Hypothesis / aims of study
The role of nitric oxide (NO•) signalling in the urinary bladder is not well understood. NO•-mediated relaxation was predominantly demonstrated in bladder neck and urethra, however, robust expression of soluble Guanylate Cyclase (sGC) and NO•-induced cyclic guanosine monophosphate (cGMP) in the urothelium and vascular endothelium/smooth muscle and the efficacy of phosphodiesterase type-5 (PDE5) inhibitors on bladder overactivity symptoms suggest additional sites of action. Since NO• activates sGC with a reduced heme iron (Fe2+) only, NO•-mediated cGMP production is abolished when the oxidation of heme iron is accelerated under oxidative stress conditions [1]. Cytochrome b5 reductase-3 (CyB5R3) has been demonstrated to be a key enzyme to maintain sGC heme in the reduced state [2], however, it can be also downregulated in the condition of oxidative stress. sGC activators, developed as treatment for pulmonary hypertension and fibrosis, can act on oxidized sGC and do not require NO• to produce cGMP. To access the hypothesis that impaired sGC signalling may adversely affect bladder function, we have studied conditional CyB5R3 smooth muscle knockout mice and the effect of sGC activator, BAY 58 2667, on bladder function in these knockout and background control mice.
Study design, materials and methods
Male four to six week old CyB5R3 smooth muscle conditional knockout and their background/age control mice were decerebrated for anesthetic-free cystometrogram (CMG) and external urethral sphincter electromyogram (EMG) recordings. BAY 58 2667 (3 mg/kg), BAY 63-2521 (3-10 mg/kg) and sildenafil (1 mg/kg) were injected intraperitoneally and their effects recorded for an hour after injection. Experiments were carried out on n ≥ 4 mice. Unpaired student t-test determined differences between knockout versus background control groups or parameters before and after treatment.
Results
Background control mouse CMG and EUS-EMG recordings demonstrate long intercontractile intervals (ICI) and low filling pressures along with EUS bursting and reduction of tonic sphincter activity during voiding (Figure 1A). Conditional smooth muscle CyB5R3 knockout mice exhibit non-voiding contractions, shortened ICI, decreased bladder compliance and voiding efficiency compared to controls (Figure 1B). BAY 58 2667 injection significantly increased ICI, voided volumes and bladder compliance and abolished non-voiding contractions in CyB5R3 knockout mice (Figure 1C). In control mice, it increased ICI and voiding volumes, however, did not significantly affect the compliance (see Table 1). There was no significant difference in external urethral sphincter activity recordings in control versus knockout mice before and after the treatment. BAY 63-2521 and sildenafil did not have any significant effect on bladder function in CyB5R3 knockout mice (not shown).
Interpretation of results
Our results demonstrate that smooth muscle specific conditional knockout mice for CyB5R3 have the symptoms of bladder overactivity including shortened ICI and non-voiding contractions. sGC activator, BAY 58 2667, acting through an NO-independent pathway, improves bladder function in these mice.
Concluding message
While the NO•-sGC signalling pathway is known to be involved in relaxation of the bladder neck, our finding supports a role for this pathway in detrusor function. Impaired sGC signalling, due to the knockout of CyB5R3, adversely affects bladder function resulting in non-voiding contractions and shortened ICI. While these symptoms were unresponsive to exogenous NO•, PDE5 inhibitors or sGC stimulators, they were ameliorated following administration of sGC activators.