The proposed study will investigate a) mechanisms which control airway permeability, b) intracellular sites of early injury following inhalation exposure of rats to 03 and N02 and c) means to block or reverse pollutant induced increased permeability. Pilot studies from our laboratory demonstrate an increase in tracheal and bronchoalveolar permeability to macromolecules following inhalation exposure of rats to 03. The increased permeability appears to result, at least in part, from the perturbation of tight junctions. Based on our preliminary observations on the distribution of actin and myosin in the apical cytoplasm and in close vicinity of tight junctions of ciliated epithelial cells and also based on the similarities in the actions of pollutant gases and cytoskeleton-disrupting drugs, we propose that the cytoskeleton is a likely intracellular target site for pollutant and that it plays a critical role in the modulation of tight junction permeability. We also anticipate the role of cytoskeleton in vesicular transport in airways as in other systems. The proposed studies take into account an extensive comparison of cytoskeleton-active drugs with pollutant gases and possible reversal of pollutant effects by the drugs. A variety of indicators will be analyzed to achieve the projected goals. Rats will be exposed to 0.2-0.8 ppm O3 or 5-25 ppm NO2. Treatments with cytoskeleton-destabilizing (colchicine and cytochalasin B) or stabilizing (Phalloidin and kinetin) drugs will be carried out either alone or in combination with pollutant exposures. Changes in mucosal permeabilities to macromolecules (mol wt 469 to 69,000) will be followed using isotope labeling procedures which were introduced in this laboratory about two years ago and are now well established. Kinetics of molecular transport under various experimental conditions will be compared. Immunocytochemistry by light and electron microscopy will be employed to characterize various cytoskeletal components and their relation to transport through tight junctions or endocytic vesicles. Freeze fracture replicas will be analyzed morphometrically to detect fine changes in tight junctional depth and intramembranous strands following drug treatments or pollutant exposures. Surface morphology of epithelial cells, injuries to cell membranes and formation of intercellular spaces resulting from retraction of adjacent cells will be monitored buy scanning electron microscopy.
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