Muscarinic blockade of β-adrenoceptor-stimulated adenylyl cyclase: the role of stimulatory and inhibitory guanine-nucleotide binding regulatory proteins (Gs and Gi)

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Abstract
1 The functional antagonism that exists between muscarinic and β-adrenoceptor function in guinea-pig tracheal smooth muscle was investigated by assessing Gs and Gi regulated adenylyl cyclase activity in isolated membranes. 2 Membranes from guinea-pig tracheal smooth muscle contain both Giα and Gsα 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 Giα. 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 Gsα. 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 Gi-dependent mechanism induces a functional inactivation of Gs activity. These results together may explain the functional antagonism that exists between increased muscarinic tone and the ability of β-adrenoceptor agonists to provoke excitation-contraction uncoupling.

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