Characterization of the interaction between muscarinic M2 receptors and β‐adrenoceptor subtypes in guinea‐pig isolated ileum

Abstract

Contraction of guinea‐pig ileum to muscarinic agonists is mediated by M3 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 M2 muscarinic receptor (70%) and to investigate the hypothesis that M2 receptors specifically oppose β‐adrenoceptor‐mediated effects in the ileum. 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 β3‐adrenoceptor selective agonist, increased cyclic AMP accumulation with – log EC50 values of 6.6 ± 0.1 and 5.8 ± 0.1 respectively. Maximal stimulation by BRL 37344 (10 μm) was 26.4 ± 5.2% of that observed with isoprenaline (10 μm). Isoprenaline (10 μm)‐stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 μm), 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 β1‐ and β3‐adrenoceptors activate adenylyl cyclase in guinea‐pig ileum. Isoprenaline (10 μm)‐stimulated cyclic AMP accumulation was inhibited (67.4 ± 0.9%) by the muscarinic agonist (+)‐cis‐dioxolane (‐log EC50 = 7.3 ± 0.1). The rank order of antagonist affinities against the (+)‐cis‐dioxolane response was (‐log KB 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 μm; 56.5 ± 2.4%) stimulated cyclic AMP accumulation. These data suggest that M2 receptors mediate inhibition of cyclic AMP accumulation in response to both β1‐ and β3‐adrenoceptor stimulation in guinea‐pig ileum. 5‐Hydroxytryptamine (5‐HT), vasoactive intestinal peptide, prostaglandins E2 and E1, all at 10 μm, significantly increased cyclic AMP accumulation. (+)‐cis‐Dioxolane (10 μm) inhibited both basal and agonist‐induced cyclic AMP accumulation. Thus the inhibitory effect of M2 receptor agonism does not appear to be restricted to β‐adrenoceptor‐stimulated cyclic AMP accumulation. The potential for involvement of activation of M2 receptors on responses to β‐adrenoceptor agonists was also studied functionally. Selective M3 receptor alkylation was achieved by pretreatment of tissues with 4‐DAMP mustard (40 nm), in the presence of methoctramine (1 μm; to protect M2 receptors). After washing, tissues were pre‐contracted with histamine (0.3 μm) and relaxed with isoprenaline (0.6 μm). Under these conditions, oxotremorine m caused concentration‐dependent contractions (‐log EC50 of 7.8 ± 0.1), that were surmountably antagonized by methoctramine (1 μm) with a ‐log KB estimate of 7.4 ± 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 μm), where the ‐log KB value for methoctramine was 7.8 ± 0.2. These data suggest that M2 receptors mediate a functional inhibition of relaxant responses to isoprenaline and BRL 37344. These findings are consistent with β1‐ and β3‐adrenoceptors coupling to stimulation of adenylyl cyclase in guinea‐pig ileum; a response that is inhibited by M2 receptor stimulation. Concordantly, M2 receptor stimulation also inhibits relaxation to both β1‐ and β3‐adrenoceptor stimulation. These results implicate M2 receptors in the modulation of sympathetic control of ileal motility. 1995 British Pharmacological Society