Because airway smooth muscle contraction is an important contributor to airflow obstruction in asthma, the long term objective is to characterize the mechanisms underlying contraction by muscarinic agonists and relaxation by beta-adrenergic agonists in airway smooth muscle. Several studies have indicated that airway smooth muscle from patients with asthma have reduced relaxant responsiveness to beta-adrenergic agonists and, therefore, it is important to understand molecular mechanisms that may underlie resistance to beta-adrenergic agonists. Because contractions induced by muscarinic agonists arc relatively resistant to relaxation by beta-adrenergic agonists and because decreasing cytosolic calcium concentrations is one mechanism underlying the adenylate cyclase-dependent relaxant effects beta-adrenergic agonists, it is hypothesized that muscarinic inhibition of adenylate cyclase activity is an important, and possibly physiologically regulated, determinant of the inhibitory effects that beta-adrenergic agonists have on cytosolic calcium. Using cells isolated from bovine tracheal smooth muscle, the specific aims are: 1) Characterize the receptor subtype, location, and G-protein coupling ofthe muscarinic receptors mediating increases in cytosolic calcium and inhibition of adenylate cyclase. 2) Show that inhibiting muscarinic regulation of adenylate cyclase increases the sensitivity of cytosolic calcium concentrations to the inhibitory effects of beta-adrenergic agonists. 3) Determine whether prolonged exposure to muscarinic agonists decreases muscarinic receptor expression, muscarinic inhibition of adenylate cyclase activity, or alters calcium concentration sensitivity to beta-adrenergic agonists. The methods used to accomplish these specific aims for isolated cells will be digital imaging of fura-2 fluorescence in single cells to measure cytosolic calcium, the development of an intact cell radioligand binding assay for isolated trachealis cells, the development of new fluorescent probes for the muscarinic receptor, and the measurement of cAMP in trachealis cells. The results of these experiments will characterize the molecular mechanisms underlying adenylate cyclase regulation in airway smooth muscle cells and will, demonstrate the functional consequences that inhibiting adenylate cyclase activity has for the regulation of calcium by beta-adrenergic agonists. The results of these studies will provide new information on the relative resistance that muscarinic responses have to the inhibitory effects of beta-adrenergic agonists.
