CHARACTERIZATION OF GTP-BINDING PROTEINS COUPLED TO INHIBITION OF ADENYLYL-CYCLASE IN GUINEA-PIG TRACHEAL EPITHELIAL-CELLS

Many important airway epithelial cell functions are regulated by intra cellular cAMP. Adenylyl cyclase, the enzyme that synthesizes cAMP, is under dual regulation in many cells, but muscarinic agonists have not been shown to inhibit adenylyl cyclase in human and dog epithelial cel ls, despite the presence of muscarinic receptors. We question whether the lack of inhibition was related to the absence of a component of th e inhibitory pathway or a lack of coupling between the components. The GTP-binding regulatory proteins (G proteins) that regulate adenylyl c yclase activity in airway epithelium have not been well characterized. We used primary cultures of guinea pig tracheal epithelial cells as a model system and identified the G proteins that modulate adenylyl cyc lase activity. Immunoblot analysis demonstrated the presence of alpha subunits corresponding to stimulatory (G(s)alpha) and inhibitory [G(i) alpha(2) and G(i)alpha(3)] G proteins as well as beta chains. These G proteins were functionally coupled to stimulation and inhibition of ad enylyl cyclase in epithelial membrane preparations. Pertussis toxin-ca talyzed [P-32]ADP-ribosylation of G(i)alpha was significantly reduced by 100 muM GTPgammaS (78.4 +/- 3.6% of control), by 100 mM NaF (41.9 /- 9.1% of control), and by carbachol (100 muM) (29.2 +/- 9.0% of cont rol). Atropine (10 muM) inhibited the carbachol effect by greater than 90%, suggesting that the muscarinic receptors were functionally coupl ed to G(i) proteins. Beta-adrenergic agonists increased adenylyl cycla se activity, but muscarinic agonists failed to inhibit this enzyme. In summary, guinea pig tracheal epithelial membranes contain muscarinic receptors, G(i)alpha(2) and adenylyl cyclase, which are appropriately coupled. However, muscarinic agonists do not inhibit adenylyl cyclase in these membranes.