The purinergic system regulates bladder function through the action of ATP or its metabolites. ATP is a functional excitatory neurotransmitter and contractile agonist in most animals and also in human overactive bladders, via P2X1 receptor activation [1]. Adenosine, a breakdown product of ATP via endonucleotidases, is a P1 receptor agonist that mediates relaxation and thus offers a modulatory influence on the effect of ATP. The P1 class of adenosine receptors (A1, A2A, A2B and A3) mediate their actions via different G-protein-coupled intracellular pathways. Nerve-mediated detrusor contractions are modulated by adenosine, via the A1 receptor subtype [2]. We tested the hypothesis that A1 receptor activation reduces directly nerve-mediated ATP release and hence contractions.

Young (12 weeks) male C57BL/6 mice were used. Mice were euthanised and the bladder removed through a midline laparotomy. Bladder strips from the dome (detrusor + mucosa) were tied to isometric force transducers in a horizontal chamber superfused with Tyrode’s solution at 37°C. Nerve-mediated contractions were generated by electrical field stimulation (EFS: 0.1ms pulses, 1-40 Hz, 3-s train every 90-s), interventions were delivered via the superfusate and the effect on contractions measured. Tension amplitude was normalised to strip weight (mN.mg-1). Tension amplitude plotted as a function of stimulation frequency was fitted to force-frequency relationships to determine maximum tension (Tmax, mN.mg-1), the frequency required to attain Tmax/2 (f1/2, Hz), and the ratio of tension at low (2 Hz) and high (24 Hz) frequency stimulation (T2/T24). Nerve-mediated ATP release was measured from a 100 µl superfusate sample taken from the same site close to the preparation using a luciferin-luciferase assay, data are fmoles ATP per µl superfusate sample. Data are means ± SD, n=number of preparations, one from each animal. Differences between data sets were subjected to repeated measures two-way ANOVA followed by parametric post hoc tests, or a Student’s paired t-test; the null hypothesis was rejected at p<0.05. The number of repeats in each set was based on a power calculation to reject the null hypothesis at p<0.05 and a power of 80%, with variance of data based on previous data with these methods.

Adenosine (1 mM) significantly reduced nerve-mediated contractions (p<0.01). Contractions were attenuated more at low frequencies of stimulation compared to higher frequencies.  Thus, there was no effect on Tmax values (p>0.05), a significant increase of the f1/2 value  (p<0.01), and a significant decrease of the T2/T24 ratio ). Adenosine also significantly reduced nerve-mediated ATP release (p<0.001) – see Table 1 and Figure 1. 
Addition of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 μM) abolished the effect of adenosine on contractions and ATP release; the antagonist had no effect by itself on any variable (p>0.05). 
The A1 receptor agonist N6-cyclopentyladenosine (CPA, 10μM) also significantly reduced nerve-mediated contractions and ATP release (p<0.001). As with adenosine, there was no effect on the Tmax value (p>0.05); however there was a significant increase of the f1/2 value (p<0.001), and a significant decrease of T2/T24 (p<0.01). Addition of CPA also significantly reduced nerve-mediated ATP release (p<0.001.
The A1/A2 receptor agonist N-ethylcarboxamidoadenosine (NECA, 10μM) had similar actions to CPA.  There was no effect on Tmax values (p>0.05), however there was a significant increase in f1/2 values ( p<0.01) and a significant decrease of T2/T24 values (p<0.01). NECA also significantly reduced nerve-mediated ATP release (p<0.001).

The addition of adenosine, CPA and NECA, resulted in the frequency-dependent attenuation of nerve-mediated contractions. The significant increase of f1/2 values, the significant decrease of T2/T24 values, with no effect on Tmax, demonstrate that greater effects were at lower compared to higher stimulation frequencies. DCPCX abolished the effects of adenosine.  The selective activity of CPA and DCPCX on A1 receptors and partial selectivity of NECA on A1/A2 receptors, are consistent with the hypothesis that adenosine acts to reduce nerve-mediated ATP release via A1 receptor activation. Moreover the dynamic range of nerve-mediated ATP release is over the lower range of frequencies used in these experiments.

Nerve-mediated ATP release is a feature of detrusor contractile activation from overactive human bladders.  The selective attenuation of this component of contractile activation by A1 receptor agonists suggests that this receptor can be a target to reduce overactive bladder contractions.

Fry CH, Bayliss M, Young JS, Hussain M (2011). Influence of age and bladder dysfunction on the contractile properties of isolated detrusor smooth muscle. BJU Int 108:E91-E96.
Pakzad M, Ikeda Y, McCarthy C, Kitney DG, Jabr RI, Fry CH (2016). Contractile effects and receptor analysis of adenosine-receptors in human detrusor muscle from stable and neuropathic bladders. Naunyn Schmiedebergs Arch Pharmacol 389(8):921-9.