Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

Abstract

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release.

Muscarinic acetylcholine receptors in the mouse esophagus: focus on intraganglionic laminar endings (IGLEs)

“…69 In both guinea-pig ileum and human colon, M1 and M3 have been described in subpopulations of ascending and descending interneurons as well as intrinsic sensory neurons 26,44,[70][71][72][73] probably involved in peristalsis regulation via activation of inhibitory pathways 26 while M2 was mostly found presynaptic on nerve fibers and varicosities. 45,70 In the mouse esophagus we found M1-M3 in both cholinergic and nitrergic myenteric neurons, suggesting occurrence on interneurons and motorneurons, probably also involved in enteric co-innervation of striated esophageal muscle. 74 Muscarinic receptors in motor endplates of striated esophageal and skeletal muscles…”

“…Knowledge about the localization of mAChR in the enteric nervous system (ENS) and their role in neuronal circuits controlling intestinal reflexes is fragmentary. M1‐M3 have been shown to influence intestinal motility and secretion and were localized in the intestines of several species, variably distributed over smooth muscle and ganglia . M1 have further been suggested to be selectively involved in mediating transmission from gastric mechanoreceptors to vagal preganglionic neurons .…”

“…M1-M3 have been shown to influence intestinal motility and secretion 39,40 and were localized in the intestines of several species, variably distributed over smooth muscle [41][42][43] and ganglia. [43][44][45][46][47][48][49][50][51] M1 have further been suggested to be selectively involved in mediating transmission from gastric mechanoreceptors to vagal preganglionic neurons. 52 Yet, the role of neuronal mAChRs and their location within the ENS, in particular in the esophagus, is not well characterized.…”

Role of interstitial cells of Cajal in the generation and modulation of motor activity induced by cholinergic neurotransmission in the stomach

“…M2r is extensively expressed in the smooth muscle cells of gastrointestinal tract, but it is also observed on the enteric neurons. 49 In the guineapig, M2r is present in the ICC of small intestine and colon, but not in stomach. 50 The M2r positive cells in our study were likely smooth muscle cells and enteric nerves according to their locations.…”

“…Our immunostaining proved a remarkable increase in M2r immunoreactivity in Ws/Ws antrum. M2r is extensively expressed in the smooth muscle cells of gastrointestinal tract, but it is also observed on the enteric neurons 49 . In the guinea‐pig, M2r is present in the ICC of small intestine and colon, but not in stomach 50 .…”