Soluble Guanylate Cyclase Activator, BAY 58-2667, Decreases Neurogenic Detrusor Overactivity and Relaxes Detrusor Smooth Muscle in Mice with Radiation Cystitis

Ikeda Y1, Zabbarova I1, Drake M2, Fry C2, Birder L1, Kanai A1

Research Type

Basic Science / Translational

Abstract Category

Pharmacology

Abstract 393
E-Poster 2
Scientific Open Discussion ePoster Session 18
Thursday 5th September 2019
13:50 - 13:55 (ePoster Station 10)
Exhibition Hall
Animal Study Basic Science Pathophysiology Pre-Clinical testing Pharmacology
1.University of Pittsburgh, 2.University of Bristol
Presenter
Y

Youko Ikeda

Links

Poster

Abstract

Hypothesis / aims of study
The role of nitric oxide (NO•) signalling in the urinary bladder is incompletely understood.  It has been demonstrated that NO• mediates relaxation of the smooth muscle in the bladder neck and urethra [1], and that the detrusor is relaxed by stimulation of β3-adrenergic receptors in humans and rats and β2 receptors in mice.  However, there is also robust expression of soluble guanylate cyclase (sGC) and NO•-induced cyclic guanosine monophosphate (cGMP) in afferent nerves, urothelial cells, myofibroblasts and vascular smooth muscle in the bladder.  There have been mixed reports on the relaxation effect of NO• in detrusor smooth muscle, however, there is still the potential for a functional effect as conditional knockdown of smooth muscle sGC and cGMP generation by 90% can still elicit NO•-mediated relaxation [2]. Therefore, it could be hypothesized that the detrusor may express sGC and cGMP at levels below detection but still be influenced by NO•. The efficacy of phosphodiesterase type-5 (PDE5) inhibitors (e.g., sildenafil/Viagra) in reducing overactive bladder symptoms has been demonstrated.  However, in situation where there is nitrergic nerve damage or chronic oxidative stress conditions, there may be little to no NO• available to activate sGC, thus, rendering PDE5 inhibitors ineffective.  As sGC activators do not require NO• and stimulates sGC with oxidized heme (or even in the absence of haem), sGC activators such as BAY 58-2667 may be able to restore cGMP to physiological levels. There is evidence that BAY 58-2667 ameliorates cyclophosphamide induced bladder overactivity [3] however, the sites of action were not fully elucidated.  The aim of our study was to investigate the acute effects of BAY 58-2667 on the bladder afferent activity and detrusor smooth muscle in mice with irradiation-induced cystitis; a chronic inflammatory condition.
Study design, materials and methods
Selective bladder irradiation: Adult female C57BL/6 mice were anesthetized with avertin (2,2,2-tribromoethanol, 300 mg/kg via intraperitoneal injection) and a lower midline incision was made into the abdomen.  Mice had their urinary bladders externalized and selectively exposed to a collimated beam of ionizing irradiation (10 Gy, 320 KV x-ray irradiator) to prevent exposure to other pelvic structures and prevent cross-sensitization.  The surgical wound was sutured, and mice were allowed to recover on prophylactic antibiotics and analgesics.  
In vitro single-unit bladder afferent nerve recordings: Three days following irradiation, mice were euthanized and bladders with associated spinal (L6-S2) nerves were dissected. The preparation was placed in a tension recording chamber with oxygenated Krebs solution for recording bladder wall tension and the spinal nerves passed into adjacent oil chambers for afferent nerve recordings.  Mechanosensitive afferent firing was elicited in response to varying stretches applied through a computer-controlled stepper motor in line with the tension transducer.   Experiments were carried out on n ≥ 4 mice.  Unpaired student t-test determined differences between irradiated versus control groups or parameters before and after treatment.
Results
In vitro afferent recordings were obtained using mouse bladder preparations where nerve firing was elicited in response to stepper-motor controlled stretch.  Afferent recordings from irradiated mouse bladders exhibited spontaneous discharges not associated with changes in bladder tension.  Spontaneous firing was not observed in non-irradiated mouse bladders.  The addition of BAY 58-2667 (0.01-1 μM) to the perifusate dose-dependently dampened afferent activity, but at higher concentrations, significantly decreased stretch-induced tension changes (Figure 1).
Interpretation of results
At three days post-bladder irradiation, there was increased sensitivity of afferent nerves and the evidence of spontaneous firing.  There was no indication of spontaneous detrusor contractions as a result of irradiation at the time point examined.  The decrease of spontaneous and stretch-induced afferent firing as well as baseline tension in vitro demonstrates the direct action of BAY 58-2667 on afferent nerves and detrusor smooth muscle.
Concluding message
sGC is highly expressed in the bladder neck and urethra where it is responsible for relaxation, whereas detrusor smooth muscle relaxes in response to β-adrenergic receptor stimulation.  Our data demonstrate that activation of sGC in the urinary bladder wall decreases afferent excitability and relaxes detrusor smooth muscle via a mechanism yet to be determined.  These data support the therapeutic potential of sGC activator for bladder afferent sensitization and detrusor overactivity.
Figure 1 Figure 1. Acute application of BAY 58-26687 decreases neurogenic detrusor overactivity and relaxes detrusor smooth muscle in mice with radiation cystitis.
References
  1. Andersson KE., Int Neurourol J, 21(1):6-11, 2017
  2. Groneburg D et al., Circulation, 121:401-409, 2010
  3. de Oliveir M et al., AJP-Renal, 311(1):F85-93, 2016
Disclosures
Funding Awards from NIH/NIDDK; R01 DK071085 (Kanai), R01 DK098361 (Kanai and Drake), P01 DK093424 (Kanai) and Department of defense SC170171 (Kanai and Ikeda) Clinical Trial No Subjects Animal Species mouse Ethics Committee University of Pittsburgh Institutional Animal Care and Use Committee