Yeh H I, Rothery S, Dupont E, Coppen S R, Severs N J
National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, England, UK.
Circ Res. 1998;83(12):1248-63. doi: 10.1161/01.res.83.12.1248.
Gap-junctional intercellular communication in endothelial cells is implicated in the coordination of growth, migration, and vasomotor responses. Up to 3 connexin types, connexin40 (Cx40), Cx37, and Cx43 may be expressed in vascular endothelium according to vascular site, species, and physiological conditions. To establish how these connexins are organized at the level of the individual endothelial gap junction, we used affinity-purified connexin-specific antibodies raised in 3 different species to permit double and triple immunolabeling in combination with confocal and electron microscopy. Using HeLa cells transfected with Cx37 and Cx40 for characterization, the anti-Cx37 antibody (raised in rabbit) and the anti-Cx40 antibody (raised in guinea pig) were shown to recognize single bands of 37 and 40 kDa, respectively, on Western blots and to give prominent punctate labeling at the cell borders, specifically in the corresponding transfectant. By applying these antibodies together with mouse monoclonal anti-Cx43 for double and triple immunofluorescence labeling at confocal microscopy, rat aortic and pulmonary arterial endothelia were found to express all 3 connexin types, whereas coronary artery endothelium expressed Cx40 and Cx37 but lacked Cx43. High-resolution en face confocal viewing of the aortic endothelium after double labeling demonstrated frequent colocalization of connexins, with distinct variation in the expression pattern within a given cell, where it made contact with different neighbors. Triple immunogold labeling at the electron-microscopic level revealed that aortic endothelial gap junctions commonly contain all 3 connexin types. This represents the first definitive demonstration of any cell type in vivo expressing 3 different connexins organized within the same gap-junctional plaque.
内皮细胞中的间隙连接细胞间通讯与生长、迁移和血管舒缩反应的协调有关。根据血管部位、物种和生理条件，血管内皮中可能表达多达3种连接蛋白类型，即连接蛋白40（Cx40）、Cx37和Cx43。为了确定这些连接蛋白在单个内皮间隙连接水平上是如何组织的，我们使用了在3种不同物种中产生的亲和纯化的连接蛋白特异性抗体，以便结合共聚焦显微镜和电子显微镜进行双重和三重免疫标记。用转染了Cx37和Cx40的HeLa细胞进行表征，抗Cx37抗体（兔源）和抗Cx40抗体（豚鼠源）在蛋白质免疫印迹上分别识别出37 kDa和40 kDa的单一条带，并在细胞边界处产生明显的点状标记，特别是在相应的转染细胞中。通过将这些抗体与小鼠抗Cx43单克隆抗体一起应用于共聚焦显微镜下的双重和三重免疫荧光标记，发现大鼠主动脉和肺动脉内皮表达所有3种连接蛋白类型，而冠状动脉内皮表达Cx40和Cx37，但缺乏Cx43。双重标记后对主动脉内皮进行高分辨率的共聚焦表面观察显示，连接蛋白频繁共定位，在给定细胞内与不同邻居接触的部位，表达模式有明显差异。电子显微镜水平的三重免疫金标记显示，主动脉内皮间隙连接通常包含所有3种连接蛋白类型。这是体内任何细胞类型中首次明确证明在同一个间隙连接斑块内表达3种不同连接蛋白的情况。
