Abstract
While acetylcholine is regarded to be the main directly contractile transmitter substance in the urinary bladder, interactions with other transmitters likely occur. Presently, the interplay between purinergic and cholinergic signalling was investigated to unravel the involvement of the urothelium and efferent neurons in the functionally important purinergically evoked release of acetylcholine in vitro. Functional characterization of receptor subtypes involved in this interplay was also performed. In vitro organ bath experiments with electrical field stimulation (EFS) or administration of agonist were performed in the absence and presence of the neurotoxin tetrodotoxin (TTX; 5×10-7M) and/or receptor antagonists, in intact and urothelium-denuded full thickness rat bladder strip preparations. Interestingly, functional contractions to ATP (10-6-10-3M) remained unaffected by TTX, but were significantly lowered in the presence of the muscarinic antagonist atropine (10-6M). However, in urothelium-denuded strip preparations, this latter phenomenon was not present and the ATP response remained unaltered. To rule out purinergic interference caused by break-down of ATP, experiments were performed in which the stable ATP-analogue αβMeATP (10-7-10-5M) gave rise to functional atropine-sensitive contractions. Furthermore, contractions to ATP were not affected by P2Y6 purinoceptor blockade (by MRS2578; 10-7, 10-5M), nor were relaxatory responses to ATP sensitive to atropine, PPADS (3×10-5M) or αβMeATP. Lastly, relaxations to ADP (10-6-10-3M) or NECA (10-8-10-5M) were unaltered by the presence of atropine. To conclude, purinergic functional contractile, but not relaxatory, responses are supported by the cholinergic transmitter system in vitro, through non-neuronal mechanisms in the urothelium. Involved purinoceptors are of the P2X-subtype, most likely P2X1 and/or P2X3.
Highlights
The parasympathetic innervation of the urinary bladder transmits the signals in the micturition phase, and acetylcholine acting on muscarinic M3 receptors evokes the major part of the contractile detrusor responses in most species (Chess-Williams et al, 2001; Matsui et al, 2000)
The present study aims to further unravel the involvement of the urothelium and efferent neurons in the functional purinergically evoked release of acetylcholine in vitro
Similar responses were seen in urothelium-denuded bladders, where the contraction to electrical field stimulation (EFS) was reduced from 23.97 ± 9.01 to 1.41 ± 0.52 mN at 40 Hz (p < 0.001, n = 5, Fig. 1b)
Summary
The parasympathetic innervation of the urinary bladder transmits the signals in the micturition phase, and acetylcholine acting on muscarinic M3 receptors evokes the major part of the contractile detrusor responses in most species (Chess-Williams et al, 2001; Matsui et al, 2000). Previous studies have suggested the anticholinergic drugs to be effective both during the voiding and the storage phase of micturition The latter may imply an important role for non-neuronal acetylcholine, e.g. from the urothelium or suburothelium (Andersson and Yoshida, 2003; Yokoyama et al, 2005). In vitro studies suggest this atropine sensitive part of the ATP-induced contractile responses to emanate from the urothelium (Stenqvist et al, 2018; Stenqvist et al, 2017) This is further supported by transmitter release studies in both urothelial cells and urinary bladder tissue, where the P2Y6 purinoceptor has been suggested to be involved in urothelial release of acetylcholine (Hanna-Mitchell et al, 2007; Silva et al, 2015). Relevant receptor subtypes in this functional interaction will be investigated
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