REGULATION OF TIGHT JUNCTION PERMEABILITY BY CALCIUM MEDIATORS AND CELL CYTOSKELETON IN RABBIT TRACHEAL EPITHELIUM

The present study investigates the mechanisms controlling tight juncti on permeability of the tracheal epithelium, with an emphasis on the re gulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their juncti onal permeability barrier was investigated in vitro by means of transe pithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, C-14-mannitol. The effects of in tra- and extracellular calcium were studied using the calcium ionophor e A 23187 and EGTA, and that of the cytoskeleton was investigated usin g cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a subs tantial decrease in transepithelial electrical resistance and an incre ase in transepithelial flux of C-14-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other ha nd, tissues treated with the calcium ionophore demonstrate a partial o r no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular cal cium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irre versibility of the membrane resistance. These results suggest that hig h extracellular calcium levels and low intracellular calcium levels ar e required for the normal maintenance of the junctional permeability i n the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tigh t junction permeability may exert their action through the calcium and cytoskeleton mechanisms.