Distribution of neuronal nicotinic acetylcholine receptors containing different alpha-subunits in the submucosal plexus of the guinea-pig

The subunit composition and localisation of nicotinic acetylcholine receptors (nAChRs) in the submucosal plexus of the guinea-pig ileum were studied using both affinity-purified monoclonal and polyclonal antibodies against α3, α4, α5 and α7 nAChR subunits and specific α7-containing nAChRs blocker methyllycaconitine (MLA). By means of immunohistochemistry performed in non-dissociated preparations, it was found that only 4% of submucosal ganglia expressed nAChRs. Specific staining, associated with cell membranes, was found with α3-, α5- and α7-, but not α4-specific antibodies. Double staining using α5- and α7-specific antibodies demonstrated that about one-half of the nAChR-positive ganglia contained neurons immunoreactive to both antibodies, while the others possessed either α5- or α7-immunoreactivity. Nanomolar concentrations of MLA prevented α7-specific antibody binding and did not influence the α5-specific antibody binding even when applied in micromolar concentrations. In electrophysiological experiments performed using a patch-clamp ‘whole-cell’ recording method, the neurons were identified by their sensitivity to MLA. In conclusion, submucosal neurons of the guinea-pig ileum express nAChRs containing α3-, α5- and α7-subunits. The co localisation of α5- and α7-subunits found in immunohistochemical experiments as well as kinetic characteristics of MLA-blocked receptors found by electrophysiological experiments allow us to suggest the presence of heteromeric α7-containing nAChRs in the submucosal plexus of the guinea-pig ileum.