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A novel muscarinic M4 receptor antagonist provides further evidence of an autoreceptor role for the muscarinic M2 receptor sub-type

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Abstract

Muscarinic M2 (AF-DX 384, BIBN-161) and M4 (PD102807) receptor antagonists were used to investigate the respective roles of these two receptor sub-types in the regulation of acetylcholine release in the rat hippocampus. In vivo dialysis studies revealed that only the muscarinic M2 receptor antagonists significantly and concentration-dependently facilitate acetylcholine release. The newly developed muscarinic M4 receptor antagonist was unable to regulate acetylcholine release except at the highest concentration tested. It would thus appear that the muscarinic receptor acting as negative autoreceptor in the rat hippocampus is of the muscarinic M2 sub-type, the role of the muscarinic M4 receptor being minimal in this regard.

Introduction

Five muscarinic receptors have been cloned thus far (for a recent review Wess, 1996). All are members of the seven-transmembrane G-protein coupled receptor family with the muscarinic M1, M3 and M5 sub-types linked to Gq/11 proteins and the production of inositides, whereas the muscarinic M2 and M4 receptors are associated with the Gi proteins and adenylate cyclase activity.

Most of these receptors are widely expressed throughout the body including the brain. However, their respective physiological roles remain to be fully established, primarily because of the limited availability of truly receptor sub-type selective agonists and antagonists. Among the putative roles of the muscarinic receptor sub-groups, an action as inhibitory autoreceptors regulating the release of acetylcholine has been suggested on the basis of anatomical (Levey et al., 1995), pharmacological Raiteri et al., 1984, Quirion et al., 1995 and behavioral (Quirion et al., 1995) data. However, it is not clear if these autoreceptors are of the muscarinic M2 Raiteri et al., 1984, Quirion et al., 1995 or M4 (McKinney et al., 1993) sub-type because of the lack of selective antagonists to distinguish between these two related receptor sub-types. For example, it has been shown that BIBN-99, a muscarinic M2 receptor antagonist unable to fully distinguish between muscarinic M2 and M4 receptors, is capable of potently reversing cognitive deficits in aged memory impaired rats (Quirion et al., 1995). This information suggests the possibility of targeting muscarinic M2 receptors as a means of alleviating learning deficits. It is thus of major importance to clearly establish the identity of the muscarinic receptor sub-type on which such antagonists are likely to act, in order to facilitate acetylcholine release and be of potential clinical relevance in diseases associated with altered cognition.

Recently, PD102807 has been developed as one of the very first selective muscarinic M4 receptor antagonists (Nelson et al, 1996). In the present study, we used this molecule in addition to the purported muscarinic M2 receptor antagonists AF-DX 384 and BIBN-161 (Doods et al., 1993) to further investigate the exact nature of the autoreceptor sub-type(s) regulating acetylcholine release in the rat hippocampus, as assessed by in vivo dialysis.

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Animals and drugs

Male Sprague–Dawley rats (270–300 g) were purchased from Charles River Canada (St. Constant, Quebec). Animals were kept in a temperature- (22–24°C), humidity (55±5%) and light- (12 h light–dark cycle, lights on at 07:00) regulated room with food and water available ad libitum for at least 3 days before surgery. The procedures involving animals and their care were conducted in accordance with the McGill University Animal Care Committee and the Canadian Council on Animal Care. BIBN-161 and AF-DX

Results

As shown in Fig. 1A and Table 1, the two muscarinic M2 receptor antagonists AF-DX 384 and BIBN-161 facilitated acetylcholine release in the rat hippocampus in a concentration dependent manner. Both drugs yielded a significant effect at 100 nM (Table 1) but AF-DX384 was slightly more potent than BIBN-161, as shown in Fig. 1C. Both AF-DX 384 (10 μM) and BIBN-161 (50 μM) were able to potently stimulate acetylcholine release, with 5–6 fold increments in release seen at these concentrations.

In

Discussion

In vivo acetylcholine release in the adult rat hippocampus was clearly stimulated, in a concentration-dependent manner, by AF-DX384>BIBN-161⋙PD102807. The first two molecules have higher or equal affinities for the cloned muscarinic M2 than the M4 receptor sub-types while the reverse is true for the newly developed muscarinic M4 receptor antagonist PD102807 Doods et al., 1993, Nelson et al., 1996. In fact, the apparent IC50 (nM) values of AF-DX384 are 325, 16, 150, 10, and 1860 for the M1, M2, M

Acknowledgements

This work was supported by an M.R.C. of Canada grant to R. Quirion, a chercheur-boursier of the F.R.S.Q. J. Day holds a fellowship award from the M.R.C.C. K. Kitaichi is now at the Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan.

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