Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31

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Autonomic Neuroscience: Basic and Clinical

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Functional and morphological examinations of P1A1 purinoceptors in the normal andinflamed urinary bladder of the rat

Renata Vesela a,b, Patrik Aronsson b, Gunnar Tobin b,⁎a Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, CZ-50005 Hradec Kralove, Czech Republicb Department of Pharmacology, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 431, SE-40530 Gothenburg, Sweden

⁎ Corresponding author. Department of Pharmacology431, SE-40530 Gothenburg, Sweden. Tel.: +46 31 786 34

E-mail address: gunnar.tobin@pharm.gu.se (G. Tobin

1566-0702/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.autneu.2010.07.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 May 2010Received in revised form 9 July 2010Accepted 9 July 2010

Keywords:Rat urinary bladderPurinergicCystitisP1Purinoceptor

The aim of the present study was to investigate the relaxatory function of adenosine receptor subtypes in raturinary bladder, and if it is altered in the state of inflammation. The in vitro responses to the P1 receptor agonistadenosine were investigated in the presence of the general P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 1⁎10−4 M). Experimentswere performed onpreparations fromnormal(healthy) rats and rats with cyclophosphamide (CYP; 100 mg kg−1 i. p.)-induced cystitis. The specific P1A1

antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1⁎10−5 M) decreased the adenosine relaxatoryresponse in normal bladders (−60%), but not in preparations from CYP pre-treated rats. Immunohistochemicalfindings support the hypothesis that the expression of P1A1 receptors in the rat urinary bladder is decreasedduring cystitis. The adenosine-evoked relaxation was not affected by the specific P1A2A antagonist SCH 58261(3⁎10−7 M), neither in normal nor in CYP pre-treated rats. The relaxation to adenosine was, however,significantly increased by the specific P1A3 antagonist MRS 1523 (1⁎10−5 M) in preparations from both normalandCYPpre-treated rats, suggestingP1A3 tobemediating bladder contraction. Thus, in the rat urinarybladder therelaxation to adenosine is mainly due to the P1A1 receptor, while the P1A3 receptor seems to be responsible forcontractile responses. The DPCPX-resistant part of the relaxation is possibly due to the P1A2B receptor, the fourthsubtype of the adenosine receptor family.

, University of Gothenburg, Box40; fax: +46 31 786 31 64.).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The functional effects by the autonomic nervous system in the lowerurinary tract may be substantially altered during inflammatory condi-tions (Giglio and Tobin, 2009). In experimentally induced cystitis in therat, the cholinergic contractile responses are reduced in associationwithmore pronounced nitric oxide (NO) mediated effects (Giglio et al.,2005). In patients suffering from interstitial cystitis, it has been reportedthat afferent pathways are sensitized and the release of adenosine 5′-triphosphate (ATP) and NO are associated to the condition (Andersson,2002; Logadottir et al., 2004; Sun and Chai, 2006). ATP produced in theurothelium initiates the stretch-induced micturition reflex by acting onP2X2 and P2X3 receptors on afferent nerve fibers (Wang et al., 2005).However, in the rat urinary bladder, ATP generates a transientcontraction caused by the stimulation of the P2X1 purinoceptors also(Ford et al., 2006). Following the contraction a sustained relaxationoccurs, which has been suggested to depend on the metabolite of ATP,adenosine, stimulating P1 purinoceptors (Burnstock et al., 1972; Giglioet al., 2001; Aronsson et al., 2010). However, conflicting results exist,

suggesting a direct action of ATP on G-protein coupled P2Y purinocep-tors (McMurray et al., 1998).

The purinoceptors exist in two principal types; the ones on whichATP is the primary agonist, and the others onwhich adenosineprimarilyacts. The former group consists of P2X (P2X1–P2X7 in mammals) andP2Y (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11–14) purinoceptors, being ligand-gated ion channels and G-protein coupled receptors, respectively. Thelatter group, the P1 purinoceptors, consist of 4 subtypes, A1, A2A, A2B andA3, all being G-protein coupled, and which all occur in the rat urinarybladder (Yu et al., 2006). The A1 receptor inhibits adenylyl cyclase, butalso activates phospholipase C (PLC) (Bucheimer and Linden, 2004). TheA2A receptor activates adenylyl cyclase, which the A2B receptor subtypealso does, but in addition A2A may activate the PLC-pathway as well(Feoktistov and Biaggioni, 1997). The A3 receptor, on the other hand,inhibits adenylyl cyclase and stimulates PLC, IP3 and intracellularcalcium via Gq-proteins (Gessi et al., 2008).

Besides its participation in the micturition reflex, ATP may inducepro-inflammatory effects in the human urinary tract, most likely viaP2Y1-, P2Y2-and/or P2Y11-receptor activation (Säve, 2010). Also,adenosine produced from ATP during inflammation is considered tobe an important factor participating in the regulation of inflammation(Hasko and Cronstein, 2004; Sitkovsky et al., 2004). While activation ofP1A1 and P1A3 receptors seems to evoke pro-inflammatory effects,P1A2A and P1A2B receptors elicit anti-inflammatory effects.

27R. Vesela et al. / Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31

We have previously shown that concomitantly to the inflamma-tion-induced alteration of the cholinergic responses in the rat,mucosal muscarinic M5 receptors as well as endothelial nitric oxidesynthase (eNOS) are up-regulated (Giglio et al., 2005; Andersson etal., 2008). However, altered expression of purinoceptors seems also tooccur in cystitis. While the expression of P2X1 and P2Y2 receptors isreduced in inflammation, the expression of P2X2 and P2X3 receptorsmay be increased (Birder et al., 2004; Dang et al., 2008). Likewise,results from cell cultures indicate an increase of P1A2A receptors,while P1A1 expression may be decreased (Save et al., 2009).

In view of the possible changes of purinoceptor expression, thepresent study was undertaken to investigate if the expression of P1A1

receptors and their functional effects are affected by cyclophosphamide(CYP)-induced cystitis in the rat. Furthermore, we wanted to relate thefunctional P1A1 response to that of other P1 purinoceptors. For thispurpose, we used different antagonists, pyridoxal-phosphate-6-azophe-nyl-2′,4′-disulfonic acid (PPADS), a general P2 antagonist (Lambrechtet al., 1992), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selectiveP1A1 antagonist (Haleen et al., 1987), SCH 58261, a selective P1A2A

antagonist (Zocchi et al., 1996; Belardinelli et al., 1998), andMRS 1523, aselective P1A3 antagonist (Mitchell et al., 1999; Gao et al., 2001; Ansariet al., 2007; Modis et al., 2009), in order to assess inhibitory effects onresponses evoked by the P1 purinoceptor agonist adenosine. In order toinduce experimental cystitis, rats were pre-treated with CYP, a cyto-static agent that is used in the treatment of neoplastic diseases. Themetabolite of CYP, acrolein, has been attributed as being responsible forinducing haemorrhagic cystitis after CYP treatment (Cox, 1979; Batistaet al., 2006). The P1A1 purinoceptor expression was examined byimmunohistochemistry.

2. Materials and methods

The Ethics Committee at the University of Gothenburg approved thestudy design, in which 25 adult male rats (300–350 g) of the Sprague–Dawley strain were used. 60 h prior to the experiments, rats wereadministered either a single dose of CYP (100 mg kg−1 i.p.) in order toyield a pronounced cystitis at the time of the experiment, as previouslydescribed (Giglio et al., 2005), or saline (9 mg kg−1 i.p.) serving ascontrol. The administration of both CYP and salinewas conducted in thepresence of the analgesic buprenorphinum (10 μg kg−1 i.m.).

2.1. Functional experiments

60 h after the CYP/saline pre-treatment the rats were intraperito-neally injected with medetomidin (Domitor® vet., 0.2 ml, Apoteket AB,Sweden) for anesthesia and then killed by an overdose of carbondioxide. Theurinarybladderwas removed, cut andopened. Twoor threestrips (6×2 mm) were excised from the body of the organ. To preventdamaging the tissue by drying out, the organ was kept in Krebsbicarbonate solution at all times. For the contraction experiments, thestrips were fastened between two steel rods, one of which wasadjustable. The organ baths were filled with Krebs bicarbonate solution(deionized water, NaCl 118 mM, KCl 4.6 mM, KH2PO4 1.18 mM, MgSO4

1.16 mM, NaHCO3 25 mM, glucose 5.5 mM and CaCl2 1.27 mM). TheKrebs solutionwaskept at 37 °Cand gassedwith5%CO2 inO2 during thewhole experiment to maintain a stable neutral pH and to provide thetissuewith oxygen. The strips were stretched to a tension of 15 mN andleft to equilibrate for 45 min. This resulted in gradual relaxationresulting in a stable tension of 7–9 mN. After 45 min, administrationof potassium-containing Krebs solution (containing 124 mM K+;obtained by exchanging Na+ for equimolar amounts of K+) wasperformed and the resulting contractionwasused as a viability referringresponse. The tension was then stabilized for each strip at 7–9 mN. Thisis a somewhat higher basal tension that is normally used (4–5 mN;Tobin and Sjogren, 1995). This tension turned out to amplify therelaxations. After 30 min of resting, medium potassium Krebs was

administrated (50 mM K+; obtained by exchanging Na+ for equimolaramounts of K+) to pre-contract the tissue. An observation period of20 min followed in order to establish a stable pre-contraction largerthan 2.5 mN above the basal tension level. After 20 min of pre-contraction, adenosine was added at a standard concentration shownto evoke stable relaxatory responses of the rat urinary bladder strips(5⁎10−5 M). This concentration was possible to dilute in water,whereas any higher concentration would have had to be diluted indimethyl sulphoxide (DMSO). When the maximal relaxation of thetissue was obtained, usually after approximately 150 s, the organ bathswere washed thoroughly and the strips were re-stretched to 7–9 mNand let to equilibrate for 30 min. Subsequently, an antagonistwas addedand the preparations were pre-contracted by medium potassium Krebssolution for a 20 min incubation period, after which the relaxatoryresponse to adenosine (5⁎10−5 M) was evaluated. This procedure wasrepeated for all antagonists used (i.e. PPADS, DPCPX and SCH 58261,MRS 1523, respectively). The agonist and all antagonists were added inthe volume of 125 μl.

2.2. Immunohistochemistry

Sections of rat urinary bladder were prepared (n=16; n=4 in eachgroup; normals (active and control groups) and CYP pre-treated (activeand control groups)) and were investigated by immunohistochemistryusing subtype specific purinoceptor P1A1 antibody. The specimenswerefixed in phosphate buffered 4% paraformaldehyde (pH 7.0), and thenembedded in paraffin. Transverse sections of the different specimenswere prepared at a thickness of 4 μm and adhered to glasses. Thesections were de-paraffinized by heating them to 60 °C for 15 min andthen subjecting them to two 30-min intervals in 100% xylene; thesections were then re-hydrated by serial incubations in 100%, 95%, 85%and 70% ethanol, followed by tris-buffered saline (TBS). Then the glasseswere heated four times for 6 min in a microwave, followed by blockadeof endogen peroxidase by 0.03% hydrogen peroxide for 30 min. Theblocking of non-specific background was managed by 5% bovine serumalbumin (BSA) in tris-buffered saline (TBS) for 30 min. The sectionswere incubated overnight with primary antibody (Anti-A1 AdenosineReceptor antibody produced in rabbit; Sigma Aldrich, Sweden) diluted1:10 in 1% BSA in TBS and for subsequent binding of the secondaryantibody (ABC Staining System, Santa Cruz Biotechnology, Santa Cruz,CA), the glasseswere incubated for 30 min, followed by incubationwithAB enzyme reagent for 30 min. Finally the tissues were stained withhematoxylin for 2–4 min. Thenegative control sampleswere treated thevery same way, except for not being exposed to primary antibody.

2.3. Materials

Substances used in contraction experiments were as follows:adenosine (FW=267.24; Sigma Aldrich, Sweden), pyridoxal-phos-phate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate(PPADS; FW=599.31 (anhydrous basis); Sigma Aldrich, Sweden),1,3-dipropyl-8-cyclopentylxanthine (DPCPX; FW=304.39; SigmaAldrich, Sweden), SCH 58261 (FW=345.36; Sigma Aldrich, Sweden),MRS 1523 (FW=395.55; Sigma Aldrich, Sweden), and dimethylsulphoxide (DMSO, FW=78.13; Sigma Aldrich).

For immunohistochemistry the following materials and sub-stances were used: ABC staining system sc-2018 for use with rabbitprimary antibody (Santa Cruz Biotechnology, USA), anti-A1 purinergicreceptor-developed in rabbit (Sigma Aldrich, Sweden), and Mayer'shematoxylin (Histolab, Gothenburg, Sweden).

2.4. Statistics

The relaxatory responses were normalized to pre-contractileresponses and arepresented aspercentage of pre-contraction. Statisticalsignificance was determined by Student's t-test for paired or unpaired

Fig. 1. Mean relaxatory responses to adenosine (5⁎10−6–5⁎10−3 M) in normalbladder strip preparations. The arrow marks the standard concentration (5⁎10−5 M)used in following experiments. The vertical bars represent S.E.M.

Fig. 3. Normal rats: Mean relaxatory responses to adenosine (5⁎10−5 M; ), in thepresence of PPADS (1⁎10−4 M; ), DPCPX (1⁎10−5 M; ) and both PPADS+DPCPX (1⁎10−4 M and 1⁎10−5 M respectively; ). Vertical bars represent S.E.M.*pb0.05, ***pb0.001.

28 R. Vesela et al. / Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31

data. When multiple comparisons with the same variable were made,statistical significance was determined by one-way analysis of variance(ANOVA) followed by the Bonferroni multiple comparison tests usingthe GraphPad Prism program (GraphPad Software, Inc., San Diego, US).P-values of 0.05or lesswere regarded as statistically significant. Data arepresented in the form of means±S.E.M.

3. Results

3.1. Functional experiments

The pre-contraction to medium potassium Krebs (50 mM) was5.07±0.33 mN (n=40) above baseline in the normal rats and6.50±0.44 mN (n=40) in the CYP pre-treated rats. Adenosine(5⁎10−6–5⁎10−3 M) evoked concentration dependent relaxationsof pre-contracted strip preparations (Fig. 1). However, the responseswere significantly larger in the normal than in the inflamed bladders.At 5⁎10−5 M the relaxation was −24±2% (n=21) in the normaland −12±1% (n=24) in the CYP pre-treated bladders (pb0.05).Also the latency to a stable relaxatory response varied; in normal ratsthe latency was 88±3 s and in CYP pre-treated rats 132±3 s;pb0.05. Also, the first part of the relaxation was faster in the normalstrips than in the inflamed, as indicated in Fig. 2.

For examination of the potency of the antagonists, adenosine wasadministered at a standard concentration of 5⁎10−5 M, which causedwell-preserved responseswhen administered repeatedly in the absenceof any antagonist. Adenosine5⁎10−5 Madministered in thepresence ofPPADS (1⁎10−4 M) evoked slightly increased relaxatory responses incomparisonwith adenosine administered in its absence (−15±0.8% vs.−19±1%; pb0.05). The presence of DPCPX (1⁎10−5 M) substantiallyreduced the relaxatory response (to−5±1%; pb0.001) and prolonged

Fig. 2. Representative recordings showing the latency of reaching the maximumrelaxation in normal rats (left) and CYP pre-treated rats (right). The numbers belowrefer to the mean±S.E.M. latencies for all animals.

the latency (104±4 s), and further, adenosine administration in thepresence of both antagonists evoked similar responses as with onlyDPCPX present (Fig. 3). However, neither PPADS (1⁎10−4 M) norDPCPX (1⁎10−5 M) affected the relaxatory response of strip prepara-tion from inflamed urinary bladder (Fig. 4).

In order to compare the P1A1 purinoceptor response with that ofany other P1 purinoceptor subtype, the adenosine response wasexamined in the presence of P1A2A and P1A3 antagonists. In order toavoid P2 purinoceptor effects, these experiments were carried out inthe presence of PPADS (1⁎10−4 M). The adenosine (5⁎10−5 M)responsewas not affected by SCH58261 (3⁎10−7 M) neither in normal(−8±0.5% vs. −10±2%, in the absence and presence of SCH 58261,respectively; pN0.05) nor in CYP pre-treated rats (−6±0.9% vs.−9±1%; pN0.05; Figs. 5 and 6). However, the incubation with both SCH58261 and MRS 1523 (1⁎10−5 M) caused significant amplification ofadenosine induced relaxations. Increased relaxations appeared in bothnormal (−10±2% vs. −24±1%; pb0.001) and in CYP pre-treatedurinary bladder strips (−9±1% vs. −20±4%; pb0.05; Figs. 5 and 6).

3.2. Immunohistochemistry

In the bladders from the CYP pre-treated rats, the urothelium wasslightly disrupted and an increased amount of vacuoles was seen inthe suburothelium when examined 48–60 h after injection. Thestaining for the P1A1 purinoceptor showed obvious expression ofthe receptor subtype in the urinary bladder from normal rats (n=4).However, staining was weak or absent in CYP pre-treated (n=4;Fig. 7). In the normal bladder, the staining of the P1A1 purinoceptors

Fig. 4. CYP pre-treated rats: Mean relaxatory responses to adenosine (5⁎10−5 M;) in the absence of antagonist, in the presence of PPADS (1⁎10−4 M; ),

DPCPX (1⁎10−5 M; ) and both PPADS+DPCPX (1⁎10−4 M and 1⁎10−5 Mrespectively; ). Vertical bars represent S.E.M. No statistical significant differenceoccurred.

Fig. 5. Normal rats: Mean relaxatory responses to adenosine (5⁎10−5 M) in the presenceof PPADS (1⁎10−4 M; ), PPADS+SCH 58261 (1⁎10−4 M and 3⁎10−7 Mrespectively; ) and PPADS+SCH 58261+MRS 1523 (1⁎10−4 M, 3⁎10−7 M and1⁎10−5 M respectively; ). Vertical bars represent S.E.M. ***pb0.001.

Fig. 6. CYP pre-treated rats: Mean relaxatory responses to adenosine (5⁎10−5 M) in thepresence of PPADS (1⁎10−4 M; ), PPADS+SCH 58261(1⁎10−4 M and 3⁎10−7 Mrespectively; ) and PPADS+SCH 58261+MRS 1523 (1⁎10−4 M, 3⁎10−7 M and1⁎10−5 M respectively; ). Vertical bars represent S.E.M. *pb0.05.

Fig. 7. Immunohistochemical staining of P1A1 receptors. Upper row shows bladders pre-treat(cystitis). Left column illustrates the control group which was not exposed to primary antibowith hematoxylin. The horizontal bar represents 100 μm.

29R. Vesela et al. / Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31

was particularly intense in the urothelium, but seemed to appear in allof the bladder wall layers.

4. Discussion

The present study shows that adenosine evokes relaxation of theisolated urinary bladder, whichmainly depends on the activation of P1A1

purinoceptors. No relaxation in response to P1A2A purinoceptor stimu-lation was observed. It was also found that in the presence of the P1A3

purinoceptor antagonist MRS 1523, the adenosine relaxation wasenhanced. Considering the intracellular couplingof theP1A3purinoceptorto PLC, IP3 and release of intracellular calcium, the enhancement of therelaxation seems likely to reflect a contractile adenosine P1A3 purino-ceptor effect. In the CYP pre-treated bladder, a substantial attenuation ofthe P1A1 purinoceptor relaxation occurred. This vanishing of the P1A1

purinoceptor response correlated to the immunohistochemistry findingsindicating a down-regulation of the expression of the receptor. Regardingthe other purinoceptors examined in the current study; that is the P1A2A

and P1A3 purinoceptors, no alterations of the functional effects mediatedby these receptors appeared in the state of CYP-induced inflammation.Regarding P1A2B purinoceptors it has been reported that they areinvolved in the purinergic relaxation in the normal urinary bladder of therat (Aronsson et al., 2010). The present findings indicate that the P1A2B

purinoceptor may also be responsible for a substantial part of theadenosine relaxation in the inflamed urinary bladder of the rat.

The contraction of the urinary bladder is primarily dependent on theactivation of muscarinic receptors. It has been shown that muscarinicM3 receptors are mainly responsible for the bladder contraction, whilemuscarinic M2 receptors contribute most significantly by opposingrelaxations induced by adenylate cyclase-coupled receptors such as theβ-adrenoceptors and P1 purinoceptors (Hegde et al., 1997; Giglio et al.,2001). Purines also participate in the regulation of themicturition reflex(King et al., 2004). While ATP principally causes contraction, it may in

ed with saline (normal bladders), bottom row the cyclophosphamide pre-treated tissuedy, right column the P1A1 receptor staining (positive samples). All sections are stained

30 R. Vesela et al. / Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31

addition, by its breakdown to adenosine, evoke a delayed relaxatoryresponse (Bailey and Hourani, 1994; Aronsson et al., 2010). However,both molecules may affect the inflammation, and adenosine has beenproved to have both pro- and anti-inflammatory effects in the lowerurinary tract (Save et al., 2009). Even though the neuronal purinergicresponse seems to be absent, or minute, in the urinary bladder inhealthy human, purines are produced from other sources as well(Sjogren et al., 1982; Save et al., 2009). For instance purines releasedfrom the urotheliummay still activate sensory neurons and participatein the regulation of inflammation (Andersson and Hedlund, 2002; Saveet al., 2009).

P1 purinoceptors are widely distributed in the body and in theurinary bladder large numbers occur (Volpini et al., 2003). Immunesystem cells also express P1 purinoceptors, and are responsive to themodulatory effects of adenosine in an inflammatory environment(Hasko et al., 2008). The characterization of the purinoceptors byemploying immunohistochemistry indicated that P1A1 receptors existin theurinary bladder of themale rat. The staining showed the receptorsto be localized to all parts of the urinary bladder; in the urothelium,suburothelially and even in the detrusormuscle. The urinary bladders ofthe cyclophosphamide pre-treated rats showed evidently less intensestaining. These findings support the suggestion that during theinflammationof the raturinary bladder the expressionof P1A1 receptorsis decreased. This corresponds well with results from cell cultures inwhich E. coli exposure results in a down-regulation of P1A1 purinocep-tors (Save et al., 2009). Furthermore the finding of the existence of P1A1

receptors in theurothelium is supportedbypreviousfindings (Aronssonet al., 2010), showing a somewhat smaller relaxatory response to ATP inurothelium-denuded bladders than in normal bladders.

The current functional examination revealed that approximately 2/3of the adenosine relaxationwasmediated via P1A1 purinoceptors in thenormal bladder. In order to avoid any additional relaxatory effectevoked by P2Y purinoceptors, PPADSwas added. This seemed to inducea larger adenosine relaxation, which could be interpreted as anadenosine P2Y purinoceptor mediated contraction. However, a draw-back with many of the compounds acting on the purinoceptors is thatthey often exert a number of effects, which makes the relatively small,however statistically significant, change in the presence of PPADS ofminor importance (Chen et al., 1996; Lambrecht, 2000). Concerning theselective P1 purinoceptor antagonists used in the present experiments,the concentrations used are the same as in other functional studiesclaiming selectivity (Lee and Reddington, 1986; Haleen et al., 1987;Belardinelli et al., 1998; Mitchell et al., 1999; Modis et al., 2009;Aronsson et al., 2010). The pronounced differences in the responsessupport that selective antagonism occurred. For instance, the blockadeof P1A1 purinoceptors almost abolished the adenosine relaxation, theblockade of P1A2A purinoceptors had no effect, and the blockade of P1A3

purinoceptors had the reversed effect. Also, the P1A1 immunohisto-chemistry study revealed morphological correlates to the functionalfindings according to this specific receptor subtype.

The adenosine-evoked relaxation was reduced by approximately1/3 in CYP pre-treated rats. While most of the response was abolishedby P1A1 purinoceptor blockade in healthy bladders, just a small partwas affected in the inflamed bladders. Since neither P1A2A nor P1A3

purinoceptor blockade inhibited this response, it may tentatively bethe result of P1A2B purinoceptor activation. In the state of inflamma-tion, compensational interactions between several mechanisms arelikely to occur, but still the P1A2B purinoceptor is a strong candidate tobe involved at one or another stage. Nevertheless, the relaxation wasobtained with a shorter latency in strips from normal bladders than instrips from inflamed bladders. Also the shape of the curves describingthe relaxatory responses differed, as could be judged by the latency tonadir. It is worth noting that in the presence of the P1A1 purinoceptorantagonist DPCPX the latency was prolonged. This suggests that therelaxation due to stimulation of the P1 adenosine receptor is caused toa large part by the P1A1 subtype.

5. Conclusions

The performed experiments show that P1A1 receptor is present inthe rat urinary bladder. They also show that the expression of the P1A1

receptor subtype is decreased during the cystitis, which affects thefunction of the urinary bladder. A tentative candidate for the relaxationin the CYP pre-treated rat is the P1A2B purinoceptor.

Acknowledgements

This study was supported by grants from Wilhelm och MartinaLundgrens Vetenskapsfond.

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