Investigation of the mechanism for the relaxation of rat duodenum mediated via M₁ muscarinic receptors

Abstract

1 Relaxation responses of the rat isolated duodenum to the putative M1 muscarinic receptor agonist, McN-A-343, were examined to determine whether the response was due to the release of known non-adrenergic, non-cholinergic relaxant neurotransmitters and to establish the involvement of M1 muscarinic receptors. 2 The role of ATP was examined with the P2 receptor antagonist, suramin, which at 30 mum antagonized the relaxant responses to alpha,beta-methylene ATP. The same dose, however, failed to inhibit the relaxation by McN-A-343. 3 The role of nitric oxide (NO) was examined with the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 100 microm), which failed to inhibit the responses to McN-A-343. As NO mediates relaxation of the duodenum via cGMP generation through guanylyl cyclase, whether the relaxation by McN-A-343 was also via cGMP was examined with the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxation responses to the NO donor, S-nitroso-N-acetyl penicillamine, were inhibited in the presence of ODQ (3 microm), but not those by McN-A-343. 4 Release of gamma-aminobutyric acid (GABA) was examined with the GABAA receptor antagonist, bicuculline (10 microm), which shifted the concentration-response curves for the relaxation of the duodenum by GABA to the right. There was a similar degree of shift in the concentration-response curve for McN-A-343 by bicuculline indicating that release of GABA from enteric neurones of the duodenum could explain the relaxation response to McN-A-343. 5 To test whether the muscarinic receptors mediating the relaxation of the duodenum were of the M1 subtype, the susceptibility to the selective competitive antagonist, pirenzepine and the selective muscarinic toxin from green mamba, MT7, was examined. Pirenzepine (1 microm) shifted the concentration-response for McN-A-343 to the right in a parallel fashion with a dose ratio of 33.3 +/- 20.2. This yielded a pA2 value of 7.5, which concords with those for other responses reputed to be mediated via M1 muscarinic receptors. The toxin MT7 was used as an irreversible antagonist and following incubation with the duodenum was washed from the bath. An incubation time of 30 min with 100 nm of MT7 caused a significant parallel shift in the concentration-response to McN-A-343 confirming the involvement of M1 muscarinic receptors. 6 This study has confirmed that McN-A-343 relaxes the rat duodenum via muscarinic receptors of the M1 subtype and that these receptors are probably located on enteric neurones from which their stimulation releases GABA.

In Vitro Isolated Tissue Functional Muscarinic Receptor Assays

“…Activation of neuronal M 1 receptors has been implicated in the relaxation response in rat isolated duodenum (Micheletti et al, 1988;Hamrouni et al, 2006). In this preparation, the M 1 -selective agonist McN-A-343 produces a rapid and transient relaxation, which is antagonized by pirenzipine, consistent with mediation by muscarinic M 1 receptors.…”

Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system

“…In addition, studies in the guinea pig ileum demonstrate M1rs modulate ACh release from nerve fibres and neurons (Vizi et al, 1989;Kawashima et al, 1990;Wiklund et al, 1993;Dietrich and Kilbinger, 1995;Marino et al, 1997). Muscarinic www.elsevier.com/locate/jchemneu Journal of Chemical Neuroanatomy 33 (2007) 193-201 receptors modulating release of neurotransmitters other than ACh has also been documented (Iverson et al, 1997;Hamrouni et al, 2006). M1rs have also been suggested to have a role in mucosal secretion and are present on epithelial cells in the mouse colon (Haberberger et al, 2006).…”

Selectivity of antagonists for theCys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons

“…Cys‐loop receptors antagonists like hexamethonium, ondansetron, picrotoxin and bicuculline have been used as tools for studying the properties and functions of these receptors (Galligan, ,b; Hamrouni et al ., ; Reis et al ., ). In general, these antagonists have been considered specific on one subgroup of Cys‐loop receptors: hexamethonium on nACh receptors, ondansetron on 5‐HT 3 receptors, and picrotoxin and bicuculline on GABA A receptors (Fargeas et al ., ; Roerig et al ., ; Chebib & Johnston, ; Reis et al ., ).…”