The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-α
The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-α
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DOI：
10.1006/excr.2000.5035
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发表时间：
2000-11-25
影响因子：
3.7
通讯作者：
Mullin, JM
中科院分区：
文献类型：
--
作者：
Clarke, H;Ginanni, N;Mullin, JM
本研究探讨了PKC- α在lc - pk1细胞中被TPA （12- o -十四烷酚13-醋酸酯）和苔藓抑素1激活后上皮屏障通透性改变中的作用。像TPA一样，苔藓抑素1结合并激活PKC，但与TPA不同的是，它不是肿瘤启动子。10(-7) M的TPA诱导lc - pk1上皮细胞片上的上皮电阻（R-t）持续下降95%，而10(-7)M苔藓抑素1仅导致R-t下降30%，5小时后自发逆转。同时将细胞片暴露于10(-7)M TPA和10(7)M苔藓抑素1中，细胞片与苔藓抑素1和MG-132（蛋白酶体抑制剂）共孵育，观察到10(-7)M TPA单独观察到的上皮通透性增加。导致6 h时间点R-t进一步下降，并抑制单独使用苔藓抑素1在该时间点的R-t恢复。TPA引起PKC-a从细胞质快速易位到细胞膜，在细胞膜上PKC-a保持升高。苔藓虫素1处理导致PKC- α从细胞质转移到膜的速度较慢，PKC- α的下调速度更快，仅在6小时后就从这个隔室中消失。经典的PKC抑制剂Go6976阻止了TPA所见的R-t的下降，TPA和苔藓虫素1处理细胞导致PKC- α的移位和下调，这与单独使用苔藓虫素1更接近。MG-132和苔藓抑素1对细胞的go孵育导致细胞膜部分pkc - α的下调较慢。与相应的对照细胞相比，苔藓虫素1处理表达pkc - α显性/阴性形式的细胞导致R-t下降速度较慢且范围较小。对于TPA和苔藓抑素1，处理细胞中膜相关部分pkc - α的水平与上皮通透性的增加密切相关。由于苔藓抑素1对紧密连接通透性的瞬时影响，它为研究连接生理学提供了一种新的药理学工具。(C) 2000年学术出版社。
The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial electrical resistance (R-t) across LLC-PK1 epithelial cell sheets, while 10(-7) M bryostatin 1 caused only a 30% decrease in R-t, which spontaneously reversed after 5 h, Simultaneous exposure of cell sheets to 10-7 M TPA and 10-7 M bryostatin 1 blunted the increase in epithelial permeability observed with 10(-7) M TPA alone, Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R-t at the 6-h time point and inhibited the recovery in R-t seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-a from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R-t seen with TPA, Treatment of cells with TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Go-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha resulted in a slower and less extensive decrease in R-t compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology. (C) 2000 Academic Press.