Ma T Y, Tran D, Hoa N, Nguyen D, Merryfield M, Tarnawski A
Division of Gastroenterology, Department of Medicine, DVA Medical Center, Long Beach, California 90822, USA.
Microsc Res Tech. 2000 Oct 15;51(2):156-68. doi: 10.1002/1097-0029(20001015)51:2<156::AID-JEMT7>3.0.CO;2-J.
Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohn's disease, NSAID-associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco-2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca(++) modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter-grown Caco-2 intestinal monolayers in Ca(++)-free solution (CFS), consisting of modified Krebs-buffer solution containing 0 mM Ca(++) and 1 mM EGTA, resulted in a rapid drop in Caco-2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco-2 TJ permeability was rapidly reversed by the re-introduction of Ca(++) (1.8 mM) into the incubation medium. The CFS-induced increase in Caco-2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO-1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS-induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri-junctional actin and myosin filaments. The inhibition of CFS-induced activation of Caco-2 MLCK with MLCK inhibitor (ML-7) prevented the CFS-induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS-induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco-2 MLCK activation and the resultant contraction of the peri-junctionally located actin-myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg(++)-myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS-induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca(++) is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca(++) from the incubation medium causes activation of Caco-2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability.
最近的研究表明，肠道紧密连接（TJ）通透性异常增加可能是包括克罗恩病、非甾体抗炎药相关性肠炎以及各种感染性腹泻综合征在内的多种疾病的重要病因。然而，参与调节肠道上皮TJ通透性的细胞内过程仍知之甚少。在本研究中，我们使用培养的Caco-2肠道上皮细胞来研究细胞外Ca(++)调节肠道上皮单层TJ屏障所涉及的细胞内过程。将滤膜生长的Caco-2肠道单层置于不含Ca(++)的溶液（CFS）中孵育，该溶液由含0 mM Ca(++)和1 mM乙二醇双四乙酸（EGTA）的改良克雷布斯缓冲液组成，导致Caco-2上皮电阻迅速下降，上皮对细胞旁标记物甘露醇和菊粉的通透性增加，表明TJ通透性增加。将Ca(++)（1.8 mM）重新引入孵育培养基后，Caco-2 TJ通透性的增加迅速逆转。CFS诱导的Caco-2 TJ通透性增加与细胞质和跨膜TJ蛋白（闭锁小带蛋白1和闭合蛋白）的分离以及相邻细胞间形成大的细胞间开口有关。CFS诱导的TJ屏障调节与肌球蛋白轻链激酶（MLCK）活性的激活以及连接周围肌动蛋白和肌球蛋白丝向心收缩有关。用MLCK抑制剂（ML-7）抑制CFS诱导的Caco-2 MLCK激活可防止CFS诱导的肌动蛋白和肌球蛋白丝收缩以及随后TJ屏障功能和结构的改变。我们的结果表明，CFS诱导的TJ蛋白改变和TJ通透性功能性增加是由Caco-2 MLCK激活以及由此导致的连接周围肌动蛋白-肌球蛋白丝收缩介导的。与MLCK在此过程中的作用一致，所选的Mg(++)-肌球蛋白ATP酶和代谢能量抑制剂，但不是蛋白质合成抑制剂，也可防止CFS诱导的肌动蛋白和肌球蛋白丝收缩以及随后TJ通透性的增加。总之，我们的结果表明细胞外Ca(++)对于维持肠道上皮TJ屏障功能至关重要。从孵育培养基中去除细胞外Ca(++)会导致Caco-2 MLCK激活，进而导致肠道单层TJ通透性增加。
