Yonago Acta medica 2002;45:19–26

Effects of Nitric Oxide on Bladder Outlet Obstruction in Rats
Koji Yanagi
Department of Urology, Tottori University, Faculty of Medicine, Yonago 683-8504 Japan
We investigated the changes in bladder function resulting from bladder outlet obstruction (BOO), with particular emphasis on the role of nitric oide (NO) in bladder response.
Twelve-week-old female Wistar rats (n = 36) were surgically treated in various ways by
dividing them into 6 groups each with 6 animals: 2 sham-operated groups, 2 obstructedgroups for 1 week or 6 weeks, and 2 groups obstructed and intraperitoneally injectedwith 150-mg/kg L-arginine once daily for 1 week or 6 weeks. We conducted bladder
strip stimulation studies using carbachol and KCl. Then the bladder was used formeasurement of NO synthase (NOS) activity and was also studied histologically using
immunohistochemical staining. Bladders in the obstructed groups were significantly
heavier than in the sham-operated groups. The mean weight of bladders in the groups
obstructed and treated with L-arginine was almost the same as that in the sham-operated
groups. Tissue bath studies demonstrated decreased contractility in response tocholinergic stimulation at obstruction, but contractility at obstruction was improvedby L-arginine for 1 week or 6 weeks. NOS activity in bladder tissue was lower in the
obstructed groups than in the sham groups, and higher in the L-arginine-combinedgroups than in the obstructed groups. These results indicate that bladder dysfunction
may be improved by treatment with L-arginine. This study suggests that increased NO
by treatment with L-arginine plays a role in improving bladder dysfunction caused by
BOO.
key words: bladder outlet obstruction; nitric oxide; nitric oxide synthase activity; L-argininemia), and that ischemia caused by BOO induces
bladder dysfunction (Lin et al., 1995; Buttyan etal., 1997).
Recently, several studies have revealed thatnitric oxide (NO) is an important neurotransmitter and intracellular messenger that promotes homeostasis in many tissues (Saito andMiyagawa, 1999). One of the most important
effects of NO is reported to be its role as a neurotransmitter in non-adrenergic non-cholinergic
nerves, and the L-arginine-NO pathway has
been shown to play a role in muscle relaxationand vasodilation (Arthur and Burnett, 1995).
NO stimulates the formation of guanylate cyclase in smooth muscle cells, converting GTP to
3' 5'-cyclic GMP (cGMP) (Burnett, 1997). A cascade of cGMP-dependent intracellular events

Bladder outlet obstruction (BOO) is one of themost common urological problems in elderlymen. An increase in urinary frequency and urgency are typical symptoms in the early stagesof outlet obstruction, appearing secondarily tobenign prostatic hyperplasia. Despite the highprevalence of BOO, the mechanisms responsible for bladder dysfunction induced by BOO remain poorly understood. In an animal model,
bladder weight was significantly increased(Saito et al., 1993a; Berggren and Uvelius, 1998;
Kim et al., 2000), and contractile response wassignificantly decreased by BOO. Some authors
have reported that detrusor mitochondrial damage may explain voiding dysfunction after BOO(Lu et al., 2000). Other reports have found that
BOO induces reduction in blood flow (ische-Abbreviations: BOO, bladder outlet obstruction; bNOS, brain NOS; cGMP, 3' 5'-cyclic GMP; ED50, median
effective dose; E max, maximum effective dose; eNOS, endothelial NOS; iNOS, inducible NOS; i.p.,
intraperitoneal; NO, nitric oxide; NOS, NO synthase

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K. Yanagi

then leads to a decrease in intracellular calcium,
ultimately causing smooth muscle relaxationand vasodilation, in part through changes inpotassium conductance (Seftel et al., 1996;
Burnett, 1997). Recently, it has been reported
that NO is induced by ischemia injury (Saito
and Miyagawa, 1999), and that NO synthase(NOS) isoform activity, that is, inducible NOS(iNOS) activity, was decreased in long-term obstruction for 5 weeks (Lemack et al., 1999).
On the other hand, NOS activity did notshow significant changes in short-term obstruction (Shabsigh et al., 2000). These results may
suggest that bladder dysfunction caused by
BOO is associated with loss of physiological effects of NO.
In this study, we investigated the effect ofNO on bladder dysfunction caused by BOO
with organ bath techniques. Further, we measured NOS activity in bladder tissue and studied
immunohistochemical staining. The aim of thisstudy was to determine whether or not the bladder dysfunction was due to BOO by treatment
with L-arginine.

eter was approximately 1.70 mm for obstruction (mild obstruction) (Saito et al., 1993b). Finally, the incision was closed. The remaining 12
rats were used for the sham operation. Shamoperated rats underwent a similar procedure
with a dissection around the urethra, but without a urethral ligature. After the operation, theanimals were divided into 6 groups each with 6
rats: Group C-1, 1 week after sham operation;
Group C-6, 6 weeks after sham operation;
Group OB-1, 1-week obstruction; Group OB-6,
6-week obstruction; Group Ar-1, 1-week obstruction with i.p. administration of 150-mg/kg
L-arginine once daily for 1 week and Group Ar6, 6-week obstruction with i.p. administrationof 150-mg/kg L-arginine once daily for 6
weeks. The rats who underwent the operation
were anesthetized, and in 1-week-treated rats
(Groups C-1, OB-1 and Ar-1), bladders were
removed on the 8th day from induction of theoperation. In 6-week-treated rats (Groups C-6,
OB-6 and Ar-6), bladders were removed on the
43rd day from induction of the operation. The
removed bladder specimens were weighed and
immediately used for the strip study, measurement of NOS activity and histological examination.

Materials and Methods

Animal model

Measurement of contractile response

All animal experiments were performed in accordance with the guidelines set out by the
Tottori University Committee for Animal Experimentation. Twelve-week-old female Wistar
rats, mean body weight 218 g, were used (n =36) (SLC, Shizuoka, Japan). The 36 rats wereanesthetized with intraperitoneal (i.p.) administration of 50-mg/kg sodium pentobarbital. Outof the 36 rats, 24 were used as models for partial
obstruction as follows. With the rat in the spinal position after a midline suprapubic incision,
the urethra was exposed without damaging the
bladder neck and urethra. A 2-0 silk suture waspassed behind the urethra and the catheter was
put on the urethra. Subsequently, the urethra
and catheter were ligated and the catheter was
removed. The presence of the catheter ensured
that the ligature did not significantly compress
the urethra. The outside diameter of each cath-

Each rat was anesthetized with an i.p. injectionof 50-mg/kg sodium pentobarbital, and the urinary bladder was removed as low on the urethra
as possible. After measuring the bladder weight,
the bladder dome was immediately separated
from the bladder base at the level of the ureteralorifices, and was separated into longitudinal
strips. The muscle strips (5 × 1.5 mm) were suspended in organ baths containing a 3.0-mL KrebsHenseleit’s solution at 37˚C. The tissues were
equilibrated with a mixture of 95% O2 and 5%
CO2. Isometric force was monitored with a TB612T transducer (Nihon Koden, Tokyo, Japan)
and processed by a MacLab multiport controller(AD Instruments Japan, Tokyo). Contractile
response to carbachol was expressed as force per
cross-sectional area of muscle (grams per square
millimeter). Cumulative dose-response curves
were constructed in a stepwise manner after the20

Nitric oxide in bladder outlet obstruction

response to the previous concentration had
reached a plateau. Contractile response of thesame muscle strips to 100-mmol/L KCl was
also monitored.

acted with rabbit anti-eNOS or -iNOS polyclonal
antibody (Chemicon, Temecula, CA) at a 1:200
dilution overnight at 4˚C. The specificallybound 1st antibodies were visualized by biotinylated horse anti-rabbit secondary antibody(1:200 dilution) with a Histofine SAB-PO (G)Kit (Nichirei Co., Tokyo). Mayer’s hematoxylin
was used for counterstaining. Negative controls were established for each group according
to the procedure mentioned above, but without
2nd antibodies.

Muscle NOS activity
Bladder dome tissues were homogenized inhomogenization buffer containing phosfatebuffered saline, pH 7.4. Homogenates were
centrifuged at 4˚C for 20 min at 10,000 × g, andthe next supernatant solutions were ultracentrifuged at 4˚C for 15 min at 100,000 × g. Filtrationof the 10,000 × g supernatant through a 0.45-µfilter prior to ultrafiltration can increase theultrafiltration rate. Up to 40 µL of a sample wasadded to each sample well for the microtiterplate, then 10 µL of freshly NADPH solution (1mmol/L) and 10 µL of nitrate reductase solution
were added to each well. After incubating atroom temperature for 60 min, 10 µL of the cofactor solution and 10 µL of the lactate dehydrogenase solution were added to each well.
This was followed by incubation at room temperature for 20 min, and then a 100-µL Griess
reagent was added to each well. Finally, amodel 550 microplate reader (Bio-Rad Laboratories, Hercules, CA) was used to read the absorbance at 540 nm. The values were estimatedper tissue weight and per amount of protein in
the tissue. Protein was determined using a commercial kit (BCA Protein Assay Reagent Kit,
Pierce, Rockford, IL).

Data analysis
Contractile data were calculated as grams of active force per cross-sectional area in millimeters. The cross-sectional area was calculatedusing the following equation:
cross-sectional area = weight/(length × 1.05),
where 1.05 is the assumed density of the muscle.
The dose ratio was obtained from the ratio of median effective dose (ED50) values (the concentration of agonist that produces a half-maximal contractile response) for carbachol. ED50 values
were calculated as geometric means, whereas
maximum effective dose (Emax) values were calculated as arithmetic means. NOS activity wascalculated as follows:
NOS activity = {(A540 – b)/m}(200/40)/60,
where A540 is the sample absorbance at 540
nm, b is the y-intercept, m is the slope, 200 is
the final volume, 40 is the sample volume, and
60 is the NOS reaction incubation time in minutes. Statistical comparison of differences between groups was performed using analysis ofvariance and Fisher’s test for the multiple comparison; P < 0.05 was regarded as the level ofsignificance.

Histological examination of the rat bladderMorphological changes in the rat bladder werestudied by light microscopy. The rat bladderdome of each group was immediately removed
and fixed with 3.7% formaldehyde-saline. After
fixation, the tissues were embedded in paraffin.
Sections (5 µm) were stained with hematoxylin
and eosin. The serial sections were also subjected to immunohistochemical staining forendothelial NOS (eNOS) and iNOS. In brief,
deparaffinized sections were retrieved using
DAKO Target Retrieval Solution and a DAKOLSAB Kit (Carpenteria, CA). They were then re-Drugs and chemicals
L-arginine, carbachol and pentobarbital werepurchased from Sigma (St. Louis, MO). DAKO
Target Retrieval Solution and the DAKO LSAB
Kit were from DAKO. Polyclonal antibodies
against eNOS and iNOS were from Chemicon.
The Histofine SAB-PO (G) Kits were from21

K. Yanagi

Table 1. Bladder weight

Nichirei. The NOS activity assay kit was fromOXIS International, Inc. (Portland, OR). The
BCA Protein Assay Reagent Kit was from
Pierce. All other chemicals were of reagent
grade.

Group[number of animals]

Bladder weight(g)C-1 [6]
0.109 ± 0.005*
OB-1 [6]0.174 ± 0.024
Ar-1 [6]0.136 ± 0.012
C-6 [6]
0.114 ± 0.006*
OB-6 [6]0.242 ± 0.035*
Ar-6 [6]0.149 ± 0.012
Bladder weight data are shown as mean ± SEM ineach group.
Ar, L-arginine-dosed; C, sham-operated; OB, obstructed.
* P < 0.05.

Results

Bladder weight
Bladder weight was significantly increased by
obstruction. For the long-term groups, themean bladder weight at 6 weeks of obstruction
was increased 2-fold compared to that in the
sham-operated group. However, the mean
bladder weight at 6 weeks of obstruction withtreatment by L-arginine was almost the same asthat in the sham-operated group. The tendency
in the 1-week groups was similar to that in the
6-week groups (Table 1).

operated groups. Contractile response inducedby 100-mmol/L KCl showed a pattern parallelto those of Emax values of carbachol in eachgroup.

Muscle NOS activity
At 1 week and 6 weeks, NOS activity was reduced in the obstructed groups as compared tothe sham-operated groups, and was increased in
the L-arginine groups as compared to the obstructed groups. But there was no significant
difference among the groups (Table 3).

Contractile response to carbachol and
KClConcentration-dependent contractile responsesof bladder strips to carbachol are shown in
Table 2, together with their Emax and ED50 values summarized. At both 1 week and 6 weeks,
the obstructed groups showed significantly
lower E max and ED 50 values than the shamoperated groups. Contractility of bladder muscles of rats receiving L-arginine significantly
recovered compared to that in the obstructedgroups, but was not equal to that in the sham-Histological changes in rat bladders bychemicals
A representative light microscopic photographof the bladder from an L-arginine-dosed rat isshown in Fig. 1 and another from an obstructed
rat in Fig. 2. Figure 1 shows eNOS positive inTable 2. Functional data of obstructed rat bladders
Group[number of animals]

Emax(g/mm2)ED50(µmol/L)

100-mmol/L KCl(g/mm2)C-1 [6]9.89 ± 0.88
1.60 ± 0.15
6.96 ± 0.91**
OB-1 [6]4.27 ± 0.52
0.89 ± 0.14
2.12 ± 0.26**
Ar-1 [6]8.93 ± 0.84
1.82 ± 0.26
5.93 ± 0.60
C-6 [6]11.38 ± 0.57
2.11 ± 0.54
8.53 ± 0.58**
OB-6 [6]4.61 ± 0.38
1.30 ± 0.21
3.09 ± 0.29**
Ar-6 [6]6.79 ± 0.32
1.71 ± 0.43
4.85 ± 0.30
Emax and ED50 are for carbachol. Data are shown as mean ± SEM in each group.
Ar, L-arginine-dosed; C, sham-operated; OB, obstructed.
* P < 0.05.

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*
*
*
*

Nitric oxide in bladder outlet obstruction

the mucosal region, vessels andsmooth muscle region. In Fig. 2,
iNOS is detected in the mucosal
region, and positive in the infiltrated
leukocytes. eNOS and iNOS weredetected in all groups in the samemanner.

Table 3. NOS activity in the rat bladderGroup[number of animals]

C-1
OB-1
Ar-1
C-6
OB-6
Ar-6

[6]
[6]
[6]
[6]
[6]
[6]

NOS activity(nmol/ng protein/min)

0.063 ± 0.004
0.049 ± 0.005
0.063 ± 0.012
0.105 ± 0.016
0.068 ± 0.013
0.079 ± 0.012

NOS activity data are shown as mean ± SEM in each group.
Ar, L-arginine-dosed; C, sham-operated; OB, obstructed.

Fig. 1. Sections of rat bladder tissue from the L-arginine-dosed group in 6-week-immunostained by antiendothelial nitric oxide synthase (eNOS) antibodies (× 100).

Fig. 2. Sections of rat bladder tissue from the obstructed group in 6-week-immunostained by anti-inducible
nitric oxide synthase (iNOS) antibodies (× 200).

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K. Yanagi

which may play a role in relaxing of the detrusor.
Increased levels of NO treated with L-arginine
may relax the urethra and decrease the load of
the bladder, and recover the blood flow.
Histochemical examination revealed that abrain subtype of NOS (bNOS) was distributedin the proximal urethra of the dog (Takeda andLepor, 1995), and in the rat bladder neck andurethra (Tamaki et al., 1995). Upon distribution of NOS in the bladder dome, our data demonstrated that eNOS was positive in the mucosal region, vessels and smooth muscle region, and that iNOS was detected in the mucosal region and the infiltrated leukocytes in the
bladder dome, as was reported before (Saito
and Miyagawa, 1999; Persson et al., 1999).
These observations indicate that vasodilationand detrusor relaxation may be induced bytreatment with L-arginine. As there is no significant difference in NOS activity in thesegroups, treatment with L-arginine increases NO
in the bladder.
As a result, L-arginine prevents an increasein bladder weight and a decrease in the contractile response: that is, L-arginine prevents
the bladder dysfunction caused by BOO. Because of this, the reasons for the results obtained in this experiment are speculated as follows:i) Experimentally obstructed bladder outlets in
the rats induced bladder overload and ischemia,
and resulted in an increase in bladder weightand bladder dysfunction; ii) Treatment by Larginine increased NO in the urethra and bladder, decreased bladder overload and increased
the blood flow of the bladder by vasodilation.
These changes resulted in a decrease in bladderweight and recovery from bladder dysfunction.
The present findings reveal the possibility
that bladder dysfunction caused by BOO is improved by treatment with L-arginine, which
may increase NO concentration.
However, Saito et al. (1998) reported thatreperfusion following ischemia causes more
severe dysfunction than that of ischemia alone
in the rat bladder. Reactive oxygen species,
such as the superoxide radical, hydroxyl radical
and hydrogen peroxide, are known to induce injury in several tissues. The generation of thesereactive oxygen species is attributed to ische-

Discussion
Several authors have postulated that obstructioninduced ischemia is responsible for the bladderdysfunction seen with BOO (Lin et al., 1995;
Buttyan et al., 1997).
Our data demonstrated that BOO increased
bladder weight and decreased contractility inresponse to carbachol and KCl. Bladder weightsignificantly increased in a 1- or 6-week outlet
obstruction. But by treatment with L-arginine,
bladder weight did not significantly increase
compared to that in the sham-operated groups.
The carbachol-induced developed tension was
decreased by BOO. The contractile response
recovered by treatment with L-arginine. In addition, we measured NOS activity in the experimentally obstructed bladders. At 6 weeks ofobstruction, NOS activity was decreased by theobstruction, and was increased by treatment
with L-arginine. But there was no significant
difference among the groups. In the histological examination, there were no distinct differences in immunohistochemical staining among
the groups. eNOS was positive in the mucosal
region, vessels and smooth muscle region, andiNOS was detected in the mucosal region and in
the infiltrated leukocytes in the bladder dome.
These data indicate that bladder function may
not to be associated with NOS activity. But
treatment with L-arginine may increase NO,
which may improve bladder function.
Bladder distention caused by BOO inducesischemic changes in the bladder (Lin et al., 1995).
The ischemia caused by BOO temporarily increases NO concentration in the bladder for shortterm obstruction (Saito and Miyagawa, 2001);
however, NO is gradually decreased for longterm obstruction, and the decrease in NO induces bladder dysfunction. It is reported that
NO is a mediator of relaxation of the urethra in
rats (Mumtaz et al., 2000), and that, as a result,the load of the bladder is reduced. The rat detrusor is reported to be capable of responding toNO (Chung et al., 1996). NO induces vasodilation, and the released NO maintains the necessary blood supply via the circulatory system bydilating arteries (Saito and Miyagawa, 1999),
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Nitric oxide in bladder outlet obstruction

JD, Lin VK. Physiologic sequelae of partial infravesical obstruction in the mouse: role of inducible nitric oxide synthase. J Urol 1999;161:
1015–1022.
8 Lin ATL, Chen MT, Yang CH. Blood flow of theurinary bladder: effects of outlet obstruction andcorrelation with bioenergetic metabolism. Neurourol Urodyn 1995;14:285–292.
9 Lu SH, Wei YH, Chang LS, Lin AT, Chen KK,
Yang AH. Morphological and morphometricanalysis of human detrusor mitochondria withurodynamic correlation after partial bladder outlet obstruction. J Urol 2000;163:225–229.
10 Muijsers RB, Folkerts G, Henricks PA, SadeghiHashjin G, Nijkamp FP. Peroxynitrite: a twofaced metbolite of nitric oxide. Life Sci 1997;60:
1833–1845.
11 Mumtaz FH, Khan MA, Thompson CS, Morgan
RJ, Mikhailidis DP. Nitric oxide in the lower urinary tract: physiological and pathological implications. BJU Int 2000;85:567–578.
12 Persson K, Poljakovic M, Johansson K, Larsson
B. Morphological and biochemical investigationof nitric oxide synthase and related enzymes in
the rat and pig urothelium. J Histochem Cytochem 1999;47:739–749.
13 Saito M, Longhrst PA, Tammela TLJ, Wein AJ,
Levin RM. Effects of partial outlet obstruction of
rat urinary bladder on micturition characteristics,
DNA synthesis and the contractile response to
field stimulation and pharmacological agents. J
Urol 1993a;150:1045–1051.
14 Saito M, Miyagawa I. Direct detection of nitricoxide in rat urinary bladder during ischemiareperfusion. J Urol 1999;162:1490–1495.
15 Saito M, Miyagawa I. Bladder dysfunction after
acute urinary retention in rats. J Urol 2001;165:
1745–1747.
16 Saito M, Wada K, Kamisaki Y, Miyagawa I. Effect of ischemia-reperfusion on contractile function of rat urinary bladder: possible role of nitricoxide. Life Sci 1998;62:149–156.
17 Saito M, Wein AJ, Levin RM. Effects of partial
outlet obstruction on contractility: comparison
between severe and mild obstruction. Neurourol
Urodyn 1993b;12:573–583.
18 Seftel AD, Viola KA, Kasner SE, Ganz MB. Nitric oxide relaxes rabbit corpus cavernosumsmooth muscle via a potassium-conductive pathway. Biochem Biophys Res Commun 1996;219:
382–387.
19 Shabsigh A, Hayek OR, Weiner D, Saidi J,
Kaplan SA, Kiss A, et al. Acute increase in bloodflow to the rat bladder subsequent to partial bladder outlet obstruction. Neurourol Urodyn 2000;
19:195–208.
20 Takami M, Akaike Y, Kojimabara Y, Igarashi I,
Saito M, Takeda M. Morphological analysis ofnitric oxide synthase-immunoreactive nerve

mia-reperfusion injury. These reactive oxygen
species damage many biological molecules. Peroxynitrate, reacting with NO and superoxide
radicals, is also reported to be a powerful oxidative product (Muijsers et al., 1997). Saito andMiyagawa (1999) offered the idea that peroxynitrate reacting with NO and superoxide radicals may be involved in ischemia-reperfusion
injury in the bladder. In their reports, NO andperoxynitrate induce bladder dysfunction, afinding which indicates that NO has a cytotoxic
effect in the experimentally obstructed bladder.
However, our presented data demonstrated that
NO has a cytoprotective effect in the bladder.
Therefore, NO has both damaging and protective effects in many tissues.
In summary, i) bladder weight was increased by BOO, and the increase in bladder weight
could be restrained by treatment with L-arginine; ii) the carbachol-induced developed tension was decreased by BOO, and was recovered
with treatment by L-arginine; and iii) in the histological studies, eNOS was positive in themucosal region, vessels and smooth muscle region, and iNOS was positive in the mucosal region and in the infiltrated leukocytes. It is suggested that treatment with L-arginine may
improve the bladder dysfunction caused by BOO.
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2527.
Received December 12, 2001; accepted January 11, 2002
Corresponding author: Dr. Koji Yanagi

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