0022-3565/00/2923-0877$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 292:877–885, 2000
Vol. 292, No. 3
Printed in U.S.A.
M2 and M4 Receptor Knockout Mice: Muscarinic Receptor
Function in Cardiac and Smooth Muscle In Vitro
PETER W. STENGEL, JESUS GOMEZA,1 JURGEN WESS,1 and MARLENE L. COHEN
Eli Lilly and Company, Lilly Research Laboratories, Neuroscience Research, Lilly Corporate Center, Indianapolis, Indiana
Accepted for publication November 16, 1999
This paper is available online at http://www.jpet.org
Activation of peripheral postjunctional muscarinic receptors produces biological responses such as bradycardia and
smooth muscle contraction (Brown and Taylor, 1996). However, the ability to attribute physiological roles to specific
native muscarinic receptor subtypes in different tissues has
become complicated by the important advances in molecular
biology identifying multiple muscarinic acetylcholine receptors (Levey, 1993). Difficulty associating a specific muscarinic receptor subtype to a physiological or pathophysiological response may occur as a result of the overlapping
expression pattern of the different muscarinic receptors, localization of multiple muscarinic receptors within a given
tissue, and/or the lack of ligands (agonists and antagonists)
with sufficient muscarinic receptor subtype selectivity or
specificity to permit conclusive assignment of receptor subtype (Wess, 1996). To date, molecular cloning techniques
have led to the discovery and identification of gene products
for five distinct muscarinic receptor subtypes designated M1
Received for publication September 8, 1999.
1
Address: National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Bioorganic Chemistry, Bldg. 8A, Room B1A-05, Bethesda,
MD 20892-0810.
receptors do not participate in smooth muscle contraction in
these tissues. In contrast, ⬃2-fold higher carbamylcholine concentration was required for contraction of stomach fundus,
urinary bladder, and trachea from M2 receptor knockout mice
(⫺logEC50 ⫽ 6.39 ⫾ 0.05, 6.07 ⫾ 0.06, and 6.27 ⫾ 0.12,
respectively) than from wild-type littermates (⫺logEC50 ⫽
6.68 ⫾ 0.07, 6.27 ⫾ 0.07, and 6.56 ⫾ 0.06, respectively).
Furthermore, the affinity of the M2 “selective” receptor antagonist AF-DX116 in inhibiting carbamylcholine-induced smooth
muscle contraction was significantly reduced in M2 receptor
knockout mice compared with tissues from wild-type littermates. Collectively, these results provide direct and unambiguous evidence that M2 receptors mediate muscarinic receptorinduced bradycardia and play a role in smooth muscle
contractility, whereas M4 receptors are not involved in stomach
fundus, urinary bladder, or tracheal contractility.
to M5 (Kubo et al., 1986a,b; Bonner et al., 1987, 1988; Peralta
et al., 1987a,b). Four of these receptors (M1 to M4), corresponding to the transfected muscarinic gene products for M1
to M4, have been pharmacologically characterized (for reviews, see Hulme et al., 1990; Caulfield, 1993; Eglen et al.,
1996).
mRNA for the muscarinic receptor subtypes M1 to M4, are
expressed in peripheral tissues such as atria (Hassall et al.,
1993; Hoover et al., 1994), stomach fundus, and urinary
bladder (Eglen et al., 1996). Although mammalian heart is
thought to possess predominantly M2 muscarinic receptors
(Caulfield, 1993), confirmed by the localization of M2 mRNA
in rat heart by in situ hybridization (Hoover et al., 1994),
expression of M1, M3, and M4 muscarinic receptor genes in
guinea pig and rat intrinsic intracardiac neurons by in situ
hybridization (Hassall et al., 1993) and in canine atrial tissue
by reverse transcription-polymerase chain reaction (Shi et
al., 1999) also has been reported. The role of these gene
products and/or their presence has not yet been associated
with any functional response in atria.
In most smooth muscle preparations where the muscarinic
receptor population was determined through antagonist ra-
ABBREVIATIONS: M1 to M5, muscarinic acetylcholine receptors; AF-DX 116, 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol6H-pyridol[2,3-b][1,4]benzodiazepine-6-one.
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ABSTRACT
Peripheral muscarinic receptors play key roles in the control of
heart rate and smooth muscle activity. In this study, bradycardic and smooth muscle contractile responses to the muscarinic agonist carbamylcholine were compared in isolated tissues from M2 and M4 muscarinic receptor knockout mice and
their wild-type littermates. Carbamylcholine (1 ⫻ 10⫺8-3 ⫻
10⫺5 M) produced similar concentration-dependent bradycardia in spontaneously beating atria from M4 receptor knockout
and wild-type control mice. In contrast, carbamylcholine did
not produce bradycardia in atria derived from M2 receptor
knockout mice, whereas such atria were responsive to adenosine-induced bradycardia. Carbamylcholine-induced contractile responses were similar in stomach fundus, urinary bladder, and tracheal preparations from M4 receptor knockout mice
and their wild-type littermates for each tissue (⫺logEC50 values
ranging from 6.20 ⫾ 0.10 to 6.76 ⫾ 0.08), suggesting that M4
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Stengel et al.
Materials and Methods
Animals. The generation of M2 and M4 muscarinic receptor
knockout mice has been described previously (Gomeza et al.,
1999a,b). Genetically, male mice used in the present study were
129J1/CF-1 hybrids (M2 receptor knockout mice and their wild-type
littermates, F2 generation) or 129SvEv/CF-1 hybrids (M4 receptor
knockout mice and their wild-type littermates, F2 generation). Animals were housed in polycarbonate ventilated cages. The animal
room was maintained at 22–24°C with a relative humidity of 35 to
70% and daily light/dark cycle (0600 –1800 h light). Food (laboratory
rodent diet 5001; PMI Feeds, St. Louis, MO) and water were supplied
ad libitum. Mice (33–58 g) were sacrificed by cervical dislocation and
the heart, stomach fundus, urinary bladder, and/or trachea were
quickly excised and placed in modified Krebs-bicarbonate buffer
solution of the following composition: 4.6 mM KCl, 1.2 mM KH2PO4,
1.2 mM MgSO4, 118.2 mM NaCl, 10.0 mM glucose, 1.6 mM
CaCl2(2H2O), and 24.8 mM NaHCO3. Experimental protocols and
procedures were approved by the Eli Lilly and Company Animal
Care and Use Committee.
Atrial Preparation. Spontaneously beating left and right atria
were dissected from ventricles and the left atrium was attached with
thread to a stationary glass rod, whereas the right atrium was tied
with thread to a force displacement transducer. The atria were
placed in organ baths containing 10 ml of Krebs-bicarbonate buffer
(see above-mentioned composition). The organ bath solution was
maintained at 37°C and aerated with a mixture of 95% O2/5% CO2.
The spontaneously beating left and right atria were placed under an
initial force of 0.5 g and equilibrated for 20 min during which time
the tissues were washed at 5-min intervals. Atrial rate in beats per
minute was measured with Sensotec transducers (model
MBL55140 – 02; Columbus, OH) that were coupled to a Compaq
deskpro-compatible data acquisition system (BIOPAC Systems, Inc.,
Goleta, CA). Cumulative concentration-response curves to carbamylcholine (10⫺8-10⫺4 M) or adenosine (10⫺6-10⫺3 M) were generated
and expressed as a percentage of the initial atrial rate.
In some experiments, AF-DX 116 was examined for its ability to
antagonize the negative chronotropic effect of cumulatively administered carbamylcholine. After initial responses were determined,
atria were incubated with AF-DX 116 (10⫺6 M) for 20 min. Responses
to carbamylcholine (10⫺7-10⫺4 M) were then repeated in the presence of antagonist. Carbamylcholine-induced bradycardia was identical before and after vehicle.
Smooth Muscle Preparations. A longitudinal section of stomach fundus, the urinary bladder body (cut from the urethral opening
to the dome), and a 5-mm length of trachea were prepared for in vitro
examination. One end of the stomach fundus and urinary bladder
was attached with thread to a stationary glass rod, whereas the
other end was tied with thread to the transducer. Tracheal rings
were mounted on hooks that were gently separated so that the lower
hook was attached with thread to a stationary rod, and the upper
hook was tied with thread to the transducer. All tissues were placed
in organ baths containing 10 ml of Krebs-bicarbonate buffer (see
above-mentioned composition). The organ bath solution was maintained at 37°C and aerated with a mixture of 95% O2/5% CO2.
Smooth muscle preparations were placed under an initial optimal
force of 2.0 g for tracheal rings and 4.0 g for stomach fundus and
urinary bladder (as determined in length-tension-optimizing studies
with each preparation), and equilibrated for 1 h, during which time
the tissues were washed at 15-min intervals. Isometric force in
grams was measured with Sensotec transducers. Stomach fundus,
urinary bladder, and trachea were initially challenged with 67 mM
KCl to confirm viability of the preparation. No significant differences
in contractile responses to 67 mM KCl occurred among tissues from
M2 and M4 receptor knockout mice and their wild-type littermates.
Cumulative contractile concentration-response curves to carbamylcholine (10⫺8-10⫺5 M) were generated and expressed as a percentage
of the 67 mM KCl-induced contraction determined for each tissue.
On a given day, tissues from M2 or M4 receptor knockout mice and
their wild-type littermates were used to avoid the possibility of any
daily systematic effect. Experiments were performed over multiple
days.
In some experiments, AF-DX 116 was examined for its ability to
antagonize the contraction produced by the cumulative administration of carbamylcholine. After initial contractile responses were
made, the smooth muscle preparations were incubated with AF-DX
116 (10⫺6 M) for 60 min. Contractile responses to carbamylcholine
(3.0 ⫻ 10⫺8-3.0 ⫻ 10⫺5 M) were then repeated in the presence of
antagonist. Carbamylcholine-induced contractions were identical before and after vehicle incubation in all tissues studied.
The antagonist equilibrium dissociation constant (KB) for AF-DX
116 versus carbamylcholine was determined according to the following equation (Furchgott, 1972): KB ⫽ [B]/[dose ratio ⫺ 1], where [B]
is the concentration of the antagonist and the dose ratio is the EC50
of the agonist in the presence of the antagonist divided by the control
EC50. EC50 was the concentration of agonist required to elicit 50% of
the maximal response. The antagonist equilibrium dissociation constant for AF-DX 116 was expressed as the negative logarithm of the
KB (i.e., ⫺logKB).
Statistical Analyses. Results were expressed as mean ⫾ S.E. of
3 to 14 isolated tissues obtained from 3 to 14 animals. Agonist
concentration-response curves were analyzed by a three-parameter
logistic nonlinear model (De Lean et al., 1978). The three modeled
parameters included the maximal response of the tissue, the EC50,
and the slope of the curves. Each curve was fitted with SAS (SAS
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dioligand-binding studies (Eglen et al., 1996), the M2 receptor subtype accounted for 70 to 80% of the receptor population, and the M3 receptor subtype accounted for 20 to 30% of
the receptor population. The contractile response of smooth
muscle to muscarinic agonists is thought to be primarily
mediated by activation of M3 receptors (Ehlert et al., 1997).
Although controversial (Eglen et al., 1996), M4 receptors
have been implicated in contractile responses of guinea pig
gall bladder (Ozkutlu et al., 1993; Oktay et al., 1998) and
guinea pig uterus (Dörje et al., 1990), raising the possibility
that M4 receptors may play a role in the contractile response
of other smooth muscle preparations. Thus, the availability
of mutant mouse strains that lack functional M2 and M4
receptors (Gomeza et al., 1999a,b) has provided the means to
examine the physiological role of muscarinic receptors in
native tissue in an unequivocal manner.
The present study focused on comparing muscarinic responses of isolated peripheral tissues derived from M2 and
M4 receptor knockout mice and their wild-type littermates.
Specifically, we examined carbamylcholine-induced bradycardia in isolated atria and contraction in three different
smooth muscle preparations (stomach fundus, urinary bladder, and trachea). In addition, the antagonism of carbamylcholine-induced responses by the M2 receptor “selective”
antagonist AF-DX 116 (11-[[[2-diethylamino-O-methyl]-1piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one) (Hammer et al., 1986; Giachetti et al.,
1986; Micheletti et al., 1987; Del Tacca et al., 1990), also was
assessed in these tissues. Our results indicate, in a direct and
unambiguous fashion, that M2 receptors are essential for
muscarinic receptor-dependent bradycardia and contribute
to muscarinic agonist-induced smooth muscle contraction.
These results demonstrate the usefulness of muscarinic receptor knockout mice as tools to assess the involvement of
distinct muscarinic receptor subtypes in specific physiological functions.
Vol. 292
2000
Cardiac and Smooth Muscle Responses of M2 and M4 Receptor Knockout Mice
879
Institute Inc., Cary, NC) on a Compaq (Deskpro 5133; Compaq,
Houston, TX) personal computer. Unpaired Student’s t test was used
to compare mean atrial rate, mean tissue KCl contractile response,
and mean ⫺logEC50 (EC50 was the agonist concentration for halfmaximal response) values between two groups. One-way ANOVA
was used to compare mean ⫺logEC50 values among stomach fundus,
urinary bladder, and trachea and Tukey test for all pairwise comparisons was performed when appropriate. Analyses were run with
SigmaStat for Windows (version 2.03; SPSS Science Inc., Chicago,
IL) on a Compaq personal computer (Deskpro 5133; Compaq). Comparisons were considered significant for P values of .05 or less.
Drugs. Carbamylcholine chloride and adenosine were purchased
from Sigma Chemical Co. (St. Louis, MO). AF-DX 116 was provided
by the Lilly Research Laboratories, Indianapolis, IN.
Results
TABLE 1
Basal atrial rate in M2 and M4 receptor knockout mice and wild-type
littermates
Mouse Genotype
Receptor
Wild-type
Knockout
Heart Rate (n)—beats/mina
M4
M2
372.0 ⫾ 16.7 (14)
388.6 ⫾ 17.7 (13)
n, number of mice per group.
a
Values are mean ⫾ S.E.
418.3 ⫾ 26.9 (14)
396.0 ⫾ 21.9 (7)
Fig. 1. Comparison of the negative chronotropic effect induced by carbamylcholine in spontaneously beating atria from M2 and M4 receptor
wild-type mice. Values are mean ⫾ S.E. of the number of atria in parentheses. Absence of error bars indicates that the magnitude of error was
less than the symbol size.
Fig. 2. Comparison of the negative chronotropic effect produced by carbamylcholine in spontaneously beating atria from M4 receptor knockout
mice and their wild-type littermates. Values are mean ⫾ S.E. of the
number of atria in parentheses. Absence of error bars indicates that the
magnitude of error was less than the symbol size. Asterisks denote a
significant difference between the chronotropic effects of carbamylcholine
in atria from M4 receptor knockout mice and their wild-type littermates
(*P ⬍ .05, unpaired Student’s t test).
mice were responsive to a noncholinergic bradycardic agonist, further arguing that the deletion of M2 receptors did not
exert a nonspecific effect on atrial rate.
Effect of AF-DX 116 on Carbamylcholine-Induced
Bradycardia. The M2 “selective” receptor antagonist AF-DX
116 (10⫺6 M) competitively antagonized the decrease in heart
rate produced by carbamylcholine in atria from M4 receptor
knockout mice and M4 and M2 wild-type littermates (Fig. 4).
The antagonist dissociation constant for AF-DX 116 was
similar in atria from M4 receptor knockout mice and M4 and
M2 wild-type littermates (Table 2).
Carbamylcholine-Induced Contraction in Smooth
Muscle. Carbamylcholine (10⫺8-10⫺5 M) contracted the
stomach fundus, urinary bladder, and trachea of M4 receptor
knockout mice and their wild-type littermates (Fig. 5). Car-
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Basal Atrial Rate in Receptor Knockout Mice and
Wild-Type Littermates. Basal heart rates of spontaneously
beating mouse atria derived from the M2 and M4 receptor
knockout mice were not different from atrial rates derived
from their wild-type littermates (Table 1). The similarity in
basal atrial rate among the four groups suggests a lack of
involvement of either M2 or M4 receptors in regulating basal
sinoatrial nodal function in vitro.
Carbamylcholine-Induced Bradycardia. Carbamylcholine (10⫺8-10⫺5 M)-induced bradycardia was similar in
spontaneously beating isolated atria from M2 (⫺logEC50 ⫽
6.14 ⫾ 0.06) and M4 (⫺logEC50 ⫽ 6.25 ⫾ 0.11) wild-type mice
(Fig. 1). Interestingly, carbamylcholine-induced bradycardia
in atria from M4 receptor knockout mice was significantly
reduced at lower carbamylcholine concentrations (3.0 ⫻
10⫺8-3.0 ⫻ 10⫺7 M) compared with the bradycardia in atria
from wild-type littermates (Fig. 2). The reduced efficacy of
carbamylcholine in M4 receptor knockout mice was accompanied by a modest, and almost statistically significant reduction (P ⫽ .06) in the potency of carbamylcholine (⫺logEC50 ⫽
6.02 ⫾ 0.03) relative to the potency (⫺logEC50 ⫽ 6.25 ⫾ 0.11)
in atria from wild-type mice (Fig. 2). This trend toward a
reduction in carbamylcholine-induced bradycardia in atria
from M4 receptor knockout mice suggests that M4 receptors
may be necessary to maximize the bradycardic effect produced by this agonist. Strikingly, the bradycardic activity of
carbamylcholine (10⫺8-10⫺4 M) was abolished in atria derived from M2 receptor knockout mice (Fig. 3, top), even at
the highest carbamylcholine concentration used (10⫺4 M).
Adenosine-Induced Bradycardia. Although heart rate
of M2 receptor knockout mice was not reduced by carbamylcholine, adenosine produced a similar concentration-dependent bradycardia in atria from M2 wild-type and M2 receptor
knockout mice (Fig. 3, bottom). The similar bradycardic effect
of adenosine indicated that atria from M2 receptor knockout
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Stengel et al.
Vol. 292
bamylcholine concentration-response curves determined
with smooth muscle derived from M4 receptor knockout and
wild-type mice were virtually superimposable in all three
tissues (Fig. 5). Accordingly, no differences occurred in the
potency of carbamylcholine in the stomach fundus, urinary
bladder, and trachea (Table 3). These data suggest that the
M4 muscarinic receptor is not involved in postjunctional
smooth muscle contraction in the stomach fundus, urinary
bladder, and trachea.
Likewise, carbamylcholine (10⫺8-10⫺5 M) contracted stomach fundus, urinary bladder, and trachea from M2 receptor
knockout mice and their wild-type littermates (Fig. 6). However, the potency of carbamylcholine in all three tissues from
the M2 receptor knockout mice was reduced by a factor of ⬃2
compared with responses in M2 wild-type mice. The differences in ⫺logEC50 values were statistically significant in the
stomach fundus, urinary bladder, and trachea (Table 3).
These studies suggest that M2 receptors play a role in the
contractile response of these smooth muscle preparations to
muscarinic agonists.
Fig. 4. Effect of the selective M2 antagonist AF-DX 116 (10⫺6 M) on
carbamylcholine-induced bradycardia in spontaneously beating atria
from M2 (top) and M4 (middle) receptor wild-type mice and from M4
(bottom) receptor knockout mice. Values are mean ⫾ S.E. of the number
of atria in parentheses. Absence of error bars indicates that the magnitude of error was less than the symbol size.
Antagonism of Carbamylcholine-Induced Contraction of Smooth Muscle Preparations by AF-DX 116.
AF-DX 116 (10⫺6 M) competitively inhibited carbamylcholine-induced contraction in stomach fundus, urinary bladder,
and trachea from M4 receptor knockout mice and their wildtype littermates (Fig. 7). The antagonist dissociation constant for AF-DX 116 in stomach fundus, urinary bladder, and
trachea from M4 receptor knockout mice and their wild-type
littermates did not differ significantly among tissues or genotypes (Table 3). These data are consistent with the conclusion that the M4 muscarinic receptor was not involved in
smooth muscle contraction to carbamylcholine in the stomach fundus, urinary bladder, and trachea.
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Fig. 3. Comparison of the negative chronotropic effect induced by carbamylcholine (top) and adenosine (bottom) in spontaneously beating atria
from M2 receptor knockout and M2 receptor wild-type mice. Values are
mean ⫾ S.E. of the number of atria in parentheses. Absence of error bars
indicates that the magnitude of error was less than the symbol size.
2000
Cardiac and Smooth Muscle Responses of M2 and M4 Receptor Knockout Mice
881
TABLE 2
Antagonist dissociation constant (⫺logKB) for inhibition of carbamylcholine-induced responses by AF-DX 116 in mouse atria and smooth muscle
M2 Mouse Genotype
M4 Mouse Genotype
Tissue
Wild-type
Knockout
P value
Atria
Stomach fundus
Urinary bladder
Trachea
7.23 ⫾ 0.06 (5)
6.49 ⫾ 0.17 (4)
6.47 ⫾ 0.06 (4)
6.54 ⫾ 0.07 (3)
7.28 ⫾ 0.04 (5)
6.55 ⫾ 0.07 (4)
6.64 ⫾ 0.14 (4)
6.53 ⫾ 0.07 (3)
Wild-type
Knockout
P value
⫺logKB ⫾ S.E. (n)
⫺logKB ⫾ S.E. (n)
0.71
0.75
0.32
0.88
7.45 ⫾ 0.11 (5)
6.51 ⫾ 0.10 (4)
6.48 ⫾ 0.12 (4)
6.72 ⫾ 0.11 (4)
ND
5.89 ⫾ 0.18 (4)
6.12 ⫾ 0.06 (4)
6.28 ⫾ 0.10 (4)
ND
0.025
0.036
0.025
ND, not determined.
Discussion
Fig. 5. Cumulative contractile response to carbamylcholine in stomach
fundus (top), urinary bladder (middle), and trachea (bottom) from M4
receptor knockout mice and their wild-type littermates. Values are
mean ⫾ S.E. of the number of tissues in parentheses. Absence of error
bars indicates that the magnitude of error was less than the symbol size.
Similarly, AF-DX 116 (10⫺6 M) produced a rightward shift
of carbamylcholine concentration-response curves in all three
smooth muscle preparations from M2 wild-type mice (Fig. 8).
The magnitude of this shift was similar to the one observed
Peripheral muscarinic receptors are involved in the regulation of many important physiological functions, including
the control of heart rate, the stimulation of glandular secretion, and smooth muscle contraction (Nathanson, 1987;
Hulme et al., 1990; Caulfield, 1993; Brown and Taylor, 1996).
In this study, we have used M2 and M4 receptor-deficient
mice (Gomeza et al., 1999a,b) to study the potential role of
these two muscarinic receptor subtypes in atrial bradycardia
and smooth muscle contractile responses in stomach fundus,
urinary bladder, and trachea. Previous studies with mouse
stomach fundus (Mizuguchi et al., 1997; Pheng et al., 1997),
urinary bladder (Durant et al., 1991; Lundbeck and Sjögren,
1992), and trachea (Henry et al., 1990; Garssen et al., 1993)
have demonstrated muscarinic-induced contractile responses, but have not detailed the multiple receptor subtypes
involved. Furthermore, although M1, M2, and M4 receptor
knockout mice have been studied (Hamilton et al., 1997;
Gomeza et al., 1999a,b), there are no reports of cardiac or
smooth muscle responses in isolated tissues from these mice.
Carbamylcholine was selected as a prototypic nonselective
muscarinic agonist not subjected to degradation by acetylcholinesterase (Roberts and Konjovic, 1969).
M2 and M4 muscarinic receptors are difficult to discriminate by classical pharmacological techniques because these
receptor subtypes share similar ligand-binding and G protein-coupling properties (Hulme et al., 1990; Caulfield, 1993;
Wess, 1996). The use of tissues derived from M2 and M4
receptor knockout mice therefore offers the advantage that
the potential involvement of these receptors in muscarinic
agonist-dependent responses can be assessed in a straightforward and unambiguous fashion (Gomeza et al., 1999a,b).
Such an approach combined with the use of pharmacological
tools provides a powerful means to dissect and understand
the receptor mechanisms governing muscarinic tissue responses. Furthermore, in muscarinic receptor knockout mice
generated to date, immunoprecipitation studies in brain and
heart have not revealed alteration or compensatory changes
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with the corresponding tissues from M4 receptor knockout
mice and their wild-type littermates. However, the AF-DX
116-induced rightward shift of the carbamylcholine concentration-response curves was significantly less in smooth muscle preparations derived from M2 receptor knockout mice
(Fig. 8). As shown in Table 2, ⫺logKB values for AF- DX 116
determined in stomach fundus, urinary bladder, and tracheal
tissues from M2 receptor knockout mice were significantly
lower than the corresponding values determined with smooth
muscle preparations from wild-type littermates.
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Stengel et al.
Vol. 292
TABLE 3
Comparison of ⫺logEC50 for carbamylcholine in stomach fundus, urinary bladder, and trachea from M2 and M4 receptor knockout mice and their
wild-type littermates
M2 Mouse Genotype
M4 Mouse Genotype
Tissue
Wild-type
Knockout
P value
Stomach fundus
Urinary bladder
Trachea
6.76 ⫾ 0.08 (7)
6.30 ⫾ 0.06 (7)
6.46 ⫾ 0.10 (3)
6.70 ⫾ 0.07 (7)
6.20 ⫾ 0.10 (7)
6.62 ⫾ 0.10 (3)
Wild-type
Knockout
P value
⫺logEC50 ⫾ S.E. (n)
⫺logEC50 ⫾ S.E. (n)
0.57
0.42
0.32
6.68 ⫾ 0.07 (7)
6.27 ⫾ 0.07 (7)
6.56 ⫾ 0.06 (6)
6.39 ⫾ 0.05 (7)
6.07 ⫾ 0.06 (7)
6.27 ⫾ 0.11 (6)
0.0062
0.049
0.029
n, number of mice per group.
in receptor levels or distribution (Hamilton et al., 1997; Gomeza et al., 1999a,b; Nadler et al., 1999).
Because heart rate is known to be cholinergically regulated
(Loffelholz and Pappano, 1985), the heart rate in animals
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Fig. 6. Cumulative contractile response to carbamylcholine in stomach
fundus (top), urinary bladder (middle), and trachea (bottom) from M2
receptor knockout mice and their wild-type littermates. Values are
mean ⫾ S.E. of the number of tissues in parentheses. Absence of error
bars indicates that the magnitude of error was less than the symbol size.
whose atria do not possess either the M2 or M4 receptor can
shed light on receptor mechanisms controlling this important
function. Interestingly, basal heart rate was unaltered in
isolated atria from mice lacking the M2 or M4 receptor, suggesting that these receptors do not play an important role in
sino-atrial function in vitro. These data are consistent with
the lack of effect of atropine on spontaneously beating canine
right atria in vitro (Vicenzi et al., 1995). However, the fact
that heart rate also was unaltered in ␤1-/␤2-adrenergic receptor double knockout mice (Rohrer et al., 1999) may suggest that in vitro studies will not detect changes in basal
heart rates.
Strikingly, atria derived from M2 receptor knockout mice
were unresponsive to the bradycardic effects of carbamylcholine. However, the negative chronotropic effects of adenosine
were fully retained in atria from M2 receptor knockout mice,
indicating that the biochemical pathway that links receptor
activation to reduction in atrial rate remains intact in these
animals. These observations provide direct evidence that the
presence of the M2 receptor subtype is essential for carbamylcholine-induced bradycardia. Our results are consistent
with previous observations indicating that the muscarinic
receptor population expressed by mammalian heart almost
exclusively consists of M2 receptors (Hulme et al., 1990;
Caulfield, 1993; Levey, 1993). In agreement with these findings, [3H]quinuclidinyl benzilate (a nonselective muscarinic
antagonist)-binding activity was shown to be almost completely abolished in hearts derived from M2 receptor knockout mice (Gomeza et al., 1999a). Moreover, functional studies
with subtype-“selective” muscarinic antagonists suggested
that muscarinic control of cardiac pacemaker activity is mediated by the M2 receptor subtype (Hulme et al., 1990; Caulfield, 1993). Consistent with these observations, we showed
in the present study that AF-DX 116 antagonized carbamylcholine-induced bradycardia in atria derived from wild-type
mice with an inhibitory antagonist dissociation constant consistent with M2 receptor blockade (Table 2).
Interestingly, in low concentrations, bradycardic effects of
carbamylcholine (3.0 ⫻ 10⫺8-3.0 ⫻ 10⫺7 M) were significantly reduced in atria derived from M4 receptor knockout
mice compared with atrial preparations derived from wildtype littermates. This observation raises the possibility that
M4 receptors may play a modulatory role in muscarinic regulation of cardiac pacemaker activity. However, this modulatory activity appears to require the presence of functional
M2 receptors because carbamylcholine-induced bradycardia
was completely abolished in M2 receptor knockout mice. Although mammalian heart predominantly expresses M2 receptors, M4 receptor mRNA is present in guinea pig and rat
intracardiac neurons (Hassall et al. 1993; Hoover et al., 1994)
as well as in canine atrial myocytes (Shi et al., 1999). Clearly,
2000
Cardiac and Smooth Muscle Responses of M2 and M4 Receptor Knockout Mice
883
however, the potential involvement of the M4 receptor subtype in modulating atrial rate to M2 receptor agonists (at
least in the mouse) will require more rigorous studies, including the demonstration that M4 receptors are indeed expressed in mouse heart. This issue will be addressed in
future studies.
Another major physiological function mediated by peripheral muscarinic receptors is the contraction of smooth muscle. Most smooth muscle preparations express multiple muscarinic receptor subtypes (Caulfield, 1993; Eglen et al., 1996;
Ehlert et al., 1997). In most cases, smooth muscle expressed
a major population of M2 receptors with a clearly lower
density of M3 receptors (Caulfield, 1993; Eglen et al., 1996;
Ehlert et al., 1997). However, although controversial, other
muscarinic receptor subtypes, including the M4 receptor
(Dörje et al., 1990, 1991a; Ozkutlu et al., 1993; Oktay et al.,
1998), also have been detected in some smooth muscle preparations by pharmacological and molecular techniques (for
reviews, see Levey, 1993; Eglen et al., 1996).
To understand the roles of M2 and M4 muscarinic receptors
in smooth muscle contraction, we examined carbamylcholineinduced contractile responses in stomach fundus, urinary
bladder, and tracheal smooth muscle preparations derived
from M2 and M4 muscarinic receptor knockout mice and their
wild-type littermates. Carbamylcholine concentration-response curves in tissues from M4 receptor-deficient mice
were virtually superimposable with those obtained with the
corresponding preparations derived from wild-type littermates. This finding clearly indicates that M4 receptors do not
play a role in modulating carbamylcholine-dependent contraction in the three tissues studied.
In contrast, carbamylcholine was significantly less potent
(by a factor of ⬃2) in contracting stomach fundus, urinary
bladder, and trachea from M2 receptor knockout mice compared with the corresponding preparations from wild-type
littermates. These data clearly indicate that M2 receptors
participate in smooth muscle contraction to carbamylcholine.
Note that contractile responses to 67 mM KCl were similar in
these smooth muscle tissues from M2 and M4 receptor knockout mice and their wild-type littermates. However, in spite of
the lack of functional M2 receptors in smooth muscle from M2
receptor knockout mice, carbamylcholine, although less potent, was still capable of eliciting maximal contractile responses in all three preparations. This observation is consistent with previous pharmacological studies suggesting that
muscarinic receptor-induced contraction is primarily mediated by M3 muscarinic receptors (Caulfield, 1993; Eglen et
al., 1996; Ehlert et al., 1997).
In agreement with these findings, studies with AF-DX 116
resulted in a negative logarithm antagonist dissociation constant in smooth muscle from M2 receptor knockout mice that
was significantly lower than the negative logarithm antago-
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2016
Fig. 7. Effect of the selective M2 antagonist AF-DX 116 (10⫺6 M) on carbamylcholine-induced smooth muscle contraction in stomach fundus, urinary
bladder, and trachea from M4 receptor knockout mice and their wild-type littermates. Values are mean ⫾ S.E. of the number of tissues in parentheses.
Absence of error bars indicates that the magnitude of error was less than the symbol size.
884
Stengel et al.
Vol. 292
nist dissociation constant in tissues from wild-type control
animals. The lower affinity of AF-DX 116 (⫺logKB values of
5.89 – 6.28) in smooth muscle from M2 receptor knockout mice
was consistent with the affinity of AF-DX 116 at M3 receptors
(Table 4) and with the notion that smooth muscle contraction
in these mice was mediated by the M3 subtype (Hulme et al.,
1990; Caulfield, 1993; Eglen et al., 1996; Ehlert et al., 1997).
Furthermore, if the antagonist dissociation constant for
AF-DX 116 at cloned M2 and M3 receptors approximates 100
and 1000 nM, respectively (Table 4), the intermediate value
(⬃320 nM) for the antagonist dissociation constant of AF-DX
116 in tissues from the M2 wild-type mice supports the contention that both M2 and M3 receptors participate in the
contractile response to muscarinic agonists in the stomach
fundus, urinary bladder, and trachea. Thus, use of smooth
muscle from the M2 knockout mice provides a useful model
for the study of physiological responses at M3 receptors.
In conclusion, this study highlights the usefulness of receptor knockout mice in combination with pharmacological
tools to study the role of specific muscarinic receptor subtypes present in peripheral tissues. We demonstrated, in a
direct and unequivocal manner, that muscarinic receptordependent bradycardia requires the presence of functional
M2 receptors. Our data also suggest that M4 receptors may
play a minor, facilitatory role in M2 receptor-mediated bradycardia, although this observation needs to be confirmed by
TABLE 4
Binding profile of AF-DX 116 in cloned human muscarinic receptor subtypes
Muscarinic Receptor Subtype
Ligand
Reference
M1
M2
5.89
6.37
6.24
5.72
ND
6.06
6.73
7.26
7.27
6.91
7.17
7.07
M3
M4
M5
ND
6.79
6.96
6.52
ND
6.76
5.55
5.52
5.29
ND
ND
5.45
⫺log[Ki]
[3H]NMS
[3H]NMS
[3H]NMS
[3H]QNB
[3H]MQNB
Mean
6.08
6.16
6.10
5.67
ND
6.00
NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate;MQNB, N-methyl-3-QNB.
ND, not determined.
Buckley et al. (1989)
Dorje et al. (1991b)
Esqueda et al. (1996)
Loudon et al. (1997)
Kovacs et al. (1998)
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2016
Fig. 8. Effect of the selective M2 antagonist AF-DX 116 (10⫺6 M) on carbamylcholine-induced smooth muscle contraction in stomach fundus, urinary
bladder, and trachea from M2 receptor knockout mice and their wild-type littermates. Values are mean ⫾ S.E. of the number of tissues in parentheses.
Absence of error bars indicates that the magnitude of error was less than the symbol size.
2000
Cardiac and Smooth Muscle Responses of M2 and M4 Receptor Knockout Mice
more detailed studies. Finally, the present study demonstrates that although M3 receptors are the predominant muscarinic subtype mediating contraction in stomach fundus,
urinary bladder, and trachea, M2 but not M4 muscarinic
receptors also play a role in carbamylcholine-induced contraction.
Acknowledgments
We are grateful to Dr. Christian C. Felder for arranging the
availability of the knockout mice and for helpful discussion and
encouragement.
References
muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart.
Neuroscience 56:1041–1048.
Henry PJ, Rigby PJ, Self GJ, Preuss JM and Goldie RG (1990) Relationship between
endothelin-1 binding site densities and constrictor activities in human and animal
airway smooth muscle. Br J Pharmacol 100:786 –792.
Hoover DB, Baisden RH and Xi-Moy SX (1994) Localization of muscarinic receptor
mRNAs in the rat heart and intrinsic cardiac ganglia by in situ hybridization. Circ
Res 75:813– 820.
Hulme EC, Birdsall NJM and Buckley NJ (1990) Muscarinic receptor subtypes. Ann
Rev Pharmacol Toxicol 30:633– 673.
Kovacs I, Yamamura HI, Waite SL, Varga EV and Roeske WR (1998) Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes
expressed in the murine fibroblast (B82) cell line. J Pharmacol Exp Ther 284:500 –
507.
Kubo T, Fukuda K, Mikami A, Maeda A, Takahashi H, Mishina M, Haga T, Haga K,
Ichiyama A, Kangawa K, Kojima M, Matsuo H, Hirose T and Numa S (1986a)
Cloning, sequencing and expression of complementary DNA encoding the muscarinc acetylcholine receptor. Nature (Lond) 323:411– 416.
Kubo T, Maeda A, Sugimoto K, Akiba I, Mikami A, Takahashi H, Haga T, Haga K,
Ichiyami A, Kangawa K, Matsuo H, Hirose T and Numa S (1986b) Primary
structure of porcine cardiac muscarinic acetylcholine receptor deduced from the
cDNA sequence. FEBS Lett 209:367–372.
Levey AI (1993) Immunological localization of m1–m5 muscarinic acetylcholine
receptors in peripheral tissues and brain. Life Sci 52:441– 448.
Loffelholz K and Pappano AJ (1985) The parasympathetic neuroeffector junction of
the heart. Pharmacol Rev 37:1–24.
Loudon JM, Bromidge SM, Brown F, Clark MSG, Hatcher JP, Hawkins J, Riley GJ,
Noy G and Orlek BS (1997) SB 202026: A novel muscarinic partial agonist with
functional selectivity for M1 receptors. J Pharmacol Exp Ther 283:1059 –1068
Lundbeck F and Sjögren 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.
Micheletti R, Montagna E and Giachetti A (1987) AF-DX 116, a cardioselective
muscarinic antagonist. J Pharmacol Exp Ther 241:628 – 634.
Mizuguchi T, Nishiyama M, Moroi K, Tanaka H, Saito T, Masuda Y, Masaki T, de
Wit D, Yanagisawa M and Kimura S (1997) Analysis of two pharmacologically
predicted endothelin B receptor subtypes by using the endothelin B receptor gene
knockout mice. Br J Pharmacol 120:1427–1430.
Nadler LS, Rosoff ML, Hamilton SE, Kalaydjian AE, McKinnon LA and Nathanson
NM (1999) Molecular analysis of the regulation of muscarinic receptor expression
and function. Life Sci 64:375–379.
Nathanson NM (1987) Molecular properties of the muscarinic acetylcholine receptor.
Ann Rev Neurosci 10:195–236.
Oktay S, Cabadak H, Iskender E, Goren Z, Caliskan E, Orun O, Aslan N, Karaalp A,
Tolun A, Ulusoy NB, Levey AI, El-Fakahany EE and Kan B (1998) Evidence for the
presence of muscarinic M2 and M4 receptors in guinea-pig gallbladder smooth
muscle. J Auton Pharmacol 18:195–204.
Ozkutlu U, Alican I, Karahan F, Onat F, Yegen BC, Ulusoy NB and Oktay S (1993)
Are m-cholinoceptors of guinea pig gallbladder smooth muscle of m4 subtype?
Pharmacology 46:308 –314.
Peralta EG, Ashkenazi A, Winslow JW, Smith DH, Ramachandran J and Capon DJ
(1987a) Distinct primary structure, ligand-binding properties and tissue-specific
expression of four human muscarinic acetylcholine receptors. EMBO J 6:3923–
3929.
Peralta EG, Winslow JW, Peterson GL, Smith DH, Ashkenazi A, Ramachandran J,
Schimerlik MI and Capon DJ (1987b) Primary structure and biochemical properties of an M2 muscarinic receptor. Science (Wash DC) 236:600 – 605.
Pheng LH, Nguyen-Le XK, Nsa Allogho S, Gobeil F and Regoli D (1997) Kinin
receptors in the diabetic mouse. Can J Physiol Pharmacol 75:609 – 611.
Roberts CM and Konjovic J (1969) Differences in the chronotropic and inotropic
response of the rat atrium to choline esters, cholinesterase inhibitors and certain
blocking agents. J Pharmacol Exp Ther 169:109 –119.
Rohrer DK, Chruscinski A, Schauble EH, Bernstein D and Kobilka BK (1999)
Cardiovascular and metabolic alterations in mice lacking both ␤1- and ␤2adrenergic receptors. J Biol Chem 274:16701–16708.
Shi H, Wang H and Wang Z (1999) Identification and characterization of multiple
subtypes of muscarinic acetylcholine receptors and their physiological function in
canine hearts. Mol Pharmacol 55:497–507.
Vicenzi MN, Woehlck HJ, Boban M, McCallum B, Atlee JL and Bosnjak ZJ (1995)
Muscarinic and ganglionic blocking properties of atropine compounds in vivo and
in vitro: Time dependence and heart rate effects. Can J Physiol Pharmacol 73:
483– 490.
Wess J (1996) Molecular biology of muscarinic acetylcholine receptors. Crit Rev
Neurobiol 10:69 –99.
Send reprint requests to: Peter W. Stengel, Eli Lilly and Company, Lilly
Research Laboratories, Neuroscience Research, Lilly Corporate Center, Indianapolis, IN 46285. E-mail: stengel_peter_w@lilly.com
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2016
Bonner TI, Buckley NJ, Young AC and Brann MR (1987) Identification of a family of
muscarinic acetylcholine receptor genes. Science (Wash DC) 237:527–532.
Bonner TI, Young AC, Brann MR and Buckley NJ (1988) Cloning and expression of
the human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1:403–
410.
Brown JH and Taylor P (1996) Muscarinic receptor agonists and antagonists, in The
Pharmacological Basis of Therapeutics, 9th ed. (Hardman JG and Limbird LE eds)
pp 141–160. McGraw-Hill Book Company, New York.
Buckley NJ, Bonner TI, Buckley CM and Brann MR (1989) Antagonist binding
properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol
Pharmacol 35:469 – 476.
Caulfield MP (1993) Muscarinic receptors—Characterization, coupling and function.
Pharmacol Ther 58:319 –379.
De Lean A, Munson PJ and Rodbard D (1978) Simultaneous analysis of families of
sigmoidal curves: Application to bioassay, radioligand assay and physiological
dose-response curves. Am J Physiol 235:E97–E102.
Del Tacca M, Danesi R, Blandizzi C and Bernardini MC (1990) Differential affinities
of AF-DX 116, atropine and pirenzepine for muscarinic receptors of guinea pig
gastric fundus, atria and urinary bladder: Might atropine distinguish among
muscarinic receptor subtype? Pharmacology 40:241–249.
Dörje F, Friebe T, Tacke R, Mutschler E and Lambrecht G (1990) Novel pharmacological profile of muscarinic receptors mediating contraction of the guinea-pig
uterus. Naunyn-Schmiedeberg’s Arch Pharmacol 342:284 –289.
Dörje F, Levey AI and Brann MR (1991a) Immunological detection of muscarinic
receptor subtype proteins (m1–m5) in rabbit peripheral tissues. Mol Pharmacol
40:459 – 462.
Dörje F, Wess J, Lambrecht G, Tacke R, Mutschler E and Brann MR (1991b)
Antagonist binding profiles of five cloned human muscarinic receptor subtypes.
J Pharmacol Exp Ther 256:727–733.
Durant PAC, Shankley NP, Welsh NJ and Black JW (1991) Pharmacological analysis of agonist-antagonist interactions at acetylcholine muscarinic receptors in a
new urinary bladder assay. Br J Pharmacol 104:145–150.
Eglen RM, Hegde SS and Watson N (1996) Muscarinic receptor subtypes and smooth
muscle function. Pharmacol Rev 48:531–565.
Ehlert FJ, Thomas EA, Gerstin EH and Griffin MT (1997) Muscarinic receptors and
gastrointestinal smooth muscle, in Muscarinic Receptor Subtypes in Smooth Muscle (Eglen RM ed) pp 87–147, CRC Press, Boca Raton.
Esqueda EE, Gerstin EH Jr, Griffin MT and Ehlert FJ (1996) Stimulation of cyclic
AMP accumulation and phosphoinositide hydrolysis by M3 muscarinic receptors in
the rat peripheral lung. Biochem Pharmacol 52:643– 658.
Furchgott RF (1972) The classification of adrenoceptors (adrenergic adrenoceptors).
An evaluation from the standpoint of adrenoceptor theory, in Catecholamines
(Blaschko H and Muscholl E ed), pp 285–335. Springer Verlag, Berlin.
Garssen J, Van Loveren H, Gierveld CM, Van Der Vliet H and Nijkamp FP (1993)
Functional characterization of muscarinic receptors in murine airways. Br J
Pharmacol 109:53– 60.
Giachetti A, Micheletti R and Montagna E (1986) Cardioselective profile of AF-DX
116, a muscarine M2 receptor antagonist. Life Sci 38:1663–1672.
Gomeza J, Shannon H, Kostenis E, Felder C, Zhang L, Brodkin J, Grinberg A, Sheng
H and Wess J (1999a) Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 96:1692–1697.
Gomeza J, Zhang L, Kostenis E, Felder C, Bymaster F, Brodkin J, Shannon H, Xia
B, Deng C and Wess J (1999b) Enhancement of D1 dopamine receptor-mediated
locomotor stimulation in M4 muscarinic acetylcholine receptor knockout mice.
Proc Natl Acad Sci USA 96:10483–10488.
Hamilton SE, Loose MD, Qi M, Levey AL, Hille B, McKnight GS, Idzerda RL and
Nathanson NM (1997) Disruption of the m1 receptor gene ablates muscarinic
receptor-dependent M current regulation and seizure activity in mice. Proc Natl
Acad Sci USA 94:13311–13316.
Hammer R, Giraldo E, Schiavi GB, Monferini E and Ladinsky H (1986) Binding
profile of a novel cardioselective muscarinic receptor antagonist, AF-DX 116, to
membranes of peripheral tissues and brain in the rat. Life Sci 38:1653–1662.
Hassall CJS, Stanford SC, Burnstock G and Buckley NJ (1993) Co-expression of four
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