Calcium signalling in human colonic epithelium

Lines, George (2019) Calcium signalling in human colonic epithelium. Masters thesis, University of East Anglia.

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Intracellular calcium signals within intestinal epithelial cells are a critical integrator of a multitude of extracellular stimuli that regulate intestinal stem cell (ISC) driven tissue homeostasis and almost every other cellular activity. Calcium is an important second messenger that has an emerging role in maintaining intestinal tissue homeostasis and in the development of malignant colorectal cancer (CRC). Understanding the regulation of intestinal tissue homeostasis through intracellular calcium signals is essential for the development of novel therapies. Cholinergic signals have been shown to stimulate calcium signals, but the mechanism and impact on the intestinal epithelium is not fully understood. The aims of this study were to firstly, investigate the spatiotemporal characteristics of intracellular calcium signals, using an ex vivo 3D native human colonic crypt model system. Secondly, investigate whether these calcium signals are remodelled during colon carcinogenesis, using an in vitro 3D model of organoids (health) and tumouroids (CRC).

Fura-2 calcium imaging revealed the spatiotemporal characteristics of muscarinic-induced intracellular calcium signals, through addition of Carbachol (Cch). Calcium signals evoked by Cch in human colonic crypts initiate at the apical pole in the base of crypts. Spreading to neighbouring cells as the calcium wave propagates across all cell types along the crypt axis. This study has found the extracellular environment is critical in the mobilisation of intracellular calcium signalling e.g. store operated calcium entry (SOCE). The higher the extracellular calcium concentration the greater the intracellular calcium response. Additionally, there appears to be an important role of non-neuronal ACh synthesis by Tuft cells in mobilising intracellular calcium at the base of crypts via muscarinic receptor activation. Even though pharmacological inhibition of the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA) pump substantially reduces Cch-induced calcium mobilisation there was still a significant calcium response after Thapsigargin treatment. The acidic lysosome was confirmed as the initiation site of intracellular calcium signals via 100 μM GPN stimulation with a lack of subsequent Cch-induced calcium response. Confirmed through coupling of fluorescence imaging and previous immunocytochemistry data.

In full culture media, there were intracellular calcium oscillations from a single 10 μM Cch stimulation, intensity of which was amplified using 100 μM Cch. In a perfusion chamber, oscillations are also induced through the constant addition of fresh 10 μM Cch solution. Inhibition of mechanistic target of Rapamycin (mTOR), important for TPC-mediated calcium mobilisation from lysosomes, remarkably had no effect on the generation of a calcium signal. Organoid formation assays investigated the effect of muscarinic receptor activation on the proliferative activity of ISCs, measured by organoid bud formation. Inhibition of receptors that facilitate mobilisation of calcium from lysosomal and ER stores, along with Cch and UTP, were used to determine if there was remodelling of the calcium signalling pathway in colon carcinogenesis. There appears to be some differentiation in relative store size in colon cancer.

In conclusion, this study identifies lysosomes at the apical of cells within the ISC niche as the site of intracellular calcium initiation in human colonic crypts. These propagate to the basal pole within cells as the signal travels up the crypt axis. Calcium signals are induced through muscarinic activation and the calcium concentration has an important role in this response, to be explored further. TPC-mediated calcium release from lysosomes is under the regulation of mTOR which itself is regulated by numerous different factors. Inhibition of mTOR did not significantly impact calcium mobilisation. Finally, this study potentially indicates a difference in ER and lysosomal calcium store sizes between health and disease. Therefore, the remodelling of these stores could serve as potential targets for novel treatments of CRC.

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 9280 not found.
Date Deposited: 20 Mar 2019 11:08
Last Modified: 20 Mar 2019 11:08


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