To increase lysosomal pH, we used chloroquine (CHQ), a lysosomotropic weak base which has been shown to collect in lysosomes. Once inside the lysosome, chloroquine becomes protonated, preventing it from leaving the lysosome and decreasing lysosomal pH. To demonstrate the importance of the lysosome to a given effect, we used Gly-Phe β-naphthylamide (GPN), another agent that collects preferentially into lysosomes, where it is cleaved by cathepsin C. The cleavage product of GPN collects in the lysosome, creating an osmotic gradient that causes the lysosome to swell and eventually lyse.
The cell-based portion of our study used immortalized normal human urothelial cells (TRT-HU1) at passage number 25-35. Cells were used for experiments after 1-2 days when they had reached 50-70% confluency.
To measure extracellular ATP concentrations, TRT-HU1 cells were grown in white-walled 96-well plates. First, the media supporting the cells was first replaced with 50µl of Krebs solution alone or containing GPN (20µM, 2X final concentration) and incubated at 37°C for 20 minutes. 50µl of Krebs (for non-stimulated controls) or CHQ (200µM, 2X final concentration) was then added and the plate incubated again for 30 minutes at 37°C. 50µl of the luciferin/luciferase assay mix (Sigma-Aldrich) was then added and the luminescence measured using a plate-based luminometer. Luminescence readings were converted to ATP concentrations using a standard curve with known concentrations of ATP.
To measure lysosomal pH, TRT-Cells were grown in black-walled 96-well plates. At the time of the experiment, the media was removed and replaced with 50µl of either Krebs solution alone (control) or CHQ (100µM). After a 30-minute incubation, the extracellular solution was aspirated and the cells incubated with 5 μM LysoSensor Yellow/Blue DND 160 (Invitrogen) for 3 min followed by a 15 min post-incubation in Krebs solution. Lysosomal pH was determined from the ratio of light emitted at 450 nm vs. 510 nm (365 nm ex) using a plate reader (Tecan Spark 20M) and calibrated by exposing cells to 10μM monensin and 20 μM nigericin in a solution containing (in mM) 20 MES, 110 KCl and 20 NaCl at pH 4.0–6.0 for 15 min.
Extracellular IL-1β concentrations were measured using a commercially available ELISA kit (Abcam). Briefly, TRT-HU1 cells, grown on 35mm dishes, were stimulated with CHQ (100µM in Krebs solution) for 2 hours. To measure IL-1β, 100µl samples of the extracellular solution was used in triplicate according to the manufacturer’s instructions.
For our in vivo experiments, female Sprague-Dawley rats (~200-250g) were anesthetized using isoflurane and CHQ (100µM in sterile saline, 0.5ml) was instilled in the bladder through a transurethral catheter for 1 hour. The animals were then used immediately for cystometry or plasma extravasation or allowed to recover for experiments one or three days later. For cystometry, the rats were anesthetized with urethane and catheterized through the bladder dome. Open cystometry was then performed by perfusing Krebs solution into the bladder at a rate of 0.08ml/min. For plasma extravasation, rats were anesthetized using urethane, and Evans Blue (50mg/kg) was injected through a jugular vein catheter. Fifteen minutes after dye injection, the rats were sacrificed by decapitation, exsanguinated and the bladder removed. After weighing, the bladder was placed in 3 ml formamide for 72 hours. The dye present in the formamide solution was quantified by measuring optical density and then the concentration was estimated using a standard curve.
For bladder strip experiments, bladders were removed from female Sprague Dawley rats, cut into strips longitudinally, and attached to a force displacement transducer in a tissue bath containing oxygenated Krebs solution at 37°C. Agonists and antagonists were bath applied and changes in basal tone and spontaneous contraction amplitude recorded.