Enzyme-Mediated eATP Degradation in Bladder

Samples from ELS of nondistended and distended bladder preparations were prepared as described previously [9 (link)]. Briefly, the 2.9 mL ELS collected at the end of time equivalent to the time for bladder filling (nondistended condition) or at the end of filling (distended condition) were placed in 4 mL Amicon Ultra centrifugal filter units with 10 kDa molecular weight cut-off (MWCO) pore size (Millipore Sigma, Burlington, MA, USA) to be concentrated by centrifugation. The ELS samples were concentrated to approximately 80–100 µL volume at 4000× g for 25 min using SorvallST 40R centrifuge (Thermofisher, Langenselbold, Germany). The concentrated ELS (cELS) were used to detect the degradation of eATP substrate by enzymes that were released in the ELS at rest or during bladder filling.