Measurement of Bladder Contractility

Both murine and human detrusor muscle strips were mounted on two parallel, horizontal stainless-steel tissue-holding needles of a myograph (needle diameter 200 μm, 610 M Multi Wire Myograph System, Danish Myo Technology A/S, Aarhus, Denmark). Chambers were filled with 6 ml of Krebs solution aerated with carbogen (mixture of 5% CO₂ and 95% O₂) at 37°C. Detrusor muscle contractions were recorded under isometric conditions. Every experiment started with a 60-min resting period while the strips were stretched to and stabilized at a passive tension of 5 mN (murine) or 3 mN (human). After the resting period, UBSMs were challenged twice with 124 mM K⁺-containing Krebs solution to examine the viability of the tissues. The contractile effect of 124 mM K⁺ was comparable in the detrusor strips obtained from the WT and the genetically modified mouse lines (Supplementary Figure 1 at https://doi.org/10.6084/m9.figshare.16815061.v5). After several washes with normal Krebs solution, the contractile effects of BK (10⁻¹⁰-10⁻⁴ M), Lys-[Des-Arg⁹]-bradykinin (B₁ receptor agonist, 10⁻⁵ M), [Phe⁸Ψ(CH-NH)-Arg⁹]-bradykinin (B₂ receptor agonist, 10⁻⁵ M), carbamoylcholine chloride [carbachol (CCh, 10⁻⁶ M)], α,β-methyleneadenosine 5′-triphosphate [α,β-meATP, ATP-analog (10⁻⁵ M)] was measured. Some of the strips were preincubated with one of the following inhibitors without washing out: R-715 (Ac-Lys-Arg-Pro-Pro-Gly-Phe-Ser-DβNal-Ile, B₁ receptor antagonist, 10⁻⁶ M, 20 min), HOE-140 (icatibant) (D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg, B₂ receptor antagonist, 10⁻⁶ M, 20 min), atropine [muscarinic-acetylcholine (ACh)-receptor antagonist, 10⁻⁶ M, 20 min], pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate (PPADS; P2 purinergic receptor antagonist, 10⁻⁵ M, 20 min), Y-27632 [ROCK inhibitor, 10⁻⁵ M, 20 min], indomethacin [non-isoform-selective COX inhibitor, 10⁻⁵ M, 20 min], N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide [NS-398, COX-2 inhibitor 10⁻⁵ M, 20 min]. When acetic acid, dimethyl sulfoxide (DMSO), or saline was the solvent of the inhibitor, they were applied in matched concentrations as vehicle control. The final concentration of acetic acid in the tissue bath was 0.1 mM, while that of DMSO was 0.1%. Since repeated use of BK or its analogs in the same specimen is problematic due to the rapid desensitization and the viability of the tissues may decline in a prolonged experiment, we performed unpaired, time-control experiments in this study. Finally, bladder strips were exposed to 124 mM K⁺-containing Krebs solution to retest the viability of the detrusor strips. Agonist-induced tension changes were normalized to the reference contraction induced by 124 mM K⁺-containing Krebs solution (second administration). A schematic diagram of our experimental protocol is demonstrated in Supplementary Figure 2 (at https://doi.org/10.6084/m9.figshare.16815097.v2).

MP100 system and AcqKnowledge 3.9.2 software from Biopac System (Goleta, CA) were used for the acquisition and analysis of myographic measurements. The moving average smoothening function of the software was applied on recordings solely in order to eliminate the noises arising from the bubbling of the medium and to reduce the high frequency—low amplitude spontaneous tension oscillations. The parameters of the smoothening filter were carefully chosen in order to eliminate only the noises but not to alter the amplitude of the BK-induced responses—the baseline and peak values were always compared before and after the smoothing. The sample rate of the recordings was 10 samples/s (10 Hz), the smoothing factor was between 10 and 40 samples. Spontaneous micro-contractions of the bladder strips were observed occasionally, however, they were not reproducible, thus these data were not considered for demonstration or evaluation in the present study.