MUSCARINIC BLOCKADE OF BETA-ADRENOCEPTOR-STIMULATED ADENYLYL CYCLASE - THE ROLE OF STIMULATORY AND INHIBITORY GUANINE-NUCLEOTIDE BINDING REGULATORY PROTEINS (GS AND GI)

1 The functional antagonism that exists between muscarinic and beta-adrenoceptor function in guinea-pig tracheal smooth muscle was investigated by assessing G(s) and G(i) regulated adenylyl cyclase activity in isolated membranes. 2 Membranes from guinea-pig tracheal smooth muscle contain both G(ialpha) and G(salpha) as assessed by Western blots with anti-G-protein antibodies. 3 GppNHp, a non-hydrolysable analogue of guanosine 5'-triphosphate (GTP), was shown to stimulate adenylyl cyclase activity at high concentrations (10(-6)-10(-4) m). GppNHp also produced a concentration-dependent reduction in pertussis toxin-catalysed adenosine diphosphate (ADP)-ribosylation of G(ialpha). 4 Pretreatment of tracheal smooth muscle slices with methacholine (10(-6) M) provoked a blockade of isoprenaline plus GTP, GppNHp- and GTP-stimulated adenylyl cyclase. 5 Addition of methacholine to membranes did not trigger inhibition of GTP-stimulated adenylyl cyclase activity but did block the isoprenaline-mediated augmentation of GTP-stimulated adenylyl cyclase activity. 6 Pretreatment of tracheal smooth muscle with methacholine (10(-6) M) provoked a blockade of cholera toxin-catalysed NAD+-dependent ADP-ribosylation of G(salpha). 7 Phorbol-12-myristate 13-acetate (PMA)-treatment of tracheal smooth muscle slices actually enhanced GppNHp-stimulated adenylyl cyclase activity in subsequently prepared membranes. 8 We suggest that methacholine in addition to inhibiting adenylyl cyclase via a G(i)-dependent mechanism induces a functional inactivation of G(s) activity. These results together may explain the functional antagonism that exists between increased muscarinic tone and the ability of beta-adrenoceptor agonists to provoke excitation-contraction uncoupling.