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Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse
uterine smooth muscle
Takio Kitazawa1, Ryuichi Hirama1, Kozue Masunaga1, Tatsuro Nakamura, Koichi1
Asakawa1, Jinshan Cao1, Hiroki Teraoka2, Toshihiro Unno3, Sei-ichi Komori3, Masahisa
Yamada4, Jürgen Wess5 and Tetsuro Taneike1
1. Department of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen
University, Ebetsu, Hokkaido 069-8501, Japan
2. Department of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University,
Ebetsu, Hokkaido 069-8501, Japan
3. Laboratory of Pharmacology, Faculty of Applied Biological Science, Gifu University,
Gifu 501-1193, Japan
4. Yamada Research Unit, RIKEN Brain Science Institute, Saitama 351-0198, Japan
5. Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and
Kidney Diseases, Bethesda, MD 20892, USA.
Correspondence Author
Takio Kitazawa: Department of Pharmacology, School of Veterinary Medicine, Rakuno
Gakuen University, Ebetsu, Hokkaido 069-8501, Japan
E-mail:[email protected]
Short title. Muscarinic receptor subtypes in mouse uterus
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Abstract
Functional muscarinic acetylcholine receptors present in the mouse uterus were
characterized by pharmacological and molecular biological studies using control (DDY and
wild type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO) , and M2
and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM – 100 µM)
increased muscle tonus and phasic contractile activity of uterine strips of control mice in a
concentration-dependent manner. The maximum carbachol-induced contractions (Emax)
differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle
had no significant effect on carbachol concentration-response relationships. Tetrodotoxin
did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists
(11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1
,4]benzodiazepin6-one (AF-DX116),
N-[2-[2-[(Dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3b][1,4] benzodiazepine-11-carboxamide (AF-DX384),
4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol
(p-F-HHSiD), himbacine, methoctramine, pirenzepine and tropicamide inhibited
carbachol-induced contractions in a competitive fashion. The pKb values for these
muscarinic receptor antagonists correlated well with the known pKi values of these
antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with
pertussis toxin (100 µg/kg, i.p. for 96h), Emax values for carbachol were significantly
decreased but effective concentration that caused 50% of Emax values (EC50) remained
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unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 µM),
followed by subsequent washout of AF-DX116, neither carbachol nor
N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M)
caused any contractile responses. Both M2 and M3 muscarinic receptor mRNAs were
detected in the mouse uterus via reverse transcription polymerase chain reaction (RT-PCR).
Carbachol also caused contraction of uterine strips isolated from M2KO mice, but the
concentration-response curve was shifted to the right and downward compared with that for
corresponding wild type mice. On the other hand, uterine strips isolated from M3KO and
M2/M3 double KO mice were virtually insensitive to carbachol. In conclusion, although
both M2 and M3 muscarinic receptors were expressed in the mouse uterus,
carbachol-induced contractile responses were predominantly mediated by the M3 receptor.
Activation of M2 receptors alone did not cause uterine contractions; however, M2 receptor
activation enhanced M3 receptor-mediated contractions in the mouse uterus.
Key words: mouse uterus, M2 receptor, M3 receptor, carbachol, contraction
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Introduction
Genitourinary smooth muscle tissues (ureter, urinary bladder, uterus) are innervated by
the parasympathetic nervous system, and their contractility is regulated by released
acetylcholine. Activation of postjunctional muscarinic receptors plays a dominant role in
nerve-evoked contractions of the uterus, with extensive cholinergic innervation occurring in
the uterine body and cervix from several species (Traurig and Papka 1993). Muscarinic
receptors belong to the superfamily of G protein-coupled receptors, and molecular cloning
studies have demonstrated the existence of five mammalian muscarinic receptor subtypes
(M1-M5). Based on their differential G-protein-coupling properties, the five receptors can
be subdivided into two major functional classes. The M1, M3 and M5 receptors couple with
Gq/11 proteins and stimulate phosphoinositide turnover, resulting in production of
inositol-trisphosphate (IP3) and diacylglycerol, while the M2 and M4 receptors couple with
Gi/o proteins and inhibit adenylate cyclase activity to decrease cytoplasmic cyclic AMP
contents (Caulfield and Birdsall 1998).
The muscarinic receptor subtypes responsible for
mediating contraction of the myometrium have been investigated in only a few animal
species (guinea pig, rat and pig), with contradictory results. Leiber et al. (1990) studied the
effects of different muscarinic receptor antagonists on carbachol-induced contractions and
measured accumulation of inositol phosphates and inhibition of cyclic AMP production in
the guinea-pig uterus. The authors concluded that the contractile response was triggered
mainly by M3 receptors via the inositol phosphate pathway and was modulated to a minor
extent by M2 receptor activity via the cyclic AMP pathway. However, in the same species,
M2 and/or M4 receptors have been proposed to mediate uterine contractions directly, based
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on functional and radioligand binding studies (Eglen et al. 1989; Doods et al.1993). In the
rat uterus, Abdalla et al. (2004) demonstrated the expression of both M2 and M3 muscarinic
receptors and the physiological significance of M3 subtype in cholinergic uterine
contractions. In the porcine uterus, the presence of M3 but not M2 muscarinic receptors has
been revealed by functional and radioligand binding studies (Kitazawa et al. 1999). These
findings suggest species-related variations in muscarinic receptor expression and
muscarinic receptor subtype mediating contractions of the uterus.
The heterogeneous expression of muscarinic receptor subtypes in visceral smooth
muscles and the lack of subtype-selective muscarinic receptor agonists and antagonists
have hindered studies of the functional roles of different muscarinic receptors in mediating
smooth muscle contraction. Recently, mutant mice lacking individual muscarinic receptor
subtypes have been generated by the use of gene targeting techniques (Wess 2004).
Analysis of these mutant mice has provided clear evidence that both M2 and M3 receptors
mediate gastrointestinal and airway smooth muscle contractions (Yamada et al. 2001;
Matsui et al. 2000; 2002; Stengel et al. 2002; Stengel and Cohen 2002; Struckmann et al.
2003; Unno et al. 2005; Kitazawa et al. 2007) , whereas the M2 receptor mediates negative
chronotropic action in the heart (Stengel et al. 2000). In exocrine organs, M1 and M3
receptors (salivary glands) or M3 and M5 receptors (gastric glands) have been shown to be
involved in cholinergic secretory actions (Gautam et al. 2004; Aihara et al. 2005). So far,
muscarinic receptor mutant mice have not been used to study the muscarinic receptor
subtypes involved in cholinergic contractions of mouse uterine smooth muscle.
The aim of the present study was to identify the muscarinic receptor subtypes involved
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in carbachol (a non-selective and cholinesterase-resistant muscarinic agonist)-induced
contractions of the mouse uterus. To accomplish this objective, the pharmacological
properties of carbachol-induced responses in uterine strips from control mice were first
characterized using different muscarinic receptor antagonists and in vivo treatment with
pertussis toxin. Subsequently, carbachol-induced responses in uterine strips were compared
among muscarinic receptor-deficient mice. Expression of M2 and M3 muscarinic receptors
in the mice uterus was examined by reverse transcription polymerase chain reaction
(RT-PCR) study using subtype-specific primers.
Materials and methods
Animals and tissue preparations
All experiments described were performed in accordance with institutional guidelines
approved by the Animal Ethics Committee of the School of Veterinary Medicine, Rakuno
Gakuen University, Ebetsu, Hokkaido, Japan.
Non-pregnant sexually mature female control mice (DDY mice; Sankyo Lab Service,
Sapporo, Japan), and mice lacking a single M2 (M2KO) or M3 receptor (M3KO) or both M2
and M3 receptors (M2/M3KO) and corresponding wild type (WT) mice were used in the
present experiments. The animals were housed in ventilated-polycarbonate cages. The
temperature of the animal room was maintained at 23±1℃ with relative humidity of
40-60% and a daily light/dark cycle (7:00 am-7:00 pm). Food (CRF-1, Oriental Yeast Co
Ltd, Japan) and water were given ad libitum. The generation of M2KO, M3KO and
M2/M3KO mice has been described previously (Gomeza et al. 1999; Yamada et al. 2001;
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Struckmann et al. 2003). The genetic background of the mice used in the present study was
129J1 (50%) x CF1 (50%) for M2KO and their corresponding WT mice, 129vEv (50%) x
CF1 (50%) for M3KO and their corresponding WT mice, 129J1(25%) x 129SvEv (25%) x
CF1 (50%) for M2/M3KO mice.
The mice, aged more than 3 months and weighing 23-30 g, were killed by cervical
dislocation. In control mice, the stage of the estrous cycle (estrous, diestrous and
metaestrous) was determined by microscopic examination of vaginal lavage. After midline
incision, the uterus was dissected from the body and uterine smooth muscle segments (5-8
cm in length and 4-6 mm in width) were taken from along the length of each uterine horn.
Each uterine segment was cut transversely and two tube-like pieces of uterine tissue were
prepared. Depending on the anatomical region, these preparations were defined as ovarian
strips (ovarian side) and cervical strips (cervical side). Uterine preparations were suspended
vertically in an organ bath filled with Krebs solution (NaCl, 118 mM;
KCl, 4.75 mM;
MgSO4, 1.2 mM; KH2PO4, 1.2 mM; CaCl2, 2.5 mM; NaHCO2, 25 mM and glucose, 11.5
mM) warmed at 37℃ and gassed with 95%O2 + 5%CO2 . After 1h equilibration at an
initial tension of 0.5 g, all suspended preparations showed spontaneous contractility.
Isometric tension recording
The activity of uterine strips was measured with an isometric force transducer (SB-11T,
Nihon Kohden) linked to a computer-aided data analysis system (LEG-1000, Nihon
Kohden) and also to an ink-writing recorder (U-228, Nippon Denshi Kagaku Japan).
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Non-selective muscarinic receptor agonists, carbachol or
N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M),
were applied cumulatively at 5-min intervals and the evoked responses were evaluated as
the area under the curve of contractile responses. The area under the curves for agonists
were normalized by that induced by 50 mM high-K+ (for 5 min) and expressed as percent
change. On the basis of concentration-response relationships, EC50 values (concentration of
agonist that caused 50% of maximum response) and Emax value (maximum response for
agonist) were estimated by least-squares nonlinear regression analysis of
concentration–response curves using Origin software (Version 7.0; Origin Lab, USA).
The effects of muscarinic receptor antagonists (AF-DX116, AF-DX384, 4-DAMP,
p-F-HHSiD, himbacine, methoctramine, pirenzepine and tropicamide) on
carbachol-induced contraction were investigated to identify muscarinic receptor subtypes
involved in contractile response. A tube-like piece of mouse uterus was hemisected with
fine scissors, and the two muscle preparations were suspended separately in two organ
baths. One preparation was treated with a muscarinic receptor antagonist and the other was
used as a control preparation not treated with an antagonist. Twenty min later, carbachol
was applied cumulatively, and the concentration-response relationships were determined
and EC50 and Emax were calculated. The antagonist dissociation constant (pKb) was
determined for each antagonist by the following formula: pKb = log(CR-1)-log[antagonist],
where [antagonist] denotes the mol/l concentration of the antagonist, and CR is the ratio of
the EC50 value of carbachol in the presence of the antagonist divided by that in the absence
of the antagonist.
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To examine whether pertussis toxin-sensitive G-protein pathways were involved in the
carbachol-induced uterine contraction, DDY mice were injected pertussis toxin (100 µg/kg,
i.p.) and treated for 96 h before the experiment as previously described (Unno et al. 2005).
After 96h later, uterine strips were isolated and contractile responses to carbachol were
examined.
In the presence of AF-DX116 (M2 receptor-preferring antagonist), 4-DAMP-mustard
was reported to bind with the M3 muscarinic receptor preferentially and to depress its
function irreversibly. After washout of AF-DX116, which had protected M2 muscarinic
receptors, it was then possible to investigate M2 muscarinic receptor function in smooth
muscle in the absence of M3 receptor activity (Thomas et al.1993, Ehlert and Griffin 1998).
The same procedure was applied to the mouse uterine strips to investigate the function of
the M2 muscarinic receptor. In brief, after observing spontaneous contractile responses and
50 mM high-K+-induced contraction, uterine smooth muscles were first treated with
AF-DX116 (1 µM) to protect M2 receptors, and 20 min later the muscle preparations were
treated with 4-DAMP mustard (30 nM) for 1 h to inactivate M3 muscarinic receptors. One
hour later, the muscle preparations were rinsed with fresh Krebs solution every 15 min for
90 min to dissociate AF-DX116 from M2 receptors, and finally carbachol or
oxotremorine-M was applied cumulatively to establish concentration-response relationships.
Time-matched uterine strips without antagonists treatment were also prepared and the
responsiveness of muscarinic receptor agonists was examined for comparison.
Reverse transcription polymerase chain reaction (RT-PCR) analysis for M2 and M3
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muscarinic receptor mRNAs
Whole uteri and brains were collected from female DDY control mice and immediately
immersed in RNA Later (Takara) over 12 h and then stored at -30°C until use. Total RNA
was extracted by the conventional acid-guanidine-phenol-chloroform method (Trizol
reagent, Invitrogen). RNA samples were then used as the template for first-strand cDNA
synthesis using Oligo dT primer (Gibco) and reverse transcriptase (SuperScript III,
Invitrogen). The reverse-transcribed products were screened for the presence of
muscarinic-receptor cDNA by PCR. The PCR-amplified products were subjected to
electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. The
sequences of primers used for detection of M2 and M3 muscarinic receptors and sizes of the
expected RT-PCR products are 5-gcggatcctgtggccaaccaagac-3 (forward) and
5-cgaattcacgattttgcgggcta-3 (reverse) (441 bp, Aihara et al. 2005) for the M2 receptors, and
5-gtacaacctcgcctttgtttcc-3 (forward) and 5-gacaaggatgttgccgatgatg-3 (reverse) (244 bp,
Zarghooni et al. 2007) for the M3 receptors.
Chemicals
The following chemicals were used in the present experiments: atropine sulphate (Wako),
carbamoylcholine chloride (carbachol, Sigma),
(11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1
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,4]benzodiazepin6-one (AF-DX116, Tocris), 4-diphenylacetoxy-N-methyl-piperidine
(4-DAMP, Tocris), 4-diphenylacetoxy-N-methyl-piperidine mustard (4-DAMP mustard,
Tocris),
N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b
][1,4] benzodiazepine-11-carboxamide (AF-DX384,Tocris), methoctramine (Sigma),
N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M,
Tocris),
pirenzepine dihydrochloride monohydrate (Tocris), tropicamide (Tocris),
himbacine (Sigma), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD, Tocris), pertussis
toxin (Wako) and tetrodotoxin (Wako). AF-DX116 and AF-DX384 were dissolved in
dimethyl sulfoxide (DMSO), and himbacine was dissolved in ethanol. Maximum
concentrations of both DMSO and ethanol in the bathing solution were set below 0.05%, a
concentration at which vehicles did not change uterine spontaneous contractility or
carbachol-induced contraction.
Statistics
The results of experiments are expressed as means± S.E.M of at least three
experiments using muscle strips from different mice. The significance of differences
between two or more groups was determined by Student’s t-test (paired and unpaired) or
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one-way analysis of variance using post hoc Bonferroni’s test. The significance of
differences was determined at p<0.05.
Results
Carbachol-induced responses in uterus preparations from control mice (DDY mice)
Fig. 1 shows typical contractile responses to carbachol in isolated uterine strips from
ovarian regions of control mice (diestrous). Carbachol induced an excitatory mechanical
response at concentrations from 300 nM to 100 µM. A low concentration of carbachol
increased the frequency of spontaneous contractions without affecting smooth muscle tonus,
but a high concentration increased both muscle tonus and frequency of spontaneous phasic
contractions. pEC50 and relative Emax were 5.44±0.12 and 273±28% (n=4), respectively. To
examine whether carbachol-induced contractile responses were subject to desensitization,
concentration-response curves were established 3 times at 1-h intervals in the same strips.
The contractile responses to carbachol were decreased markedly during the second and
third applications (Figs. 1 and 2A). Carbachol pEC50 and Emax values were 5.13±0.06 and
97.4±30% at the 2nd application and 4.72±0.12 and 63.0±13% (n=4) at the 3rd application.
However, 50 mM high-K+-induced contractile responses remained unaltered during these
experiments (area under the curve: first=100%, second=105±12%, n=4, third=102±15.2%,
n=4). Uterine strips of the cervical region also responded to carbachol in a
concentration-dependent manner. The carbachol pEC50 value (5.53±0.10, n=4) was
comparable with but the Emax value (176±24%, n=4) was significantly lower than that
observed with strips from the ovarian region (Fig. 2B).
The contractile response to
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carbachol of uterine strips from the cervical region was also subject to desensitization
(pEC50 and Emax values: 5.25±0.04 and 136±18% at the 2nd application, n=4; 5.29±0.03 and
98.6±12% at the 3rd application, n=4). Because of this phenomenon, only one carbachol
concentration-response curve was obtained per one mouse uterine preparation.
Fig. 2C shows a comparison of carbachol concentration-response curves of ovarian
uterine strips at different estrous stages (diestrous, estrous and metestrous). Carbachol
pEC50 and Emax values were 5.7±0.4 and 198±41% at the diestrous stage (n=5), 5.8±0.1 and
209±32% at the estrous stage (n=6), and 5.78±0.3 and 168±50% (n=4) at the metestrous
stage, indicating that the stage of the estrous cycle had no significant effect on
carbachol-induced uterine contractions in mice. For this reason, mice were used without
determining the stage of their estrous cycle.
Effects of muscarinic receptor antagonists
Carbachol-induced uterine contractions were inhibited by atropine (1 µM) but not by
tetrodotoxin (1 µM), suggesting the involvement of smooth muscle muscarinic receptors in
contraction. Atropine itself (1 µM) did not change uterine muscle tone and spontaneous
phasic contractility (data not shown). The muscarinic receptor subtype involved in this
contraction was characterized by determining the affinities of different muscarinic receptor
antagonists. AF-DX116 (10 µM) treatment led to a parallel shift to the right of the
carbachol concentration-response curve without affecting Emax values (pEC50 and E max :
5.4±0.04, 91±4.8% in the absence and 4.4±0.04, 102±9.6% in the presence of AF-DX116,
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n=4). The pKb for AF-DX116 was estimated to be 6.03±0.06 (n=4). Similarly, 4-DAMP (30
nM), p-F-HHSiD (30 nM), AF-DX384 (10 µM), methoctramine (10 µM), tropicamide (1
µM), pirenzepine (10 µM) and himbacine (100 nM) also shifted the curve for
carbachol-induced contraction in a parallel manner without affecting the Emax values.
Assuming a competitive interaction, pKb values for each muscarinic receptor antagonist
were calculated to be 9.5±0.02 (4-DAMP, n=4), 8.02±0.1 (p-F-HHSiD, n=4), 7.43±0.1
(AF-DX384, n=4), 5.89±0.06 (methoctramine, n=4), 7.13±0.08 (tropicamide, n=4),
6.93±0.1 (pirenzepine, n=4) and 6.91±0.13 (himbacine, n=4), respectively. Fig. 3 shows the
correlation of estimated pKb values in the mouse uterus with antagonist affinities (pKi) for
the M1 to M5 muscarinic receptors (Darroch et al. 2000; Wang et al. 2004). Correlation
coefficients (probability) and slopes of the regression lines were 0.86 (P=0.0064) and 0.62
for the M1 receptor, 0.17 (P=0.68) and 0.12 for the M2 receptor, 0.96 (P=0.0002) and 0.76
for the M3 receptor, 0.84 (P=0.0093) and 0.53 for the M4 receptor, and 0.86 (P=0.012) and
0.68 for the M5 receptor (Fig. 3). These data are consistent with the concept that
carbachol-induced contractions of the mouse uterus are mediated by the M3 muscarinic
receptor.
Effect of in vivo pertussis toxin treatment on carbachol-induced contraction
The spontaneous contractile activity or 50 mM high-K+-induced contractions of the
uterine strips isolated from pertussis toxin (100 µg/kg, i.p.) injected mice were almost same
compared with those of untreated control mice(data not shown).
Carbachol caused
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contraction of uterine strips of pertussis toxin-treated mice in a concentration-dependent
manner. The pEC50 value was comparable (5.7±0.1, n=5) with that of untreated mice but
the Emax value was significantly reduced (pertussis toxin=130±17% n=5, control=220±29%,
n=6) (Fig. 4).
Effects of 4-DAMP mustard and AF-DX116 treatment
To investigate M2 muscarinic receptor-mediated responses in the absence of M3 receptor
activity, uterine strips were treated with the M2 receptor-preferring antagonist, AF-DX116
and the irreversible M3 receptor-preferring antagonist, 4-DAMP-mustard, for 1 h. After
washout of AF-DX116, the function of M2 receptors in smooth muscle can be evaluated in
the absence of M3 receptor activity (see Materials and Methods). In such 4-DAMP
mustard/AF-DX116-treated uterine smooth muscle preparations, carbachol was virtually
devoid of functional activity (Fig. 5A). However, carbachol was fully active in
time-matched control strips (Fig. 5A, pEC50=5.71±0.23, Emax=136±32%, n=4). The area
under the curve of 50 mM high-K+-induced contraction was almost the same without
(100%) or with 4-DAMP mustard/AF-DX116 treatment (129±14%, n=6). The same
experiment was performed using another muscarinic receptor agonist, oxotremorine-M. In
the time-matched control preparations, oxotremorine-M caused contraction of uterine strips
in a concentration-dependent manner (1 nM-100 µM), and pEC50 and Emax values were
6.37±0.16 and 156±31% (n=4), respectively. However, as observed with carbachol,
oxotremorine-M was virtually devoid of functional activity in the 4-DAMP
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mustard/AF-DX116-treated preparations (Fig. 5B).
Expression of both M2 and M3 muscarinic receptors in the mouse uterus
To examine the expression of M2 and M3 muscarinic subtype mRNAs in the mouse
uterus, total RNA prepared from whole uteri and brains was subjected to RT-PCR using
mouse M2 and M3 muscarinic receptor-specific primers. In the uterus, like in the brain,
which served as a control tissue since it is known to express both M2 and M3 receptors, M2
and M3 muscarinic receptor transcripts were identified (Fig. 6).
Carbachol-induced responses in the uterus of muscarinic receptor KO mice
In the uterine muscle strips from WT and muscarinic receptor KO mice, force of 50 mM
high K+-induced contraction was 1.9±0.16g (n=10) in WT mice (6 of 10 being M2WT and
others being M3WT), 2.4±0.36g (n=6) in M2KO mice, 2.28±0.35g (n=9) in M3KO mice and
1.8±0.27g (n=7) in M2/M3KO mice. Uterine strips isolated from WT and muscarinic
receptor KO mice contracted spontaneously. The relative area under the curve of
spontaneous contraction (for 5 min, percentage to 50 mM high-K+-induced contraction)
was 22±4% (n=10) in WT mice, 25.2±5% (n=6) in M2KO mice, 17±3% (n=9) in M3KO
mice and 21±6% (n=7) in M2/M3KO mice, suggesting that the spontaneous uterine
contractile activity of WT and KO mice was comparable each other. Carbachol (10 nM100 µM) contracted the isolated uterine strips from WT mice and increased the contractile
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activity in a concentration dependent manner (10 nM- 100 µM) (Figs. 7 and 8). pEC50
values and Emax values were 6.01±0.18 and 75.9±10.5%, respectively (n=10, 6 of 10 mice
being M2WT and the others being M3WT) (Fig. 8). Carbachol also caused contractions of
uterine strips isolated from M2KO mice but the amplitude of contractile responses were
significantly small compared with those of WT. Carbachol ( 10 nM - 1 mM) had virtually
no effect on uterine strips from M3KO or M2/M3KO mice (Figs. 7 and 8).
Discussion
A previous study has revealed differences in subregions of the mouse uterus in the
responsiveness to uterotonic agents (U46619 and PGF2α; upper subregion > lower
subregion) (Griffiths et al. 2006). Consistent with this report, the carbachol-induced
contractions were higher in ovarian (upper subregion) than in cervical strips (lower
subregion). These functional differences may reflect heterogeneous receptor distribution
patterns and may contribute to produce the gradient of intraluminal pressure from the upper
uterus to the lower uterus for smooth transportation of luminal contents.
In the present study, we found that carbachol concentration-response curves were not
significantly affected by the stage of the estrous cycle (estrous, diestrous and metestrous) of
the mouse uterus. Although the expression of prostanoid and oxytocin receptors was
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reported to be changed by steroid hormones (estrous cycle and pregnancy) (Dong and
Yallampali 2000; Oponowicz et al. 2006), the affinity and density of muscarinic receptors
in the rat uterus were not changed by estrogen treatment (Abdalla et al. 2004), consistent
with the outcome of our functional studies.
The muscarinic receptor antagonists (AF-DX116, AF-DX384, 4-DAMP, p-F-HHSiD,
himbacine, methoctramine, pirenzepine and tropicamide) inhibited carbachol-induced
contractions of mouse uterine preparations in a manner compatible with a competitive
interaction. A comparison of functional pKb values with documented pKi values for these
antagonists at the five muscarinic receptor subtypes (Darroch et al. 2000; Wang et al. 2004)
strongly suggests that the M3 muscarinic receptor subtype mediates carbachol-induced
contractions of the mouse uterus. Expression of M3 receptors in the mouse uterus was also
confirmed via RT-PCR. Moreover, the key role of the M3 receptor in mediating
carbachol-induced contractions of mouse uterine strips was supported by the fact that
carbachol did not cause contractions in 4-DAMP mustard-treated strips and in uterine strips
from M3KO mice. M3 receptor-mediated uterine contractions have already been
demonstrated in rat (Varol et al. 1989, Abdalla et al. 2004), guinea pig (Leiber et al. 1990)
and pig (Kitazawa et al. 1999). Similar results have been obtained with other visceral
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smooth muscle organs, including different regions of gastrointestinal tract, urinary bladder
and airways (Yamada et al. 2001; Matsui et al. 2000; 2002; Stengel et al. 2002; Stengel and
Cohen 2002; Struckmann et al. 2003; Unno et al. 2005; Kitazawa et al. 2007).
Coexpression of M2 and M3 receptor subtypes in the myometrium has been
demonstrated in the rat and guinea pig (Leiber et al. 1990; Munns and Pennefather 1998;
Abdalla et al. 2004). In the present study, the existence of M2 receptors was suggested by
the following findings. First, carbachol-induced contractions were inhibited in uterine strips
from M2KO mice. Second, pertussis toxin treatment, which suppresses the function of Gi/o
proteins coupled with M2 receptors, decreased the contractile responses to carbachol.
Consistent with these functional studies, M2 receptor mRNA was detected in the mouse
uterus via RT-PCR.
In the mouse uterine strips, carbachol did not cause contraction of uterine strips treated
with 4-DAMP mustard (functional M2 receptors were protected by AF-DX116), and there
was no marked contractile responses to carbachol in uterine strips from M3KO mice. These
results suggest that activation of M2 receptors alone is insufficient to cause uterine
contractions in mice. Stengel et al. (2002), Stengel and Cohen (2002), Ehlert et al. (2005),
Unno et al. (2005) and Kitazawa et al. (2007) compared carbachol concentration-response
20/32
curves in various smooth muscle strips isolated from WT and M3KO mice. In trachea,
stomach, gallbladder and ileum strips from M3KO mice, a marked contraction response
remained (40-60% of the Emax of corresponding WT), due to M2 receptor activation
(Stengel et al. 2002; Stengel and Cohen 2002; Unno et al. 2005; Kitazawa et al. 2007). In
contrast, carbachol-induced contractions were virtually absent in urinary bladder strips
from M3KO mice, despite the presence of M2 receptors (Ehlert et al. 2005). Therefore,
although M2 and M3 receptors are generally co-expressed in smooth muscle tissues, the
extent to which M2 receptors directly contribute to the contractile response differs among
different smooth muscle tissues (high contribution in tracheal, gallbladder and
gastrointestinal smooth muscles and low contribution in urinary and uterine smooth
muscles).
However, the finding that the contractile response to carbachol was found to be
suppressed in uterine strips isolated from M2KO and pertussis toxin-treated mice suggests
that M2 receptor activation modulates M3 receptor-mediated uterine contractions. Three
mechanisms by which M2 receptor activity may enhance M3 receptor-mediated contractions
have been suggested. First, activation of M2 receptor leads to decreased cytoplasmic cyclic
AMP via activation of pertussis toxin-sensitive Gi/o proteins (Caulfield and Birdsall 1998).
21/32
Since cyclic AMP causes relaxation of smooth muscle tissues, the activation of M2 receptor
inhibits the relaxant effects of cyclic AMP-elevating substances (forskolin and
β-adrenoceptor agonists) on contractions elicited by activation of Gq/11-linked receptors
such as the M3 receptor. This mechanism represents an inhibition of relaxation and is
considered an indirect contractile response mediated by the M2 receptor (Ehlert et al. 2005).
Second, the M2 receptor has been shown to inhibit Ca2+-activated K+ channels (Wade and
Sims 1993; Nakamura et al. 2002). Activation of Ca2+-activated K+ channels attenuates the
contractile responses to Ca2+-mobilizing receptors (e.g. the M3 receptor). Therefore, the
inhibition of K+ channels caused by M2 receptor activation enhances the contractile
response mediated by the M3 receptors.
Third, activation of the M2 receptor has been
reported to inhibit the release of relaxing substance (nitric oxide), thereby enhancing
smooth muscle contractility (Liu and Lee 1999). If such a mechanism is operative in the
uterus, carbachol is predicted to activate presynaptic M2 receptors on inhibitory nerves and
decrease the release of relaxant substances, leading to enhanced contractions induced by M3
receptor activation. However, further studies are needed to elucidate the mechanisms by
which M2 receptor activation leads to enhancement of M3 receptor-mediated contractions in
the mouse uterus.
22/32
In conclusion, although both M2 and M3 muscarinic receptors are expressed in the
mouse uterus, carbachol-induced contractile responses were predominantly mediated by the
M3 receptor subtypes. Activation of M2 receptors alone did not cause contractions directly.
However, M2 receptor activation was found to enhance M3 receptor-mediated uterine
contractions indirectly.
23/32
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Figure Legends
Fig. 1
Carbachol-induced typical mechanical responses in isolated muscle strips of mouse uterus
(ovarian strip, diestrous cycle). After induction of 50 mM high-K+-induced contractions
(●), carbachol (10, 30 ,100, 300 nM, 1, 3, 10, 30, 100 µM, ▲) was applied cumulatively
at 5-min intervals for construction of concentration-response curves. Cumulative
application of carbachol was carried out three times at 1-h intervals (First, Second, Third).
Numbers under each curve indicate the concentrations of carbachol (logM).
Fig. 2
Concentration-response curves for carbachol in isolated muscle strips of the mouse uterus.
A: Concentration-response relationships for carbachol (ovarian segment, diestrous cycle)
were compared at the first (●), second (■) and third (▲) cumulative applications of
carbachol at 1-h intervals to determine the potential desensitization of the contractile
responses (see Fig.1). B: Concentration-response curves of carbachol were compared
between ovarian (●) and cervical regions (▲) of the mouse uterus (diestrous cycle). C:
Carbachol-induced contractile responses were compared among the ovarian uterine strips
isolated from mice in diestrous (■), estrous (●) and metestrous cycles（▲）. Mechanical
responses to carbachol were normalized using area under the curve of 50 mM high
K+-induced contraction (for 5 min) and expressed as relative changes in area under the
curve (ordinate). Abscissa: concentration of carbachol (logM). Points represent the means
30/32
of 4 or more experiments with SEM shown by vertical lines.
Fig. 3
Characterization of muscarinic receptors involved in the carbachol-induced contractions in
the isolated mouse uterus. Each figure indicates the correlation of eight muscarinic receptor
antagonist pKb values in the mouse uterus (abscissa) with documented pKi values of these
antagonists for the respective muscarinic receptor subtypes (A: M1, B: M2, C: M3, D: M4,
E: M5) (ordinate). Solid lines are linear regression lines and broken lines are lines with a
slope of 1.0. Equation of the regression line and correlation efficient (R) are indicated in
each figure. Documented pKi values of AF-DX116, AF-DX384, 4-DAMP, p-FHHSiD,
himbacine, methoctramine, pirenzepine and tropicamide were from Darroch et al. (2000)
and Wang et al. (2004).
Fig.4
Effects of in vivo treatment with pertussis toxin on contractile responses to carbachol in the
mouse uterus. Mice were treated with saline (●) or pertussis toxin (▲, 100 µg/kg, i.p.) for
96 h and uterine smooth muscle strips were obtained from these mice.
Concentration-response relationship for carbachol was determined by cumulative
application at 5-min intervals. Carbachol responses are expressed relative to 50 mM
high-K+-induced contractions (AUC; ordinate). Abscissa: concentration of carbachol
(logM). Points represent the means of 5-6 experiments with SEM shown by vertical lines.
*; p<0.05 compared with the corresponding control values.
31/32
Fig. 5
Inhibition of muscarinic receptor agonist-induced contraction in 4-DAMP mustard-treated
mouse uterine muscle strips. To inactivate M3 muscarinic receptors selectively, isolated
uterine muscle strips were first treated with AF-DX116 (1 µM) for 20 min and then treated
with 4-DAMP mustard (30 nM) for 1 h. After washout of AF-DX116, carbachol (A) or
oxotremorine-M (B) was applied cumulatively and concentration-response relationships
were established (■). Uterine smooth muscle strips not treated with 4-DAMP mustard and
AF-DX116 were used as control preparations (●). Agonist responses are expressed relative
to 50 mM K+-induced contractions (AUC; ordinate). Abscissa: concentration of agonists
(logM). Points represent the means of 4 experiments with SEM shown by vertical lines.
Fig. 6
Expression of M2 and M3 muscarinic receptor mRNA in the mouse uterus. RT-PCR was
used to amplify signals of isolated mRNA for M2 and M3 muscarinic receptors from whole
uteri. For a positive control, mRNA from mouse brain was used. Primer sequences and
sizes of the PCR products (M2: 441 bp, M3: 244 bp) are given in Materials and methods. M,
molecular markers.
Fig.7
Typical contractile responses to carbachol in uterine strips isolated from M2WT, M2KO,
M3KO and M2/M3KO mice. Carbachol was cumulatively applied to an organ bath at 5-min
32/32
intervals (M2WT, 10 nM-100 µM; others, 10 nM – 1mM, ▲). 50 mM high-K+-induced
contraction(●) was used to normalize the contractile responses to carbachol.
Fig.8
Comparison of concentration-response curves for carbachol in uterine muscle strips isolated
from wild type mice (WT, n=10, ●), M2KO mice (n=6, ○), M3KO mice (n=9, ■) and
M2/M3KO mice (n=7, □). Concentration-response relationships were determined by
cumulative application of carbachol at 5-min intervals. Contractile responses (for 5 min)
are expressed as percentages of those induced by 50 mM high-K+. Dotted line indicates the
average level of spontaneous contractile activity before application of carbachol (WT and
all KO mice). Values are means±SEM of 6-10 experiments. *; p<0.05 compared with the
corresponding values of WT mice.
Fig. 1
First
●
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
Second
●
▲
Third
1g
●
50 mM K
▲
-8
▲
▲
-7
▲
▲
-6
▲
▲
-5
▲
▲
-4
5 min
A
Relative AUC (% to 50 mM K)
Fig. 2
350
300
250
200
150
100
50
0
B
Relative AUC (% to 50 mM K)
First
Second
Third
-8
-7
-6
-5
-4
-3
Carbachol concentration (logM)
300
Ovarian region
Cervical region
250
200
150
100
50
0
-8
-7
-6
-5
-4
-3
Carbachol concentration (logM)
Relative AUC (% to 50 mM K)
C
300
Diestrous (n=5)
Estrous (n=6)
Metestrous (n=4)
250
200
150
100
50
0
-8
-7
-6
-5
-4
Carbachol concentration (logM)
Fig. 3
A
B
Y=0.62X+3.04 R=0.86
Y=0.12X+6.67 R=0.17
9
9
M2 receptor
10
M1 receptor
10
8
7
6
7
8
9
Mouse uterus (pKb)
10
C
6
7
8
9
Mouse uterus (pKb)
10
D
10
Y=0.76X+1.69 R=0.96
10
9
M4 receptor
M3 receptor
7
6
6
8
7
6
Y=0.53X+3.94 R=0.84
9
8
7
6
6
7
8
9
Mouse uterus (pKb)
10
E
Y=0.68X+1.88 R=0.86
10
M5 receptor
8
9
8
7
6
6
7
8
9
Mouse uterus (pKb)
10
6
7
8
9
Mouse uterus (pKb)
10
Relative AUC (% to 50 mM K)
Fig. 4
350
Control
PTX (100 µg/kg, i.p. 96h)
300
250
200
150
100
*
*
*
* * * *
50
0
-8
-7
-6
-5
-4
-3
Carbachol concentration (log M)
Fig. 5
Relatve AUC (% to 50 mM K)
A
200
Control
4-DAMP Mustard + AF-DX116
150
100
50
0
-9
-8
-7
-6
-5
-4
Carbachol concentration (logM)
-3
Relative AUC (% to 50 mM K)
B
250
Control
4-DAMP Mustard + AF-DX116
200
150
100
50
0
-9
-8
-7
-6
-5
-4
-3
Oxotremorine-M concentration (logM)
Fig.6
M2 receptor
M3 receptor
M Brain Uterus
M Brain Uterus
441 bp
244 bp
Fig. 7
M2WT
A
1g
●
▲
▲
▲
▲
▲
▲
▲
▲
▲
M2KO
1g
●
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
M3KO
1g
●
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
▲
M2/M3KO
●
50 mM K
-8
-7
-6
-5
-4
-3
1g
10 min
Relative AUC (% to 50 mM K)
Fig. 8
100
80
WT (M2WT, M3WT)
M2KO
M3KO
M2/M3KO
60
40
* *
*
*
* *
20
0
-9
-8
-7
-6
-5
-4
-3
Carbachol concentration (logM)
-2