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

Abstract

1 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. 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 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. 3 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 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 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. 4 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. 5 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 K_B estimate of 7.4 ± 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 μm), where the -log K_B 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. 6 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.