Guinea-Pig Isolated Urinary Bladder

Guinea-pigs were sacrificed by cervical dislocation, to avoid the possible effect of anesthetics on bladder smooth muscle response and/or drugs tested in experiments. The whole urinary bladder was excised and the detrusor muscle was dissected free from connective tissue and urothelium and cut into two equal strips isolated from the posterior face. Strips were mounted in 5 mL organ baths containing a Krebs–Henseleit solution and 0.3 μM {"type":"entrez-nucleotide","attrs":{"text":"GR159897","term_id":"238420493","term_text":"GR159897"}}GR159897, a selective NK₂ receptor antagonist (Beresford et al., 1995), 1 μM propranolol and 1 μM indomethacin. A resting tension of 1 g was applied and contractile responses were measured using isometric tension transducers (EMKA Technologies, Paris, France) connected to amplifiers and to a data acquisition system (PowerLab 16s, ADInstruments, Sydney, NSW, Australia). Strips were first challenged with 80 mM KCl in order to test tissue viability. A cumulative concentration-response curve (CRC) to SP-OMe was performed between 1 nM and 10 μM. After wash-outs and 30 min of resting period, tissues were incubated with netupitant at 1, 3, 10, and 30 nM, or its solvent (0.03% ethanol) for 90 min, then a second CRC to SP-OMe was performed. In another series of experiments, a cumulative CRC to carbachol (0.1–100 μM) was performed, then tissues were incubated for 90 min with netupitant (300 nM) or the corresponding solvent before a second CRC to carbachol was constructed. Results were expressed as % of the maximal response obtained in the first CRC to the agonist. EC₅₀ values for SP-OMe and carbachol in the absence and presence of each concentration of netupitant or solvent, were calculated by a non-linear regression and compared using the software Graph Pad Prism^® v 4.0 (GraphPad Softwares, La Jolla, CA, USA). Comparison between controls and treated CRC were performed. EC₅₀ Dose-Ratios (DR) for SP-OMe or carbachol in the presence and absence of each concentration of netupitant or solvent were calculated. The potency of netupitant on NK₁ receptors was calculated using the formula: