Hypothesis / aims of study
A sensation of bladder fullness is relayed by afferent nerves that originate in the mucosal layer. These can be activated by the release of chemical mediators from the urothelium, such as adenosine triphosphate (ATP), when the mucosa is stretched as the bladder fills. Increased stretch-activated ATP release has been noted in studies of human bladder disease as well as in animal models of bladder pathology, including interstitial cystitis, urinary urgency and incontinence, spinal cord injury-induced bladder dysfunction, detrusor overactivity, and bladder outlet obstruction [1]. Previous studies showed that modulating cyclic nucleotides, increasing cyclic guanosine monophosphate (cGMP) or decreasing cyclic adenosine monophosphate (cAMP), can reduce neuronal ATP release in the mouse bladder [2]. The aim of this study was to determine if these modulators can also reduce urothelial ATP release. Targeting purinergic sensory pathways may provide a potential drug target as these pathways are implicated in the pathogenesis of bladder dysfunction.
Study design, materials and methods
Male C57BL/6 mice were killed by CO2 asphyxiation and cervical dislocation. Bladders were excised, the mucosa dissected off and incubated at 37°C with trypsin-EDTA (0.5 g/l trypsin, 0.2 g/l EDTA) in phosphate-buffered solution for 90 minutes. Urothelial cells were released by gentle trituration, washed with gassed (5% CO2, 95% O2) Tyrode’s solution and a sample taken for cell counting, using an improved Neubauer haemocytometer stage, and their ability to exclude Trypan Blue (0.4% solution) as a viability assay. Samples (300 µl) of the cell suspension were then incubated in Eppendorf tubes at a known density for at least 90 minutes after isolation. Cell suspensions were incubated with interventions and baseline ATP was measured from a 100 µl sample, after which a series of 50 hydraulic shear loads, each of 0.1 kPa (≈1 cm H2O), was imposed over 30 seconds. The amount of ATP release was then measured from a further sample at four minutes after the mechanical stimulus. To determine the fraction of ATP release to total available cellular ATP, cells were permeabilised with 1% Triton to release remaining intracellular ATP, which was finally measured in a further sample. This allowed calculation of total releasable ATP (ATP release with shear stress + Triton). ATP release (pmoles/10^3 cells) was measured using a calibrated luciferin-luciferase assay. Data are means ± SD and differences between data sets were tested with Student’s paired t-tests; the null hypothesis was rejected at P<0.05. n values refer to the number of preparations, one each from separate animals. The number of repeats in each data set was based on a power calculation to reject the null hypothesis at P<0.05 and a power of 80%, with a data variance based on previous experiments.
Results
Addition of cinaciguat (10 µM, n = 5), a soluble guanylate cyclase (sGC) activator to induce cGMP synthesis, reduced shear force-activated ATP release from 5.2 ± 1.7 pmoles/10^3 cells in control to 3.0 ± 0.7 pmoles/10^3 cells (Figure 1A; P<0.05). These values represented 23.0 ± 7.5% and 19.1 ± 4.9% of the total releasable intracellular ATP pool, respectively.
Addition of sildenafil (20 µM, n = 5), a phosphodiesterase type 5 (PDE5) inhibitor which reduces the breakdown of cGMP, thus prolonging its activity, had a similar action, to reduce shear force-activated ATP release from 5.2 ± 1.7 pmoles/10^3 cells to 3.0 ± 1.2 pmoles/10^3 cells (Figure 1A; P<0.05). These values represented 23.0 ± 7.5% and 19.7 ± 5.2% of the total releasable intracellular ATP pool, respectively. Control experiments showed that DMSO (dimethylsulphoxide, the solvent for cinaciguat and sildenafil) had no effect on the magnitude of shear force-activated ATP release relative to control (Figure 1A; P>0.05).
The protein kinase G (PKG) inhibitor, Rp-8-CPT-cGMPS (10 µM, n = 4), abolished the ability of cinaciguat and sildenafil to reduce shear force-activated ATP release from urothelial cells (Figure 1B). In the presence of cinaciguat and Rp-8-CPT-cGMPS, shear force-activated ATP release was 4.7 ± 0.3 pmoles/10^3 cells and was not significantly different from control (in the absence of both agents); 5.1 ± 0.2 pmoles/10^3 cells (P>0.05). Corresponding values for sildenafil with Rp-8-CPT-cGMPS and control were 4.6 ± 0.3 pmoles/10^3 cells and 5.1 ± 0.2 pmoles/10^3 cells (P>0.05). Rp-8-CPT-cGMPS alone had no significant effect on ATP release (Figure 1B; P>0.05 vs control).
Addition of adenosine (1 mM, n = 6) also reduced shear force-activated ATP release from urothelial cells: from 5.2 ± 1.5 pmoles/10^3 cells in control to 2.3 ± 0.9 pmoles/10^3 cells (Figure 1C; P<0.001). These values represented 29.8 ± 3.1% and 31.7 ± 7.7% of the total releasable intracellular ATP pool, respectively. The adenosine A1 receptor antagonist (A1R), DPCPX (1 µM, n = 6), when added alone, increased shear force-activated ATP release from 5.2 ± 1.5 pmoles/10^3 cells in control to 8.2 ± 1.8 pmoles/10^3 cells (Figure 1C; P<0.01). Moreover, DPCPX reversed the reduction of stretch-activated ATP release by adenosine, to a value similar to that induced by the antagonist alone (Figure 1C: 8.1 ± 1.6 pmoles/10^3 cells; P>0.05).
Interpretation of results
Imposition of hydraulic shear forces on freshly isolated urothelial cells, of a magnitude similar to those in the bladder during filling, generated ATP release. This is of relevance as ATP, acting on P2X receptors, has been implicated in contributing to drive bladder afferent activity during filling [3]. Interventions designed to modulate intracellular cyclic nucleotides (cGMP and cAMP) levels altered shear-force induced ATP release and thus could regulate bladder wall afferent activity.
A sGC activator (cinaciguat) and a PDE5 inhibitor (sildenafil), both increasing cGMP levels, reduced shear force-induced ATP release. This action was absent in the presence of Rp-8-CPT-cGMPS, a PKG inhibitor, implying the sGC-PKG pathway is relevant to generate new cGMP.
Adenosine acting at A1R (antagonised by DPCPX) is implicated in regulating neuronal ATP release from parasympathetic motor fibres to the bladder wall [2]. One consequence of A1R activation is to reduce cAMP generation and subsequent protein kinase A activity. Adenosine reduced shear-force induced ATP release from urothelial cells, an effect reversed by DPCPX and consistent with an A1R-mediated effect. Moreover, DPCPX alone, or in the presence of adenosine, significantly augmented ATP release and one possibility is that adenosine is continually cleared from urothelial cells, exerting a significant autocrine effect.