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UNRAVELLING THE MECHANISM OF DYSREGULATED CALCIUM-MEDIATED ACTIVE ION TRANSPORT OBSERVED IN DEXTRAN SODIUM SULPHATE (DSS)-INDUCED COLITIS IN MICE

CL Hirota, C Fong, DM McKay
Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario; Gastrointestinal Research Group, University of Calgary, Calgary, Alberta

Proper surface hydration, as a consequence of electrolyte transport is critical for gut physiology. Colonic tissue from several models of colitis, and tissues from patients with IBD, display reduced responses to Cl^- secretagogues applied to ex vivo preparations mounted in Ussing chambers. We have shown in the DSS model of colitis (4% wt/vol oral DSS for 5 days, then 3 days of tap water), that colonic secretory responses to cholinergic agonists, which will act predominantly on epithelial M₃ muscarinic receptors, are virtually abolished. Assessing the mechanism of this event, we show here that it is not due to reductions in colonic epithelial M₃ receptor mRNA or protein expression as assessed by reverse transcriptase (RT)-PCR, immunofluorescent localization and Western blotting. Furthermore, short-circuit current responses to the non-cholinergic Ca²⁺-dependent secretagogue, histamine, the SERCA pump inhibitors thapsigargin and cyclopiazonic acid, and the calcium ionophore, A23187 were either absent or severely attenuated in colonic tissue from DSS-treated mice. These findings suggest either, i) loss or dysfunction of some or all of the specific ion channels, transporters and pumps that are asymmetrically expressed in the polarized epithelial cell membrane, or ii) a fundamental defect in epithelial intracellular Ca²⁺ signaling as a consequence of the DSS-induced colitis. Analysis of LiCl-purified mRNA extracted from isolated colonic crypts or mucosa subjected to RT-PCR showed no transcriptional differences in the Ca²⁺-regulated Cl^- channel, CLCA6, the Ca²⁺-regulated K⁺ channel, KCNN4, or the Na⁺/K⁺/Cl^- cotransporter, NKCC1, between control and DSS-treated mice (cytokeratin 19 was used as an epithelial-specific housekeeping gene in these experiments). Therefore, while DSS-induced colitis is accompanied by a dramatically reduced ability to respond to muscarinic agonists and other agents that evoke Ca²⁺-mediated active ion transport, this defect neither reflects transcriptional differences in M₃ receptor expression nor that of selected components of the transport machinery: CLCA6, KCNN4 or NKCC1. Thus, we speculate that the ion transport irregularity associated with DSS-colitis is due to either abnormal channel/transporter insertion into the cell membrane or a Ca²⁺ handling defect. Analysis of this model will enhance understanding of perturbed water movement in colitis and elucidate a targeted strategy to counter such pathophysiology.
Funded by the CCFC

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