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Functional atropine sensitive purinergic responses in the healthy rat bladder

Open AccessPublished:June 09, 2020DOI:https://doi.org/10.1016/j.autneu.2020.102693

      Highlights

      • A functional interaction between purinergic and cholinergic transmission exists in the rat urinary bladder.
      • ATP releases non-neuronal acetylcholine from the urothelium, important for contraction.
      • P2X1 and/or P2X3 purinoceptors are mediating this interaction.

      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 × 10−7 M) and/or receptor antagonists, in intact and urothelium-denuded full thickness rat bladder strip preparations.
      Interestingly, functional contractions to ATP (10−6–10−3 M) remained unaffected by TTX, but were significantly lowered in the presence of the muscarinic antagonist atropine (10−6 M). 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 (10−7–10−5 M) gave rise to functional atropine-sensitive contractions. Furthermore, contractions to ATP were not affected by P2Y6 purinoceptor blockade (by MRS2578; 10−7, 10−5 M), nor were relaxatory responses to ATP sensitive to atropine, PPADS (3 × 10−5 M) or αβMeATP. Lastly, relaxations to ADP (10−6–10−3 M) or NECA (10−8–10−5 M) 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.

      Abbreviations:

      ATP (adenosine-5′-triphosphate), ACh (acetylcholine), LUTS (lower urinary tract symptoms), NO (nitric oxide), αβMeATP (alpha-beta-methylene-adenosine-5′-triphosphate), ADP (adenosine-5′-diphosphate), NECA (5′-(N-ethylcarboxamido)adenosine), TTX (tetrodotoxin), PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid), BPS/IC (bladder pain syndrome/interstitial cystitis), EFS (electrical field stimulation)

      1. Introduction

      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 R.
      • Chapple C.R.
      • Yamanishi T.
      • Yasuda K.
      • Sellers D.J.
      The minor population of M3-receptors mediate contraction of human detrusor muscle in vitro.
      ;
      • Matsui M.
      • Motomura D.
      • Karasawa H.
      • Fujikawa T.
      • Jiang J.
      • Komiya Y.
      • Taketo M.M.
      Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype.
      ). However, the non-adrenergic, non-cholinergic (NANC) transmitter ATP supports this parasympathetically mediated contraction (
      • Burnstock G.
      Noradrenaline and ATP: cotransmitters and neuromodulators.
      ;
      • D’Agostino G.
      • Condino A.M.
      • Calvi V.
      • Boschi F.
      • Gioglio L.
      • Barbieri A.
      Purinergic P2X3 heteroreceptors enhance parasympathetic motor drive in isolated porcine detrusor, a reliable model for development of P2X selective blockers for detrusor hyperactivity.
      ). The contribution of the purinergic transmitter system in the contractile response, mainly mediated by the purinoceptor P2X1, varies between species (
      • O’Reilly B.A.
      • Kosaka A.H.
      • Chang T.K.
      • Ford A.P.
      • Popert R.
      • Rymer J.M.
      • McMahon S.B.
      A quantitative analysis of purinoceptor expression in human fetal and adult bladders.
      ;
      • Vial C.
      • Evans R.J.
      P2X receptor expression in mouse urinary bladder and the requirement of P2X(1) receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle.
      ). For instance, ATP does not appear to play a significant role in the parasympathetically mediated direct smooth muscle contraction of the healthy human bladder (
      • Birder L.
      • Andersson K.E.
      Urothelial signaling.
      ). The purinergic contribution to bladder control is often regarded to be more prominent during various disorders, where ATP may regulate the micturition reflex on other levels as well (
      • Fowler C.J.
      • Griffiths D.
      • de Groat W.C.
      The neural control of micturition.
      ). For instance, ATP has been shown to be important for initiating the micturition reflex arc (
      • Ford A.P.
      • Gever J.R.
      • Nunn P.A.
      • Zhong Y.
      • Cefalu J.S.
      • Dillon M.P.
      • Cockayne D.A.
      Purinoceptors as therapeutic targets for lower urinary tract dysfunction.
      ;
      • Kanai A.J.
      Afferent mechanism in the urinary tract.
      ). This activation of sensory afferents has been accredited to P2X3 and/or P2X2/3 receptors (
      • Cockayne D.A.
      • Dunn P.M.
      • Zhong Y.
      • Rong W.
      • Hamilton S.G.
      • Knight G.E.
      • Ford A.P.
      P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP.
      ). The hypersensibility of afferents observed during disorders such as bladder pain syndrome/interstitial cystitis (BPS/IC) has been suggested to stem from an increased expression of those receptors in combination with an increased release of non-neuronal ATP (
      • Sun Y.
      • Keay S.
      • De Deyne P.G.
      • Chai T.C.
      Augmented stretch activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis.
      ).
      Furthermore, ATP has paracrine functions and may stimulate the release of other neuromodulators from the bladder urothelium (
      • Winder M.
      • Tobin G.
      • Zupancic D.
      • Romih R.
      Signalling molecules in the urothelium.
      ). These substances may either directly stimulate smooth muscle cells, suburothelial cells, afferent or efferent neurons, or even trigger the release of other transmitters from the urothelium (
      • Andersson K.E.
      Bladder activation: afferent mechanisms.
      ;
      • Birder L.
      • Andersson K.E.
      Urothelial signaling.
      ).
      As previously mentioned, acetylcholine has a vital role in normal bladder function and may affect signalling at various levels of the micturition arc (
      • Andersson K.E.
      Bladder activation: afferent mechanisms.
      ;
      • Kullmann F.A.
      • Artim D.E.
      • Birder L.A.
      • de Groat W.C.
      Activation of muscarinic receptors in rat bladder sensory pathways alters reflex bladder activity.
      ). Therefore, anticholinergic drugs are a common treatment for lower urinary tract symptoms (LUTS), present in disorders such as overactive bladder (OAB) (
      • Gormley E.A.
      • Lightner D.J.
      • Burgio K.L.
      • Chai T.C.
      • Clemens J.Q.
      • Culkin D.J.
      • Urogenital R.
      Diagnosis and treatment of overactive bladder (non-neurogenic) in adults: AUA/SUFU guideline.
      ;
      • Robinson D.
      • Cardozo L.
      Antimuscarinic drugs to treat overactive bladder.
      ). However, the exact mechanism of action of those drugs remains elusive. 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 K.E.
      • Yoshida M.
      Antimuscarinics and the overactive detrusor—which is the main mechanism of action?.
      ;
      • Yokoyama O.
      • Yusup A.
      • Miwa Y.
      • Oyama N.
      • Aoki Y.
      • Akino H.
      Effects of tolterodine on an overactive bladder depend on suppression of C-fiber bladder afferent activity in rats.
      ). Hence, interactions between the cholinergic transmitter system and other signalling systems that may stimulate the release of non-neuronal acetylcholine is of scientific interest.
      Crosstalk between the purinergic and cholinergic transmitter systems, where ATP appears to induce the release of acetylcholine, supporting purinergic bladder contraction both in vivo and in vitro, has been reported previously (
      • Aronsson P.
      • Carlsson T.
      • Winder M.
      • Tobin G.
      A novel in situ urinary bladder model for studying afferent and efferent mechanisms in the micturition reflex in the rat.
      ;
      • Stenqvist J.
      • Winder M.
      • Carlsson T.
      • Aronsson P.
      • Tobin G.
      Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder.
      ). In vitro studies suggest this atropine sensitive part of the ATP-induced contractile responses to emanate from the urothelium (
      • Stenqvist J.
      • Carlsson T.
      • Winder M.
      • Aronsson P.
      Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder.
      ;
      • Stenqvist J.
      • Winder M.
      • Carlsson T.
      • Aronsson P.
      • Tobin G.
      Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder.
      ). 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 A.T.
      • Beckel J.M.
      • Barbadora S.
      • Kanai A.J.
      • de Groat W.C.
      • Birder L.A.
      Non-neuronal acetylcholine and urinary bladder urothelium.
      ;
      • Silva I.
      • Ferreirinha F.
      • Magalhaes-Cardoso M.T.
      • Silva-Ramos M.
      • Correia-de-Sa P.
      Activation of P2Y6 receptors facilitates nonneuronal adenosine triphosphate and acetylcholine release from urothelium with the lamina propria of men with bladder outlet obstruction.
      ). However, receptors functionally involved in this interaction are yet to be identified and possible neuronal involvement ought to be further examined.
      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. Additionally, relevant receptor subtypes in this functional interaction will be investigated.

      2. Materials and methodology

      2.1 Animal procedures

      The following experimental procedures were approved by the Animal Ethics Committee in Gothenburg (permit numbers 196-13 and 1794/18). A total number of 33 male Sprague Dawley rats (400–700 g) were presently used.

      2.2 In vitro organ bath experiments

      The rats were euthanized using an intraperitoneal overdose of pentobarbitone (>60 mg/kg; APL, Stockholm, Sweden) followed by excision of the heart. Subsequently, the urinary bladders were excised and kept in Krebs solution (CaCl2 1.25 mM, glucose 5.5 mM, KCl 4.6 mM, KH2PO4 1.15 mM, MgSO4 1.15 mM, NaCl 118 mM and NaHCO3 25 mM) at all times. The experiments were carried out using an organ bath set-up with full-thickness bladder strips (6 × 2 mm), excised proximal to the ureters and above the trigone. The bladder strip preparations were mounted in 25 mL organ baths between a fixed hook and an adjustable steel rod coupled to an isometric force transducer. AcqKnowledge software (Biopac Systems, Goleta, USA) and a MP100WSW data acquisition system were used to record bladder contractions and relaxations. The temperature of the baths was kept at a constant level of 38 °C using a warm-water circuit. Continuous gassing with 5% carbon dioxide in oxygen kept the pH of the Krebs solution at a constant level of 7.4. The bladder strip preparations were pre-stretched to 10 mN and a stable baseline at approximately 5 mN was achieved after 45 min. Administration of high potassium Krebs solution (124 mM, sodium exchanged for an equimolar amount of potassium) was used as a reference for maximal contraction and was administered at the beginning and at the end of each experimental session. All drugs were administered in a volume of 125 μL, thus being diluted 200 times resulting in the concentration specified. Agonists were administered cumulatively, and antagonists and tetrodotoxin (TTX, Tocris Bioscience, Bristol, United Kingdom) were allowed to equilibrate for 20 min before further stimulation.

      2.2.1 Neuronal and urothelial involvement

      Electrical field stimulation (EFS; 2–40 Hz, at a supramaximal voltage of 50 V and a square wave pulse duration of 0.8 ms), and pharmacologically active substances (ATP; 10−6–10−3 M or methacholine; 10−8–10−3 M, performed in separate groups) administered directly into the organ bath in a cumulative manner, were employed. Stimulation was repeated after blockage of voltage-gated sodium channels using the neurotoxin TTX (5 × 10−7 M). Finally, agonist stimulation was performed in the presence of both the muscarinic antagonist atropine (10−6 M) and TTX.
      Experiments on purinergic effects were performed using strip preparations from either intact- or denuded (collagenase I, 0.1% in saline, 30 min) rat urinary bladders, according to an established protocol previously shown to remove 80–90% of the urothelium without damaging the suburothelium (
      • Andersson M.C.
      • Tobin G.
      • Giglio D.
      Cholinergic nitric oxide release from the urinary bladder mucosa in cyclophosphamide-induced cystitis of the anaesthetized rat.
      ).

      2.2.2 Contractile receptor characterization

      The stable ATP-analogue α,β-methyleneadenosine 5′-triphosphate (αβMeATP; 10−7–10−5 M; Tocris Bioscience, Bristol, United Kingdom) was used as an agonist, i.e. administered once in a cumulative fashion, in the presence and absence of atropine (10−6 M). In order to confirm that no desensitization of the purinergic receptors had occurred after the administration of αβMeATP, experiments were conducted where this administration was repeated (in the absence and presence of atropine).
      ATP (10−6–10−3 M) was administered directly into the organ baths and repeated in the presence of the P2Y6 antagonist N,N″-1,4-butanediylbis[N′-(3-isothiocyanatophenyl)]thio urea (MRS2578; 10−7 and 10−5 M; Tocris Bioscience, Bristol, United Kingdom) alone or in combination with atropine (10−6 M). Since MRS2578 was diluted in dimethyl sulfoxide (DMSO), a subset of control experiments was conducted where the administrations of ATP were repeated in the presence of a corresponding volume of DMSO (125 μL, present for 20 min).
      Also, contractions to ATP (10−6–10−3 M) were studied in the absence and presence of the P2X antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 3 × 10−5 M) alone or in combination with atropine (10−6 M).

      2.2.3 Relaxatory receptor characterization

      Relaxatory responses were examined in bladder strip preparations pre-contracted with medium potassium Krebs solution (50 mM potassium, sodium exchanged for potassium), and the resulting relaxations were measured as change compared to baseline at pre-contraction.
      Responses to ATP (10−6–10−3 M) were recorded before and after desensitization of the (mainly) P2X1/3-receptors using αβMeATP (10−5 M, repeated 3–5 times until no further contractile response was observed) (
      • Ralevic V.
      • Burnstock G.
      Receptors for purines and pyrimidines.
      ). ATP was subsequently administered in the presence of both αβMeATP (10−5 M, repeated until no contractile response could be observed) and atropine (10−6 M).
      Relaxation experiments to ADP (10−6–10−3 M) or NECA (10−8–10−5 M; Tocris Bioscience, Bristol, United Kingdom) were also conducted and repeated in the presence of atropine (10−6 M).
      All substances used were purchased from Sigma-Aldrich, St Louis, MO, USA unless otherwise stated.

      2.3 Statistical analysis

      Statistical analysis was performed using repeated measurements analysis of variance (2-way ANOVA) and the Bonferroni post-hoc test. The graphs are presented as sigmoidal regression analysis curves and were generated using the GraphPad Prism software for Windows (GraphPad Software, Inc., San Diego, US). The data is presented as mean ± S.E.M and p-values < 0.05 were regarded as significantly different.

      3. Results

      3.1 Neuronal and urothelial involvement

      EFS induced frequency dependent contractions that were almost completely abolished in the presence of TTX (5 × 10−7 M) in both intact (control) and urothelium-denuded bladder strip preparations (e.g. from 26.72 ± 2.88 to 1.60 ± 0.11 mN in intact controls at a frequency of 40 Hz, in the absence and presence of TTX respectively, p < 0.001, n = 6, Fig. 1a, c ). The remaining response was not further affected by the addition of atropine. Similar responses were seen in urothelium-denuded bladders, where the contraction to EFS was reduced from 23.97 ± 9.01 to 1.41 ± 0.52 mN at 40 Hz (p < 0.001, n = 5, Fig. 1b).
      Fig. 1
      Fig. 1Mean contractile responses to electric field stimulation (EFS) at 2, 5, 10, 20 and 40 Hz before (□) and after TTX-treatment (500 nM; ■) in strip preparations from intact controls (a) and urothelium-denuded bladders (b), respectively. n = 5–6 for each group. ** indicates p < 0.01, *** indicates p < 0.001 and the vertical bars denote S.E.M. c) depicts representative traces of the EFS-induced responses in intact control bladder before and after the administration of TTX (500 nM).
      Administration of ATP (10−6–10−3 M) or methacholine (10−8–10−3 M) evoked concentration-dependent contractile responses. Methacholine-induced responses were not affected by the administration of TTX (maximal contraction of 46.22 ± 1.56 mN and 49.86 ± 2.12 mN before and after TTX-treatment respectively, n.s., n = 6; Fig. 2) but was, as expected, significantly reduced in the presence of atropine (from 46.22 ± 1.56 mN to 9.38 ± 2.93 mN at a concentration of 10−3 M methacholine, in the absence and presence of atropine, 10−6 M, respectively, p < 0.001, n = 6, Fig. 2). Nor were the purinergic responses affected by TTX (1.70 ± 0.60 mN in comparison to 1.56 ± 0.57 mN before and after incubation with TTX, at a concentration of 10−3 M ATP, n.s., n = 7, Fig. 3a ). However, the ATP-evoked contractile responses were significantly reduced after the subsequent addition of atropine (from 1.70 ± 0.60 mN to 0.68 ± 0.18 mN at a concentration of 10−3 M ATP, in the absence and presence of atropine respectively, p < 0.05, n = 7, Fig. 3a).
      Fig. 2
      Fig. 2Mean contractile responses to methacholine (10−8–10−3 M) before (□) and after TTX-treatment (500 nM) alone (■) or in combination (▲) with atropine (10−6 M). n = 6 for each group. *** indicates p < 0.001 and the vertical bars indicate S.E.M.
      Fig. 3
      Fig. 3Mean contractile responses to ATP (10−6–10−3 M) before (□) and after TTX-treatment (500 nM) alone (■) or in combination with atropine (▲; 10−6 M) for intact controls (a) and urothelium-denuded bladders (b) respectively. n = 7 for each group. ** indicates p < 0.01 and the vertical bars denote S.E.M.
      Similar to the observations in intact bladder strip preparations, the purinergic responses in urothelium-denuded bladders were unchanged in the presence of TTX (maximal contraction of 1.54 ± 0.59 mN and 2.25 ± 0.90 mN before and after TTX-administration respectively, n.s., n = 7; Fig. 3b). Interestingly, in contrast to controls the subsequent addition of atropine did not significantly affect the ATP-evoked responses in urothelium-denuded bladders (1.54 ± 0.59 mN in comparison to 1.90 ± 0.80 mN in the presence of atropine, at a concentration of 10−3 M ATP, n.s., n = 7, Fig. 3b).

      3.2 Contractile receptor characterization

      Cumulative administration of the stable ATP analogue αβMeATP (10−7–10−5 M) yielded concentration-dependent contractions seemingly greater than that of ATP. This purinergic contraction was sensitive to atropine (10−6 M; 13.67 ± 1.75 mN in comparison to 10.33 ± 1.33 mN in the presence of atropine, at a concentration of 10−5 M αβMeATP, p < 0.05, n = 12, Fig. 4). In these experiments αβMeATP was first re-administrated in the absence of atropine to ascertain that any reduction in contraction could not be ascribed to desensitizing effects of the agonist itself. No difference was seen between the first and second applications (αβMeATP 10−5 M; 13.67 ± 1.75 mN in comparison to 13.60 ± 1.80 mN, n.s., n = 12, Fig. 4). In separate control experiments, where no re-administration of αβMeATP was performed, the addition of atropine similarly reduced the αβMeATP-evoked contractile responses (αβMeATP 10−5 M; from 14.78 ± 3.00 mN to 11.19 ± 3.54 mN in the absence and presence of atropine 10−6 M, p < 0.05, n = 4).
      Fig. 4
      Fig. 4Mean contractile responses to αβMeATP (10−7–10−5 M) alone (□), followed by the re-administration of αβMeATP (△) and the subsequent addition of αβMeATP in the presence of atropine (■; 10−6 M). n = 12 for each group. * indicates p < 0.05 and the vertical bars indicate S.E.M.
      Contractions to ATP (10−6–10−3 M) were not significantly altered by addition of the P2Y6 purinoceptor antagonist MRS2578 (10−7 or 10−5 M). Rather, a trend towards increased contractions after P2Y6 blockade could be noted (ATP 10−3 M; from 2.76 ± 0.60 mN to 4.43 ± 1.2 mN in the absence and presence of MRS2578 10−5 M, respectively, n.s., n = 11, Fig. 5). Furthermore, atropine still significantly lowered contractions to ATP during P2Y6 blockade (ATP 10−3 M; from 2.76 ± 0.60 mN to 1.43 ± 0.37 mN in the absence and presence of MRS2578 10−5 M administered together with atropine 10−6 M, respectively, p < 0.05, n = 10–11, Fig. 5).
      Fig. 5
      Fig. 5Effect of P2Y6 blockade by MRS2578, shown as mean contractile responses to ATP (10−6–10−3 M) in the absence (□) and presence of MRS2578 (10−5 M; ■) and in the subsequent presence of MRS2578 in combination with atropine (10−6 M; ▲). n = 10–11 for each group. * indicates p < 0.05 and the vertical bars indicate S.E.M.

      3.3 Relaxatory receptor characterization

      Relaxatory responses were investigated in tissues pre-contracted with medium potassium Krebs solution (50 mM). Concentration-dependent relaxations were observed to ATP (10−6–10−3 M), which remained unchanged after desensitization of P2X purinoceptors by αβMeATP (repeated additions 3–5 times until no contractile response left). These purinergic relaxations were not sensitive to the muscarinic antagonist atropine (10−6 M; n = 6, Fig. 6a ). Similar findings were made in the presence of the P2X purinoceptor antagonist PPADS (3 × 10−5 M) alone, as well as together with atropine (10−6 M; n = 6, Fig. 6b).
      Fig. 6
      Fig. 6Mean relaxatory responses to ATP (10−6–10−3 M) a) before (open symbols) and after (closed symbols) desensitization of P2X purinoceptors with αβMeATP (10−5 M), administered alone (■) and in combination with atropine (♦; 10−6 M). b) Before (open symbols) and after (closed symbols) the blockade of P2X purinoceptors with PPADS (30 μM) administered alone (▲) and in combination with atropine (▼; 10−6 M). The data is presented as mean difference in relaxatory responses to the baseline at pre-contraction. n = 6 for each group. Vertical bars indicate S.E.M.
      Similar to the observations for ATP, relaxatory responses to ADP or the P1 adenosine purinoceptor agonist NECA were unaffected by the administration of atropine (10−6 M; n = 6, Fig. 7a and b ).
      Fig. 7
      Fig. 7Mean P2Y and P1 relaxatory responses to a) ADP (10−6–10−3 M, or b) NECA (10−8–10−5 M) in the absence (□, ○) and presence (■, ●) of atropine (10−6 M). The data is presented as mean difference in relaxatory responses to the baseline at pre-contraction. n = 6 for each group. Vertical bars indicate S.E.M.

      4. Discussion

      In the current study, the involvement of the urothelium and efferent neurons in the purinergically evoked release of acetylcholine in vitro was examined. Furthermore, the contribution of different receptor subtypes in this functional interplay was characterized.
      The findings show that an ATP-induced release of acetylcholine with functional implications exists in the rat urinary bladder in vitro, resulting in purinergic contractions being significantly reduced by the muscarinic antagonist atropine. Similar observations in the rat and guinea pig bladder have previously been made in vivo (
      • Aronsson P.
      • Carlsson T.
      • Winder M.
      • Tobin G.
      A novel in situ urinary bladder model for studying afferent and efferent mechanisms in the micturition reflex in the rat.
      ;
      • Sjogren C.
      • Andersson K.E.
      Inhibition of ATP-induced contraction in the guinea-pig urinary bladder in vitro and in vivo.
      ), but this phenomenon seems to be species specific and has not been observed in bladders from cat or rabbit (
      • Liu S.P.
      • Horan P.
      • Levin R.M.
      Effects of atropine, isoproterenol and propranolol on the rabbit bladder contraction induced by intra-arterial administration of acetylcholine and ATP.
      ;
      • Theobald Jr., R.J.
      The effect of arylazido aminopropionyl ATP on atropine resistant contractions of the cat urinary bladder.
      ). Interestingly, similar interactions between the purinergic and the cholinergic transmitter systems appear to occur in different parts of the intestine for some species, for instance in the mouse distal colon (
      • Moody C.J.
      • Burnstock G.
      Evidence for the presence of P1-purinoceptors on cholinergic nerve terminals in the guinea-pig ileum.
      ;
      • Zizzo M.G.
      • Mule F.
      • Serio R.
      Evidence that ATP or a related purine is an excitatory neurotransmitter in the longitudinal muscle of mouse distal colon.
      ).
      Previous functional studies of the ATP-evoked release of acetylcholine have not fully clarified from where this release emanates, although suggestions of an urothelial origin have been presented previously (
      • Stenqvist J.
      • Winder M.
      • Carlsson T.
      • Aronsson P.
      • Tobin G.
      Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder.
      ). This is supported by the current results. Namely, the presence of TTX, in a concentration abolishing EFS-evoked contractions, still allowed for atropine to cause a significant reduction in contractile response not only to cholinergic stimulation, but also to purinergic. The fact that the ATP-induced in vitro responses were TTX-insensitive strengthens the notion that ATP can exert its effects by stimulating the release of another neuromodulator from a non-neuronal source, in addition to direct stimulation of the detrusor (
      • Aronsson P.
      • Andersson M.
      • Ericsson T.
      • Giglio D.
      Assessment and characterization of purinergic contractions and relaxations in the rat urinary bladder.
      ;
      • McMurray G.
      • Dass N.
      • Brading A.F.
      Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle.
      ). Consequently, in urothelium-denuded bladders the ATP-induced release of acetylcholine appeared to be abolished, supporting our previous finding that this interaction may emanate from the urothelium (
      • Stenqvist J.
      • Carlsson T.
      • Winder M.
      • Aronsson P.
      Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder.
      ;
      • Stenqvist J.
      • Winder M.
      • Carlsson T.
      • Aronsson P.
      • Tobin G.
      Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder.
      ). Furthermore, an urothelial origin seems likely as the enzymes responsible for synthesis of acetylcholine; namely choline acetyltransferase (ChAT) and carnitine acetyltransferase (CRAT) respectively, are expressed both in detrusor and urothelial cells. Moreover, urothelial cells express the organic cation transporter OCT3, which is important for non-neuronal release of acetylcholine (
      • Hanna-Mitchell A.T.
      • Beckel J.M.
      • Barbadora S.
      • Kanai A.J.
      • de Groat W.C.
      • Birder L.A.
      Non-neuronal acetylcholine and urinary bladder urothelium.
      ). Previous studies have also shown an ATP-induced release of acetylcholine to occur from urothelial cells (
      • Silva I.
      • Ferreirinha F.
      • Magalhaes-Cardoso M.T.
      • Silva-Ramos M.
      • Correia-de-Sa P.
      Activation of P2Y6 receptors facilitates nonneuronal adenosine triphosphate and acetylcholine release from urothelium with the lamina propria of men with bladder outlet obstruction.
      ). However, the suburothelium cannot be excluded as a complementary source for purinergic release of acetylcholine as the myofibroblasts have been shown to express purinergic receptors and positive stainings for ChAT in the lamina propria have been presented previously (
      • Sui G.P.
      • Wu C.
      • Fry C.H.
      Characterization of the purinergic receptor subtype on guinea-pig suburothelial myofibroblasts.
      ;
      • Yoshida M.
      • Inadome A.
      • Maeda Y.
      • Satoji Y.
      • Masunaga K.
      • Sugiyama Y.
      • Murakami S.
      Non-neuronal cholinergic system in human bladder urothelium.
      ).
      In order to characterize possible receptors involved in ATP-evoked release of acetylcholine the effects of various purinoceptor ligands were examined. When administered as an agonist, the stable ATP-analogue αβMeATP evoked contractions that, similar to ATP, were sensitive to atropine. In the concentrations presently administered, this reduction in contraction can seem to be smaller than the relative effect on ATP-evoked functional response. This could be due to a number of reasons, such as possible differences between the two agonists in terms of affinity, receptor activation, magnitude of response, relaxatory components etc. It clearly shows, however, that atropine-sensitive purinergic contractions are, at least to a large extent, mediated by ATP rather than its metabolites. This since αβMeATP acts exclusively on P2X purinoceptors, with P2X1 and P2X3 being regarded as principal targets (
      • Ralevic V.
      • Burnstock G.
      Receptors for purines and pyrimidines.
      ). The potential importance of other purinoceptors stimulated by metabolites of ATP was further investigated by employing P2Y and P1 (i.e. “non-P2X”) selective agonists and/or antagonists. P2Y6 purinoceptor blockade with MRS2578 did not alter functional contractile responses to ATP, in contrast to what could possibly have been expected based on transmitter release data from previous studies (
      • Hanna-Mitchell A.T.
      • Beckel J.M.
      • Barbadora S.
      • Kanai A.J.
      • de Groat W.C.
      • Birder L.A.
      Non-neuronal acetylcholine and urinary bladder urothelium.
      ). Since atropine could readily affect the functional response to ATP, even under P2Y6 blockade, this purinoceptor cannot currently be shown to alone play a functionally important role in the purinergic-cholinergic interaction. Control experiments where ATP was administered in the presence of DMSO, to rule out any interference due to the dilution of MRS2578 in DMSO, showed no effect. Thus, DMSO itself was deemed to have no impact on these results. Noteworthy, a tendency towards an increase in purinergic contractile response was observed in the presence of MRS2578, which indicates that the P2Y6-purinoceptor may be involved in bladder relaxation.
      The finding that neither P2X purinoceptor desensitization with αβMeATP, nor the further addition of atropine altered the relaxatory response in pre-contracted bladder strip preparations confirms that the P2X purinoceptors (likely mainly of the P2X1 and P2X3 subtypes) are not fundamental for functional relaxations to ATP, which was expected as the P2X-purinoceptors are generally considered to mediate contractile responses (
      • Bolego C.
      • Pinna C.
      • Abbracchio M.P.
      • Cattabeni F.
      • Puglisi L.
      The biphasic response of rat vesical smooth muscle to ATP.
      ;
      • Ralevic V.
      • Burnstock G.
      Receptors for purines and pyrimidines.
      ). Similar observations were made after a more general P2X purinoceptor blockade with PPADS. Thus, any purinergically mediated release of acetylcholine seems to lack importance in the relaxatory response to ATP, and there is no obvious indication that other receptors than the P2X purinoceptors are involved in the interaction between ATP and acetylcholine. However to rule this out, the direct functional relaxatory effects to the metabolites of ATP, namely ADP and the P1 adenosine purinoceptor agonist NECA, were examined. Neither were sensitive to atropine.
      The fact that ATP may act as a modulator on the urothelium, initiating the release of other neurotransmitters, has previously been shown in cell cultures and urinary bladder tissue (
      • Hanna-Mitchell A.T.
      • Beckel J.M.
      • Barbadora S.
      • Kanai A.J.
      • de Groat W.C.
      • Birder L.A.
      Non-neuronal acetylcholine and urinary bladder urothelium.
      ;
      • Silva I.
      • Ferreirinha F.
      • Magalhaes-Cardoso M.T.
      • Silva-Ramos M.
      • Correia-de-Sa P.
      Activation of P2Y6 receptors facilitates nonneuronal adenosine triphosphate and acetylcholine release from urothelium with the lamina propria of men with bladder outlet obstruction.
      ). However, this is, to our knowledge, the first study that sheds some light on what receptor subtypes that are involved in the ATP-induced release of urothelial acetylcholine to such an extent that it may functionally stimulate smooth muscle contraction in vitro. Furthermore, urothelial acetylcholine is of great interest in the micturition reflex arc as it may exert its effect on afferent or efferent nerve endings located near the urothelium or suburothelium (
      • de Groat W.C.
      Integrative control of the lower urinary tract: preclinical perspective.
      ). It may also affect myofibroblasts of the lamina propria as well as stimulate muscarinic and nicotinic receptors directly on the urothelium, which in turn might lead to the release of other neuromodulators such as nitric oxide and ATP (M. C.
      • Andersson M.C.
      • Tobin G.
      • Giglio D.
      Cholinergic nitric oxide release from the urinary bladder mucosa in cyclophosphamide-induced cystitis of the anaesthetized rat.
      ;
      • Sui G.
      • Fry C.H.
      • Montgomery B.
      • Roberts M.
      • Wu R.
      • Wu C.
      Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions.
      ). Thus, the interaction between the purinergic and the cholinergic transmitter system may exist at several levels of the micturition reflex arc and further studies are needed in order to fully characterize this important functional link.

      5. Conclusions

      The purinergic contractile response is supported by the cholinergic transmitter system in vitro. This ATP-induced release of acetylcholine seems to emanate from the urothelium (or possibly the suburothelium) and appears to be independent of nerve transmission (TTX-insensitive). The purinoceptors involved in this interaction seem to be of the P2X-subtype, most likely P2X1 and/or P2X3.

      Acknowledgements

      Rådman och Fru Ernst Collianders foundation (2014), Wilhelm och Martina Lundgrens foundation (2017, 2019) and The Royal Society of Arts and Sciences in Gothenburg (2018–2019).

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