Phorbol ester treatment increases paracellular permeability across IEC-18 gastrointestinal epithelium in vitro

The phorbol ester, TPA, transiently increases the transepithelial permeabil ity across the gastrointestinal epithelium formed by IEC-18. There was a si gnificant decrease in transepithelial resistance (R-T) between 0 and 1.5 hr , accompanied by increased flux of polyethylene glycol (4000 MW), suggestin g that the increase was across the tight junction. By 2 hr, the decrease in R-T reversed and maintained control level. The transepithelial permeabilit y increase was prevented by coincubation with the protein kinase C (PKC) in hibitor bisindolyl-maleimide. There was a rapid (within 15 min) translocati on of PKC-alpha from the cytosolic to the "membrane-associated" compartment , followed by a down-regulation that was detectable within 60 min of TPA tr eatment. The down-regulation of PKC-alpha from the membrane was prevented b y either calpain inhibitor I or MG-132 and resulted in a sustained permeabi lity increase. The permeability changes were not accompanied by significant effects on the amount or localization of the tight junctional proteins, oc cludin and ZO-1. However, occludin did show a reversible increase in phosph orylation with TPA treatment. Together these data support a role for PKC-al pha -mediated regulation of barrier permeability in an in vitro model of sm all intestinal epithelium, perhaps through modulation of the phosphorylatio n state of the tight junctional protein, occludin.