Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is one of the most challenging urological diseases, due to its complex and unclear aetiology and the poor efficacy of current pharmacological treatment options. Phosphodiesterase 5 (PDE5) inhibitors have shown promising effects in the treatment of lower urinary tract symptoms. However, unwanted side effects have led many researchers to focus on alternative drugs that modulate the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signalling pathway. In the detrusor, NO causes cGMP accumulation and muscle relaxation by activation of the enzyme soluble guanylate cyclase (sGC). Oxidative stress conditions, for instance chronic inflammatory diseases like CP/CPPS, lead to the oxidation of sGC. In it oxidized state, NO is unable to activate sGC. However, sGC activators such as BAY 60-2770 can overcome this and activate the sGC enzyme in a NO independent manner [1].
The aim of the current study was to examine how innate bladder contractility as well as purinergic and cholinergic receptor expression is affected by induction of CP/CPPS and how this, in turn, is impacted by treatment with the sGC activator BAY 60-2770.

In the current study, 24 adult male Sprague-Dawley rats (350-500 g; Charles River Laboratories, Calco, Italy) were used. The animals were randomly divided into four groups (n=6 per group). In group 1 and 2, vehicle (10 µl sterile saline) was injected directly into the dorsal lobe of the prostate, serving as control. In group 3 and 4, rats were intraprostatically injected with zymosan (0.1 mg in 10 µl sterile saline), to create a functional model for CP/CPPS. Thereafter, the rats were allowed to recover for 7 days. On days 8-20, the rats were given daily subcutaneous injections with either dimethylsulfoxide (DMSO; 0.05 ml*day-1; serving as control; Gr1 & Gr3), or BAY 60-2770 (0.5 mg*kg-1*day-1; dissolved in 99.5% DMSO; Gr2 & Gr4). On day 21, the animals were euthanized, their bladder was excised, and ex vivo organ bath experiments were performed in which the contractile responses to electrical field stimulation (EFS), the cholinergic agonist methacholine (MeCh) and the purinergic agonist ATP were examined. Subsequently, the urinary bladder was examined immunohistochemically for purinergic (P2X1 & P2X3) and cholinergic (M3) receptor expression. 
Statistical calculations were performed using GraphPad Prism version 9.3.1 (GraphPad Software Inc., San Diego, USA). Two-way ANOVA followed by Bonferroni`s post-hoc test for multiple comparisons was used for statistical comparisons of organ bath data. Immunohistochemical findings were statistically compared using the Kruskal-Wallis test for non-parametric comparisons. Subsequently, Dunn`s multiple comparisons test was used to compare the difference in the sum of ranks between each group. Statistical significance was regarded for p-values < 0.05.

Induction of CP/CPPS did not significantly change the bladder contractile responses to EFS (Fig 1a) or MeCh (Fig 1b). However, induction of CP/CPPS significantly reduced ATP-induced bladder contractions, as compared to controls (Fig 1c). In tissues from animals in which CP/CPPS was induced, treatment with BAY 60-2770 led to significantly increased contractile bladder responses to EFS (Fig 2a) and MeCh (Fig 2b). Further, treatment with BAY 60-2770 restored alterations in ATP-induced bladder contractions caused by induction of CP/CPPS (Fig 2c). There were no significant differences between the groups regarding purinergic (P2X1 & P2X3) or cholinergic (M3) receptor expression in the bladder urothelium or detrusor.

The current study demonstrated that induction of chronic inflammation in the prostate led to reduced ATP-induced isolated bladder contractions. This finding is consistent with previous in vivo studies on cross-sensitization between the prostate and bladder and the potential effects of CP/CPPS on bladder function [2]. The present study demonstrated no significant differences between the groups regarding purinoceptor (P2X1 and P2X3) expression in the bladder. Thus, these findings indicate that induction of prostatitis leads to alterations in bladder purinoceptor sensitivity and/or down-stream purinergic signaling. The currently demonstrated effect of chronic prostate inflammation on ATP-induced bladder contractions may be of particular interest since it has previously been shown that ATP can act as a modulator of release of other neurotransmitters that have a role in bladder contractility and afferent nerve activity [3]. 
It has previously been shown that induction of chronic prostate inflammation can lead to reduced MeCh-induced bladder contractions [2]. However, in the current study, the induction of chronic prostatitis did not lead to significant changes in muscarinic receptor-induced bladder contractions. The discordance between the current study and previous studies could be explained by the fact that previous studies had an in vivo set up while the current study utilized an ex vivo organ bath setup to examine receptor-induced bladder contractile responses. The current study was purposely designed to examine the potential effects of CP/CPPS on muscarinic and purinergic contractile responses in the isolated bladder. In an in vivo set up, the alterations in bladder function could also be affected by possible changes in prostate or urethral contractility as well as in afferent and efferent nerve signaling. Further, the present study aimed to examine the effects of sGC activators, which was not investigated in vivo. 
The current study showed that after induction of chronic prostate inflammation, treatment with the sGC activator BAY 60-2770 led to increased EFS- and MeCh-induced bladder contractions. Likewise, treatment with BAY 60-2770 led to increased purinergic contractile responses. However, the purinergic responses are small in magnitude in comparison to the cholinergic responses. The observed alterations did not appear to be due to receptor up-regulation since there were no differences between the groups regarding receptor expression. Considering this, the increase in EFS-induced bladder contractions after BAY- 60-2770 treatment observed in animals in which chronic prostatitis was induced is likely due to changes in muscarinic receptor sensitivity and/or down-stream cholinergic signaling.

To the best of our knowledge, the current study is the first that examines how contractile properties of the isolated detrusor are affected by induction of CP/CPPS and a subsequent treatment period with a sGC activator. Induction of CP/CPPS led to reduced ATP-induced bladder contractions. This alteration was restored by treatment with the sGC activator BAY 60-2770. Even though further studies are required to better understand the mechanisms behind BAY 60-2770’s effects on purinergic and cholinergic responses in the cross-sensitized bladder, the current findings indicate that drugs targeting the NO/cGMP pathway might be a promising option to restore alterations in bladder function due to CP/CPPS.

Füllhase, C., et al., Reduction of obstruction related bladder overactivity by the guanylyl cyclase modulators BAY 41-2272 and BAY 60-2770 alone or in combination with a phosphodiesterase type 5 inhibitor. Neurourol Urodyn, 2015. 34(8): p. 787-93.
Aydogdu, O., et al., Prostate-to-bladder cross-sensitization in a model of zymosan-induced chronic pelvic pain syndrome in rats. Prostate, 2021. 81(4): p. 252-260.
Silva, I., et al., Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction. J Urol, 2015. 194(4): p. 1146-54.

Continence 2S2 (2022) 100213
DOI: 10.1016/j.cont.2022.100213