Occluding junctions in cultured epithelial monolayers

Abstract

When MDCK cells are cultured in monolayers, they synthesize, assemble, and seal occluding junctions that limit the paracellular route. These processes may be impaired by inhibitors of the protein synthesis but not by inhibitors of the synthesis of RNA. Once established, the occluding junctions confer to the monolayer an overall electrical resistance of 80–600 omega . cm2. At the microscopical level, the resistance of individual junctions have large variations along the perimeter of a given cell. This agrees with the images of freeze-fracture electron microscopy where the network of the junction varies abruptly from 1 to 10 strands. The junctions are impermeable to macromolecular tracers, have a 9 to 1 Na+/Cl- discrimination, and a cation selectivity following the order: K+ greater than Na+ greater than Rb+ greater than Cs+ greater than Li+. Sealing requires extracellular Ca2+, but the junctions open when the concentration of Ca2+ in the cytoplasm increases. The structural components of the cytoskeleton (microtubules and microfilaments) seem to be involved in the junctional events as revealed by staining with immunofluorescent specific antibodies. If the cells are treated with cytochalasin B, actin microfilaments disorganize, the junctions open, and the electrical resistance across the monolayers falls. The resealing of the tight junction is inhibited by this drug.