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59 EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CAR) STIMULATES IL-11 SECRETION FROM COLONIC MYOFIBROBLASTS WHICH STIMULATES REPAIR OF DAMAGED INTESTINAL BARRIER I Pacheco, D Pieris, C Spencer, RJ MacLeod Postprandial Ca2+ variations stimulate the extracellular calcium-sensing receptor (CaR) on colonic myofibroblasts. To understand if this stimulation could contribute to epithelial homeostasis we preformed array analysis. CaR activation upregulated transcripts for antiadipogenesis factor (IL-11). The current experiments demonstrate that CaR stimulation of 18Co cells and primary cell lines of human colonic myofibroblasts increased synthesis (RT-PCR) and secretion (ELISA) of IL-11. CaR mediated this increase as transient transfection with siRNA against CaR reduced stimulated secretion to basal levels and dominant negative CaR (R185Q) increased the EC50 for Ca2+ (3.8 mM to 7.5 mM). Pharmacologial inhibitors of MAP kinases demonstrated EGFR and erk1&2 were required for IL-11 secretion; Western blot analysis confirmed CaR activation stimulated erk1&2. Exogenous IL-11 added to T84 cells grown on transwells increased transepithelial resistance (TER) within 24 h and reduced FITC-dextran flux (3 kD, 10 kD) ~90%. CaR activation of the T84 cells upregulated transcript for the IL-11 receptor alpha subunit. Therefore to determine whether CaR-stimulated IL-11 could repair a damaged intestinal barrier, we challenged high resistance T84 cells with low or high Ca2+, then preformed a "Ca2+-switch" using 2 mM EDTA (5 min) and measured recovery of TER after IL-11 (100 ng/mL) addition. TER was reduced after EDTA (Ohms/cm2: 1368±58 vs 333±71). T84 cells treated with high Ca2+ and IL-11 generated a greater TER over the next 16 h compared with cells treated with low Ca2+ and IL-11 (Ohms/cm2: 2166±234 vs 1702±156, P<0.05, n=6). Together our results suggest that IL-11 stimulates the development of barrier function in the intestine and promotes the recovery of damaged barrier. Furthermore our results imply that the CaR orchestrates paracrine signalling in the epithelia and sub-epithelia to mediate barrier function.
GIDRU, Dept of Physiology, Queen's University, Kingston, Ontario