Ussing Chamber Experiments.

We used the most distal 2 cm of the mouse colon. After colon dissection, smooth muscle layer was removed by stripping, and a piece of mucosa was mounted in an Easy Mount Ussing chamber (Physiologic Instruments) using an insert with an aperture of 0.1 cm² (P2303A, Physiologic Instruments). The standard Krebs solution contained 119 mM NaCl, 2.7 mM KCl, 23 mM NaHCO₃, 1.25 mM Na₂HPO₄, 1.8 mM CaCl₂, 1.5 mM MgSO₄, 0.5 mM ascorbic acid, and 10 mM glucose. The reservoirs were continuously gassed with 5% CO₂ and 95% O₂ and maintained at 37 °C by water jackets. I_SC was measured using an automatic voltage clamping device (EVC-4000; Physiologic Instruments) that compensates for resistance of the solution between the potential measuring electrodes. The transepithelial current was applied across the tissue via a pair of Ag/AgCl electrodes that were kept in contact with the mucosal and serosal bathing solution using 3 M KCl-agar bridges. All experiments were done under voltage clamp at 0 mV. The I_SC is negative when positive current flows from serosa to mucosa. The tissue was placed in the apparatus and equilibrated for 30 min to stabilize I_SC before starting the experiment. The baseline value of electrical parameters was determined as the mean over the 10 min immediately prior to drug administration. A positive I_SC corresponds to the net electrogenic secretion of anions or the net electrogenic absorption of cations. FSK (10 μM) were used on the luminal side for confirming the viability of the tissues.