Hypothesis / aims of study
Among the non-adrenergic, non-cholinergic (NANC) signaling molecules that regulate bladder function, nitric oxide (NO) is perhaps the most elusive and ambiguous. We have previously reported on a novel method to study nitrergic responses in a direct manner in an organ bath setup [1]. By utilizing NO in aqueous solution, we demonstrated concentration-dependent relaxatory responses in both healthy and inflamed isolated bladder strips. Further, we could show that this can be attenuated by oxidizing the presumed target of NO, soluble guanylate cyclase (sGC). In the current study, a similar methodology was utilized to unravel the importance of an intact urothelium for nitrergic signaling in the isolated rat urinary bladder. In addition to aqueous NO, the relaxatory responses to a sGC activator, BAY 60-2770, were also examined. While NO mainly acts by binding to sGC in its reduced state, BAY 60-2770 is thought to bind to sGC in its oxidized state (i.e. when the heme group is oxidized). When searching for potential pharmacological possibilities to affect nitrergic signaling, sGC activators are of particular interest. This is partly due to their longer duration of action, but perhaps mainly due to their proposed mechanism of action which allows them to carry out NO-like functions also in a state of oxidative stress, such as during inflammation.
The aim of the current study was to (a) examine the role of the intact urothelium in nitrergic signaling and (b) assess if this is altered in the state of inflammation.
Study design, materials and methods
Currently, isolated rat bladder relaxatory responses to NO in aqueous solution and to the sGC activator BAY 60-2770 were examined in an organ bath setup. The oxygen-free aqueous solution with a distinct concentration of NO was produced as previously reported [1, 2]. This allows for NO to be added to an organ bath at specific concentrations, similar to any classic agonist.
In total, 32 adult male Sprague-Dawley rats (240-370 g; Charles River Laboratories, Calco, Italy) were used for this purpose. The rats were randomly divided into two groups, receiving an intraperitoneal injection with either vehicle (sterile saline; 1 mL/kg), serving as control, or cyclophosphamide (CYP; 100 mg/kg in sterile saline), in order to induce experimental cystitis. At the peak inflammatory timepoint, 60 h post injection, the rats were anaesthetized with pentobarbital and the bladder was filled with either saline or collagenase type I (0.1% w/v in sterile saline). The urethra was ligated, and the injected solution was left in the bladder for 30 mins. Subsequently, the bladder was emptied, the animals were euthanized and the bladders were excised. The bladders were cut open from the urethral opening to the apex and the inner surface was gently brushed with a q-tip to remove loose urothelial cells. From each bladder, two full-thickness strips (2 x 6 mm) were mounted in an organ bath. After a recovery period (45 mins), relaxatory responses to NO in aqueous solution (4-40 μM) and to the sGC activator BAY 60-2770 (5 x 10-10 – 5 x 10-5 M) were examined in the absence and presence of the sGC oxidizing agent ODQ (2.5 x 10-5 M). To allow for studies of relaxatory responses, the tissues were precontracted with methacholine (1 x 10-6 – 1 x 10-5 M). Viability was assessed by adding high K+ Krebs solution (124 mM) at the beginning and end of each experiment.
Statistical calculations were performed using GraphPad Prism version 10.1.2 (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. Statistical significance was regarded for p-values < 0.05. Relaxatory responses are shown as percentage (%) of precontraction. Data are presented as mean ± SEM.
Results
In healthy animals, removal of the urothelium did not cause any alterations in relaxatory responses to neither NO nor BAY 60-2770 (Fig 1a, b; Fig 2a, b), regardless of the absence or presence of ODQ. However, in tissues from animals with CYP-induced cystitis, removal of the urothelium led to significantly decreased relaxatory responses to NO in the absence of ODQ (Fig 1c; p = 0.040 and 0.025 at 2 x 10-5 and 4 x 10-5 M, respectively). The presence of ODQ led to lower relaxatory responses to NO overall (Fig 1b, d), and abolished any significant effects of urothelial denudation (Fig 1d).
The responses to BAY 60-2770 were higher in the presence of ODQ, but only in inflamed tissues (Fig 2). Removal of the urothelium led to significantly lower responses to BAY 60-2770 in inflamed tissues, both in the absence and presence of ODQ (Fig 2c, d; p = 0.012 and 0.020, respectively, at 1 x 10-5 M).
Interpretation of results
NO in aqueous solution, and the sGC activator BAY 60-2770, induced dose-dependent relaxatory responses in both the healthy and inflamed bladder. The current data demonstrate that the urothelium plays no, or a very small, role in nitrergic relaxatory responses in the isolated healthy rat urinary bladder. Conversely, in a state of inflammation, the intact urothelium seems to play a significant role in nitrergic bladder relaxation. Tentatively, in the healthy bladder, sGC is expressed mainly in the detrusor, since removal of the urothelium did not alter NO- or BAY 60-2770 induced relaxatory responses. However, in a state of inflammation, sGC may be expressed also in the urothelium, contributing to the currently observed relaxatory responses. Further, since removal of the urothelium attenuated both NO- and BAY 60-2770 induced responses in the inflamed bladder, urothelial sGC seems to be present in a physiologically relevant amount in both its reduced and oxidized state.
The methodology, including production and use of aqueous NO, is currently verified by the similar responses to NO and BAY 60-2770 as were previously reported [1]. As expected, oxidation of sGC with ODQ attenuated NO-induced relaxatory responses, but augmented BAY 60-2770 induced relaxation. The data thus also support the assumed mechanisms of action, that BAY 60-2770 acts by binding to and activating sGC in its oxidized state while NO carries out its actions via reduced sGC.
It should be noted that the current data is gathered from isolated tissues and therefore may not entirely mimic bladder nitrergic signaling in vivo. Several reports have indicated an important role for NO in the nervous regulation of the bladder, specifically regarding afferent signaling. However, the current report demonstrates the possibility of NO acting directly on the detrusor. Thus, the necessary machinery for bladder nitrergic relaxatory responses is present in the isolated detrusor. Even though the involvement of sGC has been established, the possibility of other pathways, and other signaling molecules, being involved still remains.