Platelet-activating factor (PAF) is a potent immune and inflammatory mediator. It has been suggested that PAF plays an important role in generation of the asthmatic response, as its production may be increased in the asthmatic lung, and several pulmonary lesions associated with asthma can be generated in experimental animals after exposure to PAF. Effects of PAF on respiratory cells and tissues in vitro (eg. bronchoconstriction) often parallel events observed in the asthmatic response. Since airways of asthmatics often become obstructed with mucus, effects of PAF on secretion of respiratory mucin were investigated in preliminary studies by the applicant. These showed PAF capable of stimulating secretion of respiratory mucin by explants of rodent trachea. In the proposed research, a new and unique airway epithelial cell culture system will be utilized to study cellular mechanisms of PAF-stimulated mucin secretion. In this in vitro system, dissociated cells from guinea pig trachea differentiate into a pseudostratified columnar morphology virtually identical to that in intact tissue, with ciliated, goblet and basal cells maintained in the same orientation as in vivo. These cultures synthesize and secrete mucin glycoproteins similar biochemically to those released by organ cultures of rodent airways. Thus, the advantages of cell cultures for in vitro studies (eg. pure population of epithelial cells) are combined with those of organ culture and in vivo systems (cells maintain columnar configuration, orientation, polarity of differentiation, and normal cell-cell interactions). The experiments are designed to address the following hypothesis: PAF stimulates respiratory mucin secretion via interactions with receptors on surface of secretary cells, provoking hydrolysis of membrane phosphoinositides. Hydrolysis products then act intracellularly to stimulate metabolism of arachidonic acid, whose metabolites, in turn, act within the cell to stimulate mucin secretion. Cellular mechanisms of respiratory mucin secretion under normal and PAF-stimulated conditions, and intracellular responses of possible target cells to PAF, a potential major mediator of inflammation and anaphylaxis in the lung, will be elucidated.
