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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 328, No. 1
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 142562/3413555
JPET 328:213–222, 2009 Printed in U.S.A.

Catecholamines Relax Detrusor through ␤2-Adrenoceptors

in Mouse and ␤3-Adrenoceptors in Man

Melinda Wuest, Birgit Eichhorn, Marc O. Grimm, Manfred P. Wirth, Ursula Ravens,
and Alberto J. Kaumann
Departments of Pharmacology and Toxicology (M.W., B.E., U.R.) and Urology (M.O.G., M.P.W.), Dresden University
of Technology, Dresden, Germany; and Department of Physiology, Development, and Neuroscience, University of Cambridge,
Cambridge, United Kingdom (A.J.K.)
Received June 23, 2008; accepted September 25, 2008

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015

ABSTRACT
(⫺)-Isoproterenol [4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-
1,2-benzene diol hydrochloride] relaxes murine detrusor through
␤-adrenoceptors (ARs); however, the ␤-AR subtypes involved are
unknown. ␤2-ARs have been associated with caveolae, plasma-
lemmal scaffolding domains that are absent in caveolin-1 (cav-1)
knockout (KO) mice. Here, we studied detrusor responses in the
absence and presence of ␤-AR subtype-selective antagonists in
wild-type (WT) and cav-1 KO mice. To inquire whether the murine
detrusor model is relevant to man, ␤-AR subtypes that mediate
(⫺)-isoproterenol-evoked human detrusor relaxation were investi-
gated. In WT mice, (⫺)-isoproterenol concentration-dependently
relaxed the KCl (40 mM)-precontracted detrusor (⫺logEC50M ⫽
8.04, Emax ⫽ 62%). The effects of (⫺)-isoproterenol were
surmountably antagonized by the ␤2-AR-selective antagonist
ICI 118,551 [(⫾)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-
3-[(1-methylethyl)amino]-2-butanol] (pKB ⫽ 9.28) but not
affected by the ␤1-AR-selective antagonist CGP 20712 [1-[2-
((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-
methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol]
and ␤ 3 -AR-selective L-748,337 [(S)-M-[4-[2-[3-[3-[acet-
amidomethyl)phenoxy)-2-hydroxypropyl]-amino]-ethyl]-phenyl-
benzsulfonamide)], suggesting involvement of ␤2-AR only. The
cav-1 KO detrusor displayed significant contractile dysfunction.
(⫺)-Isoproterenol was less potent and efficient in relaxing de-
trusor from cav-1 KO (⫺logEC50M, 7.76; Emax ⫽ 44%), but ICI
118,551 caused similar antagonism (pKB ⫽ 9.15), suggesting
that ␤2-AR function persisted in cav-1 KO. The ␤3-AR-selective
antagonist L-748,337 in the presence of ICI 118,551 and CGP
20712 caused additional blockade of (⫺)-isoproterenol effects
in cav-1 KO, consistent with a ␤3-AR involvement during relax-
ation and suppression of this effect in WT. (⫺)-Isoproterenol
relaxed human detrusor muscle precontracted with carbachol
(⫺logEC50M ⫽ 6.39, Emax ⫽ 52%). However, the effects of
(⫺)-isoproterenol in human detrusor were not blocked by CGP
20712 or ICI 118,551 but antagonized by L-748,337 (pKB ⫽
7.65). We conclude that murine detrusor relaxation occurs via
␤2-AR, and loss of caveolae does not perturb ␤2-AR function
but unmasks an additional activation of ␤3-AR. In contrast,
detrusor relaxation in man is mediated exclusively via ␤3-AR.

In the lower urinary tract, adrenoceptors (ARs) are impor-
tant for regulating detrusor contractile function and promot-
ing urinary continence (Andersson and Arner, 2004; Michel
and Vrydag, 2006). ␤-ARs mediate relaxation of bladder
smooth muscle via stimulation of adenylyl cyclase and sub-
sequent increase in cAMP. In rat and human detrusor, all
three ␤-AR subtypes (␤1-AR, ␤2-AR, and ␤3-AR) are ex-
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.108.142562.
pressed at the mRNA level (Matsubara et al., 2002; Nomiya
and Yamaguchi, 2003). ␤-AR-mediated detrusor relaxation
appears quite variable between different species; although
similar maximal relaxant effects of (⫺)-isoproterenol are re-
ported, the potency of (⫺)-isoproterenol varies. Moreover,
different ␤-AR subtypes are involved in detrusor relaxation
(Oshita et al., 1997; Yamazaki et al., 1998; Takeda et al.,
2003), with, for instance, ␤2-AR mediating relaxation in rab-
bit (Morita et al., 2000), ␤2-AR and ␤3-AR (Takeda et al.,
2003; Uchida et al., 2005) or even all three subtypes in rat

ABBREVIATIONS: AR, adrenoceptor; cav-1, caveolin-1; KO, knockout; RT, reverse transcriptase; PCR, polymerase chain reaction; WT, wild type;
L-748,337 (S)-M-[4-[2-[3-[3-[acetamidomethyl)phenoxy)-2-hydroxypropyl]-amino]-ethyl]-phenylbenzsulfonamide); ICI 118,551, (⫾)-1-[2,3-(dihy-
dro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; CGP 20712, 1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-
methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol; phentolamine hydrochloride, 2-[N-(3-hydroxyphenyl)-p-toluidinomethyl]-2-imidazolidine
hydrochloride; CHO, Chinese hamster ovary; BRL 37,344, (⫾)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid;
L-755,507, 4-[[hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4-hydroxyphenoxy)propyl]amino]ethyl]-benzenesulfonamide; SR 59,230, 1-(2-
ethylphenoxy-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride.

213
214 Wuest et al.
detrusor (Longhurst and Levendusky, 1999), ␤2-AR and
␤3-AR in pig (Yamanishi et al., 2002), and ␤3-AR in man
(Igawa et al., 1998, 2001; Takeda et al., 1999; Morita et al.,
2000; Yamanishi et al., 2006). Based on the latter findings,
␤3-AR are proposed as a potential target for therapy of over-
active bladder. Although the standard for treating this dis-
order are muscarinic receptor antagonists, additional treat-
ment options are urgently needed because therapeutic
success of antimuscarinic drugs is limited by low response
rates and frequent side effects (Andersson, 2004).
Evidence for functional interaction between muscarinic re-
ceptors and ␤-AR in controlling detrusor contractions was
obtained in mice in which muscarinic M2 receptors had been
knocked out. The relaxant response of the detrusor to (⫺)-
isoproterenol and forskolin was enhanced under some condi-
tions in M2 receptor knockout mice (Matsui et al., 2003;
Ehlert et al., 2007). However, the ␤-AR subtypes involved in
murine urinary bladder relaxation are not known. Prelimi-
nary experiments from our laboratory suggested involvement
of ␤2-AR.
␤-AR subtypes are not evenly distributed within the cell
membrane, but especially ␤2-ARs appear to be clustered in
caveolae. These flask-shaped invaginations of the plasma
membrane are stabilized by the caveolin proteins, of which
caveolin-1 (cav-1) is expressed in detrusor tissue (Razani et
al., 2002). Genetic deletion of cav-1 leads to loss of caveolae in
vascular smooth muscle cells (Drab et al., 2001) and marked
reduction of caveolae in detrusor smooth muscle cells (Wood-
man et al., 2004), which is expected to be accompanied by
functional impairment. ␤2-ARs were found to cluster in
caveolar structures (Schwencke et al., 1999; Ostrom et al.,
2000; Rybin et al., 2000). Arterial vascular smooth muscle
from cav-1 KO mice revealed, however, no change in ␤2-AR-
mediated relaxation but disappearance of ␤1-AR function and
appearance of ␤3-AR-mediated relaxation (Neidhold et al.,
2007). Detrusor muscle with greatly reduced caveolae (Wood-
man et al., 2004) displayed significant impairment of con-
tractile responses to muscarinic receptor stimulation or high
K⫹, resulting in a profound bladder dysfunction, particularly
in aged male cav-1 KO mice (Woodman et al., 2004; Lai et al.,
2007). We have investigated the contribution of ␤-AR sub-
types to detrusor relaxation in cav-1 KO mice.
The aim of our study was to identify the ␤-AR subtypes
involved in murine and human detrusor relaxation to clarify
whether mouse detrusor is a relevant model for human blad-
der. The relaxation of murine and human detrusor to several
␤-AR agonists was examined in the absence and presence of
␤-AR subtype-selective antagonists. Expression of mRNA
of ␤1-AR, ␤2-AR, and ␤3-AR was assessed in detrusor tissue
from wild-type and cav-1 KO mice and man by real-time
RT-PCR. We found that mouse detrusor is relaxed almost
exclusively via ␤2-AR, whereas human detrusor is relaxed
through ␤3-AR.
Materials and Methods
Contraction of Murine Detrusor. Male C57BL6 [ Charles
River, Margate, Kent, UK; wild type (WT)] mice and cav-1 KO mice,
weighing 20 to 30 g and 9 to 18 months old, were used. cav-1 KO mice
(Drab et al., 2001) were bred at the animal facility of the Medical
Faculty of Dresden University of Technology and genotyped before
the experiments. All mouse experiments were performed in accor-
dance to the regulations of the local legislation committee (permis-
sion 24-9168.24-1-2002-8 of the Dresden Regierungspräsidium).
Mice were sacrificed by cervical dislocation. The whole urinary blad-
der was removed at the bladder neck as described previously (Wuest
et al., 2005). After cutting off the dome of the bladder, the remaining
muscle ring was opened longitudinally, the mucosa was removed,
and the remaining muscle tissue was cut into four strips. The muscle
strips were mounted in 5-ml organ baths containing oxygenated
modified Tyrode’s solution maintained at 37°C. The modified Ty-
rode’s solution contained: 127 mM NaCl, 5.4 mM KCl, 1.05 mM
MgCl2, 1.8 mM CaCl2, 0.4 mM NaH2PO4, 22 mM Na2CO3, 0.04 mM
EDTA, 0.2 mM ascorbic acid, and 5.6 mM glucose, pH 7.4, when
equilibrated with 95% O2 and 5% CO2. Tension generated was mea-
sured with an isometric force transducer (GM 2; Föhr Medical In-
struments GmbH, Seeheim/Ober Beerbach, Germany), amplified,
and recorded with a data and recording system (Chart 4.0; ADIn-
struments Pty Ltd., Castle Hill, Australia). Resting load was set to 5
mN. During the equilibration period of 60 min, the bath solution was
changed twice. For depolarizing Tyrode’s solution, KCl was in-
creased to 40 mM without compensation for increase in osmolality.
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015
Long-lasting contractile responses were obtained with 40 mM KCl in
the presence of phentolamine (3 ␮M) to block ␣-AR. The tissue strips
were allowed to stabilize for 45 min to the KCl contracture before
cumulative concentration-response curves for the relaxing effects of
(⫺)-isoproterenol or (⫺)-epinephrine were obtained in the absence
and presence of subtype-selective ␤-AR antagonists. Each experi-
ment was terminated by measuring maximal relaxation in response
to the adenylyl cyclase activator forskolin (10 ␮M). Time-matched
control experiments of KCl-induced detrusor contractions in the
presence of phentolamine (3 ␮M) were run simultaneously in the
absence of a ␤-AR agonist or antagonist.
Determination of mRNA Expression in Mice Using Real-
Time PCR. For PCR studies, the urothelium and suburothelium
layers of murine urinary bladders were dissected, and only smooth
muscle tissue was frozen in fluid nitrogen. Tissue from seven (WT) or
six (cav-1 KO) mice were collected, and total RNA was isolated using
the E.Z.N.A. total RNA-Kit (PEQLAB Biotechnologie GmbH, Erlan-
gen, Germany). Human detrusor tissue optically free of tumor was
collected from six different patients (ages 59 –71) undergoing radical
cystectomy. Mucosa and serosa layers were removed, and prior hu-
man tissue was frozen in fluid nitrogen. Real-time PCR was carried
out in a RotorGene Thermal Cycler (Corbett Life Science, Sydney,
Australia) using the QuantiTect SYBR Green RT-PCR kit (QIAGEN
GmbH, Hilden, Germany). The specific primers used for determina-
tion of ␤-AR are listed in Table 1 (murine primers, Evans et al., 1999;
Chernogubova et al., 2005; human primers, Igawa et al., 1999).
Amplification conditions for RT-PCR and real-time PCR for murine
␤-ARs were 30 min at 50°C and 15 min at 95°C followed by 30 s at
94°C, 30 s at 60°C for ␤1-AR and 64°C for ␤2-AR and ␤3-AR, 30 s at
72°C, and 15 s at 80°C; for human ␤1-ARs, 50°C for 30 min, 95°C for
15 min, 30 s at 94°C, 30 s at 66°C, 30 s at 72°C, and 15 s at 85°C; for
human ␤2-ARs, 50°C for 30 min, 95°C for 15 min, 30 s at 94°C, 30 s
at 60°C, 40 s at 72°C, and 15 s at 80°C; and for human ␤3-ARs, 50°C
for 30 min, 95°C for 15 min, 30 s at 94°C, 30 s at 64°C, 40 s at 72°C,
and 15 s at 85°C were used. Primer/gene-specific internal cRNA
standards were synthesized by T7 in vitro transcription using the
Message Machine Kit (Ambion, Austin, TX) from RT-PCR products
obtained as described above but using a modified forward primer
containing the additional T7 promoter sequence (GGC CGC GG).
cRNA concentrations were calculated from primer/gene-specific
known RNA concentrations of internal standards in each dilution
series (105–1012 mol/␮l). Data were analyzed with RotorGene Soft-
ware version 4.6 (Corbett Life Science).
Contraction of Human Detrusor. Human urinary bladder tis-
sue was obtained from 10 male and four female patients (age range,
54 –77 years) undergoing radical cystectomy for muscle invasive
bladder cancer. All patients had given informed written consent in
accordance with the regulations of the local hospital ethical commit-
 ␤-Adrenoceptor-Induced Detrusor Relaxation in Mouse and Man
TABLE 1
Primer pairs used for real-time PCR of ␤-AR
The table specifies the forward (F) and the reverse primer (R) used for real-time PCR.
Species Gene Accession No.
Mouse Adrb1 NM_007419
Mouse Adrb1 NM_007419
Mouse Adrb2 NM_007420
Mouse Adrb2 NM_007420
Mouse Adrb3 NM_013462
Mouse Adrb3 NM_013462
Man hAdrb1 NM_000684
Man hAdrb1 NM_000684
Man hAdrb2 NM_000024
Man hAdrb2 NM_000024
Man hAdrb3 NM_000025
Man hAdrb3 NM_000025
tee (permission no. EK 194092004). Samples from tumor-free parts
of the bladder wall were taken. After careful removal of the serosa
and mucosa, four to eight muscle strips (10 –15 mm long, 4 –5 mm
wide) were dissected from each sample. Human detrusor strips were
exposed to 40 mM KCl for 10 min followed by washout before 100 nM
carbachol was added, also in the presence of phentolamine (3 ␮M), to
block ␣-AR. Human tissue strips were allowed to stabilize for 45 min
to the carbachol-induced contraction before cumulative concentra-
tion-response curves for the relaxing effects of (⫺)-isoproterenol were
obtained in the absence and presence of subtype-selective ␤-AR an-
tagonists. Each experiment was terminated by measuring maximal
relaxation in response to the adenylyl cyclase activator forskolin (10
␮M). Time-matched control experiments of carbachol-induced detru-
sor contractions in the presence of phentolamine (3 ␮M) were run
simultaneously in the absence of a ␤-AR agonist or antagonist.
Data Analysis and Statistics. Detrusor contraction was mea-
sured as force and expressed as millinewtons per milligram of wet
weight of each muscle strip. Contractile forces in response to 100 nM
carbachol or 40 mM KCl were considered as 100% contraction mea-
sured after 45 min of initial maximal stimulation, resulting in a
stable tonic response. ␤-AR-mediated relaxations are expressed as
percentage of the maximal relaxation using 10 ␮M forskolin at the
end of each experiment. Force of contraction of the concentration-
effect curves to ␤-AR agonists was corrected in each experiment for
the observed decline of the contractile response during the time-
matched control experiments. This correction was performed using
mean values for spontaneous decline as determined from prepara-
tions of 28 C57BL6 mice or 19 cav-1 KO mice and 11 patients. The
maximal relaxation (Emax) caused by ␤-AR agonists was expressed as
percentage of the forskolin-evoked relaxation. Cumulative concen-
tration-effect curves were analyzed by nonlinear regression of each
individual experiment using GraphPad Prism 4.0 (GraphPad Soft-
ware Inc., San Diego, CA), and mean ⫺log EC50 values for ␤-AR
agonists (molar concentration producing 50% of the maximal relax-
ing effect) were calculated from the concentration-effect curves in the
absence or presence of ␤-AR antagonists for each individual detrusor
strip. Results are expressed as mean ⫾ S.E.M. from n muscle strips
corresponding to the same number of investigated animals. Mean
values of multiple groups were analyzed by one-way analysis of
variance with an additional Bonferroni comparison test, and p ⬍
0.05 was considered significant. Schild plot analysis was used to
estimate apparent affinities (pKB) for L-748,337 in man and ICI
118,551 in mice (Arunlakshana and Schild, 1959).
Drugs and Solutions. All chemicals were of analytical grade and
purchased either from Merck (Darmstadt, Germany) or Sigma-
Aldrich (St. Louis, MO). The following drugs were used: CGP 20712,
(⫺)-epinephrine, (⫺)-isoproterenol, forskolin, and phentolamine hy-
drochloride were purchased from Sigma-Aldrich, ICI 118,551, and
L-748,337 were from Tocris Bioscience (Bristol, UK). All drugs were
dissolved in Milli-Q water (Millipore, Billerica, MA), with the excep-
tion of (⫺)-isoproterenol and (⫺)-epinephrine, which were dissolved
215
Primer Sequence (5⬘–3⬘)
F CCG CTG CTA CCA CGA CCC CAA G
R AGC CAG TTG AAG AAG AGC AAG AGG CG
F GGT TAT CGT CCT GGC CAT CGT GTT TG
R TGG TTC GTG AAG AAG TCA CAG CAA GTC TC
F TCT AGT TCC CAG CGG AGT TTT CAT CG
R CGC GCA CCT TCA TAG CCA TCA AAC C
F TCG TGT GCA CCG TGT GGG CC
R AGG AAA CGG CGC TCG CAG CTG TCG
F GCC TGC TGA CCA AGA ATA AGG CC
R CCC ATC CTG CTC CAC CT
F GCT CCG TGG CCT CAC GAG AA
R CCC AAC GGC CAG TGG CCA GTC AGC G
in water containing 200 mM ascorbic acid and 40 mM EDTA.
L-748,337 and forskolin were dissolved in dimethyl sulfoxide and
stock solutions (100 and 10 ␮M) were further diluted with Milli-Q
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015
water. The maximal concentration of 0.1% dimethyl sulfoxide in the
organ bath did not change detrusor contractile responses.
Results
Detrusor Relaxation in Wild-Type Mice. Mucosa-free
murine urinary bladder strips were precontracted with 40
mM KCl. Tonic contractions after 45 min ranged from 0.84 to
3.58 mN/mg wet weight in wild-type mice (for values from
individual experimental groups, see Tables 2 and 3). (⫺)-
Isoproterenol concentration-dependently relaxed murine de-
trusor with a potency (⫺logEC50M) of 8.04 ⫾ 0.07 and a
maximal relaxing effect (Emax compared with forskolin) of
62 ⫾ 2%. CGP 20712 (300 nM) and L-748,337 (100 nM) only
caused marginal shifts of the concentration-effect curve and
did not significantly reduce the potency of (⫺)-isoproterenol
(Fig. 1; Table 2). However, the ␤2-AR blocker ICI 118,551 (50
nM) potently antagonized the effects of (⫺)-isoproterenol
(Figs. 1B and 2A), reducing its potency by nearly 2 log units
(Table 2). Concurrent addition of CGP 20712 and ICI 118,551
and concurrent CGP 20712, L-748,337, and ICI 118,551 did
not reveal additional antagonism compared with ICI 118,551
alone (Fig. 1, B and D).
Detrusor relaxation with (⫺)-epinephrine was also exclu-
sively mediated by ␤2-ARs as evidenced by the concentration-
dependent shift with ICI 118,551 or without additional block-
ade by simultaneous concurrent presence of ␤1-AR-selective
CGP 20712 and ␤3-AR-selective L-748,337 (Fig. 2, B and D).
The calculated values for ⫺logEC50 and Emax are summa-
rized in Tables 2 and 3. Increasing concentrations of the
␤2-AR antagonist ICI 118,551 significantly reduced the po-
tency of both agonists (Fig. 2).
Figure 3 depicts the Schild plots for the influence of ICI
118,551 on the effect of (⫺)-isoproterenol and (⫺)-epineph-
rine (in the absence and presence of both CGP 20712 and
L-748,337). ⫺LogEC50 values from the concentration-effect
curves (see Tables 2 and 3) were used for calculation of Schild
plots; average slopes ranged between 0.71 and 1.12 under
different conditions, but none was significantly different
from slope 1 (Fig. 3). Therefore, to estimate the apparent
affinity of ICI 118,551 (pKB), a slope of 1 was forced through
the data in the Schild plot (Fig. 3). The pKB value of ICI
118,551 was not significantly different in the absence (9.28 ⫾
216 Wuest et al.

TABLE 2
Relaxing effects of (⫺)-isoproterenol on force of contraction induced by 40 mM KCl in detrusor of WT and cav-1 KO mice
Wild Type cav-1 Knockout
Murine Detrusor
n Force ⫺logEC50 Emax n Force ⫺logEC50 Emax

mN/mg wet wt. M % mN/mg wet wt. M %

(⫺)-Isoproterenol
Control 43 0.99 ⫾ 0.10 8.04 ⫾ 0.07 62 ⫾ 2 19 0.51 ⫾ 0.12 7.76 ⫾ 0.15 44 ⫾ 3
CGP, 300 nM 8 1.66 ⫾ 0.55 7.82 ⫾ 0.16 60 ⫾ 5 6 0.89 ⫾ 0.30 7.59 ⫾ 0.31 43 ⫾ 6
L, 100 nM 7 1.45 ⫾ 0.25 7.67 ⫾ 0.18 53 ⫾ 7 9 0.54 ⫾ 0.10 7.80 ⫾ 0.20 41 ⫾ 4
⫹CGP, 300 nM ⫹ ICI, 50 nM 5 1.17 ⫾ 0.22 6.24 ⫾ 0.18*** 61 ⫾ 4 7 0.35 ⫾ 0.05 6.47 ⫾ 0.33** 53 ⫾ 2
⫹ICI, 0.3 nM 6 2.53 ⫾ 1.52 7.96 ⫾ 0.11 64 ⫾ 1
⫹ICI, 1 nM 6 3.05 ⫾ 0.79 7.46 ⫾ 0.11** 61 ⫾ 5 8 0.43 ⫾ 0.08 6.72 ⫾ 0.23* 57 ⫾ 4
⫹ICI, 5 nM 5 1.13 ⫾ 0.23 6.71 ⫾ 0.06*** 66 ⫾ 1 7 0.82 ⫾ 0.25 6.21 ⫾ 0.20*** 47 ⫾ 9
⫹ICI, 15 nM 5 1.23 ⫾ 0.16 6.39 ⫾ 0.05*** 66 ⫾ 6
⫹ICI, 50 nM 11 1.26 ⫾ 0.30 6.22 ⫾ 0.09*** 65 ⫾ 2 8 0.59 ⫾ 0.14 7.85 ⫾ 0.29H 17 ⫾ 4
5.91 ⫾ 0.17***L 27 ⫾ 7
⫹CGP ⫹ L 4 3.58 ⫾ 0.64 7.69 ⫾ 0.28 59 ⫾ 7
⫹CGP ⫹ ICI, 0.1 nM ⫹ L 5 1.95 ⫾ 0.68 7.83 ⫾ 0.21 49 ⫾ 6
⫹CGP ⫹ ICI, 1 nM ⫹ L 4 1.20 ⫾ 0.16 6.71 ⫾ 0.10*** 52 ⫾ 7
⫹CGP ⫹ ICI, 5 nM ⫹ L 6 0.84 ⫾ 0.17 6.68 ⫾ 0.12*** 69 ⫾ 3
⫹CGP ⫹ ICI, 15 nM ⫹ L 5 0.95 ⫾ 0.17 6.56 ⫾ 0.26*** 64 ⫾ 5

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⫹CGP ⫹ ICI, 50 nM ⫹ L 13 0.56 ⫾ 0.08 5.97 ⫾ 0.13*** 58 ⫾ 4 6 0.25 ⫾ 0.04 5.48 ⫾ 0.14*** 52 ⫾ 6
Emax, maximal isoproterenol response in percentage of the maximal relaxing response to 10 ␮M forskolin. CGP, CGP 20712 (300 nM); ICI, ICI 118,551 (concentrations
as indicated in the table); L, L 748,337 (100 nM).
*, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001 versus control (⫺)-isoproterenol; p ⫽ 0.08 for control (⫺)-isoproterenol in wild type vs. cav-1 KO mice; p ⫽ 0.08 for
(⫺)-isoproterenol ⫹ ICI, 50 nM (L) vs. (⫺)-isoproterenol ⫹ CGP ⫹ ICI, 50 nM ⫹ L in cav-1 KO mice.
H
high-sensitivity, ICI-118,551-resistant component.
L
low-sensitivity, ICI-118,551-sensitive component.

TABLE 3
Relaxing effects of (⫺)-epinephrine on force of contraction induced by 40 mM KCl in detrusor of C57BL6 mice
Wild Type
Murine Detrusor n
Force ⫺logEC50 Emax

mN/mg wet wt. M %

(⫺)-Epinephrine
Control 17 1.79 ⫾ 0.58 6.95 ⫾ 0.16 58 ⫾ 3
⫹ICI, 1 nM 6 3.29 ⫾ 0.99 6.34 ⫾ 0.16# 52 ⫾ 5
⫹ICI, 5 nM 5 2.18 ⫾ 0.61 6.13 ⫾ 0.19* 52 ⫾ 6
⫹ICI, 15 nM 3 0.82 ⫾ 0.21 6.02 ⫾ 0.29* 53 ⫾ 4
⫹ICI, 50 nM 5 2.15 ⫾ 0.57 5.16 ⫾ 0.16** 52 ⫾ 7
⫹CGP ⫹ L 4 3.33 ⫾ 0.84 6.63 ⫾ 0.31 55 ⫾ 5
⫹CGP ⫹ ICI, 1 nM ⫹ L 10 2.31 ⫾ 0.38 6.38 ⫾ 0.15 45 ⫾ 5
⫹CGP ⫹ ICI, 5 nM ⫹ L 5 0.91 ⫾ 0.18 5.65 ⫾ 0.12** 50 ⫾ 8
⫹CGP ⫹ ICI, 15 nM ⫹ L 5 0.93 ⫾ 0.36 5.44 ⫾ 0.20** 56 ⫾ 5
⫹CGP ⫹ ICI, 50 nM ⫹ L 4 0.90 ⫾ 0.39 4.81 ⫾ 0.33** 49 ⫾ 5
Emax, maximal epinephrine response in percentage of the relaxing response to 10 ␮M forskolin. CGP, CGP 20712 (300 nM); ICI, ICI 118,551 (concentrations as indicated
in the table); L, L 748,337 (100 nM).
* p ⬍ 0.05; ** p ⬍ 0.01 versus control (⫺)-epinephrine.
#
p ⫽ 0.08 versus control (⫺)-epinephrine.

0.15) or concurrent presence of CGP 20712 and L-748,337
(9.26 ⫾ 0.18), inconsistent with an additional influence of
␤1-AR and ␤3-AR to ␤2-AR blockade. Estimated antagonist
affinities of ICI 118,551 revealed a similar pKB value against
(⫺)-epinephrine (9.08 ⫾ 0.16). As for (⫺)-isoproterenol, the
presence of concurrent CGP 20712 and L-748,337 did not
influence pKB value of ICI 118,551 against (⫺)-epinephrine
(9.06 ⫾ 0.07).
Detrusor Relaxation in cav-1 KO Mice. Detrusor strips
from cav-1 KO mice exhibited impaired contractile activa-
tion. Responses to 40 mM KCl were significantly smaller
(0.51 ⫾ 0.05 mN/mg wet weight, n ⫽ 92/27) than in WT
animals (0.98 ⫾ 0.06 mN/mg wet weight; n ⫽ 139/43; p ⬍
0.05). However, relaxation in response to forskolin (10 ␮M)
reached similar absolute values: 0.11 ⫾ 0.03 versus 0.10 ⫾
0.02 mN/mg wet weight in cav-1 KO and WT, respectively.
Comparison of ⫺log EC50 values showed that in cav-1 KO
mice, (⫺)-isoproterenol tended to be less potent (p ⫽ 0.08)
and reached a significantly lower Emax (p ⬍ 0.001) compared
with wild-type mice (Table 2). Concentration-effect curves for
(⫺)-isoproterenol were not affected by ␤1-AR blockade with
CGP 20712 (300 nM) or ␤3-AR blockade with L-748,337 (100
nM; Fig. 4; Table 2). On the other hand, the ␤2-AR antagonist
ICI 118,551 (50 nM) caused, as in WT, a concentration-
dependent rightward shift of the concentration-effect curve
for (⫺)-isoproterenol. However, the (⫺)-isoproterenol curves
were flatter in the presence of 50 nM ICI 118,551 than in the
absence of ICI 118,551, and computer fits to data values
yielded a biphasic function, suggesting a high-affinity
(⫺logEC50M ⫽ 7.85 ⫾ 0.29; Emax ⫽ 17%), ICI 118,551-resis-
tant component and a low-affinity ICI 118,551-sensitive com-
ponent (⫺logEC50M ⫽ 5.91 ⫾ 0.17; Emax ⫽ 27%) (Fig. 4B).
L-748,337 (100 nM) in the presence of concurrent ICI 118,551
(50 nM) and CGP 20712 (300 nM) blocked the ICI 118,551-
resistant component of the (⫺)-isoproterenol curve (Fig. 4D;
Table 2).
 ␤-Adrenoceptor-Induced Detrusor Relaxation in Mouse and Man
Molecular Analysis of ␤-Adrenoceptors in Murine
Detrusor. Expression of ␤-AR subtypes was investigated by
real-time PCR in detrusor tissue from WT and cav-1 KO mice
(Fig. 5). It is surprising that expression levels of ␤1-AR
mRNA were 5 times higher than ␤2-AR mRNA (26 ⫾ 2 versus
7 ⫾ 0.5 fg/1 ng total RNA; p ⬍ 0.01) and even higher than
␤3-AR mRNA (2 ⫾ 0.3 fg/1 ng total RNA). Although no
differences in the expression levels of ␤1-AR and ␤2-AR
mRNA were found between WT and cav-1 KO mice, ␤3-AR
217
Fig. 1. Concentration-effect curves for (⫺)-isopro-
terenol and antagonism by ␤-AR2-selective ICI
118,551 but not by ␤-AR1-selective CGP 20712 and
␤-AR3-selective L-748,337 in the murine detrusor of
wild-type C57BL6 mice. Numbers in parentheses
are number of mice. For ⫺logEC50 and Emax data,
see Table 2.
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015
Fig. 2. Concentration-dependent antagonism by the
␤2-AR antagonist ICI 118,551 of the effects of (⫺)-
isoproterenol (left) and (⫺)-epinephrine (right) in
the absence (top) and presence (bottom) of concur-
rent ␤1-AR-selective antagonist CGP 20712 and ␤3-
AR-selective antagonist L-748,337 in murine detru-
sor of wild-type C57BL6 mice. For ⫺logEC50 and
Emax data, see Tables 2 and 3.
mRNA expression was significantly smaller in the cav-1 KO
animals.
␤-Adrenoceptor-Mediated Urinary Bladder Relax-
ation in Man. Detrusor relaxation, mediated through ␤-AR,
was investigated with (⫺)-isoproterenol in mucosa-free, human
urinary bladder muscle strips that were precontracted with the
muscarinic receptor agonist carbachol (100 nM). After 45 min of
stimulation with carbachol, the tonic contractile responses
ranged from 0.16 to 0.39 mN/mg wet weight. Contractile forces
218 Wuest et al.
in individual experimental groups before exposure to (⫺)-iso-
proterenol are summarized in Table 4. (⫺)-Isoproterenol con-
centration-dependently relaxed human detrusor (Figs. 6 and 7).
As shown in the representative experiment of Fig. 6, the relax-
ing effects of cumulative additions of (⫺)-isoproterenol were
unchanged by 50 nM ICI 118,557, but the concentration-effect
curve shifted to the right by 100 nM L-748,337. Mean ⫺logEC50
and Emax are summarized in Table 4. Neither CGP 20712 (300
nM) nor ICI 118,557 (50 nM) blocked the effects of (⫺)-isopro-
terenol (Fig. 7, A and B). In contrast, the ␤3-AR antagonist
L-748,337 (30 nM–1 ␮M) caused concentration-dependent
shifts to the right of the concentration-effect curve for (⫺)-
Fig. 3. Schild plots of ICI 118,551 versus (⫺)-isopro-
terenol (left) and versus (⫺)-epinephrine (right) in
the absence (top) and presence (bottom) of concur-
rent CGP 20712 (CGP; 300 nM) and L-748,337 (L-
748; 100 nM) in murine detrusor of wild-type
C57BL6 mice.
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015
Fig. 4. Concentration-effect curves for (⫺)-isopro-
terenol and partial antagonism by ␤2-AR-selective
ICI 118,551 and ␤-AR3-selective L-748,337 but not
by ␤1-AR-selective CGP 20712 in the murine detru-
sor of cav-1 KO mice. For ⫺logEC50 and Emax data,
see Table 2.
isoproterenol, consistent with mediation through ␤3-AR (Fig.
7C). The slope of the resultant Schild plot was not significantly
different from 1. Estimation of apparent affinity of L-748,337
yielded a pKB value of 7.65 ⫾ 0.08 (Fig. 7D).
Figure 8 shows total mRNA expression of ␤-AR in the
human detrusor. In contrast to the expression levels in the
mouse urinary bladder, the ␤3-AR mRNA is the most abun-
dant in man. Its expression level is approximately 10 times
higher than ␤2-AR mRNA (2.56 ⫾ 0.95 versus 0.22 ⫾ 0.14
fg/1 ng total RNA; n ⫽ 6; p ⬍ 0.05), and both subtypes are
even expressed at much larger levels than ␤1-AR mRNA
(0.00014 ⫾ 0.00006 fg/1 ng total RNA; n ⫽ 6), respectively.
 ␤-Adrenoceptor-Induced Detrusor Relaxation in Mouse and Man
Fig. 5. Expression of ␤-AR subunits in murine detrusor tissue. Mean
values from five to eight different animals. Top, agarose gel electrophore-
sis of the samples after qualitative PCR of cDNA and gel electrophoresis.
Three different animals were analyzed from either WT or Cav-1 KO mice;
notice that there are two splice variants of ␤3-AR. NC, negative control.
Bottom, quantitative one-step real-time RT-PCR; amount of ␤-AR mRNA
normalized to total RNA. Data from the two ␤3-AR splice variants were
pooled.
Discussion
The aim of the present study was to investigate the ␤-AR
subtypes involved in the catecholamine-evoked relaxation of
murine detrusor smooth muscle, the role of caveolae, and the
relevance to human detrusor. We found that: 1) in mouse,
mainly the ␤2-AR mediates relaxation; 2) despite the loss of
caveolae in detrusor smooth muscle cells of cav-1 KO mice,
␤2-AR-mediated relaxation is mostly preserved, but a ␤3-AR
component also contributes to relaxation; and 3) in man, detru-
sor relaxation is exclusively mediated through ␤3-AR, as dem-
onstrated with the ␤3-AR-selective antagonist L-748,337.
Murine Detrusor Relaxation. Our experiments demon-
strate that catecholamine-evoked relaxation of murine detru-
sor is mediated through ␤2-AR. Only ␤2-AR-selective ICI
118,551, but not ␤1-AR-selective CGP 20712 and ␤3-AR-
selective L-748,337, caused significant blockade of catechol-
amine-evoked relaxation. ICI 118,551 caused concentration-
dependent antagonism of the relaxant effects of both
(⫺)-isoproterenol and (⫺)-epinephrine, with slopes of Schild
plots not different from 1 and similar pKB values of 9.06 to
9.28 in WT mice. Our estimated apparent affinities for ICI
118,551 in WT mice are similar to the binding affinity for
human ventricular ␤2-AR (pKi ⫽ 9.0; Kaumann and Lem-
oine, 1987) and recombinant human ␤2-AR (pKi ⫽ 9.15; Hoff-
man et al., 2004) but slightly lower than the affinity esti-
mated for guinea pig ␤2-AR (pKB ⫽ 9.6; Lemoine et al., 1985).
Ehlert et al. (2007) reported slightly lower potency and
219
maximal inhibition for (⫺)-isoproterenol-evoked relaxations
of 50 mM KCl-induced contractions in detrusor from WT
C57BL6 mice than observed by us. These small differences
are likely because of the slightly lower KCl concentration (40
mM) used to contract the detrusor and because of the nor-
malization of our data as percentage of maximal relaxation
induced by 10 ␮M forskolin.
Detrusor Relaxation in cav-1 KO Mice. Depletion of
caveolin-1 protein results in the subsequent loss of the scaf-
folding domains of caveolae (Drab et al., 2001). Several stud-
ies have been performed to investigate the physiological role
of caveolae and possible pharmacological interventions on
signaling molecules and signal transduction pathways. How-
ever, their precise contribution to physiology, for instance, in
the cardiovascular system has not been fully understood so
far (Insel and Patel, 2007). Evidence has been provided for
the enrichment of numerous G protein-coupled receptors, G
proteins, and G protein effectors in domains of caveolae (Os-
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015
trom and Insel, 2004; Insel at al., 2005). However, the func-
tional consequences of loss of caveolin-1 protein are variable.
For example, function of all three ␤-AR subtypes is lost in the
murine small intestine of cav-1 KO mice (El-Yazbi et al.,
2006). However, ␤2-AR function is preserved in murine sa-
phenous arteries from cav-1 KO mice, but ␤1-AR-mediated
relaxation, observed in WT mice, disappeared, compensated
by the appearance of a ␤3-AR component (Neidhold et al.,
2007). Our data on ␤-AR-mediated relaxation of the urinary
bladder clearly show similar function of ␤2-AR in WT and
cav-1 KO mice because the antagonism by the selective
␤2-AR antagonist ICI 118,551 was similar. The affinity of ICI
118,551 for detrusor ␤2-AR from cav-1 KO mice was similar
to the affinity of ␤2-AR from WT mice. The difference be-
tween the ⫺logEC50 values of (⫺)-isoproterenol in the ab-
sence (7.76) and presence (5.91 for the ICI-sensitive compo-
nent) of 50 nM ICI 118,551, yields a pKB of 9.15 in cav-1 KO,
similar to the pKB value of 9.26 of ICI 118,551 for WT mice.
It is interesting that in cav-1 KO mice, an ICI 118,551-
resistant component of (⫺)-isoproterenol-evoked relaxation
appeared, which could be blocked by the ␤3-adrenoceptor-
selective antagonist L-748,337, suggesting mediation through
␤3-AR. In contrast, (⫺)-isoproterenol relaxed the WT detrusor
only through ␤2-AR because no ICI 118,551-resistant effects
were found, and L-748,337 failed to cause blockade. Although
real-time PCR revealed a significantly lower quantitative
mRNA expression for the summary of both murine splice vari-
ants ␤3a and ␤3b (Evans et al., 1999) in cav-1 KO animals, the
receptors seemed to mediate relaxation only in detrusor from
cav-1 KO but not from WT mice, revealing that from mRNA
expression levels, one cannot conclude to a possible protein
function. We are fully aware that mRNA expression does not
allow extrapolation to expression and function of protein on
the cell surface; nevertheless, we refrained from studying
protein expression because of problems with selectivity of
commercially available antibodies to G protein-coupled re-
ceptors (M. C. Michel, AMC University of Amsterdam, The
Netherlands, personal communication). Our results suggest
that the relaxant ␤3-AR function is suppressed by caveolae
through an unknown mechanism in the WT detrusor,
through lack of expression of sufficient ␤3-AR or through
uncoupling of these receptors or both. Our findings on mu-
rine detrusor are similar to results of Neidhold et al. (2007),
who reported that ␤3-AR-mediated relaxation was absent in
220 Wuest et al.
TABLE 4
Relaxing effects of (⫺)-isoproterenol on force of contraction induced by 100 nM carbachol in human detrusor
Human Detrusor Strips/Patients
n mN/mg wet wt.
(⫺)-Isoproterenol
Control 12/11
⫹ CGP 20712, 300 nM 4/4
⫹ ICI 118,551, 5 nM 5/5
⫹ L 748,337, 30 nM 9/8
⫹ L 748,337, 100 nM 6/5
⫹ L 748,337, 300 nM 7/6
⫹ L 748,337, 1 ␮M 8/8
Emax, maximal isoproterenol response in percentage of the relaxing response to 10 ␮M forskolin.
* p ⬍ 0.05; ** p ⬍ 0.01; *** p ⬍ 0.001 versus control (⫺)-isoproterenol.
WT murine saphenous arteries, but ␤3-AR contributed to the
relaxation of arteries from cav-l KO mice.
We detected diminished contractile responses to 40 mM KCl
in cav-1 KO mice, compared with WT mice. Contractile dysfunc-
tion in cav-1 KO mice has been observed by Lai et al. (2004) and
Woodman et al. (2004), who found reduced contractile re-
sponses to the muscarinic receptor agonist carbachol.
␤-Adrenoceptor-Mediated Detrusor Relaxation in
Man. ␤-AR expression at the mRNA level revealed the pres-
ence of all three ␤-AR subtypes, ␤1, ␤2, and ␤3, in human
detrusor (Igawa et al., 1999; Takeda et al., 1999). Nomiya
and Yamaguchi (2003) reported that mRNA of ␤3-AR would
represent around 94% of mRNA from all adrenoceptor sub-
types detected in the human bladder of 10 patients, the
␤3-AR being the most abundant in the human detrusor.
Functional studies have proposed ␤3-AR to be the predomi-
nant subtype for the relaxation response in human urinary
bladder and excluded involvement of ␤1-AR or ␤2-AR (Igawa
et al., 1999, 2001; Takeda et al., 1999; Morita et al., 2000;
Yamanishi et al., 2006).
Supporting previous findings, we have also detected a con-
siderably higher mRNA expression level for ␤3-AR versus
Force ⫺logEC50 Emax
M %
0.24 ⫾ 0.02 6.39 ⫾ 0.14 52 ⫾ 3
0.26 ⫾ 0.03 6.55 ⫾ 0.20 58 ⫾ 2
0.16 ⫾ 0.02 6.44 ⫾ 0.19 53 ⫾ 7
0.39 ⫾ 0.11 5.99 ⫾ 0.06* 45 ⫾ 4
0.25 ⫾ 0.04 5.51 ⫾ 0.13** 53 ⫾ 4
0.25 ⫾ 0.07 5.21 ⫾ 0.09*** 50 ⫾ 5
0.32 ⫾ 0.05 4.94 ⫾ 0.23*** 47 ⫾ 4
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Fig. 6. Original recordings of cumulatively added
(⫺)-isoproterenol on detrusor strips from a 73-year-
old man with a muscle-invasive urothelial carci-
noma. Muscle strips were precontracted with 100
nM carbachol before addition of (⫺)-isoproterenol.
Recordings are shown in the absence of ␤-AR antag-
onist (top), in the presence of 50 nM ICI 118,551
(middle), and in the presence of 100 nM L-748,337
(bottom).
␤2-AR and ␤1-AR. It is interesting that we also found a large
difference between ␤2-AR and ␤1-AR mRNA expression, the
latter with the ␤1-AR receptor subtype being the one most
sparsely expressed in the human detrusor. Our data reveal
that the ␤3-AR subtype is the most abundant ␤-AR in the
human urinary bladder.
Our data using the ␤1-AR-selective antagonist CGP 20712
and ␤2-AR-selective antagonist ICI 118,551 support the hy-
pothesis that ␤3-AR, but not ␤1-AR and ␤2-AR, mediate cat-
echolamine-evoked relaxation of human detrusor. To date, all
functional studies performed have used ␤3-AR-selective ago-
nists such as BRL 37,344, CL 316,243, and L-755,507 (Michel
and Vrydrag, 2006), but there were no convincing data with
antagonists to further demonstrate ␤3-AR function in the
human detrusor. Selective ␤3-AR antagonist have not often
been used to study the functional role of ␤3-AR in the urinary
bladder because they are not widely available. Some studies
have used SR 59,230 (Igawa et al., 1999; Yamanishi et al.,
2006), which was initially described as a ␤3-AR-selective
antagonist. However, recent investigations revealed that this
compound is also nonselective (Michel and Vrydrag, 2006).
As measured in transfected CHO cells, SR 59,230 possesses a
 ␤-Adrenoceptor-Induced Detrusor Relaxation in Mouse and Man
Fig. 8. Expression of ␤-AR subunits in human detrusor tissue. Mean
values from six different patients. Amount of ␤-AR mRNA normalized to
total mRNA.
pKi of 6.91 for the human ␤3-AR and 7.21 for human ␤2-AR,
respectively (Hoffmann et al., 2004). Moreover, evidence was
found for a possible inhibition of ␣1-AR as well. In the present
study, we have used L-748,337, which was shown to be highly
selective for human ␤3-AR, possessing a pKi value of 8.40
versus 6.69 (␤2-AR) and 6.41 (␤1-AR) in CHO cells expressing
human ␤-AR (Candelore et al., 1999). It is interesting that in
the human detrusor, we have estimated a pKB value of 7.65
for L-748,337, a slightly lower affinity than estimated for
recombinant ␤3-AR in CHO cells (Candelore et al., 1999).
Using our affinity estimate, 100 nM L-748,337 would cause
82% occupancy of detrusor ␤3-AR instead of 96% ␤3-AR oc-
cupancy in CHO cells (Candelore et al., 1999). However, only
17% of the ␤2-AR would be occupied by 100 nM L-748,337,
consistent with mediation through ␤3-AR receptors of (⫺)-
isoproterenol-evoked relaxation in the human detrusor.
221
Fig. 7. Concentration-effect curves for (⫺)-isopro-
terenol and antagonism by L-748,337 in the human
detrusor. Lack of antagonism by CGP 20712 (300
nM) and ICI 118,551 (50 nM). For ⫺logEC50 and
Emax data, see Table 4. Bottom right, Schild plot for
the ␤3-AR-selective antagonist L-748,337. Number
of strips/number of patients in parentheses.
Downloaded from jpet.aspetjournals.org at ASPET Journals on March 17, 2015
The different ␤-AR subtypes involved in the relaxing effect
of catecholamines in mouse and human detrusor could also
be because of pathology. Indeed, ␤3-ARs were reported to be
overexpressed in obstructed human bladder versus cystec-
tomized bladder specimen because of malignancies (Nomyia
et al., 2003). However, at present, this issue cannot be re-
solved because we did not have access to detrusor tissue from
healthy probands or patients with defined diseases as for
instance bladder outlet obstruction or inflammation.
In conclusion, ␤-AR-mediated detrusor relaxation in WT
mouse is mediated exclusively through ␤2-AR. Although it is
known from studies performed in different type of tissue that
␤2-ARs are localized in caveolae, depletion of caveolin-1 pro-
tein hardly perturbed ␤2-AR-mediated detrusor relaxation in
mouse. However, ␤3-AR did also participate in the (⫺)-iso-
proterenol-evoked relaxation of detrusor from cav-1 KO but
not WT mice. The antagonism of (⫺)-isoproterenol-evoked
relaxation of human detrusor by the ␤3-AR-selective L-748,337,
but not by blockade of ␤1-AR and ␤2-AR, consolidates the
notion that only ␤3-ARs mediate catecholamine-evoked re-
laxation in the human bladder. The murine urinary bladder
is not a model for the investigation of ␤-AR-mediated human
detrusor relaxation.
Acknowledgments
We thank Sabine Kirsch and Romy Kempe for excellent technical
assistance.
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Address correspondence to: Melinda Wuest, Department of Pharmacology
and Toxicology, Medical Faculty, Dresden University of Technology, Fetscher-
strasse 74, 01307 Dresden, Germany. E-mail: melinda.wuest@tu-dresden.de