C. J. Greig1, S. J. Armenia1,2, V. R. Klump3, R. A. Cowles1 1Yale School Of Medicine,Department Of Surgery,New Haven, CT, USA 2New Jersey Medical School,Newark, NJ, USA 3Yale School Of Medicine,Department Of Dermatology,New Haven, CT, USA
Introduction:
Maintenance of the highly plastic intestinal epithelium relies upon a small population of stem cells localized to intestinal crypts. These specialized stem cells are responsible for both self-renewal and generation of all mature intestinal epithelial cell types, but the mechanisms by which these processes are controlled remain incompletely understood. Recent evidence suggests that muscarinic acetylcholine (ACh) signaling is involved in epithelial barrier function, epithelial proliferation and inhibition of apoptosis. Furthermore, all muscarinic ACh receptor (mAChR) subtypes have been documented throughout the gastrointestinal tract but the precise location of these receptors within the functional crypt-villus unit is unclear. Thus identification of ACh receptors present in the region of the stem cell niche would allow for design of therapies targeting the muscarinic cholinergic signaling system. We hypothesized that the intestinal crypt base would express only specific mAChRs that drive proliferation in this critical area of the mucosa.
Methods:
With institutional approval, proximal and distal intestinal segments from the jejunum and ileum were procured from wild type C57Bl/6 mice aged 12-14 weeks. Collected segments from each region of the bowel were divided and either formalin fixed and used to create paraffin sections or used for RNA extraction in an RNase-free environment per standard protocols. Paraffin sections on specialized membrane slides were used in capturing cells at the base of the intestinal crypt, performed on a LCM microscope at 100X magnification into RNAlater solution to allow for subsequent RNA extraction without significant degradation. Care was taken to avoid any subepithelial tissue and include only the bottom half of the crypt, where the intestinal stem cells are thought to reside. Using primers targeting the five subtypes of mAChRs (M1-M5), RT-PCR was performed using RNA from full-thickness intestinal and LCM crypt cell (LCM-CC) samples to determine the presence or absence of each subtype.
Results:
In full-thickness intestinal samples, mAChR subtypes M1-M4 were found in all regions of the jejunum and ileum examined. The M5 mAChR subtype was present in the proximal jejunum, but was absent from all other regions of the bowel. For LCM-CC samples, only the M1 mAChR subtype was present and was found in crypt cells from all regions of the intestine examined.
Conclusion:
All subtypes of muscarinic acetylcholine receptors were present throughout the small intestine with the exception of the M5 mAChR, which appears to be localized to the proximal jejunum. However, when cells from the crypt base were collected by laser capture microdissection and analyzed, only the M1 subtype was present by RT-PCR. Given the known location of the stem cell niche to this area, the cholinergic system, possibly via the M1 muscarinic acetylcholine receptor, is likely to be a key mediator of intestinal homeostasis.