Short communicationPharmacological analysis of the mode of interaction of McN-A-343 at atrial muscarinic M2 receptors
Introduction
The ability of a diverse range of compounds to allosterically modulate the binding of classical, or orthosteric, ligands at all five subtypes of the muscarinic acetylcholine receptors has been acknowledged for some time (see Lee and El-Fakahany, 1991; Tucek and Proska, 1995). To date, the majority of ligands identified as allosteric modulators have been found to act as inhibitors of functional responses mediated by muscarinic receptors. However, evidence has also been presented for a possible allosteric mode of interaction of the agonist, (4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium) (McN-A-343). Birdsall et al. (1983)employed a radioligand binding experimental paradigm in rat cerebral cortex and myocardium to explore the interaction between McN-A-343 and []N-methylscopolamine. In these experiments, where increasing concentrations of []N-methylscopolamine were utilized, McN-A-343 demonstrated a progressive inability to maximally inhibit radioligand binding, a hallmark of negative allosteric modulation (see Lee and El-Fakahany, 1991). This phenomenon was most marked in rat myocardium. In contrast, a more recent binding study by Waelbroeck (1994)found no evidence for McN-A-343 recognizing an accessory site on rat cardiac muscarinic receptors.
Although radioligand binding studies have been employed more frequently than traditional isolated tissue experiments in the delineation and quantification of allosteric phenomena, functional verification of allosteric interactions lies at the heart of the potential therapeutic exploitation of modulating agents. Furthermore, very few studies have addressed the consequences of allosteric perturbation on muscarinic receptor-G protein coupling (but see Jakubı́k et al., 1996), yet the possibility of receptor activation via the allosteric binding site would represent a novel and significant therapeutic target in disorders of impaired signalling via the orthosteric binding site.
Thus, the aim of the current study was to employ a functional experimental paradigm to further evaluate the mode of interaction of McN-A-343 at guinea-pig atrial muscarinic M2 receptors.
Section snippets
Materials and methods
Guinea-pig atria were prepared as described previously (Christopoulos and Mitchelson, 1994) and suspended between two platinum wire electrodes in a 20 ml organ bath containing Krebs–Henseleit buffer (Lanzafame et al., 1996). Inotropic responses to electrical stimulation were measured as described previously (Lanzafame et al., 1996).
Cumulative concentration–response curves for the negative inotropic effects of carbachol (Sigma, St. Louis) and McN-A-343 (Research Biochemicals, Natick) were
Results
In the electrically-driven guinea-pig atrium, McN-A-343 behaved as a partial agonist, causing a maximal inhibitory effect corresponding to approximately 30% of the carbachol maximum (Fig. 1). Also shown in Fig. 1 are the results of operational model-fitting of the data, for the mean of eight different experiments.
After partial receptor alkylation with propylbenzylcholine mustard, which resulted in the abolition of the agonistic properties of McN-A-343, the latter agent was utilized as an
Discussion
Simultaneous operational model-fitting of the carbachol and McN-A-343 concentration–response curves in guinea-pig atria confirmed the weak partial agonist activity of the latter agent. The finding of a mean log KA of −4.52 is in general agreement with previous affinity estimates for McN-A-343 at M2 receptors (Elnatan and Mitchelson, 1993), obtained using a technique whereby partial agonists modify responses to full agonists. As both agonists were tested in the same preparation, and as carbachol
Acknowledgements
The authors would like to thank Professor Esam E. El-Fakahany (USA) for critical review of the manuscript. This work was supported by a grant from the National Health and Medical Research Council of Australia.
References (17)
- et al.
Interactions of agonists with an allosteric antagonist at muscarinic acetylcholine M2 receptors
Eur. J. Pharmacol.
(1996) - et al.
Allosteric antagonists of the muscarinic acetylcholine receptor
Biochem. Pharmacol.
(1991) - et al.
Allosteric modulation of muscarinic acetylcholine receptors
Trends Pharmacol. Sci.
(1995) - et al.
The affinity and efficacy of onium salts on the frog rectus abdominus
Br. J. Pharmacol.
(1967) - et al.
The effect of McN-A-343 on muscarinic receptors in the cerebral cortex and heart
Br. J. Pharmacol.
(1983) - et al.
Operational models of pharmacological agonism
Proc. Roy. Soc. Lond. B
(1983) - et al.
Assessment of the allosteric interactions of the bisquaternary heptane-1,7-bis-(dimethyl-3′-phthalimidopropyl)-ammonium bromide at M1 and M2 muscarine receptors
Mol. Pharmacol.
(1994) Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null methods
Mol. Pharmacol.
(1988)
Cited by (17)
The pharmacology of McN-A-343
2012, Pharmacology and TherapeuticsCitation Excerpt :In the esophagus muscularis mucosa of the North American opossum (Didelphis marsupialis) transmural electrical stimulation caused a biphasic contraction consisting of a rapid phasic response due to release of Ach from cholinergic neurons followed by a tonic contraction due to the release of substance P from sensory neurons (Rowbotham et al., 1985). Muscarinic agonists, including McN-A-343, produced a contraction of the tissue by activation of muscarinic receptors on the muscularis mucosa and also inhibited release of substance P by activation of neuronal muscarinic receptors to diminish the magnitude of the tonic phase of the response to electrical stimulation (Daniel et al., 1986, 1987). The muscarinic receptor subtype involved in both the contractile response to the muscarinic agonist and the inhibition of the tonic phase of the response to electrical stimulation appeared to be the M1 subtype in that pirenzepine appeared as an effective antagonist of both responses.
Allosteric modulation of G protein-coupled receptors: A pharmacological perspective
2011, NeuropharmacologyCitation Excerpt :This compound is one of the earliest examples of a mAChR partial agonist, and has been shown to display stimulus-bias at the M2 mAChR (Griffin et al., 2007). Depending on the experimental paradigm, McN-A-343 had previously been reported to interact either competitively or allosterically at the M2 mAChR (Birdsall et al., 1983; Christopoulos and Mitchelson, 1997; May et al., 2007a; Waelbroeck, 1994). A subsequent study by (Valant et al., 2008) addressed this conundrum by demonstrating that the behavior of the molecule can be recapitulated by the combination of two fragments, tetramethylammonium (TMA), which behaves as a robust orthosteric agonist of the M2 mAChR, and 3-chlorophenylcarbamate, which is a negative allosteric modulator of TMA signaling efficacy, thus accounting for the partial agonism of the parent compound and, potentially, for its functional selectivity (Fig. 6).
A novel mechanism of G protein-coupled receptor functional selectivity: Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand
2008, Journal of Biological ChemistryCitation Excerpt :Second, McN-A-343 has recently been found to exhibit true functional selectivity at the M2 mAChR, displaying preferentially greater activity at Gα15 protein-mediated versus Gαi protein-mediated signaling pathways when compared with classic mAChR agonists (10). Third, McN-A-343 can interact allosterically with the orthosteric antagonist [3H]NMS at the M2 mAChR (11–13), although its interaction with other orthosteric ligands, in particular agonists, appears consistent with a competitive mechanism (13, 14). To reconcile these observations and because McN-A-343 shares structural similarities with the well known mAChR orthosteric agonists carbachol and oxotremorine-M (Fig. 1), we hypothesized that it may actually represent a hitherto unappreciated hybrid molecule composed of both an orthosteric and an allosteric moiety, the latter presumably mediated by regions of the molecule diverging from the classic muscarinic pharmacophore, i.e. regions containing the 3-chlorophenylcarbamate moiety; McN-A-343 analogs lacking this structure but retaining the trimethylammonium moiety have been characterized as classic mAChR agonists (15).
Transducer abstraction - A novel approach to the detection of partial agonist efficacy in radioligand binding studies
2000, Journal of Pharmacological and Toxicological Methods