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Distribution of Muscarinic Acetylcholine Receptor Subtypes in the Murine Small Intestine
Eleanor D. Muise, Neeru Gandotra, John J. Tackett, Michaela C. Bamdad, *Robert A. Cowles
Yale School of Medicine, New Haven, CT
Objective: Neuronal serotonin stimulates enterocyte turnover in the murine small intestine, and previous work suggests this is mediated by submucosal neurons that promote cell division via a cholinergic pathway. Distribution of the five known muscarinic receptor subtypes (mAChRs) in the small intestine has not been fully studied, and their potential role in intestinal adaptation is unknown. We hypothesized that mAChRs have distinct anatomic distributions within the bowel that coordinate the effects of acetylcholine on the small intestine.
Design: Basic science research, animal model.
Setting: Laboratory-based, animal model study, academic medical center.
Patients: Male C57/BL6 mice.
Intervention(s): Male C57/BL6 mice aged 2, 4, 6, and 8 weeks were euthanized by CO2 and the duodenum, jejunum, and ileum were harvested. RNA was isolated, and cDNA synthesized for analysis by RT-PCR. Subtype-specific PCR primers were used to determine the distribution of each mAChR subtype. PCR products were sequenced to confirm that the correct receptor subtype product was amplified. Tissue for immunofluorescence was fixed with Nakane fixative and embedded in epon. Antigen retrieval was performed with citrate buffer and heat, followed by 1% sodium dodecyl sulfate. Immunofluorescence was performed with polyclonal antibodies specific to each mAChR1-5.????
Main Outcome Measure(s): Mortality with survival defined as alive in-hospital at one year or discharge
Results: All five mAChR subtypes were present in the duodenum, jejunum, and ileum at all ages by RT-PCR. Immunofluorescence microscopy revealed the presence of mAChR1, mAChR3, mAChR4, and mAChR5 in association with enterocytes along the villus and within the myenteric plexus; mAChR2 staining within the crypt stem cell compartment; and mAChR4 along the central lacteal.
Conclusions: Muscarinic receptors are widely distributed along the entire alimentary tract. mAChR2 appears to localize to the crypt stem cell compartment, making it a plausible regulator of stem cell activity and an attractive target for therapies aimed at stimulating intestinal mucosal growth. The functional significance of mAChR subtypes requires further study.
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