Nerve-mediated contractions of detrusor smooth muscle are generated by adenosine triphosphate (ATP) and acetylcholine (ACh) release from efferent nerves. In humans, ACh is responsible for contractions in healthy, normal bladders, whereas ATP has an additional role in many overactive bladder pathologies [1]. Phosphodiesterase type 5 (PDE5) inhibitors like sildenafil alleviate lower urinary tract symptoms. However, the mechanism of action remains unclear, although it reduces purinergic-mediated contractions and inhibits nerve-mediated ATP release (dominant at low stimulation frequencies), whilst leaving ACh release intact (dominant at high stimulation frequencies) [2]. PDE5 inhibitors reduce cyclic guanosine monophosphate (cGMP) hydrolysis and prolong its activity. One route for cGMP formation is via activation of soluble guanylate cyclase (sGC) through generation of nitric oxide (NO). The aims of this study were to determine if agents that increase intracellular levels of cGMP, and associated downstream cellular signalling, influence detrusor contractility and nerve-mediated ATP release.

Bladders from male C57BL/6 mice (12 weeks) were used to prepare detrusor preparations. Upon electrical field stimulation (EFS; 0.1-ms pulses, 1-40 Hz, 3-s train every 90-s), ATP and ACh release were measured simultaneously (fmol.µlˉ¹.mg wet weightˉ¹) with tension (mN.mgˉ¹). Interventions were delivered via the superfusate. Force-frequency relations were generated, and the percentage reduction of force was plotted as a function of stimulation frequency. ATP was measured using a luciferin-luciferase assay, and ACh release was measured using a choline/ACh assay. Previous studies have demonstrated a significant relationship between the reduction of both tension and ATP release at low stimulation frequencies (e.g., 2 Hz), and this frequency is used to analyse specifically the effect of interventions on purinergic contractions without involvement of nerve-mediated ACh release, the latter measured here at 20 Hz stimulation [3]. Data are means ± SD and differences between data sets were tested with Student’s paired t-tests or repeated measures with ANOVA followed by parametric post-hoc tests where appropriate; 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 control and intervention set was based on a power calculation to reject the null hypothesis at P < 0.05 and a power of 80%, with a variance of data based on previous experimental data with these experiments [2, 3].

A sGC activator to induce cGMP synthesis, cinaciguat (10 µM, n=6), reduced nerve-mediated contractions at low frequencies but not high frequencies (Figure 1B-C): it also reduced ATP release across the frequency range (Figure 1D). At 2 Hz stimulation, cinaciguat reduced tension and ATP release by similar percentages (38.0 ± 5.6% and 38.3 ± 9.9%, respectively: Table 1). At 20 Hz stimulation, cinaciguat had no effect on tension (Figure 1C) or ACh release (112.5 ± 14.3 fmol.µlˉ¹.mgˉ¹ vs 108.0 ± 11.3 fmol.µlˉ¹.mgˉ¹: Figure 1E). Similarly, a cell-permeable analogue of cGMP, 8-Br-cGMP (1 and 10 µM, n=6), which directly activates protein kinase G (PKG), exerted a concentration-dependent reduction of force at low frequencies, and nerve-mediated ATP release (Table 1), mirroring the effects of cinaciguat and those previously reported with the PDE5 inhibitor, sildenafil [2]. A NO-donor, sodium nitroprusside (SNP, 10-1000 µM, n=6), to raise NO and cGMP levels, also mediated a concentration-dependent reduction of nerve-mediated tension and ATP release at 2 Hz stimulation (Table 1). Addition of PKG inhibitor, Rp-8-CPT-cGMPS (10 µM, n=6), blocked the effect of sildenafil (20 µM), cinaciguat (10 µM) and 8-Br-cGMP (10 µM) on nerve-mediated contractions and ATP release at 2 Hz stimulation (Table 1).
The sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-1]quinoxaline-1-one (ODQ, 10 µM, n=6), alone had no effect on contraction magnitude or neurotransmitter release, however the subsequent addition of cinaciguat (10 µM) in the presence of ODQ produced comparable reductions in contraction magnitude and ATP release as generated by cinaciguat alone (Table 1). The addition of sildenafil (20 µM) in the presence of ODQ (10 µM, n=6), reduced tension and ATP release at 2 Hz stimulation, but to a smaller extent compared to cinaciguat (Table 1), and when used in the absence of ODQ (Table 1 and [2]). The addition of sildenafil (20 µM), in the presence of cinaciguat (10 µM, n=6), further reduced nerve-mediated contractions and neuronal ATP release (Table 1).

Interventions designed to increase intracellular cGMP levels by: enhancing sGC activity with SNP or cinaciguat to increase its production; reducing its breakdown by PDE5 inhibition with sildenafil; or adding it directly to the intracellular pool with 8-Br-cGMP, all selectively reduced low frequency contractions and nerve-mediated ATP release. Oxidising sGC to an inactive form with ODQ did not hinder the ability of cinaciguat to enhance sGC activity and reduce nerve-mediated contractions and ATP release at low frequencies. However, ODQ did affect the ability of sildenafil to reduce low-frequency contractions and ATP release demonstrating a difference in mechanism of action between the PDE5 inhibitor and cinaciguat. This is further highlighted by the additive effects of sildenafil in the presence of cinaciguat, with the combination of cinaciguat and sildenafil having an augmented effect on nerve-mediated contractions and ATP release, in comparison to either drug alone. The intracellular target for cGMP is PKG, as its inhibition abolished the effects of cinaciguat, sildenafil and 8-Br-cGMP on nerve-mediated contractions and ATP release. Because ATP-dependent contractions are associated with those from overactive bladder pathologies, these offer a therapeutic target to selectively attenuate these contractions, whilst leaving physiological detrusor contractile function intact.

Elevation of cGMP by pathways involving NO, sGC, and PDE5, reduces ATP release from motor nerves supplying detrusor, and the downstream pathways have been characterised with involvement of PKG. This study demonstrates that differential regulation of transmitter release is possible at the detrusor-muscle junction. The ability to specifically attenuate ATP release, whilst leaving unaffected physiological contractile function through ACh release, offers a novel therapeutic target, as ATP is associated with pathological contractile function in the human bladder. Moreover, the additive effects of sildenafil and cinaciguat on tension reduction and ATP release suggests that a combination treatment of these agents may be more efficacious.

Bayliss M, Wu C, Newgreen D, Mundy AR, Fry CH. A quantitative study of atropine-resistant contractile responses in human detrusor smooth muscle, from stable, unstable and obstructed bladders. J Urol 1999.
Chakrabarty B, Ito H, Ximenes M, Nishikawa N, Vahabi B, Kanai AJ, Pickering AE, Drake MJ, Fry CH. Influence of sildenafil on the purinergic components of nerve-mediated and urothelial ATP release from the bladder of normal and spinal cord injured mice. Br J Pharmacol 2019.
Chakrabarty B, Drake MJ, Kanai AJ, Fry CH. Selective reduction of neurotransmitter release by cAMP-dependent pathways in mouse detrusor. Am J Physiol Regul Integr Comp Physiol 2022.

Continence 7S1 (2023) 100825
DOI: 10.1016/j.cont.2023.100825