Lower urinary tract symptoms (LUTS) associated with bladder diseases such as overactive bladder and neurogenic bladder dysfunction, are highly prevalent and have a severe effect on the quality of life of patients. Current treatments for LUTS have recognised limitations, including uncertain efficacy and adverse effects. PDE type 5 (PDE5) inhibitors like sildenafil (Viagra ®) have been found to alleviate LUTS; however, a complete understanding of its actions on peripheral control of bladder contractility remains unclear. Previously, we have demonstrated that sildenafil reduces low-frequency, purinergic mediated contractions and selectively inhibits neuronal ATP release in vitro [1]. In this study, the effects of sildenafil on nerve-mediated contractions of the whole mouse bladder in situ were characterised and compared to nerve-mediated contractions of the isolated mouse bladder in vitro. A comprehensive description of how sildenafil reduces contractile function should give insight into the pathology of bladder disease and may suggest a potential therapeutic mechanism of PDE5 inhibitors in their treatment.

Young (12 weeks old) female mice were administered heparin (50 IU, i.p.) and deeply anaesthetised with isoflurane (2%) until loss of paw withdrawal reflex for the following non-recovery procedure. The procedure has been modified from previously described methods for the use of in situ arterially-perfused mouse preparations [2]. The brain was removed, and the spinal cord was pithed with a blunt wire before arterial perfusion of the preparation. The ureters were ligated bilaterally to prevent natural bladder filling, the urethra was clamped, and a catheter was used to record bladder pressure and maintain isovolumetric conditions in the bladder lumen. A glass suction electrode was used to stimulate the left pelvic nerve (0.1 ms pulses, 1-24 Hz, 3-s train every 90-s; Figure 1A), once it has been identified and dissected. The amplitude of bladder pressure movements (mmHg) was analysed, and drug interventions were delivered arterially. Female mice were used due to difficulties in successfully dissecting and stimulating pelvic nerve in male mice. To investigate contractions in vitro, bladders were removed from similarly-aged mice and detrusor strips were tied to an isometric force transducer in a horizonal superfusion chamber. Nerve-mediated contractions (tetrodotoxin-sensitive) were generated by electrical field stimulation (EFS: protocol as for pelvic nerve stimulation), and drug interventions were delivered via the superfusate. Contraction amplitude was normalised for tissue preparation weight (mN.mg-1). Frequency-relationships were used to determine maximum pressure/tension (Pmax / Tmax), the frequency to attain Pmax/2 or Tmax/2 (f1/2, Hz), and the ratio at low and near maximum frequencies (P2/20 / T2/20). Data are means ± SD, n=number of experiments. Differences between data sets were analysed by ANOVA; the null hypothesis was rejected at p<0.05.

Preganglionic pelvic nerve stimulation in situ generated frequency-dependent intravesical bladder pressure transients in the bladder (n=6). Sildenafil (20 μM) reduced these pressure transients (Figure 1B) from: 2.26 ± 0.59 mmHg to 1.48 ± 0.51 mmHg at 2 Hz stimulation; 5.46 ± 1.28 mmHg to 3.57 ± 0.92 mmHg at 8 Hz stimulation; 7.01 ± 1.69 mmHg to 5.39 ± 1.21 mmHg at 20 Hz stimulation (p<0.05). The addition of sildenafil had no effect on the Pmax from 7.04 ± 3.41 mmHg to 5.66 ± 1.21 mmHg, but increased the f1/2 from 3.41 ± 0.37 to 5.21 ±  0.48 (p<0.05). Similarly, sildenafil (20 μM) reduced the amplitude of EFS-induced contractions in vitro (n=8, Figure 1C) from: 0.53 ± 0.12 mN/mg to 0.17 ± 0.04 mN/mg at 2 Hz stimulation; 1.26 ± 0.25 mN/mg to 0.59 ± 0.13 mN/mg at 8 Hz stimulation; 1.56 ± 0.31 mN/mg to 1.05 ± 0.25 mN/mg at 20 Hz stimulation (p<0.05). The addition of sildenafil had no effect on the Tmax from 1.58 ± 0.21 mN/mg to 1.30 ± 0.27 mN/mg, and increased the f1/2 from 3.37 ± 0.47 to 7.56 ± 0.92 (p<0.05).

Figure 1. (A) Schematic diagram illustrating the arterially-perfused in situ mouse model used for preganglionic pelvic nerve stimulation. (B) Pelvic nerve stimulation in situ; the effect of sildenafil (20 μM) on pressure transient magnitudes at frequencies between 1-24 Hz. (C) Nerve-mediated stimulation of isolated detrusor preparations; the effect of sildenafil (20 μM) on contraction magnitudes at frequencies between 1-24 Hz.

Parasympathetic preganglionic axons in the pelvic nerve provide the principal excitatory pathway to post-ganglionic fibres in the pelvic ganglia which ultimately result in co-ordinated detrusor smooth muscle contraction that causes voiding. In the current study, the preganglionic pelvic nerve was identified in an arterially-perfused whole mouse in situ model. Stimulation of the pelvic nerve resulted in a frequency-dependent increase of bladder pressure comparable to what is observed in in vitro contractility studies using detrusor muscle strips. Sildenafil reduced nerve-mediated bladder contractions in situ, with an increase in the f1/2 value, similar to observations in vitro. Sildenafil reduced the magnitude of pre- and post-ganglionic stimulated contractions in the mouse bladder, by similar proportions which presumes the ganglia are simple relays and do not regulate the frequency-dependence of nervous excitation to the bladder. Nerve-mediated contractions are dominated by ATP release at low frequencies, and ACh release at higher frequencies [3]. An increase in the f1/2 value further supports the predominant effect of sildenafil at the low frequency, purinergic component of nerve-mediated contractions [1]. The cellular pathways that mediate this action of sildenafil are undergoing characterisation.

Similarities between the whole bladder in situ preparation and in vitro contraction experiments further validate the translation of stimulating postganglionic nerve terminals as a model to study the dependence of bladder contraction on pelvic nerve stimulation. The description of the parasympathetic ganglia as simple one-to-one relays simplifies our understanding of the excitatory pathway. Sildenafil reduces preganglionic pelvic nerve-stimulated whole bladder contractions in situ and postganglionic nerve-mediated contractions in vitro, demonstrating an action of sildenafil at postganglionic nerve-terminals innervating detrusor smooth muscle principally by reducing nerve-mediated ATP release at postganglionic fibres.

CHAKRABARTY, B., ITO, H., XIMENES, M., VAHABI, B., PICKERING, T., KANAI, A., DRAKE M., FRY, C. 2017. The effects of sildenafil on bladder contractility and neuronal ATP release in the mouse urinary bladder. Podium short oral, 47th Annual meeting of International Continence Society (ICS) 2017 in Florence.
ITO, H., DRAKE, M. J., FRY, C. H., KANAI, A. J. & PICKERING, A. E. 2018. Characterization of mouse neuro-urological dynamics in a novel decerebrate arterially perfused mouse (DAPM) preparations. Neurourol Urodyn.
TSAI, M. H., KAMM, K. E., STULL, J. T. 2012. Signalling to contractile proteins by muscarinic and purinergic pathways in neutrally stimulated bladder smooth muscle. J Physiol, 590, 5107-21.