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 Gs and Gi regulated adenylyl cyclase activity in isolated membranes. 2. Membranes from guinea-pig tracheal smooth muscle contain both Gi alpha and Gs alpha 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 Gi alpha. 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 Gs alpha. 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 beta-adrenoceptor agonists to provoke excitation-contraction uncoupling.
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow.
N J
NJ
Pyne
M W
MW
Grady
D
D
Shehnaz
P A
PA
Stevens
S
S
Pyne
I W
IW
Rodger
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002772:Cholera Toxin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008566:Membranes / Q000187:drug effects; D008688:Methacholine Compounds / Q000494:pharmacology; D009130:Muscle, Smooth / Q000187:drug effects; D010277:Parasympathomimetics / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014132:Trachea / Q000187:drug effects
D000319:Adrenergic beta-Antagonists; D008688:Methacholine Compounds; D010277:Parasympathomimetics; D006165:Guanylyl Imidodiphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate