Collares-Buzato C B, Jepson M A, McEwan G T, Simmons N L, Hirst B H
Gastrointestinal Drug Delivery Research Centre, University of Newcastle upon Tyne Medical School, UK.
Histochemistry. 1994 Mar;101(3):185-94. doi: 10.1007/BF00269543.
Strains I and II of Madin-Darby canine kidney (MDCK) cells, which differ markedly in transepithelial resistance (RT) and paracellular permeability, have been used to investigate whether differences in the cellular content of uvomorulin/E-cadherin and phosphotyrosine may be correlated with junctional properties. Using immunocytochemistry, the strain I "tight" epithelia showed significantly stronger uvomorulin staining at regions of cell-cell contact compared with strain II "leaky" MDCK epithelia. In contrast, strain I MDCK cells showed a relatively faint phosphotyrosine staining, distributed evenly throughout the cytoplasm, while strain II MDCK cells displayed intense staining for phosphotyrosine residues in the junctional region and the lateral cell membrane with additional labelling of the cytoplasm. Exposure to vanadate in conjunction with H2O2 (which are potent inhibitors of protein tyrosine phosphatases) resulted in a dramatic increase in phosphotyrosine staining at the intercellular area and, concomitantly, induced changes in cell morphology, a significant decrease in RT, increase in paracellular inulin permeability, and time-dependent disappearance of uvomorulin from the cell-cell contact sites. Moreover, the effects of vanadate/H2O2 treatment were more dramatic in strain II compared with strain I cells, consistent with greater generation of tyrosine-modified protein in strain II cells. An inverse relationship was demonstrated between membrane-associated uvomorulin/E-cadherin and cellular phosphotyrosine content, which varied between the two strains of MDCK cells and when phosphotyrosine was directly manipulated. These data support the hypothesis that regulation of paracellular permeability may result from specific tyrosine phosphorylation of protein components of the junctional complex.
麦迪逊-达比犬肾（MDCK）细胞的I型和II型菌株，其跨上皮电阻（RT）和细胞旁通透性存在显著差异，已被用于研究uvomorulin/E-钙黏蛋白和磷酸酪氨酸的细胞含量差异是否可能与连接特性相关。使用免疫细胞化学方法，与II型“渗漏”的MDCK上皮细胞相比，I型“紧密”上皮细胞在细胞-细胞接触区域显示出明显更强的uvomorulin染色。相反，I型MDCK细胞显示出相对较弱的磷酸酪氨酸染色，均匀分布在整个细胞质中，而II型MDCK细胞在连接区域和细胞膜侧面显示出强烈的磷酸酪氨酸残基染色，细胞质也有额外的标记。与过氧化氢（蛋白质酪氨酸磷酸酶的有效抑制剂）联合暴露于钒酸盐导致细胞间区域的磷酸酪氨酸染色显著增加，同时诱导细胞形态变化、RT显著降低、细胞旁菊粉通透性增加以及uvomorulin从细胞-细胞接触位点随时间消失。此外，与I型细胞相比，钒酸盐/过氧化氢处理对II型细胞的影响更显著，这与II型细胞中酪氨酸修饰蛋白的生成更多一致。在膜相关的uvomorulin/E-钙黏蛋白和细胞磷酸酪氨酸含量之间显示出反比关系，这在两种MDCK细胞菌株之间以及直接操纵磷酸酪氨酸时有所不同。这些数据支持这样的假设，即细胞旁通透性的调节可能源于连接复合体蛋白质成分的特定酪氨酸磷酸化。
