Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB115

Poster Communications

Non-neuronal secretion of acetylcholine by human colonic crypts triggers calcium signals in LGR5-positive intestinal stem cells and promotes crypt progenitor cell proliferation

N. Pelaez-Llaneza1, A. lee1, A. parris1, V. jones1, G. Lines1, J. Tang1, M. R. williams1

1. School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom.

The human colonic epithelium is highly innvervated by cholinergic neurons and acetylcholine (ACh) has been implicated in regulating intestinal fluid/mucus secretion, stem cell-driven tissue renewal, epithelial integrity and regeneration, and colon carcinogenesis. Recent studies have suggested that murine Tuft cells express choline acetyltransferase (ChAT) and provide a source of non-neuronal of ACh. The aim of this study was to determine whether the human colonic epithelium can synthesise ACh and regulate intestinal stem/progenitor cell biology. Methods: 3D cultures of human colonic crypts were generated from colorectal tissue samples obtained at surgical resection (NRES approval). Intracellular calcium was monitored by Fura2/Fluo4 fluorescence imaging. Transcript expression was assessed by RT-PCR and protein localisation was visualised by immunofluorescence labelling. The concentration of ACh secreted into the media of cultured human colonic crypts was quantified by HILIC-coupled tandem mass spectroscopy (HILIC-MS/MS). Cultured human colonic crypt cell proliferation was assessed by nuclear EdU incorporation. Results: Bath application of ACh, carbachol or muscarinic agonists methacholine and oxotremorine triggered a calcium signal at the apical pole of slender cells located at the very base of human colonic crypts. Live cell immunofluorescence labelling with antibodies specific for LGR5 and PTK7, confirmed that the cholinergic calcium signal orginated in intestinal stem cells. Co-immunofluorescence labelling of markers for ER (e.g. KDEL), endolysosomes (e.g. LAMP1 and lysotracker), InsP3Rs, RyRs, and two-pore channels (TPCs) suggested that calcium was moblised from endolysosomes via TPCs located at the apical pole of intestinal stem cells. This was confirmed by pharmacological inhibition of calcium signal generation by TPC inhibitors NED-19 (100 μM) and diltiazem (100 μM), with only partial inhibition by ryanodine (50 μM), 8-bromo cADP ribose (30 μM), xestospongin C (2 μM) and 2-APB (50 μM). NED-19 autofluorescence also co-localised with the cholinergic calcium signal trigger site. RT-PCR demonstrated the expression of CHAT mRNA and the protein co-immunolocalised with markers of intestinal Tuft cells, which predominated within the stem cell niche. Media conditioned for 12-24 hours with human colonic crypts was found by HILIC-MS/MS to contain an ACh concentration of 90 ±10 nM (N = 5 subjects). Accordingly, crypt-conditioned media initiated a calcium signal in the stem cell niche of similar amplitude to that invoked by addition of exogenous ACh (100 nM, N = 3 subjects). Finally, exogenous ACh stimulated an increase in EdU positive nuclei from 18 ± 2% to 44 ± 5% (n>5 crypts in each group; representative of N>5 subjects) in the human colonic stem cell niche. Conclusions: Non-neuronal secretion of ACh by human colonic crypts triggers calcium signals in LGR5-positive intestinal stem cells and promotes crypt progenitor cell proliferation.

Where applicable, experiments conform with Society ethical requirements