Marano C W, Garulacan L A, Ginanni N, Mullin J M
Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096, USA.
Dig Dis Sci. 2001 Jul;46(7):1490-9. doi: 10.1023/a:1010696005958.
The phorbol ester, TPA, transiently increases the transepithelial permeability across the gastrointestinal epithelium formed by IEC-18. There was a significant decrease in transepithelial resistance (R(T)) between 0 and 1.5 hr, accompanied by increased flux of polyethylene glycol (4000 MW), suggesting that the increase was across the tight junction. By 2 hr, the decrease in R(T) reversed and maintained control level. The transepithelial permeability increase was prevented by coincubation with the protein kinase C (PKC) inhibitor bisindolylmaleimide. There was a rapid (within 15 min) translocation of PKC-alpha from the cytosolic to the "membrane-associated" compartment, followed by a down-regulation that was detectable within 60 min of TPA treatment. The down-regulation of PKC-alpha from the membrane was prevented by either calpain inhibitor I or MG-132 and resulted in a sustained permeability increase. The permeability changes were not accompanied by significant effects on the amount or localization of the tight junctional proteins, occludin and ZO-1. However, occludin did show a reversible increase in phosphorylation with TPA treatment. Together these data support a role for PKC-alpha-mediated regulation of barrier permeability in an in vitro model of small intestinal epithelium, perhaps through modulation of the phosphorylation state of the tight junctional protein, occludin.
佛波酯TPA可短暂增加由IEC-18形成的胃肠道上皮的跨上皮通透性。在0至1.5小时之间，跨上皮电阻（R(T)）显著降低，同时聚乙二醇（4000MW）通量增加，这表明增加是通过紧密连接实现的。到2小时时，R(T)的降低逆转并维持在对照水平。与蛋白激酶C（PKC）抑制剂双吲哚马来酰胺共同孵育可阻止跨上皮通透性的增加。PKC-α在15分钟内迅速从胞质转移至“膜相关”区室，随后在TPA处理60分钟内可检测到下调。钙蛋白酶抑制剂I或MG-132可阻止PKC-α从膜上的下调，并导致通透性持续增加。通透性变化并未对紧密连接蛋白occludin和ZO-1的量或定位产生显著影响。然而，TPA处理后occludin的磷酸化确实出现了可逆性增加。这些数据共同支持了PKC-α介导的调节小肠上皮体外模型中屏障通透性的作用，可能是通过调节紧密连接蛋白occludin的磷酸化状态来实现的。
