癌细胞逆转过程中N-cadherin与间隙连接蛋白Cx43功能表达的关系

钙粘合蛋白 (N cadherin ,N cad)在人肺癌细胞的表达明显低于正常人肺细胞 .肺癌细胞的间隙连接通讯功能缺陷 ,连接蛋白Cx43表达抑制 .Cx43cDNA转染肺癌细胞的 4个阳性克隆其Cx43蛋白表达升高水平相近 ,但通讯功能有差别 ,与各克隆N cad的表达水平有正相关性 .N cad表达高的克隆Cx43在膜间隙连接的分布和通讯功能最明显 ,细胞分化改善 ,在裸鼠体内生长抑制 (抑制率 75 % )有显著性 .反之N cad表达低的克隆Cx43在膜间隙连接不明显 ,细胞通讯功能弱 ,恶性表型无逆转 .提示N cad与Cx43转录后表达过程的调节密切相关 ,两者介导的粘合和通讯功能有协同促进肺癌细胞逆转的作用 .     

迷路损伤增强大鼠前庭内侧核生长相关蛋白mRNA水平

前庭代偿是研究神经可塑性的一个理想模型。生长相关蛋白（ＧＡＰ－４３）在神经再生和突触重组中起重要作用。用ＤＩＧ标记的ＧＡＰ－４３ ｃＤＮＡ片段作探针进行原位杂交,检测了大鼠迷路损伤５、１２、２０和３０ｄ后前庭内侧核ＧＡＰ－ｍＲＮＡ表达的变化。结果表明,迷路损毁后两侧前庭内侧核ＧＡＰ－４３ｍＲＮＡ的水平以不同的幅度和时程明显升高。这一结果表示,ＧＡＰ－４３ｍＲＮＡ水平的提高可能与前庭代偿中突触重组和     

缺氧和DDPH对人胚肺成纤维细胞前胶原mRNA表达的影响

用核酸原位杂交和图像分析等方法,观察直接缺氧（Ｈ）和缺氧猪肺动脉内皮细胞条件培养液（ＨＥＣＣＭ）对人胚肺成纤维细胞（ＫＭＢ１７）的前胶原ｐｒｏα１（Ⅰ）,ｐｒｏα１（Ⅲ）ｍＲＮＡ表达和抗高血压药１－（２,６－二甲基苯氧基）－２－（３,４－二甲氧基苯乙氨基）丙烷盐酸盐（ＤＤＰＨ）对此过程的影响。结果发现,Ｈ和ＨＥＣＣＭ均可使ＫＭＢ１７的两型前胶原ｍＲＮＡ表达量增高,明显高于对照组（Ｐ＜０．０１）。ＤＤＰＨ对ＨＥＣＣＭ组细胞的Ⅰ,Ⅲ两型前胶原ｍＲＮＡ表达增高均有显著的抑制作用（抑制率分别为－４３．９７％和－５６．２２％）,而对Ｈ组仅抑制ｐｒｏα１（Ⅰ）前胶原ｍＲＮＡ的过量表达（－５３．５８％）。提示缺氧可直接或通过肺动脉内皮细胞的介导,促进人胚肺成纤维细胞的Ⅰ、Ⅲ两型前胶原ｍＲＮＡ表达,ＤＤＰＨ在基因转录水平上对此过程有抑制作用     

人肝癌细胞株QGY—7703的p53基因及其表达研究

本文利用等位基因分析,ＰＣＲ－ＳＳＣＰ,ＲＴ－ＣＰＲ／序列分析,Ｎｏｒｔｈｅｒｎ印迹,免疫组织化学,Ｗｅｓｔｅｒｎ印迹,免疫沉淀等方法对人肝癌细胞株ＱＧＹ－７７０３的Ｐ５３基因背景进行了研究,发现１７号染色体短臂可能存在等位基因缺失,在Ｐ５３基因的编码序列上没有发现任何突变,但发现其ｍＲＮＡ和蛋白表达水平很低,表明在ＱＧＹ－７７０３细胞中Ｐ５３基因的低水平表达可能同细胞的恶性程度有关。     

表面活性物质相关蛋白表达的调控研究进展

特异性的肺表面活性物质相关蛋白（ＳＰ）包括亲水性的ＳＰ－Ａ、ＳＰ－Ｄ和疏水性的ＳＰ－Ｂ、ＳＰ－Ｃ．它们的表达与合成受众多生理、病理因素影响。本文综述了该领域的研究进展。１．ＳＰ表达的组织细胞特异性调控：只有肺内某些细胞（肺泡Ⅱ型细胞、Ｃｌａｒａ细胞等）能合成分泌ＳＰ,这可能是由ＳＰ基因中特定序列决定的,如ＳＰ－Ｂ基因的细胞特异性表达的调控成分。２．ＳＰ表达的发育期调控：ＳＰ基因属发育控制基因家族。在人类妊娠前３ｍｏｎ胎肺中,ＳＰ基本不表达：妊娠１５－１８ｗｋ时,气管、支气管上皮细胞即可见ＳＰ－Ｂ、ＳＰ－ＣｍＲＮＡｓ和表达蛋白,它们可能比ＳＰ－Ａ出现早：妊娠１９－２０ｗｋ时可见ＳＰ－ＡｍＲＮＡ和表达蛋白,胎肺组织在体外无激素条件下培养可很快诱导ＳＰ表达,在妊娠后３ｍｏｎ内,各ＳＰｍＲＮＡｓ及其表达蛋白水平与磷脂水平平行升高,也与ＳＰ降低表面张力的特性逐渐增强相关,羊水中可检出这些蛋白,板层体的出现与ＳＰ－Ｂ的表达密切相关,而比ＳＰ－Ａ的表达早,胎肺发育过程中ＳＰｍＲＮＡｓ增加至少部分是由于其基因转录率升高,可能同时也与翻译增加有关。３．糖皮质激素对ＳＰ表达的调控：糖皮质激素对ＳＰ－Ａ表达的调控极复杂,且与剂量     

重组含有Epstein—Barr病毒潜伏膜蛋白1（LMP1）基因的杆状病毒在昆虫？…

用杆状病毒表达系统重组病毒,在昆虫细胞中表达了完整的含有ＥＢＶ－ＬＭＰ１基因３个外显子开放读码框架的长２．３ｋｂ的ｃＤＮＡ片段。用重组病毒感染Ｓｆ９细胞,用免疫荧光染色,结果表明：４８小时表达重组蛋白,７２小时细胞较完整,免疫荧光染色强阳性,９６小时后细胞出现破碎。我们采集７２小时的组织培养上清和细胞破碎裂解液,分别采用ＳＤＳ－ＰＡＧＡＥ、ＨＰＬＣ分子筛法,用免疫蛋白印迹法实验证明,表达的蛋白能被     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

低氧对肺动脉平滑肌细胞PDGF mRNA表达的影响

应用原位杂交技术,研究了低氧对单层培养的猪肺动脉平滑肌细胞血小板源性生长因子（ＰＤＧＦ）－Ａ和－Ｂ链ｍＲＮＡ表达的影响。用全自动图像分析仪检测两组细胞ＰＤＧＦ－Ａ和－Ｂ链杂交产物的平均光密度值。结果表明：常氧条件下无血清培养的肺动脉平滑肌细胞ＰＤＧＦ－Ａ和－Ｂ链ｍＲＮＡ表达阳性颗粒稀少,低氧条件下血清培养的肺的动脉平滑肌细胞ＰＤＧＦ－Ｂ链ｍＲＮＡ表达阳性颗粒明显增多,较密集地分布于整个细胞内,为常     

血管平滑肌细胞增殖与Cdk抑制蛋白p27的表达

ｐ２７蛋白是细胞周期素依赖性激酶（Ｃｄｋ）抑制蛋白家族中的一种,主要对外部促进或抑制细胞增殖的信号起反应。本研究应用流式细胞仪（ＦＣＭ）双标记的方法观察血管紧张素Ⅱ（ＡｎｇⅡ）、血管加压素（ＡＶＰ）和血小板源生长因子（ＰＤＧＦ）对血管平滑肌细胞（ＶＳＭＣｓ）细胞周期百分比和ｐ２７蛋白表达量的影响。静止状态培养的ＶＳＭＣｓ加入ＡｎｇⅡ,ＡＶＰ,ＰＤＧＦＢＢ后,在不同时间收集细胞,用碘化丙啶（ＰＩ）标记细胞ＤＮＡ,以确定细胞所处的周期。用ｐ２７蛋白的单抗和标记了ＦＩＴＣ的二抗标记细胞,通过流式细胞仪测定被激发出的荧光量来确定细胞ｐ２７蛋白表达的相对量。结果显示,ＡｎｇⅡ刺激ＶＳＭＣｓ增生,其蛋白含量增加了４３６％（Ｐ＜００１）,但不抑制ｐ２７蛋白的表达;ＡＶＰ可轻度抑制ｐ２７的表达,有轻度促进ＶＳＭＣｓ增殖和增生的作用（Ｐ＜００５）;ＰＤＧＦ明显抑制ｐ２７的表达,引起细胞增殖。本研究结果提示,ｐ２７蛋白抑制ＶＳＭＣｓ通过Ｇ１期进入Ｓ期,是抑制ＶＳＭＣｓ增殖的重要调节因子。     

脑啡肽增强胶质细胞的神经营养作用与NO生成减少有关

本文在ＳＤ大鼠大脑皮层胶质细胞神经元共培养模式上,以神经元存活、突起生长、生长相关蛋白４３（ｇｒｏｗｔｈａｓｏｃｉａｔｅｄｐｒｏｔｅｉｎ４３,ＧＡＰ４３）ｍＲＮＡ的表达为指标,观察了脑啡肽对胶质细胞神经营养作用的影响,并对其机理作了初步探讨。结果表明,经脑啡肽处理的胶质细胞能使神经元的存活计数增加２８％（Ｐ＜００５）,单个神经元突起总长度增加１１％（Ｐ＜００５）,最长突起长度增加１６％（Ｐ＜００５）,ＧＡＰ４３ｍＲＮＡ的表达增加２６％（Ｐ＜００５）。然后又观察了脑啡肽（１０－６～１０－１２ｍｏｌ／Ｌ）对培养胶质细胞生成一氧化氮（ＮＯ）的影响。结果表明,浓度为１０－８,１０－１０ｍｏｌ／Ｌ的脑啡肽能明显抑制其生成（Ｐ＜００５）。结果提示,脑啡肽可能增强胶质细胞的神经营养作用,其机制之一可能是通过抑制胶质细胞ＮＯ的生成。     

Correlation between expression of cadherin and gap junctional communication in human lung carcinoma cells

ＣａｄｈｅｒｉｎｓａｒｅｍｅｍｂｒａｎｏｕｓＣａ2+ｄｅｐｅｎｄｅｎｔ,ｈｏｍｏｐｈｉｌｉｃ,ａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅｓｔｈａｔａｒｅａｓｓｕｍｅｄｔｏｐｌａｙｅｓｓｅｎｔｉａｌｒｏｌｅｓｉｎｃｅｌｌｒｅｃｏｇｎｉｔｉｏｎ,ｃｅｌｌｓｏｒｔｉｎｇ,ｅｍｂｒｙｏｎｉｃｍｏｒｐｈｏｇｅｎｅｓｉｓａｎｄｓｉｇｎａｌｔｒａｎｓｄｕｃｔｉｏｎ[1,2].ＴｈｅｓｅｔｒａｎｓｍｅｍｂｒａｎｅｍｏｌｅｃｕｌｅｓｆｏｒｍａｐｌａｑｕｅｔｈｒｏｕｇｈｉｔｓｃｙｔｏｐｌａｓｍｉｃｄｏｍａｉｎａｔＣｔａｉｌｗｉｔｈｔｈｅ…     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

Blockade of connexin 43 expression by stable transfection of antisense cDNA in cultured vascular smooth muscle cells

Gap junctional communication is involved in embryogenesis, cell growth control, and coordinated contraction of cardiac myocytes. It has been hypothesized that gap junctions coordinate responses of vascular cells to constrictor or dilator stimulation. Three connexin (Cx) proteins, 37, 40, and 43, are found in the vasculature. Cx43 gap junctions are widely distributed along the vascular tree, although a precise physiologic role in vascular function is unknown because of a lack of specific functional inhibitors and of suitable animal models. To investigate the role of Cx43 in intercellular communication among vascular smooth muscle (VSM) cells, we selectively modified the expression of the Cx43 gene using antisense cDNA stable transfections in culture. Results show that in cells stably transfected with antisense Cx43 cDNA, gene expression of Cx43 could be reduced to 20% of that observed in vector-transfected cells. In spite of the mRNA and protein reduction, the antisense Cx43 cDNA-transfected cells did not show a significant reduction in dye transfer or a difference in cell growth rate as compared with control. These results suggest either that the residual amount of Cx43 protein is sufficient for dye transfer and growth control or that the dye transfer in these cells can be mediated by Cx40 or other connexin proteins. Therefore, more potent approaches, such as dominant negative and gene knockout, are required to fully block gap junctional communication in VSM cells.     

Deletion of a single allele of Cx43 is associated with a reduction in the gap junctional intercellular communication and increased cell proliferation of mouse lung pneumocytes type II

OBJECTIVES: Connexins (Cx) are proteins that form the gap junctional channels at neighbouring plasma membranes between adjacent cells. Cxs are involved in cell communication, which is reportedly correlated with cell proliferation and differentiation. Alterations in connexin expression and/or gap junctional intercellular communication (GJIC) capacity have long been postulated to be important in a number of pathological conditions including cancer. This study was performed to determine the consequences of the deletion of a single allele of Gja1 (Cx43 gene) in Alveolar Type II cells (APTIIs), and its impact on GJIC and cell proliferation. MATERIAL AND METHODS: In order to do so, APTIIs from wild type (Cx43(+/+)) and heterozygous (Cx43(+/-)) mice were harvested and cultured for 4 days. The GJIC capacity was evaluated by scrape-loading method, with the transfer of lucifer yellow dye. The expression of Cx43 was evaluated by immunofluorescence method and Western blotting. Cell proliferation was evaluated by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: It was observed that GJIC capacity was significantly reduced and cell proliferation index was significantly higher in Cx43(+/-) cells compared to Cx43(+/+) cells. CONCLUSIONS: These results show that knocking out one allele of Cx43 leads to a lower cell to cell communication capacity, and consequently induces a higher cell proliferation. Because chemically induced lung adenomas in mice are known to originate from APTIIs, these alterations may play a critical role in their susceptibility to lung carcinogenesis.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.     

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

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. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.     

Connexin43 Reduces Melanoma Growth within a Keratinocyte Microenvironment and during Tumorigenesis in Vivo

Connexins (Cx) have been identified as tumor suppressors or enhancers, a distinction that appears to be dependent on the type and stage of disease. However, the role of connexins in melanoma tumorigenesis and their status during cancer onset and progression remain controversial and unclear. Here, we show that the aggressive B16-BL6 mouse melanoma cell line expresses low basal levels of Cx26 and Cx43, rendering them gap junctional intercellular communication-deficient as elucidated by immunofluorescence, Western blotting, and dye transfer studies. Following ectopic expression of green fluorescent protein-tagged Cx26 and Cx43 in these connexin-deficient melanomas, punctate gap junction-like plaques were evident at sites of cell-cell apposition, and the incidence of dye transfer was significantly increased similar to connexin-rich keratinocytes. We found that the expression of Cx43, but not Cx26, significantly reduced cellular proliferation and anchorage-independent growth from control melanomas, whereas migration was unaffected. Additionally, melanomas expressing Cx43 displayed significantly reduced growth within the in situ-like microenvironment of keratinocytes, despite a lack of heterocellular gap junctional intercellular communication between the two cell types. Furthermore, when grown in vivo in the chicken chorioallantoic membrane, primary tumors derived from Cx43-expressing melanomas were significantly smaller than controls, whereas Cx26-expressing melanomas produced tumors similar to controls. Collectively, these results suggest that Cx43, and not Cx26, can act as a tumor suppressor during melanoma tumorigenesis.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.