We are using the frog bladder and monolayers of epithelial cells in culture as model systems to study the permeability of tight junctions. We have demonstrated that apical Ca2+ is important for the resealing of tight junctions in the frog urinary bladders. Mg2+ is not able to replace Ca2+ in promoting junction resealing. We have observed that protein kinase C (PKC) inhibitors and activators markedly affect tight junction recovery after they are disrupted. These results confirm previous reports that suggest that the formation and permeability of tight junctions are actively regulated by second messenger-generating systems involving PKC and G proteins. A possible specific target for these regulatory proteins is the tight junction-associated protein ZO-1. A6, a kidney epithelial cell line, cultured in either the presence or absence of collagen on the substrate, forms confluent monolayers similar in overall morphology within 5-7 days. However, the monolayers grown on collagen coated substrate have a 10 fold higher transepithelial electrical resistance (TER) than those without collagen. We followed the changes in distribution and phosphorylation states of the protein ZO-1 during junction formation in these cells. While the amount of ZO-1 is the same, this protein is phosphorylated to a greater extent in cells grown on collagen substrate during the initial stages of confluence. For one day-old monolayers this difference is as high as three fold and progressively decreases as the culture becomes totally confluent and the TER stabilizes. We conclude that phosphorylation levels of A6 cells at the first stages of cell monolayer establishment may influence the determination of the final molecular structure and permeability of tight junctions. Using immunocytochemical techniques we determined that G alpha12 and PKC zeta precisely with ZO-1 and may be involved in locally regulating tight junctions.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
National Institute on Deafness and Other Communication Disorders
Department
Type
DUNS #
City
State
Country
United States
Zip Code