Mechanism and role of calcium in excitation-mucus secretion coupling in human colonic epithelium

Defective mucus barrier function is one of the contributing factors for inflammatory bowel disease (IBD) pathogenesis. Understanding the regulation of mucus secretion is essential for developing new therapies. Cholinergic signals have been shown to stimulate mucus secretion, but the mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism of calcium (Ca2+) coupling mucus (MUC2) secretion in colonic goblet cells in situ, using an ex-vivo 3D model native human colonic crypt model system. Immunohistochemistry revealed the presence of cholinergic neurons in close proximity to the basal membrane of the crypts, where the muscarinic receptor (M3AchR) was expressed. Fura-2 Ca2+ imaging demonstrated that Carbachol (Cch) evoked Ca2+ signals at the human colonic crypt base via M3AchR activation, which then spread to all cell types along the crypt-axis. Surprisingly, pharmacological inhibition of the canonical 2nd messenger IP3 pathway had no major effect on Ca2+ signal generation, and depleting the ER Ca2+ store demonstrated Cch-induced Ca2+ mobilisation from other intracellular organelles, including lysosomes. Inhibition of pathways and 2nd messengers which mobilise Ca2+ from lysosomal stores – including chemicals targeting CD38, NOX1, NAADP and TPC – profoundly suppressed Cch-induced Ca2+ signals. Furthermore, inhibition of this cholinergic coupled calcium signalling pathway prevented MUC2 mucus secretion from colonic crypt goblet cells in situ. This is the first study to demonstrate that M3AchR activation mobilises Ca2+ from acidic lysosomes in the human colonic epithelium. These findings also implicate NAADP as the key 2nd messenger which mediates this pathway via activation of two pore channels. Modulation of Ca2+ release from acidic stores may serve as a potential target for treating IBD and other gastrointestinal pathologies.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of East Anglia

Year dc:date.issued

2015

Author dc:creator

• Kam, Christy

Language dc:language

en