Immunohistochemical localisation shows that muscarinic receptors M1R, M2R and M3R mediate both cholinergic and nitrergic pathways in human colon

Also cited as a journal article: Gastroenterology, 2010; 135(Suppl. 1):S-572BACKGROUND: Acetylcholine is the most common neurotransmitter in the GI tract and is released by more than 50% of enteric neurons. The specificity of its action is controlled by the location and characteristics of the receptors it binds to. Muscarinic acetylcholine receptors (MR) are involved in multiple intestinal reflexes. There are 5 subtypes with 1, 3 & 5 having excitatory while 2 and 4 have inhibitory effects on cells. The location of different subtypes has been predicted from physiological, pharmacological and ligand-binding studies. This study aimed to localise the three most abundant subtypes of MRs (M1R, M2R and M3R) in human colon using antibodies, fluorescence immunohistochemistry and confocal microscopy. The location of MR subtypes on different types of neurons was also determined. METHODS: RT-PCR was used to determine expression levels of M1Rs, M2Rs and M3Rs in human colon. Indirect immunofluorescence and confocal microscopy was used to localise MRs in cryostat-cut sections of human colon. Sections were double labelled for synaptophysin, calbindin, cChAT, NOS, SP, and VAChT to determine the subtypes of neurons and nerve fibres. Western blotting was used to confirm specificity of the muscarinic antisera. RESULTS: The three MR subtypes were differentially expressed in human colon. Immunoreactivity (IR) for M2Rs and M3Rs was abundant on the surface of circular and longitudinal muscle cells with M2R more abundant than M3R. IR for all 3 receptors was present on cholinergic and nitrergic nerve fibres in the circular muscle. M1R-IR and M3R-IR were present in cholinergic and nitrergic myenteric neurons with M3R on more nitrergic than cholinergic neurons. M1R-IR was also on neurons containing calbindin. M2R was not present in cell bodies but was located presynaptically on cholinergic and nitrergic nerve fibres in the myenteric ganglia and in the muscle layers. Only M1R-IR was present on submucosal neurons. In the submucosa, M3R-IR was on vascular smooth muscle cells and M1R-IR was on endothelial cells of blood vessels. M3R-IR was present on the cell membrane of mucosal epithelial cells located at the apex of the crypts. CONCLUSIONS: In the human colon, subtypes of MRs were present on multiple cell types underlying motility, secretory and vasoactive reflexes. MRs were present on cholinergic neurons and nerve fibres but they were also abundant on nitrergic cells and fibres. In these locations they modulate both excitatory and inhibitory pathways. The cellular distribution for MRs found in this study agrees with data from functional studies.Andrea M. Harrington, Cristal J. Peck, Lu Liu, Elizabeth Burcher, John M. Hutson, Bridget R. Southwel