Characterization of the interaction between muscarinic M 2 receptors and β‐adrenoceptor subtypes in guinea‐pig isolated ileum

1 Contraction of guinea‐pig ileum to muscarinic agonists is mediated by M 3 receptors, even though they account for only 30% of the total muscarinic receptor population. The aim of this study was to characterize the biochemical and functional effects of stimulation of the predominant M 2 muscarinic receptor (70%) and to investigate the hypothesis that M 2 receptors specifically oppose β‐adrenoceptor‐mediated effects in the ileum. 2 In guinea‐pig ileal longitudinal smooth muscle slices, isoprenaline, a non‐selective β‐adrenoceptor agonist, and BRL 37344 (sodium‐4‐[2‐[2‐hydroxy‐2‐(3‐chlorophenyl)ethylamino]propyl]‐phenoxyacetate sesquihydrate), a β 3 ‐adrenoceptor selective agonist, increased cyclic AMP accumulation with – log EC 50 values of 6.6 ± 0.1 and 5.8 ± 0.1 respectively. Maximal stimulation by BRL 37344 (10 μ m ) was 26.4 ± 5.2% of that observed with isoprenaline (10 μ m ). Isoprenaline (10 μ m )‐stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 μ m ), with a propranolol‐resistant component of 28.2 ± 6.8% of the maximal stimulation to isoprenaline. In contrast, basal and BRL 37344 responses were resistant to this antagonist. These data provide evidence that both β 1 ‐ and β 3 ‐adrenoceptors activate adenylyl cyclase in guinea‐pig ileum. 3 Isoprenaline (10 μ m )‐stimulated cyclic AMP accumulation was inhibited (67.4 ± 0.9%) by the muscarinic agonist (+)‐ cis ‐dioxolane (‐log EC 50 = 7.3 ± 0.1). The rank order of antagonist affinities against the (+)‐ cis ‐dioxolane response was (‐log K B values in parentheses): atropine (9.0 ± 0.2) > methoctramine (7.1 ± 0.1) > p ‐fluoro‐hexa‐hydrosilaphenidol ( p ‐F‐HHSiD; 6.5 ± 0.2) ≥ pirenzepine (6.3 ± 0.2). (+)‐ cis ‐dioxolane also significantly inhibited BRL 37344 (10 μ m ; 56.5 ± 2.4%) stimulated cyclic AMP accumulation. These data suggest that M 2 receptors mediate inhibition of cyclic AMP accumulation in response to both β 1 ‐ and β 3 ‐adrenoceptor stimulation in guinea‐pig ileum. 4 5‐Hydroxytryptamine (5‐HT), vasoactive intestinal peptide, prostaglandins E 2 and E 1 , all at 10 μ m , significantly increased cyclic AMP accumulation. (+)‐ cis ‐Dioxolane (10 μ m ) inhibited both basal and agonist‐induced cyclic AMP accumulation. Thus the inhibitory effect of M 2 receptor agonism does not appear to be restricted to β‐adrenoceptor‐stimulated cyclic AMP accumulation. 5 The potential for involvement of activation of M 2 receptors on responses to β‐adrenoceptor agonists was also studied functionally. Selective M 3 receptor alkylation was achieved by pretreatment of tissues with 4‐DAMP mustard (40 n m ), in the presence of methoctramine (1 μ m ; to protect M 2 receptors). After washing, tissues were pre‐contracted with histamine (0.3 μ m ) and relaxed with isoprenaline (0.6 μ m ). Under these conditions, oxotremorine m caused concentration‐dependent contractions (‐log EC 50 of 7.8 ± 0.1), that were surmountably antagonized by methoctramine (1 μ m ) with a ‐log K B estimate of 7.4 ± 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 μ m ), where the ‐log K B value for methoctramine was 7.8 ± 0.2. These data suggest that M 2 receptors mediate a functional inhibition of relaxant responses to isoprenaline and BRL 37344. 6 These findings are consistent with β 1 ‐ and β 3 ‐adrenoceptors coupling to stimulation of adenylyl cyclase in guinea‐pig ileum; a response that is inhibited by M 2 receptor stimulation. Concordantly, M 2 receptor stimulation also inhibits relaxation to both β 1 ‐ and β 3 ‐adrenoceptor stimulation. These results implicate M 2 receptors in the modulation of sympathetic control of ileal motility.