The applicant will spend 3 years in formal research training in the laboratories of the sponsors, Drs. Steven Kelsen, and Robert Colman. Training will be primarily acquired by hand-on performance of experiments described in this proposal which will examine the regulation of the phosphodiesterase (PDE) expressed in human tracheobronchial cells. The applicant will characterize the PDE system in a transformed cell line (BEAS-2B) and in cells from normal and asthmatic humans, in both freshly dissociated and primary cultures. The following specific aims will be carried out: 1) Purify and characterize the soluble and particulate PDEs in a human airway epithelial cell line (BEAS-2B), and in freshly isolated normal human airway epithelial cells; 2) Sequence cAS-PDE (type Iv) PDE cDNA from BEAS-2B cells and compare sequences with that of known cAS-PDE; 3) Compare the regulation of cGI-PDE and cAS-PDE gene expression in cultured airway epithelial cells and a human monocyte cell line (U937) in response to inflammatory stimuli; and 4) Compare the regulation of cAS-PDE gene expression in freshly isolated human tracheal epithelial cells from normal and mild asthmatic subjects.
