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Pharmacological Research 65 (2012) 129– 136

Contents lists available at SciVerse ScienceDirect

Pharmacological Research

jo ur n al hom epage: www.elsev ier .com/ locate /yphrs

urinergic P2X3 heteroreceptors enhance parasympathetic motor drive insolated porcine detrusor, a reliable model for development of P2X selectivelockers for detrusor hyperactivity

ianluigi D’Agostinoa,∗ , Anna Maria Condinoa , Valentina Calvia , Federica Boschia , Luciana Giogliob ,nnalisa Barbieri a

Experimental Pharmacology Unit, Department of Drug Sciences, University of Pavia, 27100 Pavia, ItalyDepartment of Experimental Medicine, University of Pavia, 27100 Pavia, Italy

r t i c l e i n f o

rticle history:eceived 20 June 2011eceived in revised form 12 October 2011ccepted 14 October 2011

eywords:urinergic heteroreceptorsarasympatheticorcine detrusoretrusor hyperactivity

a b s t r a c t

Various forms of low urinary tract symptoms (LUTS) seem dependant upon dysregulation of the puriner-gic pathway which produces sensory- or motor-activated incontinence. A body of evidence in humanurinary bladders supports a link between up-regulation of purinergic activity and the pathogenesisof detrusor instability. This study investigated the potential role of adenosine 5′-triphosphate (ATP)in the control of detrusor motor drive in a model of porcine urinary bladder. The involvement of ATPon excitatory activity was assessed by measuring neurally-evoked [3H]-acetylcholine (ACh) release andsmooth muscle contraction in detrusor strips. Epithelium-deprived preparations were used to mini-mize the influence of non-neural sources of ACh and ATP on parasympathetic neurotransmission. AChrelease and smooth muscle contractility were not significantly affected by neural ATP in normal detru-sor, but markedly enhanced when ATP hydrolysis was reduced by ectoATPase inhibitors, as well as by�,�-methylene-ATP (ABMA), agonist resistant to ecto-enzymes degradation. Prejunctional P2X receptorslocated on cholinergic nerves are involved in such potentiating effect. These purinergic heteroreceptorswere characterized as P2X3 subunits by means of the putative antagonists: NF449 (P2X1,3 selective),NF023 (P2X1,3 selective), PPNDS (P2X1 selective) and A-317491 (P2X3 selective). In porcine detrusor,

P2X3 receptors are functionally expressed at neural site facilitating neurogenic ACh release. When purinebreakdown is experimentally down-regulated to mimicking the impaired purinergic pathway observedin pathological human bladders, endogenous ATP can markedly enhance detrusor contractility throughactivation of these receptors. Since P2X3 blockade represents a potential therapeutic approach for dis-eases of the urinary tract, isolated porcine detrusor represents a reliable model for development of novelselective P2X3 antagonists ben

Abbreviations: [3H]-ACh, acetylcholine tritiated; A-317491, 5-[[[(3-phen-xyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino] carbonyl]-,2,4-benzenetricarboxylic acid sodium salt hydrate; ABMA, �,�-methyl-neadenosine 5′-triphosphate trisodium salt; ARL67156, 6-N,N-diethyl-d-,�-dibromomethyleneATP trisodium salt; ATP, adenosine 5′-triphosphateisodium salt; Bz-ATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphateri(triethylammonium) salt; CRC, concentration–response curve; DPCPX,,3-dipropyl-8-cyclopentylxanthine; EFS, electrical field stimulation; NF023,,8′-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-tri-ulfonicacid, hexasodium salt; NF449, 4,4′ ,4′′ ,4′′ ′-[carbonylbis(imino-5,1,3-enzenetriyl-bis(carbonylimino))]tetrakis-1,3 benzenedisulfonic acid, octasodiumalt; PPNDS, pyridoxal-5′-phosphate-6-(2′-naphthyl azo-6′-nitro-4′ ,8′-disulfonate)etrasodium salt; TTX, tetrodotoxin; �-CTX, �-conotoxin GVIA; ZM 241385, 4-(2-7-amino-2-(2-furyl) [1,2,4]triazolo[2,3-a] [1,3,5]triazin-5-ylamino]ethyl)phenol.∗ Corresponding author at: Department of Drug Sciences, School of Pharmacy,niversity of Pavia, Viale Taramelli 12, I-27100 Pavia, Italy. Tel.: +39 0382 987398;

ax: +39 0382 987405.E-mail address: dagg@unipv.it (G. D’Agostino).

043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2011.10.002

eficial in the treatment of detrusor hyperactivity.© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Adenosine 5′-triphosphate (ATP), a co-transmitter in mostnerves in the peripheral nervous system, represents a prime candi-date between modulators determining up- and down-regulationof synaptic transmission [1]. Although in such mechanism thebreakdown product adenosine seems partly involved, a substan-tial body of evidence supports the notion that ATP can act directlythrough specific receptor types, ranging from P2Y to P2X recep-tor families [2]. This last family includes seven (P2X1–7) receptorsubunits which can form either homomultimeric or heteromulti-meric structures related to nonselective calcium channel opening[3]. Different P2X subunits were identified in a wide variety of tis-

sues in peripheral organs [4], including the urinary bladder whereP2X1,2,4 subunits are present on detrusor muscle-cells, P2X1–3,5on bladder nerves and P2X2,3,7 subunits are located on urothelialcells [5]. In this organ, ATP is co-stored and released together with

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he main neurotransmitter acetylcholine (ACh) [6], contributing toerve-mediated bladder contraction and urine elimination [7,8].he relative importance of the purinergic component, however, ispecies dependent, being dominant in cat, mouse and rabbit, tooderate in guinea pig, rat and dog, to less pronounced in pig

nd humans [9]. Although negligible in normal human bladder10], the role of ATP has recently attracted considerable atten-ion in the pathophysiology of abnormal detrusors [11]. In fact,he purinergic component seems responsible for the increasedtropine-resistance of parasympathetically-evoked contractionsbserved in aging [12] and in subjects with urological diseasetates such as obstructed bladder (OB) [10,13] and interstitial cys-itis (IC) [14], or idiopathic detrusor overactivity (IDO) [15], wherehe magnitude is greatest [16]. For those patients suffering from aigh prevalence of detrusor instability, current treatment options

nclude muscarinic antagonists, drugs with challenging tolerabil-ty profile and marginal efficacy [17]. This suboptimal managementeaves a relevant opportunity for the development of superior ther-peutics surfaced through the discovery of novel receptor targets.

In this respect, prejunctional heteroceptors that serve a com-ined function in sensory and motor pathway of the human bladderave been suggested as potential pharmacological targets [18–20].ecently, neural P2X3 receptors have been suggested to exert arominent role in the control of bladder fullness sensation andfferent transmitter release in pathophysiological circumstances21], in particular in human bladders with detrusor overactivityDO) where an increase in P2X3 receptor expression was observedoo [22].

Consequently, the blockade of these receptors might be clin-cally beneficial [21,23,24], but the development of novel P2X3ntagonists requires a first screening of their pharmacologicalctivity in an animal model in which compelling evidence has sur-aced that a dysregulation of the purinergic pathway promotingonditions of functional neurologic hyperactivity relates to motorefferent) urologic abnormalities.

For this purpose we used the urinary bladder of the pig, con-idered a reliable animal model for urological research from manyspects [25–29] but, in particular, from the notion that the quantityf released ATP, proportion of ATP release from neuronal (parasym-athetic terminals) and non-neural sources (urothelium) as well ascto-ATPase activity affecting purine levels are remarkably similaretween pig and humans [8].

The aim of the present study was to provide direct evidencehat neurogenic ATP and P2X receptors are involved in the con-rol of detrusor motor drive, determining detrusor smooth muscleyperactivity in conditions of up-regulation of purinergic pathway.ccordingly, [3H]-ACh release and contractile experiments werearried out in the isolated detrusor of the pig, epithelium-deprivedo minimize the influence of non-neural sources of ACh and ATP inhe characterization of neural P2X receptors.

. Materials and methods

.1. Materials

The following compounds were purchased: [methyl-3H]-holine chloride (2.89 TBq/mmol) from Amersham (Arlingtoneights, IL, USA); TTX, �-conotoxin GVIA, Suramin, 4,4′,4′′,4′′ ′-

carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]etrakis-1,3 benzenedisulfonic acid, octasodium salt (NF449),nd 4-(2-[7-amino-2-(2-furyl) [1,2,4]triazolo[2,3-a] [1,3,5]triazin-

-ylamino]ethyl)phenol (ZM 241385) from Tocris Cook-on Ltd. (Cabot Park Bristol, UK); atropine sulfate,examethonium bromide, hemicolinium-3, 6-N,N-diethyl-d-�,�-ibromomethyleneATP trisodium salt (ARL67156), ATP, 2′-3′-O-(4

l Research 65 (2012) 129– 136

-benzoylbenzoyl)-ATP (Bz-ATP), �,�-methylene-ATP lithium salt(ABMA), 5-[[[(3-phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino] carbonyl]-1,2,4-benzenetricarboxylic acid,sodium salt hydrate (A-317491), 8,8′-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulfonicacid,hexasodium salt (NF023), pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and potato’s apyrasegrade III from Sigma-RBI (St. Louis, MO, USA).

2.2. Preparation of porcine detrusor strips

Specimens from the anterior part of the urinary bladder dome ofDumeco strain male pigs (>5 months, carcass weight 180–200 kg),obtained from local abattoir, were transported to the laboratory ingassed (95% O2 and 5% CO2) Krebs’ solution containing (mM): NaCl120, KCl 4.7, MgSO4 0.6, NaHCO3 25, KH2PO4 1.2, CaCl2 2.0 and glu-cose 10 at 6 ◦C. Muscle strips (20 mm long, 4 mm wide), prepared byremoving serosal and mucosal layers, were mounted isometricallyunder an initial tension of 2 g in 2 ml perfusion chamber superfusedwith Krebs’ solution at 37 ◦C. Electrical field stimulation (EFS) wasapplied by means of two platinum electrodes placed parallel to thepreparation.

The study was approved by the Animal Ethics Committee ofUniversity of Pavia and complies with the current Italian laws.

2.3. Release experiments in porcine detrusor strips

After a 30 min equilibration period, neuronal release of [3H]-ACh was assessed according to the procedure previously describedfor the human bladder [20]. Briefly, the preparation was incubatedfor 45 min with [methyl-3H] choline (92 kBq/ml) and stimulated by10 s pulse trains delivered at 10 Hz (0.2 ms duration, 60 V/cm, 60 sapart) to label neuronal ACh stores. Following loading, the prepara-tions were washed out for 120 min by superfusion at a constant rateof 2 ml/min (Minipulse 2HP8 flow inducer, Gilson Medical Electron-ics, Middleton, WI, USA). Hemicholinium-3 (10 �M) was presentin the superfusion solution throughout the experiment to preventcholine uptake. Starting at the 121st min (zero time), the fluid wascollected continuously in 3 min periods (6 ml samples) and aliquots(1 ml) were measured in 3 ml of Ultima Gold (Packard BioScience,Groningen, The Netherlands) scintillation cocktail by liquid scin-tillation spectrometry (Tri-Carb 2700TR, PerkinElmer, Shelton, CT,USA). Quench correction curves were established and external stan-dardization was used for counting efficiency (% counting efficiency:59 ± 1.8). Tritium content was expressed in disintegration per s(Becquerel) for gram of dry weight of the tissue (Bq/g) determinedat the end of the experiment.

The strips were stimulated 2 times by EFS (S1 and S2) beginning9 (S1) and 54 (S2) min after zero time with 6 trains of 9 s, deliveredat 20 Hz (0.1 ms durations, 90 V/cm) 33 s apart. The EFS (S1 and S2)caused an increase of [3H]-outflow that was calculated as differencebetween the total tritium outflow during 3 min stimulation plus thefollowing 12 min (stimulation outflow period) and the calculatedspontaneous outflow. The decline for the spontaneous outflow wascalculated by fitting a linear regression line to the values (expressedin Bq/g) of 3 min-samples before and after the stimulation outflowperiod.

The exposure of P2X agonists was allowed either 2 minbefore the onset of S2 (preliminary set of experiments) orduring the second train of S2 (along the study). Other drugs

were added to the superfusional fluid 10 min (P2X antagonists,ecto-ATPase and ecto-ATPase inhibitors) and 20 min (TTX, �-conotoxin GVIA, hexamethonium, atropine) before the onset ofS2.

G. D’Agostino et al. / Pharmacological Research 65 (2012) 129– 136 131

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ig. 1. Time course of contractile responses and [3H]ACh release evoked by EFS (S1

espectively). The effects of 5 �M TTX (C and D) and of 30 �M hexamethonium (E aoint represents the radioactivity per gram of tissue in the superfusate collected in

The change in the ratio between the [3H]-outflow evoked duringhe two stimulation periods (S1/S2) compared to that observed inontrol situations (S1/S2 in the absence of tested drugs) was takens a measure of the effect of tested drugs.

Accordingly, concentration–response curves (CRCs) for ATP and,�-meATP (ABMA) were constructed in the absence and in theresence of P2X antagonists.

.4. Contractile assessments in porcine detrusor strips

In the same experiment, the EFS effectiveness in producingCh release was also assessed by measuring muscular contractileffects on a polygraph (Battaglia Rangoni, Bologna, Italy). Smooth

uscle contractions (C1 and C2) were assessed as the sum of the

ix contractions evoked by S1 and S2, respectively. The changen the ratio C2/C1 was taken as a measure of the effect of testedrugs.

2 applied 45 min apart) in pig detrusor strips in control conditions (A and B panels,are shown in middle and bottom panels, respectively. In B, D and F diagrams, each

samples.

2.5. Statistical analysis

Values from individual experiments were averaged, and the SEMvalues were calculated. Drug potency estimates were evaluated as−log EC50 (negative log of the molar concentration-producing half-maximal effect) by nonlinear curve fitting (GraphPad Prism, version5; GraphPad Software Inc., San Diego, CA). Apparent affinity (pKi)estimates were calculated according to Kenakin [30]. Differencesbetween means were analyzed with one-way analysis of variance(ANOVA) followed by Dunnett’s multiple comparison test. Valuesof p < 0.01 were taken as statistically significant.

3. Results

3.1. Neural and muscular effects evoked by EFS

The experimental approach used in this study allowed to recordsimultaneously in each experiment both prejunctional (neural

132 G. D’Agostino et al. / Pharmacologica

Fig. 2. Residual [3H]-ACh release evoked by EFS in pig detrusor strips in comparisontcs

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o control conditions in the presence of selective Na+-channel toxin (TTX) and Ca2+-hannel toxin (�-CTX). Data are the mean ± SEM of 3–5 experiments. (**) p < 0.001ignificance vs controls.

3H]-ACh release) and postjunctional (smooth muscle contraction)ffects induced by EFS.

In pig isolated detrusor strips, EFS produced a contractileesponse (C1; 7.66 ± 2.09 g, n = 20) and a marked 3H-outflow (S1;591 ± 860 Bq/g dry tissue) (Fig. 1A and B). In control experiments,he second EFS induced similar contractile and outflow patterns,howing a C2/C1 and S2/S1 ratio value of 0.93 ± 0.03 and 0.82 ± 0.02,espectively.

In the presence of 5 �M TTX, EFS-evoked contractile responsend [3H]-outflow were reduced of 92 ± 3% and 84 ± 8%, respectivelyn = 3; Figs. 1C, D and 2), but not affected by the ganglionic blockerexamethonium (10 �M) (n = 3; Fig. 1E and F).

At variance, atropine mostly abolished the EFS-evoked con-

ractile effect (by about 91% at 1 �M; not shown). The N-typea2+-channel blocker �-conotoxin GVIA (0.1–3 �M) reducedoncentration-dependently the [3H]-outflow (83 ± 5% maximalnhibition; Fig. 2). Taken together, these data indicate that the

ig. 3. Comparison of the EFS-evoked contractions in control conditions (A) and in the prection 2.

l Research 65 (2012) 129– 136

3H-outflow mostly reflects the release of neural [3H]-ACh fromcholinergic terminals, as previously observed in human detrusor[20].

Purinergic ligands affected the EFS-evoked effects to differentextents, depending on the used experimental conditions.

When exposed 2 min before S2, the purinergic agonists ATPand ABMA (1 �M to 1 mM) failed to affect both contraction and[3H]-ACh release evoked by EFS, but increased the smooth musclebasal tension of the strip without any significant change of basalspontaneous [3H]-outflow. The musculature was concentration-dependently contracted, but in a reproducible manner only forABMA with a maximal extent at 300 �M (45 ± 8% of EFS-evokedcontraction; n = 6; Fig. 3B). The partial agonist Bz-ATP (1 �M to1 mM) revealed to be ineffective.

Conversely, when added during S2, ATP produced a facilitation ofEFS-evoked [3H]-ACh release that was measurable (8.3 ± 2% maxi-mal enhancement, n = 3; Fig. 4) but not significant (p = 0.048), likelydue to a fast-desensitization [5] and competition [31] induced byendogenous ATP on P2X receptors.

To minimizing these effects, a 10 min pretreatment withapyrase, an ATP/ADPase that dephosphorylates ATP to ADP thanto AMP, was used. In the presence of 4 U/ml apyrase, exoge-nously applied ATP during S2, but not the P2X1,7 selective agonistBz-ATP (p = 0.05; n = 5; Fig. 5), induced a significant concentration-dependent facilitation on EFS-evoked [3H]-ACh release (maximaleffect of 50.7 ± 2.8%; p = 0.0015; Figs. 4 and 5), as well as on neuro-genic contraction by about 20% (n = 6; Fig. 5).

A more effective facilitation was induced by ABMA (1 �Mto 1 mM), agonist resistant to ecto-enzymes degradation, inreproducible CRCs for [3H]-ACh release (maximal facilitationof 214 ± 20%; n = 9; p = 0.0007; Figs. 5 and 7A). At the sametime, neurogenic contractions were markedly enhanced by ABMA

in concentration-dependent manner (maximal facilitation of252 ± 29% (n = 8; p = 0.0044; Figs. 3C, 5 and 7A)).

Similarly, EFS produced a marked increase in [3H]-ACh release(144 ± 4%, n = 4; p = 0.0008; Fig. 5) following the exposure of 300 �M

esence of 300 �M of ABMA, 120 s before EFS (B) and during EFS (C). For details see

G. D’Agostino et al. / Pharmacological Research 65 (2012) 129– 136 133

Fig. 4. Facilitatory effect on the [3H]-ACh release induced in pig detrusor strips byATP and ABMA in the absence and in the presence of apyrase (4 U/ml). Data are them

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Table 1Comparison of pEC50/pIC50 at P2X homomeric receptors and pKi/pEC50 values cal-culated for P2X ligands at prejunctional level in porcine detrusor.

Ligands Selectivity apEC50/pIC50 pEC50 pKi

ABMA(�,�-me ATP)

P2X1 5.54.06 ± 0.09P2X3 5.7

ATPP2X1 7.0

4.56 ± 0.10P2X3 5.9

Bz-ATPP2X1 4.6

nvP2X3 –P2X7 5.2

SuraminP2X1 5.7

6.74 ± 0.10P2X3 5.4

NF449P2X1 9.5

5.45 ± 0.20P2X3 5.6

NF023P2X1 6.7

5.03 ± 0.14P2X3 5.0

PPNDS P2X1 7.8 <6

A-317491P2X1 4.9

8.06 ± 0.06P2X3 8.0

Fs

ean ± SEM of 3–9 experiments.

RL67156. In the presence of this drug at a concentration whichikely prevents, albeit not totally, ATP conversion without affectingdenosine levels [32], the neurogenic contraction was enhancedoo (by 72 ± 7, n = 4; p = 0.004; Fig. 5).

These results suggest that the observed inability of endoge-ous ATP to induce a significant facilitatory effect on cholinergicctivity in normal detrusor strips is likely the result of thereak down rate of ATP occurring in the parasympatheticleft.

To characterize the type(s) of purinergic receptors affectingFS-evoked [3H]-ACh release and contraction, the CRC of ATP wasarried out in the presence of adenosine receptors antagonists.he blockade of inhibitory A1 and excitatory A2A adenosine recep-ors with 100 �M DPCPX and ZM 241385, respectively selective

ntagonists for A1 and A2A receptor subtypes, did not produce anyignificant variation on both [3H]-ACh release and contraction (dataot shown).

ig. 5. Comparison of EFS-evoked effects ([3H]-ACh release and contraction) induced by Atrips. Apy (apyrase 4 U/ml). Data are the mean ± SEM of 3–9 experiments. (*) p < 0.01; (**

a Data are derived from the references [31,33–35].pEC50/pKi values are means ± SEM of 5–7 experiments; nv: not valuable.

Conversely, the behaviour of CRCs of ABMA was affected in thepresence of P2X receptors antagonists, whose selectivity is listed inTable 1 [31,33–35].

The CRC of ABMA was antagonized by 1 �M suramin (Fig. 6A),but was not affected by 10 �M NF023 (Fig. 6B) nor by 100 nM PPNDSor by NF449 at nanomolar concentrations (not shown). At vari-ance, the CRC of ABMA was competitively antagonized by NF449at micromolar concentrations (Fig. 6C) and by 100 nM A-317491,putative potent antagonist for P2X3 monomer (Fig. 6D).

From CRCs the pharmacodynamic parameters of the used lig-ands were calculated and listed in Table 1 together with theirpublished pEC50/pIC50 values at P2X subunits. The comparison ofthe obtained pIC50/pKi of any selective antagonists with their affin-ity values at P2X subunits is consistent with the notion that P2Xheteroceptors located at cholinergic side belong to P2X3 receptors.

The EFS-evoked contractile effect in normal detrusor strips wascholinergic in origin since it was mostly abolished by atropinein control experiments (by about 91% at 1 �M; not shown). At

TP (1 mM), ABMA (300 �M), Bz-ATP (1 mM) and ARL67156 (300 �M) in pig detrusor) p < 0.001 significance vs controls.

134 G. D’Agostino et al. / Pharmacological Research 65 (2012) 129– 136

Fig. 6. Facilitatory effect on the [3H]-ACh release in pig detrusor strips induced by ABMA in the absence and in the presence of P2X antagonists: (A) Suramin (1 �M); (B)NF023 (10 �M); (C) NF449 (3 �M, 10 �M, 30 �M); and (D) A317491 (100 nM). All experiments were performed in presence of apyrase. Data are the mean ± SEM of 5–7e

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ariance, an atropine-resistant component was present in thenhanced EFS-evoked contractions induced by ABMA. In fact, theseeurogenic contractions were sensitive but not suppressed by 1 �Mtropine, which produced only a right shift of CRC of ABMA, withoutbolishing it (Fig. 7B).

In an attempt to characterize such non-cholinergic component,he neurogenic contractions enhanced in the presence ABMA weretudied in a separate set of experiments after the complete blockadef P2X1 receptors with 100 nM NF449 (pKi 9.5 at P2X1 receptors).n this case too, the CRC of ABMA was shifted to the right but notattened by NF449.

. Discussion

It is widely accepted that parasympathetic postganglionicerves supplying mammals urinary bladder use ACh and ATP aso-transmitters, in proportions variable in different pathophysio-ogical circumstances [6]. Our findings, corroborating the notionhat parasympathetically-released ACh and ATP represent exci-atory neurotransmitters to smooth muscle of porcine detrusor8,36,37], demonstrate that the functional motor drive in physio-ogical conditions prevalently depends on ACh. One account of theegligible participation of neurogenic ATP in eliciting smooth mus-le contraction could be due to the accelerated breakdown of theurine that occurs in the synaptic cleft of normally active porcineladder. Our data likely support the above suggestion, since the

urinergic component reemerges to play a significant role in thefferent signalling pathway when the enzymatic hydrolysis of ATPas experimentally reduced. As of the matter of fact, in the pres-

nce of the potent ecto-ATPase inhibitor ARL67156, endogenous

ATP released in response to nerve stimulation can significantlyenhance [3H]-ACh release and, in turn, smooth muscle contraction.In consideration of the use of ARL67156 at a fixed concentration,which prevents but not able to produce total inhibition of the ecto-ATPase activity [32], agents resistant to ecto-ATPases degradationwould be expected to induce a more effective enhancement. Really,a marked potentiation (by about 250%) was produced in normaldetrusor strips by the non-hydrolyzable ATP analog, ABMA.

Based on the notion that ABMA is a potent agonist to P2X1 andP2X3 receptors [3], the purinergic excitatory motor drive seems tobe essentially dependent on the activation of these P2X subtypes.On the other hand, the involvement of the breakdown productadenosine has to be excluded, as the blockade of specific inhibitoryA1 and excitatory A2A receptors with selective antagonists (namely,DPCPX and ZM 241385, respectively) did not produce any signifi-cant variation on [3H]-ACh release.

The inefficiency of Bz-ATP (P2X1 partial agonist) likely bars theparticipation of P2X1 subunit, but a definitive characterization ofthe involved receptors was carried out by means of selective P2X3receptor antagonists. The comparison of their pharmacodynamicparameters obtained in this study with those published at P2X1–3human recombinant homomeric receptors (see Table 1) clearly sug-gests that the purine receptors affecting ACh release belong to P2X3subunit.

In conditions of up-regulation of the purinergic pathway (i.e.experimentally-reduced rate of ATP hydrolysis or stimulation with

a non-hydrolyzable ATP analog), this P2X3-dependent prejunc-tional excitatory mechanism raises to a relevant role in producingsmooth muscle hyperactivity. Such smooth muscle potentiationis likely due to the concomitant activation of muscarinic M3 [28]

G. D’Agostino et al. / Pharmacologica

Fig. 7. (A) Concentration–response curve for EFS-evoked [3H]-ACh release (©)and smooth muscle contraction (�) induced by ABMA in pig detrusor strips. (B)Concentration–response curve for EFS-evoked smooth muscle contraction inducedbN

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y ABMA in the absence (�) and in the presence of 1 �M atropine (�) or 100 nMF449 (�). Data are the mean ± SEM of 8–9 experiments.

nd P2X1 muscular receptors [5]. Indeed, the enhancement of EFS-nduced contraction was atropine-resistant to a great extent (morehan 50%) as well as only partially blocked by the P2X1 antagonistF449.

In this respect, it is noteworthy that atropine-resistance phe-omenon is described in isolated strips of bladders from patientsuffering for DO [5] where purinergic activity is enhanced dueo a reduced degree of purine breakdown [3,38]. Taken together,ur data suggest that in a suitable animal model for urologicalesearch the purinergic-receptor mechanisms involved in poten-iating detrusor motor drive comprise excitatory muscular P2X1 asell as neural P2X3 subunits facilitating the release of ACh, and

heir blockade can efficaciously contribute in reducing detrusoryocytes overexcitation in experimentally-matched pathological

onditions.

[

l Research 65 (2012) 129– 136 135

5. Conclusion

The presence of P2X3 subunits have been previously revealedby immunohistochemistry on the urinary bladder of mammals,prevailingly on sensory nerves and in nerve bundles beneath theurothelium [5]. Our data support the notion that P2X3 recep-tors exert the role of excitatory heteroceptors on parasympatheticterminals and provide direct evidence that in pig detrusor thedown-regulation of purine breakdown produces smooth musclehyperactivity, a condition resembling that observed in humaninstable detrusors.

Since the up-regulation of P2X3 receptors, predominant subunitin human bladder nerve profiles [5], seems to be involved in gen-erating symptoms of bladder dysfunctions [39], orally bioavailableantagonists with selectivity for P2X3 receptors have been suggestedas potential clinical candidates in the treatment of pain-relateddisorders of the lower urinary tract [40]. If our findings couldbe extrapolated to humans, the blockade of P2X3 heteroceptorson cholinergic terminals might represent an additional puriner-gic target in the control of pathological conditions of the detrusor,particularly when associated with motor (efferent) abnormalities.Therefore, since obtaining a large supply of human bladder speci-mens for functional assessments is not so feasible, isolated porcinedetrusor provides a reliable animal model to develop novel selec-tive P2X3 antagonists for human use.

Acknowledgments

This work was supported by grants to G.D. from BML Foundation(Milan, Italy). Portions of this work were previously presented atthe 2007 SIF Congress, 6–9 June, Cagliari, Italy.

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