2.3 Organ-bath experiments
All samples were taken from the bladder body. From each patient, 5–10
longitudinal bladder strips (length = 20 ± 1.5 mm; width = 8–9 mm) were
studied. Bladder strips were placed in warm Krebs solution (composition
in mM: NaCl, 118; KCl, 4.7; CaCl2, 1.9;
MgSO4, 1.2; NaHCO3, 24.9;
KH2PO4, 1.2; glucose, 11.7; pH = 7.4)
and bubbled with 95% O2 and 5% CO2.
Strips were tied at each end using a fine thread, mounted in a vertical
organ bath in oxygenated Krebs solution (volume = 20 mL), and heated to
37 °C in a circulating warm water-bath. The longitudinal tension of the
isolated preparations was recorded continuously with an isometric
transducer and then processed with Lab Chart 7 (AD Instruments, New
South Wales, Australia). Tissues were stretched to a baseline tension of
15mN (1.5 g). Contractions were allowed to equilibrate for 30–40 min,
until the appearance of stable SBCs. In strips with no SBCs, carbachol
(1–10nM) was applied to initiate SBCs. Strips that had a spontaneous or
carbachol-evoked SBCs were considered as having “good” viability. All
the strips from the 26 patients had good viability.
In a pilot study, we found that repeated application of high
concentrations (≥100μM) of
H2O2 in the same strip induced
desensitization (supplementary figure 1). Thus, the noncumulative
concentration–response relationship of
H2O2was determined. On completion of experiments, threads were removed and
strips were weighed after water absorption with tissue paper.