Phorbol ester treatment increases paracellular permeability across IEC-18 gastrointestinal epithelium in vitro.
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.
Marano
C W
CW
Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096, USA.
Garulacan
L A
LA
Ginanni
N
N
Mullin
J M
JM
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004068:Digestive System Physiological Phenomena; D015536:Down-Regulation; D004848:Epithelium / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D010539:Permeability / Q000187:drug effects*; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats
D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D010703:Phorbol Esters; D011092:Polyethylene Glycols; D011493:Protein Kinase C