The purinergic transmitter system plays a vital role in bladder signalling and is involved at various levels of the micturition reflex arc. For instance, ATP may have direct effects on both the detrusor muscle as well as a modulatory role on the urothelium and the sensory nerves. These effects may be altered by interactions with other transmitter systems such as the cholinergic- and nitrergic signalling systems. Evidence for such an interaction with the cholinergic transmitter system have been presented previously where ATP-induced smooth muscle contractions were shown to be significantly reduced in the presence of the non-selective muscarinic antagonist atropine, suggesting an ATP-induced release of acetylcholine in vitro (1). This release of acetylcholine was suggested to be of urothelial origin and is likely important for healthy bladder function. However, this mechanism needs to be further investigated and this study will examine the ATP-induced release of acetylcholine in vivo where, aside from the urothelium, also the involvement of efferent nerve signalling can be examined in an intact system. Furthermore, different parts of the ATP-evoked detrusor contraction in vivo will be identified, such as neuronal, urothelial and immediate smooth muscle stimulation.

The experimental procedures were approved by the local ethics committee (#196-2013 and 5.8.18-12484/2018). Sprague Dawley rats (Charles River, Germany, 450-750 g) were deeply anesthetized using isoflurane and placed on a thermo-regulated heating pad. After control of the depth of anaesthesia the femoral artery and vein were uncovered and catheterized for blood pressure monitoring and drug administration respectively. Afterwards a laparotomy was performed to uncover the bladder which was catheterized for cystometric measurements via a pressure transducer. Differences in bladder pressure were recorded for concentration series of the cholinergic agonist methacholine (1, 2 and 5 µg/kg, i.v.) and the purinergic agonist ATP (5, 10 and 100 µg/kg, i.v.). The measurements were compared to responses after denervation of the pelvic nerves (by cutting the isolated pelvic nerves) as well as in the presence of the muscarinic antagonist atropine (1000 µg/kg, i.v., 20 min). The involvement of the urothelium was investigated by performing additional experiments in enzymatically denuded bladders (treated with collagenase I, 0.1%, for 30 min).

The maximal change in intravesical pressure in response to intravenously administered ATP was observed in urothelium intact bladders (3.04 ± 0.65 mm/Hg, 100 µg/kg) and was lowered in the denuded group (1.55 ± 0.21 mm/Hg, 100 µg/kg, p<0.05). Disruption of the pelvic innervation indicated a trend towards a lowered contractile response for both purinergic and cholinergic responses. Furthermore, both cholinergic and ATP-evoked contractile responses were significantly reduced in the presence of atropine in intact bladder (e.g from 1.63 ± 0.21 mm/Hg to 0.55 ± 0.15 mm/Hg for ATP, 10 µg/kg, in the absence and presence of atropine respectively, n=7, p<0.05). In urothelium denuded animals no significant alteration of the purinergic responses was observed in the presence of the muscarinic antagonist (e.g from 0.74 ± 0.24 mm/Hg to 0.85 ± 0.36 mm/Hg before and after administration atropine respectively at 10 µg/kg, n=6-7).

The effects of purinergic signalling in the urinary bladder is complex and the ability of ATP to stimulate neuromodulator release from the urothelium is well established as a concept. 
As has been previously observed, the purinergic contractile responses in vivo appear to possess an atropine sensitive part. This suggests an ATP-induced release of acetylcholine which seems to emanate from the urothelium and may thus directly stimulate the muscarinic receptors on the detrusor muscle. Parts of the purinergic signalling is, of course, also evoked via the pelvic nerve efferents. However, disruption of the pelvic innervation did not significantly affect the atropine sensitive parts of the ATP-induced contractile responses, indicating the involvement of acetylcholine in the efferent part of the purinergic response in vivo to be minute or absent. While purinoceptor subtypes have previously been mentioned in the context of cholinergic function (1, 2) and transmitter release (3), this is, to the best of our knowledge, the first study to show the ATP-induced release of urothelial acetylcholine to be of a functionally relevant extent, stimulating direct smooth muscle contraction in vivo.

A cholinergic part directly linked to the purinergic response in vivo has been confirmed and been determined to be of mostly urothelial origin with less to none involvement of the pelvic efferent nerves. This may be an important mechanism involved in micturition and its effect on other levels of the micturition reflex arc might be of great interest for further studies.

Stenqvist, J., et al., Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder. Eur J Pharmacol, 2017. 809: p. 253-260.
Stenqvist, J., et al., Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder. Auton Neurosci, 2018. 213: p. 60-70.
Silva, I., et al., Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction. J Urol, 2015. 194(4): p. 1146-54.