In vitro detrusor contractility measurements.

In vitro detrusor smooth muscle (DSM) contractility measurements were performed as previously described (22). Briefly, freshly isolated urinary bladders from mice in each group at 6 wk old (N = 10–12 per group) were cut into two halves longitudinally. Each strip (~3 mm × 6 mm, n = 20 per group) was placed in organ baths (Radnoti, Monrovia, CA) filled with oxygenated Tyrode’s buffer (in mM; 125 NaCl, 2.5 KCl, 23.8 NaHCO₃, 0.5 MgCl₂, 0.4 NaH₂PO₄, 1.8 CaCl₂, and 5.5 glucose) at 37°C. Tissues were equilibrated for 45 min and then stretched to their optimum length for muscle contraction (Lo) in which the maximum force for muscle contraction was produced by electrical field stimulation (EFS; 70 V, 32 Hz). Once Lo was determined, each muscle strip was allowed to equilibrate a further 30 min in fresh Tyrode’s buffer. The tissues were subjected to several tests, including the contractile responses to EFS (70 V, 0.5–32 Hz), the acetylcholine receptor (AChR) agonist carbachol (CCh, 0.1 to 100 µM), high KCl (125 mM replaced NaCl in Tyrode’s buffer), 5 mM ATP, and α,β-methylene ATP [α,β-meATP, purinoceptor P2X_1/3 agonist, 3 µM]. Contractile responses to EFS were also recorded after 15 min of incubation of the following substances: 1) 2,2,6,6-tetramethylpiperidin-4-yl heptanoate [TMPH; antagonist of neuronal nicotinic AChR (nAChR) formed by the combination of the α and β subunits (α3, α4, β2, and β4), 0.3 µM], the combination of 2) TMPH and atropine [muscarinic (mAChR) antagonist, 1 µM], 3) atropine and α,β-meATP (desensitizes P2X_1/3), 4) atropine, α,β-meATP, and methyllycaconitine citrate [MLA; α7 nAChR blocker, 10 µM], and 5) tetrodotoxin [TTX; Na⁺ channel blocker, 1 µM]. Contractile parameters were measured using PowerLab Laboratory-Chart version 8.1.9 (AD Instruments, Colorado Springs, CO). Force measurements were performed and analyzed as previously described (22).