Parasympathetic nerve terminals to detrusor muscle release two excitatory neurotransmitters, acetylcholine (ACh) and ATP. In the normal human bladder, ACh fully supports contraction due to rapid extracellular breakdown of ATP, whereas in bladder pathologies such as overactive bladder ATP also contributes to contractile activation [1]. However, in most animals, including mice, detrusor from normal bladders also has a dual nerve-mediated ATP/ACh component to contraction. Thus, in this context, mouse detrusor offers a good model for overactive human tissue. Phosphodiesterase type 5 (PDE5) inhibitors like sildenafil inhibit cGMP breakdown and reduce the purinergic component of nerve-mediated contractions [2]. We hypothesise that increasing intracellular cGMP levels by different pathways decreases nerve-mediated ATP release. However, the effect on ACh release is not known, and this will also be tested. cGMP levels were raised by: i) increasing soluble guanylate cyclase activity with the activator cinaciguat; ii) reducing its breakdown with sildenafil; iii) by addition of a cell permeable cGMP analogue. The involvement in any effect on the enzyme target for cGMP, protein kinase G (PKG), was also investigated.

Bladders were dissected from 12-week male C57BL/6 mice, and tissue strips, with an intact mucosa, were attached to an isometric force transducer and superfused with Tyrode’s solution (pH 7.4, with 24 mM NaHCO3/5% CO2 buffer, 36°C). Contractions were generated by electrical field stimulation (EFS; 0.1-ms pulses, 1-40 Hz, 3-s train every 90 s) that were inhibited by tetrodotoxin (1 µM). Drug interventions were delivered by the superfusate and the effects on nerve-mediated contractions measured. Tension amplitude (mN) was normalised to preparation weight (mN.mg-1). Nerve-mediated amplitude, plotted as a function of stimulation frequency, was analysed to generate Tmax, the maximum tension at high frequencies and f1/2, the frequency to attain Tmax/2. A reduction of Tmax implies an action on force mainly via ACh-dependent pathways; an increase of f1/2 implies a force reduction mainly via ATP-dependent pathways.  Superfusate samples were taken from a fixed point near the preparation with minimal mechanical disturbance [2]. ATP release was measured across the frequency range using a luciferin-luciferase assay, and ACh release was measured at 20 Hz stimulation using a choline/ACh assay kit (both Sigma). Data are mean±SEM and differences between data sets were tested with repeated measures two-way ANOVA followed by parametric post-hoc tests or Student’s paired t-tests where appropriate; the null hypothesis was rejected at P<0.05.

Cinaciguat (10 µM, Fig. 1A) and sildenafil (20 µM, Fig. 1B) both reduced nerve-mediated ATP release, but had no significant effects on ACh release.  Nerve-mediated contraction data were consistent with this result, with no effect of either compound on Tmax, but each an increase of the f1/2 value (Fig 1A, B).  The cGMP analogue 8-bromo-cGMP (1 µM) had a similar effect on ATP dynamics: nerve-mediated ATP release was reduced, f1/2 increased (Fig. 2A) and no effect on Tmax. The PKG inhibitor, Rp-8-CPT-cGMPS (10µM), had no effect itself on nerve-mediated contractions and ATP release.  It also inhibited the effects of sildenafil so that there was now no decrease of nerve-mediated ATP release and no increase of f1/2 values (Fig 2B).

Three interventions were designed to increased intracellular cGMP levels: to increase its production by enhancing guanylate cyclase activity, reduce its breakdown by PDE5 inhibition, or add directly to the intracellular pool. All were similarly efficacious to reduce nerve-mediated ATP release and selectively reduced low frequency contraction (increase f1/2 values). Two of these compounds, cinaciguat and sildenafil were tested on nerve-mediated ACh release where there was no significant effect, or on the contractile equivalent, Tmax values. The intracellular target for cGMP in regulating transmitter release is PKG, as its separate inhibition, abolished contractile changes associated with sildenafil and its attenuation of ATP release. This is the first demonstration of selective inhibition of neurotransmitter, namely ATP, release from nerves supplying detrusor smooth muscle. Because ATP is associated with pathological contractions in the human bladder it offers a therapeutic target to selectively attenuate these contractions whilst leaving physiological contractile regulation intact.

Elevation of intracellular cGMP by a number of different routes selectivity reduces transmitter release to detrusor smooth muscle; attenuating ATP release whilst leaving ACh release intact. This offers a specific target for pathological mechanisms associated with abnormal functional bladder activity but leaving normal physiological pathways intact.

McCarthy CJ, Ikeda Y, Skennerton D, Chakrabarty B, Kanai AJ, Jabr RI, Fry CH (2019). Characterisation of nerve-mediated ATP release from bladder detrusor muscle and its pathological implications. Br J Pharmacol 2019; 176: 4720-4730.
Chakrabarty B, Ito H, Ximenes M, Nishikawa N, Vahabi B, Kanai AJ, Pickering AE, Drake MJ, Fry CH (2019). Influence of sildenafil on the purinergic components of nerve-mediated and urothelial ATP release from the bladder of normal and spinal cord injured mice. Br J Pharmacol 176:2227-2237.