Characterization of the interaction between muscarinic M2 receptors and beta-adrenoceptor subtypes in guinea-pig isolated ileum.

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 beta-adrenoceptor-mediated effects in the ileum. 2. In guinea-pig ileal longitudinal smooth muscle slices, isoprenaline, a non-selective beta-adrenoceptor agonist, and BRL 37344 (sodium-4-[2-[2-hydroxy-2-(3- chlorophenyl)ethylamino]propyl]-phenoxyacetate sesquihydrate), a beta 3-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 microM) was 26.4 +/- 5.2% of that observed with isoprenaline (10 microM). Isoprenaline (10 microM)-stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 microM), 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 beta 1- and beta 3-adrenoceptors activate adenylyl cyclase in guinea-pig ileum. 3. Isoprenaline (10 microM)-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 IM; 56.5 +/-2.4%) stimulated cyclic AMP accumulation. These data suggest that M2 receptors mediate inhibition of cyclic AMP accumulation in response to both beta l- and beta 3-adrenoceptor stimulation in guinea-pig ileum.4. 5-Hydroxytryptamine (5-HT), vasoactive intestinal peptide, prostaglandins E2 and E1, all at 10 micro M,significantly increased cyclic AMP accumulation. (+)-cis-Dioxolane (10 micro M) inhibited both basal and agonist-induced cyclic AMP accumulation. Thus the inhibitory effect of M2 receptor agonism does not appear to be restricted to beta-adrenoceptor-stimulated cyclic AMP accumulation.5. The potential for involvement of activation of M2 receptors on responses to beta-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 micro M; to protect M2 receptors). After washing, tissues were pre-contracted with histamine (0.3 micro M) and relaxed with isoprenaline (0.6 micro M).Under these conditions, oxotremorine M caused concentration-dependent contractions (-log EC50 of 7.8 +/- 0.1), that were surmountably antagonized by methoctramine (1 microM) with a - log KB estimate of 7.4 +/- 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 micro M), 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 37344.6. These findings are consistent with beta l- and beta 3-adrenoceptors coupling to stimulation of a denylylcyclase in guinea-pig ileum; a response that is inhibited by M2 receptor stimulation. Concordantly, M2 receptor stimulation also inhibits relaxation to both beta l- and beta 3-adrenoceptor stimulation. These results implicate M2 receptors in the modulation of sympathetic control of ileal motility.