Measuring Colon Muscle Contractility

Strips of longitudinal (6mm in length) muscle from the 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. Next, the 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, pH7.4) for 15 minutes. The resting tension was set to approximately 0.5 g prior to force measurement. The isotonic contractions of longitudinal muscle were recorded for 1 hour, with H-T buffer being changed every 15 minutes. The mean amplitude of the basal tension and frequency of phasic contractions were measured for 30 minutes after experiments. The detailed force assays were performed according to our previously described methods^{54 (link), 55 (link)}.