Full text of DOI:10.1038/sj.bjp.0704165

British Journal of Pharmacology (2001) 133, 1035 ± 1040
ã
2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00
www.nature.com/bjp
Pharmacological characterization of muscarinic receptors in mouse
isolated urinary bladder smooth muscle
*,1A. Choppin & 1,2R.M. Eglen
1Genitourinary-Pharmacology, Neurobiology Unit, Roche Bioscience, Palo Alto, California, CA 94304, U.S.A.
1
The pharmacological characteristics of muscarinic receptors in the male mice urinary bladder
smooth muscle were studied.
(+)-Cis-dioxolane, oxotremorine-M, acetylcholine, carbachol and pilocarpine induced concen-
2
tration-dependent contractions of the urinary bladder smooth muscle (pEC50=6.6+0.1, 6.9+0.1,
6.7+0.1, 5.8+0.1 and 5.8+0.1, EMax=3.2+0.8 g, 2.7+0.4 g, 1.0+0.1 g, 2.7+0.3 and 0.9+0.2 g,
respectively, n=4). These contractions were competitively antagonized by a range of muscarinic
receptor antagonists (pKB values): atropine (9.22+0.09), pirenzepine (6.85+0.08), 4-DAMP
(8.42+0.14), methoctramine (5.96+0.05), p-F-HHSiD (7.48+0.09), tolterodine (8.89+0.13), AQ-
RA 741 (7.04+0.12), s-secoverine (8.21+0.09), zamifenacin (8.30+0.17) and darifenacin
(8.70+0.09).
3
In this tissue, the pKB values correlated most favourably with pKi values for these compounds at
human recombinant muscarinic M3 receptors. A signi®cant correlation was also noted at human
recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between M3
and m5 receptors.
4 In recontraction studies, in which the muscarinic M3 receptor population was decreased, and
conditions optimized to study M2 receptor activation, methoctramine exhibited an anity estimate
consistent with muscarinic M3 receptors (pKB=6.23+0.14; pA2=6.16+0.03).
5
Overall, these data study suggest that muscarinic M3 receptors are the predominant, if not the
exclusive, subtype mediating contractile responses to muscarinic agonists in male mouse urinary
bladder smooth muscle.
British Journal of Pharmacology (2001) 133, 1035 ± 1040
Keywords: Muscarinic receptors; M3-receptor; urinary bladder smooth muscle
Abbreviations: AQ-RA 741, (11-({4-[4-(diethylamino)butyl]-1-piperidinyl}acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiaze-
pine-6-one); 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine; p-F-HHSiD, para ¯uoro hexahydrosiladifenidol
Introduction
Muscarinic receptors are pharmacologically classi®ed into
four subtypes, M1, M2, M3 and M4 which equate with four of
the known muscarinic receptor gene products (m1, m2, m3
and m4) (Hulme et al., 1990; Cau®eld, 1993; Eglen et al.,
1996 for reviews). A ®fth gene has been identi®ed, m5, for
which a role has yet to be unambiguously de®ned (see Eglen
& Nahorski, 2000 for review).
Being widely distributed, muscarinic receptors play a key
physiological role in peripheral organs, including the urinary
bladder. In most smooth muscles, the muscarinic M2
receptor subtype accounts for 70 ± 80% of the receptor
population whereas the M3 receptor subtype forms only 20 ±
30% (Eglen et al., 1996). In this tissue from rat, it was
proposed that muscarinic M3 receptor activation primarily
causes direct contraction of the smooth muscle and the
muscarinic M2 receptor contracts the tissue indirectly, by
reversing sympathetically mediated relaxation (Hegde et al.,
1997). Pharmacological characterization of muscarinic re-
ceptors mediating contraction of detrusor muscle has been
well established in rat (Longhurst et al., 1995; Hegde et al.,
1997), rabbit (Tobin & Sjogren, 1995; Choppin et al., 1998),
guinea-pig (Noronha-Blob et al., 1989) and human (New-
green
& Naylor, 1996). Several investigations of the
muscarinic receptors mediating contractions of mouse
bladder have been undertaken (Durant et al., 1991;
Paravicini et al., 2000; Stengel et al., 2000; Welsh et al.,
2000), and most suggest a major role of the muscarinic M3
receptor in the contractile response, with the role of the M2
receptor, if any, being unresolved. Recently, the situation has
become clearer with the use of transgenic mice that lack
either the muscarinic M2 (Stengel et al., 2000) or M3
receptor (Matsui et al., 2000). These data collectively indicate
a minimal role for the former and that the latter mediates
most of the contractile response. The objective of the present
study was therefore to examine, using a range of de®ning
antagonists, the pharmacological characteristics of muscari-
nic receptors present in male mouse urinary smooth muscle
using isolated tissue studies.
*Author for correspondence at: Deltagen, Inc., 1003 Hamilton Avenue,
Menlo Park, CA 94025, U.S.A. E-mail: Achoppin@Deltagen.com
2Current address: DiscoveRx Corporation, 42501 Albrae Street, Suite
100, Fremont, CA 94538, USA
A preliminary account of the ®ndings has been presented
previously to the 9th international symposium on subtypes of
muscarinic receptors (Eglen & Choppin, 2001).

1036
A. Choppin & R.M. Eglen
Muscarinic receptors in mice urinary bladder smooth muscle
Methods
Data analysis
In vitro contractile studies
Contractions were recorded as changes in tension from
baseline and expressed as a percentage of the maximum
response of the ®rst agonist concentration-e€ect curve.
Agonist concentration-response curves were ®tted using a
nonlinear iterative ®tting program (Origin, Microcal Soft-
ware, Inc., Northampton, MA, U.S.A.) using the relationship
of Parker & Waud (1971). Agonist potencies and maximum
response are expressed as pEC50 (7 logarithm of the molar
concentration of agonist producing 50% of the maximum
response) and Emax, respectively. Concentration-ratios (CRs)
were determined from EC50 values in the presence and
absence of antagonist. Antagonist anity estimates (pKB
values) were determined with the equation described by
Furchgott (1972) (pKB=7log ([antagonist]/CR-1)) or using
the method of Arunlakshana & Schild (1959) using at least
three concentrations of the antagonist (pA2 values). In cases
where the slope of the linear regression was not signi®cantly
di€erent from unity, the slope was constrained to unity and
the data expressed as the pKB value. All data are expressed as
mean+s.e.mean. Pearson correlation coecients (r) and
associated P-values were calculated using the method
described by Dixon & Massey (1983). The sum of squares
of di€erences in anity estimates for each plot (S (y ± x)2,
noted ssq) de®nes the proximity of the data points to the line
of identity (y=x).
Male C57BL6 mice (25 ± 30 g) were euthanized by CO2
asphyxiation. The urinary bladder was isolated, cleared of
adhering adipose tissue and placed in oxygenated Krebs
solution (composition in mM: NaCl 118.2, KCl 4.6, CaCl2
2.5, MgSO4.7H2O 1.2, KH2PO4 1.2, NaHCO3 24.8 and
dextrose 10.0). The physiological solution contained in-
domethacin (10 mM) in order to reduce prostaglandin-
induced spontaneous activity of the tissues. Four strips of
urinary bladder smooth muscle were cut from the
supratrigonal portion of the bladder (longitudinal section).
The tissues were mounted in 10 ml organ baths containing
Krebs solution, maintained at 378C and constantly aerated
with 95% O2/5% CO2 (pH=7.4). Grass FT03 transducers
were used to measure changes in isometric tension of the
tissues, which were displayed on a Grass 7E polygraph. The
tissues were maintained at a resting tension of 1 g during
an equilibration period of 60 min. Tension adjustments
were made as necessary. The tissues were washed every
15 min.
The viability of each tissue was assessed by determining the
contractile response to KCl (30 mM) at the start of the
experimental protocol. After washing, tissues were re-
equilibrated for 10 min and allowed to regain baseline
tension. Cumulative concentration-e€ect curves to agonists
((+)-cis-dioxolane, oxotremorine-M, acetylcholine, carbachol
and pilocarpine; 1 nM ± 0.1 mM) were then constructed in
each tissue. Thereafter, tissues were equilibrated in either the
absence (time control) or presence of antagonist for a 90 min
period during which tissues were washed every 10 min.
Subsequently, a second concentration-e€ect curve to the
same agonist was constructed.
Compounds used
Atropine sulphate, indomethacin and oxybutynin chloride
were obtained from Sigma Chemical Co (MO, U.S.A.). (+)-
Cis-dioxolane, acetylcholine, carbachol, oxotremorine-M,
pilocarpine, pirenzepine dihydrochloride, methoctramine
hydrochloride, 4-diphenylacetoxy-N-methylpiperidine (4-
DAMP) methiodide, 4-DAMP mustard and para ¯uoro
hexahydrosiladifenidol (p-F-HHSiD) hydrochloride were
obtained from Research Biochemicals Inc. (MA, U.S.A.).
Darifenacin hydrobromide and zamifenacin fumerate were
generously provided by P®zer Central Research (Sandwich,
Kent, U.K.). AQ-RA 741 (11-({4-[4-(diethylamino)butyl]-1-
piperidinyl}acetyl)-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzo-
diazepine-6-one) was donated by Boehringer Ingelheim
Pharmaceuticals, Inc. (Ridge®eld, CT, U.S.A.). Isoproterenol,
tolterodine and s-secoverine hydrochloride were synthesized
at Roche Bioscience (Palo Alto, U.S.A.).
Recontraction experiments
After an initial concentration-response curve to (+)-cis-
dioxolane was established, the tissues were washed and
equilibrated with 4-DAMP mustard (40 nM) for 60 min in the
presence of methoctramine (0.3 mM). This procedure enabled
selective alkylation of M3 but not M2 receptors (Hegde et al.,
1997). 4-DAMP mustard was then removed from the tissues
by over¯ow with Krebs solution containing methoctramine
(0.3 mM) every 10 min for 60 min and subsequently with
methoctramine-free Krebs solution every 10 min for 90 min.
The tissues were then contracted with 90 mM of KCl and
subsequently relaxed with isoproterenol (30 mM). Once the
tissues had relaxed to baseline, a cumulative concentration-
e€ect curve to (+)-cis-dioxolane (1 nM ± 0.1 mM) was con-
structed.
All compounds were diluted in distilled water except
indomethacin which was diluted in polyethylene glycol.
Results
Characterization of muscarinic receptors mediating
contractions of the mice isolated urinary bladder smooth
muscle
Effects of an M2 antagonist (methoctramine) on the
recontractile responses to (+)-cis-dioxolane
After constructing two concentration-e€ect curves to (+)-cis-
dioxolane under conditions described above,
cumulative concentration e€ect curve to (+)-cis-dioxolane
(1 nM ± 0.3 mM) was constructed after equilibration of tissue
in absence (time control) or presence of methoctramine (0.1 ±
1.0 mM) for 90 min.
(+)-Cis-dioxolane, oxotremorine-M, acetylcholine, carbachol
and pilocarpine induced concentration-dependent contrac-
tions of the mice urinary bladder smooth muscle
(pEC50=6.6+0.1, 6.9+0.1, 6.7+0.1, 5.8+0.1 and 5.8+0.1,
a
third
E
max=3.2+0.8 g, 2.7+0.4 g, 1.0+0.1 g, 2.7+0.3 and
0.9+0.2 g, respectively, n=4). Time-control experiments
British Journal of Pharmacology vol 133 (7)

A. Choppin & R.M. Eglen
Muscarinic receptors in mice urinary bladder smooth muscle
1037
showed that two consecutive concentration-e€ect curves to
these agonists could be constructed in the same tissue with no
signi®cant temporal change in the agonist potency and
maximum response (Figure 1).
Pharmacological characterization of the muscarinic recep-
tor mediating direct contractions was done by determination
of antagonist anities. Several antagonists (atropine, pir-
enzepine, 4-DAMP, methoctramine, p-F-HHSiD, tolterodine,
AQ-RA 741, s-secoverine, zamifenacin and darifenacin) were
tested for their ability to inhibit (+)-cis-dioxolane-induced
responses and their functional anity estimates (pKB) are
muscle and the anities at human recombinant muscarinic
receptors showed signi®cant correlation (r=0.94,
P50.0001, ssq=1.42) at m3 but also at m5 receptors
(r=0.84, P=0.002, ssq=3.70). In contrast, poor correlations
were observed at m1, m2 and m4 (r=0.75, ssq=4.38; r=0.32,
ssq=12.59; r=0.67, ssq=5.42) respectively (Figure 2).
a
Characterization of muscarinic receptors mediating the
recontractions in mouse urinary bladder smooth muscle
Under control conditions, (+)-cis-dioxolane produced con-
centration-dependent contractions of the mouse urinary
bladder smooth muscle (pEC50=6.57+0.05, n=4). After
preferential alkylation of M3 receptor (exposure to 4-DAMP
mustard in presence of methoctramine), (+)-cis-dioxolane
produced recontractile (reversal of contraction) responses
(pEC50=6.01+0.05, n=4) of KCl-precontracted tissues,
which were relaxed with isoproterenol. The maximum
recontractile response (expressed as per cent of the control
curve) was 26+2% (n=4). No time-dependent changes in
agonist sensitivity were observed during the construction of
two consecutive concentration-recontractile e€ect curves. As
shown in Figure 3, methoctramine produced surmountable
antagonism of the recontractile response to (+)-cis-dioxo-
lane. The anity estimate (pA2) for methoctramine was
6.16+0.03 and the slope of the Schild plot was not
signi®cantly di€erent than unity.
summarized in Table 1. All these compounds, in
a
concentration-dependent fashion, with parallel rightward
displacements, surmountably antagonized cumulative agonist
concentration-response curves. The rank order of antagonist
anities (pKB) was: atropine (9.22+0.09), tolterodine (8.89+
0.13), darifenacin (8.70+0.09), 4-DAMP (8.42+0.14), zami-
fenacin (8.30+0.17), s-secoverine (8.21+0.09), p-F-HHSiD
(7.48+0.09), AQ-RA 741 (7.04+0.12), pirenzepine (6.85+
0.08) and methoctramine (5.96+0.05).
Comparison of functional data for mice urinary bladder
smooth muscle with binding data at human recombinant
muscarinic receptors
Correlation analysis between the anities of the antagonists
at muscarinic receptors in the mice urinary bladder smooth
Figure 1 E€ects of muscarinic agonists on mouse urinary bladder smooth muscle. Contractile e€ects were expressed as percentages
of the maximum response of the control curve. The values shown are means+s.e.mean, n54 animals.
British Journal of Pharmacology vol 133 (7)

1038
A. Choppin & R.M. Eglen
Muscarinic receptors in mice urinary bladder smooth muscle
Table 1 Anity estimates for muscarinic antagonists in mouse urinary bladder smooth muscle
m1
pKi
m2
pKi
m3
pKi
m4
pKi
m5
pKi
Bladder
pKB
Antagonist
Atropine (3 nM)
Pirenzepine (1 mM)
4-DAMP (10 nM)
Methoctramine (10 mM)
p-F-HHSiD (0.3 mM)
Tolterodine (10 nM)
AQ-RA 741 (0.3 mM)
s-secoverine (10 nM)
Zamifenacin (0.1 mM)
Darifenacin (10 nM
9.1a
8.0
8.9a
6.3
9.5a
6.8
9.2a
7.1
9.1a
6.9
9.22+0.09
6.85+0.08
8.42+0.14
5.96+0.05
7.48+0.09
8.89+0.13
7.04+0.12
8.21+0.09
8.30+0.17
8.70+0.09
9.2a
6.7
8.1a
7.7
9.3a
6.1
8.4a
7.0
8.9a
6.3
7.3a
8.5b
7.6c
8.5c
7.6
6.6a
8.4b
8.9c
8.9c
7.2
7.5a
8.5b
7.5c
8.3c
7.9
7.2a
8.3b
7.9c
8.6c
6.9
6.7a
8.5b
6.0c
7.0c
7.3
7.8
7.0
8.8
7.7
8.0
Values shown are means+s.e.mean., n=4. pKi values are from Loury et al. (1999). apKi values taken from Hedge et al. (1997). bpKi
values taken from Nilvebrant et al. (1996). cpKi values taken from Choppin et al. (1999).
Figure 2 Correlation between the functional anities (pKB values) of muscarinic antagonists at muscarinic receptor in mouse
isolated urinary bladder smooth muscle and binding anities (pKi values) at human recombinant muscarinic receptors (m1-m5; a ± e
respectively). The binding data were taken from Dorje et al., 1991; Eglen et al., 1997; Hegde et al., 1997; Nilvebrant et al., 1996. The
broken line is the line of identity (x=y) while the solid line is the correlation plot (the inserts give the correlation factors (r) and the
sum of squares values (ssq)).
Mouse urinary bladder smooth muscle
Discussion
Inspection of the agonist potencies and maximal responses
Previous studies using mouse urinary bladder (Durant et al.,
1991; Lundbeck & Sjogren, 1992) have demonstrated a
muscarinic-induced contractile response but did not char-
acterize the receptor subtype(s) involved. The present study
has examined in detail the pharmacological characteristics of
muscarinic receptors in this tissue.
suggest that the muscarinic receptor mediating contraction
was associated with a low ecacy. Thus, the partial agonist,
pilocarpine, yielded a potency similar to the anity and gave
a lower maximal response than seen with the full agonists.
Similar observations have been seen in urinary bladder tissue
from rat (Hegde et al., 1997). (+)-Cis-dioxolane produced
British Journal of Pharmacology vol 133 (7)

A. Choppin & R.M. Eglen
Muscarinic receptors in mice urinary bladder smooth muscle
1039
Figure 3 Recontraction experiments in mouse urinary bladder smooth muscle: e€ect of methoctramine on the recontractile
concentration-e€ect to (+)-cis-dioxolane obtained after elevation of adenylyl cyclase activity following preferential alkylation of
muscarinic M3 receptors (n=4). (a) Time control; (b) +0.3 mM methoctramine; (c) +1.0 mM methoctramine; (d) +3.0 mM
methoctramine.
concentration-dependent contractions, which were inhibited
in a concentration-dependent and competitive fashion by
muscarinic antagonists. The apparent anity estimates of
these antagonists correlated most strikingly with the binding
anities of the antagonists at m3 recombinant muscarinic
receptors (pKi are: atropine 9.5; 4-DAMP 9.3; AQ-RA 741,
7.5; darifenacin 8.8; methoctramine 6.1; tolterodine 8.5;
pirenzepine 6.8; s-secoverine 8.3; p-F-HHSiD 7.5 and
zamifenacin 7.9; r=0.94, ssq=1.42; Dorje et al., 1991; Eglen
et al., 1997; Hegde et al., 1997; Nilvebrant et al., 1996) and
are consistent with the exclusive involvement of M3
muscarinic receptors in the direct contractile response to
muscarinic agonists. This accords with ®ndings in the rabbit
(Tobin, 1995; Tobin & Sjogren, 1995), rat (Longhurst et al.,
1995; Hegde et al., 1997), human (Newgreen & Naylor, 1996)
and preliminary results in female mice (Paravicini et al., 2000)
bladder. It should be noted, however, that a signi®cantly
good correlation (r=0.84, ssq=3.70) was also obtained with
the binding anities of the antagonists at m5 recombinant
muscarinic receptors (pKi values: atropine 9.1; 4-DAMP 8.9;
AQ-RA 741, 6.0; darifenacin 8.0; methoctramine 6.3;
tolterodine 8.5; pirenzepine 6.9; s-secoverine 7.0; p-F-HHSiD
6.7 and zamifenacin 7.3; Dorje et al., 1991; Eglen et al., 1997;
Hegde et al., 1997; Nilvebrant et al., 1996). This is
unsurprising since many ligands discriminate poorly between
M3 and m5 receptors, and highlights the diculty of
excluding a role for the latter in M3 mediated responses.
In contrast to the rat urinary bladder and several
gastrointestinal smooth muscle tissues, functional studies
(recontraction experiments) in the mouse bladder revealed
no indirect contractile role of M2 receptors, and only M3
receptor activation induced bladder contraction. The low
anity of methoctramine (pA2=6.16) argues against the
involvement of an M2 receptor and the remaining 26% of the
maximal response observed with (+)-cis-dioxolane after
recontraction are likely due to an incomplete alkylation of
muscarinic M3 receptors. Data from studies performed in M2
knockout mice (Stengel et al., 2000) suggest that muscarinic
M2 receptors play
a minor role in carbachol induced
contraction of isolated bladder smooth muscle, since the
potency of muscarinic agonists is only modestly reduced, and
the maximal response una€ected. Concordantly, data from
transgenic mice lacking the muscarinic M3 receptor also
suggest predominant involvement of muscarinic M3 receptor,
as the contraction in vitro was virtually abolished in these
mice (Matsui et al., 2000). In vivo, urinary retention was
marked in these animals, suggesting that a dominant, if not
exclusive, role of this subtype prevails when voiding re¯exes
are intact (Matsui et al., 2000). The data obtained in the
present study are consistent with these ®ndings. Parasympa-
thetic nerves innervating the urinary bladder are endowed
with prejunctional inhibitory muscarinic receptors, which
have been classi®ed as muscarinic M2 receptors in the rabbit
(Tobin & Sjogren, 1995) and rat (Somogyi & De Groat,
British Journal of Pharmacology vol 133 (7)

1040
A. Choppin & R.M. Eglen
Muscarinic receptors in mice urinary bladder smooth muscle
1992) urinary bladder but M4 in the guinea-pig urinary
bladder. M2 receptors may also act prejunctionally in the
mouse bladder but given the diculty to distinguish between
these two subtypes, this function has not been investigated in
the present study.
mouse bladder equates most closely with the M3 muscarinic
receptor. Moreover, these data suggest that only M3
receptors play a role in both direct and indirect contraction
in accord with emerging data from knockout animals. It thus
appears that the mouse urinary bladder di€ers from the
mouse ileum and urinary bladder tissue from other species,
including rat and possibly human. It therefore remains to be
established if mouse tissue represents an optimal species to
provide a useful model for disorders of human urinary
bladder function.
Conclusions
The present study has shown that the pharmacological
antagonist pro®le of the muscarinic receptors present in the
References
ARUNLAKSHANA, O. & SCHILD, H.O. (1959). Some quantitative
uses of drug antagonists. Br. J. Pharmacol., 14, 48 ± 58.
CAUFIELD, M.P. (1993). Muscarinic receptors-characterization,
coupling and function. Pharmacol. Ther., 58, 319 ± 379.
CHOPPIN, A., EGLEN, R.M. & HEGDE, S.S. (1998). Pharmacological
characterisation of muscarinic receptors in rabbit isolated iris
sphincter muscle and urinary bladder smooth muscle. Br. J.
Pharmacol., 124, 883 ± 888.
LUNDBECK, F. & SJOGREN, C. (1992). A pharmacological in vitro
study of the mouse urinary bladder at the time of acute change in
bladder reservoir function after irradiation. J. Urol., 148, 179 ±
182.
MATSUI, M., MOTOMURA, D., KARASAWA, H., FUJIKAWA, T.,
JIANG, J., KOMIYA, Y., TAKAHASHI, S. & TAKETO, M.M. (2000).
Multiple functional de®cits in peripheral autonomic organs in
mice lacking muscarinic acetylcholine receptor gene for the M3
subtype. Proc. Natl. Acad. Sci., 97, 9579 ± 9584.
CHOPPIN, A., LOURY, D.N., WATSON, N., HEGDE, S.S. & EGLEN,
R.M. (1999). S-secoverine: a de®ning ligand in muscarinic M5
receptors characterization. Br. J. Pharmacol., 128, 33P.
NEWGREEN, D.T.
& NAYLOR, A.M. (1996). Characterization of
functional muscarinic receptors in human bladder. Br. J.
Pharmacol., 119, 45P.
DIXON, W.J.
& MASSEY, F.J. (1983). Introduction to statistical
analysis, 4th edition, New York: McGraw-Hill Publishing
Company.
DORJE, F., WESS, J., LAMBRECHT, G., TACKE, R., MUTSCHLER, E.
& BRANN, M.R. (1991). Antagonist binding pro®les of ®ve cloned
human muscarinic receptor subtypes. J. Pharmacol. Exp. Ther.,
256, 727 ± 733.
DURANT, P.A., SHANKLEY, N.P., WELSH, N.J. & BLACK, J.W. (1991).
Pharmacological analysis of agonist-antagonist interactions at
acetylcholine muscarinic receptors in a new urinary bladder assay
(1991). Br. J. Pharmacol., 104, 145 ± 150.
NILVEBRANT, L., SUNDQUIST, S. & GILLBERG, P.-G. (1996).
Tolterodine is not subtype (m1-m5) selective but exhibits
functional bladder selectivity in vivo. Neurourol. Urodyn., 15, 34
(abstract).
NORONHA-BLOB, L., LOWE, V.C., PATTON, A., CANNING, B.,
COSTELLO, D. & KINNIER, W.J. (1989). Muscarinic receptors:
relationships among phosphoinositide breakdown, adenylate
cyclase inhibition, in vitro detrusor muscle contractions and in
vivo cystometrogram studies in guinea-pig bladder. J. Pharma-
col. Exp. Ther., 249, 843 ± 851.
EGLEN, R.M., BONHAUS, D.W., CALIXTO, J.J., CHOPPIN, A.,
PARAVICINI, T., PENNEFATHER, J.N., LAU, W.A.K., MA, S. &
LEUNG, E., LOEB, M., LOURY, D., MOY, T., WILDA, M.
&
PATAK, E. (2000). Muscarinic receptors mediating contraction
of the urinary bladder from the female mouse. Proc. Aust. Soc.
Clin. Exp. Pharmacol. Toxicol., 7, 1 ± 12P.
HEGDE, S.S. (1997). Characterization of the interaction of
tolterodine at muscarinic receptor subtypes in vitro and in vivo.
Br. J. Pharmacol., 120, 63P.
PARKER, R.B. & WAUD, D.R. (1971). Pharmacological estimation of
drug-receptor dissociation constants. Statistical evaluation. I.
Agonists. J. Pharmacol. Exp. Ther., 177, 1 ± 12.
SOMOGYI, G.T. & DE GROAT, W.C. (1992). Evidence for inhibitory
nicotinic and facilitatory muscarinic receptors on cholinergic
nerve terminals of the rat urinary bladder. J. Auton. Nerv. Syst.,
37, 89.
EGLEN, R.M. & CHOPPIN, A. (2001). Pharmacological characterisa-
tion of muscarinic receptors in mouse urinary bladder smooth
muscle. Life Sci., 68, 2634.
EGLEN, R.M., HEGDE, S.S.
& WATSON, N. (1996). Muscarinic
receptor subtypes and smooth muscle function. Pharmacol.
Rev., 48, 531 ± 565.
EGLEN, R.M. & NAHORSKI, S.R. (2000). The muscarinic M5 receptor:
a silent or emerging subtype? Br. J. Pharmacol., 130, 13 ± 21.
FURCHGOTT, R.F. (1972). The classi®cation of adrenoceptors
(adrenergic receptors). An evaluation from the standpoint of
receptor theory. In Catecholamines, Handbook of Experimental
Pharmacology, Vol. 33. Ed. Blaschko, H. & Muscholl, E. pp.
283 ± 335. Berlin, Heidelberg, New York: Springer.
HEGDE, S.S., CHOPPIN, A., BONHAUS, D., BRIAUD, S., LOEB, M.,
MOY, T.M., LOURY, D. & EGLEN, R.M. (1997). Functional role of
M2 and M3 muscarinic receptors in the urinary bladder of rats in
vitro and in vivo. Br. J. Pharmacol., 120, 1409 ± 1418.
STENGEL, P.W., GOMEZA, J., WESS, J. & COHEN, M.L. (2000). M2
and M4 receptor knockout mice: muscarinic receptor function in
cardiac and smooth muscle in vitro. J. Pharmacol. Exp. Ther.,
292, 877 ± 885.
TOBIN, G. (1995). Muscarinic receptor subtypes in the submandib-
ular gland and the urinary bladder of the rabbit: in vivo and in
vitro functional comparisons of receptor antagonists. J. Auton.
Pharmacol., 15, 451 ± 463.
TOBIN, G. & SJOGREN, C. (1995). In vivo and in vitro e€ects of
muscarinic receptor antagonists on contractions and release of
[3H]-acetylcholine in the rabbit urinary bladder. Eur. J.
Pharmacol., 281, 1 ± 8.
HULME, E.C., BIRDSALL, N.J.M.
& BUCKLEY, N.J. (1990).
Muscarinic receptor subtypes. Ann. Rev. Pharmacol. Toxicol.,
30, 633 ± 673.
WELSH, N.J., EGLEN, R.M. & SHANKLEY. (2000). Pharmacological
comparison of the muscarinic receptors mediating contraction of
the guinea-pig left atrium, gastric smooth muscle and mouse
urinary bladder. Br. J. Pharmacol., 131, 57P.
LONGHURST, P.A., LEGGETT, R.E.
& BRISCOE, J.A.K. (1995).
Characterization of functional muscarinic receptors in the rat
urinary bladder. Br. J. Pharmacol., 116, 2279 ± 2285.
LOURY, D.N., HEGDE, S.S., BONHAUS, D.W. & EGLEN, R.M. (1999).
Ionic strength of assay bu€ers in¯uences antagonist binding
anity estimates at muscarinic M1-M5 cholinoceptors. Life Sci.,
64, 6P.
(Received February 19, 2001
Revised May 11, 2001
Accepted May 11, 2001)
British Journal of Pharmacology vol 133 (7)