Phosphodiesterase type 5 (PDE5) inhibitors, used clinically to treat erectile dysfunction, reduce cGMP hydrolysis and thus elevate intracellular levels. One route for cyclic guanosine monophosphate (cGMP) formation is via activation of soluble guanylate cyclases (sGCs) through generation of nitric oxide (NO). PDE5 inhibitors such as sildenafil also alleviate lower urinary tract symptoms, however the mechanism of action remains unclear, although it reduces purinergic-mediated contractions and inhibits nerve-mediated ATP release [1]. The aims of the study were: i) to determine if agents that increase intracellular levels of cGMP influence detrusor contractility and nerve-mediated ATP release and ii) if the NO/cGMP pathway has a role in detrusor.

Young (12 weeks) male C57BL/6 mice were used. Mice were euthanised and the bladder was removed through a midline laparotomy. Bladder strips from the dome (detrusor + mucosa) were tied to isometric force transducers in a horizontal chamber superfused with Tyrode’s solution at 37°C. Nerve-mediated contractions were generated by electrical field stimulation (EFS: 0.1ms pulses, 1-40 Hz, 3-s train every 90-s), and drug interventions were delivered via the superfusate and the effect on nerve-mediated contractions measured. Tension amplitude (mN) was normalised for strip weight (mN.mg-1). Nerve-mediated amplitude plotted as a function of stimulation frequency was fitted to an equation to generate force-frequency relationships to determine maximum tension (Tmax, mN.mg-1). Nerve-mediated ATP release was measured, over a range of frequencies from 1-40 Hz, from a 100 µl superfusate sample taken from the same site close to the preparation using a luciferin-luciferase assay, data are fmoles ATP per µl superfusate sample. Data are means ± SD, n=number of preparations, one each from separate animals and were n=6 in all experiments. Differences between data sets were subject to a repeated measures two-way ANOVA followed by a parametric post hoc tests, or a Student’s paired t-test; the null hypothesis was rejected at p<0.05. 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 variance of data based on previous data with these methods.

A cell-permeable analogue of cGMP, 8-bromo-cGMP (8-Br-cGMP, 1μM), was used to directly increase cGMP levels. Nerve-mediated contractions were significantly decreased (two-way ANOVA, p<0.001), and nerve-mediated ATP release was also reduced (p<0.001) – Table 1.  These effects were mirrored by addition of sildenafil (20 µM) which had a larger effect on nerve-mediated contractions and almost abolished ATP release. 
By contrast, manipulation of a putative NO/sGC pathway had none or very small effects on nerve-mediated contractions or ATP release.  Two inhibitors of enzymes in the pathway were used that if active would reduce NO and hence cGMP production. The NO-synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 10 μM) had no effect on contractions or ATP release.  The sGC inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10μM) also had no significant effects on either nerve-mediated contractions or ATP release.
Finally, a NO-donor, Na nitroprusside (SNP, 10 µM) was used that would in principle raise NO and cGMP levels.  SNP also had no significant effect on nerve-mediated contractions although there was a small reduction of nerve-mediated ATP release.

Sildenafil, a PDE5 inhibitor, and addition of a cell permeable cGMP analogue, 8-Br-cGMP both resulted in a decrease of nerve-mediated contraction and ATP release.  Sildenafil had a greater proportional effect compared to 8-Br-cGMP and the actual increase of cGMP generated by these interventions remains to be established.  
By contrast, inhibition of putative enzymes in the NO/cGMP pathway – NO-synthase and sGC, had no effect on nerve-mediated contractions and ATP release and it may be concluded that this route to synthesise cGMP was not active under the conditions of these experiments.  Direct addition of NO through a donor, Na-nitroprusside, also had no action on tension but did reduce nerve-mediated ATP release and this mechanism remains to be elucidated.

The data are consistent with the hypothesis that an increase of cGMP levels in detrusor preparations reduces force and nerve-mediated ATP release.  It remains to be established if a rise of cGMP in efferent nerve endings is solely responsible for these phenomena. However, the involvement of an NOS/NO/sGC pathway to mediate these effects does not seem to be central to these findings. The data open the possibility of a new target to regulate detrusor contractions, namely efferent nerve endings to detrusor.

Chakrabarty B, Ito H, Ximenes, M, Nishikawa N, Vahabi B, Kanai AJ, Pickering AE, Drake MJ, Fry CH (2019). 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.