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Functional and morphological examinations of P1A1 purinoceptors in the normal and inflamed urinary bladder of the rat
Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31
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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Functional and morphological examinations of P1A1 purinoceptors in the normal and inflamed urinary bladder of the rat Renata Vesela a,b, Patrik Aronsson b, Gunnar Tobin b,⁎ a b
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, CZ-50005 Hradec Kralove, Czech Republic Department of Pharmacology, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 431, SE-40530 Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 5 May 2010 Received in revised form 9 July 2010 Accepted 9 July 2010 Keywords: Rat urinary bladder Purinergic Cystitis P1 Purinoceptor
a b s t r a c t The aim of the present study was to investigate the relaxatory function of adenosine receptor subtypes in rat urinary bladder, and if it is altered in the state of inflammation. The in vitro responses to the P1 receptor agonist adenosine were investigated in the presence of the general P2 receptor antagonist pyridoxal-phosphate-6azophenyl-2′,4′-disulfonic acid (PPADS; 1 ⁎ 10− 4 M). Experiments were performed on preparations from normal (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 relaxatory response in normal bladders (−60%), but not in preparations from CYP pre-treated rats. Immunohistochemical findings support the hypothesis that the expression of P1A1 receptors in the rat urinary bladder is decreased during 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 normal and CYP pre-treated rats, suggesting P1A3 to be mediating bladder contraction. Thus, in the rat urinary bladder the relaxation to adenosine is mainly due to the P1A1 receptor, while the P1A3 receptor seems to be responsible for contractile responses. The DPCPX-resistant part of the relaxation is possibly due to the P1A2B receptor, the fourth subtype of the adenosine receptor family. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The functional effects by the autonomic nervous system in the lower urinary tract may be substantially altered during inflammatory conditions (Giglio and Tobin, 2009). In experimentally induced cystitis in the rat, the cholinergic contractile responses are reduced in association with more pronounced nitric oxide (NO) mediated effects (Giglio et al., 2005). In patients suffering from interstitial cystitis, it has been reported that 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 the urothelium initiates the stretch-induced micturition reflex by acting on P2X2 and P2X3 receptors on afferent nerve fibers (Wang et al., 2005). However, in the rat urinary bladder, ATP generates a transient contraction caused by the stimulation of the P2X1 purinoceptors also (Ford et al., 2006). Following the contraction a sustained relaxation occurs, which has been suggested to depend on the metabolite of ATP, adenosine, stimulating P1 purinoceptors (Burnstock et al., 1972; Giglio et al., 2001; Aronsson et al., 2010). However, conflicting results exist,
⁎ Corresponding author. Department of Pharmacology, University of Gothenburg, Box 431, SE-40530 Gothenburg, Sweden. Tel.: +46 31 786 34 40; fax: +46 31 786 31 64. E-mail address: [email protected] (G. Tobin). 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.07.008
suggesting a direct action of ATP on G-protein coupled P2Y purinoceptors (McMurray et al., 1998). The purinoceptors exist in two principal types; the ones on which ATP is the primary agonist, and the others on which adenosine primarily acts. The former group consists of P2X (P2X1–P2X7 in mammals) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11–14) purinoceptors, being ligandgated ion channels and G-protein coupled receptors, respectively. The latter group, the P1 purinoceptors, consist of 4 subtypes, A1, A2A, A2B and A3, all being G-protein coupled, and which all occur in the rat urinary bladder (Yu et al., 2006). The A1 receptor inhibits adenylyl cyclase, but also activates phospholipase C (PLC) (Bucheimer and Linden, 2004). The A2A receptor activates adenylyl cyclase, which the A2B receptor subtype also 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 intracellular calcium via Gq-proteins (Gessi et al., 2008). Besides its participation in the micturition reflex, ATP may induce pro-inflammatory effects in the human urinary tract, most likely via P2Y1-, P2Y2-and/or P2Y11-receptor activation (Säve, 2010). Also, adenosine produced from ATP during inflammation is considered to be an important factor participating in the regulation of inflammation (Hasko and Cronstein, 2004; Sitkovsky et al., 2004). While activation of P1A1 and P1A3 receptors seems to evoke pro-inflammatory effects, P1A2A and P1A2B receptors elicit anti-inflammatory effects.
R. Vesela et al. / Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31
We have previously shown that concomitantly to the inflammation-induced alteration of the cholinergic responses in the rat, mucosal muscarinic M5 receptors as well as endothelial nitric oxide synthase (eNOS) are up-regulated (Giglio et al., 2005; Andersson et al., 2008). However, altered expression of purinoceptors seems also to occur in cystitis. While the expression of P2X1 and P2Y2 receptors is reduced in inflammation, the expression of P2X2 and P2X3 receptors may 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, the present 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 the functional P1A1 response to that of other P1 purinoceptors. For this purpose, we used different antagonists, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a general P2 antagonist (Lambrecht et al., 1992), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective P1A1 antagonist (Haleen et al., 1987), SCH 58261, a selective P1A2A antagonist (Zocchi et al., 1996; Belardinelli et al., 1998), and MRS 1523, a selective P1A3 antagonist (Mitchell et al., 1999; Gao et al., 2001; Ansari et al., 2007; Modis et al., 2009), in order to assess inhibitory effects on responses evoked by the P1 purinoceptor agonist adenosine. In order to induce experimental cystitis, rats were pre-treated with CYP, a cytostatic agent that is used in the treatment of neoplastic diseases. The metabolite of CYP, acrolein, has been attributed as being responsible for inducing haemorrhagic cystitis after CYP treatment (Cox, 1979; Batista et al., 2006). The P1A1 purinoceptor expression was examined by immunohistochemistry. 2. Materials and methods The Ethics Committee at the University of Gothenburg approved the study design, in which 25 adult male rats (300–350 g) of the Sprague– Dawley strain were used. 60 h prior to the experiments, rats were administered either a single dose of CYP (100 mg kg− 1 i.p.) in order to yield a pronounced cystitis at the time of the experiment, as previously described (Giglio et al., 2005), or saline (9 mg kg− 1 i.p.) serving as control. The administration of both CYP and saline was conducted in the presence 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 intraperitoneally injected with medetomidin (Domitor® vet., 0.2 ml, Apoteket AB, Sweden) for anesthesia and then killed by an overdose of carbon dioxide. The urinary bladder was removed, cut and opened. Two or three strips (6× 2 mm) were excised from the body of the organ. To prevent damaging the tissue by drying out, the organ was kept in Krebs bicarbonate solution at all times. For the contraction experiments, the strips were fastened between two steel rods, one of which was adjustable. 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). The Krebs solution was kept at 37 °C and gassed with 5% CO2 in O2 during the whole experiment to maintain a stable neutral pH and to provide the tissue with oxygen. The strips were stretched to a tension of 15 mN and left to equilibrate for 45 min. This resulted in gradual relaxation resulting in a stable tension of 7–9 mN. After 45 min, administration of potassium-containing Krebs solution (containing 124 mM K+; obtained by exchanging Na+ for equimolar amounts of K+) was performed and the resulting contraction was used as a viability referring response. The tension was then stabilized for each strip at 7–9 mN. This is a somewhat higher basal tension that is normally used (4–5 mN; Tobin and Sjogren, 1995). This tension turned out to amplify the relaxations. After 30 min of resting, medium potassium Krebs was
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administrated (50 mM K+; obtained by exchanging Na+ for equimolar amounts of K+) to pre-contract the tissue. An observation period of 20 min followed in order to establish a stable pre-contraction larger than 2.5 mN above the basal tension level. After 20 min of precontraction, adenosine was added at a standard concentration shown to 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 in dimethyl sulphoxide (DMSO). When the maximal relaxation of the tissue was obtained, usually after approximately 150 s, the organ baths were washed thoroughly and the strips were re-stretched to 7–9 mN and let to equilibrate for 30 min. Subsequently, an antagonist was added and the preparations were pre-contracted by medium potassium Krebs solution for a 20 min incubation period, after which the relaxatory response to adenosine (5⁎ 10− 5 M) was evaluated. This procedure was repeated for all antagonists used (i.e. PPADS, DPCPX and SCH 58261, MRS 1523, respectively). The agonist and all antagonists were added in the volume of 125 μl. 2.2. Immunohistochemistry Sections of rat urinary bladder were prepared (n= 16; n = 4 in each group; normals (active and control groups) and CYP pre-treated (active and control groups)) and were investigated by immunohistochemistry using subtype specific purinoceptor P1A1 antibody. The specimens were fixed in phosphate buffered 4% paraformaldehyde (pH 7.0), and then embedded in paraffin. Transverse sections of the different specimens were prepared at a thickness of 4 μm and adhered to glasses. The sections were de-paraffinized by heating them to 60 °C for 15 min and then subjecting them to two 30-min intervals in 100% xylene; the sections were then re-hydrated by serial incubations in 100%, 95%, 85% and 70% ethanol, followed by tris-buffered saline (TBS). Then the glasses were heated four times for 6 min in a microwave, followed by blockade of endogen peroxidase by 0.03% hydrogen peroxide for 30 min. The blocking of non-specific background was managed by 5% bovine serum albumin (BSA) in tris-buffered saline (TBS) for 30 min. The sections were incubated overnight with primary antibody (Anti-A1 Adenosine Receptor antibody produced in rabbit; Sigma Aldrich, Sweden) diluted 1:10 in 1% BSA in TBS and for subsequent binding of the secondary antibody (ABC Staining System, Santa Cruz Biotechnology, Santa Cruz, CA), the glasses were incubated for 30 min, followed by incubation with AB enzyme reagent for 30 min. Finally the tissues were stained with hematoxylin for 2–4 min. The negative control samples were treated the very 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-phosphate-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; Sigma Aldrich, Sweden), SCH 58261 (FW = 345.36; Sigma Aldrich, Sweden), MRS 1523 (FW = 395.55; Sigma Aldrich, Sweden), and dimethyl sulphoxide (DMSO, FW = 78.13; Sigma Aldrich). For immunohistochemistry the following materials and substances were used: ABC staining system sc-2018 for use with rabbit primary antibody (Santa Cruz Biotechnology, USA), anti-A1 purinergic receptor-developed in rabbit (Sigma Aldrich, Sweden), and Mayer's hematoxylin (Histolab, Gothenburg, Sweden). 2.4. Statistics The relaxatory responses were normalized to pre-contractile responses and are presented as percentage of pre-contraction. Statistical significance was determined by Student's t-test for paired or unpaired
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Fig. 3. Normal rats: Mean relaxatory responses to adenosine (5 ⁎ 10− 5 M; ), 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 M respectively; ). Vertical bars represent S.E.M. *p b 0.05, ***p b 0.001. Fig. 1. Mean relaxatory responses to adenosine (5 ⁎ 10− 6–5 ⁎ 10− 3 M) in normal bladder strip preparations. The arrow marks the standard concentration (5 ⁎ 10− 5 M) used in following experiments. The vertical bars represent S.E.M.
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 using the GraphPad Prism program (GraphPad Software, Inc., San Diego, US). P-values of 0.05 or less were regarded as statistically significant. Data are presented in the form of means ± S.E.M. 3. Results 3.1. Functional experiments The pre-contraction to medium potassium Krebs (50 mM) was 5.07 ± 0.33 mN (n = 40) above baseline in the normal rats and 6.50 ± 0.44 mN (n = 40) in the CYP pre-treated rats. Adenosine (5 ⁎ 10− 6–5 ⁎ 10− 3 M) evoked concentration dependent relaxations of pre-contracted strip preparations (Fig. 1). However, the responses were significantly larger in the normal than in the inflamed bladders. At 5 ⁎ 10− 5 M the relaxation was − 24 ± 2% (n = 21) in the normal and − 12 ± 1% (n = 24) in the CYP pre-treated bladders (p b 0.05). Also the latency to a stable relaxatory response varied; in normal rats the latency was 88 ± 3 s and in CYP pre-treated rats 132 ± 3 s; p b 0.05. Also, the first part of the relaxation was faster in the normal strips than in the inflamed, as indicated in Fig. 2. For examination of the potency of the antagonists, adenosine was administered at a standard concentration of 5 ⁎ 10− 5 M, which caused well-preserved responses when administered repeatedly in the absence of any antagonist. Adenosine 5 ⁎ 10− 5 M administered in the presence of PPADS (1⁎ 10− 4 M) evoked slightly increased relaxatory responses in comparison with adenosine administered in its absence (−15 ± 0.8% vs. −19± 1%; p b 0.05). The presence of DPCPX (1⁎ 10− 5 M) substantially reduced the relaxatory response (to −5 ± 1%; p b 0.001) and prolonged
Fig. 2. Representative recordings showing the latency of reaching the maximum relaxation in normal rats (left) and CYP pre-treated rats (right). The numbers below refer to the mean ± S.E.M. latencies for all animals.
the latency (104 ± 4 s), and further, adenosine administration in the presence of both antagonists evoked similar responses as with only DPCPX present (Fig. 3). However, neither PPADS (1⁎ 10− 4 M) nor DPCPX (1⁎ 10− 5 M) affected the relaxatory response of strip preparation from inflamed urinary bladder (Fig. 4). In order to compare the P1A1 purinoceptor response with that of any other P1 purinoceptor subtype, the adenosine response was examined in the presence of P1A2A and P1A3 antagonists. In order to avoid P2 purinoceptor effects, these experiments were carried out in the presence of PPADS (1 ⁎ 10− 4 M). The adenosine (5 ⁎ 10− 5 M) response was not affected by SCH 58261 (3⁎ 10− 7 M) neither in normal (−8 ± 0.5% vs. −10± 2%, in the absence and presence of SCH 58261, respectively; p N 0.05) nor in CYP pre-treated rats (−6 ± 0.9% vs. −9 ± 1%; p N 0.05; Figs. 5 and 6). However, the incubation with both SCH 58261 and MRS 1523 (1⁎ 10− 5 M) caused significant amplification of adenosine induced relaxations. Increased relaxations appeared in both normal (−10± 2% vs. −24± 1%; p b 0.001) and in CYP pre-treated urinary bladder strips (−9 ± 1% vs. −20± 4%; p b 0.05; Figs. 5 and 6).
3.2. Immunohistochemistry In the bladders from the CYP pre-treated rats, the urothelium was slightly disrupted and an increased amount of vacuoles was seen in the suburothelium when examined 48–60 h after injection. The staining for the P1A1 purinoceptor showed obvious expression of the 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 M respectively; ). Vertical bars represent S.E.M. No statistical significant difference occurred.
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was particularly intense in the urothelium, but seemed to appear in all of the bladder wall layers. 4. Discussion
Fig. 5. Normal rats: Mean relaxatory responses to adenosine (5 ⁎ 10− 5 M) in the presence of PPADS (1 ⁎ 10− 4 M; ), PPADS + SCH 58261 (1 ⁎ 10− 4 M and 3 ⁎ 10− 7 M respectively; ) and PPADS + SCH 58261 + MRS 1523 (1 ⁎ 10− 4 M, 3 ⁎ 10− 7 M and 1 ⁎ 10− 5 M respectively; ). Vertical bars represent S.E.M. ***pb 0.001.
Fig. 6. CYP pre-treated rats: Mean relaxatory responses to adenosine (5⁎ 10− 5 M) in the presence of PPADS (1 ⁎ 10− 4 M; ), PPADS + SCH 58261(1 ⁎ 10− 4 M and 3 ⁎ 10− 7 M respectively; ) and PPADS + SCH 58261 + MRS 1523 (1⁎ 10− 4 M, 3 ⁎ 10− 7 M and 1 ⁎ 10− 5 M respectively; ). Vertical bars represent S.E.M. *pb 0.05.
The present study shows that adenosine evokes relaxation of the isolated urinary bladder, which mainly depends on the activation of P1A1 purinoceptors. No relaxation in response to P1A2A purinoceptor stimulation was observed. It was also found that in the presence of the P1A3 purinoceptor antagonist MRS 1523, the adenosine relaxation was enhanced. Considering the intracellular coupling of the P1A3 purinoceptor to PLC, IP3 and release of intracellular calcium, the enhancement of the relaxation seems likely to reflect a contractile adenosine P1A3 purinoceptor effect. In the CYP pre-treated bladder, a substantial attenuation of the P1A1 purinoceptor relaxation occurred. This vanishing of the P1A1 purinoceptor response correlated to the immunohistochemistry findings indicating a down-regulation of the expression of the receptor. Regarding the other purinoceptors examined in the current study; that is the P1A2A and P1A3 purinoceptors, no alterations of the functional effects mediated by these receptors appeared in the state of CYP-induced inflammation. Regarding P1A2B purinoceptors it has been reported that they are involved in the purinergic relaxation in the normal urinary bladder of the rat (Aronsson et al., 2010). The present findings indicate that the P1A2B purinoceptor may also be responsible for a substantial part of the adenosine relaxation in the inflamed urinary bladder of the rat. The contraction of the urinary bladder is primarily dependent on the activation of muscarinic receptors. It has been shown that muscarinic M3 receptors are mainly responsible for the bladder contraction, while muscarinic M2 receptors contribute most significantly by opposing relaxations induced by adenylate cyclase-coupled receptors such as the β-adrenoceptors and P1 purinoceptors (Hegde et al., 1997; Giglio et al., 2001). Purines also participate in the regulation of the micturition reflex (King et al., 2004). While ATP principally causes contraction, it may in
Fig. 7. Immunohistochemical staining of P1A1 receptors. Upper row shows bladders pre-treated with saline (normal bladders), bottom row the cyclophosphamide pre-treated tissue (cystitis). Left column illustrates the control group which was not exposed to primary antibody, right column the P1A1 receptor staining (positive samples). All sections are stained with hematoxylin. The horizontal bar represents 100 μm.
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addition, by its breakdown to adenosine, evoke a delayed relaxatory response (Bailey and Hourani, 1994; Aronsson et al., 2010). However, both molecules may affect the inflammation, and adenosine has been proved to have both pro- and anti-inflammatory effects in the lower urinary tract (Save et al., 2009). Even though the neuronal purinergic response seems to be absent, or minute, in the urinary bladder in healthy human, purines are produced from other sources as well (Sjogren et al., 1982; Save et al., 2009). For instance purines released from the urothelium may still activate sensory neurons and participate in the regulation of inflammation (Andersson and Hedlund, 2002; Save et al., 2009). P1 purinoceptors are widely distributed in the body and in the urinary bladder large numbers occur (Volpini et al., 2003). Immune system cells also express P1 purinoceptors, and are responsive to the modulatory effects of adenosine in an inflammatory environment (Hasko et al., 2008). The characterization of the purinoceptors by employing immunohistochemistry indicated that P1A1 receptors exist in the urinary bladder of the male rat. The staining showed the receptors to be localized to all parts of the urinary bladder; in the urothelium, suburothelially and even in the detrusor muscle. The urinary bladders of the cyclophosphamide pre-treated rats showed evidently less intense staining. These findings support the suggestion that during the inflammation of the rat urinary bladder the expression of P1A1 receptors is decreased. This corresponds well with results from cell cultures in which E. coli exposure results in a down-regulation of P1A1 purinoceptors (Save et al., 2009). Furthermore the finding of the existence of P1A1 receptors in the urothelium is supported by previous findings (Aronsson et al., 2010), showing a somewhat smaller relaxatory response to ATP in urothelium-denuded bladders than in normal bladders. The current functional examination revealed that approximately 2/3 of the adenosine relaxation was mediated via P1A1 purinoceptors in the normal bladder. In order to avoid any additional relaxatory effect evoked by P2Y purinoceptors, PPADS was added. This seemed to induce a larger adenosine relaxation, which could be interpreted as an adenosine P2Y purinoceptor mediated contraction. However, a drawback with many of the compounds acting on the purinoceptors is that they often exert a number of effects, which makes the relatively small, however statistically significant, change in the presence of PPADS of minor importance (Chen et al., 1996; Lambrecht, 2000). Concerning the selective P1 purinoceptor antagonists used in the present experiments, the concentrations used are the same as in other functional studies claiming 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 responses support that selective antagonism occurred. For instance, the blockade of P1A1 purinoceptors almost abolished the adenosine relaxation, the blockade of P1A2A purinoceptors had no effect, and the blockade of P1A3 purinoceptors had the reversed effect. Also, the P1A1 immunohistochemistry study revealed morphological correlates to the functional findings according to this specific receptor subtype. The adenosine-evoked relaxation was reduced by approximately 1/3 in CYP pre-treated rats. While most of the response was abolished by P1A1 purinoceptor blockade in healthy bladders, just a small part was affected in the inflamed bladders. Since neither P1A2A nor P1A3 purinoceptor blockade inhibited this response, it may tentatively be the result of P1A2B purinoceptor activation. In the state of inflammation, compensational interactions between several mechanisms are likely to occur, but still the P1A2B purinoceptor is a strong candidate to be involved at one or another stage. Nevertheless, the relaxation was obtained with a shorter latency in strips from normal bladders than in strips from inflamed bladders. Also the shape of the curves describing the relaxatory responses differed, as could be judged by the latency to nadir. It is worth noting that in the presence of the P1A1 purinoceptor antagonist DPCPX the latency was prolonged. This suggests that the relaxation due to stimulation of the P1 adenosine receptor is caused to a large part by the P1A1 subtype.
5. Conclusions The performed experiments show that P1A1 receptor is present in the rat urinary bladder. They also show that the expression of the P1A1 receptor subtype is decreased during the cystitis, which affects the function of the urinary bladder. A tentative candidate for the relaxation in the CYP pre-treated rat is the P1A2B purinoceptor.
Acknowledgements This study was supported by grants from Wilhelm och Martina Lundgrens Vetenskapsfond.
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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Functional and morphological examinations of P1A1 purinoceptors in the normal and inflamed urinary bladder of the rat Renata Vesela a,b, Patrik Aronsson b, Gunnar Tobin b,⁎ a b
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, CZ-50005 Hradec Kralove, Czech Republic Department of Pharmacology, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 431, SE-40530 Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 5 May 2010 Received in revised form 9 July 2010 Accepted 9 July 2010 Keywords: Rat urinary bladder Purinergic Cystitis P1 Purinoceptor
a b s t r a c t The aim of the present study was to investigate the relaxatory function of adenosine receptor subtypes in rat urinary bladder, and if it is altered in the state of inflammation. The in vitro responses to the P1 receptor agonist adenosine were investigated in the presence of the general P2 receptor antagonist pyridoxal-phosphate-6azophenyl-2′,4′-disulfonic acid (PPADS; 1 ⁎ 10− 4 M). Experiments were performed on preparations from normal (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 relaxatory response in normal bladders (−60%), but not in preparations from CYP pre-treated rats. Immunohistochemical findings support the hypothesis that the expression of P1A1 receptors in the rat urinary bladder is decreased during 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 normal and CYP pre-treated rats, suggesting P1A3 to be mediating bladder contraction. Thus, in the rat urinary bladder the relaxation to adenosine is mainly due to the P1A1 receptor, while the P1A3 receptor seems to be responsible for contractile responses. The DPCPX-resistant part of the relaxation is possibly due to the P1A2B receptor, the fourth subtype of the adenosine receptor family. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The functional effects by the autonomic nervous system in the lower urinary tract may be substantially altered during inflammatory conditions (Giglio and Tobin, 2009). In experimentally induced cystitis in the rat, the cholinergic contractile responses are reduced in association with more pronounced nitric oxide (NO) mediated effects (Giglio et al., 2005). In patients suffering from interstitial cystitis, it has been reported that 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 the urothelium initiates the stretch-induced micturition reflex by acting on P2X2 and P2X3 receptors on afferent nerve fibers (Wang et al., 2005). However, in the rat urinary bladder, ATP generates a transient contraction caused by the stimulation of the P2X1 purinoceptors also (Ford et al., 2006). Following the contraction a sustained relaxation occurs, which has been suggested to depend on the metabolite of ATP, adenosine, stimulating P1 purinoceptors (Burnstock et al., 1972; Giglio et al., 2001; Aronsson et al., 2010). However, conflicting results exist,
⁎ Corresponding author. Department of Pharmacology, University of Gothenburg, Box 431, SE-40530 Gothenburg, Sweden. Tel.: +46 31 786 34 40; fax: +46 31 786 31 64. E-mail address: [email protected] (G. Tobin). 1566-0702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2010.07.008
suggesting a direct action of ATP on G-protein coupled P2Y purinoceptors (McMurray et al., 1998). The purinoceptors exist in two principal types; the ones on which ATP is the primary agonist, and the others on which adenosine primarily acts. The former group consists of P2X (P2X1–P2X7 in mammals) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11–14) purinoceptors, being ligandgated ion channels and G-protein coupled receptors, respectively. The latter group, the P1 purinoceptors, consist of 4 subtypes, A1, A2A, A2B and A3, all being G-protein coupled, and which all occur in the rat urinary bladder (Yu et al., 2006). The A1 receptor inhibits adenylyl cyclase, but also activates phospholipase C (PLC) (Bucheimer and Linden, 2004). The A2A receptor activates adenylyl cyclase, which the A2B receptor subtype also 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 intracellular calcium via Gq-proteins (Gessi et al., 2008). Besides its participation in the micturition reflex, ATP may induce pro-inflammatory effects in the human urinary tract, most likely via P2Y1-, P2Y2-and/or P2Y11-receptor activation (Säve, 2010). Also, adenosine produced from ATP during inflammation is considered to be an important factor participating in the regulation of inflammation (Hasko and Cronstein, 2004; Sitkovsky et al., 2004). While activation of P1A1 and P1A3 receptors seems to evoke pro-inflammatory effects, P1A2A and P1A2B receptors elicit anti-inflammatory effects.
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We have previously shown that concomitantly to the inflammation-induced alteration of the cholinergic responses in the rat, mucosal muscarinic M5 receptors as well as endothelial nitric oxide synthase (eNOS) are up-regulated (Giglio et al., 2005; Andersson et al., 2008). However, altered expression of purinoceptors seems also to occur in cystitis. While the expression of P2X1 and P2Y2 receptors is reduced in inflammation, the expression of P2X2 and P2X3 receptors may 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, the present 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 the functional P1A1 response to that of other P1 purinoceptors. For this purpose, we used different antagonists, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a general P2 antagonist (Lambrecht et al., 1992), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective P1A1 antagonist (Haleen et al., 1987), SCH 58261, a selective P1A2A antagonist (Zocchi et al., 1996; Belardinelli et al., 1998), and MRS 1523, a selective P1A3 antagonist (Mitchell et al., 1999; Gao et al., 2001; Ansari et al., 2007; Modis et al., 2009), in order to assess inhibitory effects on responses evoked by the P1 purinoceptor agonist adenosine. In order to induce experimental cystitis, rats were pre-treated with CYP, a cytostatic agent that is used in the treatment of neoplastic diseases. The metabolite of CYP, acrolein, has been attributed as being responsible for inducing haemorrhagic cystitis after CYP treatment (Cox, 1979; Batista et al., 2006). The P1A1 purinoceptor expression was examined by immunohistochemistry. 2. Materials and methods The Ethics Committee at the University of Gothenburg approved the study design, in which 25 adult male rats (300–350 g) of the Sprague– Dawley strain were used. 60 h prior to the experiments, rats were administered either a single dose of CYP (100 mg kg− 1 i.p.) in order to yield a pronounced cystitis at the time of the experiment, as previously described (Giglio et al., 2005), or saline (9 mg kg− 1 i.p.) serving as control. The administration of both CYP and saline was conducted in the presence 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 intraperitoneally injected with medetomidin (Domitor® vet., 0.2 ml, Apoteket AB, Sweden) for anesthesia and then killed by an overdose of carbon dioxide. The urinary bladder was removed, cut and opened. Two or three strips (6× 2 mm) were excised from the body of the organ. To prevent damaging the tissue by drying out, the organ was kept in Krebs bicarbonate solution at all times. For the contraction experiments, the strips were fastened between two steel rods, one of which was adjustable. 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). The Krebs solution was kept at 37 °C and gassed with 5% CO2 in O2 during the whole experiment to maintain a stable neutral pH and to provide the tissue with oxygen. The strips were stretched to a tension of 15 mN and left to equilibrate for 45 min. This resulted in gradual relaxation resulting in a stable tension of 7–9 mN. After 45 min, administration of potassium-containing Krebs solution (containing 124 mM K+; obtained by exchanging Na+ for equimolar amounts of K+) was performed and the resulting contraction was used as a viability referring response. The tension was then stabilized for each strip at 7–9 mN. This is a somewhat higher basal tension that is normally used (4–5 mN; Tobin and Sjogren, 1995). This tension turned out to amplify the relaxations. After 30 min of resting, medium potassium Krebs was
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administrated (50 mM K+; obtained by exchanging Na+ for equimolar amounts of K+) to pre-contract the tissue. An observation period of 20 min followed in order to establish a stable pre-contraction larger than 2.5 mN above the basal tension level. After 20 min of precontraction, adenosine was added at a standard concentration shown to 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 in dimethyl sulphoxide (DMSO). When the maximal relaxation of the tissue was obtained, usually after approximately 150 s, the organ baths were washed thoroughly and the strips were re-stretched to 7–9 mN and let to equilibrate for 30 min. Subsequently, an antagonist was added and the preparations were pre-contracted by medium potassium Krebs solution for a 20 min incubation period, after which the relaxatory response to adenosine (5⁎ 10− 5 M) was evaluated. This procedure was repeated for all antagonists used (i.e. PPADS, DPCPX and SCH 58261, MRS 1523, respectively). The agonist and all antagonists were added in the volume of 125 μl. 2.2. Immunohistochemistry Sections of rat urinary bladder were prepared (n= 16; n = 4 in each group; normals (active and control groups) and CYP pre-treated (active and control groups)) and were investigated by immunohistochemistry using subtype specific purinoceptor P1A1 antibody. The specimens were fixed in phosphate buffered 4% paraformaldehyde (pH 7.0), and then embedded in paraffin. Transverse sections of the different specimens were prepared at a thickness of 4 μm and adhered to glasses. The sections were de-paraffinized by heating them to 60 °C for 15 min and then subjecting them to two 30-min intervals in 100% xylene; the sections were then re-hydrated by serial incubations in 100%, 95%, 85% and 70% ethanol, followed by tris-buffered saline (TBS). Then the glasses were heated four times for 6 min in a microwave, followed by blockade of endogen peroxidase by 0.03% hydrogen peroxide for 30 min. The blocking of non-specific background was managed by 5% bovine serum albumin (BSA) in tris-buffered saline (TBS) for 30 min. The sections were incubated overnight with primary antibody (Anti-A1 Adenosine Receptor antibody produced in rabbit; Sigma Aldrich, Sweden) diluted 1:10 in 1% BSA in TBS and for subsequent binding of the secondary antibody (ABC Staining System, Santa Cruz Biotechnology, Santa Cruz, CA), the glasses were incubated for 30 min, followed by incubation with AB enzyme reagent for 30 min. Finally the tissues were stained with hematoxylin for 2–4 min. The negative control samples were treated the very 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-phosphate-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; Sigma Aldrich, Sweden), SCH 58261 (FW = 345.36; Sigma Aldrich, Sweden), MRS 1523 (FW = 395.55; Sigma Aldrich, Sweden), and dimethyl sulphoxide (DMSO, FW = 78.13; Sigma Aldrich). For immunohistochemistry the following materials and substances were used: ABC staining system sc-2018 for use with rabbit primary antibody (Santa Cruz Biotechnology, USA), anti-A1 purinergic receptor-developed in rabbit (Sigma Aldrich, Sweden), and Mayer's hematoxylin (Histolab, Gothenburg, Sweden). 2.4. Statistics The relaxatory responses were normalized to pre-contractile responses and are presented as percentage of pre-contraction. Statistical significance was determined by Student's t-test for paired or unpaired
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R. Vesela et al. / Autonomic Neuroscience: Basic and Clinical 159 (2011) 26–31
Fig. 3. Normal rats: Mean relaxatory responses to adenosine (5 ⁎ 10− 5 M; ), 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 M respectively; ). Vertical bars represent S.E.M. *p b 0.05, ***p b 0.001. Fig. 1. Mean relaxatory responses to adenosine (5 ⁎ 10− 6–5 ⁎ 10− 3 M) in normal bladder strip preparations. The arrow marks the standard concentration (5 ⁎ 10− 5 M) used in following experiments. The vertical bars represent S.E.M.
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 using the GraphPad Prism program (GraphPad Software, Inc., San Diego, US). P-values of 0.05 or less were regarded as statistically significant. Data are presented in the form of means ± S.E.M. 3. Results 3.1. Functional experiments The pre-contraction to medium potassium Krebs (50 mM) was 5.07 ± 0.33 mN (n = 40) above baseline in the normal rats and 6.50 ± 0.44 mN (n = 40) in the CYP pre-treated rats. Adenosine (5 ⁎ 10− 6–5 ⁎ 10− 3 M) evoked concentration dependent relaxations of pre-contracted strip preparations (Fig. 1). However, the responses were significantly larger in the normal than in the inflamed bladders. At 5 ⁎ 10− 5 M the relaxation was − 24 ± 2% (n = 21) in the normal and − 12 ± 1% (n = 24) in the CYP pre-treated bladders (p b 0.05). Also the latency to a stable relaxatory response varied; in normal rats the latency was 88 ± 3 s and in CYP pre-treated rats 132 ± 3 s; p b 0.05. Also, the first part of the relaxation was faster in the normal strips than in the inflamed, as indicated in Fig. 2. For examination of the potency of the antagonists, adenosine was administered at a standard concentration of 5 ⁎ 10− 5 M, which caused well-preserved responses when administered repeatedly in the absence of any antagonist. Adenosine 5 ⁎ 10− 5 M administered in the presence of PPADS (1⁎ 10− 4 M) evoked slightly increased relaxatory responses in comparison with adenosine administered in its absence (−15 ± 0.8% vs. −19± 1%; p b 0.05). The presence of DPCPX (1⁎ 10− 5 M) substantially reduced the relaxatory response (to −5 ± 1%; p b 0.001) and prolonged
Fig. 2. Representative recordings showing the latency of reaching the maximum relaxation in normal rats (left) and CYP pre-treated rats (right). The numbers below refer to the mean ± S.E.M. latencies for all animals.
the latency (104 ± 4 s), and further, adenosine administration in the presence of both antagonists evoked similar responses as with only DPCPX present (Fig. 3). However, neither PPADS (1⁎ 10− 4 M) nor DPCPX (1⁎ 10− 5 M) affected the relaxatory response of strip preparation from inflamed urinary bladder (Fig. 4). In order to compare the P1A1 purinoceptor response with that of any other P1 purinoceptor subtype, the adenosine response was examined in the presence of P1A2A and P1A3 antagonists. In order to avoid P2 purinoceptor effects, these experiments were carried out in the presence of PPADS (1 ⁎ 10− 4 M). The adenosine (5 ⁎ 10− 5 M) response was not affected by SCH 58261 (3⁎ 10− 7 M) neither in normal (−8 ± 0.5% vs. −10± 2%, in the absence and presence of SCH 58261, respectively; p N 0.05) nor in CYP pre-treated rats (−6 ± 0.9% vs. −9 ± 1%; p N 0.05; Figs. 5 and 6). However, the incubation with both SCH 58261 and MRS 1523 (1⁎ 10− 5 M) caused significant amplification of adenosine induced relaxations. Increased relaxations appeared in both normal (−10± 2% vs. −24± 1%; p b 0.001) and in CYP pre-treated urinary bladder strips (−9 ± 1% vs. −20± 4%; p b 0.05; Figs. 5 and 6).
3.2. Immunohistochemistry In the bladders from the CYP pre-treated rats, the urothelium was slightly disrupted and an increased amount of vacuoles was seen in the suburothelium when examined 48–60 h after injection. The staining for the P1A1 purinoceptor showed obvious expression of the 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 M respectively; ). Vertical bars represent S.E.M. No statistical significant difference occurred.
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was particularly intense in the urothelium, but seemed to appear in all of the bladder wall layers. 4. Discussion
Fig. 5. Normal rats: Mean relaxatory responses to adenosine (5 ⁎ 10− 5 M) in the presence of PPADS (1 ⁎ 10− 4 M; ), PPADS + SCH 58261 (1 ⁎ 10− 4 M and 3 ⁎ 10− 7 M respectively; ) and PPADS + SCH 58261 + MRS 1523 (1 ⁎ 10− 4 M, 3 ⁎ 10− 7 M and 1 ⁎ 10− 5 M respectively; ). Vertical bars represent S.E.M. ***pb 0.001.
Fig. 6. CYP pre-treated rats: Mean relaxatory responses to adenosine (5⁎ 10− 5 M) in the presence of PPADS (1 ⁎ 10− 4 M; ), PPADS + SCH 58261(1 ⁎ 10− 4 M and 3 ⁎ 10− 7 M respectively; ) and PPADS + SCH 58261 + MRS 1523 (1⁎ 10− 4 M, 3 ⁎ 10− 7 M and 1 ⁎ 10− 5 M respectively; ). Vertical bars represent S.E.M. *pb 0.05.
The present study shows that adenosine evokes relaxation of the isolated urinary bladder, which mainly depends on the activation of P1A1 purinoceptors. No relaxation in response to P1A2A purinoceptor stimulation was observed. It was also found that in the presence of the P1A3 purinoceptor antagonist MRS 1523, the adenosine relaxation was enhanced. Considering the intracellular coupling of the P1A3 purinoceptor to PLC, IP3 and release of intracellular calcium, the enhancement of the relaxation seems likely to reflect a contractile adenosine P1A3 purinoceptor effect. In the CYP pre-treated bladder, a substantial attenuation of the P1A1 purinoceptor relaxation occurred. This vanishing of the P1A1 purinoceptor response correlated to the immunohistochemistry findings indicating a down-regulation of the expression of the receptor. Regarding the other purinoceptors examined in the current study; that is the P1A2A and P1A3 purinoceptors, no alterations of the functional effects mediated by these receptors appeared in the state of CYP-induced inflammation. Regarding P1A2B purinoceptors it has been reported that they are involved in the purinergic relaxation in the normal urinary bladder of the rat (Aronsson et al., 2010). The present findings indicate that the P1A2B purinoceptor may also be responsible for a substantial part of the adenosine relaxation in the inflamed urinary bladder of the rat. The contraction of the urinary bladder is primarily dependent on the activation of muscarinic receptors. It has been shown that muscarinic M3 receptors are mainly responsible for the bladder contraction, while muscarinic M2 receptors contribute most significantly by opposing relaxations induced by adenylate cyclase-coupled receptors such as the β-adrenoceptors and P1 purinoceptors (Hegde et al., 1997; Giglio et al., 2001). Purines also participate in the regulation of the micturition reflex (King et al., 2004). While ATP principally causes contraction, it may in
Fig. 7. Immunohistochemical staining of P1A1 receptors. Upper row shows bladders pre-treated with saline (normal bladders), bottom row the cyclophosphamide pre-treated tissue (cystitis). Left column illustrates the control group which was not exposed to primary antibody, right column the P1A1 receptor staining (positive samples). All sections are stained with hematoxylin. The horizontal bar represents 100 μm.
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addition, by its breakdown to adenosine, evoke a delayed relaxatory response (Bailey and Hourani, 1994; Aronsson et al., 2010). However, both molecules may affect the inflammation, and adenosine has been proved to have both pro- and anti-inflammatory effects in the lower urinary tract (Save et al., 2009). Even though the neuronal purinergic response seems to be absent, or minute, in the urinary bladder in healthy human, purines are produced from other sources as well (Sjogren et al., 1982; Save et al., 2009). For instance purines released from the urothelium may still activate sensory neurons and participate in the regulation of inflammation (Andersson and Hedlund, 2002; Save et al., 2009). P1 purinoceptors are widely distributed in the body and in the urinary bladder large numbers occur (Volpini et al., 2003). Immune system cells also express P1 purinoceptors, and are responsive to the modulatory effects of adenosine in an inflammatory environment (Hasko et al., 2008). The characterization of the purinoceptors by employing immunohistochemistry indicated that P1A1 receptors exist in the urinary bladder of the male rat. The staining showed the receptors to be localized to all parts of the urinary bladder; in the urothelium, suburothelially and even in the detrusor muscle. The urinary bladders of the cyclophosphamide pre-treated rats showed evidently less intense staining. These findings support the suggestion that during the inflammation of the rat urinary bladder the expression of P1A1 receptors is decreased. This corresponds well with results from cell cultures in which E. coli exposure results in a down-regulation of P1A1 purinoceptors (Save et al., 2009). Furthermore the finding of the existence of P1A1 receptors in the urothelium is supported by previous findings (Aronsson et al., 2010), showing a somewhat smaller relaxatory response to ATP in urothelium-denuded bladders than in normal bladders. The current functional examination revealed that approximately 2/3 of the adenosine relaxation was mediated via P1A1 purinoceptors in the normal bladder. In order to avoid any additional relaxatory effect evoked by P2Y purinoceptors, PPADS was added. This seemed to induce a larger adenosine relaxation, which could be interpreted as an adenosine P2Y purinoceptor mediated contraction. However, a drawback with many of the compounds acting on the purinoceptors is that they often exert a number of effects, which makes the relatively small, however statistically significant, change in the presence of PPADS of minor importance (Chen et al., 1996; Lambrecht, 2000). Concerning the selective P1 purinoceptor antagonists used in the present experiments, the concentrations used are the same as in other functional studies claiming 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 responses support that selective antagonism occurred. For instance, the blockade of P1A1 purinoceptors almost abolished the adenosine relaxation, the blockade of P1A2A purinoceptors had no effect, and the blockade of P1A3 purinoceptors had the reversed effect. Also, the P1A1 immunohistochemistry study revealed morphological correlates to the functional findings according to this specific receptor subtype. The adenosine-evoked relaxation was reduced by approximately 1/3 in CYP pre-treated rats. While most of the response was abolished by P1A1 purinoceptor blockade in healthy bladders, just a small part was affected in the inflamed bladders. Since neither P1A2A nor P1A3 purinoceptor blockade inhibited this response, it may tentatively be the result of P1A2B purinoceptor activation. In the state of inflammation, compensational interactions between several mechanisms are likely to occur, but still the P1A2B purinoceptor is a strong candidate to be involved at one or another stage. Nevertheless, the relaxation was obtained with a shorter latency in strips from normal bladders than in strips from inflamed bladders. Also the shape of the curves describing the relaxatory responses differed, as could be judged by the latency to nadir. It is worth noting that in the presence of the P1A1 purinoceptor antagonist DPCPX the latency was prolonged. This suggests that the relaxation due to stimulation of the P1 adenosine receptor is caused to a large part by the P1A1 subtype.
5. Conclusions The performed experiments show that P1A1 receptor is present in the rat urinary bladder. They also show that the expression of the P1A1 receptor subtype is decreased during the cystitis, which affects the function of the urinary bladder. A tentative candidate for the relaxation in the CYP pre-treated rat is the P1A2B purinoceptor.
Acknowledgements This study was supported by grants from Wilhelm och Martina Lundgrens Vetenskapsfond.
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