A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion.
A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion.
复制标题
DOI：
10.1152/ajprenal.1999.277.4.f524
复制
发表时间：
1999-10
期刊：
影响因子：
--
通讯作者：
Jiuming Ye;T. Tsukamoto;A. Sun;S. Nigam
中科院分区：
文献类型：
--
作者：
Jiuming Ye;T. Tsukamoto;A. Sun;S. Nigam
紧密连接（TJ）的完整性，这是负责的极化上皮细胞的渗透性屏障，在缺血性损伤过程中被破坏，必须重建恢复。最近，在Madin-Darby犬肾细胞中使用缺血和再灌注损伤的ATP消耗-补充模型，显示TJ蛋白如闭合小带-1（ZO-1）可逆地形成大的复合物并与细胞骨架蛋白缔合（T. Tsukamoto和S. K. Nigam，J.Biol.Chem.272：16133-16139，1997）。在这项研究中，我们研究了细胞内钙离子的作用，在TJ重组ATP耗竭-补充后，通过使用细胞渗透钙螯合剂1，2-双（2-氨基苯氧基）乙烷- N，N，N '，N'-四乙酸-AM（BAPTA-AM）。在ATP耗竭期间降低细胞内钙与ATP补充后渗透性屏障重建的显著抑制相关，如通过跨上皮电阻和甘露醇通量测量的，通过免疫荧光分析测定的闭合蛋白亚细胞定位的显著改变，以及通过Triton X-100提取测定的ZO-1和其他TJ蛋白的溶解度降低，这表明降低细胞内钙增强了TJ蛋白与细胞骨架的相互作用。免疫共沉淀研究表明，溶解度降低可能部分是由于大TJ蛋白与胞衬蛋白的复合物的稳定。尽管离子型去污剂（SDS和脱氧胆酸盐）似乎会导致含有ZO-1的复合物从细胞骨架上解离，但对溶解蛋白质的蔗糖梯度分析表明，钙螯合导致这些复合物的自缔合。总之，这些结果提高了细胞内钙离子在缺血性损伤损伤的TJ重组中起重要促进作用的可能性。钙似乎是必要的TJ-细胞骨架复合物的解离，从而允许功能TJ重组和细胞旁通透性屏障恢复。
The integrity of the tight junction (TJ), which is responsible for the permeability barrier of the polarized epithelium, is disrupted during ischemic injury and must be reestablished for recovery. Recently, with the use of an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney cells, TJ proteins such as zonula occludens-1 (ZO-1) were shown to reversibly form large complexes and associate with cytoskeletal proteins (T. Tsukamoto and S. K. Nigam, J. Biol. Chem. 272: 16133-16139, 1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N', N'-tetraacetic acid-AM (BAPTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability barrier following ATP repletion as measured by transepithelial electrical resistance and mannitol flux, marked alterations in the subcellular localization of occludin by immunofluorescent analysis, and decreased solubility of ZO-1 and other TJ proteins by Triton X-100 extraction assay, suggesting that lowering intracellular calcium potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that decreased solubility may partly result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) appeared to cause a dissociation of ZO-1-containing complexes from the cytoskeleton, sucrose gradient analyses of the solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium plays an important facilitatory role in the reassembly of the TJ damaged by ischemic insults. Calcium appears to be necessary for the dissociation of TJ-cytoskeletal complexes, thus permitting functional TJ reassembly and paracellular permeability barrier recovery.