The inhibition of adenylyl cyclase activity in isolated lung membranes by muscarinic and α-adrenoreptor agonists: Role of G-protein α and βγ sub-units

Abstract

Forskolin (10 μM) failed to abolish the GppNHp- (0.1 nM) dependent inhibition of adenylyl cyclase activity in isolated membranes. Whilst clonidine (1 μM), an α 2-adrenoceptor agonist, inhibited adenylyl cyclase activity it did not protentiate the GppNHp-dependent inhibition. This indicates that low concentrations of the guanine-nucleotide activate sufficient G i to inhibit adenylyl cyclase activity maximally and that clonidine inhibits this enzyme via a similar route. These data support a role of α i sub-unit inhibition of adenylyl cyclase activity. In contrast, forskolin (10 μM) abolished the GppNHp (0.001–0.1 nM) dependent inhibition of adenylyl cyclase activity in membranes where adenylyl cyclase activity is limited, i.e. where activity has been depleted by approximately 80%. In this case, inhibition of adenylyl cyclase by βγ sub-units is implicated and only becomes evident under these conditions. Adenylyl cyclase is also inhibited by muscarinic receptor agonist, methacholine and by the α 2-adrenoreptor agonists, clonidine and nor-adrenaline. Both classes of agonist also elicit an increase in the cholera toxin-catalysed ADP-ribosylation of the splice variant forms of G s α and of a polypeptide of 41,000 M r. The ADP-ribosylation of the 41,000 M r polypeptide is inhibited by GTP (100 μM) and therefore displays characteristics similar to G i α. Muscarinic receptor and α 2-adrenoceptor agonists appear to inhibit adenylyl cyclase activity in lung membranes predominantly via G i α. Lung expresses both type II and IV adenylyl cyclase which are stimulated by direct interaction with βγ sub-units and this is conditional upon the co-incident activation of G s α. Inhibition of adenylyl cyclase by G i α provides a means by which muscarinic receptor and α 2-adrenoceptor agonists can inhibit these isoforms independently of the βγ sub-unit effects.