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Type: Conference paper
Title: Localisation of muscarinic receptor M1-M3 immunoreactivity in human colon
Author: Harrington, A.
Citation: Proceedings of the Joint International Society Meetng in Neurogastroenterology and GI Motility, held in Boston, September, 14-17 2006: pp.777
Publisher: Wiley-Blackwell Publishing Ltd.
Publisher Place: United Kingdom
Issue Date: 2006
ISSN: 1350-1925
Conference Name: Joint International Society Meeting in Neurogastroenterology and GI Motility (2006 : Boston, Massachusetts)
Statement of
A. M. Harrington, J. M. Hutson and B. R. Southwell
Abstract: Physiological, pharmacological, radioligand binding and in-situ hybridisation studies show human intestine has M1, M2 and M3 muscarinic receptor subtypes, but the precise localisation of receptor subtypes is not known due to lack of specific antibodies for immunhistological studies. This study used new antibodies, fluorescence immunohistochemistry and confocal microscopy to determine the precise cellular location of cholinergic muscarinic receptor subtypes M1-M3 in human colon. Methods: Mid transverse colon removed from children with familial adenomatous polyposis was fixed, frozen, sectioned and incubated with M1, M2 or M3 antisera followed by fluorescent secondary antibodies. Sections were double labelled with antibodies against synaptophysin to display nerve varicosities and cKit to identify interstitial cells of Cajal. Images were collected on a Leica LSM confocal microscope to allow the subcellular location of receptors to be defined. Preabsorption with the immunising peptide and western blotting were used to confirm antibody specificity. Colon was also snap frozen, RNA extracted and RT-PCR performed to confirm expression of all receptor subtypes. Results: In paediatric human colon, mRNA was abundant for all 3 receptors (M1-M3). M1-immunoreactivity (-IR) was present in myenteric and submucosal neuron cell bodies and in endothelial cells of submucosal blood vessels. M2-IR was present on the surface of smooth muscle cells in both muscle layers and in varicosities on nerve fibres in myenteric ganglia and in the circular muscle. Strong M2-IR was present on endothelial cells in submucosal and serosal blood vessels. M3-IR was present on smooth muscle cells in both muscle layers, at lower abundance than M2. M3-IR was also present in some myenteric neuron cell bodies. Immunoreactivity was absent following preabsorption with immunising peptide. Western blotting confirmed the antibodies bound to proteins of the expected size. Conclusion: Consistent with pharmacological studies M2-IR and M3-IR were present on muscle with M2 more abundant thanM3. Nerves contained all 3 receptors with different distributions. M1-IR was present in cell bodies of many myenteric and submucosal neurons suggesting thatM1 receptors mediate cholinergic neuro-neuronal transmission in human colon. M3 was present in only a small subset of myenteric neurons. M2-IR was located presynaptically on nerve fibres suggesting that M2 receptors act as autoreceptors regulating acetylcholine release. M1-IR and M2-IR were present on endothelial cells in blood vessels suggesting they mediate cholinergic regulated vaso-activity.
Rights: Copyright 2006 The Authors. Journal Compilation Copyright 2006 Blackwell Publishing Ltd.
RMID: 0020109113
DOI: 10.1111/j.1365-2982.2006.00826.x
Appears in Collections:Medicine publications

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