Ussing Chamber Experiments

The distal colon was cut longitudinally along the mesenteric border. The serosa, muscularis and submucosa layers were stripped away with fine forceps to prepare a mucosa sample. Stripped mucosa was mounted in a modified Ussing Chamber in a tissue holder (Easy Mount Chamber; Physiologic Instruments, San Diego, CA, United States) with an aperture surface area of 0.3 cm². Each sample was bathed bilaterally in Krebs-Henseleit solution (KHS). A short-circuit current was measured in vitro in Ussing chambers. Transepithelial PD was then clamped at 0 mV, and the short-circuit current (I_SC) was recorded with a VCCMC6 voltage-current clamp amplifier (Physiologic Instruments, San Diego, CA, United States). Transepithelial resistance (TR) (Ω^∗cm²) was measured by altering the membrane potential in a stepwise fashion (0.1 mV) and calculated from the ohmic relationship.