European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats Antonio Celso S. Ramos-Filho, Julio Alejandro Rojas Moscoso, Fabiano Camasini, Juliana de Almeida Faria, Gabriel Forato Anhê, Fabíola Z. Mónica, Edson Antunes n a

Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), 13084-971 Campinas, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 24 March 2014 Received in revised form 20 May 2014 Accepted 21 May 2014

Association between hypertension and bladder symptoms has been described. We hypothesized that micturition dysfunction may be associated with renin–angiotensin system (RAS) acting in urethra. The effects of the anti-hypertensive drugs losartan (AT1 antagonist) and captopril (angiotensin-converting enzyme inhibitor) in comparison with atenolol (β1-adrenoceptor antagonist independently of RAS blockade) have been investigated in bladder and urethral dysfunctions during renovascular hypertension in rats. Two kidney-1 clip (2K-1C) rats were treated with losartan (30 mg/kg/day), captopril (50 mg/kg/ day) or atenolol (90 mg/kg/day) for eight weeks. Cystometric study, bladder and urethra smooth muscle reactivities, measurement of cAMP levels and p38 MAPK phosphorylation in urinary tract were determined. Losartan and captopril markedly reduced blood pressure in 2K-1C rats. The increases in non-voiding contractions, voiding frequency and bladder capacity in 2K-1C rats were prevented by treatments with both drugs. Likewise, losartan and captopril prevented the enhanced bladder contractions to electrical-field stimulation (EFS) and carbachol, along with the impaired relaxations to β-adrenergic-cAMP stimulation. Enhanced neurogenic contractions and impaired nitrergic relaxations were observed in urethra from 2K-1C rats. Angiotensin II also produced greater urethral contractions that were accompanied by higher phosphorylation of p38 MAPK in urethral tissues of 2K-1C rats. Losartan and captopril normalized the urethral dysfunctions in 2K-1C rats. In contrast, atenolol treatment largely reduced the blood pressure in 2K-1C rats but failed to affect the urinary tract smooth muscle dysfunction. The urinary tract smooth muscle dysfunction in 2K-1C rats takes place by local RAS activation irrespective of levels of arterial blood pressure. & 2014 Published by Elsevier B.V.

Keywords: 2K-1C Urethra Losartan Captopril P38 MAPK cAMP

1. Introduction Normal bladder function includes a storage phase and a voiding phase, which are controlled by complex interactions of efferent and afferent fibers from the autonomic nervous system and somatic innervation (Andersson and Arner, 2004). Precise coordination between bladder and urethra are required to normal urinary tract function (Birder, 2013). Bladder smooth muscle contraction by acetylcholine released from parasympathetic nerves together with urethral relaxation by release of nitric oxide (NO) from nitrergic fibers are important components for an efficient bladder emptying. Abnormalities of any component of these neural pathways result in micturition dysfunction, clinically expressed as symptoms of urgency, with or without urge incontinence, frequency and nocturia (Yoshimura et al., 2008).

n

Corresponding author. Tel.: þ 55 19 3521 9556; fax: þ55 19 3289 2968. E-mail addresses: [email protected], [email protected] (E. Antunes).

Vascular risk factors including hypertension have been reported to play a role for the development of lower urinary tract symptoms (LUTS) (Ponholzer et al., 2006). In animal models, cystometric changes and bladder overactivity have also been reported in hypertensive rats due to chronic nitric oxide (NO) blockade (Mónica et al., 2008) and spontaneously hypertensive rats (SHR) (Jin et al., 2009). Renovascular hypertensive rats (2K-1C) also display cystometric alterations including increased bladder capacity and non-voiding contractions, as well as lower intercontraction micturition intervals (Ramos-Filho et al., 2011). Functionally, the bladder from 2K-1C rats shows greater contractions to muscarinic agonist stimulation and decreased β-adrenoceptormediated cAMP signal transduction. Angiotensin II (ANG II), an octopeptide product of the renin– angiotensin system (RAS), is an important regulator of the vascular system (Patel and Mehta, 2012). Increased serum ANG II induces vasoconstriction, vascular remodeling and endothelial dysfunction (Voors et al., 2005). The AT1 receptor activation by ANG II leads to mitogen-activated protein kinase-38 phosphorylation (p38 MAPK) that has been associated with impairment of vascular endothelial

http://dx.doi.org/10.1016/j.ejphar.2014.05.038 0014-2999/& 2014 Published by Elsevier B.V.

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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cell function and leads to arterial hypertension (Yang et al., 2014), as well as with cardiac hypertrophy in renovascular hypertensive mice (Pellieux et al., 2000). Outside the cardiovascular system, including the lower urinary tract, evidence indicates that serum ANG II exerts a functional role in the maintenance of urethral tone and stress continence (Phull et al., 2007). Accordingly, hypertensive patients treated with ANG II receptor antagonists are less susceptible to urinary tract symptoms (Ito et al., 2013). Besides, ANG II has been implicated in the pathogenesis of bladder dysfunction in models of bladder outlet obstruction (BOO) (Cho et al., 2012; Comiter and Phull, 2012) and ovariectomy (RamosFilho et al., 2013). The present study aimed to test if the hyperactivity of the renin–angiotensin system (RAS) and the resulting arterial hypertension contribute to the micturition dysfunction in 2K-1C rats. For that purpose, 2K-1C and age-matched SHAM rats were chronically treated with either losartan (AT1 receptor antagonist) or captopril (angiotensin-converting enzyme inhibitor), after which cystometry and in vitro bladder and urethra smooth muscle reactivities were performed. Western blot and ELISA assay in bladder and urethra tissues were performed to explore the mechanisms by which RAS inhibition improves urinary function in the hypertensive rats. Treatment of rats with the β1-adrenoceptor antagonist atenolol was also employed as an antihypertensive agent acting independently of RAS blockade.

2.4. Cystometric study After 8 weeks of renovascular hypertension, male rats were subjected to cystometric study under urethane anesthesia (1.2 g/ kg, i.p), according to a previous study (Ramos-Filho et al., 2013). Briefly, 1-cm incision was made along the midline of the rat abdomen. The bladder was exposed and a butterfly cannula (25 G) was inserted into the bladder dome. The cannula was connected to a three-way tap, one port of which was connected to a pressure transducer and the other to the infusion pump through a catheter (PE50). Before starting the cystometry, the bladder was emptied via the third port. An equilibration period of 5 min was allowed before saline infusion and recording the cystometric parameters. Continuous cystometry was carried out by infusing saline into the rat bladder at a rate of 4 ml/h, and lasted 35 min. Bladder pressures were recorded using computer software (Power Lab v.7.0 system, ADInstruments, Sydney-NSW, Australia). The following parameters were assessed: threshold pressure (TP; intravesical pressure immediately before micturition), voiding pressure (VP; pressure reached during micturition), frequency of voiding, capacity (CP; volume of saline needed to induce the first micturition), basal pressure (BP), and frequency of non-voiding contraction (NVC; spontaneous bladder contractions greater than 4 mmHg from the baseline pressure that did not result in a void). One rat was used for each cystometrogram (CMG). Bladders from cystometric study were not used in the other experiments.

2. Materials and methods 2.1. Animals All animal procedures and the experimental protocols were approved by the Ethical Principles in Animal Research adopted by Brazilian College for Animal Experimentation (COBEA). Male and Female Wistar rats were housed at constant room temperature with 12-h light and dark cycles. Food and water were available ad libitum.

2.2. Induction of renovascular hypertension Female and male rats with average age of 10 weeks (body weight between 250 and 300 g) underwent renovascular hypertension induction, as described previously (Moreno et al., 1996). Briefly, rats were anesthetized with thiopental sodium (Tiopentaxs 20 mg/kg, i.p.). A silver clip (0.2 mm i.d.) was placed around the left renal artery to produce a partial occlusion. Control agematched rats (SHAM group) were submitted to similar procedures with the exception of the silver clip placement.

2.3. Drug treatments Immediately after renovascular hypertension procedure, SHAM and 2K-1C rats were randomly grouped to receive losartan (30 mg/ kg/day; n ¼19), captopril (50 mg/kg/day; n ¼19), atenolol (90 mg/ kg/day; n ¼19) or tap water alone (control group; n¼ 19) for 8 weeks. These doses of losartan, captopril and atenolol have been chosen according to previous studies in rats (Boshra et al., 2011; Nobre et al., 2006; Oron-Herman et al., 2005). Drugs were dissolved daily in tap water at a final concentration calculated as a function of body weight and volume of water consumed the day before. The treatments were started immediately after the recovery period from the anesthesia. Systolic blood pressure (SBP) was achieved using a tail-cuff method in conscious animals. SBP and body weight were measured before the beginning of treatment and every week during treatment until week 8.

2.5. Bladder smooth muscle preparations and concentration– response curves Male rats were stunned by inhalation of CO2, euthanized by decapitation and exsanguinated. Bladder dome was removed and cut into two longitudinal strips. Bladder smooth muscle strips with intact urothelium (detrusor and mucosa) were mounted in 10-ml organ baths containing Krebs–Henseleit solution with the following composition (mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose, pH 7.4, at 37 1C and bubbled with a gas mixture of 95% O2 and 5% CO2. Changes in isometric force were recorded using a computer Software (Power Lab v.7.0 system, ADInstruments, Sydney-NSW, Australia). The resting tension was adjusted to 20 mN at the beginning of the experiments. The equilibration period was 60 min and the bathing medium was changed every 15 min until the start of the experiments. Cumulative concentration–response curves to the full muscarinic agonist carbachol (10  9–10  3 M) were constructed by using one-half log unit. Bladder relaxations to the non-selective βadrenoceptor agonist isoproterenol (10  9–10  3 M) were also evaluated in bladder pre-contracted with KCl (80 mM). Nonlinear regression analysis to determine the pEC50 was carried out using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA) with the constraint that F¼ 0. All concentration–response data were evaluated for a fit to a logistics function in the form: E ¼Emax/ ([1 þ(10c/10x)n] þF), where E is the maximum response produced by agonists; c is the logarithm of the EC50, the concentration of drug that produces a half-maximal response; x is the logarithm of the concentration of the drug; the exponential term, n, is a curvefitting parameter that defines the slope of the concentration– response line, and F is the response observed in the absence of added drug. Data were normalized to the wet weight of the respective urinary bladder strips, and the values of Emax were represented by mN/mg. Relaxing responses were calculated as percentages of the maximal changes from the steady-state contraction produced by KCl (80 mM) in each tissue.

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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2.6. Urethral smooth muscle preparations and concentration– response curves To evaluate the functional responses in urethral smooth muscle we have used female rats. Male rats were not used because there exist an anatomical limitation in removing prostatic urethra (i.e. the part of the urethra, which runs through the prostate). Briefly, female rats were stunned by inhalation of CO2, euthanized by decapitation and exsanguinated. Urethras were dissected out and cut into two rings of approximately 3 mm in length after removal of surrounding fat and connective tissues. Each ring was suspended between two wire hooks and mounted in 10-ml organ chambers filled with Krebs' solution at the same conditions showed for bladder preparations. To record the development of isometric tension, hooks were fixed to the bottom of the chamber and to a force transducer (AdInstruments, MA, USA) connected to a PowerLab 4/30 data-acquisition system (Software Chart, version 7.0; ADInstruments, Colorado Springs, MA, USA). The resting tension was adjusted to 3 mN at the beginning of the experiments. The equilibration period was 45 min and the bathing medium was changed every 15 min until the start of the experiments. Cumulative concentration–response curves to ANG II (10  10–10  6 M) were constructed by using one-half log units. Values of Emax were represented by mN and pEC50 were calculated as described above for bladder. 2.7. Electrical-field stimulation (EFS) in bladder and urethra EFS was applied in bladder strips or urethral rings placed between two platinum ring electrodes (3 mm diameter) connected to a Grass S88 stimulator (Astro-Med, Industrial Park,RI). Frequency–response curves (4–32 Hz) were elicited by stimulating the tissues for 10 s with pulses of 1 ms width at 80 V, with 3 min interval between stimulations (see further details in Section 3). 2.8. Measurement of cAMP levels in bladder Bladder strips were equilibrated for 30 min in warmed (37 1C) and oxygenated (95% O2/5% CO2) Krebs solution. Tissues were stimulated for 15 min with isoproterenol (10 μM) or left unstimulated (basal). Next, tissues were immediately frozen in liquid nitrogen. Frozen tissues were pulverized, homogenized in trichloroacetic acid (TCA, 5% wt/vol), centrifuged for 10 min at 4 1C at 1500g, and the supernatant was collected. The pellet was dried and weighted. TCA was extracted from the supernatant with three washes of water saturated ether. Preparation of tracer, samples, standards and incubation with antibody were performed as described in commercially available kits (Cayman Chemical Cyclic AMP EIA kit, Ann Arbor, MI, USA). The assays were performed in duplicates, and the pellet weight was used to normalize the data that were calculated as pmol/mg tissue. Data was expressed as % of cAMP generated between mean baseline and after stimulation with isoproterenol.

polyacrylamide gels and then electrotransfered to nitrocellulose membrane, performed for 3 h at 45 V (constant) in a semi-dry device (Bio-Rad, Hercules, CA, USA). Nonspecific protein binding to nitrocellulose was reduced by pre-incubating the membrane overnight at 4 1C in blocking buffer (0.5% non-fat dried milk, 10 mM Tris, 100 mM NaCl, and 0.02% Tween 20). Detection using specific antibodies, HRP-conjugated secondary antibodies, and luminol solution was performed. Anti-p38 MAPK (9212) and Antiphospho-p38 MAPK (9216) were obtained from Cell Signaling technology (Cell Signaling, MA, USA). Densitometry was performed using the Scion Image Software (Scion Corporation, Frederick, MD), and results represented as the ratio of the density of phosphorylated p38 MAPK to the density of the total p38 MAPK.

2.10. Drugs and chemicals Angiotensin II, atenolol, atropine, carbachol, captopril, guanethidine, isoproterenol, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), losartan potassium, phentolamine, tetrodotoxin and vasopressin were purchased from Sigma-Aldrich (St Louis, MO, USA). All reagents used were of analytical grade. Stock solutions were prepared in deionized water.

2.11. Statistical analysis All measurements are expressed as means 7S.E.M. Comparisons among the groups were evaluated using ANOVA and the Tukey post hoc analysis (StatMate). The significance level was chosen as P o0.05.

3. Results 3.1. Tail-cuff pressure and body weight 2K-2C rats exhibited higher tail-cuff pressure compared with SHAM group (P o0.05). Treatment with captopril (50 mg/kg/day), losartan (30 mg/kg/day) and atenolol (90 mg/kg/day) significantly attenuated the increased tail-cuff pressure in 2K-1C rats (Table 1). 2K-1C rats also exhibited a higher ratio bladder/body weight compared with SHAM group (Po 0.05), which was prevented by treatment with captopril or losartan, but not with atenolol (n ¼10). The body weight did not change significantly between groups (not shown). Table 1 Tail-cuff pressure (TCP), ratio between bladder/body weight and cystometric parameters (basal pressure, threshold pressure and voiding pressure) in SHAM and 2K-1C rats, treated or not with captopril (CAP; 50 mg/kg/day), losartan (LOS; 30 mg/kg/day) or atenolol (ATE; 90 mg/kg/day) for 8 weeks.

2.9. Western blotting Urethras were isolated, washed in Krebs–Henseleit solution and homogenized in SDS lysis buffer with Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Inc., Westbury, NY) operated at maximum speed for 30 s and centrifuged (12,000g, 4 1C, 20 min) to remove insoluble material. Protein concentrations of the supernatants were determined by the Bradford assay, and equal amount of protein from each sample (100 μg) was treated with Laemmli buffer containing dithiothreitol (100 mM). Samples were heated in a boiling water bath for 10 min and resolved by SDS-PAGE. The proteins were separated by 10%

3

SHAM 2K-1C 2K-1C – CAP 2K-1C – LOS 2K-1C – ATE

TCP (mmHg)

Bladder/BW (mg/g)

BP (mmHg)

TP (mmHg)

VP (mmHg)

118 78.3 176 714.7a 113 76.3b 110 78.6b 137 75.8b

0.31 0.38a 0.32b 0.32b 0.39a

5.6 7 0.2 6.2 7 0.1 5.8 7 0.2 5.7 7 0.4 6.4 7 0.2

18.3 7 2.9 16.0 7 4.8 20.0 7 2.9 21.5 7 3.3 20.6 7 1.4

28.0 7 12.5 25.4 7 4.5 22.5 7 2.6 22.3 7 2.2 29.0 7 3.6

Data represent the means 7S.E.M. (n ¼5 for cystometric parameters; n¼ 10 for tailcuff and ratio between bladder/body weight). BW, body weight; BP, basal pressure; TP, threshold pressure; VP, voiding pressure. a

Po 0.05 compared with SHAM group (ANOVA followed by a Tukey test). P o 0.05 compared with untreated 2K-1C rats (ANOVA followed by a Tukey test). b

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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contractions seen in 2K-1C rats (Fig. 2A). The voltage-gated sodium channel blocker tetrodotoxin (1 mM) nearly abolished the EFSinduced contractions in all groups, confirming the neurogenic nature of the responses (not shown). Carbachol (10  9–10  3 M) produced concentration-dependent bladder contractions, which were markedly greater in 2K-1C compared with SHAM group (Po 0.05; n¼ 5 each group). Treatment with captopril and losartan did not change the responses to carbachol in SHAM group, but normalized the contractions in 2K-1C rats (Fig. 2B). Treatment with atenolol failed to affect the increased carbachol-induced contractions in 2K-1C rats (Fig. 2B). The potency (pEC50) for carbachol did not significantly change between groups (Table 2).

3.2. Cystometric study The micturition pattern in 2K-1C rats was irregular and characterized by significant increases in the frequency of voiding, capacity and NVCs (P o0.05; Fig. 1) compared with SHAM group. Treatments with captopril or losartan prevented these micturition changes in 2K-1C, whereas atenolol treatment had no significant effect (Fig. 1; n ¼5 each group). The basal pressure, threshold pressure and voiding pressure did not significantly change between groups (Table 1). 3.3. Contractions to electrical-field stimulation (EFS) and carbachol in bladder Electrical-field stimulation (EFS) produced frequencydependent bladder contractions in both groups, which were greater in 2K-1C rats (P o0.05) at 4–32 Hz (Fig. 2A; n ¼ 5 each group). Captopril and losartan treatments did not change the EFSinduced contractions in SHAM group (not shown), but normalized the increased contractions in 2K-1C rats in all frequencies tested. In contrast, atenolol failed to affect the increased EFS-induced

3.4. Bladder relaxations to isoproterenol and cAMP levels in bladder tissues The non-selective β-adrenoceptor agonist isoproterenol (10  9– 10  3 M) produced concentration-dependent bladder relaxations in KCl (80 mM)-pre-contracted strips (Fig. 2C; n ¼5 each group). Maximal relaxations to isoproterenol were significantly lower in

SHAM SHAM

Frequency of Voiding (number/min)

2K1C

2K1C

0.8

2K1C+CAP 2K1C+LOS

0.6

2K1C+ATE

0.4 0.2 0.0

2K1C + LOS Capacity (mL)

2.0

5 mmHg

2K1C + CAP

1.5 1.0 0.5 0.0

NVCs (number / min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

2K1C + ATE

0.4 0.3 0.2 0.1 0.0

t: min 0

5

10

15

20

25

30

35

Fig. 1. Cystometric study in SHAM and 2K-1C male rats, treated or not with losartan (LOS; 30 mg/kg/day), captopril (CAP; 50 mg/kg/day) or atenolol (ATE; 90 mg/kg/day) for 8 weeks. (A) Typical cystometric traces; (B) frequency of voiding; (C) capacity; (D) non-voiding contractions (NVCs). Data represent the mean 7S.E.M. for 5 rats each group. *Po 0.05 compared with SHAM group; #P o0.05 compared with untreated 2K-1C group (one-way ANOVA followed by a Tukey test). Arrows in the cystometric trace indicate the micturition peaks.

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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4

SHAM 2K1C 2K1C+CAP

3

2K1C+LOS 2K1C+ATE

*

#

# #

*

*

*

*

* *

2

*

# #

#

# #

1 0

Contraction (mN / mg)

5

Contraction (mN / mg)

4

SHAM 2K1C 2K1C+CAP

3

2K1C+LOS 2K1C+ATE

2

1

0 4

8

16

32

-9

Frequency (Hz)

-8

-7

-6

-5

-4

-3

Log [carbachol] : M

Fig. 2. Contractile responses to electrical-field stimulation (EFS; 4–32 Hz) (A) and concentration–response curve to carbachol (10  9–10  3 M, B) in bladders from SHAM and 2K-1C rats, treated or not with losartan (LOS; 30 mg/kg/day), captopril (CAP; 50 mg/kg/day) and atenolol (ATE; 90 mg/kg/day). Data represent the means7 S.E.M. for 5 rats each group. *Po 0.05 compared with SHAM group; #Po 0.05 compared with untreated 2K-1C group (one-way ANOVA followed by a Tukey test). Table 2 Potency (pEC50) and maximal responses (Emax) to both carbachol (CCh) and isoproterenol (ISO) in bladder, and to angiotensin II (Ang II) in urethra smooth muscle. Data were obtained in SHAM and 2K-1C rats, treated or not with captopril (CAP; 50 mg/kg/day), losartan (LOS; 30 mg/kg/day) or atenolol (ATE; 90 mg/kg/day) for 8 weeks. Groups

SHAM 2K-1C 2K-1C – CAP 2K-1C – LOS 2K-1C – ATE

Bladder

Urethra

CCh pEC50

Emax

ISO pEC50

Emax

Ang II pEC50

Emax

5.717 0.06 5.58 7 0.08 5.59 7 0.06 5.83 7 0.07 5.677 0.07

2.5 7 0.1 3.3 7 0.1b 2.5 7 0.1d 2.3 7 0.1d 3.17 0.1b

6.107 0.09 6.26 7 0.09 6.23 7 0.08 6.29 7 0.06 5.82 7 0.10a

61.17 2.2 48.87 2.1a 61.0 7 1.6c 61.2 7 1.3c 48.57 4.0a

7.81 7 0.09 7.83 7 0.08 7.58 7 0.06 7.337 0.09a 7.667 0.05

0.4 7 0.1 1.6 7 0.1b 0.3 7 0.1d 0.3 7 0.1d 1.6 7 0.1b

Data represent the means 7 S.E.M. (n¼ 5). a Po 0.05 and b P o0.001 compared with SHAM (ANOVA followed by a Tukey test). c P o0.05 and dPo 0.001 compared with untreated 2K-1C rats (ANOVA followed by a Tukey test).

0

125

20

100

AMPc (% increase)

Relaxation (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

5

40 SHAM 2K1C 2K1C+CAP 2K1C+LOS 2K1C+ATE

60

80 -9

-8

-7

75 #

50 25

*

# *

0 -6

-5

-4

-3

Log [isoproterenol] : M

Fig. 3. Concentration–response curve to isoproterenol (10  9–10  3 M, A) and % of cAMP generated after isoproterenol (10 mM) stimulation in bladders (B) from SHAM and 2K-1C rats, treated or not with losartan (LOS; 30 mg/kg/day), captopril (CAP; 50 mg/kg/day) and atenolol (ATE; 90 mg/kg/day). Data represent the means7 S.E.M. for 5 rats each group. *Po 0.05 compared with SHAM group; #Po 0.05 compared with untreated 2K-1C group (one-way ANOVA followed by a Tukey test).

2K-1C compared with SHAM group (P o0.05; Table 2). Captopril and losartan normalized the decreased relaxations in 2K-1C rats, whereas atenolol failed to affect the decreased bladder relaxations (Fig. 2C; Table 2). The potency (pEC50) for isoproterenol was not significantly changed in any experimental group, except in 2K-1C rats receiving atenolol where a significant reduction in this parameter was observed (Table 2). The signaling pathway of β-adrenergic receptors involves the activation of adenylyl cyclase with consequent formation of the nucleotide cAMP (Longhurst et al., 1997; Uchida et al., 2005). Since isoproterenol-induced bladder relaxations were reduced in 2K-1C rats, we next evaluated the cAMP levels in the bladder tissues. Incubation of bladder tissues from SHAM group with isoproterenol

(10 μM) significantly elevated the cAMP levels (P o0.05; Fig. 2D). However, isoproterenol nearly failed to significantly elevate the cAMP levels in bladder tissues from 2K-1C rats. Treatment with captopril and losartan significantly restored the isoproterenolinduced cAMP increase in 2K-1C rats. The reduced cAMP levels in bladders from 2K-1C rats were not changed by treatment with atenolol (Fig. 2D). 3.5. Contractile responses to EFS and angiotensin II in urethra smooth muscle We next explored the functional responses in urethra smooth muscle in 2K-1C rats, treated or not with losartan, captopril and

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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6

0.8 0.6

2.0

SHAM 2K1C 2K1C+CAP 2K1C+LOS 2K1C+ATE

*

*

0.4 * 0.2

*

*

# #

# #

2

4

* #

*

*

#

# #

# #

0.0

1.5

1.0

0.5

0.0 8

16

32

-10

Frequency (Hz)

40

30

SHAM 2K1C 2K1C+ CAP 2K1C+LOS 2K1C+ATE

*

Contraction (mN)

Contraction (mN)

1.0

Relaxation (%)

SHAM 2K1C 2K1C+CAP 2K1C+LOS 2K1C+ATE #

#

# ## * *

-8

-7

-6

#

20

10

-9

Log [angiotensin II]: M

*

*

#

#

*

*

*

*

*

*

0 2

4

8 Frequency (Hz)

16

32

Fig. 4. Evaluation of the contractile and relaxant responses of urethra smooth muscle from SHAM and 2K-1C rats. Contractile responses to electrical-field stimulation (EFS; 4–32 Hz, A) and angiotensin II (10  10–10  6 M, B), as well as to relaxant responses to electrical-field stimulation (EFS; 4–32 Hz, C) from SHAM and 2K-1C rats, treated or not with losartan (LOS; 30 mg/kg/day), captopril (CAP; 50 mg/kg/day) or atenolol (ATE; 90 mg/kg/day). Relaxant responses were calculated relative to the maximal changes from the contraction produced by vasopressin (0.1 mM) in each tissue, which was taken as 100%. Data represent the mean 7 S.E.M. of 5 rats each group. *Po 0.05 compared with SHAM group, #Po 0.05 compared with untreated 2K-1C group (one-way ANOVA followed by a Tukey test).

atenolol. We have used urethra from female rats because there is an anatomical limitation in isolating urethras from male rats related to the male anatomy. Electrical-field stimulation (EFS; 2–32 Hz) produced frequency-dependent urethral contractions, which were significantly greater in 2K-1C compared with SHAM group (Fig. 3A; P o0.05). Treatment with captopril and losartan prevented the increased contractions in 2K-1C animals, whereas atenolol had no effect (Fig. 3A). Incubation with either tetrodotoxin (1 mM) or phentolamine (non-selective α-adrenergic antagonist) markedly reduced EFS-induced urethral contractions (not shown). Cumulative addition of ANG II (10  10–10  6 M) produced concentration-dependent contractions in isolated urethral rings, achieving maximal responses at 300 nM (Fig. 3B). The maximal contractions to ANG II were markedly greater in 2K-1C group (P o0.01), which were completely prevented by captopril and losartan treatments (Fig. 3B; Table 2). Atenolol treatment did not change the enhanced ANG II-induced responses in 2K-1C group. The potency (pEC50) for ANG II was not significantly changed in any experimental group, except in 2K-1C rats receiving losartan where a reduction was observed (Table 2). 3.6. Relaxant responses to nitrergic stimulation in urethra smooth muscle Bladder emptying involves loss of urethral smooth muscle sphincter tone due to the release of NO from the nitrergic fibers (Andersson, 2001). The nitrergic relaxations were evaluated according to a previous study (Triguero et al., 2009). Briefly, urethral preparations were pretreated with a mixture of guanethidine (50 mM; to deplete the catecholamine stores of adrenergic fibers), atropine (1 mM; to produce muscarinic antagonism) and D-tubocurarine (10 mM; to block the activation of urethral striated muscle). Next, urethral preparations were pre-contracted with

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Fig. 5. Western blotting analysis of p38 MAPK phosphorylation (P-p38 MAPK) in isolated urethra from SHAM and 2K-1C female rats, treated or not with losartan (LOS; 30 mg/kg/day), captopril (CAP; 50 mg/kg/day) and atenolol (ATE; 90 mg/kg/ day) for 8 weeks. Data represent the mean 7 S.E.M. of 4 rats each group. *Po 0.05 compared with control values, #Po 0.05 compared with untreated 2K-1C group (one-way ANOVA followed by a Tukey test).

vasopressin (0.1 mM), and when a stable contraction level was attained, a series of EFS (2–32 Hz) were applied to allow full nitrergic relaxations. Fig. 3C shows that 2K-1C rats exhibited decreased nitrergic relaxations in all frequencies tested compared with SHAM group (P o0.05). Treatment with losartan or captopril (but not with atenolol) significantly prevented the reductions of nitrergic relaxation in 2K-1C rats (Fig. 3C). As expected, incubation of urethral preparations with the nitric oxide inhibitor L-NAME

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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(100 mM) reduced by 95% (P o0.001) the nitrergic relaxations evoked by EFS in all groups. 3.7. Western blotting in urethral tissues Total p38 MAPK protein expressions in urethral tissues were not different among groups (Fig. 4). However, phosphorylated p38 MAPK expression was significantly higher in urethral tissues from 2K-1C compared with SHAM group (P o0.05). Treatment with captopril and losartan (but not with atenolol) fully prevented the p38 MAPK phosphorylation in 2K-1C (Figs. 4 and 5).

4. Discussion The RAS is well known to play a pivotal role in arterial hypertension in renovascular hypertensive rats. However, little is known about the role of RAS in the lower urinary tract smooth muscle and micturition dysfunction. The goal of this work was to test if RAS hyperactivity and the resulting arterial hypertension contribute to micturition dysfunction in 2K-1C rats. Long-term treatments with captopril and losartan all prevented the elevated systolic blood pressure in 2K-1C rats. Moreover, losartan and captopril normalized the bladder and urethral dysfunction in 2K-1C rats, as evidenced by both functional (cystometry and in vitro smooth muscle reactivities) and molecular assays (p38 MAPK phosphorylation). However, our findings that atenolol reduced blood pressure, but had no effect in the functional and molecular alterations in 2K-1C rats strongly suggest that arterial hypertension per se does not account for the resulting micturition dysfunction in 2K-1C rats. Therefore, micturition dysfunction in 2K-1C rats may be secondary to local RAS overactivation in the urinary tract smooth muscle. The voiding cycle comprises the filling phase where bladder relaxes and the outflow tract offers a high resistance, and the emptying phase where the outflow resistance falls and the bladder wall generates a high wall tension to raise intravesical pressure (Andersson and Arner, 2004; Birder, 2013). Renovascular hypertensive (2K-1C) rats exhibit bladder dysfunction characterized by increases in the voiding frequency, capacity and NVC (Ramos-Filho et al., 2011). We show here that long-term treatment with captopril and losartan fully normalized the voiding dysfunction in 2K-1C rats, whereas atenolol had no protective effect. Acetylcholine released from cholinergic fibers and its binding to muscarinic receptors represents the main mechanism of bladder contraction (Yoshimura et al., 2008). On the other hand, activation of β2- and β3-adrenoceptors in the bladder is the major mechanism accounting for the filling phase (Deba et al., 2009; Takeda et al., 1999). Our study showed that EFS- and carbachol-induced bladder contractions were greater in 2K-1C, an effect significantly attenuated by treatments with captopril and losartan, but not with atenolol. Impairment of β-adrenergic bladder relaxation has been shown in 2K-1C rats (Ramos-Filho et al., 2011). Activation of β-adrenergic receptors stimulates adenylyl cyclase, consequently increasing cAMP concentration and relaxing the bladder smooth muscle (Longhurst et al., 1997; Uchida et al., 2005). Treatment with captopril and losartan normalized the intracellular cAMP levels in bladders from hypertensive rats, restoring consequently the impaired isoproterenol-induced bladder relaxations to the level of control animals. On the other hand, treatment with atenolol had no effect on isoproterenol-induced responses and cAMP levels in bladder tissues. Therefore, RAS inhibition by captopril and losartan prevents the bladder hyperactivity by both decreasing bladder contractility and reestablishing the relaxation. Accordingly, losartan has been shown to improve urodynamic parameters in BOO mouse model (Comiter and Phull, 2012).

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Urethra smooth muscle contraction by sympathetic fibers stimulation is the main physiological pathway that maintains continence. Noradrenaline released from sympathetic fibers activates post-junctional α1-adrenoceptors leading to urethral contractions (Andersson, 2001). Electrical-field stimulation (1 and 32 Hz) produced noradrenaline-dependent urethral contractions (tetrodotoxin-sensitive responses), which were greater in 2K-1C rats. Captopril and losartan (but not atenolol) restored these neurogenic contractions in the 2K-1C group. The sympathetic nervous system interacts with RAS in vascular smooth muscle where ANG II enhances the release of norepinephrine from sympathetic nerve terminals, and in turn sympathetic nervous system may promote RAS activation and ANG II formation (Stegbauer et al., 2008; Zimmerman et al., 1987). Exogenous ANG II has no effect in the rat isolated bladder (Ramos-Filho et al., 2013), but it is described to exert a functional role in the maintenance of urethral tone and stress continence (Phull et al., 2007). Moreover, urethral hypercontractility and up-regulation of angiotensin receptors in urethral tissue contribute to micturition dysfunction in female rats under prolonged estrogen deprivation (Ramos-Filho et al., 2013). However, to the best of our knowledge, no previous study investigated the role of ANG II in the urinary tract smooth muscle in conditions of renovascular hypertension. In our study, exogenous ANG II produced concentration-dependent urethral contractions that were greater in 2K-1C compared with SHAM rats. Treatment with captopril and losartan reduced the ANG II-induced urethral contractions in 2K-1C group to the level of controls, whereas atenolol had no effect. Angiotensin II, via AT1 receptors, activates downstream signaling pathways including MAP kinases, such as ERK 1/2, JNK and p38 (Mehta and Griendling, 2007). MAPKs are a family of serine/threonine kinases which are classically associated with vascular smooth muscle cell contraction, migration, adhesion, collagen deposition, cell growth, differentiation, and survival. In the lower urinary tract, p38 MAPK is expressed in bladder smooth muscle cells, and when stimulated by ANG II promotes an increase in p38 MAPK phosphorylation (Nguyen et al., 2000). In our study, an increase in p38 MAPK phosphorylation was observed in the urethral tissues of 2K-1C rats, which was normalized by captopril and losartan, but not by atenolol. The mechanisms by which p38 MAPK contributes to smooth muscle contraction are unclear. However, some evidences suggest that p38 activates MAPKAP kinases 2 and 3, which phosphorylate heat shock protein (HSP27). The functions of HSP27 in smooth muscle are not fully defined, but there is evidence that HSP27 acts as a chaperone or binding partner for Rho-kinase and protein kinase C (PKC), directly increasing smooth muscle contraction (Patil et al., 2004). Therefore, we believe that a similar effect might exist in urethra, that is, ANG II activating p38 MAPK phosphorylation in smooth muscle, increasing consequently Rho and PKC activity, leading to greater urethral contractions in 2K1C rats. Micturition is initiated by a loss of urethral smooth muscle tone that is mediated by the release of nitric oxide (NO) from nitrergic fibers supplying the urethral tissues (Fry et al., 2010; Yoshida et al., 1998). Our results in urethra preparations showed that EFS produces frequency-dependent nitrergic relaxations, which are largely decreased in 2K-1C rats. In addition, urethra smooth muscle from 2K-1C rats relaxed markedly less to nitrergic stimulation, and this nitrergic impairment was fully restored by treatments with captopril and losartan, but not by atenolol. It is known that ANG II significantly decreases endogenous NO bioavailability (Schulman et al., 2006). A reduced expression of neuronal NO synthase (nNOS) was also recently found in mesenteric vascular beds from 2K-1C rats (Koyama et al., 2010). In conclusion, this work showed that long-term treatment with losartan and captopril (but not atenolol) prevents the micturition dysfunctions in 2K-1C rats, suggesting that RAS overactivity takes

Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i

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part in the pathophysiology of micturition dysfunction associated with renovascular hypertension. Increased urethral contractions to ANG II via p38 MAPK phosphorylation may be an important mechanism leading to bladder outlet resistance that timely progress to overactivity bladder. The main limitation of our study is that caution should be exercised when using data obtained from animal studies. Usually, drug metabolism greatly differs between humans and rodents. In our study in rats, doses used of losartan, captopril and atenolol (30, 50 and 90 mg/kg, respectively) are markedly higher than those in humans. Therefore, although this work shows that angiotensin-converting enzyme inhibition and/or angiotensin AT1 receptor blockade improve micturition dysfunction in renovascular hypertensive rats, these beneficial results need to be corroborated by clinical trials in humans.

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Please cite this article as: Ramos-Filho, A.C.S., et al., Blockade of renin–angiotensin system prevents micturition dysfunction in renovascular hypertensive rats. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.05.038i