Proximal Colon Muscle Contractility Assay

Strips of longitudinal (6 mm in length) muscle from proximal colon were mounted in a 37 °C organ bath with a force transducer (MLT0202; AD Instruments, Spain) connected to a PowerLab (AD Instruments, Australia) recording device. Briefly, Ca²⁺-free HEPES-Tyrode (H-T) buffer (140.6 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl₂, 10 mmol/L HEPES, and 5.6 mmol/L glucose, pH 7.4) was used to wash out the lumen contents of proximal colon segments. Then, segments were transferred to the organ bath and equilibrated in H-T buffer (137.0 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl₂, 1.8 mmol/L CaCl₂, 10 mmol/L HEPES, and 5.6 mmol/L glucose, pH 7.4) for 15 min. The resting tension was set to 0.5 g prior to force measurement. The isotonic contractions of longitudinal muscle was recorded for 1 h, with H-T buffer being changed every 15 min. The mean amplitude of the basal tension and the frequency of phasic contractions were measured for 30 min after experiments. The detailed force assays were performed according to our previously described methods [26 (link), 27 (link)].