2.1. Preparation and treatments
All the experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.
Female Sprague-Dawley rats weighing 250-300 grams were used for these experiments. After the bladder was removed from rats anaesthetized with isoflurane, the animals were euthanized in CO2 chambers. The bladders were cut in half; one half was left intact while the other half was gently swabbed with a Q-tip in single longitudinal sweeps to remove the urothelium. Two longitudinal strips were prepared from each half and placed into a tissue bath containing oxygenized Krebs solution.
2.2. Histological examination of bladder strips
Pieces of intact and swabbed strips were transferred to a Petri-dish containing 4% paraformaldehyde and soaked overnight. The tissues were then embedded in paraffin, and 5 μm slices were cut and stained with hematoxilin-eosin. The slides were examined and photographed under a light microscope with 200 times magnification.
2.3. Measurement of bladder contractility
Intact and swabbed strips were mounted in organ baths of 5 mL in Krebs solution containing (mM): NaCl (113.0); KCl (4.7), CaCl2 (1.25), MgSO4 (1.20), NaHCO3 (25.0), KH2PO4 (1.2); d-Glucose (11.5) and bubbled with 95% O2/ 5% CO2 at 37° C. Trains of square wave pulses (25 V/cm; 0.25 ms, 10 Hz 100 shocks in every 100 s) were delivered by a Grass S88 stimulator (Astromed, RI) via platinum wire electrodes inserted from the top and the bottom of the baths. The isometric contractions were measured with force transducers (F100, WPI) connected to a bridge amplifier (TBM4, WPI, Sarasota, FL), and the contractile data were collected with a computerized data acquisition system. An initial 10 mN pretension was applied at the beginning of the experiments. After a 30 min equilibrium time, we started the stimulation and data collection. Drugs were injected into the bath. The contractile force (mN) was standardized on the basis of the cross-sectional area of the bladder strips expressed as mN/mm2 that was calculated from the strip weight, length, and specific density (1.06 kg/L) as described earlier .
2.4. Measurement of ATP and NO release
Bladder strips (either intact or with swabbed urothelium) were mounted in a heated tissue bath of 1 mL at 37°C and superfused with oxygenated Krebs at a rate of 1 ml/min. After a 30-minute equilibrium period, one-minute effluents were collected with a fraction collector (Gilson, Middletown MI) for a period of 16 min. The bladder strips were electrically stimulated (50 V, 10 Hz) starting from the 3rd minute for a period of one minute then pharmacologically stimulated with 50 μM carbachol or substance P (SP) for one minute started from the 11th minute of the perfusion. The collected samples were stored in -80°C until the ATP and NO assays were done.
The released amount of ATP and its metabolic derivatives were assayed with HPLC connected to a fluorescent detector (Dynamax, Rainin). The release was expressed as total purine release (i.e. sum of the measured ATP, ADP, AMP, and adenosine release) based on the assumption that all of the metabolites derive from ATP . An aliquot (500 μL) of the effluents was derivatized with chloro-acetaldehyde at 90°C to transform the purines to fluorescent 6-eteno-derivatives using the method described by Todorov et al. . After the precolumn derivatization, 200 μL aliquot of the effluents was injected into a Nova- Pak, Phenyl, 8 × 10 HPLC column (Waters, Milford, MA). The fluorescent detector was set at 230 μm excitation and 420 μm emission wavelengths. The separation of purines was performed with a two-pump gradient system (Gilson 306) with a pump speed of 2 mL/min applying 50 μM phosphate buffer (Buffer A) and 25% methanol phosphate buffer (Buffer B). A linear gradient was run between 3 and 15 minutes from 0% - 100% of Buffer B. Both the pump control and the peak integration were carried out with HPLC software (Unipoint, Gilson, Midtown, WI). The peak areas of the samples were compared with those of standards (ATP, ADP, AMP and adenosine). The quantity of released purines was expressed on the basis of the strip weight in pmol/mg.
NO release was measured with a nitric oxide analyzer (NOAH; GE) by reducing the nitrate and nitrite to NO in the effluents injected into the reaction chamber with vanadium chloride at 95°C. At the beginning of every assay a nitrate standard curve was constructed. Then, 20 μL effluents were injected into the reaction chamber, and the area of the obtained peak was compared to that of the standard curve. The corresponding release values of NO were normalized by the weight of the bladder strip.
2.5. Statistical analysis
For statistical analyses one way ANOVA followed by multiple comparisons post-test (Dunnett test) was used. Data were presented as mean ± S.E.; P < 0.05 was considered statistically significant. Statistical analysis and graphics were performed with Prism 4 (Graph Pad Software, San Diego CA) computer program.
Alpha-beta methylene ATP (α, β meATP), carbachol, atropine, Substance P, vanadium chloride, ATP, ADP, AMP, adenosine, sodium nitrate, as well as all constituents of Krebs solution, were purchased from Sigma-Aldrich (St Louis, MO).