Bile acid modulation of calcium signals & crypt cell proliferation in the native human colonic epithelium

Previous studies have led to the proposal that elevated levels of secondary bile acids contribute to the pathogenesis of conditions such as secretory diarrhoea, inflammatory bowel disease and colon cancer. Increasing evidence suggests that these conditions arise in part from bile acid modulation of cellular signalling pathways. Studies on intestinal cell lines suggest that secondary bile acids, taurolithocholate (LCT) and taurodeoxycholate (OCT) in particular, may act directly on the intestinal epithelium by modulating calcium signalling, possibly via activation of acetylcholine receptors. AIMS: To investigate secondary bile acid modulation of calcium signalling, the canonical Wnt pathway and crypt cell proliferation in the native human colonic epithelium. METHODS: Colonic crypts were isolated from tissue biopsies obtained at sigmoidoscopy from healthy subjects (Ethical approval). Isolated crypts were attached to collagen-coated coverslips and cultured for 24 hours - 3 days in serum-free OMEM (5%C02/3rC). For calcium experiments colonic crypts were loaded with the calcium-sensitive dye Fura2-AM. For proliferation experiments crypts were incubated at 5%C02/37°C for 24 hours in the presence of BrdU. RESULTS: Application of LCT or LCA (1 00-300 ~M) failed to elicit a calcium response in any part of the crypt or modulate ACh- induced calcium signals (n=16). In contrast, OCT (0.5-1 mM) elicited oscillations of intracellular calcium levels along the entire crypt-axis (n=27). The highest oscillation frequencies were observed in the basal regions of the crypt. OCT-induced calcium oscillations were still evident in the absence of calcium in the bathing medium (n=3). Depletion of calcium from intracellular stores by thapsigargin (2 ~M) (n=3) abolished the onset of OCT-induced calcium oscillations as did pre-incubation with 2-APB (1 00 ~M) (n=3), TMB-8 (1 00 ~M) (n=3) and caffeine (20 mM) (n=3). The pacemaker region for OCT- induced oscillations did not reside at the crypt base. In contrast, acetylcholine (ACh) (1 0 ~M) induced non-oscillatory biphasic calcium signals at the crypt base that propagated in a unidirectional manner along the crypt axis (n=57). Atropine (1 ~M) inhibited the ACh response (n=3), but did not affect OCT- induced calcium oscillations. Finally, crypts treated with OCT exhibited increased levels of nuclear l3-catenin and Ki67 labelling suggesting activation of the proliferative Wnt signalling pathway. CONCLUSION: (Patho)physiologicallevels of OCT are sufficient to set in to motion a train of intracellular calcium oscillations along the crypt axis and stimulate crypt cell proliferation. The consequences to human colonic crypt fluid secretion and tissue renewal are expected to be significant and will impact on conditions such as secretory diarrhoea and colon cancer. The molecular target for OCT remains to be determined, but is unlikely to be a member of the muscarinic receptor family.EThOS - Electronic Theses Online ServiceGBUnited Kingdo