Peixoto Elisa Bouçada Mauro Inácio, Collares-Buzato Carla Beatriz
Department of Histology and Embryology, Institute of Biology, State University of Campinas, Brazil.
Cell Struct Funct. 2005 Feb;29(5-6):165-78. doi: 10.1247/csf.29.165.
Natural and synthetic polycationic proteins, such as protamine, have been used to reproduce the tissue injury and changes in epithelial permeability caused by positively charged substances released by polymorphonuclear cells during inflammation. Protamine has diverse and often conflicting effects on epithelial permeability. The effects of this polycation on the distribution and expression of tight junction (TJ)-associated proteins have not yet been investigated. In this work, we examined the influence of protamine on paracellular barrier function and TJ structure using two strains of the epithelial Madin-Darby canine kidney (MDCK) cell line that differed in their TJ properties ("tight" TJ-strain I and "leaky" TJ-strain II). Protamine induced concentration-, time- and strain-dependent alterations in transepithelial electrical resistance (Rt) only when applied to apical or apical+basolateral monolayer surfaces, indicating a polarity of action. In MDCK II cells, protamine (50 microg/ml) caused a significant increase in Rt that returned to control values after 2 h. However, the treatment of this MDCK strain with a higher concentration of protamine (250 microg/ml) significantly decreased the Rt after 30 min. In contrast, treated MDCK I monolayers showed a significant decrease in Rt after apical treatment with protamine at both concentrations. The protamine-induced decrease in Rt was paralleled by an increase in the phenol red basal-to-apical flux in both MDCK strains, suggesting disruption of the paracellular barrier. Marked changes in cytoskeletal F-actin distribution/polymerization and a significant reduction in the junctional expression of the tight junctional proteins occludin and claudin-1 but subtle alterations in ZO-1 were observed following protamine-elicited paracellular barrier disruption. In conclusion, protamine induces alterations in the epithelial barrier function of MDCK monolayers that may involve the cytoskeleton and TJ-associated proteins. The various actions of protamine on epithelial function may reflect different degrees of interaction of protamine with the plasma membrane and different intracellular processes triggered by this polycation.
天然和合成的聚阳离子蛋白，如鱼精蛋白，已被用于重现炎症期间多形核细胞释放的带正电物质所引起的组织损伤和上皮通透性变化。鱼精蛋白对上皮通透性有多种且常常相互矛盾的影响。这种聚阳离子对紧密连接（TJ）相关蛋白的分布和表达的影响尚未得到研究。在这项工作中，我们使用两种紧密连接特性不同的上皮性犬肾（MDCK）细胞系（“紧密”TJ - I系和“渗漏”TJ - II系）研究了鱼精蛋白对细胞旁屏障功能和TJ结构的影响。仅当鱼精蛋白应用于顶端或顶端 + 基底外侧单层表面时，才会诱导跨上皮电阻（Rt）出现浓度、时间和细胞系依赖性改变，表明其作用具有极性。在MDCK II细胞中，鱼精蛋白（50微克/毫升）使Rt显著增加，2小时后恢复到对照值。然而，用更高浓度的鱼精蛋白（250微克/毫升）处理该MDCK细胞系30分钟后，Rt显著降低。相反，在两种浓度下，经顶端用鱼精蛋白处理的MDCK I单层细胞的Rt均显著降低。在两种MDCK细胞系中，鱼精蛋白诱导的Rt降低与酚红从基底到顶端通量的增加平行，表明细胞旁屏障被破坏。在鱼精蛋白引起细胞旁屏障破坏后，观察到细胞骨架F - 肌动蛋白分布/聚合有明显变化，紧密连接蛋白occludin和claudin - 1的连接表达显著降低，但ZO - 1仅有细微改变。总之，鱼精蛋白诱导MDCK单层细胞的上皮屏障功能发生改变，这可能涉及细胞骨架和TJ相关蛋白。鱼精蛋白对上皮功能的各种作用可能反映了鱼精蛋白与质膜相互作用的不同程度以及这种聚阳离子引发的不同细胞内过程。
