Basic Science / Translational
Christopher H Fry
Atropine-resistant, nerve-mediated contractions occur in human detrusor only from bladders with detrusor overactivity (DO) regardless of pathology (idiopathic, neuropathic, obstructive ), and also in detrusor of most small animals. Such residual contractions are abolished by pre-treatment with α,β methylene ATP (ABMA) to de-sensitise ionotropic purinergic receptors, so it is presumed that ATP is the neurotransmitter, additional to acetylcholine. However, the mechanism whereby ATP is an additional transmitter in human DO has not been clarified. Furthermore, preliminary data suggest nerve-mediated ATP release is independent of acetylcholine release , which implies that therapeutic strategies may be devised to reduce selectively ATP release in overactive bladders. This study aimed to test the hypotheses: i) atropine-resistant contractions result from incomplete ATP breakdown in the nerve-muscle junction; ii) ATP and acetylcholine are preferentially released at different frequencies from motor nerves.
Human and guinea-pig detrusor was used. Human biopsies were obtained at open-surgery from patients with idiopathic (n=6; 50±15yr) or neuropathic (n=6; 34±10yr) detrusor overactivity (human DO), or those undergoing cystectomy with no DO symptoms (human stable, n=9; 57±14yr). NDO and IDO data were not significantly different in any variable and were merged. Patient ages of the merged DO and stable groups were not statistically different. Guinea-pig detrusor was obtained immediately after animals were euthanised.
Nerve-mediated contractions. Detrusor strips (<1mm diam, mucosa removed), superfused with Tyrode's solution at 37°C (pH 7.4) were field stimulated with 3-s trains (0.1 ms pulses) between 1 and 32 Hz; contractions were abolished by tetrodotoxin (1 µmol.l-1). Atropine (1 µmol.l-1), ABMA (1 µmol.l-1) or apyrase (10U.ml-1) were added to the superfusate as required.
Measurement of ecto-ATPase activity. Detrusor tissue homogenates (7 mg.ml-1) were added to Ca2+-free Tyrode's with initial [ATP] 0.2, 0.5, 1.0, 2.0 or 5.0 mmol.l-1. Two sets of tubes at each [ATP] were used, with the ecto-ATPase inhibitor ARL-67156 (100 µmol.l-1) added to one set. Aliquots were tested for [ATP] every two minutes with a luciferin-luciferase assay to obtain initial rate of breakdown, and the rate plotted as a function of [ATP]. Maximum ectoATPase activity, VATP,max and the Km value were calculated from a plot of ARL-dependent rate of ATP degradation as a function of initial [ATP].
Gene expression of ecto-ATPases. High-quality total RNA was isolated from detrusor tissue homogenate (RNeasy, Qiagen) and used to generate cDNA using reverse-transcription. cDNA was used as a template for qPCR reactions with selectively-designed primers for ectonucleoside triphosphate diphospho-hydrolase (ENTPD) -1, -2, -3, -5 (TaqMan, Applied Biosystems, UK). Gene expressions levels were normalised to 18S ribosomal RNA as an internal control.
Nerve-mediated ATP release. This was measured using amperometric ATP electrodes placed on the surface of detrusor preparations, with reference to a null electrode (Sarissa, UK).
Data presentation and analysis. All data are mean±SEM (n=number of preparations), all variables were measured at least three times in each preparation. Comparison of data sets used ANOVA with post hoc Bonferroni tests. The null hypothesis was rejected at p<0.05(*).
Atropine-resistance (AR) was absent in human stable detrusor, most in guinea-pig and intermediate in human DO. AR contractions were further abolished by ABMA. Reduction of contractions by the non-specific extracellular-acting ATPase apyrase showed the same trend (Table 1). The maximum ARL-67516 dependent ATPase activity (VATP,max) was greatest in human stable detrusor homogenates, least in guinea-pig tissue and intermediate in human OA detrusor (Table 1). The Km of ecto-ATPase activity was similar in all three groups.
For nerve-mediated contractions, the similarity of the AR proportion and that which is reduced by apyrase gives confirmatory evidence that both represent purinergic components of contraction. Figure 1A plots the relationship between maximum ecto-ATPase activity and either the percentage AR or reduction by apyrase; in both cases there was a significant inverse relationship.
ENTPdase-1 expression was significantly less in human OA detrusor compared to that from human stable bladders (Table 1). Expressions of ENTPDase-2, -3 and -5 were much less and no difference between stable and OA human (stable vs OA bladder x10-4/18S: ENTPDase-2; 0.035±0.008 vs 0.029±0.010: ENTPDase-3; 0.090±0.025 vs 0.047±0.018: ENTPDase-5, 0.11±0.018 vs 0.14±0.044).
Figure 1B shows ATP-transients associated with nerve-mediated contractions. However, the frequency-dependence of the two phenomena was different – the frequency for half-maximal response, f1/2, was lower for ATP-transients than for nerve-mediated contractions (figure 1B inset). Also f1/2 values for ATP release and AR contractions (2.5±0.4 vs 3.5±0.6 Hz, respectively) were not significantly different, consistent with AR contractions being due to ATP release.
The apyrase data provide more direct evidence that atropine-resistant contractions are due to ATP release. The inverse relation between the magnitude of ATP-dependent contractions and ecto-ATPase activity in the three experimental groups implies that atropine-resistance is due to incomplete breakdown of ATP at the nerve-muscle junction in human OA and guinea-pig detrusor, but there is complete breakdown in human detrusor from stable bladders. This was consistent with reduced expression of the main ecto-ATPase subtype (ENTPD-1) in human OA detrusor. Nerve-mediated release of ATP has been measured for the first time in detrusor, and one observation is that ATP is released preferentially at low frequencies and by inference acetylcholine (ACh) at higher frequencies.
A molecular explanation for atropine-resistant contractions associated with pathological bladders is provided by reduced expression of the major ecto-ATPase subtype. The different frequency-dependence of ATP and ACh release provides a targeted drug model to modulate the release of a transmitter (ATP) associated with detrusor overactivity in the human bladder.