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 P-adrenoceptormediated effects in the ileum.2 In guinea-pig ileal longitudinal smooth muscle slices, isoprenaline, a non-selective P-adrenoceptor agonist, and BRL 37344 (sodium-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylaminolpropyl]-phenoxyacetate sesquihydrate), a P3-adrenoceptor selective agonist, increased cyclic AMP accumulation with -log EC";, values of 6.6 ± 0.1 and 5.8 ± 0.1 respectively. Maximal stimulation by BRL 37344 (10 gM) was 26.4 ± 5.2% of that observed with isoprenaline (10 iM). Isoprenaline (10 gM)-stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 PM), with a propranololresistant 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 Pl-and B3-adrenoceptors activate adenylyl cyclase in guinea-pig ileum.3 Isoprenaline (10 gM)-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 Pl-and P3-adrenoceptor stimulation in guinea-pig ileum. appear to be restricted to P-adrenoceptor-stimulated cyclic AMP accumulation. 5 The potential for involvement of activation of M2 receptors on responses to fradrenoceptor 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 riM; to protect M2 receptors). After washing, tissues were pre-contracted with histamine (0.3 gM) and relaxed with isoprenaline (0.6 AM).Under these conditions, oxotremorine M caused concentration-dependent contractions (-log EC50 of 7.8 ± 0.1), that were surmountably antagonized by methoctramine (1 gM) with a -log KB estimate of 7.4 ± 0.1. Similar observations were seen versus relaxation produced by BRL 37344 (1 tM), 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 Pl-and P3-adrenoceptors coupling to stimulation of adenylyl cyclase in guinea-pig ileum; a response that is inhib...

Characterization of muscarinic receptors mediating contractions of circular and longitudinal muscle of human isolated colon

“…Eglen & Harris (1993) demonstrated that in the absence of elevated cyclic AMP and with an intact M3 receptor population, responses to (+)-cis-dioxolane in guinea-pig ileum did not involve M2 receptors. However, under experimental conditions where intracellular cyclic AMP is elevated, M2 receptor stimulation can elicit contractions or modulate adrenoceptorinduced relaxation by inhibition of adenylate cyclase in rat and guinea-pig ileum (Candell et al, 1990;Thomas et al, 1993;Reddy et al, 1994). Additionally, in other tissues such as the guinea-pig uterus it is the M2 muscarinic receptors through which a functional contractile response is mediated (Eglen et al, 1989).…”

The role of M2‐muscarinic receptors in mediating contraction of the pig urinary bladder in vitro

“…It has been suggested that the conditions in vitro are not favourable to the demonstration of an M 2 -response which involves the inhibition of adenylate cyclase. Indeed in guinea-pig ileum, responses normally mediated via M 3 -receptor can involve M 2 -activity following M 3 inactivation and elevation of cyclic AMP levels, the response then being regarded as a re-contraction or reversal of relaxation (Thomas et al, 1993;Reddy et al, 1995). This has also been shown in the rat bladder, but in this species the M 2 -receptor population is large (Hegde et al, 1997;Braverman & Ruggieri, 1999) and the M 2 : M 3 ratio is far greater than found in other species.…”

“…The urinary bladder, like the majority of other smooth muscles from many species, exhibit heterogeneous populations of muscarinic receptors (Hegde et al, 1997;Reddy et al, 1995). A predominance of the M 2 muscarinic receptor subtype, with a minor population of M 3 -receptors, has been reported for urinary bladder smooth muscle for several species.…”

“…Although no direct contractile response to M 2 receptor activation can be demonstrated, an indirect in¯uence on contraction via inhibition of cyclic AMP-mediated smooth muscle relaxation by b-adrenoceptors, forskolin, 5-hydroxytryptamine or vasoactive intestinal peptide has been reported (Grin & Ehlert, 1992;Caul®eld, 1993;Reddy et al, 1995;Eglen et al, 1996). M 2 -receptors have also been suggested to be able to inhibit smooth muscle relaxation through decreasing the opening probability of K + -channels (Caul®eld, 1993;Kume & Kotliko, 1991).…”

“…Recently, an M 2 -mediated contraction to muscarinic stimulation has been demonstrated following selective M 3 -receptor inactivation and elevation of cyclic AMP levels in guinea-pig ileum (Thomas et al, 1993;Reddy et al, 1995) and in the rat bladder (Hegde et al, 1997), an eect which manifests as a re-contraction.…”