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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2012 Nov 29;167(8):1737–1752. doi: 10.1111/j.1476-5381.2012.02127.x

Effects of kinin B1 and B2 receptor antagonists on overactive urinary bladder syndrome induced by spinal cord injury in rats

Stefânia Forner 1, Edinéia L Andrade 1, Alessandra C Martini 1, Allisson F Bento 1, Rodrigo Medeiros 1,*, Janice Koepp 1, João B Calixto 1
PMCID: PMC3525875  PMID: 22862305

Abstract

Background and Purpose

Kinin B1 and B2 receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive.

Experimental Approach

We investigated the role of kinin B1 and B2 receptors in OAB after SCI in rats.

Key Results

SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B1 receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B1 protein in the urinary bladder and B1 and B2 receptor protein in spinal cord. Interestingly, both B1 and B2 protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B1 or B2 agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B2 selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B1 antagonist des-Arg9-[Leu8]-bradykinin reduced the number of NVCs while the non-peptide B1 antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume.

Conclusions and Implications

Taken together, these data show the important roles of B1 and B2 receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.

Keywords: B1 and B2 kinin receptors, overactive bladder, spinal cord injury, pro-inflammatory interleukins

Introduction

Spinal cord injury (SCI) is a devastating condition that affects about 2.5 million people worldwide; it has a profound impact on quality of life and life expectancy, causing a great economic burden to society (Thuret et al., 2006).

The disruption of coordinated control between the brain, spinal cord and peripheral nervous system caused by SCI leads to several secondary pathological conditions, including lower urinary tract dysfunctions. As a consequence, individuals develop overactive urinary bladder (OAB), a syndrome characterized by exacerbated contractions of the urinary bladder during the filling phase, associated with detrusor sphincter dyssynergia (DSD) and inefficient voiding (de Groat et al., 1993; Yoshimura and de Groat, 1997; Yoshimura and Chancellor, 2003).

Overactive urinary bladder syndrome affected approximately 450 million people worldwide in 2008. It has been projected that this number will increase by 100 000 by 2018 (Irwin et al., 2011). Following SCI, OAB is triggered by the emergence of a micturition reflex at the spinal level. In this context, C-fibre urinary bladder afferents present remarkable plasticity and become the predominant afferent route carrying impulses from the spinal tract to control the micturition reflex (de Groat, 1990; Kruse et al., 1995). In addition to C-fibre sensitization to various stimuli, a local effector function of afferent C-fibre endings contributes to neurogenic inflammation (Juszczak et al., 2009). These two phenomena are pivotal for the development of OAB.

Kinins are a group of peptides formed in plasma and tissues during different physiological states and in response to inflammation, infection and tissue trauma. Once formed and released, kinins exert their biological effects through the activation of two G-protein coupled receptors, the kinin B1 and B2 receptors (Calixto et al., 2000; Moreau et al., 2005). The B2 receptor is constitutively expressed in most tissues, whereas the B1 receptor is generally not constitutively expressed to a great extent under normal conditions but its expression is up-regulated following tissue damage/inflammation (Ni et al., 2003; Calixto et al., 2004; Ferreira et al., 2005; Fox et al., 2005; Leeb-Lundberg et al., 2005; Kayashima et al., 2011). In this context, it was shown that B1 receptor expression is induced in response to the B1 ligand Lys-des-Arg9-bradykinin (BK; Phagoo et al., 1999) and inflammatory cytokines such as IL-1β and TNF-α (Campos et al., 1998; Passos et al., 2004; Fernandes et al., 2005). The expression of B1 receptors might also be regulated by B2 receptors through the activation of NF-κB and MAP kinases (Phagoo et al., 1999; Passos et al., 2004).

At the level of the urinary bladder, B2 receptor expression is firmly established in detrusor muscle and urothelial cells (Chopra et al., 2005). The activation of B2 receptors present in pelvic afferent fibres stimulates the contraction of the urinary bladder detrusor muscle (Meini et al., 2000). In this regard, it has been shown that the B2 receptor plays a key role in the excitatory motor innervation of the urinary bladder (Lecci et al., 1995; Patra and Westfall, 1996; Belichard et al., 1999). On the other hand, the constitutive expression of B1 receptors in the urinary bladder is still controversial. B1 receptor mRNA was undetectable in control rat detrusor muscle and urothelium (Chopra et al., 2005). These findings are consistent with previous binding studies in control urinary bladder homogenates (Lecci et al., 1999). Nonetheless, Belichard and collaborators (1999) found a basal expression of mRNA coding for B1 receptors in the rat urinary bladder. Supporting this hypothesis, specific rat B1 immunoreactivity was observed in nerve terminals innervating the urinary bladder (Wotherspoon and Winter, 2000). Interestingly, stimulation of the B1 receptor induces the release of chemical mediators that affect afferent fibres, causing urinary bladder contraction (Lecci et al., 1999).

Under pathological states, such as cystitis, both B1 and B2 receptors have been reported to be involved in the regulation of urinary bladder functionality (Marceau et al., 1980; Maggi, 1997). In this context, BK can evoke an inflammatory response and changes in the urinary bladder reflex by directly activating B2 receptors on afferent fibres and/or indirectly by release of ATP and other neurotransmitters of the urothelium (Chopra et al., 2005). With regard to the B1 receptor, it has been shown that its activation evokes the release of ATP and increases intracellular calcium levels in urothelial cells in the inflamed urinary bladder of the rat (Chopra et al., 2005).

Currently, the treatment options available for OAB syndrome are limited, only modestly effective and have serious side effects that can limit their usefulness (Gulur and Drake, 2010). Hence, there is an enormous and urgent medical need for the development of novel therapeutic strategies that target improvements in OAB, especially following SCI. After considering the data implicating kinin receptors in the pathophysiology of the urinary bladder, we investigated the role exerted by B1 and B2 receptors on SCI-induced OAB.

Methods

Animals

Male Wistar rats (270–300 g) from the animal house of the Department of Pharmacology, Federal University of Santa Catarina (UFSC, Florianópolis, Brazil), were used in the experiments. The animals were healthy and were housed in a room maintained under a controlled temperature (22 ± 2°C) and a 12 h light/12 h dark cycle and 60–80% humidity. Food and water were provided ad libitum. The experimental procedures were approved by the ethics committee of the Federal University of Santa Catarina (number 016/CEUA/PRPe/2008. Procedure PP00158) and followed the National Institutes of Health Animal Care Guidelines (NIH Publications N°80-23). The results of all studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).

SCI procedure

The surgical procedure was performed as previously described (Vanický et al., 2001; Andrade et al., 2011). The animals were anaesthetized by an i.p. injection of a 1:1 solution containing xylazine (10 mg·kg−1) and ketamine (70 mg·kg−1), and they also received a wide spectrum antibiotic (oxytetracycline chloride 20%). A dorsal midline incision was made from the T9-T12 spinal process. A laminectomy was performed at the T11 vertebra level using an operating microscope and a dental drill followed by the insertion of an embolectomy catheter (2-French Fogarty, Lemaitre Catheters, Burlington, VT, USA) previously calibrated for a 4.5 mm diameter.

The catheter was introduced into the epidural space and advanced to the rostral direction in such a way that the catheter balloon rested on the T10 vertebra level. Subsequently, the catheter was maintained inflated at a diameter of 4.5 mm until the spinal reflexes disappeared, then it was deflated and carefully removed. The muscle and skin were sutured. Sham-operated animals were only subjected to laminectomy.

The SCI-induced animals had their urinary bladder manually emptied twice a day by means of abdominal compression until the restoration of partial spontaneous micturition. The pre- and post-surgical care followed standard protocols as well as the regulations of the Multicenter Animal Spinal Cord Injury Study (Santos-Benito et al., 2006).

The SCI rats were divided into separate groups: (i) early phase of recovery, 2 days; (ii) intermediate phase of recovery, 7 and 14 days; (iii) late phase of recovery, 28 days, in accordance with a previous study (Lee et al., 2011).

Evaluation of motor behaviour

Motor behaviour of the sham-operated and SCI animals was assessed in an open-field arena using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale (Basso et al., 1995). The animals were evaluated every two days from day 0 to day 28 following the surgical procedure.

Determination of cytokine levels

The urinary bladders were homogenized in phosphate buffer containing 0.05% Tween 20, 0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA and 2 ng·mL−1 aprotinin A (all from Sigma-Aldrich, St. Louis, MO, USA). The homogenate was centrifuged at 5000 × g for 15 min at 4°C, and the supernatants were stored at −70°C until analysis. IL-1β, IL-6 and TNF-α levels were measured using ELISA kits from R&D Systems (Minneapolis, MN, USA), according to the manufacturer's recommendations. The amount of protein in each sample was measured using the standard Bradford method (Bradford, 1976).

RNA extraction and real-time PCR

Total RNA from the whole urinary bladder, dorsal root ganglion (DRG, L6-S1) and corresponding segment of the spinal cord was extracted using TRizol® reagent (Invitrogen Corp., Carlsbad, CA, USA) and its concentration was determined by NanoDrop 1100 (Nanodrop Technologies, Wilmington, DE, USA) as previously described by Bento et al. (2011). The 3’ quencher MGB and FAM-labelled probes for rat B1 and B2 receptors (Rn00570261_m1 and Rn01430057_m1, respectively) and the 3’ quencher MGB and VIC-labelled probe for rat β-actin ACTB (Rn00667869_m1), which was used as an endogenous control for normalization. The PCR reactions were performed in a 96-well Optical Reaction Plate (Applied Biosystems, Foster City, CA, USA). The thermocycler parameters were as follows: 50°C for 2 min, 95°C for 10 min, 50 cycles of 95°C for 15 sec and 60°C for 1 min. Expression of the target genes was calibrated against conditions found in control animals.

Western blot analysis

Urinary bladder and spinal cord tissue samples were removed 2, 7, 14 and 28 days after surgery and homogenized in complete RIPA and the total protein concentration was determined using a NanoDrop 1100 (NanoDrop Technologies, Wilmington, DE, USA). Equivalent amounts of protein for each sample were loaded per lane and electrophoretically separated using 10% denaturing PAGE (SDS-PAGE). After being transferred to a polyvinylidene fluoride membrane, the blots were assembled directly into a Snap i.d. Protein Detection System (Millipore Corporation, Billerica, MA, USA) blot holder for immunodetection. Filters were blocked with 1% BSA – tris-buffered saline-tween 20 buffer and then probed with specific primary antibodies anti-B1 receptor and anti-B2 receptor, according to the manufacturer's recommendations (1:200, all from Alomone Labs Ltd, Jerusalem, Israel), for 10 min at room temperature. Control experiments were carried out by pre-absorbing antibodies with the respective peptide antigen according to the manufacturer's instructions (Alomone Labs). After being washed three times with TBS-T buffer, the membranes were incubated with HRP conjugated donkey anti-rabbit IgG secondary antibody (1:800, Promega Corporation, Madison, WI, USA) for 10 min at room temperature. After being washed, the immunocomplexes were visualized using the SuperSignal West Pico Chemiluminescent Substrate Detection System (Thermo Fisher Scientific, Rockford, IL, USA). Band density measurements were made using Scion Image Software (Scion Corporation, Frederick, MD, USA).

Immunohistochemical analysis

On days 2, 7, 14 and 28 after surgery, immunohistochemical detection of B1 and B2 receptors was performed in the bladder (5 μm) using rabbit anti-B1 receptor polyclonal antibody (1:200; Alomone Labs Ltd) and rabbit anti-B2 receptor polyclonal antibody (1:200; Alomone Labs Ltd) respectively. Control experiments were carried out by pre-absorbing antibodies with the respective peptide antigen according to the manufacturer's instructions (Alomone Labs). We followed the method previously described by Andrade et al. (2011). In order to evaluate the difference in B1 and B2 receptors staining between urothelial cells and detrusor muscle, digital images of the whole bladder were captured, the specific area of each tissue was delimited, the intensity of receptor staining was measured and the resulting value was divided by the surrounding area. The results are presented as arbitrary units per area.

Isolated tissue preparations

Naive, sham-operated and SCI animals were killed by an overdose of barbiturate i.p. (100 mg·kg−1, pentobarbital sodium). The urinary bladders were carefully removed and set up in 5 mL organ bath as previously described by Andrade et al. (2011).

Following stabilization, complete cumulative concentration-response curves (CRCs) were plotted for des-Arg9-BK (B1 receptor agonist: 0.001–10 μM) and BK (B2 receptor agonist: 0.001–10 μM). Only one agonist was tested in each preparation. In a different set of experiments, we assessed the effect of selective kinin receptor antagonists on urinary bladder strips obtained 28 days after the induction of SCI. The CRCs for des-Arg9-BK (0.001–10 μM) and BK (0.001–10 μM) were obtained in the absence or presence of the B1 receptor antagonist des-Arg9-[Leu8]-BK (30 μM) or the B2 receptor antagonist icatibant (0.03 μM). The concentrations of antagonist were chosen on the basis of previous studies (El Sayah et al., 2006; Hara et al., 2008). The results obtained in all experiments are expressed as g tension.

Cystometric parameters

The urodynamic studies were carried out on the 28th day, at which time all of the animals showed partial spontaneous micturition restoration as previously described by Andrade et al. (2011).

We assessed the micturition pressure (MP, maximum urinary bladder pressure during micturition), basal pressure (BP, the lowest urinary bladder pressure between micturitions), threshold pressure (TP, urinary bladder pressure immediately before micturition) and the intercontraction interval (ICI). The number and mean amplitude of non-voiding contractions (NVCs) were also measured. The NVCs were defined as rhythmic intravesical pressure increases of greater than 5 mmHg from baseline pressure without the release of saline from the urethra.

Saline voided from the urethral meatus was collected and measured to determine the voided volume (VV). The saline infusion was stopped at the beginning of the voiding contraction and the residual volume (RV) was measured by withdrawing saline through the intravesical catheter and then manually expressing the remaining intravesical contents by exerting pressure on the urinary bladder abdominal wall. The urinary bladder capacity (BC) was calculated as the VV plus the RV. The BC was assessed and the voiding efficiency (VE) was determined as a percentage using the following equation: VE = [(VV/BC) × 100].

When investigating the effects of kinin receptor antagonists, reproducible micturition cycles were recorded before (used as baseline values) and 45 min after administration of the B2 receptor antagonist icatibant (100 μmol·kg−1, i.v., caudal), the B1 receptor peptide antagonist des-Arg9-[Leu8]-BK (0.3 μmol·kg−1, i.v., caudal), the B1 non-peptide antagonist SSR240612 (0.4 μmol·kg−1, i.v., caudal) or vehicle (saline). The doses of the antagonists were selected on the basis of previous studies (Meini et al., 2000; Su et al., 2009).

Drugs and chemical reagents

The nomenclature of drugs and molecular targets adopted in this study is that of Alexander et al. (2011). The following drugs and reagents were used: pentobarbital sodium, urethane (Sigma Chemical Co., St. Louis, MO, USA), des-Arg9-[Leu8]-BK, des-Arg9-BK and BK (Bachem, Bubendorf, Switzerland), ketamine and xylazine (Virbac, São Paulo, SP, Brazil), oxytetracyclin chloridrate (Terramicin; Pfizer, São Paulo, Brazil), icatibant (Aventis Pharma Deutschland, Frankfurt Main, Germany) and SSR240612 was kindly donated by Sanofi-Synthelabo (France) respectively; des-Arg9-BK, icatibant, des-Arg9-[Leu8]-BK, BK and the SSR240612 solution were prepared in saline. SSR240612 was placed in ultrasom bath until it became soluble. The vehicle used had no pharmacological effects on the tonus of the preparations or on agonist-induced contractions or cystometric parameters.

Statistical analysis

All values are expressed as the mean ± SEM, except for the EC50 values (i.e. the concentration of agonist that induced 50% of the response between the baseline and maximum effect) which are given as geometric means accompanied by their respective 95% confidence limits. The EC50 values were estimated by using concentrations of each drug that gave an effect between the minimum and maximum using the linear regression for individual experiments with GraphPad Prism® 4 software (GraphPad Software Inc., San Diego, CA, USA). The statistical significance between the groups was assessed using one or two-way anova followed by the Bonferroni test or by Student's paired or unpaired t-tests. A P-value of less than 0.05 (P < 0.05) was considered to be statistically significant.

Results

SCI-induced significant motor impairments

Sham-operated animals showed normal hindlimb motor activity after the surgery. However, all SCI animals developed complete paraplegia corresponding to a BBB score of 0–1 in the first week after surgery, followed by modest motor improvements in the next 3 weeks post-SCI, reaching a final score of 3. In order to guarantee OAB development in all SCI animals, rats that scored over 4 were not used in subsequent experiments (data not shown).

Levels of pro-inflammatory cytokines in the urinary bladder increased after SCI

In order to determine urinary bladder inflammatory conditions in response to SCI, we evaluated the levels of pro-inflammatory cytokines in the urinary bladder. Figure 1 (A and B) shows that the levels of IL-1β and IL-6 were 7- and 42-fold higher respectively, in the SCI urinary bladder compared to the sham-operated urinary bladder, and this increase was only observed on the second day after surgery. The level of TNF-α remained unchanged in both groups (Figure 1C).

Figure 1.

Figure 1

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The levels of IL-1β, IL-6 and TNF-α were measured in the urinary bladders of sham-operated, SCI and naive animals. Two days after SCI, the levels of IL-1β and IL-6 (A and B, respectively) were significantly higher in the SCI urinary bladder compared to sham-operated urinary bladders. The levels of TNF-α remained unchanged in both groups (C). Each column represents the mean ± SEM of four animals. The asterisks denote the levels of significance. *P < 0.05 compared with the sham-operated group (two-way anova followed by a Bonferroni test).

B1 and B2 receptor mRNA levels increased after SCI

In order to evaluate the involvement of kinins and their receptors in OAB triggered by SCI, we first determined the mRNA levels for B1 and B2 receptors by real-time PCR in the urinary bladder, DRG (L6-S1), and corresponding segments of spinal cord from naive, sham-operated, and SCI animals taken 2, 7 and 14 days after surgery. The mRNA level of B1 receptor was increased in the urinary bladder (day 2, 12-fold; day 7, 6-fold), DRG (day 2, 6-fold) and spinal cord (days 2 and 7, 3-fold) after SCI when compared to the respective sham-operated groups (Figure 2 A, C and E respectively). The mRNA levels of B2 receptor were reduced (day 14, 14%) in DRG (Figure 2 D), but were not significantly altered in the urinary bladder and spinal cord (Figure 2B and F respectively).

Figure 2.

Figure 2

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SCI caused a significant increase in B1 receptor mRNA levels in the urinary bladder and spinal cord on days 2 and 7 (A and E, respectively) and in DRG (C) on day 2 after SCI. B2 receptor mRNA levels were significantly decreased in DRG on day 14 (D). Each column represents the mean ± SEM of three animals. *P < 0.05 compared with the sham-operated group (one-way anova followed by a Bonferroni test).

SCI induced alterations in B1 and B2 receptor protein levels in the urinary bladder and spinal cord

Due to significant alterations in mRNA expression, we next investigated possible changes in B1 and B2 receptor protein expression in the urinary bladder collected 2, 7, 14 and 28 days after the surgical procedure. Our results showed a constitutive expression of both B1 and B2 receptors in the urinary bladder and spinal cord of naive group (Figure 3). Interestingly, when compared to the respective sham-operated group, SCI induced an up-regulation of B1 receptors on day 2 after surgery in the urinary bladder (62.3 ± 15.7%; Figure 3A). However, the B2 receptor expression was unaffected in this tissue in all the periods assessed (Figure 3B). The SCI procedure resulted in an up-regulation of the expression of the B1 receptor on day 7 (95.5 ± 27.1%) and B2 receptor on days 2 (36.3 ± 8.3%) and 7 (73.8 ± 4.5%) after the surgery in the spinal cord (Figure 3C and D respectively).

Figure 3.

Figure 3

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SCI caused a significant up-regulation in B1 receptor expression in the urinary bladder on day 2 (A) and in the spinal cord on day 7 (C) after SCI. B2 receptor expression was significantly increased in the spinal cord on days 2 and 7 after SCI (D) but it did not induce any significant alterations in the urinary bladder(B). Total levels of B1 and B2 receptors were determined by Western blot analysis using specific antibodies. Each column represents the mean ± SEM of four animals. *P < 0.05 compared with the sham-operated-operated group (one-way anova followed by a Bonferroni test).

No band was observed in the control experiments (Figure 3).

B1 and B2 receptor expression in the urothelial cells and detrusor muscle of the urinary bladder

We next determined the expression of B1 and B2 receptors in the detrusor muscle and urothelium after SCI. The immunohistochemical analysis, illustrated in Figure 4, showed that B1 receptor expression was equally distributed in detrusor muscle and urothelium of naive and sham-operated animals (Figure 4A), while B2 receptor expression was higher in urothelium that in detrusor muscle of naive (fourfold) and sham-operated animals on days 2 (26-fold), 7 (19-fold), 14 (16-fold), and 28 (570-fold) after surgery (Figure 4C). Moreover, B1 and B2 receptor expression were equally distributed in detrusor muscle and urothelium in all the analysed time-points post-SCI (Figure 4B and D respectively). No staining was observed in the control experiments (Figure 4 E and H).

Figure 4.

Figure 4

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Immunohistochemical analysis of B1 and B2 receptor expression in the urinary bladder detrusor muscle and urothelium of naive and sham-operated (A and C, respectively) and SCI (B and D, respectively) rats assessed 2, 7, 14 and 28 days after surgery. Representative images of control group (E and H, respectively) and B1 (F and G) and B2 (I and J) receptors immunostaining in the urinary bladder detrusor muscle (arrowhead) and urothelium (arrow) of sham-operated and SCI rats respectively (scale bar = 100 μm). The results are represented as arbitrary units per area. Each column represents the mean and vertical lines show the SEM of three to four animals. *P < 0.05 compared with the respective urothelium group (one-way anova followed by a Bonferroni test).

SCI increased the functional contractile response to both B1 and B2 agonists in the urinary bladder

Once the overexpression of both B1 and B2 receptors was observed in the urinary bladder of SCI animals, we next evaluated the reactivity of isolated urinary bladders to selective agonists of these receptors. Sham-operated urinary bladder strips stimulated with the B1 receptor agonist des-Arg9-BK (0.0001–10 μM) showed quite moderate contractile responses compared with the urinary bladder strips of naive animals when assessed on day 2 post-surgery. In addition, on days 7, 14 and 28 after surgery, no significant changes in the contractile response were observed between urinary bladder strips from sham-operated and naive rats (Figure 5). Compared to sham-operated urinary bladders, the SCI urinary bladders showed a significantly higher contractile response to des-Arg9-BK (Figure 5A–D). The calculated mean EC50 values obtained for des-Arg9-BK in the SCI group were 0.3 (0.08–1.2); 0.4 (0.09–2.4); 0.2 (0.009–3.9) and 0.04 (0.02–0.1) μM, and the tension-at-maximum-concentration values were 2.1 ± 0.3, 1.3 ± 0.2, 1.5 ± 0.1, and 1.3 ± 0.5 g for 2, 7, 14 and 28 days after surgery respectively. After SCI, des-Arg9-BK was more potent on day 28 and more efficacious on day 2 after SCI, according to the EC50 and maximal effect analyses respectively. The contractile response induced by des-Arg9-BK in the SCI urinary bladder strips on day 28 after surgery was significantly reduced (28.6 ± 12.5%) in the presence of the selective B1 receptor antagonist des-Arg9-[Leu8]-BK (30 μM; Figure 5E).

Figure 5.

Figure 5

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Contractile response induced by the B1 receptor agonist (des-Arg9-BK; 0.0001–10 μM) in isolated urinary bladders 2 (A), 7 (B), 14 (C) and 28 (D) days after surgery. The B1 receptor antagonist (des-Arg9-[Leu8]-BK) reduced the contractile response induced by des-Arg9-BK (0.0001–10 μM) in isolated urinary bladders of SCI animals assessed 28 days after surgery (E). The results are expressed as g of tension. Each point represents the mean ± SEM of six animals for each group. The asterisks denote the significance levels. *P < 0.05 compared with the sham-operated-operated group (two-way anova followed by a Bonferroni test).

When naive and sham-operated urinary bladders strips were exposed to the B2 receptor agonist BK (0.0001–10 μM), similar contractile responses were shown in all the periods analysed (Figure 6). In contrast, the SCI urinary bladder strips showed significantly higher contractile responses to BK when compared with sham-operated urinary bladder strips. The EC50 values for BK were 0.09 (0.03–0.2), 3.6 (0.8–17) 0.09 (0.03–0.2) and 0.1 (0.06–0.3) μM and the tension-at-maximum-concentration values were 2.3 ± 0.4, 3.3 ± 0.4, 4.3 ± 0.7 and 5.3 ± 0.7 g for 2, 7, 14 and 28 days after SCI respectively. After SCI, BK was more potent on days 2 and 14 and more efficacious on day 28 at inducing the contractile response, according to the EC50 and maximal effect analyses respectively.

Figure 6.

Figure 6

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Contractile response induced by the B2 receptor agonist bradykinin (0.0001–10 μM) in isolated urinary bladders 2 (A), 7 (B), 14 (C), and 28 (D) days after surgery. The B2 receptor antagonist (icatibant) reduced the contractile response induced by bradykinin (0.0001–10 μM) in isolated urinary bladders of SCI animals assessed 28 days after surgery (E). The results are expressed as g of tension. Each point represents the mean ± SEM of four animals for each group. The asterisks denote the levels of significance. *P < 0.05 compared with the sham-operated-operated group (two-way anova followed by a Bonferroni test).

The BK-induced contractile response in the SCI urinary bladder strips obtained 28 days after surgery was significantly reduced (89.5 ± 2.5%) in the presence of the selective B2 receptor antagonist icatibant (0.03 μM; Figure 6E).

Effect of the B1 or B2 receptors antagonist on urodynamic alterations induced by SCI

Finally, we investigated the urodynamic behaviour of urine voiding by measuring the intravesical pressure and volume in sham-operated and SCI animals. During the urinary bladder filling phase, SCI animals showed more NVCs (10.0 ± 2.6) than sham-operated animals (0.5 ± 0.3). In comparison to sham-operated animals, the SCI animals showed significant alterations such as reductions in the ICI (49.0 ± 7.1%), VV (18.9 ± 1.5%) and VE (2.4 ± 0.3%), as well as an increase in TP (212.7 ± 30.6%) and urinary BC (655.2 ± 72.4%; Table 1). No significant alterations in the urodynamic parameters were observed between naive and sham-operated groups (data not shown).

Table 1.

Changes in cystometric parameters induced by SCI

Parameters Sham operated (n = 6–8) SCI (n = 6–8)
Mean amplitude of NVCs (mmHg) 8.20 ± 5.30 22.60 ± 1.90*
Number of NVCs 0.50 ± 0.30 10.00 ± 2.60*
Basal pressure (mmHg) 7.20 ± 3.30 15.70 ± 2.70
Pressure threshold (mmHg) 8.30 ± 2.40 17.70 ± 2.50*
Maximum voiding pressure (mmHg) 28.70 ± 6.10 30.40 ± 5.70
Intercontraction interval (min) 1.40 ± 0.20 0.60 ± 0.10*
Voided volume (mL) 0.20 ± 0.02 0.04 ± 0.00*
Urinary bladder capacity (mL) 0.40 ± 0.08 2.60 ± 0.30*
Voiding efficiency (%) 63.70 ± 11.00 1.50 ± 0.20*
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*

P < 0.05 compared with sham operated (Student's unpaired t-test). Values are expressed as mean ± SEM.

Acute systemic treatment with the B1 receptor antagonist des-Arg9-[Leu8]-BK (0.3 μmol·kg−1, i.v.) only reduced the number of NVCs (54.6 ± 18.8%; Figure 7B–E and Table 2), whereas the acute systemic treatment of SCI animals with the B1 receptor non-peptide antagonist SSR240612 (0.4 μmol·kg−1) not only reduced the NVCs (77.3 ± 10.8%) and the urinary BC (55.1 ± 3.6%), but it also increased VV (89.3 ± 34.6%) and the VE (223.81 ± 137.1%; Figure 8 and Table 2). On the other hand, the B2 receptor antagonist icatibant (100 μmol·kg−1, i.v., 45 min pretreatment) significantly reduced the amplitude (21.0 ± 9.2%) and the number (59.0 ± 5.8%) of NVCs (Figure 9B–E and Table 2). No significant changes were found in the intercontraction interval, VE, TP or urinary BC before or after the administration of vehicle or kinin receptor antagonists (Table 2).

Figure 7.

Figure 7

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Representative cystometric trace of sham-operated, SCI and DALBK-treated SCI rats on day 28 after surgery. During the filling phase, the sham-operated and SCI rats showed a large number of voiding (A; arrow) and non-voiding (B; double arrow) contractions respectively. The number of NVCs (C and D; double arrow) were reduced by DALBK (des-Arg9-[Leu8]-BK (0.3 μmol·kg−1) treatment, but not the amplitude (C and E; arrowhead). Each column represents the mean and the vertical lines indicate the SEM of six to eight animals. *P < 0.05 compared with pretreatment values (Student's paired t-test).

Table 2.

Changes in cystometric parameters induced by SCI and the effect of i.v. administration of vehicle (saline), icatibant (100 μmol·kg−1), DALBK (0.3 μmol·kg−1) or SSR240612 (0.4 μmol·kg−1)

SCI
Vehicle (n = 6–8) Icatibant (n = 6–8) DALBK (n = 6–8) SSR240612 (n = 6–8)
Parameters Pre Post Pre Post Pre Post Pre Post
Mean amplitude of NVCs (mmHg) 20.50 ± 3.20 22.90 ± 4.40 20.60 ± 2.66 16.30 ± 1.90* 16.70 ± 2.70 10.90 ± 3.90 11.66 ± 1.99 7.66 ± 1.22
Number of NVCs 10.40 ± 2.60 8.20 ± 2.60 12.20 ± 2.60 5.40 ± 0.50 * 8.30 ± 2.50 3.80 ± 1.50* 11.00 ± 2.27 2.50 ± 1.19*
Basal pressure (mmHg) 17.40 ± 4.10 17.90 ± 4.20 13.50 ± 3.10 11.60 ± 2.00 9.20 ± 3.40 7.90 ± 3.10 1.90 ± 0.44 0.87 ± 0.38
Pressure threshold (mmHg) 20.20 ± 4.20 20.60 ± 4.20 15.50 ± 3.10 13.10 ± 2.00 9.90 ± 3.60 8.60 ± 3.20 3.74 ± 0.42 2.60 ± 0.09
Maximum voiding pressure (mmHg) 27.90 ± 3.60 26.50 ± 2.50 27.30 ± 5.80 20.30 ± 2.40 17.00 ± 3.30 15.70 ± 2.70 16.77 ± 1.81 14.73 ± 3.46
Intercontraction interval (min) 0.50 ± 0.02 0.50 ± 0.04 0.60 ± 0.06 0.50 ± 0.03 0.80 ± 0.10 0.70 ± 0.10 0.71 ± 0.13 0.94 ± 0.24
Voided volume (mL) 0.05 ± 0.06 0.05 ± 0.03 0.04 ± 0.00 0.05 ± 0.00 0.02 ± 0.00 0.03 ± 0.00 0.46 ± 0.009 0.87 ± 0.016*
Urinary bladder capacity (mL) 2.70 ± 0.30 2.70 ± 0.40 2.80 ± 0.40 2.60 ± 0.20 2.90 ± 0.40 2.80 ± 0.30 2.17 ± 0.16 0.97 ± 0.08*
Voiding efficiency (%) 2.10 ± 0.40 2.15 ± 0.40 1.50 ± 0.20 2.10 ± 0.30 1.10 ± 0.40 1.30 ± 0.40 2.25 ± 0.57 9.17 ± 2.03*
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*

P < 0.05 compared with pretreatment (Student's paired t-test). Values are expressed as mean ± SEM.

Figure 8.

Figure 8

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Representative cystometric trace of sham-operated, SCI and SSR240612-treated SCI rats on day 28 after surgery. During the filling phase, the sham-operated and SCI rats showed a large number of voiding (A; arrow) and non-voiding (B; double arrow) contractions respectively. The number of NVCs (C and D; double arrow) were reduced by SSR240612 (0.4 μmol·kg−1) treatment, but not the amplitude (C and E; arrowhead). Each column represents the mean and the vertical lines indicate the SEM of six to eight animals. *P < 0.05 compared with pretreatment values (Student's paired t-test).

Figure 9.

Figure 9

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Representative cystometric traces of sham-operated, SCI and icatibant-treated SCI rats on day 28 after surgery. During the filling phase, the sham-operated and SCI rats showed a large number of voiding (A; arrow) and non-voiding (B; double arrow) contractions respectively. The number (C and D; double arrow) and amplitude (C and E; arrowhead) of NVCs were reduced by icatibant (100 μmol·kg−1) treatment. Each column represents the mean and the vertical lines indicate the SEM of six to eight animals. *P < 0.05 compared with pretreatment values (Student's paired t-test).

Discussion

Herein, we report the novel finding indicating that SCI induces the overexpression of B1 receptors in the neurogenic urinary bladder of rat. Notably, the in vivo inhibition of B1 or B2 receptors by selective antagonists provides improvements in urodynamic parameters altered in OAB. Thus, the altered expression and sensitivity of B1 and B2 receptors could contribute to the emergence of OAB in SCI rats.

Spinal cord injury leads to important pathologies in the lower urinary tract, including OAB syndrome. SCI-induced OAB is known to produce discomfort in patients and it has become an economic challenge. The current treatment options for OAB syndrome include antimuscarinic agents and β3-adrenoceptor agonists, which have been associated with moderate to ineffective responses in some patients (Lecci and Maggi, 2005; Gulur and Drake, 2010; Limberg et al., 2010; Smith and Wein, 2010). Alterations in the properties, innervations and structure of the detrusor muscle and an increase in the expression and/or sensitivity of urothelial-sensory molecules/receptors that lead to afferent sensitization have been documented as being possible mechanisms for the genesis of SCI-induced OAB (Brading, 1997).

In the present study, we demonstrated the constitutive expression of both B1 and B2 receptors in the rat urinary bladder. The existence of the constitutive expression of the B1 receptor in the urinary bladder is still controversial. Chopra et al. (2005) failed to detect B1 receptor mRNA in the rat urinary bladder, whereas Belichard et al. (1999) and our group reported a basal expression of mRNA coding for the B1 receptor in the rat urinary bladder while Wotherspoon and Winter (2000) detected rat B1 receptor immunoreactivity in peripheral nerve terminals innervating the urinary bladder. Of note, our present data showed that both B1 receptor mRNA and protein were overexpressed in SCI urinary bladders in the early phase of recovery, while B2 receptor mRNA and protein expression were not changed following SCI. Consistent with these observations, an up-regulation of B1 receptor mRNA levels was reported in the detrusor muscle and urothelium following chemical cystitis in rats (Chopra et al., 2005). In contrast to the B1 receptor, B2 receptor expression was higher in urothelium than in detrusor muscle of naive and sham groups, suggesting the significant role exerted by the urothelial B2 receptor in the BK-induced contractile response of the urinary bladder under physiological conditions. Of particular interest is the finding that both B1 and B2 receptors protein expression was equally distributed in the detrusor muscle and urothelium through the different phases of recovery after SCI. Therefore, B1 and B2 receptors present in both structures could actively contribute to the exacerbated contractile response of the urinary bladder to des-Arg9-BK and BK stimulus caused by SCI.

It should be noted that while B1 receptor expression was not up-regulated on day 28 after SCI, the contractile response to des-Arg9-BK was exacerbated in this late phase of recovery. Of note, the contractile response to BK was exacerbated in all the analysed periods after SCI, but B2 receptor expression remained unchanged. Thus, the enhanced functional activity of both B1 and B2 receptor in the absence of significant B1 and B2 receptor up-regulation could be explained by receptor sensitization following tissue damage/inflammation.

A basal constitutive expression of the B1 receptor was demonstrated in DRG ganglia in rats, mice and monkeys (Ma et al., 2000; Wotherspoon and Winter, 2000; Shughrue et al., 2003; Rashid et al., 2004). Herein, we showed a significant amount of B1 receptor mRNA in the DRG (dorsal root ganglion, L6-S1) in the sham-operated group and its up-regulation in the SCI group on day 2 after surgery. Thus, B1 receptors could have been synthesized in the DRG during the early phase of recovery and moved towards the peripheral afferent terminal, reflecting an increase in B1 receptor protein on day 2 following SCI in the urinary bladder. However, part of the B1 receptor synthesis could have been carried out by epithelial and muscle cells of the urinary bladder since that B1 receptor mRNA levels are increased in urinary bladder in this period.

It has been documented that both B1 and B2 receptors are constitutively present in the mammalian spinal cord (Calixto et al., 2000; Couture and Lindsey, 2000; Wotherspoon and Winter, 2000; Ma and Heavens, 2001; Shughrue et al., 2003). In accordance with that, we observed the constitutive expression of B1 and B2 receptor mRNA and protein in the rat spinal cord. Relatively high levels of B1 receptor mRNA found in the spinal cord on days 2 and 7 after SCI were accompanied by an increased rate of B1 receptor protein synthesis on day 7 after SCI. Furthermore, we observed a marked up-regulation of the B2 receptor protein, but not of mRNA, in the spinal cord on days 2 and 7 after SCI, indicating that the basal levels of mRNA seem to be sufficient for the synthesis of the B2 receptor protein in the spinal cord. Therefore, these data suggest that spinal B1 and B2 receptors significantly contribute to central modulation of the reflex micturition after SCI, as the B1 and B2 receptor proteins were up-regulated in the corresponding portion of the spinal cord.

It is now well established that pro-inflammatory cytokines such as IL-1β induce the expression of B1 and B2 receptors (Campos et al., 1999; Calixto et al., 2004). Interestingly, IL-1β may induce the up-regulation of B1 receptors via the MAP-kinase pathways and NF-κB stimulation in several systems (Schanstra et al., 1998; Sjuve et al., 2000; Calixto et al., 2004; Moreau et al., 2005). In addition, Lee et al. (2008) have shown that B2 receptor, PLC and PKC pathways are required for the production of BK-induced IL-6. In accord with these data, we showed increased levels of the pro-inflammatory cytokines IL-1β and IL-6 in SCI urinary bladders 2 days after injury. Even though we did not evaluate the effect of the B1 and B2 receptors antagonists on cytokine levels, it is plausible that the overexpression of both B1 and B2 receptors in the SCI urinary bladder could be caused, at least in part, by the enhancement of cytokine levels observed in the early phase of recovery, which seems to contribute to the afferent sensitization observed in the OAB.

Herein, we evaluated the effect of the B1 receptor antagonist des-Arg9-[Leu8]-BK (DALBK) and the B2 receptor antagonist icatibant on the contractile activity 28 days after surgery, a period in which the contractile response to BK and des-Arg9-BK became stabilized. Icatibant is a known selective and potent B2 receptor antagonist that exhibits some anti-inflammatory properties such as reducing plasma protein extravasation, as well as reducing the hyper-reflexia in chemically-induced cystitis models (Hock et al., 1991; Lembeck et al., 1991; Giuliani et al., 1993; Ahluwalia et al., 1994; Smith and Wein, 2010). The peptide B1 receptor antagonist DALBK has a rapid onset of action as well as a rapid reversibility and it has been shown to have a good affinity for the B1 receptor rabbit urinary bladder (Hall and Morton, 1997; Regoli et al., 1998). Our present results showed that the incubation of the urinary bladder preparations with DALBK reduced the contractile response induced by the lower concentration of des-Arg9-BK. This phenomenon could be explained by the competitive antagonism exerted by DALBK on the des-Arg9-BK-induced contractile response, which is reversible on exposure of the preparations to higher concentrations of the agonist. Otherwise, the incubation of isolated SCI urinary bladders with icatibant consistently reduced the contractile response to BK. This finding is in agreement with the results of several studies showing that icatibant is a non-competitive antagonist of B2 receptors (Hock et al., 1991; Wirth et al., 1991; Rhaleb et al., 1992; Félétou et al., 1994; Kajekar and Myers, 2000; Levy and Zochodne, 2000).

Similar to the analysis of the effect of kinin receptor antagonists on contractile activity of the isolated urinary bladder, urodynamic analyses of the micturition process were assessed on day 28 after SCI, a period in which the external urethral sphincter (EUS) activity was partially improved, enabling the release and measure of fluid through the urethra. A previous study showed that the urinary bladder is areflexic during the first 4 days after SCI, and that following this period NVCs were present (Takahara et al., 2007). The NVCs are represented by increased spontaneous activity of the detrusor smooth muscle cells during the filling phase, which may cause OAB. Therefore, NVCs are believed to play a central role in the pathophysiology of OAB. It has been shown that the spontaneous phasic activity of the detrusor tissue is myogenic in origin (Liu et al., 1998) and that the urothelium plays a significant role in modulating the nature of these contractions (Buckner et al., 2002). Our results showed that SCI caused a significant increase in the amplitude and number of NVCs during the urinary bladder filling phase. With regard to this parameter, icatibant seems to be more effective than DALBK and SSR240612 since those B1 receptor antagonists reduced only the number of NVCs while icatibant reduced both the amplitude and number of NVCs. These findings suggest that both B1 and B2 receptors expressed in the detrusor muscle and urothelium are involved in the emergence of NVCs.

SCI disrupts the phasic activity of the EUS resulting in decreased VE and increased (RV; Dolber et al., 2007). The dysfunction of the EUS triggers the development of detrusor-sphincter DSD. In our study, we observed marked alterations in the voiding caused by the EUS dysfunction such as reduced VV and VE and greater urinary BC due to the excessive stretching of the detrusor muscle triggered by overloading the urinary bladder. In that sense, acute systemic treatment with SSR240612, a non-peptide B1 receptor antagonist, was more effective than the treatment with DALBK, a peptide B1 receptor antagonist, because SSR240612 significantly increased the VV and the VE and reduced the urinary BC. The action of SSR240612 on the VE suggests that B1 receptor blockade might have an effect on the spinal motor neurons that control the impaired activity of the EUS after SCI. Moreover, the greater efficacy of SSR240612 compared to DALBK on the voiding function could be explained by their long-lasting efficacy and good bioavailability (Gougat et al., 2004). Therefore, B1 receptor inhibition seems to exert beneficial actions on OAB and DSD.

In summary, we provided evidence for the basal expression of B2 receptors in the urinary bladder, which suggests that BK may play a role in normal urinary bladder functioning. The up-regulation of B1 receptors in SCI urinary bladders might be associated with the alterations in urinary bladder reflex pathways in OAB caused by SCI. Moreover, the B1 and B2 receptor antagonism improves urodynamic parameters associated with the OAB in SCI animals. Thus, kinin receptor-selective antagonists might constitute an attractive pharmacological tool for the treatment of urinary bladder overactivity in neurogenic conditions such as SCI.

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Programa de Apoio aos Núcleos de Excelência (PRONEX) and by Fundação de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC) (Brazil). SF received a fellowship from CAPES; ELA, ACM and AFB received fellowships from CNPq; JK received a grant from FAPESC.

Glossary

BC

urinary bladder capacity

CRC

cumulative concentration-response curves

DALBK

des-Arg9-[Leu8]-bradykinin

DRG

dorsal root ganglion

NVCs

non-voiding contractions

OAB

overactive urinary bladder

RV

residual volume

SCI

spinal cord injury

SSR240612

(2R)-2-((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride

VE

voiding efficiency

VV

voided volume

Conflict of interest

The authors declare no conflict of interest.

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