The contribution of Clara cells to the balance of cell types in airway epithelium depends on the stimulus used; for example, NO2 damage of ciliated cells leads mainly to Clara cell differentiation along a pathway which gives rise specifically to ciliated cells. Beta-adrenergic agonists have several effects on the development of airway epithelium, including mitogenic stimulation, release of secretory products, and the conversion of Clara cells into various other cells within the bronchiolar epithelium. The Beta2-subclass of adrenergic agonists stimulates cAMP production, leading to the activation of cAMP-dependent protein kinase (cAK). To understand the mechanism by which Clara cells generate such a diversity of differentiative responses, important intracellular mediators of the response to environmental changes--protein kinase enzymes--will be studied during normal and hormonally accelerated fetal lung development, and during the regenerative repair of NO2 damage in adult lung. Immunohistochemical analysis of the amount, cellular location, and subcellular localization of the regulatory subunits, RI and RII, of cAK will be done in the proximal epithelium of fetal lung in untreated mice, and in fetuses where the mother was injected with dexamethasone (DEX) or the Beta-agonist albuterol (ALB). A fluorescent probe of a protein kinase inhibitor protein (F:PKI) which binds to activated cAK catalytic subunit will be used to estimate the degree of cAK dissociation in different parts of this epithelium. Antibody to cGMP-dependent protein kinase (cGK) will be used to assess the amount and location of this enzyme after DEX and acetylcholine treatment; cGK is in much higher concentration in fetal than in adult lung. Similar studies will be done during stages of repair of adult bronchiolar epithelium in response to NO2 treatment. Clara cells will be isolated from these adult mice, and the phosphorylation of proteins in intact cells determined. The functional status of RI, RII ATP-binding proteins, and GTP-binding proteins, will also be assessed in these Clara cell extracts using nucleotide photoaffinity labeling techniques. Finally, the presence of receptors for glucocorticoids, Beta-agonists, and cholinergic/muscarinic agonists will be determined in isolated Clara cells and in cell lines cloned from Clara cell-derived tumors.
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