Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB093

Poster Communications

Ligand/receptor-specific human colonic crypt calcium signatures are conferred in part by membrane receptor location and differential coupling to intracellular calcium stores.

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

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

A human colonic crypt is the unitary structure of the large intestinal epithelium. The crypt cell population (approx. 1000 cells per crypt) is topologically organised into an exquisite hierarchy: immature stem cells remain located within the stem cell niche at the crypt-base, while their progeny mature into functional epithelial cell types as they migrate along the crypt-axis. On reaching the surface epithelium, they are shed into the gut lumen. A number of morphogenetic gradients that were first shown to exhibit a spatial correlation with the differentiation hierarchy represented by the intestinal crypt(-villus) axis have subsequently been demonstrated to play major roles in regulating intestinal tissue renewal in health and disease. The aim of this study was to gain novel insights into the coordination of human colonic crypt physiology by correlating the spatio-temporal characteristics of receptor-mediated calcium signals with the physiological function of polarised epithelial cells along the crypt-axis. Methods: Human colonic crypts were isolated from colorectal tissue samples obtained at surgical resection (NRES approval) and placed in a 3D culture system. The spatio-temporal characteristics of intracellular calcium was monitored by Fura2/Fluo-4 imaging and the mechanism of receptor-mediated calcium mobilisation was characterised by a pharmacological approach. Calcium signalling toolkit components were visualised by fluorescence immunolabelling. Results: Muscarinic receptor activation initiated a colonic crypt calcium signal at the apical pole of intestinal stem cells (ISCs) located at the crypt base. ISCs were enriched with M3AChRs on the basal membrane and the apical site of calcium signal initiation correlated with a high concentration of endolysosmes endowed with two pore calcium channels (TPCs) and CD38 (known to synthesise NAADP). Cholinerigic calcium signals proceeded to propagate across the polarised epithelial cell to the basal pole and up the crypt-axis by following an apical cell-to-cell path. Conversely, UTP (an agonist of P2Y receptor subtypes), intiated calcium signals in the upper crypt region, which propagated downward towards the crypt base. Unlike for cholinergic calcium signals, the cellular subtype responsible for purinergic calcium signal initiation was not identified, nor did there appear to be a concentrated hot spot of a specific type of intracellular calcium channel. However, whereas the muscarinic receptor coupled calcium signal was abolished by TPC inhibitors (NED-19 and diltiazem, both 250 μM), the purinergic receptor-coupled calcium signal was blocked by the InsP3R inhibitor (2-APB, 100 μM), but not by TPC inhibitors. Conclusions: Ligand/receptor-specific human colonic crypt calcium signatures are conferred in part by membrane receptor location and differential coupling to intracellular calcium stores, and provide novel insights into 'bottom-up' and 'top-down' co-ordination of colonic crypt physiology.

Where applicable, experiments conform with Society ethical requirements