 expression but decreased in MCF-7 cells after elevating miR-206 expression, which demonstrated a significantly negative correlation between miR-206 and Cx43 expression (P =0.03).MiR-206 can drastically decrease Cx43 expression of MCF-7 cells but exerts no effects on Cx43 expression in 293 cells transfected with the Cx43 coding region but the lack of Cx43-3'UTR, suggesting that Cx43-3'UTR may be the key in Cx43 regulated by miR-206.Luciferase expression showed that the inhibition efficiency was

  12. Effects of Cx43 gene modification on the proliferation and migration of the human lung squamous carcinoma cell line NCI-H226.

    Science.gov (United States)

    Zang, J-P; Wei, R

    2015-10-27

    In this study, the human lung squamous carcinoma cell line NCI-H226 was transfected with the recombinant plasmid pBudCE4.1_Cx43 to explore the role of the Cx43 gene in cell growth, cell cycle, and tumor migration. pBudCE4.1-Cx43 was transfected into human lung squamous carcinoma NCI-H226 cells using Lipofectamine TM2000. The mRNA and protein expressions of Cx43 in the transfected cells were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The cell-cell communication was detected using the scratch dye tracer method and the cell cycle was detected by flow cytometry. The CCK-8 proliferation, scratch healing, and cell invasion assays were performed to evaluate the effect of the Cx43 gene transfection on the proliferation, migration, and invasive abilities of NCI-H226 cells. Cx43 mRNA and protein expressions and the fluorescence intensity in the scratch healing test were significantly higher in the experimental group than those in the control and blank groups (P migration, respectively, in the experimental group, compared to the control and blank groups (P migration of human lung squamous carcinoma cell line NCI-H226, thereby inhibiting tumor cell proliferation.

  13. Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina

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    Elena eIvanova

    2015-10-01

    Full Text Available Retinal degeneration (RD encompasses a family of diseases that lead to photoreceptor death and visual impairment. Visual decline due to photoreceptor cell loss is further compromised by emerging spontaneous hyperactivity in inner retinal cells. This aberrant activity acts as a barrier to signals from the remaining photoreceptors, hindering therapeutic strategies to restore light sensitivity in RD. Gap junctions, particularly those expressed in AII amacrine cells, have been shown to be integral to the generation of aberrant activity. It is unclear whether gap junction expression and coupling are altered in RD. To test this, we evaluated the expression and phosphorylation state of connexin36, the gap junction subunit predominantly expressed in AII amacrine cells, in two mouse models of RD, rd10 (slow degeneration and rd1 (fast degeneration. Using Ser293-P antibody, which recognizes a phosphorylated form of connexin36, we found that phosphorylation of connexin36 in both slow and fast RD models was significantly greater than in wildtype controls. This elevated phosphorylation may underlie the increased gap junction coupling of AII amacrine cells exhibited by RD retina.

  14. Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling.

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    Watkins, Marcus; Grimston, Susan K; Norris, Jin Yi; Guillotin, Bertrand; Shaw, Angela; Beniash, Elia; Civitelli, Roberto

    2011-04-15

    Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

  15. Effects of Losartan on expression of connexins at the early stage of atherosclerosis in rabbits

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    Li-ming Ruan, Wei Cai, Jun-zhu Chen, Jin-feng Duan

    2010-01-01

    Full Text Available Aim: to investigate effects of Losartan on expression of connexin 40 and 43 (Cx40 and Cx43, in arteries at the early stage of atherosclerosis in a rabbit model. Methods: A total of 28 male New Zealand white rabbits were divided into following groups: control group, high fat diet group, and Losartan group (10 mg/kg/day. Losartan was administrated in food for two weeks. Iliac arteries were obtained for immunohistochemistry, transmission electron microscopy, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR. Results: Transmission electron microscopy revealed abundant gap junctions between neointimal smooth muscle cells (SMCs, which were markedly reduced by treatment. RT-PCR and Western blot assay showed that the mRNA and protein expression of Cx40 and Cx43 were elevated in the neointimal area at the early stage of atherosclerosis. The mRNA and protein expression of Cx43 were significantly down-regulated by losartan treatment but those of Cx40 were not markedly changed. Conclusion: Cx40 and Cx43 in the neointimal SMCs were up-regulated at the early stage of atherosclerosis. Losartan (an angiotensin-converting enzyme inhibitor could reduce neointima proliferation and down-regulate the elevated protein expression of Cx43, suggesting the rennin-angiotensin system (RAS plays an important role in the remodeling of gap junction between ventricular myocytes under pathological conditions.

  16. Connexin36 Gap Junction Blockade Is Ineffective at Reducing Seizure-Like Event Activity in Neocortical Mouse Slices

    Directory of Open Access Journals (Sweden)

    Logan J. Voss

    2010-01-01

    Full Text Available Despite much research, there remains controversy over the role of gap junctions in seizure processes. Many studies report anticonvulsant effects of gap junction blockade, but contradictory results have also been reported. The aim of this study was to clarify the role of connexin36 (Cx36 gap junctions in neocortical seizures. We used the mouse neocortical slice preparation to investigate the effect of pharmacological (mefloquine and genetic (Cx36 knockout mice (Cx36KO manipulation of Cx36 gap junctions on two seizure models: low-magnesium artificial cerebrospinal fluid (ACSF and aconitine perfusion in low-magnesium ACSF. Low-magnesium- (nominally zero and aconitine- (230 nM induced seizure-like event (SLE population activity was recorded extracellularly. The results were consistent in showing that neither mefloquine (25 μM nor genetic knockdown of Cx36 expression had anticonvulsant effects on SLE activity generated by either method. These findings call into question the widely held idea that open Cx36 gap junctions promote seizure activity.

  17. Connexin hemichannels explain the ionic imbalance and lead to atrophy in denervated skeletal muscles.

    Science.gov (United States)

    Cisterna, Bruno A; Vargas, Aníbal A; Puebla, Carlos; Sáez, Juan C

    2016-11-01

    Denervated fast skeletal muscles undergo atrophy, which is associated with an increase in sarcolemma permeability and protein imbalance. However, the mechanisms responsible for these alterations remain largely unknown. Recently, a close association between de novo expression of hemichannels formed by connexins 43 and 45 and increase in sarcolemma permeability of denervated fast skeletal myofibers was demonstrated. However, it remains unknown whether these connexins cause the ionic imbalance of denervates fast myofibers. To elucidate the latter and the role of hemichannels formed by connexins (Cx HCs) in denervation-induced atrophy, skeletal myofibers deficient in Cx43 and Cx45 expression (Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice) and control (Cx43(fl/fl)Cx45(fl/fl) mice) were denervated and several muscle features were systematically analyzed at different post-denervation (PD) times (1, 3, 5, 7 and 14days). The following sequence of events was found in denervated myofibers of Cx43(fl/fl)Cx45(fl/fl) mice: 1) from day 3 PD, increase in sarcolemmal permeability, 2) from day 5 PD, increases of intracellular Ca(2+) and Na(+) signals as well as a significant increase in protein synthesis and degradation, yielding a negative protein balance and 3) from day 7 PD, a fall in myofibers cross-section area. All the above alterations were either absent or drastically reduced in denervated myofibers of Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice. Thus, the denervation-induced Cx HCs expression is an early event that precedes the electrochemical gradient dysregulation across the sarcolemma and critically contributes to the progression of skeletal muscle atrophy. Consequently, Cx HCs could be a therapeutic target to drastically prevent the denervation-induced atrophy of fast skeletal muscles. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Chronic exposure to hexachlorobenzene results in down-regulation of connexin43 in the breast.

    Science.gov (United States)

    Delisle, Ariane; Ferraris, Emanuelle; Plante, Isabelle

    2015-11-01

    Decreased expression of connexins has been associated with cancer, but the underlying mechanisms are poorly understood. We have previously shown that a 5 day exposure to hexachlorobenzene (HCB) resulted in decreased connexins expression in hepatocytes 45 days later, and that this down-regulation was linked to activation of Akt through the ILK pathway. Because HCB promotes cancer in both the liver and breast, the present study aimed to determine if the mechanisms are similar in both tissues. MCF-12A breast cells were thus transfected with vectors coding for either Akt or a constitutively active form of Akt. In those cells, activation of Akt was correlated with decreased Cx43 levels. Female rats were then exposed to HCB by gavage either following the same protocol used previously for the liver or through a chronic exposure. While no changes were observed after the 5 days exposure protocol, chronic exposure to HCB resulted in increased Akt levels and decreased Cx43 levels in breast cells. In vitro, Akt was activated in MCF-12A cells exposed to HCB either for 7 days or chronically, but no changes were observed in junctional proteins. Together, these results suggested that, while activation of Akt can decrease Cx43 expression in breast cells in vitro, other mechanisms are involved during HCB exposure, leading to a decrease in Cx43 levels in a model- and duration-dependent manner. Finally, we showed that HCB effects are tissue specific, as we did not observe the same results in breast and liver tissues.

  19. Expression of gap junction proteins connexins 26, 30, and 43 in Dupuytren's disease.

    Science.gov (United States)

    Holzer, Lukas A; Cör, Andrej; Holzer, Gerold

    2014-02-01

    Dupuytren's disease (DD) is a benign fibroproliferative process of the palmar aponeurosis showing similarities to wound healing. Communication of cells involved in wound healing is mediated by the composition of gap junction (GJ) proteins. We investigated the expression of 3 GJ proteins, connexins 26, 30, and 43 (Cx26, Cx30, and Cx43) in DD. Fragments of Dupuytren's tissue from 31 patients (mean age 56 (30-76) years, 24 male) were analyzed immunohistochemically and compared to control tissue for expression of the GJ proteins Cx26, Cx30, and Cx43 and also alfa-smooth muscle actin (α-SMA). 14 of 31 samples could be attributed to the involutional phase (α-SMA positive) whereas 17 samples had to be considered cords in the residual phase (α-SMA negative). Expression of Cx26 and Cx43 was seen in 12 of the 14 samples from the involutional phase, and Cx30 was seen in 7 of these. Only 4 of the 17 samples from the residual phase showed any Cx, and there was none in the controls. The high expression of GJ proteins Cx26, Cx30, and Cx43 in α-SMA positive myofibroblast-rich nodules, which are characteristic of the active involutional phase of DD, suggests that connexins could be a novel treatment target for the treatment of DD.

  20. Expression of connexins in human preimplantation embryos in vitro

    Directory of Open Access Journals (Sweden)

    Leese Henry J

    2004-06-01

    Full Text Available Abstract Intercellular communication via gap junctions is required to coordinate developmental processes in the mammalian embryo. We have investigated if the connexin (Cx isoforms known to form gap junctions in rodent preimplantation embryos are also expressed in human embryos, with the aim of identifying species differences in communication patterns in early development. Using a combination of polyA PCR and immunocytochemistry we have assessed the expression of Cx26, Cx31, Cx32, Cx40, Cx43 and Cx45 which are thought to be important in early rodent embryos. The results demonstrate that Cx31 and Cx43 are the main connexin isoforms expressed in human preimplantation embryos and that these isoforms are co-expressed in the blastocyst. Cx45 protein is expressed in the blastocyst but the protein may be translated from a generally low level of transcripts: which could only be detected in the PN to 4-cell embryos. Interestingly, Cx40, which is expressed by the extravillous trophoblast in the early human placenta, was not found to be expressed in the blastocyst trophectoderm from which this tissue develops. All of the connexin isoforms in human preimplantation embryos are also found in rodents pointing to a common regulation of these connexins in development of rodent and human early embryos and perhaps other species.

  1. Connexin mutant embryonic stem cells and human diseases

    Institute of Scientific and Technical Information of China (English)

    Kiyomasa; Nishii; Yosaburo; Shibata; Yasushi; Kobayashi

    2014-01-01

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  2. Connexin mutant embryonic stem cells and human diseases.

    Science.gov (United States)

    Nishii, Kiyomasa; Shibata, Yosaburo; Kobayashi, Yasushi

    2014-11-26

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  3. Linoleic Acid Permeabilizes Gastric Epithelial Cells by Increasing Connexin43 Levels in the Cell Membrane Via a GPR40- and Akt-Dependent Mechanism

    Science.gov (United States)

    Puebla, Carlos; Cisterna, Bruno A.; Salas, Daniela P.; Delgado-López, Fernando; Lampe, Paul D.; Sáez, Juan C.

    2016-01-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintain the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt. PMID:26869446

  4. Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism.

    Science.gov (United States)

    Puebla, Carlos; Cisterna, Bruno A; Salas, Daniela P; Delgado-López, Fernando; Lampe, Paul D; Sáez, Juan C

    2016-05-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.

  5. Loss of connexin36 in rat hippocampus and cerebellar cortex in persistent Borna disease virus infection.

    Science.gov (United States)

    Köster-Patzlaff, Christiane; Hosseini, Seyed Mehdi; Reuss, Bernhard

    2009-03-01

    Neonatal Borna disease virus (BDV) infection of the Lewis rat leads to progressive degeneration of dentate gyrus granule cells, and cerebellar Purkinje neurons. Our aim here was to clarify whether BDV interfered with the formation of electrical synapses, and we, therefore, analysed expression of the neuronal gap junction protein connexin36 (Cx36) in the Lewis rat hippocampal formation, and cerebellar cortex, 4 and 8 weeks after neonatal infection. Semiquantitative RT-PCR, revealed a BDV-dependent decrease in Cx36 mRNA in the hippocampal formation 4 and 8 weeks post-infection (p.i.), and in the cerebellar cortex 8 weeks p.i. Correspondingly, immunofluorescent staining revealed reduced Cx36 immunoreactivity in both dentate gyrus, and ammons horn CA3 region, 4 and 8 weeks post-infection. In the cerebellar cortex, Cx36 immunoreactivity was detected only 8 weeks post-infection in the molecular layer, where it was down regulated by BDV. Our findings demonstrate, for the first time, distinct BDV-dependent reductions in Cx36 mRNA and protein in the rat hippocampal formation and cerebellar cortex, suggesting altered neuronal network properties to be an important feature of persistent viral brain infections.

  6. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

    Science.gov (United States)

    Li, Nan; Mruk, Dolores D; Mok, Ka-Wai; Li, Michelle W M; Wong, Chris K C; Lee, Will M; Han, Daishu; Silvestrini, Bruno; Cheng, C Yan

    2016-04-01

    Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to

  7. Connexins in the early development of the African clawed frog Xenopus laevis (Amphibia: The role of the connexin43 carboxyl terminal tail in the establishment of the dorso-ventral axis

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    Jaime Cofre

    2007-03-01

    Full Text Available Connexins are a family of related proteins identified in vertebrate forming gap junctions, which mediate cell-to-cell communication in early embryos, with an important role in establishing embryonic asymmetry and ‘communication compartments’. By in situ hybridization, immunocytochemistry, reverse transcriptase PCR (RT-PCR and western blotting we show that a Cx43-like molecule is present in oocytes and embryos of the African clawed frog Xenopus laevis, with specific localization in the animal-vegetal axis. This specific distribution is suggestive for an important role for this protein in the establishment of the dorso-ventral axis. Antisense RNA and antibodies directed against rat carboxyl terminal tail of the Cx43 (CT-Cx43 and injected in 1-cell stage Xenopus embryos, induced pronounced alterations in nervous system development, with a severe ventralization phenotype. Coherently, the overexpression of CT-Cx43 produced a dorsalization of the embryos. In antisense treated embryos, the expression of the beta-catenin gene is eliminated from the Nieuwkoop center, the pattern expression of the Chordin, Xnot and Xbra is modified, with no effect in expression of the Goosecoid gene. In CT-Cx43 mRNA treated embryos the pattern of expression of the beta-catenin, Chordin, Goosecoid, Xnot and engrailed-2 genes is modified. The expression of beta-catenin is increased in the Nieuwkoop center, the expression pattern of Chordin and Goosecoid is expanded to the posterior neural plate and engrailed-2 presents ectopic expression in the ventral region. Taken together our data suggest a role for CT-Cx43 as a maternal determinant with a critical function in the formation of the dorso-ventral axis in Xenopus laevis. The Cx43 may be one of the earliest markers of the dorso-ventral axis in these embryos and could possibly be acting through regionalization of factors responsible for the establishment of this axis.

  8. Histopathological analysis of neonatal mouse hearts with connexin43 gene defects%Connexin43基因缺陷新生小鼠心脏组织病理学研究

    Institute of Scientific and Technical Information of China (English)

    谢利剑; 黄国英; 赵晓晴; 沈媛; 周国民

    2005-01-01

    目的观察不同程度Connexin43基因缺陷对新生小鼠心脏发育的影响.方法采用Cx43基因敲除(Cx43 KO)和CMV43CT两种Cx43基因缺陷的小鼠模型,聚合酶链反应鉴定基因型.然后将新生小鼠心脏分离固定,HE染色观察心脏的形态结构.普通C57BL6/SJ小鼠作为对照.结果所有11只纯合型Cx43 KO小鼠生后1 d内均死亡,心脏表现为严重右室流出道梗阻.在20只纯合型CMV43CT新生小鼠中,有12只生后2 d内死亡,其心脏不仅表现有右室流出道梗阻,还出现了房间隔缺损、室间隔缺损等其他心脏畸形;而另外8只未见心脏异常.所有杂合型Cx43 KO和CMV43CT基因缺陷的小鼠生后均存活,心脏病理学检测未见异常.结论 Cx43基因缺陷与心脏发育异常有密切的关系,但不同类型和不同程度的Cx43基因功能缺失对心脏发育的影响也不尽相同.

  9. Connexins and the kidney.

    Science.gov (United States)

    Hanner, Fiona; Sorensen, Charlotte Mehlin; Holstein-Rathlou, Niels-Henrik; Peti-Peterdi, János

    2010-05-01

    Connexins (Cxs) are widely-expressed proteins that form gap junctions in most organs, including the kidney. In the renal vasculature, Cx37, Cx40, Cx43, and Cx45 are expressed, with predominant expression of Cx40 in the endothelial cells and Cx45 in the vascular smooth muscle cells. In the tubules, there is morphological evidence for the presence of gap junction plaques only in the proximal tubules. In the distal nephron, Cx30, Cx30.3, and Cx37 are expressed, but it is not known whether they form gap junctions connecting neighboring cells or whether they primarily act as hemichannels. As in other systems, the major function of Cxs in the kidney appears to be intercellular communication, although they may also form hemichannels that allow cellular secretion of large signaling molecules. Renal Cxs facilitate vascular conduction, juxtaglomerular apparatus calcium signaling, and tubular purinergic signaling. Accordingly, current evidence points to roles for these Cxs in several important regulatory mechanisms in the kidney, including the renin angiotensin system, tubuloglomerular feedback, and salt and water reabsorption. At the systemic level, renal Cxs may help regulate blood pressure and may be involved in hypertension and diabetes.

  10. Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2014-01-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia. - Highlights: • DMBA increases Cx37 and Cx43 expression prior to follicle loss. • During follicle loss both Cx37 and Cx43 expressions are reduced. • CX43 protein is absent in follicle remnants lacking an oocyte.

  11. HIF-1 and c-Src mediate increased glucose uptake induced by endothelin-1 and connexin43 in astrocytes.

    Directory of Open Access Journals (Sweden)

    José Carlos Valle-Casuso

    Full Text Available In previous work we showed that endothelin-1 (ET-1 increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43, the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

  12. Blocking connexin43 expression caused abnormal zebrafish posterior somites development%Connexin43基因表达下调导致斑马鱼胚胎后部体节发育异常

    Institute of Scientific and Technical Information of China (English)

    刘东

    2011-01-01

    目的:验证斑马鱼胚胎体节的发育是否受到间隙连接蛋白connexin43(cx43)基因表达调控.方法:利用吗啉修饰的反义寡核苷酸(morpholino antisense oligos)下调cx43基因的表达;单克隆抗体CH1进行全胚胎的免疫荧光来标记体节;全胚胎的TUNEL实验检测细胞凋亡.结果:研究发现cx43基因表达下调的斑马鱼胚胎呈现为尾部向侧下弯曲,并且后部体节排列紊乱.进一步的研究证实cx43下调诱导了后部体节细胞的异常凋亡.结论:cx43与斑马鱼后部体节的正常发育有关.

  13. Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study.

    OpenAIRE

    Maria Sobaniec-Lotowska; Mariusz Koda; Stanislaw Sulkowski; Luiza Kanczuga-Koda; Mariola Sulkowska

    2005-01-01

    Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of c...

  14. The action of mimetic peptides on connexins protects fibroblasts from the negative effects of ischemia reperfusion

    Directory of Open Access Journals (Sweden)

    Beverley J. Glass

    2015-11-01

    Full Text Available Connexins have been proposed as a target for therapeutic treatment of a variety of conditions. The main approaches have been by antisense or small peptides specific against connexins. Some of these peptides enhance communication while others interfere with connexin binding partners or bind to the intracellular and extracellular loops of connexins. Here, we explored the mechanism of action of a connexin mimetic peptide by evaluating its effect on gap junction channels, connexin protein levels and hemichannel activity in fibroblast cells under normal conditions and following ischemia reperfusion injury which elevates Cx43 levels, increases hemichannel activity and causes cell death. Our results showed that the effects of the mimetic peptide were concentration-dependent. High concentrations (100-300 μM significantly reduced Cx43 protein levels and GJIC within 2 h, while these effects did not appear until 6 h when using lower concentrations (10-30 μM. Cell death can be reduced when hemichannel opening and GJIC were minimised.

  15. 间隙连接蛋白Connexin43对力刺激下人牙周膜细胞成骨相关转录因子表达的影响%Effect of Connexin43 on Mechanical Tension-stimulated Osteogenic Transcription Factors of Human Periodontal Ligament Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    李盛楠; 张华菁; 霍波; 张丁

    2016-01-01

    目的 研究间隙连接蛋白Connexin43 (Cx43)在牵张力刺激下对牙周膜成纤维细胞成骨相关转录因子表达的影响.方法 对体外培养的人牙周膜成纤维细胞(hPDLFs)施加变形量为5%的牵张力,在受力1、2、4、8、24 h后检测成骨相关转录因子Osterix、RUNX2及Cx43 mRNA的表达.分别采用间隙连接的阻断剂18α-GA和基因沉没Cx43的方法阻断Cx43后观察Osteix、RUNX2 mRNA表达的变化.结果 对hPDLFs施加牵张力时,hPDLFs内的Cx43、Osterix和RUNX2mRNA水平随加力时间增加表达均明显增强(p均<O.05),在24h达到最高值.分别阻断间隙连接通道和抑制间隙连接蛋白Cx43,结果显示Osterix和RUNX2 mRNA表达水平均受到明显抑制(P均<O.05).结论 力刺激下间隙连接蛋白Cx43及成骨转录因子Osterix和RUNX2在hPDLFs中的表达呈现时间依赖性增加.间隙连接蛋白Cx43可能参与了这两种成骨转录因子表达的调节.

  16. c-Src Kinase Inhibition Reduces Arrhythmia Inducibility and Connexin43 Dysregulation after Myocardial Infarction

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    Rutledge, Cody A.; Ng, Fu Siong; Sulkin, Matthew S.; Greener, Ian D.; Sergeyenko, Artem M.; Liu, Hong; Gemel, Joanna; Beyer, Eric C.; Sovari, Ali A.; Efimov, Igor R.; Dudley, Samuel C.

    2014-01-01

    Objectives The aim of this study was to evaluate the role of c-Src inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI). Background MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation. Methods MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry ECG monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative PCR, and immunohistochemistry. Results Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p=0.003) and distal ventricle (346%, p=0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p=0.95) and distal ventricle (94%, p=1.0). PP1 raised Cx43 expression by 69% in the scar border (p=0.048) and by 73% in distal ventricle (p=0.043) compared to PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, ECG pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1. Conclusions c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI. PMID:24361364

  17. Gap junction connexins in female reproductive organs: implications for women's reproductive health.

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    Winterhager, Elke; Kidder, Gerald M

    2015-01-01

    Connexins comprise a family of ~20 proteins that form intercellular membrane channels (gap junction channels) providing a direct route for metabolites and signalling molecules to pass between cells. This review provides a critical analysis of the evidence for essential roles of individual connexins in female reproductive function, highlighting implications for women's reproductive health. No systematic review has been carried out. Published literature from the past 35 years was surveyed for research related to connexin involvement in development and function of the female reproductive system. Because of the demonstrated utility of genetic manipulation for elucidating connexin functions in various organs, much of the cited information comes from research with genetically modified mice. In some cases, a distinction is drawn between connexin functions clearly related to the formation of gap junction channels and those possibly linked to non-channel roles. Based on work with mice, several connexins are known to be required for female reproductive functions. Loss of connexin43 (CX43) causes an oocyte deficiency, and follicles lacking or expressing less CX43 in granulosa cells exhibit reduced growth, impairing fertility. CX43 is also expressed in human cumulus cells and, in the context of IVF, has been correlated with pregnancy outcome, suggesting that this connexin may be a determinant of oocyte and embryo quality in women. Loss of CX37, which exclusively connects oocytes with granulosa cells in the mouse, caused oocytes to cease growing without acquiring meiotic competence. Blocking of CX26 channels in the uterine epithelium disrupted implantation whereas loss or reduction of CX43 expression in the uterine stroma impaired decidualization and vascularization in mouse and human. Several connexins are important in placentation and, in the human, CX43 is a key regulator of the fusogenic pathway from the cytotrophoblast to the syncytiotrophoblast, ensuring placental growth

  18. Higher Incidence of Lung Adenocarcinomas Induced by DMBA in Connexin 43 Heterozygous Knockout Mice

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    Krishna Duro de Oliveira

    2013-01-01

    Full Text Available Gap junctions are communicating junctions which are important for tissue homeostasis, and their disruption is involved in carcinogenic processes. This study aimed to verify the influence of deletion of one allele of the Connexin 43 gene on cancer incidence in different organs. The 7, 12-dimethylbenzanthracene (DMBA carcinogenic model, using hebdomadary doses by gavage of 9 mg per animal, was used to induce tumors in Connexin 43 heterozygous or wild-type mice. The experiment began in the eighth week of the mice life, and all of them were euthanized when reaching inadequate physical condition, or at the end of 53 weeks. No statistical differences occurred for weight gain and cancer survival time (P=0.9853 between heterozygous and wild-type mice. Cx43+/− mice presented significantly higher susceptibility to lung cancer (P=0.0200 which was not evidenced for benign neoplasms (P=0.3449. In addition, incidence of ovarian neoplasms was 2.5-fold higher in Cx43+/− mice, although not statistically significant. Other organs showed a very similar cancer occurrence between Cx43 groups. The experiment strengthens the evidence of the relationship between Connexin 43 deficiency and carcinogenesis.

  19. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice.

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    Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F

    2015-01-01

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1mg/kg; ip) for 14days and ovaries collected 3days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (Pobese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (Pobesity while total CX37 protein was reduced (Pobese ovaries. Cx43 mRNA and total protein levels were decreased (Pobese ovaries while basal protein staining intensity was reduced (Pobese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (Pobesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Sertoli-cell-specific knockout of connexin 43 leads to multiple alterations in testicular gene expression in prepubertal mice

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    Sarah Giese

    2012-11-01

    A significant decline in human male reproductive function has been reported for the past 20 years but the molecular mechanisms remain poorly understood. However, recent studies showed that the gap junction protein connexin-43 (CX43; also known as GJA1 might be involved. CX43 is the predominant testicular connexin (CX in most species, including in humans. Alterations of its expression are associated with different forms of spermatogenic disorders and infertility. Men with impaired spermatogenesis often exhibit a reduction or loss of CX43 expression in germ cells (GCs and Sertoli cells (SCs. Adult male transgenic mice with a conditional knockout (KO of the Gja1 gene [referred to here as connexin-43 (Cx43] in SCs (SCCx43KO show a comparable testicular phenotype to humans and are infertile. To detect possible signaling pathways and molecular mechanisms leading to the testicular phenotype in adult SCCx43KO mice and to their failure to initiate spermatogenesis, the testicular gene expression of 8-day-old SCCx43KO and wild-type (WT mice was compared. Microarray analysis revealed that 658 genes were significantly regulated in testes of SCCx43KO mice. Of these genes, 135 were upregulated, whereas 523 genes were downregulated. For selected genes the results of the microarray analysis were confirmed using quantitative real-time PCR and immunostaining. The majority of the downregulated genes are GC-specific and are essential for mitotic and meiotic progression of spermatogenesis, including Stra8, Dazl and members of the DM (dsx and map-3 gene family. Other altered genes can be associated with transcription, metabolism, cell migration and cytoskeleton organization. Our data show that deletion of Cx43 in SCs leads to multiple alterations of gene expression in prepubertal mice and primarily affects GCs. The candidate genes could represent helpful markers for investigators exploring human testicular biopsies from patients showing corresponding spermatogenic deficiencies and for

  1. Roles of gap junctions, connexins and pannexins in epilepsy

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    Shanthini eMylvaganam

    2014-05-01

    Full Text Available Enhanced gap junctional communication (GJC between neurons is considered a major factor underlying the neuronal synchrony driving seizure activity. In addition, the hippocampal sharp wave ripple complexes, associated with learning and seizures, are diminished by GJC blocking agents. Although gap junctional blocking drugs inhibit experimental seizures, they all have other nonspecific actions. Besides interneuronal GJC between dendrites, inter-axonal and inter-glial GJC is also considered important for seizure generation. Interestingly, in most studies of cerebral tissue from animal seizure models and from human patients with epilepsy, there is up-regulation of glial, but not neuronal gap junctional mRNA and protein. Significant changes in the expression and post-translational modification of the astrocytic connexin Cx43, and Panx1 were observed in an in vitro Co++ seizure model, further supporting a role for glia in seizure-genesis, although the reasons for this remain unclear. Further suggesting an involvement of astrocytic GJC in epilepsy, is the fact that the expression of astrocytic Cx mRNAs (Cxs 30 and 43 is several fold higher than that of neuronal Cx mRNAs (Cxs 36 and 45, and the number of glial cells outnumber neuronal cells in mammalian hippocampal and cortical tissue. Pannexin expression is also increased in both animal and human epileptic tissues. Specific Cx43 mimetic peptides, Gap 27 and SLS, inhibit the docking of astrocytic connexin Cx43 proteins from forming intercellular gap junctions, diminishing spontaneous seizures. Besides GJs, Cx membrane hemichannels in glia and Panx membrane channels in neurons and glia are also inhibited by gap junctional pharmacological blockers. Although there is no doubt that connexin-based gap junctions and hemichannels, and pannexin-based membrane channels are related to epilepsy, the specific details of how they are involved and how we can modulate their function for therapeutic purposes remain to

  2. Modulation of connexin 36 expression in basal ganglia and motor cortex in rat model of Parkinson's disease

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    CHEN Xian-wen

    2013-08-01

    Full Text Available Objective To observe the expression of connexin 36 (Cx36 in the striatum and motor cortex of rat model of Parkinson's disease (PD in order to explore whether gap junction is involved in the pathogenesis of the cortex-basal ganglia circuit disturbances in PD. Methods Hemi-parkinsonian rat model was produced by stereotaxically injecting 6-hydroxydopamine (6-OHDA to right medial forebrain bundle (MFB. Immunohistochemical staining and Western blotting analysis were used to observe the expression changes of Cx36 in the striatum and motor cortex. Double immunofluorescence labeling was used to analyze the expression of Cx36 in enkephalin (ENK positive medium spiny neurons and Parvalbumin (PV positive interneurons in the striatum. Results Immunohistochemical staining showed Cx36 expression was elevated in the right striatum as well as right motor cortex of PD group compared with normal control group (t = 2.474, P = 0.048; t = 2.614, P = 0.040. Double immunofluorescence labeling staining revealed that ENK-positive striatum neurons were elevated (t = 3.987, P = 0.007 and Cx36 expression was increased in ENK-positive striatum neurons (t = 3.271, P = 0.017 in PD group compared with normal control group. While PV-positive interneurons decreased (t = 2.777, P = 0.032 and Cx36 expression was down-regulated in PV-positive interneurons (t = 2.624, P = 0.039 compared with the normal control group. Western blotting indicated that the 6-OHDA lesion induced a significant upregulation of Cx36 to (119.31 ± 8.92% in comparison with the normal group [(104.05 ± 3.82%] in right striatum (t = 3.516, P = 0.024. In right motor cortex Cx36 increased to (138.20 ± 17.88% , induced a significant upregulation of Cx36 in the right motor cortex in comparison with the normal control group [(105.27 ± 2.82%; t = 4.068, P = 0.015]. Conclusion The Cx36 expression was generally increased in the striatum and motor cortex of PD rat model, with upregulation in ENK-positive striatum

  3. The connexin43 carboxyl terminus and cardiac gap junction organization.

    Science.gov (United States)

    Palatinus, Joseph A; Rhett, J Matthew; Gourdie, Robert G

    2012-08-01

    The precise spatial order of gap junctions at intercalated disks in adult ventricular myocardium is thought vital for maintaining cardiac synchrony. Breakdown or remodeling of this order is a hallmark of arrhythmic disease of the heart. The principal component of gap junction channels between ventricular cardiomyocytes is connexin43 (Cx43). Protein-protein interactions and modifications of the carboxyl-terminus of Cx43 are key determinants of gap junction function, size, distribution and organization during normal development and in disease processes. Here, we review data on the role of proteins interacting with the Cx43 carboxyl-terminus in the regulation of cardiac gap junction organization, with particular emphasis on Zonula Occludens-1. The rapid progress in this area suggests that in coming years we are likely to develop a fuller understanding of the molecular mechanisms causing pathologic remodeling of gap junctions. With these advances come the promise of novel approach to the treatment of arrhythmia and the prevention of sudden cardiac death. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Connexin 43 Is a Potential Prognostic Biomarker for Ewing Sarcoma/Primitive Neuroectodermal Tumor

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    Marilyn M. Bui

    2011-01-01

    Full Text Available Connexins (Cxs are building unit proteins of gap junctions (GJs that are prognostic markers in carcinomas. To investigate the role of Cx in Ewing sarcoma (EWS/primitive neuroectodermal tumor (PNET, we examined the expression of Cx43 and Cx26 in 36 EWS/PNETs and found (1 cytoplasmic Cx43 reactivity in 28/36 (78% cases. (2 Cx43 score was significantly correlated with overall survival (P=.025. The average scores for patients alive and dead at 3 years are 46.08 and 96.98 (P=.004 at 5 years are 46.06 and 96.42 (P=.002. (3 Metastasis had a significant effect on the overall survival (P=.003. (4 Cytoplasmic Cx26 reactivity was detected in 2 of 36 (6% patients who died with metastasis. Our results suggest a possible oncogenic and prognostic role for Cx43 and Cx26 in EWS/PNET. The lack of membranous immunoreactivity suggests that the effect of Cx in EWS/PNET is via a GJ function-independent mechanism.

  5. Role of connexin-43 hemichannels in the pathogenesis of Yersinia enterocolitica.

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    Velasquez Almonacid, L A; Tafuri, S; Dipineto, L; Matteoli, G; Fiorillo, E; Della Morte, R; Fioretti, A; Menna, L F; Staiano, N

    2009-12-01

    Connexin (Cx) channels are sites of cytoplasmic communication between contacting cells. Evidence indicates that the opening of hemichannels occurs under both physiological and pathological conditions. In this paper, the involvement of Cx-43 hemichannels is demonstrated in the pathogenesis of Yersinia. Parental HeLa cells and transfected HeLa cells stably expressing Cx-43 (HCx43) were infected with Yersiniaenterocolitica, and bacterial uptake was measured by the colony-forming unit method. Bacterial uptake was higher in HCx43 cells than in parental cells and was inhibited by the Cx channel blocker, 18-alpha-glycyrrhetinic acid (AGA). The inhibitory effect of AGA was more pronounced on the Y. enterocolitica uptake by HCx43 cells than by parental cells. The ability of HCx43 cells to incorporate the permeable fluorescent tracer Lucifer Yellow (LY) was assessed. Dye incorporation was inhibited by AGA, whereas Y. enterocolitica infection of HCx43 cells increased LY incorporation. Western blotting analysis demonstrated that Y. enterocolitica infection of HCx43 cells induced tyrosine phosphorylation of Cx-43, thus supporting a critical role for Cx-43 in the strategies exploited by bacterial pathogens to invade non-phagocytic cells.

  6. Classical swine fever virus down-regulates endothelial connexin 43 gap junctions.

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    Hsiao, Hsiang-Jung; Liu, Pei-An; Yeh, Hung-I; Wang, Chi-Young

    2010-07-01

    Classical swine fever is a contagious disease of pigs characterized by fatal hemorrhagic fever. Classical swine fever virus (CSFV) induces the expression of pro-inflammatory and pro-coagulant factors of vascular endothelial cells and establishes a long-term infection. This study aimed to understand the effect of CSFV on endothelial connexin 43 (Cx43) expression and gap junctional intercellular coupling (GJIC). Porcine aortic endothelial cells were infected with CSFV at different multiplicity of infection for 48 h. Semi-quantitative RT-PCR, immunoconfocal microscopy, and Western blotting showed that the transcription and translation of Cx43 were reduced, and this was associated with an attenuation of GJIC. This decrease occurred in a time-dependent manner. An ERK inhibitor (PD98059), a JNK inhibitor (SP600125), and proteasome/lysosome inhibitors all significantly reversed the reduction in Cx43 protein levels without any influence on the titer of progeny virus. In addition, CSFV activated ERK and JNK in a time-dependent manner and down-regulated Cx43 promoter activity, mainly through decreased AP2 binding. This effect was primarily caused by the replication of CSFV rather than a consequence of cytokines being induced by CSFV infection of endothelial cells.

  7. The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.

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    Ahir, Bhavesh K; Pratten, Margaret K

    2016-07-01

    Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 μm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Contribution of intracellular calcium and pH in ischemic uncoupling of cardiac gap junction channels formed of connexins 43, 40, and 45: a critical function of C-terminal domain.

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    Giriraj Sahu

    Full Text Available Ischemia is known to inhibit gap junction (GJ mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD condition. 5 minutes of OGD decreased the junctional conductance (Gj of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca(2+]i and pH (pHi with potassium phosphate buffer. Clamping of either [Ca(2+]i or pHi, through BAPTA (2 mM or HEPES (80 mM respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT truncated Cx43 (Cx43-Δ257. Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249, and Cx45 (Cx45-Δ272 helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca(2+]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.

  9. Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts.

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    Marco Heinrich

    Full Text Available BACKGROUND: The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC and overactive bladder syndrome (OAB. Gap junctional intercellular communication (GJIC is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC and suburothelial myofibroblasts (hsMF. Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL 4, IL6, IL10, tumor necrosis factor-alpha (TNFα and transforming growth factor-beta1 (TGFβ1 on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC and human suburothelial myofibroblasts (hsMF. METHODOLOGY/PRINCIPAL FINDINGS: HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP. Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFβ1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC. CONCLUSIONS/SIGNIFICANCE: Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that

  10. The role of spinal interleukin-1β and astrocyte connexin 43 in the development of mirror-image pain in an inflammatory pain model.

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    Choi, Hoon-Seong; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kwon, Soon-Gu; Choi, Sheu-Ran; Kang, Suk-Yun; Moon, Ji-Young; Han, Ho-Jae; Kim, Hyun-Woo; Beitz, Alvin J; Lee, Jang-Hern

    2016-10-20

    Although we have recently demonstrated that carrageenan-induced inflammation upregulates the expression of spinal interleukin (IL)-1β, which inhibits spinal astrocyte activation and results in the delayed development of Mirror-Image Pain (MIP), little is known regarding the mechanisms that underlie how spinal IL-1β inhibits the astrocyte activation. In this study, we examined the effect of spinal IL-1β on astrocyte gap junctions (GJ) and the development of MIP. Following unilateral carrageenan (CA) injection, mechanical allodynia (MA) was evaluated at various time points. Immunohistochemistry and Western blot analysis were used to determine changes in the expression of GFAP and connexins (Cx) in the spinal cord dorsal horn. Carrageenan rats showed a delayed onset of contralateral MA, which mimicked the temporal expression pattern of spinal Cx43 (an astrocyte gap junctional protein) and GFAP. Intrathecal administration of an interleukin-1 receptor antagonist (IL-1ra) twice-a-day on post-carrageenan injection days 0 to 3 caused a significant increase in contralateral MA and spinal Cx43 and GFAP expression. In addition, co-administration of IL-1β with IL-1ra blocked the IL-1ra-induced increase in contralateral MA and the upregulated expression of spinal Cx43 and GFAP. Finally, co-administration of carbenoxolone (CBX; a GJ decoupler) or Gap26 (a specific Cx43 mimetic blocking peptide) with IL-1ra significantly blocked the IL-1ra-induced early development of contralateral MA and the associated upregulation of spinal Cx43 and GFAP expression. These results demonstrate that spinal IL-1β suppresses Cx43 expression and astrocyte activation during the early phase of CA-induced inflammation resulting in the delayed onset of contralateral MA. These findings imply that spinal IL-1β can inhibit astrocyte activation and regulate the time of induction of contralateral MA through modulation of spinal Cx43 expression.

  11. Carbon Monoxide (CO) Is a Novel Inhibitor of Connexin Hemichannels*

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    León-Paravic, Carmen G.; Figueroa, Vania A.; Guzmán, Diego J.; Valderrama, Carlos F.; Vallejos, Antonio A.; Fiori, Mariana C.; Altenberg, Guillermo A.; Reuss, Luis; Retamal, Mauricio A.

    2014-01-01

    Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or “free HCs” at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions. PMID:25384983

  12. Effects of Banxia Xiexin Decoction and Its Components on Protein Expression of Cx43 in Gap Junction in Gastric Tissue of Electrogastric Dysrhythmias Rats%半夏泻心汤及其拆方对胃电节律失常大鼠胃组织缝隙连接蛋白Cx43表达的影响

    Institute of Scientific and Technical Information of China (English)

    李翠; 张冬梅; 娄利霞; 赵明镜; 李丽娜; 陈萌

    2015-01-01

    Objective To discuss compatibility regularity of Banxia Xiexin Decoction adjusting gastric mobility. Methods Ninty healthy SD male rats were divided into normal group (10 rats) and model group (80 rats). Rat models of electrogastric dysrhythmias were established. After the evaluation of gastric electrophysiology, Banxia Xiexin Decoction were divided into Xinkai, Kujiang, Ganbu, Xinkaikujiang, Xinkaiganbu, and Kujiangganbu groups according to the compatibility regularity of Banxia Xiexin Decoction, 10 rats for each group. Rats in each medication administration group received gavage with same concentration but different volume for 4 weeks. Each group was analyzed for gastric electrical parameters after administration. The expressions of Cx43 and Cx43 mRNA in the gastric tissue of rats in each group were analyzed by immunohistochemistry and real-time fluorescent quantitative PCR. Results Compare with the normal group, the expressions of Cx43 and Cx43 mRNA in the gastric tissue of rats increased significantly in the model group. Compared with the model group, variable coefficient of electrogastric slow wave frequency in all medication administration groups decreased obviously. Banxia Xiexin Decoction and its components had decreased the expression of Cx43 and Cx43 mRNA to some extent, among which Xinkaiganbu group had best effects. Conclusion The mechanism of Banxia Xiexin Decoction adjusting gastric motility maybe related to its function of decreasing the expressions of Cx43 and Cx43 mRNA, as to adjust disordered electrogastric rhythm.%目的:探讨半夏泻心汤调节胃运动的配伍规律。方法选取90只健康SD雄性大鼠,分为正常组10只和模型组80只。复制大鼠胃电节律失常模型,经胃电生理学指标评价后,根据半夏泻心汤的配伍特点,将其拆分为辛开、苦降、甘补、辛开苦降、辛开甘补、苦降甘补组,每组10只。各给药组按相同药物浓度不等体积连续灌胃4周。给药后进

  13. Aberrant distributions and relationships among E-cadherin, beta-catenin, and connexin 26 and 43 in endometrioid adenocarcinomas.

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    Wincewicz, Andrzej; Baltaziak, Marek; Kanczuga-Koda, Luiza; Lesniewicz, Tomasz; Rutkowski, Ryszard; Sobaniec-Lotowska, Maria; Sulkowski, Stanislaw; Koda, Mariusz; Sulkowska, Mariola

    2010-07-01

    During carcinogenesis, loss of intracellular cohesion is observed among cancer cells with altered expression of such adhesion molecules as E-cadherin and beta-catenin, and aberrant expression and cellular location of intercellular gap junction proteins-connexins. The aim of this study was to evaluate immunohistochemically the expression and relationship between E-cadherin and beta-catenin, and the connexins Cx26 and Cx43 in 86 endometrioid adenocarcinomas. The aberrant cytoplasmic translocation of the studied proteins was a predominant finding, whereas only a minority of cases showed normal, nuclear beta-catenin labeling or membranous distribution of the remaining molecules. E-cadherin was positively and significantly associated with beta-catenin (P=0.001, r=0.366), as was Cx26 with Cx43 (P<0.001, r=0.719), E-cadherin with Cx26 (P<0.001, r=0.413), and E-cadherin and Cx43 (P<0.001, r=0.434) in all cancers. A subgroup of endometrioid adenocarcinomas (FIGO IB+II) exclusively showed a positive significant association between the expression of beta-catenin and Cx26 (P=0.038, r=0.339). In addition, there were significantly more beta-catenin-positive carcinomas among superficially spreading cancers (FIGO IA) than among deeper invading neoplasms (FIGO IB+II) (P=0.056). The altered location of the studied proteins indicates impairment of their physiological functions. In particular, normal membranous distribution of E-cadherin and connexins is lost and replaced by abnormal cytoplasmic accumulation in most cancers, and thus intercellular ties are expected to be weakened and loosened as a consequence. In contrast, the lack of relationship between beta-catenin and connexins, E-cadherin seems to be closely associated with the expression of Cx26 and Cx43 in endometrioid adenocarcinomas.

  14. Increased Connexin 43 Expression Improves the Migratory and Proliferative Ability of H9c2 Cells by Wnt-3a Overexpression

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    Xiaoyu LIU; Wen LIU; Ling YANG; Beili XIA; Jinyan LI; Ji ZUO; Xiaotian LI

    2007-01-01

    The change of connexin 43 (Cx43) expression and the biological behaviors of Cx43 in rat heart cell line H9c2, expressing Wnt-3a (wingless-type MMTV integration site family, member 3A), were evaluated in the present study. Plasmid pcDNA3.1/Wnt-3a was constructed and transferred into H9c2 cells.The cell model Wnt-3a+-H9c2 steadily expressing Wnt-3a was obtained. Compared with H9c2 and pcDNA3.1-H9c2 cells, the expression of Cx43 in Wnt-3a+-H9c2 cells was clearly increased, the proliferation of Wnt-3a+-H9c2 cells was significantly changed, and cell migration abilities were also improved (P<0.05).In comparison with H9c2 and pcDNA3.1-H9c2 cells, the G2 phase of the cell cycle increased by 11% in Wnt-3a+-H9c2 cells. Thus, Wnt-3a overexpression is associated with an increase in Cx43 expression and altered migratory and proliferative activity in H9c2 cells. Cx43 might be one of the downstream target genes regulated by Wnt-3a.

  15. Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis.

    Science.gov (United States)

    Cea, Luis A; Puebla, Carlos; Cisterna, Bruno A; Escamilla, Rosalba; Vargas, Aníbal A; Frank, Marina; Martínez-Montero, Paloma; Prior, Carmen; Molano, Jesús; Esteban-Rodríguez, Isabel; Pascual, Ignacio; Gallano, Pía; Lorenzo, Gustavo; Pian, Héctor; Barrio, Luis C; Willecke, Klaus; Sáez, Juan C

    2016-07-01

    Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca(2+) levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice was significantly better than that of control mdx Cx43(fl/fl)Cx45(fl/fl) mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

  16. THE FEATURES OF CONNEXINS EXPRESSION IN THE CELLS OF NEUROVASCLAR UNIT IN NORMAL CONDITIONS AND HYPOXIA IN VITRO

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    A. V. Morgun

    2014-01-01

    Full Text Available The aim of this research was to assess a role of connexin 43 (Cx43 and associated molecule CD38 in the regulation of cell-cell interactions in the neurovascular unit (NVU in vitro in physiological conditions and in hypoxia.Materials and methods. The study was done using the original neurovascular unit model in vitro. The NVU consisted of three cell types: neurons, astrocytes, and cerebral endothelial cells derived from rats. Hypoxia was induced by incubating cells with sodium iodoacetate for 30 min at37 °C in standard culture conditions.Results. We investigated the role of connexin 43 in the regulation of cell interactions within the NVU in normal and hypoxic injury in vitro. We found that astrocytes were characterized by high levels of expression of Cx43 and low level of CD38 expression, neurons demonstrated high levels of CD38 and low levels of Cx43. In hypoxic conditions, the expression of Cx43 and CD38 in astrocytes markedly increased while CD38 expression in neurons decreased, however no changes were found in endothelial cells. Suppression of Cx43 activity resulted in down-regulation of CD38 in NVU cells, both in physiological conditions and at chemical hypoxia.Conclusion. Thus, the Cx-regulated intercellular NAD+-dependent communication and secretory phenotype of astroglial cells that are the part of the blood-brain barrier is markedly changed in hypoxia.

  17. Effects of reperfusion arrhythmias and myocardial connexin 43 by Compound Astragalus Mixture Nourishing Heart in a rat model of acute myocardial ischemic reperfusion injury%复方黄芪养心合剂对大鼠缺血再灌注心肌缝隙连接蛋白43的影响

    Institute of Scientific and Technical Information of China (English)

    陈启兰; 龚一萍; 祝光礼; 齐国安; 赫小龙; 任兴昌; 王骋

    2013-01-01

    目的:观察复方黄芪养心合剂对SD大鼠缺血再灌注心肌细胞缝隙连接蛋白43(Cx43)分布的改变和表达的影响.方法:应用复方黄芪养心合剂按每天14g/kg剂量、琥珀酸美托洛尔缓释片按每天(MSSRT) 9.5mg/kg剂量灌胃2周后,对SD大鼠行冠状动脉左前降支结扎30min后再灌注60min造成心肌缺血再灌注(I/R)损伤模型,记录Ⅱ导联心电图,采用免疫组织化学法(IHC)观察心肌细胞Cx43分布的改变;运用Image Pro Plus 6.0图像分析软件对Cx43的表达进行半定量分析.结果:Cx43平均光密度(AOD)I/R组心肌内膜下缺血区Cx43的AOD显著低于假手术组(P<0.01),且MSSRT组、复方组较1/R组升高(P<0.05).结论:对冠状动脉左前降支结扎术后再灌注SD大鼠,复方黄芪养心合剂对心肌细胞Cx43分布的改变有改善作用,有促进心肌内膜下缺血区域Cx43表达的作用,其作用与MSSRT相近.%Objective: To evaluate the effects of Compound Astragalus Mixture Nourishing Heart (CAMNH) antiarrhythmia and myocardial connexin 43 (Cx43) in a rat model of acute myocardial ischemic reperfusion injury. Methods: Myocardial infarction (MI) was induced by ligating left anterior descending coronary artery (LAD) for 30 minutes, followed by reperfusion of 60 minutes in rats. Rats were treated with CAMNH (14g·kg-1·d-1) or metoprolol succinate sustained-release tablets (MSSRT, 9.5mg·kg-1d-1) for 14 days before MI. The distribution of myocardial Cx43 was observed by Immunohistochemistry (IHC), and the expression of myocardial Cx43, which were represented by average optical density (AOD), was measured by Image Pro Plus 6.0. Results: The expression of myocardial Cx43 in infarction region was significantly reduced, disordered and even all disappearing, the expression of myocardial Cx43 from infarction region into ischemic region and normal region had a gradual recovery of transitional variability. Ischemia/reperfusion(I/R) group vs sham-operated (SO

  18. Opening of pannexin and connexin based-channels increases the excitability of nodose ganglion sensory neurons.

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    Mauricio Antonio Retamal

    2014-06-01

    Full Text Available Satellite glial cells (SGCs are the main glia in sensory ganglia. They surround neuronal bodies and form a cap that prevents the formation of chemical or electrical synapses between neighboring neurons. SGCs have been suggested to establish bidirectional paracrine communication with sensory neurons. However, the molecular mechanism involved in this cellular communication is unknown. In the central nervous system, astrocytes present connexin43 (Cx43 hemichannels and pannexin1 (Panx1 channels, and their opening allows the release of signal molecules, such as ATP and glutamate. We propose that these channels could play a role in the glia-neuron communication in sensory ganglia. Therefore, we studied the expression and function of Cx43 and Panx1 in rat and mouse nodose-petrosal-jugular complex (NPJc by confocal immunofluorescence, molecular and electrophysiological techniques. Cx43 and Panx1 were detected in SGCs and sensory neurons, respectively. In the rat and mouse, the electrical activity of vagal nerve increased significantly after nodose neurons were exposed to Ca2+/ Mg2+-free solution, a condition that increases the open probability of Cx hemichannels. This response was partially mimicked by a cell-permeable peptide corresponding to the last 10 amino acids of Cx43 (TAT-Cx43CT. Enhanced neuronal activity was reduced by Cx hemichannel, Panx1 channel and P2X7 receptor blockers. Moreover, the role of Panx1 was confirmed in NPJc, because Panx1 knockout mouse showed a reduced increase of neuronal activity induced by Ca2+/Mg2+-free extracellular conditions. Data suggest that Cx hemichannels and Panx channels serve as paracrine communication pathways between SGCs and neurons by modulating the excitability of sensory neurons.

  19. Opening of pannexin- and connexin-based channels increases the excitability of nodose ganglion sensory neurons.

    Science.gov (United States)

    Retamal, Mauricio A; Alcayaga, Julio; Verdugo, Christian A; Bultynck, Geert; Leybaert, Luc; Sáez, Pablo J; Fernández, Ricardo; León, Luis E; Sáez, Juan C

    2014-01-01

    Satellite glial cells (SGCs) are the main glia in sensory ganglia. They surround neuronal bodies and form a cap that prevents the formation of chemical or electrical synapses between neighboring neurons. SGCs have been suggested to establish bidirectional paracrine communication with sensory neurons. However, the molecular mechanism involved in this cellular communication is unknown. In the central nervous system (CNS), astrocytes present connexin43 (Cx43) hemichannels and pannexin1 (Panx1) channels, and the opening of these channels allows the release of signal molecules, such as ATP and glutamate. We propose that these channels could play a role in glia-neuron communication in sensory ganglia. Therefore, we studied the expression and function of Cx43 and Panx1 in rat and mouse nodose-petrosal-jugular complexes (NPJcs) using confocal immunofluorescence, molecular and electrophysiological techniques. Cx43 and Panx1 were detected in SGCs and in sensory neurons, respectively. In the rat and mouse, the electrical activity of vagal nerve increased significantly after nodose neurons were exposed to a Ca(2+)/Mg(2+)-free solution, a condition that increases the open probability of Cx hemichannels. This response was partially mimicked by a cell-permeable peptide corresponding to the last 10 amino acids of Cx43 (TAT-Cx43CT). Enhanced neuronal activity was reduced by Cx hemichannel, Panx1 channel and P2X7 receptor blockers. Moreover, the role of Panx1 was confirmed in NPJc, because in those from Panx1 knockout mice showed a reduced increase of neuronal activity induced by Ca(2+)/Mg(2+)-free extracellular conditions. The data suggest that Cx hemichannels and Panx channels serve as paracrine communication pathways between SGCs and neurons by modulating the excitability of sensory neurons.

  20. Connexin43- and Pannexin-Based Channels in Neuroinflammation and Cerebral Neuropathies

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    Denis Sarrouilhe

    2017-10-01

    Full Text Available Connexins (Cx are largely represented in the central nervous system (CNS with 11 Cx isoforms forming intercellular channels. Moreover, in the CNS, Cx43 can form hemichannels (HCs at non-junctional membrane as does the related channel-forming Pannexin1 (Panx1 and Panx2. Opening of Panx1 channels and Cx43 HCs appears to be involved in inflammation and has been documented in various CNS pathologies. Over recent years, evidence has accumulated supporting a link between inflammation and cerebral neuropathies (migraine, Alzheimer’s disease (AD, Parkinson’s disease (PD, major depressive disorder, autism spectrum disorder (ASD, epilepsy, schizophrenia, bipolar disorder. Involvement of Panx channels and Cx43 HCs has been also proposed in pathophysiology of neurological diseases and psychiatric disorders. Other studies showed that following inflammatory injury of the CNS, Panx1 activators are released and prolonged opening of Panx1 channels triggers neuronal death. In neuropsychiatric diseases, comorbidities are frequently present and can aggravate the symptoms and make therapeutic management more complex. The high comorbidity between some neuropathies can be partially understood by the fact that these diseases share a common etiology involving inflammatory pathways and Panx1 channels or Cx43 HCs. Thus, anti-inflammatory therapy opens perspectives of targets for new treatments and could have real potential in controlling a cerebral neuropathy and some of its comorbidities. The purpose of this mini review is to provide information of our knowledge on the link between Cx43- and Panx-based channels, inflammation and cerebral neuropathies.

  1. Bisphosphonates improve trabecular bone mass and normalize cortical thickness in ovariectomized, osteoblast connexin43 deficient mice.

    Science.gov (United States)

    Watkins, Marcus P; Norris, Jin Yi; Grimston, Susan K; Zhang, Xiaowen; Phipps, Roger J; Ebetino, Frank H; Civitelli, Roberto

    2012-10-01

    The gap junction protein, connexin43 (Cx43) controls both bone formation and osteoclastogenesis via osteoblasts and/or osteocytes. Cx43 has also been proposed to mediate an anti-apoptotic effect of bisphosphonates, potent inhibitors of bone resorption. We studied whether bisphosphonates are effective in protecting mice with a conditional Cx43 gene deletion in osteoblasts and osteocytes (cKO) from the consequences of ovariectomy on bone mass and strength. Ovariectomy resulted in rapid loss of trabecular bone followed by a slight recovery in wild type (WT) mice, and a similar degree of trabecular bone loss, albeit slightly delayed, occurred in cKO mice. Treatment with either risedronate (20 μg/kg) or alendronate (40 μg/kg) prevented ovariectomy-induced bone loss in both genotypes. In basal conditions, bones of cKO mice have larger marrow area, higher endocortical osteoclast number, and lower cortical thickness and strength relative to WT. Ovariectomy increased endocortical osteoclast number in WT but not in cKO mice. Both bisphosphonates prevented these increases in WT mice, and normalized endocortical osteoclast number, cortical thickness and bone strength in cKO mice. Thus, lack of osteoblast/osteocyte Cx43 does not alter bisphosphonate action on bone mass and strength in estrogen deficiency. These results support the notion that one of the main functions of Cx43 in cortical bone is to restrain osteoblast and/or osteocytes from inducing osteoclastogenesis at the endocortical surface. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Connexin-43 channels are a pathway for discharging lactate from glycolytic pancreatic ductal adenocarcinoma cells.

    Science.gov (United States)

    Dovmark, T H; Saccomano, M; Hulikova, A; Alves, F; Swietach, P

    2017-08-10

    Glycolytic cancer cells produce large quantities of lactate that must be removed to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H(+)-coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility that other venting mechanisms become important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was strong between Colo357 cells, weaker yet hypoxia-inducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with levels of connexin-43 (Cx43), a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H(+) ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150 μm). MiaPaCa2 cells, which are Cx43 negative in monolayer culture, showed markedly increased Cx43 immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma), which can explain the advantage of junctional transmission over MCT in vivo. We propose that Cx43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Furthermore

  3. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    Science.gov (United States)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  4. A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells.

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    Valentina Cigliola

    Full Text Available Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36, which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D. GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123 associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.

  5. Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue.

    Science.gov (United States)

    Sampaio da Silva, Andréa Moreira; Bruno, Jamily Bezerra; de Lima, Laritza Ferreira; Ribeiro de Sá, Naíza Arcângela; Lunardi, Franciele Osmarini; Ferreira, Anna Clara Accioly; Vieira Correia, Hudson Henrique; de Aguiar, Francisco Léo Nascimento; Araújo, Valdevane Rocha; Lobo, Carlos Henrique; de Alencar Araripe Moura, Arlindo; Campello, Cláudio Cabral; Smitz, Johan; de Figueiredo, José Ricardo; Ribeiro Rodrigues, Ana Paula

    2016-05-01

    Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P  0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not

  6. 不同剂量香烟烟雾提取物对阴茎海绵体NOS活性和Cx43蛋白的影响%Cigarette smoke extract reduces NOS activity and CX43 expression in the corporal cavernosum

    Institute of Scientific and Technical Information of China (English)

    刘小兵; 吴天鹏; 詹运运; 王朝阳

    2011-01-01

    目的:探讨不同剂量的香烟烟雾提取物对雄性大鼠勃起功能的影响,进一步研究吸烟导致ED的发病机制.方法:75只健康雄性SD大鼠(8周龄),随机分为5组(15只/组).A组为对照组;B组为皮下注射二甲基亚砜(DMSO)组;C组为皮下注射香烟烟雾提取物(CSE)低剂量组;D组为皮下注射CSE中剂量组;E组为皮下注射CSE高剂量组.应用SD雄性大鼠建立皮下注射CSE模型60 d后,经皮下注射阿朴吗啡(APO)后观察大鼠阴茎勃起情况,处死大鼠取阴茎海绵体组织.一部分标本采用激光共聚焦扫描显微镜方法检测缝隙连接蛋白43(Cx43)的表达;另一部分标本采用比色法测定大鼠阴茎海绵体组织中NOS活性.结果:不同剂量CSE组阴茎勃起次数、阴茎海绵体组织NOS活性和海绵体平滑肌中Cx43表达与DMSO组和对照组比较均明显减少(P0.05).结论:CSE使阴茎海绵体组织NOS活性明显降低、海绵体平滑肌中Cx43蛋白表达明显减少并且严重影响阴茎勃起功能,并且CSE剂量越大,其影响越明显.提示,Cx43蛋白表达下调、NOS活性降低可能是CSE导致ED的发病机制之一.%Objective: To study the effects of different doses of cigarette smoke extract (CSE) on the erectile function of male rats and the mechanism of smoking-induced erectile dysfunction (ED).Methods: A total of 75 healthy male SD rats were randomly divided into Groups A (control), B ( dimethyl sulphoxide [DMSO]), C ( low-dose CSE), D ( medium-dose CSE) and E ( high-dose CSE).CSE models were established in male SD rats by hypodermic injection, and 60 days later observed for penile erection following subcutaneous injection of apomorphine.Then the rats were killed and the penile cavernous body obtained for the examination of NOS activity by chromatometry and the determination of Cx43 expression by laser scanning confocal fluorescence microscopy (LCSM).Results: Compared with the control and DMSO groups, penile erection frequency, NOS

  7. Beta Lactams Antibiotic Ceftriaxone Modulates Seizures, Oxidative Stress and Connexin 43 Expression in Hippocampus of Pentylenetetrazole Kindled Rats

    Science.gov (United States)

    Hussein, Abdelaziz M.; Ghalwash, Mohammed; Magdy, Khaled; Abulseoud, Osama A.

    2016-01-01

    Background and Purpose: This study aimed to investigate the effect of ceftriaxone on oxidative stress and gap junction protein (connexin 43, Cx-43) expression in pentylenetetrazole (PTZ) induced kindling model. Methods: Twenty four Sprague dawely rats were divided into 3 equal groups (a) normal group: normal rats. (b) PTZ kindled group: received PTZ at the dose of 50 mg/kg via intraperitoneal injection (i.p.) every other day for 2 weeks (c) ceftriaxone treated group: received ceftriaxone at the dose 200 mg\\kg/12 hrs via i.p. injection daily from the 6th dose of PTZ for 3 days. Racine score, latency before beginning the first myoclonic jerk and duration of the jerks used as parameters of behavioral assessment. Immunohistopathological study for Cx-43 expression in hippocampus and measurement of markers of oxidative stress (malondialdehyde [MDA], low reduced glutathione [GSH] and catalase [CAT]) in hippocampal neurons were done. Results: PTZ kindling was associated with behavioral changes (in the form high stage of Racine score, long seizure duration and short latency for the first jerk), enhanced oxidative stress state (as demonstrated by high MDA, low GSH and CAT) and up regulation of Cx43 in hippocampal regions. While, ceftriaxone treatment ameliorated, significantly, PTZ-induced convulsions and caused significant improvement in oxidative stress markers and Cx-43 expression in hippocamal regions (p < 0.05). Conclusions: These findings support the anticonvulsive effects of some beta-lactams antibiotics which could offer a possible contributor in the basic treatment of temporal lobe epilepsy. This effect might be due to reduction of oxidative stress and Cx43 expression. PMID:27390674

  8. Connexin abundance in resistance vessels from the renal microcirculation in normo- and hypertensive rats

    DEFF Research Database (Denmark)

    Braunstein, Thomas Hartig; Sørensen, Charlotte Mehlin; Holstein-Rathlou, Niels-Henrik

    2009-01-01

    The expression of connexins in renal arterioles is believed to have a profound impact on conducted responses, regulation of arteriolar tonus and renal blood flow. We have previously shown that in renal preglomerular arterioles, conducted vasomotor responses are 40% greater in spontaneously...... with SD and SHR. This high abundance of Cx37 was not related to blood pressure because normotensive SD demonstrated a level of Cx37 similar to that of SHR. Additionally, we found no evidence for an increased abundance of Cx40 and Cx43 in renal arterioles of SHR when compared with normotensive counterparts....

  9. Absence of venous valves in mice lacking Connexin37.

    Science.gov (United States)

    Munger, Stephanie J; Kanady, John D; Simon, Alexander M

    2013-01-15

    Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell-cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels.

  10. A simple RT-PCR-based strategy for screening connexin identity

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    M. Urban

    1999-08-01

    Full Text Available Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

  11. Connexin 36 is expressed in beta and connexins 26 and 32 in acinar cells at the end of the secondary transition of mouse pancreatic development and increase during fetal and perinatal life.

    Science.gov (United States)

    Pérez-Armendariz, Elia Martha; Cruz-Miguel, Lourdes; Coronel-Cruz, Cristina; Esparza-Aguilar, Marcelino; Pinzon-Estrada, Enrique; Rancaño-Camacho, Elizabeth; Zacarias-Climaco, Gerardo; Olivares, Paola Fernández; Espinosa, Ana Maria; Becker, Ingeborg; Sáez, Juan C; Berumen, Jaime; Pérez-Palacios, Gregorio

    2012-06-01

    To identify when during fetal development connexins (Cxs) 26 (Cx26) 32 (Cx32), and 36 (Cx36) begin to be expressed, as well as to characterize their spatial distribution, real time polymerase chain reaction and immunolabeling studies were performed. Total RNA from mouse pancreases at 13 and 18 days postcoitum (dpc) and 3 days postpartum (dpp) was analyzed. In addition, pancreatic sections of mouse at 13, 14, 15, 16, 18 dpc and 3 dpp and of rat at term were double labeled with either anti-insulin or anti-α-amylase and anti-Cx26 or -Cx32 or -Cx36 antibodies and studied with confocal microscopy. From day 13 dpc, Cxs 26, 32, and 36 transcripts were identified and their levels increased with age. At 13-14 dpc, Cxs 26 and 32 were localized in few acinar cells, whereas Cx36 was distributed in small beta cell clumps. From day 14 dpc onwards, the number of labeled cells and relative immunofluorescent reactivity of all three Cxs at junctional membranes of the respective cell types increased. Cxs 26 and 32 colocalized in fetal acinar cells. In rat pancreas at term, a similar connexin distribution was found. Relative Cxs levels evaluated by immunoblotting also increased (two-fold) in pancreas homogenates from day 18 dpc to 3 dpp. The early cell specific, wide distribution, and age dependent expression of Cxs 26, 32, and 36 during fetal pancreas ontogeny suggests their possible involvement in pancreas differentiation and prenatal maturation.

  12. Silencing of desmoplakin decreases connexin43/Nav1.5 expression and sodium current in HL‑1 cardiomyocytes.

    Science.gov (United States)

    Zhang, Qianhuan; Deng, Chunyu; Rao, Fang; Modi, Rohan M; Zhu, Jiening; Liu, Xiaoying; Mai, Liping; Tan, Honghong; Yu, Xiyong; Lin, Qiuxiong; Xiao, Dingzhang; Kuang, Sujuan; Wu, Shulin

    2013-09-01

    Desmosomes and gap junctions are situated in the intercalated disks of cardiac muscle and maintain the integrity of mechanical coupling and electrical impulse conduction between cells. The desmosomal plakin protein, desmoplakin (DSP), also plays a crucial role in the stability of these interconnected components as well as gap junction connexin proteins. In addition to cell‑to‑cell junctions, other molecules, including voltage‑gated sodium channels (Nav1.5) are present in the intercalated disk and support the contraction of cardiac muscle. Mutations in genes encoding desmosome proteins may result in fatal arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). Therefore, the aim of the present study was to determine whether the presence of DSP is necessary for the normal function and localization of gap junction protein connexin43 (Cx43) and Nav1.5. To examine this hypothesis, RNA interference was utilized to knock down the expression of DSP in HL‑1 cells and the content, distribution and function of Cx43 and Nav1.5 was assessed. Western blotting and flow cytometry experiments revealed that Cx43 and Nav1.5 expression decreased following DSP silencing. In addition, immunofluorescence studies demonstrated that a loss of DSP expression led to an abnormal distribution of Cx43 and Nav1.5, while scrape‑loading dye/transfer revealed a decrease in dye transfer in DSP siRNA‑treated cells. The sodium current was also recorded by the whole‑cell patch clamp technique. The results indicated that DSP suppression decreased sodium current and slowed conduction velocity in cultured cells. The present study indicates that impaired mechanical coupling largely affects electrical synchrony, further uncovering the pathogenesis of ARVC.

  13. 3,5,4′-tri-O-acetylresveratrol Ameliorates Seawater Exposure-Induced Lung Injury by Upregulating Connexin 43 Expression in Lung

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    Lijie Ma

    2013-01-01

    Full Text Available The aim of the present study was to examine the effects of 3,5,4′-tri-O-acetylresveratrol on connexin 43 (Cx43 in acute lung injury (ALI in rats induced by tracheal instillation of artificial seawater. Different doses (50, 150, and 450 mg/kg of 3,5,4′-tri-O-acetylresveratrol were administered orally for 7 days before modeling. Four hours after seawater inhalation, histological changes, contents of TNF-α, IL-1β and IL-10, and the expression of Cx43 in lungs were detected. Besides, the gap junction communication in A549 cells and human umbilical vein endothelial cells (HUVECs challenged by seawater was also evaluated. Histological changes, increased contents of inflammatory factors, upregulation in gene level, and deregulation in protein level of Cx43 in lungs stimulated by seawater were observed. On the other hand, pretreatment with 3,5,4′-tri-O-acetylresveratrol significantly inhibited infiltration of inflammation, development of pulmonary edema, and contents of inflammatory mediators in lungs. Above all, 3,5,4′-tri-O-acetylresveratrol upregulated the expression of Cx43 in both gene and protein levels, and its intermediate metabolite, resveratrol, also enhanced the gap junction communication in the two cell lines. The results of the present study suggested that administration of 3,5,4′-tri-O-acetylresveratrol may be beneficial for treatment of inflammatorycellsin lung.

  14. Effects of ferulic acid on oxidative stress, heat shock protein 70, connexin 43, and monoamines in the hippocampus of pentylenetetrazole-kindled rats.

    Science.gov (United States)

    Hussein, Abdelaziz M; Abbas, Khaled M; Abulseoud, Osama A; El-Hussainy, El-Hussainy M A

    2017-06-01

    The present study investigated the effects of ferulic acid (FA) on pentylenetetrazole (PTZ)-induced seizures, oxidative stress markers (malondialdehyde (MDA), catalase, and reduced glutathione (GSH)), connexin (Cx) 43, heat shock protein 70 (Hsp 70), and monoamines (serotonin (5-HT) and norepinephrine (NE)) levels in a rat model of PTZ-induced kindling. Sixty Sprague Dawley rats were divided into 5 equal groups: (a) normal group; (b) FA group: normal rats received FA at a dose of 40 mg/kg daily; (c) PTZ group: normal rats received PTZ at a dose of 50 mg/kg i.p. on alternate days for 15 days; (d) FA-before group: treatment was the same as for the PTZ group, except rats received FA; and (e) FA-after group: rats received FA from sixth dose of PTZ. PTZ caused a significant increase in MDA, Cx43, and Hsp70 along with a significant decrease in GSH, 5-HT, and NE levels and CAT activity in the hippocampus (p < 0.05). Pre- and post-treatment with FA caused significant improvement in behavioral parameters, MDA, CAT, GSH, 5-HT, NE, Cx43 expression, and Hsp70 expression in the hippocampal region (p < 0.05). We conclude that FA has neuroprotective effects in PTZ-induced epilepsy, which might be due to attenuation of oxidative stress and Cx43 expression and upregulation of neuroprotective Hsp70 and neurotransmitters (5-HT and NE).

  15. Role of gap junction and connexin-43 in hypoxic-ischemic brain damage

    Institute of Scientific and Technical Information of China (English)

    Jieying Lin; Niyang Lin

    2006-01-01

    OBJECTEVE:Gap junctin (GJ)is the structural basis for direct intercellular communication of nerve cells . Connexin(Cx) is the protein subunit for constructling GJ channel. Among them, Cx43is closely related with nervous system. Both Cx43 and nervous system play an important role in the pathophysiological development of hypoxic-ischemic injury. We are in attempt to investigate GJ,Cx43 and their correlations with hypoxic-ischemic brain damage by research.DATA SOURCES:Using the terms "brain gap junction"in English and "gap junction"in Chinese, we searched the Medline database and Chinese BioMedical Literature Database as well as China Hospital Knowledge Database to identify the articles published from 1996 to 2006 about GJ and brain hypoxic-ischemic injury.STUDY SELECTION:The articles were selected firstly and abstracts of 250 articles were read thuugh.Articles in which the experimental design met randomized controlled principle were included,and study articles and case reports with repetitve contents were excluded.DATA EXTRACTION:Among 53 included correlative articles, 23 were excluded for repetitive contents and the other 30 were analyzed.DATA SYNTHESIS:GJ,widely esistling in nervous system,plays a key role in maintainling normal differentiation and development as well as physiological function brain tissue.GJ channel is a hydrophilic,low-selectivity and lowohmic channel, which can provide direct channel for intercellular substance transmission and information communication. It plays an important role in the differentiation and development of nerve cells and regulation of physiological function,The funtions of GJ channel are regulated by many factors,which invilved intracellular Ph value, Ca2+concentration, ATP concentration, phosphorylation of Cx, transchannel pressure,some neurohormonal factors,regulatory factors of protein and so on. Cx43 is the main component of GJ channel in the brain tissues. Its expression in the brain tissue of mammal is the strongest

  16. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

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    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-01-01

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.

  17. Anti-arrhythmic effect of U50,488H is mediated by preserving Cx43 protein via intracellular calcium regulation%U50,488H抗心肌缺血性心律失常与调节钙-Cx43通路有关

    Institute of Scientific and Technical Information of China (English)

    林家骥; 周贺; 裴建明

    2012-01-01

    AIM:To test the hypothesis that antiarrhythmic properties of U50,488H may be mediated by preserving Cx43 protein via regulating intracellular calcium.METHODS:Using an in vivo arrhythmia study,arrhythmia was induced by temporary occlusion of the left anterior descending (LAD) coronary artery with a silk suture for 30 min in Sprague Dawley rats weighing 250 to 350 g.U50,488H,a κ-opioid receptor (κ-OR) agonist,Nor-BNI,a κ-opioid receptor antagonist,Bay K8644,a calcium channel agonist,nifedipine,a calcium channel inhibitor and heptanol,a Cx43 inhibitor were chosen to be intravenously ( i.v.) injected into a femoral vein prior to ischemia.A sham group underwent the same surgical procedures except that the suture underneath the LAD was left untied.In an in vitro arrhythmia study,each heart was quickly removed and underwent an initial 10 min of normal baseline perfusion and was subjected to perfusion at 37℃ for 30 min (for analysis of arrhythmia) or 90 min (for Western blotting).The hearts were then randomly divided into three groups:Con (normal calcium perfusion,1.5 mmol/L),high calcium (high calcium perfusion,3.3 mmol/L) and low calcium ( low calcium perfusion,0.5 mmol/L).Before and during the ischemia period,electrocardiogram (ECG) was used to measure the incidence of arrhythmias.RESULTS:By performing ECG monitoring and immunoblotting in isolated Langendorff-perfused rat hearts,high concentrations of calcium-perfused rat hearts exhibited increased cardiac arrhythmias.Diminished expression of Cx43 protein was observed.U50,488H dose-dependently inhibited L-type calcium current in single ventricular myocytes of rats using whole-cell patch clamp techniques.These effects were blocked by nor-BNI,a selective κ-OR antagonist.Administration of U50,488H before myocardial ischemia attenuated total arrhythmia scores.This effect was blocked by nor-BNI,antagonized by Bay K8644,an L-type calcium channel agonist,and by the Cx43 uncoupler heptanol.Finally,immunoblotting data

  18. Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

    Science.gov (United States)

    Voytenko, L P; Lushnikova, I V; Savotchenko, A V; Isaeva, E V; Skok, M V; Lykhmus, O Yu; Patseva, M A; Skibo, G G

    2015-08-07

    The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in

  19. Connexin 32 and 43 promoter methylation in Helicobacter pylori-associated gastric tumorigenesis.

    Science.gov (United States)

    Wang, Yu; Huang, Li-Hua; Xu, Can-Xia; Xiao, Jing; Zhou, Li; Cao, Dan; Liu, Xiao-Min; Qi, Yong

    2014-09-07

    To explore the mechanism of abnormal Connexin (Cx) 32 and Cx43 expression in the gastric mucosa after Helicobacter pylori (H. pylori) infection. Biopsy specimens of gastric mucosa in different gastric carcinogenesis stages with H. pylori infection, that is, non-atrophic gastritis (NAG; n = 24), chronic atrophic gastritis (CAG; n = 25), intestinal metaplasia (IM; n = 28), dysplasia (DYS; n = 24), and gastric cancer (GC; n = 30), as well as specimens of normal gastric mucosa without H. pylori infection (NGM; n = 25), were confirmed by endoscopy and pathological examination. Cx32 and Cx43 mRNA expression was detected by real-time polymerase chain reaction (PCR). Cx32 and Cx43 promoter CpG island methylation status was determined by methylation-specific PCR (MSP), bisulfite PCR sequencing (BSP) and MassArray methods. The relative mRNA expression levels in the gastric mucosa of patients with NGM, NAG, CAG, IM, DYS and GC were 0.146 ± 0.011, 0.133 ± 0.026, 0.107 ± 0.035, 0.039 ± 0.032, 0.037 ± 0.01 and 0.03 ± 0.011 for Cx32; and 0.667 ± 0.057, 0.644 ± 0.051, 0.624 ± 0.049, 0.555 ± 0.067, 0.536 ± 0.058 and 0.245 ± 0.121 for Cx43, respectively, which were gradually decreasing and significantly different (GC vs NGM: P infection-associated gastric carcinogenesis, and it is associated with hypermethylation of these genes' promoter.

  20. Cellular and deafness mechanisms underlying connexin mutation induced hearing loss – A common hereditary deafness

    Directory of Open Access Journals (Sweden)

    Jeffrey C Wingard

    2015-05-01

    Full Text Available Hearing loss due to mutations in the connexin gene family which encodes gap junctional proteins is a common form of hereditary deafness. In particular, connexin 26 (Cx26, GJB2 mutations are responsible for ~50% of nonsyndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate that congenital deafness mainly results from cochlear developmental disorders rather than hair cell degeneration and endocochlear potential (EP reduction, while late-onset hearing loss results from reduction of active cochlear amplification, even though cochlear hair cells have no connexin expression. Moreover, new experiments further demonstrate that the hypothesized K+-recycling disruption is not a principal deafness mechanism for connexin deficiency induced hearing loss. Additionally, there is no clear relationship between specific changes in connexin (channel functions and the phenotypes of mutation-induced hearing loss. Cx30, Cx29, Cx31, and Cx43 mutations can also cause hearing loss with distinct pathological changes in the cochlea. These new studies provide invaluable information about deafness mechanisms underlying connexin mutation induced hearing loss and also provide important information for developing new protective and therapeutic strategies for this common deafness. However, the detailed cellular mechanisms underlying these pathological changes and pathogeneses of specific-mutation induced hearing loss remain unclear. Finally, little information is available for humans. Further studies to address these deficiencies are urgently required.

  1. 连续性血液净化对肺泡上皮细胞Connexin43的影响%Effects of continuous blood purification on Connexin43 in human alveolar epi-thelial cells

    Institute of Scientific and Technical Information of China (English)

    杨溢; 甘华; 李正荣; 文以君; 王喜超

    2009-01-01

    AIM: To investigate the effect of continuous blood purification (CBP) on the expression of Connexin43 (Cx43) in human alveolar epithelial cells (AECs) induced by the serum of patients with severe acute pancreatitis (SAP) accompanied with acute lung injury (ALI). METHODS: Fasting serum of healthy volunteers and serum of patients with SAP and ALI at pre-CBP, 6-hour-CBP and 20-hour-CBP were collected, Immunofluores-cence staining and RT-PCR were used to detect the expression of Cx43 and Cx43 mRNA in AECs separately cultured by the serum from different groups for 48 h in vitro. ELISA was used to detect the level of serum TNF-α in each group. RESULTS: Cx43 posi-tive staining in each patient group was significantly less than that in healthy control group. With the increase of the treatment time, the positive staining gradually increased. The relative expression of Cx43 mRNA in pre-CBP group(0.08±0.01) was significantly leas than that in healthy control group (0.57±0.02) (P<0.01). With the increase of the treatment time, the expression gradually increased in 6-hoar-CBP group(0.23±0.02) and 20-hour-CBP group(0.36±0.02) (P<0.01). The level of serum TNF-α in pre-CBP group (59.43±4.50) ng/L was significantly higher than that in healthy control group(16.06±3.68) ng/L(P<0.01). With the increase of the treatment time, the level gradually decreased in 6-hour-CBP group (41.16±3.49) ng/L and 20-hour-CBP group (34.65±3.22) ng/L (P<0.01). The expression of Cx43 mRNA was negatively correlated with the level of TNF-α. CONCLUSION: The decrease of Cx43 in AECs plays a part in the process of SAP patients with secondary ALI. CBP up-regulates the expression of Cx43 by removing TNF-α and thus protects the respiratory function.%目的:研究连续性血液净化(CBP)对重症急性胰腺炎(SAP)伴急性肺损伤(ALI)患者血清诱导的人肺泡上皮细胞(AECs)间隙连接蛋白Connexin43(Cx43)表达的影响.方法:采集健康志愿者清晨空腹及SAP伴ALI患者CBP

  2. Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin 43 hemichannels

    Science.gov (United States)

    Orellana, Juan A.; Hernández, Diego E.; Ezan, Pascal; Velarde, Victoria; Bennett, Michael V. L.; Giaume, Christian; Sáez, Juan C.

    2009-01-01

    Brain ischemia causes more extensive injury in hyperglycemic than normoglycemic subjects, and the increased damage is to astroglia as well as neurons. In the present work, we found that in cortical astrocytes from rat or mouse, reoxygenation after hypoxia in a medium mimicking interstitial fluid during ischemia increases hemichannel activity and decreases cell-cell communication via gap junctions as indicated by dye uptake and dye coupling, respectively. These effects were potentiated by high glucose during the hypoxia in a concentration-dependent manner (and by zero glucose) and were not observed in connexin 43−/− astrocytes. The responses were transient or persistent after short and long periods of hypoxia, respectively. The persistent responses were associated with a progressive reduction in cell viability that was prevented by La3+ or peptides that block connexin 43 (Cx43) hemichannels or by inhibition of p38 MAP kinase prior to hypoxia-reoxygenation but not by treatments that block pannexin hemichannels. Block of Cx43 hemichannels did not affect the reduction in gap junction mediated dye coupling observed during reoxygenation. Cx43 hemichannels may be a novel therapeutic target to reduce cell death following stroke, particularly in hyperglycemic conditions. PMID:19705457

  3. Reversal of TRESK Downregulation Alleviates Neuropathic Pain by Inhibiting Activation of Gliocytes in the Spinal Cord.

    Science.gov (United States)

    Zhou, Jun; Chen, Hongtao; Yang, Chengxiang; Zhong, Jiying; He, Wanyou; Xiong, Qingming

    2017-02-03

    Despite the consensus that activation of TWIK-related spinal cord K(+) (TRESK) might contribute to the pathogenesis of chronic pain, the specific mechanisms underlying the transfer and development of pain signals still remain obscure. In the present study, we validated that TRESK was expressed in neurons instead of glial cells. Furthermore, in the SNI model of neuropathic pain (NP), downregulation of TRESK in spinal cord neurons resulted in upregulation of connexin 36 (Cx36) and connexin 43 (Cx43), both being subtypes of gap junctions in the spinal cord, with gliocytes in the spinal cord activated ultimately. Compared with SNI rats, intrathecal injection of TRESK gene recombinant adenovirus significantly downregulated the expression levels of Cx36 and Cx43 and suppressed the activation of gliocytes in the spinal cord, with hyperalgesia significantly reduced. In conclusion, TRESK contributes to the pathogenesis of NP by upregulation of synaptic transmission and activation of gliocytes.

  4. Targeting connexin 43 in diabetic wound healing: Future perspectives

    Directory of Open Access Journals (Sweden)

    Bajpai S

    2009-01-01

    Full Text Available The unknown mechanisms of impaired tissue repair in diabetes mellitus are making this disease a serious clinical problem for the physicians worldwide. The lacuna in the knowledge of the etiology of diabetic wounds necessitates more focused research in order to develop new targeting tools with higher efficacy for their effective management. Gap-junction proteins, connexins, have shown some promising results in the process of diabetic wound healing. Till now the role of connexins has been implicated in peripheral neuropathy, deafness, skin disorders, cataract, germ cell development and treatment of cancer. Recent findings have revealed that gap junctions play a key role in normal as well as diabetic wound healing. The purpose of this review is to provide the information related to etiology, epidemiology, clinical presentation of diabetic wounds and to analyze the role of connexin 43 (Cx43 in the diabetic wound healing process. The current control strategies and the future research challenges have also been discussed briefly in this review.

  5. Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36

    Science.gov (United States)

    Corsini, Silvia; Tortora, Maria; Rauti, Rossana; Nistri, Andrea

    2017-01-01

    Motoneuron disease including amyotrophic lateral sclerosis may be due, at an early stage, to deficit in the extracellular clearance of the excitatory transmitter glutamate. A model of glutamate-mediated excitotoxic cell death based on pharmacological inhibition of its uptake was used to investigate how activation of neuronal nicotinic receptors by nicotine may protect motoneurons. Hypoglossal motoneurons (HMs) in neonatal rat brainstem slices were exposed to the glutamate uptake blocker DL-threo-β-benzyloxyaspartate (TBOA) that evoked large Ca2+ transients time locked among nearby HMs, whose number fell by about 30% 4 h later. As nicotine or the gap junction blocker carbenoxolone suppressed bursting, we studied connexin 36 (Cx36), which constitutes gap junctions in neurons and found it largely expressed by HMs. Cx36 was downregulated when nicotine or carbenoxolone was co-applied with TBOA. Expression of Cx36 was preferentially observed in cytosolic rather than membrane fractions after nicotine and TBOA, suggesting protein redistribution with no change in synthesis. Nicotine raised the expression of heat shock protein 70 (Hsp70), a protective factor that binds the apoptotic-inducing factor (AIF) whose nuclear translocation is a cause of cell death. TBOA increased intracellular AIF, an effect blocked by nicotine. These results indicate that activation of neuronal nicotinic receptors is an early tool for protecting motoneurons from excitotoxicity and that this process is carried out via the combined decrease in Cx36 activity, overexpression of Hsp70 and fall in AIF translocation. Thus, retarding or inhibiting HM death may be experimentally achieved by targeting one of these processes leading to motoneuron death. PMID:28617431

  6. Gap junctions in the inner ear: comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals.

    Science.gov (United States)

    Forge, Andrew; Becker, David; Casalotti, Stefano; Edwards, Jill; Marziano, Nerissa; Nevill, Graham

    2003-12-08

    The distribution and size of gap junctions (GJ) in the sensory epithelia of the inner ear have been examined in a reptile (gecko), birds (chicken and owl), and mammals (mouse, guinea pig, gerbil, and bat), and the connexin composition of GJs in the mammalian inner ear has been assessed. Freeze fracture revealed a common pattern of GJ distribution in auditory and vestibular sensory epithelia in the different vertebrate classes. In all these tissues, GJs are numerous, often occupying more than 25% of the plasma membrane area of supporting cells and sometimes composed of more than 100,000 channels. Screening for 12 members of the connexin family in the mammalian inner ear by RT-PCR, Western blotting, and immunohistochemistry revealed four connexin isotypes, cx26, cx30, cx31, and cx43, in the cochlea and three, cx26, cx30, and cx43, in the vestibular organs. With antibodies characterised for their specificity, cx26 and cx30 colocalised in supporting cells of the organ of Corti, in the basal cell region of the stria vascularis, and in type 1 fibrocytes of the spiral ligament. No other connexin was detected in these regions. Cx31 was localised among type 2 fibrocytes below the spiral prominence, a region where cx30 was not expressed and cx26 expression appeared to be low. Cx43 was detected only in the region of "tension fibrocytes" lining the inner aspect of the otic capsule. This suggests separate functional compartments in the cochlea. In addition to cx26 and cx30, cx43 was detected in supporting cells of the vestibular sensory epithelia. Where cx26 and cx30 were colocalised, double immunogold labelling of thin sections showed both cx26 and cx30 evenly distributed in individual GJ plaques, a pattern consistent with the presence of heteromeric connexons. Coimmunoprecipitation of cochlear membrane proteins solubilised with a procedure that preserves the oligomeric structure of connexons confirmed the presence of heteromeric cx26/cx30 connexons. Heteromeric cx26/cx30

  7. Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

    Directory of Open Access Journals (Sweden)

    P. Yahuaca

    2000-04-01

    Full Text Available The carboxyl-terminal (CT domain of connexin43 (Cx43 has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.

  8. Human articular chondrocytes express multiple gap junction proteins: differential expression of connexins in normal and osteoarthritic cartilage.

    Science.gov (United States)

    Mayan, Maria D; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J

    2013-04-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Connexins in Prostate Cancer Initiation and Progression

    Science.gov (United States)

    2013-11-01

    Endogenous Cx43 in RWPE-1 and PZ- HPV -7 cell lines. To explore the role of GJs composed of Cx43 and Cx32 regulates the acinar differentiation of RWPE...1 and PZ- HPV -7 cells in 3-D cutlures, we have knocked down the expression of endogenous Cx43 using Sh-Cx43RNA described in our previous studies...5271 and 5242, Brinkmann Instrument , Westbury, NY) mounted on a Leica DMIRE2 microscope as described previously (24). The micro- injected cells were

  10. Gap junction proteins in the light-damaged albino rat.

    Science.gov (United States)

    Guo, Cindy X; Tran, Henry; Green, Colin R; Danesh-Meyer, Helen V; Acosta, Monica L

    2014-01-01

    Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression.

  11. Independence of connexin expression and vasomotor conduction from sympathetic innervation in hamster feed arteries.

    Science.gov (United States)

    Looft-Wilson, Robin C; Haug, Sara J; Neufer, P Darrell; Segal, Steven S

    2004-01-01

    Vasomotor responses can travel along the wall of resistance microvessels by two distinct mechanisms: cell-to-cell conduction through gap junctions or the release of neurotransmitter along perivascular nerves. It is unknown whether vascular innervation influences the expression of connexin molecules which comprise gap junctions, or the conduction of vasomotor responses. In feed arteries of the hamster retractor muscle (RFA), the authors tested whether sympathetic denervation would alter the expression of connexin isoforms and the conduction of vasomotor responses. Using intact vessels with sympathetic innervation and those 7-8 days following denervation surgery, mRNA expression was quantified using real-time PCR, cellular localization of Cx protein was characterized using immunohistochemistry, and vasomotor responses to dilator and constrictor stimuli were evaluated in isolated pressurized RFA. Connexin protein localization and mRNA expression were similar between innervated and denervated vessels. mRNA levels were Cx43 = Cx37 > Cx45 > Cx40. Vasodilation to acetylcholine conducted >/=2000 microm along innervated and denervated vessels, as did the biphasic conduction of vasoconstriction and vasodilation in response to KCl. Vasoconstriction to phenylephrine conducted <500 microm and was attenuated (p <.05) in denervated vessels. The profile of connexin expression and the conduction of vasomotor responses are largely independent of sympathetic innervation in feed arteries of the hamster retractor muscle (RFA).

  12. Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication

    Science.gov (United States)

    Li, Bin; Wu, Yingya; Liu, Xijuan; Tan, Yuhui; Du, Biaoyan

    2017-01-01

    Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling. PMID:27903977

  13. Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels.

    Science.gov (United States)

    Chi, Yuan; Gao, Kun; Zhang, Hui; Takeda, Masayuki; Yao, Jian

    2014-07-01

    Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well-known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non-junctional connexin43 (Cx43) hemichannels, pore-forming α-haemolysin and channels involved in ATP release under hypotonic conditions. Hemichannels were activated by removing extracellular Ca(2+) . The influences of suramin on hemichannel activities were evaluated by its effects on influx of fluorescent dyes and efflux of ATP. The membrane permeability and integrity were assessed through cellular retention of preloaded calcein and LDH release. Suramin blocked Cx43 hemichannel permeability induced by removal of extracellular Ca(2+) without much effect on Cx43 expression and gap junctional intercellular communication. This action of suramin was mimicked by its analogue NF023 and NF449 but not by another P2 purinoceptor antagonist PPADS. Besides hemichannels, suramin also significantly blocked intracellular and extracellular exchanges of small molecules caused by α-haemolysin from Staphylococcus aureus and by exposure of cells to hypotonic solution. Furthermore, it prevented α-haemolysin- and hypotonic stress-elicited cell injury. Suramin blocked membrane channels and protected cells against toxin- and hypotonic stress-elicited injury. Our finding provides novel mechanistic insights into the pharmacological actions of suramin. Suramin might be therapeutically exploited to protect membrane integrity under certain pathological situations. © 2014 The British Pharmacological Society.

  14. 白藜芦醇上调心肌缝隙连接蛋白43的表达降低大鼠心肌梗死后心律失常%Resveratrol exerts anti-arrhythmic effects in rats with myocardial infarction through up-regulation of cardiac connexin43 expression

    Institute of Scientific and Technical Information of China (English)

    王光宇; 毕亚光; 叶婷婷; 张庆勇; 魏盟

    2016-01-01

    目的:探讨白藜芦醇对大鼠心肌梗死( MI)后室性心律失常的影响。方法将造模成功的心肌梗死SD大鼠随机分为心肌梗死组(MI组,n=8)和白藜芦醇组(Res组,n=8),另设假手术组(Sham组,n=8),其中Res组给予10 mg・ kg-1・ d-1剂量的灌胃(1次/d),MI组和sham组给予5%羧甲基纤维素钠灌胃。4周后,对大鼠行在体程序性电生理刺激实验;Masson ’ s染色观察心肌梗死面积变化;免疫组织化学法检测心肌缝隙连接蛋白43(connexin43,Cx43)和 Cx43的368位丝氨酸磷酸化(P368Cx43)分布水平;免疫印迹法检测心肌 Cx43、P368Cx43和蛋白激酶 Cβ2(PKCβ2)表达水平。结果与MI组相比,白藜芦醇能显著降低室性心律失常的诱发率(87.5%对37.5%,P<0.05),增加心室颤动(室颤)阈值[(3.2±0.3)V对(5.2±0.8)V,P<0.05];与sham组相比,MI组的Cx43表达量显著降低(P<0.05),然而P368Cx43和PKCβ2的表达量却显著升高(P<0.05)。白藜芦醇能明显增加梗死心肌Cx43表达量(P<0.05),降低P368Cx43表达和PKCβ2表达(P<0.05)。结论白藜芦醇能有效改善大鼠MI心肌电生理学特性,提高心肌Cx43表达,减少室性心律失常的发生率。%Objective To investigate effect of resveratrol on ventricular arrhythmias in rats with myo-cardial infarction .Methods Twenty-four adult male Sprague-Dawley rats were randomly divided into myocar-dial infarction group(MI group,n=8), resveratrol group (Res group,n=8) after successful establishment of MI model,and sham group (n=8) which underwent thoracotomy and pericardiotomy without the left anterior descending ligation.Res group was given a dose of 10 mg・ kg-1・ d-1 resveratrol (i.g.once a day) for 4 weeks. MI group and sham group received 5%sodium carboxymethyl cellulose gavage .At 4 weeks after induced MI the programmed electrical

  15. Cellular and Deafness Mechanisms Underlying Connexin Mutation-Induced Hearing Loss - A Common Hereditary Deafness.

    Science.gov (United States)

    Wingard, Jeffrey C; Zhao, Hong-Bo

    2015-01-01

    Hearing loss due to mutations in the connexin gene family, which encodes gap junctional proteins, is a common form of hereditary deafness. In particular, connexin 26 (Cx26, GJB2) mutations are responsible for ~50% of non-syndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate that congenital deafness mainly results from cochlear developmental disorders rather than hair cell degeneration and endocochlear potential reduction, while late-onset hearing loss results from reduction of active cochlear amplification, even though cochlear hair cells have no connexin expression. However, there is no apparent, demonstrable relationship between specific changes in connexin (channel) functions and the phenotypes of mutation-induced hearing loss. Moreover, new experiments further demonstrate that the hypothesized K(+)-recycling disruption is not a principal deafness mechanism for connexin deficiency induced hearing loss. Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), and Cx43 (GJA1) mutations can also cause hearing loss with distinct pathological changes in the cochlea. These new studies provide invaluable information about deafness mechanisms underlying connexin mutation-induced hearing loss and also provide important information for developing new protective and therapeutic strategies for this common deafness. However, the detailed cellular mechanisms underlying these pathological changes remain unclear. Also, little is known about specific mutation-induced pathological changes in vivo and little information is available for humans. Such further studies are urgently required.

  16. hESC expansion and stemness are independent of connexin forty-three-mediated intercellular communication between hESCs and hASC feeder cells.

    Directory of Open Access Journals (Sweden)

    Jin-Su Kim

    Full Text Available BACKGROUND: Human embryonic stem cells (hESCs are a promising and powerful source of cells for applications in regenerative medicine, tissue engineering, cell-based therapies, and drug discovery. Many researchers have employed conventional culture techniques using feeder cells to expand hESCs in significant numbers, although feeder-free culture techniques have recently been developed. In regard to stem cell expansion, gap junctional intercellular communication (GJIC is thought to play an important role in hESC survival and differentiation. Indeed, it has been reported that hESC-hESC communication through connexin 43 (Cx43, one of the major gap junctional proteins is crucial for the maintenance of hESC stemness during expansion. However, the role of GJIC between hESCs and feeder cells is unclear and has not yet been reported. METHODOLOGY/PRINCIPAL FINDINGS: This study therefore examined whether a direct Cx43-mediated interaction between hESCs and human adipose-derived stem cells (hASCs influences the maintenance of hESC stemness. Over 10 passages, hESCs cultured on a layer of Cx43-downregulated hASC feeder cells showed normal morphology, proliferation (colony growth, and stemness, as assessed by alkaline phosphatase (AP, OCT4 (POU5F1-Human gene Nomenclature Database, SOX2, and NANOG expression. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that Cx43-mediated GJIC between hESCs and hASC feeder cells is not an important factor for the conservation of hESC stemness and expansion.

  17. Genomic instability induced in distant progeny of bystander cells depends on the connexins expressed in the irradiated cells.

    Science.gov (United States)

    de Toledo, Sonia M; Buonanno, Manuela; Harris, Andrew L; Azzam, Edouard I

    2017-06-15

    To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells. A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or (137)Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20-25 population doublings). Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing ((137)Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA

  18. Effect of calcium channel blocker on gap junctional connexin 43 in infarcted myocardium in rats%钙通道阻滞剂对梗死心肌中不同部位心肌连接蛋白43表达的影响

    Institute of Scientific and Technical Information of China (English)

    杨永健; 速晓华; 唐兵; 李德; 杨大春

    2011-01-01

    目的:研究L和L/T型钙通道阻制剂对梗死心脏不同部位(坏死区域、肥厚区域等)心肌组织中连接蛋白43(Cx43)的影响.方法:随机将大鼠48只分为假手术组、心肌梗死(MI)组、阿莫地平(L型钙通道阻滞剂)组和米贝拉地尔(L/T型钙通道阻滞剂)组(每组n=12只).通过结扎大鼠左冠状动脉建立MI模型,术前7 d,上述4个组分别用安慰剂、L型钙通道阻滞剂阿莫地平4 mg/(kg·d)和L/T型钙通道阻滞剂米贝拉地尔10 mg/(kg·d).术后1、3、7 d,分别检测左心室游离壁(LVFW,梗死区)、心室间隔(IS,肥厚区)和右心室壁(Rv),正常心肌组织中Cx43蛋白的表达.术后7 d显微直视下测LVFW处MI病灶的大小、IS的厚度及左心室的大小.结果:IS中Cx43蛋白表达于术后1、3、7 d呈逐渐增加的趋势;LVFW中Cx43蛋白的表达于术后1、3、7 d时均处于低水平,与对照组相比差异显著(P<0.05).RV中Cx43蛋白的表达于术后1、3、7 d无显著差异,与对照组相比也无显著性差异.米贝拉地尔能明显地抑制LVFW心肌组织中Cx43表达的下调,缩小MI病灶;阿莫地平则抑制肥厚心肌中Cx43蛋白的表达,明显抑制IS的肥厚.结论:MI病理过程中,梗死病灶内Cx43的表达下调,肥厚组织中Cx43的表达上调.L和L/T型钙通道阻滞剂均能减轻心肌重构与选择性地调节心肌组织中Cx43的表达有关.%AIM: To investigate the effect of cardiac L- and L/T-type Ca2+ channels on gap junctional connexin 43 ( Cx43 ) in myocardium infarcted heart remodeling of rats. METHODS: Rat myocardium infarction model was established by permanent ligation of the left coronary artery. Infarcted rats were treated with oral placebo, amlodipine [ L-channel blockade, 4 mg/( kg · day) ] or mibefradil [ L/T-channel blockade, 10 mg/(kg·day] beginning 7 days before induction of myocardial infarction (MI). Protein levels of Cx43 were measured 1, 3 and 7 days postcoronary occlusion in the noninfarcted and

  19. Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

    Directory of Open Access Journals (Sweden)

    Hussein Mansour

    Full Text Available We investigated age-associated changes in retinal astrocyte connexins (Cx by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC. We compared Wistar rat retinal wholemounts in animals aged 3 (young adult, 9 (middle-aged and 22 months (aged. We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05 but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05. With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.

  20. Effect of doxycycline on connexin 43 protein expression of cardiomyoblast cells in anoxic rat%多西环素对缺氧大鼠心肌细胞缝隙连接蛋白43表达的影响

    Institute of Scientific and Technical Information of China (English)

    罗刚; 巫相宏; 安迪; 黄文

    2013-01-01

    目的 探讨多西环素对缺氧大鼠H9c2心肌细胞缝隙连接蛋白43 (connexin 43,Cx 43)表达的影响及其可能的机制.方法 培养大鼠H9c2心肌细胞,用Western Blotting法检测缺氧6h、12h、24h后Cx43总蛋白的表达量及基质金属酶抑制剂(多西环素)、PI3K抑制剂(LY294002)、ERK1/2抑制剂(U0126)干预缺氧H9c2心肌细胞后Cx43总蛋白的表达量改变.结果 缺氧组较空白对照组Cx43总蛋白表达量明显降低,差异有统计学意义(P<0.01).缺氧6h时,多西环素和U0126干预组较缺氧组Cx43总蛋白表达量明显增高,差异有统计学意义(P<0.05);缺氧12h时,U0126干预组Cx43总蛋白表达量仍较缺氧组增高,差异有统计学意义(P<0.05);缺氧24 h时,多西环素和U0126干预后Cx43总蛋白表达量与缺氧组比较,差异无统计学意义(P>0.0.05).LY294002干预组Cx43总蛋白表达量在观察时间范围内均较缺氧组显著降低,差异有统计学意义(P<0.01).结论 在培养的大鼠H9c2心肌细胞中,多西环素通过抑制基质金属酶增加缺氧心肌细胞Cx43总蛋白表达量,该作用至少部分抑制由ERK1/2信号介导的传导通路.%Objectives To investigate the effects and the possible mechanisms of doxycycline on the expression of connexin 43 (Cx43) in rat H9c2 cardiomyoblast cells under hypoxic conditions.Methods H9c2 cardiomyoblast cells were cultured with hypoxia treatment for 6 h,12 h and 24 h.Expressions of Cx43 in cardiomyocytes were quantified by Western Blotting,as well as the changes intervened by matrix metalloproteinases (MMPs) inhibitor (doxycycline),PI3K inhibitor (LY294002),ERK1/2 inhibitor (U0126) under hypoxic conditions.Results Expression of Cx43 in hypoxia group was obviously lower than that in normal control group (P<0.01).Expressions of Cx43 in doxycycline group and U0126 group were obviously higher than that in hypoxia group after 6 h of hypoxia (P<0.05).Expression of Cx43 in U0126 group was still obviously

  1. Activation, Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

    DEFF Research Database (Denmark)

    Hansen, Daniel Bloch; Ye, Zu-Cheng; Calloe, Kirstine

    2014-01-01

    . Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small...... atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed...

  2. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring.

    Science.gov (United States)

    Rossini, Kamila Fernanda; Oliveira, Camila Andrea de; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-07-01

    The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de

  3. Connexin 30.2 is expressed in mouse pancreatic beta cells.

    Science.gov (United States)

    Coronel-Cruz, C; Hernández-Tellez, B; López-Vancell, R; López-Vidal, Y; Berumen, J; Castell, A; Pérez-Armendariz, E M

    2013-09-06

    Nowadays, connexin (Cx) 36 is considered the sole gap junction protein expressed in pancreatic beta cells. In the present research we investigated the expression of Cx30.2 mRNA and protein in mouse pancreatic islets. Cx30.2 mRNA and protein were identified in isolated islet preparations by qRT-PCR and Western blot, respectively. Immunohistochemical analysis showed that insulin-positive cells were stained for Cx30.2. Confocal images from double-labeled pancreatic sections revealed that Cx30.2 and Cx36 fluorescence co-localize at junctional membranes in islets from most pancreases. Abundant Cx30.2 tiny reactive spots were also found in cell cytoplasms. In beta cells cultured with stimulatory glucose concentrations, Cx30.2 was localized in both cytoplasms and cell membranes. In addition, Cx30.2 reactivity was localized at junctional membranes of endothelial or cluster of differentiation 31 (CD31) positive cells. Moreover, a significant reduction of Cx30.2 mRNA was found in islets preparations incubated for 24h in 22mM as compared with 3.3mM glucose. Therefore, it is concluded that Cx30.2 is expressed in beta and vascular endothelial cells of mouse pancreatic islets.

  4. An atrial-fibrillation-linked connexin40 mutant is retained in the endoplasmic reticulum and impairs the function of atrial gap-junction channels

    Directory of Open Access Journals (Sweden)

    Yiguo Sun

    2014-05-01

    Full Text Available Connexin40 (Cx40-containing gap-junction channels are expressed in the atrial myocardium and provide a low-resistance passage for rapid impulse propagation. A germline mutation in the GJA5 gene, which encodes Cx40, resulting in a truncated Cx40 (Q49X was identified in a large Chinese family with lone (idiopathic atrial fibrillation (AF. This mutation co-segregated with seven AF probands in an autosomal-dominant way over generations. To test the hypothesis that this Cx40 mutant affects the distribution and function of atrial gap junctions, we studied the Q49X mutant in gap-junction-deficient HeLa and N2A cells. The Q49X mutant, unlike wild-type Cx40, was typically localized in the cytoplasm and failed to form gap-junction plaques at cell-cell interfaces. When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43. Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43. Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum. These findings provide evidence that the Q49X mutant is capable of impairing gap-junction distribution and function of key atrial connexins, which might play a role in the predisposition to and onset of AF.

  5. Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus.

    Science.gov (United States)

    Esen, Nilufer; Shuffield, Debbie; Syed, Mohsin M D; Kielian, Tammy

    2007-01-01

    Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits. Copyright 2006 Wiley-Liss, Inc.

  6. [Dynamics of local expression of connexin-43 and basic fibroblast growth factor receptors in patients with skin and soft-tissue infections against the background of diabetes mellitus type II].

    Science.gov (United States)

    Vinnik, Iu S; Salmina, A B; Tepliakova, O V; Drobushevskaia, A I; Malinovskaia, N A; Pozhilenkova, E A; Morgun, A V; Gitlina, A G

    2014-01-01

    Clinical results of wound healing dynamics were studied in 60 patients with soft-tissue infection against the background of diabetes mellitus type II. At the same time the study considered indices of intercellular contacts protein tissue expression such as connexin 43 (Cx43) and basic fibroblast growth factor receptors (bFGFR). The basic therapy of biopsy material of wound borders was applied. The reduction of bFGFR expression and the minor growth of Cx43 expression were observed. The pain syndrome proceeded for a long time and there were signs of perifocal inflammation, retard wound healing with granulation tissue. The application of combined method of ozone therapy which included autohemotherapy with ozone and an external management of wound by ozone-oxygen mixture facilitated to considerable shortening of inflammatory phase and regeneration. It was associated with increased Cx43 expression (in 1.9 times) in comparison with initial level and bFGFR was enlarged in 1.7 times to eighth day of postoperative period.

  7. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

    Science.gov (United States)

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2015-10-12

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.

  8. Asymmetric expression of connexins between luminal epithelial- and myoepithelial- cells is essential for contractile function of the mammary gland.

    Science.gov (United States)

    Mroue, Rana; Inman, Jamie; Mott, Joni; Budunova, Irina; Bissell, Mina J

    2015-03-01

    Intercellular communication is essential for glandular functions and tissue homeostasis. Gap junctions couple cells homotypically and heterotypically and co-ordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and the distribution of some Cx subtypes in the heterotypic gap junctions is not symmetrical; in the murine mammary gland, Cx26, Cx30 and Cx32 are expressed only in the luminal epithelial cells and Cx43 is expressed only in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy and throughout lactation suggesting essential roles for these proteins in the functional secretory activity of the gland. Transgenic (Tg) mice over-expressing Cx26 driven by keratin 5 promoter had an unexpected mammary phenotype: the mothers were unable to feed their pups to weaning age leading to litter starvation and demise in early to mid-lactation. The mammary gland of K5-Cx26 female mice developed normally and produced normal levels of milk protein, suggesting a defect in delivery rather than milk production. Because the mammary gland of K5-Cx26 mothers contained excessive milk, we hypothesized that the defect may be in an inability to eject the milk. Using ex vivo three-dimensional mammary organoid cultures, we showed that tissues isolated from wild-type FVB females contracted upon treatment with oxytocin, whereas, organoids from Tg mice failed to do so. Unexpectedly, we found that ectopic expression of Cx26 in myoepithelial cells altered the expression of endogenous Cx43 resulting in impaired gap junction communication, demonstrated by defective dye coupling in mammary epithelial cells of Tg mice. Inhibition of gap junction communication or knock-down of Cx43 in organoids from wild-type mice impaired contraction in response to oxytocin, recapitulating the observations from the mammary glands of Tg mice. We conclude that Cx26 acts as a trans-dominant negative for Cx43 function in

  9. Niflumic acid relaxes mesenteric small artery through downregulating connexin 43 expression in smooth muscle cells from spontaneously hypertensive rat%尼氟灭酸对自发性高血压大鼠肠系膜微小动脉平滑肌细胞连接蛋白43的下调作用

    Institute of Scientific and Technical Information of China (English)

    刘卫东; 张雯; 司军强; 马克涛; 李丽

    2014-01-01

    目的 探讨尼氟灭酸(niflumic acid,NFA)对自发性高血压大鼠(SHR)肠系膜微小动脉平滑肌细胞缝隙连接中连接蛋白43(connexin43,Cx43)表达量的影响.方法 通过尾动脉无创血压测定Wistar大鼠和SHR的血压值.应用压力肌动图检测不同浓度NFA对Wistar大鼠和SHR肠系膜微小动脉舒缩功能的影响.Western blot检测Wistar大鼠和SHR肠系膜动脉Cx43表达的差异.Western blot检测不同浓度NFA处理原代培养的SHR肠系膜微小动脉平滑肌细胞Cx43的蛋白表达水平.结果 苯肾上腺素(phenylephrine,PE)可以引起肠系膜微小动脉血管预收缩至(193±13.5) μm,而3×10-4mol/L的NFA可以使其舒张至初始水平(275±17.1) μm,且Wistar大鼠的舒张反应显著高于SHR,差异有统计学意义(F=55.47,P<0.05,n=6).SHR肠系膜动脉血管一级分支和三级分支Cx43蛋白的表达水平分别高于Wistar大鼠相应血管分支,且肠系膜三级分支血管Cx43蛋白的表达水平高于一级分支,差异有统计学意义(F=1 014.43,P<0.01).不同浓度的NFA处理的肠系膜微小动脉培养的平滑肌细胞,其Cx43蛋白的表达水平均低于对照,且呈浓度依赖性,差异有统计学意义(F=1 480.20,P<0.01).结论 Cx43可能介导了SHR肠系膜微小动脉平滑肌细胞间的通讯,从而影响血管的舒缩反应.而NFA可以降低平滑肌细胞Cx43蛋白的表达,并在一定程度上舒张血管.%Objective To explore the impact of niflumic acid (NFA) on connexin 43 (Cx43)expression in smooth muscle cells of mesenteric small artery from spontaneously hypertensive rats (SHR).Methods Blood pressure of Wistar rats and spontaneously hypertensive rats (SHR) was measured by the tail cuff method.Relaxation and contraction of mesenteric small artery from Wistar rat and SHR were evaluated by pressure myograph system under various concentrations of NFA.Protein Cx43 expression on primary cultured mesenteric smooth muscle cells from Wistar rats and SHR was

  10. Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera).

    Science.gov (United States)

    Wang, Li; Li, Gang; Wang, Jinhong; Ye, Shaohui; Jones, Gareth; Zhang, Shuyi

    2009-04-01

    Gap junction protein connexin43 (Cx43), encoded by the GJA1 gene, is the most abundant connexin in the cardiovascular system and was reported as a crucial factor maintaining cardiac electrical conduction, as well as having a very important function in facilitating the recycling of potassium ions from hair cells in the cochlea back into the cochlear endolymph during auditory transduction processes. In mammals, bats are the only taxon possessing powered flight, placing exceptional demand on many organismal processes. To meet the demands of flying, the hearts of bats show many specialties. Moreover, ultrasonic echolocation allows bat species to orientate and often detect and locate food in darkness. In this study, we cloned the full-length coding region of GJA1 gene from 12 different species of bats and obtained orthologous sequences from other mammals. We used the maximum likelihood method to analyse the evolution of GJA1 gene in mammals and the lineage of bats. Our results showed this gene is much conserved in mammals, as well as in bats' lineage. Compared with other mammals, we found one private amino acid substitution shared by bats, which is located on the inner loop domain, as well as some species-specific amino acid substitutions. The evolution rate analyses showed the signature of purifying selection on not only different classification level lineages but also the different domains and amino acid residue sites of this gene. Also, we suggested that GJA1 gene could be used as a good molecular marker to do the phylogenetic reconstruction.

  11. Adenovirus vector E4 gene regulates connexin 40 and 43 expression in endothelial cells via PKA and PI3K signal pathways.

    Science.gov (United States)

    Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K; Vincent, Loïc; Hackett, Neil R; Wang, Shiyang; Young, Lauren M; Hempstead, Barbara; Crystal, Ronald G; Rafii, Shahin

    2005-05-13

    Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4-, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intra-tracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways.

  12. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    Eric C Beyer

    2013-04-01

    Full Text Available The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8 have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death and formation of cytoplasmic accumulations (that may act as light scattering particles. These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

  13. 星形胶质细胞的缝隙连接蛋白43参与冷冻伤后脑水肿的形成∗%Astrocytic connexin 43 is involved in development of cryoinjury-induced cerebral edema

    Institute of Scientific and Technical Information of China (English)

    章科娜; 丁悦敏; 杜月光; 龚婉; 张雄

    2016-01-01

    AIM: To discuss the change of astrocytic connexin 43 (Cx43) expression in cerebral edema and the potential roles of Cx43 in the development of cerebral edema. METHODS:Cryoinjury-induced cerebral edema model was established in the right parietal lobe cortex in the rats. The animals were divided into sham group, cryoinjury-induced cerebral edema model group, and cryoinjury-induced cerebral edema model injected with carbenoxolone ( or octanol ) group. Dry and wet weight method was used to measure the content of brain water. Methanamide method was used to evalu-ate the permeability of blood-brain barrier ( BBB) after cryoinjury. HE staining showed the physiological changes in injured brain cortex. The protein expression of Cx43 was detected by Western blot and immunohistochemical staining. RESULTS:The brain water content in the injured cortex of rats was increased and cerebral edema developed to the peak at 24 h after cryoinjury. The permeability of BBB was increased in the injured cortex of the rats, and the area where BBB was damaged was larger than injured area. HE staining showed that many cells were dead in the central area of cryoinjury, and at the same time cerebral edema was appeared obviously in the surrounding area of injured brain cortex. The protein expression of Cx43 was up-regulated in the surrounding area of injured brain cortex, while Cx43 expression decreased in the injured cor-tex at different periods after cryoinjury. Both the content of brain water in the injured cortex and the permeability of BBB at 24 h after cryoinjury were reduced after injecting carbenoxolone or octanol intraperitoneally to block Cx43. CONCLU-SION:The cerebral edema up-regulates the expression of Cx43 and also strengthens its function. Once Cx43 is blocked, the degree of cerebral edema in rats is attenuated.%目的::探讨脑水肿后星形胶质细胞缝隙连接蛋白43(Cx43)的表达及其在脑水肿的发生发展过程中所起的作用。方法:采用颅骨外液

  14. The relationship between CRH、cortisol and the expression of Cx-43 on myometrium before and during term labor%子宫平滑肌间隙连接蛋白43、促肾上腺皮质激素释放激素、皮质醇与分娩发动的研究

    Institute of Scientific and Technical Information of China (English)

    刘志强; 王豫黔; 张丹; 朱慧玲

    2005-01-01

    目的:探讨临产前、后子宫平滑肌细胞间隙连接蛋白Cx-43的表达以及母血、脐血、羊水中促肾上腺皮质激素释放激素(CRH)、皮质醇(Cortisol)的水平变化,分析它们与分娩发动的关系.方法:应用免疫组化SABC法结合计算机图象分析技术分析60例足月妊娠产妇子宫下段平滑肌Cx-43的表达;用放射免疫方法测定产妇的静脉血、羊水及胎儿脐静脉血中CRH、Cortisol的含量.结果:临产组子宫肌细胞Cx-43的表达明显强于未临产组,差异有显著性(P<0.01).临产组母血、脐血中CRH、Cortisol及羊水中Cortisol与临产前比较显著升高(P<0.01),且与子宫肌Cx-43蛋白的表达呈显著正相关(P<0.01);结论:CRH、Cortisol在分娩发动中起重要作用,并可能通过促进子宫平滑肌Cx-43的表达参与分娩发动.

  15. Connexin-Based Therapeutics and Tissue Engineering Approaches to the Amelioration of Chronic Pancreatitis and Type I Diabetes: Construction and Characterization of a Novel Prevascularized Bioartificial Pancreas

    Directory of Open Access Journals (Sweden)

    J. Matthew Rhett

    2016-01-01

    Full Text Available Total pancreatectomy and islet autotransplantation is a cutting-edge technique to treat chronic pancreatitis and postoperative diabetes. A major obstacle has been low islet cell survival due largely to the innate inflammatory response. Connexin43 (Cx43 channels play a key role in early inflammation and have proven to be viable therapeutic targets. Even if cell death due to early inflammation is avoided, insufficient vascularization is a primary obstacle to maintaining the viability of implanted cells. We have invented technologies targeting the inflammatory response and poor vascularization: a Cx43 mimetic peptide that inhibits inflammation and a novel prevascularized tissue engineered construct. We combined these technologies with isolated islets to create a prevascularized bioartificial pancreas that is resistant to the innate inflammatory response. Immunoconfocal microscopy showed that constructs containing islets express insulin and possess a vascular network similar to constructs without islets. Glucose stimulated islet-containing constructs displayed reduced insulin secretion compared to islets alone. However, labeling for insulin post-glucose stimulation revealed that the constructs expressed abundant levels of insulin. This discrepancy was found to be due to the expression of insulin degrading enzyme. These results suggest that the prevascularized bioartificial pancreas is potentially a tool for improving long-term islet cell survival in vivo.

  16. 缝隙连接蛋白43在偏头痛大鼠三叉神经颈髓复合体的表达及其对痛觉超敏的影响%Connexin43 expression in trigeminocervical complex and its effect on allodynia in rats with migraine

    Institute of Scientific and Technical Information of China (English)

    武茜; 黄琳; 郭永涛; 荣蓉; 史兆春; 燕兰云; 万琪

    2013-01-01

    Objective To investigate the efficacy of connexin43 (Cx43) in trigeminocervical complex on allodynia in rat models of migraine.Methods SD rats were randomly divided into 9 groups (n=6):blank group,sham-operated group,saline group,improved inflammatory soup for 3 d group,improved inflammatory soup for 7 d group,carbenoxolone (CBX) prevention group,CBX prevention control group,CBX treatment group and CBX treatment control group.Improved inflammatory soup was used to stimulate the dual matter adjacent to the superior sagittal sinus of rats repeatedly to induce migraine models in the later 7 groups.The pain threshold of periorbital skin was measured by Von-Frey hairs.On the 11th postoperative day,the spinal cord was removed for detection of Cx43 expression in the trigeminocervical complex by immunofluorescence.Results Von-Frey hairs study showed that:the pain threshold of improved inflammatory soup groups had a daily drop; rats in the improved inflammatory soup for 3 d group suffered allodynia,while those in the CBX prevention group did not; the pain threshold in the CBX treatment group ascended after using CBX.Immunofluorescence of nucleus and Cx43 showed that:the mean optical density (OD) of improved inflammatory soup for 3 d group and for 7 d group significantly incresed (P<0.05); nucleus and Cx43 abundantly expressed in lamina Ⅰ and Ⅱ of dorsal horn in the improved inflammatory soup for 3 d group and lamina Ⅰ-Ⅳ of dorsal horn in improved inflammatory soup for 7 d group; significant difference was noted on the mean OD values between of CBX prevention/treatment groups and their control groups (P<0.05).Conclusion Repeatedly infusing improved inflammatory soup to dual matter can effectively induce allodynia in rat models of migraine and Cx43 abundantly expresses in trigeminocervical complex after being given the soup; allodynia in migraine model of rats can be prevented and relieved by intraperitoneal administrating of CBX,which suggests that Cx43 may play

  17. Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas.

    Science.gov (United States)

    Wu, Jie; Gao, Ming; Rice, Stephen G; Tsang, Candy; Beggs, John; Turner, Dharshaun; Li, Guohui; Yang, Bo; Xia, Kunkun; Gao, Fenfei; Qiu, Shenfeng; Liu, Qiang; Kerrigan, John F

    2016-06-01

    Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

  18. Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas

    Directory of Open Access Journals (Sweden)

    Jie Wu

    2016-06-01

    Full Text Available Human hypothalamic hamartoma (HH is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36 and connexin-43 (Cx43 in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

  19. Localization of connexin 30 in the luminal membrane of cells in the distal nephron.

    Science.gov (United States)

    McCulloch, Fiona; Chambrey, Régine; Eladari, Dominique; Peti-Peterdi, János

    2005-12-01

    Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

  20. Neuroprotection in the treatment of glaucoma--A focus on connexin43 gap junction channel blockers.

    Science.gov (United States)

    Chen, Ying-Shan; Green, Colin R; Danesh-Meyer, Helen V; Rupenthal, Ilva D

    2015-09-01

    Glaucoma is a form of optic neuropathy and a common cause of blindness, affecting over 60 million people worldwide with an expected rise to 80 million by 2020. Successful treatment is challenging due to the various causes of glaucoma, undetectable symptoms at an early stage and inefficient delivery of drugs to the back of the eye. Conventional glaucoma treatments focus on the reduction of elevated intraocular pressure (IOP) using topical eye drops. However, their efficacy is limited to patients who suffer from high IOP glaucoma and do not address the underlying susceptibility of retinal ganglion cells (RGC) to degeneration. Glaucoma is known as a neurodegenerative disease which starts with RGC death and eventually results in damage of the optic nerve. Neuroprotective strategies therefore offer a novel treatment option for glaucoma by not only preventing neuronal loss but also disease progression. This review firstly gives an overview of the pathophysiology of glaucoma as well as current treatment options including conventional and novel delivery strategies. It then summarizes the rational for neuroprotection as a novel therapy for glaucomatous neuropathies and reviews current potential neuroprotective strategies to preserve RGC, with a focus on connexin43 (Cx43) gap junction channel blockers.

  1. The potential prognostic value of connexin 26 and 46 expression in neoadjuvant-treated breast cancer

    Directory of Open Access Journals (Sweden)

    Teleki Ivett

    2013-02-01

    Full Text Available Abstract Background Several classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST are still missing. Deregulation of gap junction channel forming connexins (Cx has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems. Methods The expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA. Cx plaques of Results In our cohort dominated by hormone receptor (ER/PR positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1–2 had significantly better overall survival (p=0.015 than cases with scores 3–5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (20% pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (ppre-chemo=0.006; Sataloff TB (ppre-chemo=0.005; ppost-chemo=0.029 and in Miller-Payne G3 (ppre-chemo=0.002; ppost-chemo=0.012 classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups. Conclusion Our results suggest that Cx46 and Cx26 expression

  2. Protein kinase C-dependent regulation of connexin43 gap junctions and hemichannels

    DEFF Research Database (Denmark)

    Alstrøm, Jette Skov; Stroemlund, Line Waring; Nielsen, Morten Schak

    2015-01-01

    and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current...

  3. The Connexin40A96S mutation from a patient with atrial fibrillation causes decreased atrial conduction velocities and sustained episodes of induced atrial fibrillation in mice.

    Science.gov (United States)

    Lübkemeier, Indra; Andrié, René; Lickfett, Lars; Bosen, Felicitas; Stöckigt, Florian; Dobrowolski, Radoslaw; Draffehn, Astrid M; Fregeac, Julien; Schultze, Joachim L; Bukauskas, Feliksas F; Schrickel, Jan Wilko; Willecke, Klaus

    2013-12-01

    Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and a major cause of stroke. In the mammalian heart the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) are strongly expressed in the atrial myocardium mediating effective propagation of electrical impulses. Different heterozygous mutations in the coding region for Cx40 were identified in patients with AF. We have generated transgenic Cx40A96S mice harboring one of these mutations, the loss-of-function Cx40A96S mutation, as a model for atrial fibrillation. Cx40A96S mice were characterized by immunochemical and electrophysiological analyses. Significantly reduced atrial conduction velocities and strongly prolonged episodes of atrial fibrillation were found after induction in Cx40A96S mice. Analyses of the gating properties of Cx40A96S channels in cultured HeLa cells also revealed significantly lower junctional conductance and enhanced sensitivity voltage gating of Cx40A96S in comparison to Cx40 wild-type gap junctions. This is caused by reduced open probabilities of Cx40A96S gap junction channels, while single channel conductance remained the same. Similar to the corresponding patient, heterozygous Cx40A96S mice revealed normal expression levels and localization of the Cx40 protein. We conclude that heterozygous Cx40A96S mice exhibit prolonged episodes of induced atrial fibrillation and severely reduced atrial conduction velocities similar to the corresponding human patient.

  4. Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK-ERK pathway.

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    Lee, Kyung Mi; Kwon, Jung Yeon; Lee, Ki Won; Lee, Hyong Joo

    2009-01-15

    Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H(2)O(2))-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H(2)O(2); instead, it inhibited GJIC synergistically with H(2)O(2). AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK-ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary

  5. Vitamin E and caloric restriction promote hepatic homeostasis through expression of connexin 26, N-cad, E-cad and cholesterol metabolism genes.

    Science.gov (United States)

    Santolim, Leonardo Vinícius; Amaral, Maria Esméria Corezola do; Fachi, José Luís; Mendes, Maíra Felonato; Oliveira, Camila Andréa de

    2017-01-01

    Connexins (Cx) and cadherins are responsible for cell homeostasis. The Cx activity is directly related to cholesterol. The present work investigates whether vitamin E, with or without caloric restriction (CR), alters the mRNA expression of Cx26, Cx32, Cx43, N-cadherins (N-cads), E-cadherins (E-cads) and alpha-smooth muscle actin (α-SMA), and evaluates their relation to cholesterol metabolism in rat liver. Animals were divided into different groups: control with ad libitum diet (C), control+vitamin E (CV), aloric restriction with intake to 60% of group C (CR), and the intake of group CR+vitamin E (RV). There were increases of manganese superoxide dismutase (Mn-SOD) and glutathione S-transferase mu 1, indicating antioxidant effects of CR and vitamin E. An increase of nitric oxide in the CR group was in agreement with the Mn-SOD data. Supplementation with vitamin E, with or without CR, upregulated the expression of Cx26 mRNA and increased low-density lipoprotein cholesterol (LDL-c) in the CV group. Reductions of Cx32 and Cx43 were associated with lower LDL-c. Increases in Hmgcr and low-density lipoprotein receptor (LDLr) in the CV and RV groups could be explained by the effect of vitamin E. A reduction of LDLr in the CR group was due to the reduced dietary intake. Increases in cadherins in the CV, CR and RV groups were indicative of tissue maintenance, which was also supported by increases of α-SMA in groups CV and RV. Finally, vitamin E, with or without CR, increased Cx26, probably modulated by expression of the Hmgcr and LDLr genes. This suggests important relationship of Cxs and cholesterol metabolism genes.

  6. Connexins, E-cadherin, Claudin-7 and β-catenin transiently form junctional nexuses during the post-natal mammary gland development.

    Science.gov (United States)

    Dianati, Elham; Poiraud, Jérémy; Weber-Ouellette, Anne; Plante, Isabelle

    2016-08-01

    Gap junctions are intercellular channels made of connexins (Cxs) that allow direct communication between adjacent cells. Modulation of Cxs has been associated with abnormal development and function of the mammary gland and breast cancer. However, the mechanisms underlying their expression during normal mammary gland are not yet known. Cxs interact with components of tight and adherens junctions. Thus, we hypothesized that the expression levels of Cxs vary during mammary gland development and are regulated through stage-dependent interactions with members of the tight and adherens junctions. Our specific objectives were to: 1) determine the expression of Cxs and tight and adherens junction proteins throughout development and 2) characterize Cxs interactions with components of tight and adherens junctions. Murine mammary glands were sampled at various developmental stages (pre-pubescent to post-weaning). RT-qPCR and western-blot analyses demonstrated differential expression patterns for all gap (Cx43, Cx32, Cx26, Cx30), tight (Claudin-1, -3, -4, -7) and adherens (β-catenin, E- and P-cadherins) junctions throughout development. Interestingly, co-immunoprecipitation demonstrated interactions between these different types of junctions. Cx30 interacted with Cx26 just at the late pregnancy stage. While Cx43 showed a persistent interaction with β-catenin from virginity to post-weaning, its interactions with E-cadherin and Claudin-7 were transient. Cx32 interacted with Cx26, E-cadherin and β-catenin during lactation. Immunofluorescence results confirmed the existence of a junctional nexus that remodeled during mammary gland development. Together, our results confirm that the expression levels of Cxs vary concomitantly and that Cxs form junctional nexuses with tight and adherens junctions, suggesting the existence of common regulatory pathways.

  7. Altered expression of connexin43 and its possible role in endothelin - 1 - induced contraction in rabbit basilar artery%缝隙连接蛋白43在内皮素诱导的脑基底动脉收缩中的表达变化

    Institute of Scientific and Technical Information of China (English)

    赵万巨; 王海涛; 洪涛

    2011-01-01

    目的 探讨Cx43在内皮素诱导的脑基底动脉收缩中的表达变化及其可能的作用.方法 血管环张力实验检测内皮素诱导的脑基底动脉的收缩变化并应用Western blot检测基底动脉Cx43蛋白的表达变化,染料传输实验用来检测脑基底动脉收缩过程中平滑肌细胞间缝隙连接的功能变化.结果 浓度递增的内皮素导致脑基底动脉呈显著浓度依赖性的收缩,一定浓度缝隙连接阻断剂苷珀酸显著缓解该收缩;收缩过程中,Cx43的蛋白表达呈显著时间依赖性的升高,苷珀酸减弱该表达的升高;内皮素刺激下,血管平滑肌细胞间的染料传输呈时间依赖性的升高,苷珀酸显著减少染料在细胞间的传输.结论 脑血管痉挛过程中,通过增加Cx43的表达,血管细胞间缝隙连接的功能被内皮素激活并在血管痉挛病理过程中发挥重要作用;抑制缝隙连接的功能是有效缓解蛛网膜下腔出血后脑血管痉挛的新途径.%Objective To investigate the role of connexin43 in ET- 1 -induced contraction in rabbit basilar artery.Methods The ET - 1 - induced contraction without or with carbenoxolone was studied with an isometric tension system.The expression of connexin43 protein in ET - t stimulated basilar arteries was studied with Western blot.Scrape/scratch method was used to analyze the function of gap junction in cultured rabbit cerebrovascular smooth muscle ceils.Results ET - 1 produced a concentration - dependent contraction.Carbenoxolone inhibited ET- 1 induced contraction.The connexin43 protein level was increased in ET- 1stimulated basilar arteries.Carbenoxolone decreased the connexin43 protein level increased by ET - 1.Cells treated with ET - 1 appeared positive communication and the dye transfer was increased in a time - dependent fashion.Carbenoxolone suppressed the ET - 1 - induced increasement of dye transfer.Conclusions The enhancement of gap junction intercellular communication is

  8. Connexin channels and phospholipids: association and modulation

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    Harris Andrew L

    2009-08-01

    Full Text Available Abstract Background For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood. Results Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred. Conclusion

  9. Cleft lip in oculodentodigital dysplasia suggests novel roles for connexin43.

    Science.gov (United States)

    Amano, K; Ishiguchi, M; Aikawa, T; Kimata, M; Kishi, N; Fujimaki, T; Murakami, A; Kogo, M

    2012-07-01

    Oculodentodigital Dysplasia (ODDD) is a rare syndrome involving anomalies in eye, tooth, and digit formation, caused by mutations in CX43/GJA1. In addition to classic dental features, ODDD includes oral and craniofacial accessory symptoms such as characteristic facial appearance and cleft palate. However, there have been no reports of ODDD accompanied by cleft lip. Herein we report, for the first time, a male, sporadic, Asian proband presenting bilateral cleft lip. By direct sequence analysis, our proband was diagnosed as having ODDD with a heterozygous mutation, codon 142 G>A in GJA1 and CX43E48K. We excluded the possibility of pathogenic mutations in B3GALTL, BMP4, TFAP2A, PVRL1, IRF6, and MSX1. To address how CX43/GJA1 is related to cleft lip, we performed immunohistochemistry using mouse and human mid-facial tissue. CX43 expression was detected in the nasal compartment and nasal and maxillary processes at murine developmental stage E12.5. Furthermore, CX43 expression was found in the epithelial tissue inside the human subepithelial cleft lip that completes epithelial fusion. Therefore, we suggest that CX43/GJA1 is involved in lip formation. Our case report of ODDD with a bilateral cleft lip suggests that CX43/GJA1 might be a novel candidate gene for syndromic cleft lip.

  10. Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry.

    Science.gov (United States)

    Azanza, M J; Pes, N; Pérez-Bruzón, R N; Aisa, J; Raso, M; Junquera, C; Lahoz, J M; Maestú, C; Martínez-Ciriano, C; Pérez-Castejón, C; Vera-Gil, A; Del Moral, A

    2007-05-01

    The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.

  11. Laminin-332 alters connexin profile, dye coupling and intercellular Ca2+ waves in ciliated tracheal epithelial cells

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    Olsen Colin E

    2006-08-01

    Full Text Available Abstract Background Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin α3β3γ2 (LM-332 proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC. Methods Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca2+ waves. Expression of connexin (Cx mRNA and proteins were assayed by reverse transcriptase – polymerase chain reaction and immunocytochemistry, respectively. Results When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular Ca2+ waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca2+waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins. Conclusion Alterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function.

  12. Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

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    Shuo Liu

    Full Text Available Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases

  13. Reciprocal regulation of epileptiform neuronal oscillations and electrical synapses in the rat hippocampus.

    Science.gov (United States)

    Kinjo, Erika R; Higa, Guilherme S V; Morya, Edgard; Valle, Angela C; Kihara, Alexandre H; Britto, Luiz R G

    2014-01-01

    Gap junction (GJ) channels have been recognized as an important mechanism for synchronizing neuronal networks. Herein, we investigated the participation of GJ channels in the pilocarpine-induced status epilepticus (SE) by analyzing electrophysiological activity following the blockade of connexins (Cx)-mediated communication. In addition, we examined the regulation of gene expression, protein levels, phosphorylation profile and distribution of neuronal Cx36, Cx45 and glial Cx43 in the rat hippocampus during the acute and latent periods. Electrophysiological recordings revealed that the GJ blockade anticipates the occurrence of low voltage oscillations and promotes a marked reduction of power in all analyzed frequencies.Cx36 gene expression and protein levels remained stable in acute and latent periods, whereas upregulation of Cx45 gene expression and protein redistribution were detected in the latent period. We also observed upregulation of Cx43 mRNA levels followed by changes in the phosphorylation profile and protein accumulation. Taken together, our results indisputably revealed that GJ communication participates in the epileptiform activity induced by pilocarpine. Moreover, considering that specific Cxs undergo alterations through acute and latent periods, this study indicates that the control of GJ communication may represent a focus in reliable anti-epileptogenic strategies.

  14. Cardiac connexins and impulse propagation

    NARCIS (Netherlands)

    J.A. Jansen; T.A.B. van Veen; J.M.T. de Bakker; H.V.M. van Rijen

    2010-01-01

    Gap junctions form the intercellular pathway for cell-to-cell transmission of the cardiac impulse from its site of origin, the sinoatrial node, along the atria, the atrioventricular conduction system to the ventricular myocardium. The component parts of gap junctions are proteins called connexins (C

  15. Acute inflammation decreases the expression of connexin 40 in mouse lung.

    Science.gov (United States)

    Rignault, Stéphanie; Haefliger, Jacques-Antoine; Waeber, Bernard; Liaudet, Lucas; Feihl, François

    2007-07-01

    Transmigration of neutrophil polymorphonuclear leukocytes through the microvascular endothelium is a cardinal event of acute inflammation. In vitro, this process can be restricted by gap junctional intercellular communication, but whether it also occurs in vivo is unknown. Connexin 40 (Cx40) is a gap junctional protein abundantly present in the lung, notably in vascular endothelium. We hypothesized that acute lung inflammation would be aggravated in knockout mice genetically deficient in Cx40. This hypothesis was tested in two different models: 1) intranasal instillation of LPS at either supramaximal (50 microg/mouse) or inframaximal dose (0.01 microg/mouse) and 2) pulmonary inflammation as a distant consequence of an abdominal infection caused by cecal ligation and perforation. Pulmonary transmigration of neutrophils was assessed by counting these cells in bronchoalveolar lavage fluid (LPS model) or with the myeloperoxidase assay in homogenates of blood-free tissue (cecal ligation and perforation model). Pulmonary content in Cx40 and Cx43 was evaluated with immunoblots. In wild-type mice, there was a time-dependent decrease of Cx40 expression in both models. The time points for studies with the knockout mice were chosen in such a manner that inflammation was clearly present and Cx40 still largely expressed in wild-type animals. In either model, the development of lung inflammation did not differ between wild-type and Cx40-deficient mice. In conclusion, the pulmonary expression of the Cx40 protein is progressively and markedly decreased in two different murine models of acute lung inflammation, but there is no causal relationship between this process and the pulmonary transmigration of neutrophils.

  16. Role of connexins in human congenital heart disease: the chicken and egg problem

    Directory of Open Access Journals (Sweden)

    Aida eSalameh

    2013-06-01

    Full Text Available Inborn cardiac diseases are among the most frequent congenital anomalies and are the main cause of death in infants within the first year of age in industrialized countries when not adequately treated. They can be divided into simple and complex cardiac malformations. The former ones, for instance atrial and ventricular septal defects, valvular or subvalvular stenosis or insufficiency account for up to 80% of cardiac abnormalities. The latter ones, for example transposition of the great vessels, Tetralogy of Fallot or Shone's anomaly often do not involve only the heart but also the great vessels and although occurring less frequently these severe cardiac malformations will become symptomatically within the first months of age and have a high risk of mortality if the patients remain untreated. In the last decade there is increasing evidence that cardiac gap junction proteins, the connexins (Cx, might have an impact on cardiac anomalies. In the heart mainly three of them (Cx40, Cx43 and Cx45 are differentially expressed with regard to temporal organogenesis and to their spatial distribution in the heart. These proteins, forming gap junction channels, are most important for a normal electrical conduction and coordinated synchronous heart muscle contraction and also for the normal embryonic development of the heart. Animal and also some human studies revealed that at least in some cardiac malformations alterations in certain gap junction proteins are present but until today no particular gap junction mutation could be assigned to a specific cardiac anomaly. As gap junctions transmit growth and differentiation signals from cell to cell it is reasonable to assume that they are somehow involved in misdirected growth present in many inborn heart diseases playing a primary or contributory role. This review addresses potential role of gap junctions in the development of inborn heart anomalies like the conotruncal heart defects.

  17. Role of connexins in human congenital heart disease: the chicken and egg problem.

    Science.gov (United States)

    Salameh, Aida; Blanke, Katja; Daehnert, Ingo

    2013-01-01

    Inborn cardiac diseases are among the most frequent congenital anomalies and are the main cause of death in infants within the first year of age in industrialized countries when not adequately treated. They can be divided into simple and complex cardiac malformations. The former ones, for instance atrial and ventricular septal defects, valvular or subvalvular stenosis or insufficiency account for up to 80% of cardiac abnormalities. The latter ones, for example transposition of the great vessels, Tetralogy of Fallot or Shone's anomaly often do not involve only the heart, but also the great vessels and although occurring less frequently, these severe cardiac malformations will become symptomatic within the first months of age and have a high risk of mortality if the patients remain untreated. In the last decade, there is increasing evidence that cardiac gap junction proteins, the connexins (Cx), might have an impact on cardiac anomalies. In the heart, mainly three of them (Cx40, Cx43, and Cx45) are differentially expressed with regard to temporal organogenesis and to their spatial distribution in the heart. These proteins, forming gap junction channels, are most important for a normal electrical conduction and coordinated synchronous heart muscle contraction and also for the normal embryonic development of the heart. Animal and also some human studies revealed that at least in some cardiac malformations alterations in certain gap junction proteins are present but until today no particular gap junction mutation could be assigned to a specific cardiac anomaly. As gap junctions have often been supposed to transmit growth and differentiation signals from cell to cell it is reasonable to assume that they are somehow involved in misdirected growth present in many inborn heart diseases playing a primary or contributory role. This review addresses the potentional role of gap junctions in the development of inborn heart anomalies like the conotruncal heart defects.

  18. Loss of functional endothelial connexin40 results in exercise-induced hypertension in mice.

    Science.gov (United States)

    Morton, Susan K; Chaston, Daniel J; Howitt, Lauren; Heisler, Jillian; Nicholson, Bruce J; Fairweather, Stephen; Bröer, Stefan; Ashton, Anthony W; Matthaei, Klaus I; Hill, Caryl E

    2015-03-01

    During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.

  19. Connexins and pannexins in liver damage

    Science.gov (United States)

    Crespo Yanguas, Sara; Willebrords, Joost; Maes, Michaël; da Silva, Tereza Cristina; Veloso Alves Pereira, Isabel; Cogliati, Bruno; Zaidan Dagli, Maria Lucia; Vinken, Mathieu

    2016-01-01

    Connexins and pannexins are key players in the control of cellular communication and thus in the maintenance of tissue homeostasis. Inherent to this function these proteins are frequently involved in pathological processes. The present paper reviews the role of connexins and pannexins in liver toxicity and disease. As they act both as sensors and effectors in these deleterious events connexins and pannexins could represent a set of novel clinical diagnostic biomarkers and drug targets. PMID:27065778

  20. Activation, permeability, and inhibition of astrocytic and neuronal large pore (hemi)channels.

    Science.gov (United States)

    Hansen, Daniel Bloch; Ye, Zu-Cheng; Calloe, Kirstine; Braunstein, Thomas Hartig; Hofgaard, Johannes Pauli; Ransom, Bruce R; Nielsen, Morten Schak; MacAulay, Nanna

    2014-09-19

    Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.

  1. Methamphetamine compromises gap junctional communication in astrocytes and neurons.

    Science.gov (United States)

    Castellano, Paul; Nwagbo, Chisom; Martinez, Luis R; Eugenin, Eliseo A

    2016-05-01

    Methamphetamine (meth) is a central nervous system (CNS) stimulant that results in psychological and physical dependency. The long-term effects of meth within the CNS include neuronal plasticity changes, blood-brain barrier compromise, inflammation, electrical dysfunction, neuronal/glial toxicity, and an increased risk to infectious diseases including HIV. Most of the reported meth effects in the CNS are related to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters, especially dopamine, norepinephrine, and epinephrine. However, little is known about the effects of meth on connexin (Cx) containing channels, such as gap junctions (GJ) and hemichannels (HC). We examined the effects of meth on Cx expression, function, and its role in NeuroAIDS. We found that meth altered Cx expression and localization, decreased GJ communication between neurons and astrocytes, and induced the opening of Cx43/Cx36 HC. Furthermore, we found that these changes in GJ and HC induced by meth treatment were mediated by activation of dopamine receptors, suggesting that dysregulation of dopamine signaling induced by meth is essential for GJ and HC compromise. Meth-induced changes in GJ and HC contributed to amplified CNS toxicity by dysregulating glutamate metabolism and increasing the susceptibility of neurons and astrocytes to bystander apoptosis induced by HIV. Together, our results indicate that connexin containing channels, GJ and HC, are essential in the pathogenesis of meth and increase the sensitivity of the CNS to HIV CNS disease. Methamphetamine (meth) is an extremely addictive central nervous system stimulant. Meth reduced gap junctional (GJ) communication by inducing internalization of connexin-43 (Cx43) in astrocytes and reducing expression of Cx36 in neurons by a mechanism involving activation of dopamine receptors (see cartoon). Meth-induced changes in Cx containing channels increased extracellular levels of glutamate and resulted in higher

  2. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  3. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the

  4. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  5. Mutations in connexin genes and disease.

    Science.gov (United States)

    Pfenniger, Anna; Wohlwend, Annelise; Kwak, Brenda R

    2011-01-01

    Connexins are a family of transmembrane proteins that are widely expressed in the human body. Connexins play an important role in cell-cell communication and homeostasis in various tissues by forming gap junction channels, which enable a direct passage of ions or metabolites from one cell to another. Twenty-one different connexins are expressed in humans, each having distinct expression patterns and regulation properties. Knowledge on this family of proteins can be gained by making an inventory of mutations and associated diseases in human. PubMed and other relevant databases were searched. In addition, key review articles were screened for relevant original publications. Sections of representative organs were photographed and annotated. The crucial role of connexins is highlighted by the discovery of mutations in connexin genes which cause a variety of disorders such as myelin-related diseases, skin disorders, hearing loss, congenital cataract, or more complex syndromes such as the oculodendrodigital dysplasia. This review systematically addresses current knowledge on mutations in connexin genes and disease, focusing on the correlation between genetic defects, cellular phenotypes and clinical manifestations. The review of diseases caused by mutations in connexin genes highlights the essential nature of connexin function and intercellular communication in tissue homeostasis. © 2010 The Authors. European Journal of Clinical Investigation © 2010 Stichting European Society for Clinical Investigation Journal Foundation.

  6. Connexin43 in retinal injury and disease.

    Science.gov (United States)

    Danesh-Meyer, Helen V; Zhang, Jie; Acosta, Monica L; Rupenthal, Ilva D; Green, Colin R

    2016-03-01

    Gap junctions are specialized cell-to-cell contacts that allow the direct transfer of small molecules between cells. A single gap junction channel consists of two hemichannels, or connexons, each of which is composed of six connexin protein subunits. Connexin43 is the most ubiquitously expressed isoform of the connexin family and in the retina it is prevalent in astrocytes, Müller cells, microglia, retinal pigment epithelium and endothelial cells. Prior to docking with a neighboring cell, Connexin43 hemichannels have a low open probability as open channels constitute a large, relatively non-specific membrane pore. However, with injury and disease Connexin43 upregulation and hemichannel opening has been implicated in all aspects of secondary damage, especially glial cell activation, edema and loss of vascular integrity, leading to neuronal death. We here review gap junctions and their roles in the retina, and then focus in on Connexin43 gap junction channels in injury and disease. In particular, the effect of pathological opening of gap junction hemichannels is described, and hemichannel mediated loss of vascular integrity explained. This latter phenomenon underlies retinal pigment epithelium loss and is a common feature in several retinal diseases. Finally, Connexin43 channel roles in a number of retinal diseases including macular degeneration, glaucoma and diabetic retinopathy are considered, along with results from related animal models. A final section describes gap junction channel modulation and the ocular delivery of potential therapeutic molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Connexins in lymphatic vessel physiology and disease.

    Science.gov (United States)

    Meens, Merlijn J; Sabine, Amélie; Petrova, Tatiana V; Kwak, Brenda R

    2014-04-17

    Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (Gap junction channels allow diffusion of similar molecules between the cytoplasms of adjacent cells. The expression and function of connexins in blood vessels has been intensely studied in the last few decades. In contrast, only a few studies paid attention to lymphatic vessels; convincing in vivo data with respect to expression patterns of lymphatic connexins and their functional roles have only recently begun to emerge. Interestingly, mutations in connexin genes have been linked to diseases of lymphatic vasculature, most notably primary and secondary lymphedema. This review summarizes the available data regarding lymphatic connexins. More specifically it addresses (i) early studies aimed at presence of gap junction-like structures in lymphatic vessels, (ii) more recent studies focusing on lymphatic connexins using genetically engineered mice, and (iii) results of clinical studies that have reported lymphedema-linked mutations in connexin genes. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin.

    Directory of Open Access Journals (Sweden)

    Richard J Miron

    Full Text Available In recent years, enamel matrix derivative (EMD has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43 and N-cadherin (N-cad as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

  9. Immunohisto- and cytochemistry analysis of connexins

    Science.gov (United States)

    Cogliati, Bruno; Maes, Michaël; Pereira, Isabel Veloso Alves; Willebrords, Joost; da Silva, Tereza Cristina; Crespo Yanguas, Sara; Vinken, Mathieu

    2016-01-01

    Summary Immunohistochemistry (IHC) is a ubiquitous used technique to identify and analyze protein expression in the context of tissue and cell morphology. In the connexin research field, IHC is applied to identify the subcellular location of connexin proteins, as this can be directly linked to their functionality. The present chapter describes a protocol for fluorescent IHC to detect connexin proteins in tissues slices and cells, with slight modifications depending on the nature of biological sample, histological processing and/or protein expression level. Basically, fluorescent IHC is a short, simple and cost-effective technique, which allows the visualization of proteins based on fluorescent-labeled antibody-antigen recognition. PMID:27207286

  10. GAP junctional communication in brain secondary organizers.

    Science.gov (United States)

    Bosone, Camilla; Andreu, Abraham; Echevarria, Diego

    2016-06-01

    Gap junctions (GJs) are integral membrane proteins that enable the direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells. They are formed by the apposition of two connexons belonging to adjacent cells. Each connexon is formed by six proteins, named connexins (Cxs). Current evidence suggests that gap junctions play an important part in ensuring normal embryo development. Mutations in connexin genes have been linked to a variety of human diseases, although the precise role and the cell biological mechanisms of their action remain almost unknown. Among the big family of Cxs, several are expressed in nervous tissue but just a few are expressed in the anterior neural tube of vertebrates. Many efforts have been made to elucidate the molecular bases of Cxs cell biology and how they influence the morphogenetic signal activity produced by brain signaling centers. These centers, orchestrated by transcription factors and morphogenes determine the axial patterning of the mammalian brain during its specification and regionalization. The present review revisits the findings of GJ composed by Cx43 and Cx36 in neural tube patterning and discuss Cx43 putative enrollment in the control of Fgf8 signal activity coming from the well known secondary organizer, the isthmic organizer. © 2016 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.

  11. Connexins in wound healing; perspectives in diabetic patients.

    Science.gov (United States)

    Becker, David L; Thrasivoulou, Christopher; Phillips, Anthony R J

    2012-08-01

    Skin lesions are common events and we have evolved to rapidly heal them in order to maintain homeostasis and prevent infection and sepsis. Most acute wounds heal without issue, but as we get older our bodies become compromised by poor blood circulation and conditions such as diabetes, leading to slower healing. This can result in stalled or hard-to-heal chronic wounds. Currently about 2% of the Western population develop a chronic wound and this figure will rise as the population ages and diabetes becomes more prevalent [1]. Patient morbidity and quality of life are profoundly altered by chronic wounds [2]. Unfortunately a significant proportion of these chronic wounds fail to respond to conventional treatment and can result in amputation of the lower limb. Life quality and expectancy following amputation is severely reduced. These hard to heal wounds also represent a growing economic burden on Western society with published estimates of costs to healthcare services in the region of $25B annually [3]. There exists a growing need for specific and effective therapeutic agents to improve healing in these wounds. In recent years the gap junction protein Cx43 has been shown to play a pivotal role early on in the acute wound healing process at a number of different levels [4-7]. Conversely, abnormal expression of Cx43 in wound edge keratinocytes was shown to underlie the poor rate of healing in diabetic rats, and targeting its expression with an antisense gel restored normal healing rates [8]. The presence of Cx43 in the wound edge keratinocytes of human chronic wounds has also been reported [9]. Abnormal Cx43 biology may underlie the poor healing of human chronic wounds and be amenable therapeutic intervention [7]. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  12. Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus).

    Science.gov (United States)

    Lavrov, Igor; Fox, Lyle; Shen, Jun; Han, Yingchun; Cheng, Jianguo

    2016-01-01

    Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

  13. Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus.

    Directory of Open Access Journals (Sweden)

    Igor Lavrov

    Full Text Available Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32, connexin 36 (Cx36, connexin 37 (Cx37, and connexin 43 (Cx43. Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

  14. Mixed Electrical-Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36.

    Science.gov (United States)

    Hamzei-Sichani, Farid; Davidson, Kimberly G V; Yasumura, Thomas; Janssen, William G M; Wearne, Susan L; Hof, Patrick R; Traub, Roger D; Gutiérrez, Rafael; Ottersen, Ole P; Rash, John E

    2012-01-01

    Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for "mixed" (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328-1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons.

  15. Mixed electrical-chemical synapses in adult rat hippocampus are primarily glutamatergic and coupled by connexin-36

    Directory of Open Access Journals (Sweden)

    Farid eHamzei-Sichani

    2012-05-01

    Full Text Available Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in the mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for mixed (electrical/chemical synapses in adult rat hippocampus on both principal cells and interneurons. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr, apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into four weakly-fixed CA3pyr was detected in MF axons that contacted the injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold-labeling revealed diverse sizes and morphologies of connexin36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328-1140 connexons, three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin section images of a CA3pyr, but none found by immunogold-labeling were at GABAergic mixed synapses, suggesting their rarity. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal

  16. Role of connexins and pannexins in cardiovascular physiology.

    Science.gov (United States)

    Meens, Merlijn J; Kwak, Brenda R; Duffy, Heather S

    2015-08-01

    Connexins and pannexins form connexons, pannexons and membrane channels, which are critically involved in many aspects of cardiovascular physiology. For that reason, a vast number of studies have addressed the role of connexins and pannexins in the arterial and venous systems as well as in the heart. Moreover, a role for connexins in lymphatics has recently also been suggested. This review provides an overview of the current knowledge regarding the involvement of connexins and pannexins in cardiovascular physiology.

  17. Effect of inhibiting connexin 43 expression on the expression of rat glial cell growth factors in prolactinomas%抑制缝隙连接蛋白43表达对大鼠催乳素腺瘤中胶质细胞生长因子表达的影响

    Institute of Scientific and Technical Information of China (English)

    张亚菊; 王海涛; 张冉; 靳峰

    2016-01-01

    Objective To investigate the effect of inhibiting connexin 43 (Cx43) expression on the expression of rat glial cell growth factor in prolactinomas.Methods Forty-eight female rats were randomly assigned to normal, simple estradiol, estradiol + carbenoxolone, and simple carbenoxolone groups by the completely randomized method (n =12 in each group).A model of rat prolactinoma was induced by estradiol.The normal group did not accept drug treatment;the simple estradiol group was injected estradiol intramuscularly (3 000 mg/kg, once a week, for 13 weeks);10 weeks after injection of estradiol, the estradiol + carbenoxolone group injected carbenoxolone via subarachnoid (once a week, for 3 weeks);and the simple carbenoxolone group only injected carbenoxolone for 3 weeks.The changes of body mass of pituitary and histomorphology were observed.Western blot was used to detect the expression levels of Cx43 and glial growth factor (GGF).Results The pituitary weights of the normal, simple estradiol, estradiol + carbenoxolone and simple carbenoxolone groups were 51.0 ±0.4 mg, 93.3 ± 1.0 mg, 52.7 ±2.1 mg and 68.5 ± 1.3 mg, respectively.There were significant differences (F =786.73, P < 0.01);the relative expression quantities of Cx43 were 8.0 ± 2.2% , 34.7 ± 4.0,.9.3 ± 3.1% , and 15.3 ± 3.8% ,respectively, and the relative expression quantities of GGF were 17.7 ± 5.2%, 46.9 ± 1.4%, 15.8 ± 3.6%, and 20.0 ±2.3%, respectively.There were significant differences (F =1051.51.P <0.01;F =806.58, P < 0.01).The pituitary histopathological morphology of the estradiol + carbenoxolone group was close to the normal group and the simple carbenoxolone group.Conclusions Inhibition of pituitary Cx43 expression may inhibit the GGF expression, thereby inhibiting the growth of pituitary prolactinomas in rats.%目的 探讨在催乳素腺瘤中,抑制缝隙连接蛋白43(Cx43)的表达对大鼠胶质细胞生长因子表达的影响.方法 采用

  18. Morphology and the expression of connexin 43 and 40 in bundles of transition cells in the adult atrioventricular junction%成人房室交界区过渡细胞束形态学及连接蛋白43和40的表达

    Institute of Scientific and Technical Information of China (English)

    宋慧芳; 吕华; 范益民; 夏仲年; 阎八一; 高雨仁; 杨桂姣

    2011-01-01

    目的 观察临床上心律失常常见发生部位及射频消融治疗靶点部位--房室交界区和邻近区域的形态学特点及连接蛋白(Cx)43和40的表达,为心律失常发生机制及可能的有效治疗部位提供形态学依据.方法 10例正常成人心脏,选取房室交界区及其邻近部位,常规石蜡包埋,HE、Masson染色,选定目标部位行Cx43、Cx40免疫组织化学染色,并进行图像及统计学分析.结果 峡部、冠状窦口周围和远端、二尖瓣纤维环处及与右房瓣上肌环邻近的中心纤维体等处可见较多过渡细胞束走行,并从不同方向与房室结及结后延伸不同部位相连.过渡细胞组成、排布、心肌纤维走行和相互间的联系有别于普通工作心肌.Cx43、Cx40在过渡细胞束1~4及右房瓣上肌环中分布范围小但集中,而在左房近膜部处的过渡细胞中表达较多;峡部Cx43、Cx40表达较集中.结论 房室交界区及其邻近区域存在双(多)径传导的形态学基础,且与目前常用的射频消融治疗心律失常靶点部位具有对应关系.%Objective To observe the morphological feature and the expression of connexin( Cx )43 and Cx40 in the common arrhythmogenesis and radiofrequenc:y ahlation( RFA ) region, the atrioventricular junction and its surrounding area, in order to provide the morphological basis for understanding the mechanism of arrhythmogenesis and possible sites for therapy.Methods Ten fresh normal adult hearts were used in this study.The specimens containing the atrioventricular node( AVN ) was harvested, embedded in paraffin and sectioned.The sections were stained with HE.Masson staining.Expression of Cx43 and Cx40 was examined by immunohistochemistry and image analysis.Results There were the bundles of transition cells at cavo-tricuspid isthmus, around the opening and distal end of coronary sinus, at the annulus fibrosus of bicuspid valve, and at the central fibrous body( CFB ) near the annulus of right

  19. CONNEXIN-47 AND CONNEXIN-32 IN GAP JUNCTIONS OF OLIGODENDROCYTE SOMATA, MYELIN SHEATHS, PARANODAL LOOPS AND SCHMIDT-LANTERMAN INCISURES: IMPLICATIONS FOR IONIC HOMEOSTASIS AND POTASSIUM SIPHONING

    OpenAIRE

    KAMASAWA, N.; SIK, A.; Morita, M; Yasumura, T; Davidson, K. G. V.; Nagy, J.I.; RASH, J.E.

    2005-01-01

    The subcellular distributions and co-associations of the gap junction-forming proteins connexin47 and connexin32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin47 was co-localized with astrocytic connexin43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin32 without connexin47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron ...

  20. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    Science.gov (United States)

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

  1. Connexins, hearing and deafness: clinical aspects of mutations in the connexin 26 gene.

    Science.gov (United States)

    Lefebvre, P P; Van De Water, T R

    2000-04-01

    Congenital deafness is a very frequent disorder occurring in approximately I in 1000 live births. Mutations in GJB2 encoding for gap junction protein connexin-26 (Cx26) have been established as the basis of autosomal recessive non-syndromic hearing loss and proposed in some rare cases of autosomal dominant form of deafness. Connexin are gap-junction proteins which constitute a major system of intercellular communication important in the exchange of electrolytes, second messengers and metabolites. In the inner ear, connexin 26 expression was demonstrated in the stria vascularis, basement membrane, limbus and the spiral prominence of the human cochlea. The loss of connexin 26 in the gap junction complex would expect to disrupt the recycling of potassium from the synapses at the base of hair cells through the supporting cells and fibroblasts of potassium ions back to the high potassium containing endolymph of the cochlear duct and therefore would result in a local intoxication of the Corti s organ by potassium, leading to the hearing loss. The discovery of the genes responsible of hearing loss in particular the identification of mutations in the gene coding for connexin 26 allows to hope some tremendous help in genetic counseling. The possible implication of the mutation of the connexin gene in the pathophysiology of some progressive adult deafness opens new prospects in the fine diagnostic of the ear diseases and eventually may lead to new therapeutic strategies applied to the cochlea.

  2. Biphasic Effect of Basic Fibroblast Growth Factor on Anterior Pituitary Folliculostellate TtT/GF Cell Coupling, and Connexin 43 Expression and Phosphorylation.

    Science.gov (United States)

    Vitale, M L; Barry, A

    2015-10-01

    Basic fibroblast growth factor (bFGF) is a mitogenic and differentiating cytokine. In the anterior pituitary, folliculostellate (FS) cells constitute the major source of bFGF. bFGF affects endocrine cell proliferation and secretion in the anterior pituitary. In addition, bFGF increases its own expression by acting directly on FS cells. FS cell Cx43-mediated gap junction intercellular communication allows the establishment of an intrapituitary network for the transmission of information. In the present study, we assessed how bFGF regulates FS cell coupling. Time course studies were carried out on the FS cell line TtT/GF. Short-term bFGF treatment induced a transient cell uncoupling and the phosphorylation in Ser368 of membrane-bound Cx43 without modifying Cx43 levels. We demonstrated the involvement of the protein kinase C (PKC) isoform α in the phosphorylation of Cx43 in S368. Moreover, we showed that bFGF induced PKCα activation by stimulating its expression, phosphorylation and association with the plasma membrane. The long-term incubation with bFGF increased TtT/GF cell coupling, total Cx43 levels and Cx43 accumulation at the cell membrane of cytoplasmic projections. The Cx43 level increase was a result of the stimulation of Cx43 gene transcription as mediated by the extracellular-regulated kinase 1/2 signalling pathway. Taken together, the data show that bFGF modulates TtT/GF cell coupling by activating different pathways that lead to opposite effects on Cx43 phosphorylation and expression depending on the duration of the exposure of the cells to bFGF. A short-term bFGF exposure reduces cell-to-cell communication as a mean of desynchronising FS cells. By contrast, long-term exposure to bFGF enhances cell-to-cell communication and facilitates coordination among FS cells. © 2015 British Society for Neuroendocrinology.

  3. Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning.

    Science.gov (United States)

    Kamasawa, N; Sik, A; Morita, M; Yasumura, T; Davidson, K G V; Nagy, J I; Rash, J E

    2005-01-01

    The subcellular distributions and co-associations of the gap junction-forming proteins connexin 47 and connexin 32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin 47 was co-localized with astrocytic connexin 43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin 32 without connexin 47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron microscopy, connexin 47 immunolabeling was on the oligodendrocyte side of gap junctions between oligodendrocyte somata and astrocytes. By freeze-fracture replica immunogold labeling, large gap junctions between oligodendrocyte somata and astrocyte processes contained much more connexin 47 than connexin 32. Along surfaces of internodal myelin, connexin 47 was several times as abundant as connexin 32, and in the smallest gap junctions, often occurred without connexin 32. In contrast, connexin 32 was localized without connexin 47 in newly-described autologous gap junctions in Schmidt-Lanterman incisures and between paranodal loops bordering nodes of Ranvier. Thus, connexin 47 in adult rodent CNS is the most abundant connexin in most heterologous oligodendrocyte-to-astrocyte gap junctions, whereas connexin 32 is the predominant if not sole connexin in autologous ("reflexive") oligodendrocyte gap junctions. These results clarify the locations and connexin compositions of heterologous and autologous oligodendrocyte gap junctions, identify autologous gap junctions at paranodes as potential sites for modulating paranodal electrical properties, and reveal connexin 47-containing and connexin 32-containing gap junctions as conduits for long-distance intracellular and intercellular movement of ions and associated osmotic water. The autologous gap junctions may regulate paranodal electrical properties during saltatory conduction. Acting in series and in parallel, autologous and

  4. Impact of the controlled release of a connexin 43 peptide on corneal wound closure in an STZ model of type I diabetes.

    Directory of Open Access Journals (Sweden)

    Keith Moore

    Full Text Available The alpha-carboxy terminus 1 (αCT1 peptide is a synthetically produced mimetic modified from the DDLEI C-terminus sequence of connexin 43 (Cx43. Previous research using various wound healing models have found promising therapeutic effects when applying the drug, resulting in increased wound healing rates and reduced scarring. Previous data suggested a rapid metabolism rate in vitro, creating an interest in long term release. Using a streptozotocin (STZ type I diabetic rat model with a surgically induced corneal injury, we delivered αCT1 both directly, in a pluronic gel solution, and in a sustained system, using polymeric alginate-poly-l-ornithine (A-PLO microcapsules (MC. Fluorescent staining of wound area over a 5 day period indicated a significant increase in wound closure rates for both αCT1 and αCT1 MC treated groups, withαCT1 MC groups showing the most rapid wound closure overall. Analysis of inflammatory reaction to the treatment groups indicated significantly lower levels of both Interferon Inducible T-Cell Alpha Chemoattractant (ITAC and Tumor Necrosis Factor Alpha (TNFα markers using confocal quantification and ELISA assays. Additional analysis examining genes selected from the EMT pathway using RT-PCR and Western blotting suggested αCT1 modification of Transforming Growth Factor Beta 2 (TGFβ2, Keratin 8 (Krt8, Estrogen Receptor 1 (Esr1, and Glucose Transporter 4 (Glut4 over a 14 day period. Combined, this data indicated a possible suppression of the inflammatory response by αCT1, leading to increased wound healing rates.

  5. The gap junction cellular internet: connexin hemichannels enter the signalling limelight

    National Research Council Canada - National Science Library

    Evans, W Howard; De Vuyst, Elke; Leybaert, Luc

    2006-01-01

    Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs...

  6. Voltage regulation of connexin channel conductance.

    Science.gov (United States)

    Oh, Seunghoon; Bargiello, Thaddeus A

    2015-01-01

    Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque. This rectification likely underpins the electrical rectification observed in some electrical synapses. Both intercellular and undocked hemichannels also display two distinct forms of voltage-dependent gating, termed Vj (fast)-gating and loop (slow)-gating. This review summarizes our current understanding of the molecular determinants and mechanisms underlying these conformational changes derived from experimental, molecular-genetic, structural, and computational approaches.

  7. Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

    Directory of Open Access Journals (Sweden)

    Beyer E.C.

    2000-01-01

    Full Text Available Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43 in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel, we stably transfected connexin43-containing normal rat kidney (NRK cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

  8. Cardiac to cancer: connecting connexins to clinical opportunity.

    Science.gov (United States)

    Grek, Christina L; Rhett, J Matthew; Ghatnekar, Gautam S

    2014-04-17

    Gap junctions and their connexin components are indispensable in mediating the cellular coordination required for tissue and organ homeostasis. The critical nature of their existence mandates a connection to disease while at the same time offering therapeutic potential. Therapeutic intervention may be offered through the pharmacological and molecular disruption of the pathways involved in connexin biosynthesis, gap junction assembly, stabilization, or degradation. Chemical inhibitors aimed at closing connexin channels, peptide mimetics corresponding to short connexin sequences, and gene therapy approaches have been incredibly useful molecular tools in deciphering the complexities associated with connexin biology. Recently, therapeutic potential in targeting connexins has evolved from basic research in cell-based models to clinical opportunity in the form of human trials. Clinical promise is particularly evident with regards to targeting connexin43 in the context of wound healing. The following review is aimed at highlighting novel advances where the pharmacological manipulation of connexin biology has proven beneficial in animals or humans. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Connexin's Connection in Breast Cancer Growth and Progression.

    Science.gov (United States)

    Banerjee, Debarshi

    2016-01-01

    Gap junctions are cell-to-cell junctions that are located in the basolateral surface of two adjoining cells. A gap junction channel is composed of a family of proteins called connexins. Gap junction channels maintain intercellular communication between two cells through the exchange of ions, small metabolites, and electrical signals. Gap junction channels or connexins are widespread in terms of their expression and function in maintaining the development, differentiation, and homeostasis of vertebrate tissues. Gap junction connexins play a major role in maintaining intercellular communication among different cell types of normal mammary gland for proper development and homeostasis. Connexins have also been implicated in the pathogenesis of breast cancer. Differential expression pattern of connexins and their gap junction dependent or independent functions provide pivotal cross talk of breast tumor cells with the surrounding stromal cell in the microenvironment. Substantial research from the last 20 years has accumulated ample evidences that allow us a better understanding of the roles that connexins play in the tumorigenesis of primary breast tumor and its metastatic progression. This review will summarize the knowledge about the connexins and gap junction activities in breast cancer highlighting the differential expression and functional dynamics of connexins in the pathogenesis of the disease.

  10. Connexins and Cadherin Crosstalk in the Pathogenesis of Prostate Cancer

    Science.gov (United States)

    2015-09-01

    and challenges of connexin connections to cancer. Nat Rev Cancer, 10, 435-441. 5. Plante ,I., Stewart,M.K.G., Barr,K., Allan,A.L., and Laird,D.W...Gap junction protein connexin32 interacts with the Src homology 3/hook do- main of Discs large homolog 1. J. Biol. Chem. 282, 9789–9796 45. Stauch, K

  11. Expression of connexin 43 gene during early dental development in zebra fish%细胞间隙连接蛋白43基因在斑马鱼牙齿早期发育中的表达

    Institute of Scientific and Technical Information of China (English)

    徐智云; 刘鑫; 黄兴; 杨德琴

    2015-01-01

    Objective This study aims to investigate the expression of connexin 43 (cx43) gene during early development in zebra fish and provide a foundation for further research of cx43 gene in tooth development. Methods   Total RNA was extracted within 72 h after fertilization of zebra fish embryos and then reversed transcribed to generate the cDNA library. The specific fragments of the cx43 gene were then cloned and connected to the PGEMT vector. After confirming the constructed plasmid, the corresponding RNA polymerase was chosen, and the digoxin-labeled anti-sense mRNA probe of cx43 was syn-thesized in vitro. The cx43 gene expression of zebra fish indifferent stages was carried out by in situ hybridization. The rela-tionship of the cx43 gene expression and anatomy of the pharyngeal teeth were compared by alizarin red staining. Results The mRNA antisense probe of cx43 was acquired. The positive signal of sepia was observed in the different stages of zebra fish pharyngeal teeth after fertilization. After fertilization for 9 days, the expression site of cx43 in situ hybridization was over-lapped in accordance with the anatomical site of the pharyngeal teeth. Conclusion   cx43 gene participates in tooth develop-ment and mineralization process and plays a crucial role in later mineralization.%目的:探讨细胞间隙连接蛋白43(cx43)基因在斑马鱼早期胚胎中的表达模式,为进一步研究其在牙齿发育过程中的功能奠定基础。方法提取受精后72 h的斑马鱼胚胎总RNA,逆转录合成cDNA,特异性扩增cx43基因片段,连接到PGEMT

  12. Characterization of connexin30.3-deficient mice suggests a possible role of connexin30.3 in olfaction.

    Science.gov (United States)

    Zheng-Fischhöfer, Qingyi; Schnichels, Marc; Dere, Ekrem; Strotmann, Jörg; Loscher, Nadine; McCulloch, Fiona; Kretz, Markus; Degen, Joachim; Reucher, Harald; Nagy, James I; Peti-Peterdi, Janos; Huston, Joseph P; Breer, Heinz; Willecke, Klaus

    2007-12-01

    We have generated connexin30.3-deficient mice in which the coding region of the connexin30.3 gene was replaced by the lacZ reporter gene. The expression pattern of this connexin was characterized using beta-galactosidase staining and immunoblot analyses. In skin, beta-galactosidase/connexin30.3 protein was expressed in the spinous and granulous layers of the epidermis. Specific beta-galactosidase/connexin30.3 expression was also detected in the thin ascending limb of Henle's loop in the kidney. In addition, we found beta-galactosidase/connexin30.3 in progenitor cells of the olfactory epithelium and in a subpopulation of cells in the apical layer of the vomeronasal organ. Connexin30.3-deficient mice were fertile and displayed no abnormalities in the skin or in the chemosensory systems. Furthermore, they showed normal auditory thresholds as measured by brain stem evoked potentials. These mice did, however, exhibit reduced behavioural responses to a vanilla scent.

  13. The Increased Expression of Connexin and VEGF in Mouse Ovarian Tissue Vitrification by Follicle Stimulating Hormone

    Directory of Open Access Journals (Sweden)

    Yanzhou Yang

    2015-01-01

    Full Text Available Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. And appropriate FSH plays an important role in antiapoptosis during ovarian follicle development. Therefore, in this study, 0.3 IU/mL FSH was administered into medium during mouse ovarian cryopreservation by vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation. The results suggested that the expressions of Cx37, Cx43, apoptotic molecular caspase-3, and angiogenesis molecular VEGF were confirmed using immunohistochemistry, western blotting, and real-time PCR, and the results suggested that the treatment with FSH remarkably increased the number of morphologically normal follicles in vitrified/warmed ovaries by upregulating the expression of Cx37, Cx43, VEGF, and VEGF receptor 2, but downregulating the expression of caspase-3. In addition, the vitrified/warmed ovaries were transplanted, and the related fertility was analyzed, and the results suggested that the fertility, neoangiogenesis, and follicle reserve were remarkably increased in the FSH administrated group. Taken together, administration of 0.3 IU/mL FSH during ovarian cryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply with avascular transplantation via upregulation of Cx43, Cx37, and VEGF/VEGFR2, as well as through its antiapoptotic effects.

  14. Connexins in respiratory and gastrointestinal mucosal immunity.

    Science.gov (United States)

    Bou Saab, Joanna; Losa, Davide; Chanson, Marc; Ruez, Richard

    2014-04-17

    The mucosal lining forms the physical and chemical barrier that protects against pathogens and hostile particles and harbors its own population of bacteria, fungi and archea, known as the microbiota. The immune system controls tolerance of this population of microorganisms that have proven to be beneficial for its host. Keeping its physical integrity and a correct balance with the microbiota, the mucosa preserves its homeostasis and its protective function and maintains host's health. However, in some conditions, pathogens may succeed in breaching mucosal homeostasis and successfully infecting the host. In this review we will discuss the role the mucosa plays in the defense against bacterial pathogens by considering the gap junction protein connexins. We will detail their implication in mucosal homeostasis and upon infection with bacteria in the respiratory and the gastrointestinal tracts. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Retroviral expression of connexins in embryonic chick lens.

    Science.gov (United States)

    Jiang, J X; Goodenough, D A

    1998-03-01

    To develop an in vivo model system in which exogenous proteins can be expressed in embryonic chick lens and to further understand the function of connexin-mediated gap junction intercellular communication in lens cell biology. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells. Retroviral constructs were prepared containing alkaline phosphatase (AP) and FLAG-tagged connexins. Chick lenses were infected in situ by injecting virus into the lumen of lens vesicles at stage 18, cultures were taken at various periods. The lenses were then dissected, and the expressed proteins were visualized by AP histochemical examination and immunostaining. Twenty-four hours after infection, alkaline phosphatase could be seen in epithelia and fibers. As lens fiber maturation progressed, however, the alkaline phosphatase staining was lost as the fibers matured, presumably because of the proteolytic removal of the enzyme. By 72 hours, alkaline phosphatase staining could still be observed in epithelial cells and in differentiating fibers in the bow region but not in the mature lens fibers. FLAG-tagged exogenous lens connexins were also abundantly expressed by viral infection. The exogenous connexins were localized at the cell surfaces in junctional maculae and showed the same cell-type specific distribution as that of their endogenous connexin counterparts. An in vivo model system has been developed in the chick that provides opportunities to study the expression of wild-type and mutant proteins during lens differentiation. Expression of wild-type connexins has revealed that the characteristic distribution of the three different lens connexins is maintained even when expression is driven by a viral promoter.

  16. NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides

    Directory of Open Access Journals (Sweden)

    Yuksel Batir

    2016-12-01

    Full Text Available In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26, Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in “Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy” (Y. Batir, T.A. Bargiello, T.L. Dowd, 2016 [1]. The mutation G12R affects the structure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structure propensity (SSP score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cβ protons. This data supplements the calculated NMR structures from NOESY data [1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition, we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, “Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG” (G. Merutka, H.J. Dyson, P.E. Wright, 1995 [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds.

  17. Downregulation of Rho associated coiled-coil forming protein kinase 1 in the process of delayed myocardialization of cardiac proximal outflow tract septum in connexin 43 knockout mice embryo

    Institute of Scientific and Technical Information of China (English)

    QI Chun-hua; ZHAO Xiao-qing; MA Duan; MA Xiao-jing; ZHOU Guo-min; HUANG Guo-ying

    2011-01-01

    Background The connexln43 knockout (Cx43 KO) mouse dies at birth with an enlarged conotruncal region, which leads to the obstruction of the right outflow tract (OFT). Since myocardialization of the proximal OFT septum is one of the key events during heart development, we investigated the process in the Cx43 KO embryo hearts. Rho associated coiled-coil forming protein kinase 1 (ROCK1), is a recently found key molecule to regulate the myocardialization of OFT, but its spatiotemporal expression pattern during myocardialization remains unknown. The objective of this study was to investigate the differentially expressed pattern of ROCK1 between Cx43 KO and wild type embryo hearts, and its relationship with the delayed myocardialization in Cx43 KO embryo hearts.Methods Using immunohistochemistry, the processes of myocardiolization were investigated both in Cx43 KO and wild type embryo hearts. The differentially expressed pattern of ROCK1 between Cx43 KO and wildtype embryo hearts was evaluated both at the mRNA and protein level by real-time RT-PCR and immunohistochemistry.Results The expression of α-sarcomeric actin (α-SCA) in the proximal OFT septum of Cx43 KO embryos was delayed. Meanwhile, it was shown that the downregulation of ROCK1 coincided with delayed myocardialization. The expression of ROCK1 protein was mainly limited to the proximal outflow tract septum from embryo day (E) E11.5 to E15.5. Its expression pattern was similar with that of α-SCA. Real-time RT-PCR found that the expression level of Rock-1 mRNA began at a low level on E11.5 and reached peak at E13.5 and E14.5.Conclusions ROCK1 may have an important role in the process of myocardialization of the proximal OFT septum. Downregulation of ROCK1 is likely to contribute to the aberrant myocardialization in Cx43 KO embryo hearts.

  18. Role of connexin 43 in ischemia-reperfusion injury effect of genetic connexin 43 manipulation on myocardial cell death and arrhythmias /

    OpenAIRE

    Sánchez García, José Antonio

    2013-01-01

    El daño miocárdico por isquemia-reperfusión es una de las principales causas de morbilidad y mortalidad. La reperfusión es el tratamiento de elección en pacientes con infarto de miocardio. Sin embargo, ésta ocasiona un daño adicional por mecanismos provocados por la restauración del flujo, que puede ser prevenido, al menos en parte y en el escenario experimental, por sustancias aplicadas en el momento de la reperfusión. Una de las posibles dianas terapéuticas es la conexina 43 (Cx43) y los ca...

  19. Oligodendrocytes in Mouse Corpus Callosum are Coupled Via Gap Junction Channels Formedby Connexin47 and Connexin32

    Institute of Scientific and Technical Information of China (English)

    Marta Maglione; Oliver Tress; Brigitte Haas; Khalad Karram; Jacqueline Trotter; Klaus Willecke; Helmut Kettenmann

    2010-01-01

    已有的超微结构研究显示,白质中的少突胶质细胞和星形胶质细胞之间存在缝隙连接,但少突胶质细胞之间不存在缝隙连接,虽然体外培养的少突胶质细胞可形成功能性缝隙连接.本文研究新生小鼠胼胝体急性脑片中的少突胶质细胞的功能性连接.以全细胞膜片钳技术用生物胞素(一种可渗透的缝隙连接示踪剂)标记少突胶质细胞.平均61个细胞为链霉亲和素-Cy3标记的生物胞素阳性.约77%的连结细胞表达少突胶质细胞标志蛋白CNPase阳性染色,9%表达星形胶质细胞标志蛋白GFAP阳性,14%为CNPase和GFAP阴性.后者的大部分表达Olig2和一些NG2(少突胶质细胞前体细胞的标志物).少突胶质细胞表达Cx47、Cx32和Cx29,星形胶质细胞表达Cx43和Cx30.在Cx47敲除小鼠中,连结细胞的数量减少80%.单独删除Cx32或Cx29并不能显著减少连结细胞的数量,但Cx32/Cx47双缺陷小鼠中没有观察到相互连结的细胞.Cx47敲除完全消除了少突胶质细胞与星形胶质细胞间的耦联.在Cx43敲除动物中,少突胶质细胞-星形胶质细胞间连接仍然存在,但与少突胶质细胞前体细胞间的耦联没有被观察到.在Cx43/Cx30双敲除小鼠中,少突胶质细胞-星形胶质细胞连接几乎不存在.解开连结的少突胶质细胞显示为较高的膜输入电阻.本文认为,白质中的少突胶质细胞依靠Cx47和Cx32的表达形成功能性的合胞体,而星形胶质蛋白缝隙连接蛋白的表达能提升此网络的大小.

  20. Connexins form functional hemichannels in porcine ciliary epithelium.

    Science.gov (United States)

    Shahidullah, Mohammad; Delamere, Nicholas A

    2014-01-01

    The expression of connexins in the ciliary epithelium is consistent with gap junctions between the pigmented (PE) and nonpigmented ciliary epithelium (NPE) that form when connexon hemichannels from adjacent cells pair to form a channel. Here we present evidence that suggests undocked connexons may form functional hemichannels that permit exchange of substances between NPE and the aqueous humor. Intact porcine eyes were perfused via the ciliary artery and propidium iodide (PI) (MW 668) was added to the aqueous humor compartment as a tracer. After calcium-free solution containing PI was introduced into the aqueous humor compartment for 30 min, fluorescence microscopy revealed PI in the NPE cell layer. PI entry into the NPE was inhibited by calcium and by the connexin antagonist 18α-glycyrrhetinic acid (18-AGA). Studies also were carried out with cultured porcine NPE. Under normal conditions, little PI entered the cultured cells but calcium-free medium stimulated PI accumulation and the entry was inhibited by 18-AGA. In cells loaded with calcein (MW 622), calcium-free solution stimulated calcein exit. 18-AGA partially suppressed calcein exit in calcium-free medium. Connexin 43 and connexin 50 proteins were detected by western blot analysis in both native and cultured NPE. In the intact eye, immunolocalization studies revealed connexin 50 at the basolateral, aqueous humor-facing, margin of the NPE. In contrast, connexin 43 was observed at the junction of the PE and NPE layer and on the basolateral membrane of PE. The results point to functional hemichannels at the NPE basolateral surface. It is feasible that hemichannels might contribute to the transfer of substances between the ciliary epithelium cytoplasm and aqueous humor.

  1. 急性心肌缺血时连接蛋白43迅速降解的非均一性研究%Heterogeneous degradation of connexin43 during acute myocardial ischemia in dog hearts

    Institute of Scientific and Technical Information of China (English)

    李玉光; 林吉进; 王东明; 张元春; 卢成志

    2002-01-01

    目的探讨急性心肌缺血时心肌细胞连接蛋白43(Cx43)含量和分布的改变.方法 20只犬随机分为4组,通过结扎冠状动脉造成分别为0、1、3、6 h的急性心肌缺血,应用激光共聚焦显微镜技术和荧光免疫组织化学方法对缺血心肌Cx43含量和分布的改变进行定量研究.结果急性心肌缺血1h ,Cx43像素密度较对照组减少22.2%(P<0.01),3h减少40.0%(P<0.01),6h减少53.8%(P<0.01).对照组心肌细胞端对端连接处Cx43含量约为侧对侧连接处的1.36倍,缺血6 h后,侧对侧连接处Cx43含量反而为端对端连接处的1.6倍.对照组各层心肌细胞Cx43含量无显著差异,缺血后,中间层心肌Cx43含量较其他层心肌Cx43含量下降更明显,差异有显著性(P<0.05或P<0.01).结论急性心肌缺血时Cx43迅速降解,分布模式也发生明显的改变,各层心肌Cx43的降解程度明显不均一.

  2. Connexins and pannexins: New insights into microglial functions and dysfunctions

    Directory of Open Access Journals (Sweden)

    Rosario Gajardo-Gómez

    2016-09-01

    Full Text Available In a physiological context, microglia adopt a resting phenotype that is associated with the production of anti-inflammatory and neurotrophic factors. In response to a wide variety of insults, they shift to the activated phenotype that is necessary for the proper restoration of brain homeostasis. When the intensity of the threat is relatively high, microglial activation can worsen the damage progression instead of providing protection, with potentially significant consequences for neuronal survival. Coordinated interactions among microglia and with other brain cells, including astrocytes and neurons, is critical for the development of timely and optimal inflammatory responses in the brain parenchyma. Tissue synchronization is in part mediated by connexins and pannexins, which are protein families that form different plasma membrane channels to communicate with neighboring cells. At one end, the gap junction channels (which are exclusively formed by connexins in vertebrates connect the cytoplasm of contacting cells to coordinate electrical and metabolic coupling. At the other end, hemichannels and pannexons (which are formed by connexins and pannexins, respectively communicate via intra- and extracellular compartments and serve as diffusion pathways for the exchange of ions and small molecules. In this review, we discuss the evidence available concerning the functional expression and regulation of connexin- and pannexin-based channels in microglia and their contribution to microglial function and dysfunction. We focus on the possible implications of these channels in microglia-to-microglia, microglia-to-astrocyte and neuron-to-microglia interactions in the inflamed brain.

  3. Role of connexin43-interacting proteins at gap junctions

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2006-01-01

    Gap junctions are arrays of cell-to-cell channels that allow diffusion of small molecules between neighboring cells. The individual channels are formed by the four-transmembrane connexin (Cx) proteins. Recently, multiple proteins have been found to interact at the cytoplasmic site with the most abun

  4. Role of connexin43-interacting proteins at gap junctions

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2006-01-01

    Gap junctions are arrays of cell-to-cell channels that allow diffusion of small molecules between neighboring cells. The individual channels are formed by the four-transmembrane connexin (Cx) proteins. Recently, multiple proteins have been found to interact at the cytoplasmic site with the most

  5. The double life of connexin channels: single is a treat.

    Science.gov (United States)

    Bruzzone, Roberto

    2015-04-01

    Although several genetic diseases are caused by mutations in channels made by connexin family members, there has been little progress in the development and validation of therapeutic options. An in vitro study in this issue of JID suggests that an anti-malarial drug may be beneficial in keratitis-ichthyosis deafness, a severe conexin channel disease associated with potentially fatal recurrent infections.

  6. Connexins in endothelial barrier function - novel therapeutic targets countering vascular hyperpermeability.

    Science.gov (United States)

    Soon, Allyson Shook Ching; Chua, Jia Wang; Becker, David Laurence

    2016-10-28

    Prolonged vascular hyperpermeability is a common feature of many diseases. Vascular hyperpermeability is typically associated with changes in the expression patterns of adherens and tight junction proteins. Here, we focus on the less-appreciated contribution of gap junction proteins (connexins) to basal vascular permeability and endothelial dysfunction. First, we assess the association of connexins with endothelial barrier integrity by introducing tools used in connexin biology and relating the findings to customary readouts in vascular biology. Second, we explore potential mechanistic ties between connexins and junction regulation. Third, we review the role of connexins in microvascular organisation and development, focusing on interactions of the endothelium with mural cells and tissue-specific perivascular cells. Last, we see how connexins contribute to the interactions between the endothelium and components of the immune system, by using neutrophils as an example. Mounting evidence of crosstalk between connexins and other junction proteins suggests that we rethink the way in which different junction components contribute to endothelial barrier function. Given the multiple points of connexin-mediated communication arising from the endothelium, there is great potential for synergism between connexin-targeted inhibitors and existing immune-targeted therapeutics. As more drugs targeting connexins progress through clinical trials, it is hoped that some might prove effective at countering vascular hyperpermeability.

  7. Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules.

    Science.gov (United States)

    Bevans, C G; Kordel, M; Rhee, S K; Harris, A L

    1998-01-30

    Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

  8. Nonspecific effects of the gap junction blocker mefloquine on fast hippocampal network oscillations in the adult rat in vitro.

    Science.gov (United States)

    Behrens, C J; Ul Haq, R; Liotta, A; Anderson, M L; Heinemann, U

    2011-09-29

    It has been suggested that gap junctions are involved in the synchronization during high frequency oscillations as observed during sharp wave-ripple complexes (SPW-Rs) and during recurrent epileptiform discharges (REDs). Ripple oscillations during SPW-Rs, possibly involved in memory replay and memory consolidation, reach frequencies of up to 200 Hz while ripple oscillations during REDs display frequencies up to 500 Hz. These fast oscillations may be synchronized by intercellular interactions through gap junctions. In area CA3, connexin 36 (Cx36) proteins are present and potentially sensitive to mefloquine. Here, we used hippocampal slices of adult rats to investigate the effects of mefloquine, which blocks Cx36, Cx43 and Cx50 gap junctions on both SPW-Rs and REDs. SPW-Rs were induced by high frequency stimulation in the CA3 region while REDs were recorded in the presence of the GABA(A) receptor blocker bicuculline (5 μM). Both, SPW-Rs and REDs were blocked by the gap junction blocker carbenoxolone. Mefloquine (50 μM), which did not affect stimulus-induced responses in area CA3, neither changed SPW-Rs nor superimposed ripple oscillations. During REDs, 25 and 50 μM mefloquine exerted only minor effects on the expression of REDs but significantly reduced the amplitude of superimposed ripples by ∼17 and ∼54%, respectively. Intracellular recordings of CA3 pyramidal cells revealed that mefloquine did not change their resting membrane potential and input resistance but significantly increased the afterhyperpolarization following evoked action potentials (APs) resulting in reduced probability of AP firing during depolarizing current injection. Similarly, mefloquine caused a reduction in AP generation during REDs. Together, our data suggest that mefloquine depressed RED-related ripple oscillations by reducing high frequency discharges and not necessarily by blocking electrical coupling.

  9. Connexins: a myriad of functions extending beyond assembly of gap junction channels

    Directory of Open Access Journals (Sweden)

    Mroue Rana M

    2009-03-01

    Full Text Available Abstract Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.

  10. Connexins: a myriad of functions extending beyond assembly of gap junction channels.

    Science.gov (United States)

    Dbouk, Hashem A; Mroue, Rana M; El-Sabban, Marwan E; Talhouk, Rabih S

    2009-03-12

    Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.

  11. Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

    Institute of Scientific and Technical Information of China (English)

    Ling Liu; Haiyan Liu; Zhenping Gao; Linbo Zhang; Lue Su; Guojun Dong; Haiyang Yu; Jiayi Tian; Hang Zhao; Yanyan Xu

    2012-01-01

    Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system.Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases.To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1, 2.5 and 5 μg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-α and connexin 43 mRNA, C6 cells were treated with 5 μg/mL lipopolysaccharide for 3-48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-α mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopolysaccharide-inducedneuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatorymechanism.

  12. Detection of connexins in liver cells using sodiumdodecylsulfate polyacrylamide gel electrophoresis and immunoblot analysis

    Science.gov (United States)

    Willebrords, Joost; Maes, Michaël; Yanguas, Sara Crespo; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Since connexin expression is partly regulated at the protein level, immunoblot analysis represents a frequently addressed technique in the connexin research field. The present chapter describes the set-up of an immunoblot procedure, including protein extraction and quantification from biological samples, gel electrophoresis, protein transfer and immunoblotting, which is optimized for analysis of connexins in liver tissue. In essence, proteins are separated on a polyacrylamide gel using sodiumdodecylsulfate followed by transfer of proteins on a nitrocellulose membrane. The latter allows specific detection of connexins with antibodies combined with revelation through enhanced chemiluminescence. PMID:27207285

  13. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    Science.gov (United States)

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  14. Modulation of connexin signaling by bacterial pathogens and their toxins.

    Science.gov (United States)

    Ceelen, Liesbeth; Haesebrouck, Freddy; Vanhaecke, Tamara; Rogiers, Vera; Vinken, Mathieu

    2011-09-01

    Inherent to their pivotal tasks in the maintenance of cellular homeostasis, gap junctions, connexin hemichannels, and pannexin hemichannels are frequently involved in the dysregulation of this critical balance. The present paper specifically focuses on their roles in bacterial infection and disease. In particular, the reported biological outcome of clinically important bacteria including Escherichia coli, Shigella flexneri, Yersinia enterocolitica, Helicobacter pylori, Bordetella pertussis, Aggregatibacter actinomycetemcomitans, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium species, Streptococcus pneumoniae, and Staphylococcus aureus and their toxic products on connexin- and pannexin-related signaling in host cells is reviewed. Particular attention is paid to the underlying molecular mechanisms of these effects as well as to the actual biological relevance of these findings.

  15. Glial connexins and gap junctions in CNS inflammation and disease.

    Science.gov (United States)

    Kielian, Tammy

    2008-08-01

    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.

  16. Connexins and Cadherin Crosstalk in the Pathogenesis of Prostate Cancer

    Science.gov (United States)

    2014-09-01

    vitamin D3 synthesis in the skin , a role for this vitamin in decreasing the risk of developing PCA has been suggested [5,6]. Numerous in vitro studies...role of connexins in ear and skin physiology- Functional insights from disease-associated mutations. Biochimica et Biophysica Acta (BBA... Antioxidant and Redox Signaling 11, 283-296 4. Laird, D. W. (2010) The gap junction proteome and its relationship to disease. Trends In Cell Biology 20

  17. Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice.

    Science.gov (United States)

    Maes, Michaël; Crespo Yanguas, Sara; Willebrords, Joost; Weemhoff, James L; da Silva, Tereza Cristina; Decrock, Elke; Lebofsky, Margitta; Pereira, Isabel Veloso Alves; Leybaert, Luc; Farhood, Anwar; Jaeschke, Hartmut; Cogliati, Bruno; Vinken, Mathieu

    2017-08-15

    Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. [The effect of Connexin43 downregulation on biological functions of HUVEC].

    Science.gov (United States)

    Zhang, Cai-zhen; Mu, Xiao-feng; Xu, Xian-xiang; Qiu, Fei; Lin, Jun-sheng; Diao, Yong

    2015-03-01

    Connexin43 has been shown to play a pivotal role in wound healing process. Wound repair is enhanced by acute downregulation of connexin43, by increasing proliferation and migration of keratinocyte and fibroblast. Angiogenesis is also a central feature of wound repair, but little is known about the effects of connexin43 modulation on functions of endothelial cells. We used connexin43 specific small interference RNA (siRNA) to reduce the expression of connexin43 in human umbilical vein endothelial cell (HUVEC), and investigated the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVEC. Treatment of siRNA markedly reduced the expression of connexin43 by -80% in HUVEC (P HUVEC decreased significantly (P < 0.05), compared with that of the normal cells. The results suggest that temporally downregulation of connexin43 expression at early stage of wound to inhibit the abnormal angiogenesis characterized with leaky and inflamed blood vessels, maybe a prerequisite for coordinated normal healing process.

  19. Expression of connexin 36 in central nervous system and its role in epileptic seizure

    Institute of Scientific and Technical Information of China (English)

    PENG Yu-fen; WU Jiong-xing; YANG Heng; DONG Xuan-qi; ZHENG Wen; SONG Zhi

    2012-01-01

    Objective This review discusses the experimental and clinical studies those show the expression of connexin 36 in the central nervous system and the possible role of connexin 36 in epileptic seizure.Data sources All articles used in this review were mainly searched from PubMed published in English from 1996 to 2012.Study selection Odginal articles and reviews were selected if they were related to the expression of connexin 36 in the central nervous system and its role in epilepsy.Results The distribution of connexin 36 is developmentally regulated,cell-specific and region-specific.Connexin 36 is involved in some neuronal functions and epileptic synchronization.Changes in the connexin 36 gene and protein were accompanied by seizures.Selective gap junction blockers have exerted anticonvulsant actions in a variety of experiments examined in both humans end experimental animals.Conclusions Connexin 36 plays an important role in both physiological and pathological conditions in the central nervous system.A better understanding of the role of connexin 36 in seizure activity may contribute to the development of new therapeutic approaches to treating epilepsy.

  20. The Renal Connexome and Possible Roles of Connexins in Kidney Diseases.

    Science.gov (United States)

    Sala, Gabriele; Badalamenti, Salvatore; Ponticelli, Claudio

    2016-04-01

    Connexins are membrane-spanning proteins that allow for the formation of cell-to-cell channels and cell-to-extracellular space hemichannels. Many connexin subtypes are expressed in kidney cells. Some mutations in connexin genes have been linked to various human pathologies, including cardiovascular, neurodegenerative, lung, and skin diseases, but the exact role of connexins in kidney disease remains unclear. Some hypotheses about a connection between genetic mutations, endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) in kidney pathology have been explored. The potential relationship of kidney disease to abnormal production of connexin proteins, mutations in their genes together with ER stress, or the UPR is still a matter of debate. In this scenario, it is tantalizing to speculate about a possible role of connexins in the setting of kidney pathologies that are thought to be caused by a deregulated podocyte protein expression, the so-called podocytopathies. In this article, we give examples of the roles of connexins in kidney (patho)physiology and propose avenues for further research concerning connexins, ER stress, and UPR in podocytopathies that may ultimately help refine drug treatment.

  1. [Polymorphism of connexin 40 gene-- a novel genetic marker of the sick sinus node syndrome].

    Science.gov (United States)

    Chernova, A A; Nikulina, S Iu; Shul'man, V A; Kukushkina, T S; Voevoda, M I; Maksimov, V N

    2011-01-01

    In this work we have demonstrated for the first time on the clinico-genetic material association between hereditary sick sinus node syndrome and connexin 40 gene polymorphism. We have revealed that heterozygous variant of connexin 40 gene variant is more frequent among patients with sick sinus node syndrome and their healthy relatives than in persons of control group.

  2. Connexins and pannexins in the integumentary system: the skin and appendages.

    Science.gov (United States)

    Faniku, Chrysovalantou; Wright, Catherine S; Martin, Patricia E

    2015-08-01

    The integumentary system comprises the skin and its appendages, which includes hair, nails, feathers, sebaceous and eccrine glands. In this review, we focus on the expression profile of connexins and pannexins throughout the integumentary system in mammals, birds and fish. We provide a picture of the complexity of the connexin/pannexin network illustrating functional importance of these proteins in maintaining the integrity of the epidermal barrier. The differential regulation and expression of connexins and pannexins during skin renewal, together with a number of epidermal, hair and nail abnormalities associated with mutations in connexins, emphasize that the correct balance of connexin and pannexin expression is critical for maintenance of the skin and its appendages with both channel and non-channel functions playing profound roles. Changes in connexin expression during both hair and feather regeneration provide suggestions of specialized communication compartments. Finally, we discuss the potential use of zebrafish as a model for connexin skin biology, where evidence mounts that differential connexin expression is involved in skin patterning and pigmentation.

  3. The Role of Connexins in Wound Healing and Repair: Novel Therapeutic Approaches

    Science.gov (United States)

    Wong, Pui; Tan, Teresa; Chan, Catherine; Laxton, Victoria; Chan, Yin Wah Fiona; Liu, Tong; Wong, Wing Tak; Tse, Gary

    2016-01-01

    Gap junctions are intercellular proteins responsible for mediating both electrical and biochemical coupling through the exchange of ions, second messengers and small metabolites. They consist of two connexons, with (one) connexon supplied by each cell. A connexon is a hexamer of connexins and currently more than 20 connexin isoforms have been described in the literature thus far. Connexins have a short half-life, and therefore gap junction remodeling constantly occurs with a high turnover rate. Post-translational modification, such as phosphorylation, can modify their channel activities. In this article, the roles of connexins in wound healing and repair are reviewed. Novel strategies for modulating the function or expression of connexins, such as the use of antisense technology, synthetic mimetic peptides and bioactive materials for the treatment of skin wounds, diabetic and pressure ulcers as well as cornea wounds, are considered. PMID:27999549

  4. Expression of gap junction protein connexin 43 in bovine urinary bladder tumours.

    Science.gov (United States)

    Corteggio, A; Florio, J; Roperto, F; Borzacchiello, G

    2011-01-01

    The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). The oncogenic activity of BPV is largely associated with the major oncoprotein E5. Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis. The present study investigated biochemically and immunohistochemically the expression of connexin 43 in samples of normal (n=2), dysplastic (n=3) and neoplastic (n=23) bovine urothelium. The tumours included 10 carcinomas in situ, five papillary urothelial carcinomas and eight invasive urothelial carcinomas. Normal and dysplastic urothelium had membrane expression of connexin 43, but this was reduced in samples of carcinoma in situ. Papillary urothelial carcinomas showed moderate cytoplasmic and membrane labelling, while invasive carcinoma showed loss of connexin 43 expression. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. TGF-β1 inhibits connexin-43 expression in cultured smooth muscle cells of human bladder

    Institute of Scientific and Technical Information of China (English)

    Chi Qiang; Zhou Fenghai; Wang Yangmin

    2009-01-01

    Objective: In this research, we studied the TGF-β1 effects on connexin-43 expression in cultured human bladder smooth muscle cells. Methods: Human bladder smooth muscle cells primary cultures, with bladder tissue obtained from patients undergoing cystectomy, were intervened by recombinant human TGF-β1. Connexin-43 expression in human bladder smooth muscle cells was then examined by Western blotting and immunocytochemistry. Results: Stimulation with TGF-β1 led to significant reduction of cormexin-43 immunoreactivity and coupling (P<0.0001). Connexin-43 protein expression was significantly downregnlated (P<0.05). Simultaneously, low phosphorylation species of connexin-43 were particularly affected. Conclusion: Our experiments demonstrated a significant downregulation of connexin-43 by TGF-β1 in cultured human bladder smooth muscle cells. These findings support the view that TGF-β1 is involved in the pathophysiology of urinary bladder dysfunction.

  6. Sox4 mediates Tbx3 transcriptional regulation of the gap junction protein Cx43

    NARCIS (Netherlands)

    Boogerd, C.J.; Wong, L.Y.; van den Boogaard, M.; Bakker, M.A.J.; Tessadori, F.; Bakkers, J.; 't Hoen, P.A.C.; Moorman, A.F.; Christoffels, V.M.; Barnett, P.

    2011-01-01

    Tbx3, a T-box transcription factor, regulates key steps in development of the heart and other organ systems. Here, we identify Sox4 as an interacting partner of Tbx3. Pull-down and nuclear retention assays verify this interaction and in situ hybridization reveals Tbx3 and Sox4 to co-localize

  7. Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

    NARCIS (Netherlands)

    Soares, Ana Rosa; Martins-Marques, Tania; Ribeiro-Rodrigues, Teresa; Ferreira, Joao Vasco; Catarino, Steve; Pinho, Maria Joao; Zuzarte, Monica; Anjo, Sandra Isabel; Manadas, Bruno; Sluijter, Joost P. G.; Pereira, Paulo; Girao, Henrique

    2015-01-01

    Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, m

  8. Arrhythmogenesis in the remodeled heart : the role of spatially dispersed Cx43 expression

    NARCIS (Netherlands)

    Boulaksil, M.

    2010-01-01

    The heart is able to adapt to new, often pathologic, conditions, so-called cardiac remodeling. Although initially adequate, these adaptations could can become maladaptive over time. One of the adaptations of the heart during pathology is ventricular hypertrophy, which may go hand in hand with an inc

  9. Induction of connexin 32 expression by potential embryonic signals in rabbit uterine epithelium.

    Science.gov (United States)

    Antoskiewicz, B; Müller, G; Grümmer, R; Winterhager, E

    1996-12-01

    Connexin 32 induction is found in rabbit uterine epithelium as a response to embryo recognition. Here we have chosen this connexin 32 expression as a cell biological marker to define the type of a locally acting embryonic signal. 17 beta-estradiol, onapristone, catechol estrogen (4-hydroxy-estradiol), prostaglandins E2 and F2 alpha, db-cAMP, and glass beads as mechanical stimuli were given to pseudopregnant animals on day 4, 5 or 6 posthuman chorionic gonadotropin (hCG). The induction of connexin 32 corresponded to the time of implantation at days 6-8 post-hCG by immunohistochemistry and Northern blot analysis. Untreated pseudopregnant animals started to express connexin 32 on day 8 post-hCG. In animals treated with 4-hydroxy-estradiol, 17 beta-estradiol or prostaglandins, connexin 32 expression started 1 day earlier (day 7 post-hCG) and led to an enhanced connexin 32 expression on day 8 post-hCG compared to control animals. The antigestagen, onapristone, as well as cAMP did not alter the endogenous program. Mechanical stimuli led to a high expression of connexin 32 starting at day 7 post-hCG whereas in pregnancy the blastocyst induces connexin 32 expression from day 6 postcoitum onwards. Combination of mechanical stimuli with 17 beta-estrogen advanced the induction to day 6 post-hCG. We conclude that a mechanical stimulus in combination with 17 beta-estradiol induces connexin 32 synthesis in a similar manner as compared to the blastocyst during pregnancy.

  10. Short-term pacing in the mouse alters cardiac expression of connexin43

    Directory of Open Access Journals (Sweden)

    Rindler Michael J

    2008-05-01

    Full Text Available Abstract Background Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. Results The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. Conclusion Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.

  11. Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro

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    Eugenín, Eliseo A.; González, Hernán E.; Sánchez, Helmuth A.; Brañes, María C.; Sáez, Juan C.

    2007-01-01

    Connexin43 (Cx43), a gap junction protein subunit, has been previously detected in Kupffer cells (KCs) during liver inflammation, however, KCs phagocytose cell debris that may include Cx43 protein, which could explain the detection of Cx43 in KCs. We determined that KCs express Cx43 and form gap junctions both in vivo and in vitro. In liver sections of animals treated with LPS, Cx43 was detected at ED2+ cells interfaces, indicating formation of GJ between KCs in vivo. In vitro, unstimulated KCs cultures did not form functional GJs, and expressed low levels of Cx43 that showed a diffuse intracellular distribution. In contrast, KCs treated with LPS plus IFN-γ, expressed a greater amount of Cx43 at both the, protein and mRNA levels, and showed Cx43 at cell-cell contacts associated with higher dye coupling. In conclusion, activation of KCs in vivo or in vitro resulted in enhanced Cx43 expression levels and formation of GJ that might play relevant roles during liver inflammation. PMID:17900549

  12. Connexin37: a potential modifier gene of inflammatory disease.

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    Chanson, Marc; Kwak, Brenda R

    2007-08-01

    There is an increasing appreciation of the importance of gap junction proteins (connexins) in modulating the severity of inflammatory diseases. Multiple epidemiological gene association studies have detected a link between a single nucleotide polymorphism in the human connexin37 (Cx37) gene and coronary artery disease or myocardial infarction in various populations. This C1019T polymorphism causes a proline-to-serine substitution (P319S) in the regulatory C terminal tail of Cx37, a protein that is expressed in the vascular endothelium as well as in monocytes and macrophages. Indeed, these three cell types are key players in atherogenesis. In the early phases of atherosclerosis, blood monocytes are recruited to the sites of injury in response to chemotactic factors. Monocytes adhere to the dysfunctional endothelium and transmigrate across endothelial cells to penetrate the arterial intima. In the intima, monocytes proliferate, mature, and accumulate lipids to progress into macrophage foam cells. This review focuses on Cx37 and its impact on the cellular and molecular events underlying tissue function, with particular emphasis of the contribution of the C1019T polymorphism in atherosclerosis. We will also discuss evidence for a potential mechanism by which allelic variants of Cx37 are differentially predictive of increased risk for inflammatory diseases.

  13. Functional hemichannels formed by human connexin 26 expressed in bacteria.

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    Fiori, Mariana C; Krishnan, Srinivasan; Cortes, D Marien; Retamal, Mauricio A; Reuss, Luis; Altenberg, Guillermo A; Cuello, Luis G

    2015-03-18

    Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.

  14. Viral regulation of aquaporin 4, connexin 43, microcephalin and nucleolin.

    Science.gov (United States)

    Fatemi, S Hossein; Folsom, Timothy D; Reutiman, Teri J; Sidwell, Robert W

    2008-01-01

    The current study investigated whether human influenza viral infection in midpregnancy leads to alterations in proteins involved in brain development. Human influenza viral infection was administered to E9 pregnant Balb/c mice. Brains of control and virally-exposed littermates were subjected to microarray analysis, SDS-PAGE and western blotting at three postnatal stages. Microarray analysis of virally-exposed mouse brains showed significant, two-fold change in expression of multiple genes in both neocortex and cerebellum when compared to sham-infected controls. Levels of mRNA and protein levels of four selected genes were examined in brains of exposed mice. Nucleolin mRNA was significantly decreased in day 0 and day 35 neocortex and significantly increased in day 35 cerebellum. Protein levels were significantly upregulated at days 35 and 56 in neocortex and at day 56 in cerebellum. Connexin 43 protein levels were significantly decreased at day 56 in neocortex. Aquaporin 4 mRNA was significantly decreased in day 0 neocortex. Aquaporin 4 protein levels decreased in neocortex significantly at day 35. Finally, microcephalin mRNA was significantly decreased in day 56 neocortex and protein levels were significantly decreased at 56 cerebellum. These data suggest that influenza viral infection in midpregnancy in mice leads to long-term changes in brain markers for enhanced ribosome genesis (nucleolin), increased production of immature neurons (microcephalin), and abnormal glial-neuronal communication and neuron migration (connexin 43 and aquaporin 4).

  15. Extracellular cysteine in connexins: role as redox sensors

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    Mauricio Antonio Retamal

    2016-01-01

    Full Text Available Connexin-based channels comprise hemichannels and gap junction channels. The opening of hemichannels allow for the flux of ions and molecules from the extracellular space into the cell and vice versa. Similarly, the opening of gap junction channels permits the diffusional exchange of ions and molecules between the cytoplasm and contacting cells. The controlled opening of hemichannels has been associated with several physiological cellular processes; thereby unregulated hemichannel activity may induce loss of cellular homeostasis and cell death. Hemichannel activity can be regulated through several mechanisms, such as phosphorylation, divalent cations and changes in membrane potential. Additionally, it was recently postulated that redox molecules could modify hemichannels properties in vitro. However, the molecular mechanism by which redox molecules interact with hemichannels is poorly understood. In this work, we discuss the current knowledge on connexin redox regulation and we propose the hypothesis that extracellular cysteine could be important for sensing changes in redox potential. Future studies on this topic will offer new insight into hemichannel function, thereby expanding the understanding of the contribution of hemichannels to disease progression.

  16. Role of Connexins and Pannexins in the Pancreas.

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    Cigliola, Valentina; Allagnat, Florent; Berchtold, Lukas Adrian; Lamprianou, Smaragda; Haefliger, Jacques-Antoine; Meda, Paolo

    2015-11-01

    The pancreas produces enzymes with a digestive function and hormones with a metabolic function, which are produced by distinct cell types of acini and islets, respectively. Within these units, secretory cells coordinate their functioning by exchanging information via signals that flow in the intercellular spaces and are generated either at distance (several neural and hormonal inputs) or nearby the pancreatic cells themselves (inputs mediated by membrane ionic-specific channels and by ionic- and metabolite-permeant pannexin channels and connexin "hemichannels"). Pancreatic secretory cells further interact via the extracellular matrix of the pancreas (inputs mediated by integrins) and directly with neighboring cells, by mechanisms that do not require extracellular mediators (inputs mediated by gap and tight junction channels). Here, we review the expression and function of the connexins and pannexins that are expressed by the main secretory cells of the exocrine and endocrine pancreatic cells. Available data show that the patterns of expression of these proteins differ in acini and islets, supporting distinct functions in the physiological secretion of pancreatic enzymes and hormones. Circumstantial evidence further suggests that alterations in the signaling provided by these proteins are involved in pancreatic diseases.

  17. Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

    Science.gov (United States)

    Mansour, Hussein; McColm, Janet R; Cole, Louise; Weible, Michael; Korlimbinis, Anastasia; Chan-Ling, Tailoi

    2013-01-01

    We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (Pgap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.

  18. Connexin mutations in X-linked Charcot-Marie-Tooth disease

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    Bergoffen, J. (Univ. of Pennsylvania Medical School, Philadelphia, PA (United States)); Scherer, S.S.; Wang, S.; Scott, M.; Bone, L.J.; Chen, K.; Lensch, M.W.; Fischbeck, K.H. (Univ. of Pennsylvania Medical School, PA (United States)); Paul, D.L. (Harvard Medical School, Boston, MA (United States)); Change, P.F. (Univ. of Pennsylvania Medical School and Neurology Division, Philadelphia, PA (United States))

    1993-12-24

    X-linked Charcot-Marie-Tooth disease (CMTX) is a form of hereditary neuropathy with demyelination. Recently, this disorder was mapped to chromosome Xq13.1. The gene for the gap junction protein connexin32 is located in the same chromosomal segment, which led to its consideration as a candidate gene for CMTX. With the use of Northern (RNA) blot and immunohistochemistry techniques, it was found that connexin32 is normally expressed in myelinated peripheral nerve. Direct sequencing of the connexin32 gene showed seven different mutations in affected persons from eight CMTX families. These findings, a demonstration of inherited defects in a gap junction protein, suggest that connexin32 plays an important role in peripheral nerve.

  19. Connexin: a potential novel target for protecting the central nervous system?

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    Hong-yan Xie

    2015-01-01

    Full Text Available Connexin subunits are proteins that form gap junction channels, and play an important role in communication between adjacent cells. This review article discusses the function of connexins/hemichannels/gap junctions under physiological conditions, and summarizes the findings regarding the role of connexins/hemichannels/gap junctions in the physiological and pathological mechanisms underlying central nervous system diseases such as brain ischemia, traumatic brain and spinal cord injury, epilepsy, brain and spinal cord tumor, migraine, neuroautoimmune disease, Alzheimer′s disease, Parkinson′s disease, X-linked Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher-like disease, spastic paraplegia and maxillofacial dysplasia. Connexins are considered to be a potential novel target for protecting the central nervous system.

  20. Connexin 26 mutations in congenital SNHL in Indian population

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    S S Nayyar

    2011-01-01

    Full Text Available Introduction: Hearing impairment is a sensory disability that affects millions of people all over the world. Fifty percent of these cases are hereditary. Two genes have been localized to DFNB locus (GJB2 & GJB6 on chromosome 13 which have been commonly implicated in autosomal recessive causes of deafness among which the Connexin 26 mutation is the most common. Materials and Methods: Twenty-seven unrelated Indian patients between the ages of 1 and 23 years with nonsyndromic congenital sensorineural hearing loss for GJB2 mutations, specifically for W24X. Analysis was done by the polymerase chain reaction (PCR Restriction fragment length polymorphism RFLP and sequencing methods. Results: Seven out of these 27 subjects were found to have the W24X mutation, implying a frequency of 26% (7/27. Conclusion: Our results are in concordance with what has been reported in world literature. We also showed a 100% concordance between the PCR RFLP and sequencing methods.

  1. Regulation of Connexin-Based Channels by Fatty Acids

    Science.gov (United States)

    Puebla, Carlos; Retamal, Mauricio A.; Acuña, Rodrigo; Sáez, Juan C.

    2017-01-01

    In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood. PMID:28174541

  2. Regulation of Connexin-Based Channels by Fatty Acids.

    Science.gov (United States)

    Puebla, Carlos; Retamal, Mauricio A; Acuña, Rodrigo; Sáez, Juan C

    2017-01-01

    In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood.

  3. Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

    Science.gov (United States)

    Rash, John E; Kamasawa, Naomi; Davidson, Kimberly G V; Yasumura, Thomas; Pereda, Alberto E; Nagy, James I

    2012-06-01

    Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

  4. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

    NARCIS (Netherlands)

    Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

    2002-01-01

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.

  5. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

    NARCIS (Netherlands)

    Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

    2002-01-01

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.

  6. Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue

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    Malmström Anders

    2006-05-01

    Full Text Available Abstract Background Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. Methods Uterine isthmic biopsies were collected from non-pregnant (n = 7, term pregnant women not in labour (n = 14, in normal labour (n = 7 and in prolonged labour (n = 7. mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. Results In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p Conclusion The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility.

  7. Connexins and cyclooxygenase-2 crosstalk in the expression of radiation-induced bystander effects

    Science.gov (United States)

    Zhao, Y; de Toledo, S M; Hu, G; Hei, T K; Azzam, E I

    2014-01-01

    Background: Signalling events mediated by connexins and cyclooxygenase-2 (COX-2) have important roles in bystander effects induced by ionising radiation. However, whether these proteins mediate bystander effects independently or cooperatively has not been investigated. Methods: Bystander normal human fibroblasts were cocultured with irradiated adenocarcinoma HeLa cells in which specific connexins (Cx) are expressed in the absence of endogenous Cx, before and after COX-2 knockdown, to investigate DNA damage in bystander cells and their progeny. Results: Inducible expression of gap junctions composed of connexin26 (Cx26) in irradiated HeLa cells enhanced the induction of micronuclei in bystander cells (Pbystander response due to connexin expression. However, COX-2 knockdown resulted in enhanced micronucleus formation in the progeny of the bystander cells (P<0.001). COX-2 knockdown delayed junctional communication in HeLa Cx26 cells, and reduced, in the plasma membrane, the physical interaction of Cx26 with MAPKKK, a controller of the MAPK pathway that regulates COX-2 and connexin. Conclusions: Junctional communication and COX-2 cooperatively mediate the propagation of radiation-induced non-targeted effects. Characterising the mediating events affected by both mechanisms may lead to new approaches that mitigate secondary debilitating effects of cancer radiotherapy. PMID:24867691

  8. The connexin 30.3 of zebrafish homologue of human connexin 26 may play similar role in the inner ear.

    Science.gov (United States)

    Chang-Chien, Ju; Yen, Yung-Chang; Chien, Kuo-Hsuan; Li, Shaun-Yow; Hsu, Tsai-Ching; Yang, Jiann-Jou

    2014-07-01

    The intercellular gap junction channels formed by connexins (CXs) are important for recycling potassium ions in the inner ear. CXs are encoded by a family of the CX gene, such as GJB2, and the mechanism leading to mutant connexin-associated diseases, including hearing loss, remains to be elucidated. In this study, using bioinformatics, we found that two zebrafish cx genes, cx27.5 and cx30.3, are likely homologous to human and mouse GJB2. During embryogenesis, zebrafish cx27.5 was rarely expressed at 1.5-3 h post-fertilization (hpf), but a relatively high level of cx27.5 expression was detected from 6 to 96 hpf. However, zebrafish cx30.3 transcripts were hardly detected until 9 hpf. The temporal experiment was conducted in whole larvae. Both cx27.5 and cx30.3 transcripts were revealed significantly in the inner ear by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). In the HeLa cell model, we found that zebrafish Cx27.5 was distributed intracellularly in the cytoplasm, whereas Cx30.3 was localized in the plasma membrane of HeLa cells stably expressing Cx proteins. The expression pattern of zebrafish Cx30.3 in HeLa cells was more similar to that of cells expressing human CX26 than Cx27.5. In addition, we found that Cx30.3 was localized in the cell membrane of hair cells within the inner ear by immunohistochemistry (IHC), suggesting that zebrafish cx30.3 might play an essential role in the development of the inner ear, in the same manner as human GJB2. We then performed morpholino knockdown studies in zebrafish embryos to elucidate the physiological functions of Cx30.3. The zebrafish cx30.3 morphants exhibited wild-type-like and heart edema phenotypes with smaller inner ears at 72 hpf. Based on these results, we suggest that the zebrafish Cx30.3 and mammalian CX26 may play alike roles in the inner ear. Thus, zebrafish can potentially serve as a model for studying hearing loss disorders that result from human CX

  9. Connexin 26 facilitates gastrointestinal bacterial infection in vitro.

    Science.gov (United States)

    Simpson, Charlotte; Kelsell, David P; Marchès, Olivier

    2013-01-01

    Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.

  10. Overexpression of connexin 26 in carcinoma of the pancreas.

    Science.gov (United States)

    Kyo, Naganori; Yamamoto, Hirofumi; Takeda, Yutaka; Ezumi, Koji; Ngan, Chew Yee; Terayama, Motokazu; Miyake, Masakazu; Takemasa, Ichiro; Ikeda, Masataka; Doki, Yuichiro; Dono, Keizo; Sekimoto, Mitsugu; Nojima, Hiroshi; Monden, Morito

    2008-03-01

    Contrary to the previously purported role of gap junction (GJ) associated-protein connexin 26 (Cx26) as a tumor suppressor, increased expression of Cx26 has recently been demonstrated in several human malignancies. Surprisingly, this high expression is reportedly related to poor prognosis in squamous cell lung carcinoma and breast cancer. In this study, we examined levels of Cx26 in various human gastrointestinal (GI) carcinomas, with a focus on pancreatic carcinomas, using immunohistochemistry. Many GI carcinomas displayed abundant Cx26 expression, predominantly in the cytoplasm. Cx26 was detected in 5/8 gastric cancers (62.5%), 6/8 squamous cell carcinomas of the esophagus (75.0%), 7/8 pancreatic cancers (87.5%) and 7/8 colon cancer cases (87.5%). However, Cx26 expression was not present in hepatocellular carcinoma (HCC, 0/8). Extensive immunohistochemical examination was performed on pancreatic carcinomas, revealing strong expression of Cx26 protein in 30/43 cases (70%), weak expression in 6/43 (14%) and no expression in 7/43 (16%). The present study demonstrated up-regulated Cx26 expression in a considerable percentage of GI carcinomas, with the exception of HCC. Our findings suggest that Cx26 may be involved in some of the malignant processes of GI cancers, and especially in pancreatic carcinomas.

  11. Intracerebroventricular injection of lipopolysaccharide increases gene expression of connexin32 gap junction in rat hippocampus.

    Science.gov (United States)

    Abbasian, Mohammad; Sayyah, Mohammad; Babapour, Vahab; Mahdian, Reza

    2013-01-01

    Gap junctions are intercellular membrane channels that provide direct cytoplasmic continuity between adjacent cells. This communication can be affected by changes in expression of gap junctional subunits called Connexins (Cx). Changes in the expression and function of connexins are associated with number of brain neurodegenerative diseases. Neuroinflammation is a hallmark of various central nervous system (CNS) diseases, like multiple sclerosis, Alzheimer's disease and epilepsy. Neuroinflammation causes change in Connexins expression. Hippocampus, one of the main brain regions with a wide network of Gap junctions between different neural cell types, has particular vulnerability to damage and consequent inflammation. Cx32 - among Connexins- is expressed in hippocampal Olygodandrocytes and some neural subpopulations. Although multiple lines of evidence indicate that there is an association between neuroinflammation and the expression of connexin, the direct effect of neuroinflammation on the expression of connexins has not been well studied. In the present study, the effect of neuroinflammation induced by the Lipopolysaccharide (LPS) on Cx32 gene and protein expressions in rat hippocampus is evaluated. LPS (2.5µg/rat) was infused into the rat cerebral ventricles for 14 days. Cx32 mRNA and protein levels were measured by Real Time PCR and Western Blot after 1st, 7th and 14th injection of LPS in the hippocampus. Significant increase in Cx32 mRNA expression was observed after 7th injection of LPS (P < 0.001). However, no significant change was observed in Cx32 protein level. LPS seems to modify Cx32 GJ communication in the hippocampus at transcription level but not at translation or post-translation level. In order to have a full view concerning modification of Cx32 GJ communication, effect of LPS on Cx32 channel gating should also be determined.

  12. Immunohistochemistry using an antibody to unphosphorylated connexin 43 to identify human myometrial interstitial cells

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    Van Lommel Alfons

    2008-09-01

    Full Text Available Abstract Background Myometrial smooth myocytes contract as a result of electrical signalling via a process called excitation-contraction coupling. This process is understood in great detail at the cellular level but the generation and coordination of electrical signals throughout the myometrium are incompletely understood. Recent evidence concerning the vital role of interstitial cells of Cajal in tissue-level signalling in gastrointestinal tract, and the presence of similar cells in urinary tract smooth muscle may be relevant for future research into myometrial contractility but there remains a lack of evidence regarding these cells in the myometrium. Methods Single stain immunohistochemical and double stain immunofluorescence techniques visualised antibodies directed against total connexin 43, unphosphorylated connexin 43, KIT, alpha-SMA and prolyl 4-hydroxylase in myometrial biopsies from 26 women representing all stages of reproductive life. Results Myometrial smooth myocytes from term uterine biopsies expressed connexin 43 in a punctate pattern typical of gap junctions. However, on the boundaries of the smooth muscle bundles, cells were present with a more uniform staining pattern. These cells continued to possess the same staining characteristics in non-pregnant biopsies whereas the smooth myocytes no longer expressed connexin 43. Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells. Double-stain immunofluorescence for unphosphorylated connexin 43 and KIT, an established marker for interstitial cells, revealed a complete match indicating these cells are myometrial interstitial cells (MICs. MICs had elongated cell processes and were located mainly on the surface of the smooth muscle bundles and within the fibromuscular septum. No particular arrangement of cells as plexuses was observed. Antibody to prolyl 4-hydroxylase

  13. Connexin 26 and autosomal recessive non-syndromic hearing loss

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    Mukherjee Monisha

    2003-01-01

    Full Text Available Prelingual deafness occurs with a frequency of 1 in 1000 live births and is divided into syndromic and non-syndromic forms contributing 40 and 60% respectively. Autosomal recessive non-syndromic hearing loss (ARNSHL is responsible for 80% cases of childhood deafness. Nearly all genes localized for ARNSHL cause prelingual, severe to profound, sensorineural hearing impairment. ARNSHL is genetically heterogeneous and at least 39 loci have been identified. The most significant finding to date has been the discovery of mutations in GJB2 gene at the DFNB1 locus on chromosome 13q12 as the major cause of profound prelingual deafness. This was first reported in a Tunisian family in 1994 and thereafter in many different countries. GJB2 gene encodes the gap-junction protein, connexin 26 (Cx26, mutations in which have become the first genetic marker of inherited hearing loss. Allele-specific polymerase chain reaction (AS-PCR, single stranded conformation polymorphism (SSCP and sequencing methods have been developed for the detection of mutations in Cx26 gene. In India as well, the Cx26 mutations are being screened in families with hearing impaired children using these molecular methods. Therefore, in order to create awareness among the clinicians and the affected families; we have attempted to review the Cx26 gene mutations responsible for autosomal recessive type of non-syndromic hearing loss. The efficacy and utility of Cx26 gene analysis might open the path to proper counseling of families for carrier detection and prenatal diagnosis. It may even facilitate the development of strategies in future for the treatment of this common genetic disorder.

  14. Connexin32 expression in central and peripheral nervous systems

    Energy Technology Data Exchange (ETDEWEB)

    Deschenes, S.M.; Scherer, S.S.; Fischbeck, K.H. [Univ. of Pennslylvania, PA (United States)

    1994-09-01

    Mutations have been identified in the gap junction gene, connexin32 (Cx32), in patients affected with the X-linked form of the demyelinating neuropathy, Charcot-Marie-Tooth disease (CMTX). Gap junctions composed of Cx32 are present and developmentally regulated in a wide variety of tissues. In peripheral nerve, our immunohistochemical analysis localized Cx32 to the noncompacted myelin of the paranodal regions and the Schmidt-Lantermann incisures, where previous studies describe gap junctions. In contrast to the location of Cx32 in peripheral nerve and the usual restriction of clinical manifestations to the peripheral nervous system (PNS) (abstract by Paulson describes an exception), preliminary studies show that Cx32 is present in the compacted myelin of the central nervous system (CNS), as demonstrated by radial staining through the myelin sheath of oligodendrocytes in rat spinal cord. Analysis of Cx32 expression in various regions of rat CNS during development shows that the amount of Cx32 mRNA and protein increases as myelination increases, a pattern observed for other myelin genes. Studies in the PNS provide additional evidence that Cx32 and myelin genes are coordinately regulated at the transcriptional level; Cx32 and peripheral myelin gene PMP-22 mRNAs are expressed in parallel following transient or permanent nerve injury. Differences in post-translational regulation of Cx32 in the CNS and PNS may be indicated by the presence of a faster migrating form of Cs32 in cerebrum versus peripheral nerve. Studies are currently underway to determine the unique role of Cx32 in peripheral nerve.

  15. Connexin32 mutations and Xq13 physical map

    Energy Technology Data Exchange (ETDEWEB)

    Johanson, R.A.; Bone, L.J. [Univ. of Pennsylvania School of Medicine, Philadelphia (United States); Neystat, M. [Columbia Univ., New York, NY (United States)] [and others

    1994-09-01

    Mutations in the gap junction protein connexin32 (Cx32) are associated with X-linked Charcot-Marie-Tooth disease (CMTX). We and others have identified 24 separate mutations in 27 families. The third transmembrane domain of Cx32 has two mutations that occur in multiple families: three unrelated families have the same missense mutation at codon 139 (Val to Met), and two additional unrelated families have a single base change in codon 142 (Arg to Trp). In order to physically map the region surrounding the Cx32 gene and identify regulatory sequences that could be affected by additional mutations, we are developing a contiguous set of YAC clones for the region (Xq13.1-13.3). The YACs were identified primarily by PCR-based screening of total genomic YAC libraries from CEPH and Genethon. Additional YACs were obtained from collaborating investigators and a local library. A total of 41 overlapping YACs were identified with the STSs DXS106, DXS453, DXS348, IL2RG, GJB1(Cx32), CCG1, DXS559, DXS227, DXS986, DXS1197, and DXS128. Overlap between the YACs was determined by shared STS content and by appearance of identical segments in inter-Alu PCR. The set of overlapping YAC clones helps to define the relationship between CMTX/Cx32 and the nearby gene for severe combined immunodeficiency syndrome (SCIDX/IL2RG), and it is now being used in localization of the gene for dystonia-parkinsonism syndrome (lubag disease, DYT3), which also maps to this region.

  16. Interplay between connexin40 and nitric oxide signaling during hypertension.

    Science.gov (United States)

    Le Gal, Loïc; Alonso, Florian; Mazzolai, Lucia; Meda, Paolo; Haefliger, Jacques-Antoine

    2015-04-01

    Connexins (Cxs) and endothelial nitric oxide synthase (eNOS) contribute to the adaptation of endothelial and smooth muscle cells to hemodynamic changes. To decipher the in vivo interplay between these proteins, we studied Cx40-null mice, a model of renin-dependent hypertension which displays an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels. These mice, which were either untreated or subjected to the 1-kidney, 1-clip (1K1C) procedure, a model of volume-dependent hypertension, were compared with control mice submitted to either the 1K1C or the 2-kidney, 1-clip (2K1C) procedure, a model of renin-dependent hypertension. All operated mice became hypertensive and featured hypertrophy and altered Cx expression of the aorta. The combination of volume- and renin-dependent hypertension in Cx40-/- 1K1C mice raised blood pressure and cardiac weight index. Under these conditions, all aortas showed increased levels of Cx40 in endothelial cells and of both Cx37 and Cx45 in smooth muscle cells. In the wild-type 1K1C mice, the interactions between Cx40 and Cx37 with eNOS were enhanced, resulting in increased NO release. The Cx40-eNOS interaction could not be observed in mice lacking Cx40, which also featured decreased levels of eNOS. In these animals, the volume overload caused by the 1K1C procedure resulted in increased phosphorylation of eNOS and in a higher NO release. The findings provide evidence that Cx40 and Cx37 play an in vivo role in the regulation of eNOS.

  17. Connexin- and pannexin-based channels in normal skeletal muscles and their possible role in muscle atrophy.

    Science.gov (United States)

    Cea, Luis A; Riquelme, Manuel A; Cisterna, Bruno A; Puebla, Carlos; Vega, José L; Rovegno, Maximiliano; Sáez, Juan C

    2012-08-01

    Precursor cells of skeletal muscles express connexins 39, 43 and 45 and pannexin1. In these cells, most connexins form two types of membrane channels, gap junction channels and hemichannels, whereas pannexin1 forms only hemichannels. All these channels are low-resistance pathways permeable to ions and small molecules that coordinate developmental events. During late stages of skeletal muscle differentiation, myofibers become innervated and stop expressing connexins but still express pannexin1 hemichannels that are potential pathways for the ATP release required for potentiation of the contraction response. Adult injured muscles undergo regeneration, and connexins are reexpressed and form membrane channels. In vivo, connexin reexpression occurs in undifferentiated cells that form new myofibers, favoring the healing process of injured muscle. However, differentiated myofibers maintained in culture for 48 h or treated with proinflammatory cytokines for less than 3 h also reexpress connexins and only form functional hemichannels at the cell surface. We propose that opening of these hemichannels contributes to drastic changes in electrochemical gradients, including reduction of membrane potential, increases in intracellular free Ca(2+) concentration and release of diverse metabolites (e.g., NAD(+) and ATP) to the extracellular milieu, contributing to multiple metabolic and physiologic alterations that characterize muscles undergoing atrophy in several acquired and genetic human diseases. Consequently, inhibition of connexin hemichannels expressed by injured or denervated skeletal muscles might reduce or prevent deleterious changes triggered by conditions that promote muscle atrophy.

  18. RXP-E: a connexin43-binding peptide that prevents action potential propagation block

    DEFF Research Database (Denmark)

    Lewandowski, Rebecca; Procida, Kristina; Vaidyanathan, Ravi

    2008-01-01

    Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and ...

  19. Connexin32 gene mutations in X-linked dominant Charcot-Marie-Tooth disease (CMTX1)

    NARCIS (Netherlands)

    Janssen, EAM; Kemp, S; Hensels, GW; Sie, OG; deDieSmulders, CEM; Hoogendijk, JE; deVisser, M; Bolhuis, PA

    Single-strand conformational polymorphisms (SSCP) of the connexin32 gene were analyzed in 121 patients possibly affected by Charcot-Marie-Tooth (CMT) disease. The 121 patients were selected from 443 possible CMT/HNPP (hereditary neuropathy with liability to pressure palsies) patients based on

  20. Atrial fibrillation-linked germline GJA5/connexin40 mutants showed an increased hemichannel function.

    Directory of Open Access Journals (Sweden)

    Yiguo Sun

    Full Text Available Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40 have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca2+]o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.

  1. Connexin32 gene mutations in X-linked dominant Charcot-Marie-Tooth disease (CMTX1)

    NARCIS (Netherlands)

    Janssen, EAM; Kemp, S; Hensels, GW; Sie, OG; deDieSmulders, CEM; Hoogendijk, JE; deVisser, M; Bolhuis, PA

    1997-01-01

    Single-strand conformational polymorphisms (SSCP) of the connexin32 gene were analyzed in 121 patients possibly affected by Charcot-Marie-Tooth (CMT) disease. The 121 patients were selected from 443 possible CMT/HNPP (hereditary neuropathy with liability to pressure palsies) patients based on geneti

  2. Connexin32 gene mutations in X-linked dominant Charcot-Marie-Tooth disease (CMTX1)

    NARCIS (Netherlands)

    Janssen, EAM; Kemp, S; Hensels, GW; Sie, OG; deDieSmulders, CEM; Hoogendijk, JE; deVisser, M; Bolhuis, PA

    1997-01-01

    Single-strand conformational polymorphisms (SSCP) of the connexin32 gene were analyzed in 121 patients possibly affected by Charcot-Marie-Tooth (CMT) disease. The 121 patients were selected from 443 possible CMT/HNPP (hereditary neuropathy with liability to pressure palsies) patients based on geneti

  3. Mind the gap: connexins and cell-cell communication in the diabetic kidney.

    Science.gov (United States)

    Hills, Claire E; Price, Gareth W; Squires, Paul E

    2015-02-01

    Connexins, assembled as a hexameric connexon, form a transmembrane hemichannel that provides a conduit for paracrine signalling of small molecules and ions to regulate the activity and function of adjacent cells. When hemichannels align and associate with similar channels on opposing cells, they form a continuous aqueous pore or gap junction, allowing the direct transmission of metabolic and electrical signals between coupled cells. Regulation of gap junction synthesis and channel activity is critical for cell function, and a number of diseases can be attributed to changes in the expression/function of these important proteins. Diabetic nephropathy is associated with several complex metabolic and inflammatory responses characterised by defects at the molecular, cellul