S. J. Armenia1,2, C. J. Greig1, R. A. Cowles1 1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2New Jersey Medical School,Newark, NJ, USA
Introduction: Growth of the intestinal epithelium is critical during intestinal adaptation and after intestinal mucosal injury. While several peptide hormones have been shown to stimulate intestinal mucosal growth, the role of enteric neurotransmitters, such as acetylcholine (ACh), in mucosal homeostasis remains poorly defined. Previous work documenting serotonergic regulation of enterocyte proliferation implicated muscarinic ACh receptors (mAChRs) in the signaling mechanism. The distribution of the five mAChR subtypes on dividing enterocytes has not been reported but may assist in the development of targeted therapies aimed at stimulating intestinal mucosal growth. In order to establish an in vitro model for study of cholinergic signaling in the intestinal mucosa, we aimed to document the presence of mAChRs in cultured enterocytes. We hypothesized that while mAChRs would be widely distributed in the small bowel, the IEC-6 cell line would express enterocyte-specific muscarinic receptors allowing these cells to respond to muscarinic receptor agonists in a well-controlled model system.
Methods: The IEC-6 small intestine epithelial cell line was cultured under standard conditions and, with institutional approval, small intestinal segments were obtained from male wild type C57Bl/6 mice between the ages of 12-14 weeks. RNA was extracted from IEC-6 cells and whole intestinal segments in an RNase-free environment per standard protocols. RT-PCR was performed using targeted primers to document the presence or absence of each of the five mAChRs. PCR products were separated by gel electrophoresis and visualized using ethidium bromide staining. To assess the responses of epithelial cells to agonists, cultured IEC-6 cells were treated with serial dilutions of bethanechol, a muscarinic receptor agonist, for 48 hours. Cellular viability and proliferation were assessed using an MTT assay. Statistical analysis was performed using Student’s t-test, with significance assumed when p<0.05.
Results: In whole intestinal segments, the presence of RNA from all five mAChRs was demonstrated by RT-PCR. In cultured IEC-6 cells, RNA for two of the five mAChR subtypes (M1 and M4) was amplified by RT-PCR. Treatment of IEC-6 cells with bethanechol stimulated cellular proliferation in a dose-dependent manner.
Conclusion: Muscarinic ACh receptors are widely distributed in the small intestine. While all mAChR subtypes can be documented in whole small intestinal segments, only M1 and M4 mAChRs appear to be expressed in IEC-6 cells. The ability of bethanechol to stimulate IEC-6 cell proliferation suggests that ACh may play an important role in enterocyte proliferation in-vivo. The specific mAChR responsible for this action and the subsequent intracellular signaling pathways warrant further study of this model.