Functional and binding studies with muscarinic M₂‐subtype selective antagonists

Abstract

1 The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies. 2 On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF-DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium. 3 [3H]-N-methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97). 4 [3H]-pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4-DAMP, HSD, AF,-DX1 16 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on Ml sites than on the gland.5 Himbacine had a 20 fold lower affinity at M1 binding sites than at heart sites, and it should therefore be an important tool in identifying M1 sites. 6 Inhibition of [3H]-N-methyl scopolamine binding to rat ileum and rat brainstem by M2-selective antagonists was best described by a two-site model. In both cases the major population of sites (70-90%) appeared to be similar to sites found on the heart (correlation coefficients = 0.95 and 0.97). The other site appeared to be similar to that on the submandibular gland (correlation coefficients = 0.96 and 1.00). 7 The correlations observed in these studies in which a range of selective muscarinic antagonists was used lend weight to previous studies indicating the presence of three functionally important muscarinic receptor subtypes, typified by the binding sites studied in the cerebral cortex, submandibular gland and heart. 8 We propose that the sub-classification of the M2 muscarinic receptor into M2 and M3 subtypes on the basis of ligand binding studies should be extended to cover functionally-defined receptors as well.

Novel pharmacological profile of muscarinic receptors mediating contraction of the guinea-pig uterus

“…The affinities obtained at uterine muscarinic receptors of immature guinea-pigs were compared to those determined at M 1 (rabbit vas deferens), M 2 (guinea-pig atria) and M 3 receptors (guinea-pig ileum). The antimuscarinic potencies of the antagonists at Mb M2 and M 3 receptors (pArvalues, Table 1) werein good agreement with their affinity estimates determined in radioligand binding studies (Lazareno and Roberts 1989;Waelbroeck et al 1989) and previous functional studies (Barlow et al 1976;Gilani and Cobbin 1986;Micheletti et al 1987;Eltze 1988;Eltze et al 1988;Waelbroeck et al 1989;. A comparison of these affinity values with those determined at uterine muscarinic receptors suggests that the muscarinic receptor mediating contractions of the guinea-pig uterus is pharmacologically unique ( Table 1 ).…”

“…M 2 and M 3 receptors are primarily found in lower brain areas and in peripheral effector argans such as heart (M 2 ), smooth muscle (M 3 ) and glands (M 3 ) and display low affinity for pirenzepine. They can be distinguished by the use of selective antagonists such as methoctramine and AF-DX 116 (M 2 > M 1 > M 3 ) (Melchiorre et al 1987;Hammer et al 1986;Micheletti et al 1987), himbacine (M 1 ~ M 2 > M 3 ) (Gilani and Cobbin 1986;Lazareno and Roberts 1989), sila-hexocyclium (M 1 ~ M 3 > M 2 ) Waelbroeck et al 1989), 4-DAMP and hexahydro-sila-difenidol (M 3 ::? :: M 1 > M 2 ) (Barlow et al 1976;Mutschier and Lambrecht 1984;.…”

Characterization of muscarinic receptorsmediating vasodilation in rat perfused kidney

“…The failure of himbacine, AF-DX 384 and AQ-RA 741 to discriminate between functional responses at muscarinic M 1 and M 2 receptors in rabbit vas deferens contrasts with reports on the ability of the antagonists to distinguish between these receptor types in binding sturlies (Lazareno and Roberts, 1989;Doods et al, 1991;Dörje et al, 1991b). However, although pharmacological (Eltze, 1988) and immunological techniques using subtype-specific antisera (Dörje et al, 1991a) have shown that rabbit vas deferens is endowed only with M 1 and M 2 receptors, the inability of these antagonists to discriminate M 1 from M 2 receptor-mediated responses in rabbit vas deferens remains unclear and deserves further investigation.…”

Pharmacology of the putative M4 muscarinic receptor mediating Ca‐current inhibition in neuroblastoma × glioma hybrid (NG 108–15) cells

“…Thus, the absolute and relative potencies of himbacine, pirenzepine and methoctramine measured against muscarinic inhibition of ICa are very similar to the ligand displacement potencies on putative M4 receptors in both NG 108-15 cells and rabbit lung (Lazareno et al, 1990), and clearly differ from their potencies at M1, M2 or M3 binding sites (Lazareno et al, 1990) or at the corresponding receptor subtypes in functional assays (e.g. Brown et al, 1980;Lazareno & Roberts, 1989). Although the potency of pirenzepine against ICa inhibition (pKB 7.74) was slightly higher than its binding potency in NG 108-15 cells (pKB 7.2;Lazareno et al, 1990), the accuracy of functional pKB calculations is probably limited by the non-parallel shift of the dose-response curve (see below).…”