Nagy J I, Ochalski P A, Li J, Hertzberg E L
Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Neuroscience. 1997 May;78(2):533-48. doi: 10.1016/s0306-4522(96)00584-2.
Gap junctions between astrocytes as well as between astrocytes and oligodendrocytes in rat brain were immunohistochemically labelled with a monoclonal and an affinity-purified polyclonal antibody generated against connexin-26. By light microscopy, the immunolabelling patterns obtained were, with a few exceptions, remarkably similar to previously described distribution patterns of the gap junctional protein connexin-43, which is expressed by astrocytes and is localized at astrocytic gap junctions. By electron microscopy, immunoreactivity with these two anti-connexin-26 antibodies was restricted to astrocytes; inter-astrocytic gap junctional membranes were symmetrically labelled, heterologous oligo-astrocytic junctional membranes were asymmetrically labelled only on the astrocyte side and oligo-oligodendrocyte junctions were unlabelled. Two additional anti-connexin-26 antibodies that were found to produce punctate labelling in leptomeninges and liver failed to do so in brain parenchyma, consistent with reports indicating the absence of authentic connexin-26 in this tissue. Antibodies that labelled astrocytic gap junctions exhibited no cross-reaction with connexin-43 or connexin-32, as demonstrated by western blotting, but recognized liver connexin-26 as well as several brain proteins, including an approximately 32000 mol. wt protein that did not correspond to connexin-32 and a 26000 mol. wt protein that co-migrated with liver connexin-26. These results suggest that connexin-26, or more likely a protein having sequence homology with connexin-26, is targeted to astrocytic gap junctions and raise the possibility of the existence of connexins that may be co-expressed with connexin-43 in most, but perhaps not all, astrocytes.
用针对连接蛋白-26产生的单克隆抗体和亲和纯化的多克隆抗体，对大鼠脑内星形胶质细胞之间以及星形胶质细胞与少突胶质细胞之间的缝隙连接进行免疫组织化学标记。通过光学显微镜观察，除少数例外，所获得的免疫标记模式与先前描述的缝隙连接蛋白连接蛋白-43的分布模式非常相似，连接蛋白-43由星形胶质细胞表达并定位于星形胶质细胞的缝隙连接处。通过电子显微镜观察，这两种抗连接蛋白-26抗体的免疫反应性仅限于星形胶质细胞；星形胶质细胞间的缝隙连接膜被对称标记，异源性少突胶质细胞-星形胶质细胞连接膜仅在星形胶质细胞一侧被不对称标记，而少突胶质细胞-少突胶质细胞连接未被标记。另外两种抗连接蛋白-26抗体在软脑膜和肝脏中产生点状标记，但在脑实质中未产生，这与该组织中不存在真正的连接蛋白-26的报道一致。如蛋白质印迹法所示，标记星形胶质细胞缝隙连接的抗体与连接蛋白-43或连接蛋白-32无交叉反应，但能识别肝脏连接蛋白-26以及几种脑蛋白，包括一种分子量约为32000道尔顿的蛋白（与连接蛋白-32不符）和一种分子量为26000道尔顿的蛋白（与肝脏连接蛋白-26共迁移）。这些结果表明，连接蛋白-26，或者更可能是与连接蛋白-26具有序列同源性的一种蛋白，定位于星形胶质细胞的缝隙连接，并增加了在大多数（但可能不是所有）星形胶质细胞中可能与连接蛋白-43共表达的连接蛋白存在的可能性。
