The purinergic system regulates bladder function at several sites by the action of ATP or its metabolites. ATP is a co-transmitter with acetylcholine (ACh) at parasympathetic nerve terminals, and with detrusor from most animal bladders it has an excitatory role in generating nerve-mediated contractions. ATP is released over lower stimulation frequencies (<~12 Hz), whilst ACh release predominates at higher frequencies. However, with human bladder, ATP only has such a role in bladder pathologies, such as overactive bladder; but with normal human detrusor ACh is the sole functional transmitter, explained by variable rates of extracellular ATP hydrolysis by endonucleotidases [1]. It would therefore be highly desirable to selectively reduce ATP release from nerve terminals, leaving ACh release intact. Adenosine, an ATP metabolite, reduces nerve-mediated contractions, with a large effect at low stimulation frequencies, but very much less at higher frequencies. The action is mediated by A1 receptors on nerve terminals and will reduce intracellular cyclic AMP (cAMP) levels and similarly reduce activity of its major target, protein kinase A (PKA) [2].  Thus, it may be inferred that adenosine selectively reduces nerve-mediated ATP release but there has been no direct demonstration. We hypothesised that A1 receptor agonists do reduce nerve-mediated ATP, but not ACh, release, an action mediated by an A1 receptor-cAMP-PKA pathway.

Bladders were dissected from 12-week male C57BL/6 mice, and tissue strips with mucosa intact, were mounted to record contractions generated by electrical field stimulation (EFS; 0.1-ms pulses, 1-40 Hz, 3-s train every 90 s) and inhibited by 1 µM tetrodotoxin. Drug interventions were delivered by the superfusate, Tyrode’s solution (24 mM NaHCO3:5%CO2, pH 7.4, 36°C). Peak tension (mN) was normalised to preparation weight (mN.mg-1). Tension was plotted as a function of stimulation frequency and analysed to estimate the maximum tension at high frequencies (Tmax, mN.mg-1) and the frequency to generate Tmax/2, f1/2 (Hz). 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. Nerve-mediated ATP and ACh release was measured in superfusate 100 µl samples taken immediately before and during EFS. ATP was measured using a luciferin-luciferase assay [3] at 8 Hz stimulation, and ACh release was measured at 20 Hz stimulation using a choline/ACh assay kit (Sigma); the frequencies were chosen to reflect when ATP and ACh release was near optimal [3]. Data are presented are mean±SEM; 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.

Adenosine (1 mM) reduced nerve-mediated contractions at low frequencies (f1/2 increased) and reduced ATP release (Fig 1A, B). However, there was no effect on higher frequency contractions (Tmax) and there was no effect on ACh release (Fig 1C, D). Further experiments were undertaken to find the cellular pathway underlying these effects of adenosine on contractile function and ATP release (Fig 2). Similar results to adenosine on f1/2 and ATP release were recorded with the A1 receptor selective agonist CPA (N6-cyclopentyladenosine, 10 µM, Fig 2A) and the non-selective A1/A2 receptor agonist NECA (5'-(N-ethylcarboxamido) adenosine, 10 µM, Fig 2B). The effect of adenosine was blocked by the A1 receptor antagonist, DCPCX (dipropyl-cyclopentylxanthine, 1 µM, Fig 2C). Direct inhibition of PKA by CAMPs-Rp (10 µM) also increased f1/2 and decreased ATP release (Fig 2D). Two agents that increase cAMP, the adenylate cyclase activator forskolin (1 µM) to activate and the cell permeable cAMP analogue 6-MB-cAMP (100 µM) had no effect on f1/2 values and ATP release (data not shown). An additional target for intracellular cAMP, over PKA, is EPAC (exchange protein directly activated by cAMP) that may regulate ATP release. An EPAC activator (007-AM, 10 µM) and EPAC inhibitor (ESI-09, 20 µM) both had no effect on f1/2 values and ATP release (data not shown). None of the interventions listed above had a significant effect on Tmax values.

Adenosine and A1 receptor agonists generated a frequency-dependent attenuation of nerve-mediated contractions. The significant increase on f1/2 values, with no effect on Tmax demonstrate that greater effects were at lower stimulation frequencies where ATP release is more predominant.  This was corroborated by direct measurement of reduced nerve-mediated ATP release. DPCPX abolished the effects of adenosine, consistent with the hypothesis that adenosine acts via A1 receptor activation. The main target for cAMP in mediating nerve-mediated ATP release is via PKA and not by the EPAC route. An increase of cAMP by forskolin or 6-MB-cAMP had no effect which implies that normal levels of intracellular cAMP are sufficient to maintain nerve-mediated ATP release. By contrast adenosine had no effect on ACh release or Tmax. This the first direct demonstration that differential regulation of transmitter release is possible at the detrusor nerve-muscle junction. Because ATP is associated with pathological contractile function in the human bladder, the ability to specifically attenuate its release offers a novel therapeutic drug target.

This study has shown for the first time that adenosine selectively reduces ATP release from motor nerves supplying detrusor smooth muscle. The pathway of this action has also been characterised: initially by activation of an A1 receptor, with downstream inhibition of adenylate cyclase, cAMP generation and PKA. Modulation of cyclic nucleotide levels, such as cAMP, provides a novel target of pathological purinergic motor pathways.

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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.
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.