of Work: Our studies on the mechanisms of regulation of permeability barriers focused on exploring the role of phosphorylation of the membrane protein, occludin and on testing small synthetic peptides, homologous to segments of occludin on the opening and closing of tight junctions (TJs). The organization and phosphorylation of occludin was analyzed in MDCK cells following several procedures that disrupt and reform TJs. Western blot analysis shows that the 61-63 kDa bands of occludin do not change when the monolayers are made permeable by removing extracellular Ca++, but that they disappear after 20 hrs of Ca++ starvation. In vivo phosphorylation studies showed that occludin is phosphorylated on serine and threonine under normal conditions; however cells treated with phenylarsine oxide (a specific tyrosine phosphatase inhibitor) showed a reduced transepithelial resistance (TER) and phosphorylation of the occludin on tyrosine residues. These results support the hypothesis that occludin phosphorylation is involved in the regulation of TJs. In experiments carried out using A6 cell monolayers we observed that custom-made small oligopeptides homologous to segments of the first external loop of chick occludin molecule (i.e., SNYYGSGLSY corresponding to the residues 100 to 109 and SNYYGSGLS corresponding to residues 100 to 108) impaired junction resealing when introduced into the apical bathing fluid at concentrations in the range of 0.5 to 1.5 mg/ml. Changes in localization of Z0-1, a cytoplasmic protein that underlies the membrane at the TJs, were monitored immunocytochemically during both TJ opening and resealing. The presence or absence of the oligopeptides showed no influence on the pattern of change of Z0-1 localization. These observations support the hypothesis that TJ resealing results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, they indicate that small peptides homologous to the occludin sequence can be useful as specific reagents for transiently opening the tight junctions for effective drug delivery across epithelial barriers.
