0022-3565/02/3012-643–650$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 301:643–650, 2002
Vol. 301, No. 2
4598/981652
Printed in U.S.A.
Muscarinic Receptor Knockout Mice: Role of Muscarinic
Acetylcholine Receptors M2, M3, and M4 in CarbamylcholineInduced Gallbladder Contractility
PETER W. STENGEL and MARLENE L. COHEN
Eli Lilly and Company, Neuroscience Research, Lilly Corporate Center, Indianapolis, Indiana
Received October 2, 2001; accepted January 21, 2002
This article is available online at http://jpet.aspetjournals.org
tor knockout mice, with affinities consistent with M3 receptor
interaction. In addition, maximal contraction to carbamylcholine
was reduced in gallbladder from M2 receptor knockout mice and
affinities for AF-DX 116 and pirenzepine in gallbladder from M3
receptor knockout mice were consistent with their affinities at M2
receptors. In M4 receptor knockout mice, contraction to carbamylcholine was dextrally shifted, although the affinities for AF-DX
116 and pirenzepine in gallbladder from M2 or M3 knockout mice
were not similar to their affinities at M4 receptors. The M4 receptor
may serve as an accessory protein necessary for optimal potency
of M2 and M3 receptor-mediated responses. Thus, muscarinic
receptor knockout mice provided direct and unambiguous evidence that M3, and to a lesser extent, M2 receptors are the
predominant muscarinic receptors mediating gallbladder contractility, and M4 receptors appear necessary for optimal potency of
carbamylcholine in gallbladder contraction.
Messenger RNA for M1, M2, M3, and M4 receptors has been
identified in gallbladder (Heinig et al., 1997), raising the
possibility that one or more of these receptors may be responsible for cholinergically mediated contractility in this tissue.
In fact, multiple studies using pharmacological tools have
implicated a role for M3 receptors on gallbladder contractility
in guinea pig (von Schrenck et al., 1993; Takahashi et al.,
1994; Eltze et al., 1997) and cat (Chen et al., 1995). In
addition, M2 receptor activation has been linked to contractility in the cat (Chen et al., 1995) and guinea pig gallbladder
(Oktay et al., 1998), although this receptor may serve as an
inhibitory presynaptic autoreceptor in guinea pig gallbladder
(Parkman et al., 1999) rather than possessing a direct role in
gallbladder smooth muscle contractility (Akici et al., 1999).
The possibility of a prominent role for M3 and a more modest
role for M2 receptors in muscarinic-induced contraction of
gallbladder is consistent with previous observations documenting a similar role for these receptors in carbamylcholine-induced contraction of other smooth muscles such as
mouse trachea, stomach fundus, and urinary bladder (Sten-
gel et al., 2000). In addition to these muscarinic receptors,
the M4 receptor has also been proposed to play an important
role in contractility of the guinea pig gallbladder (Ozkutlu et
al., 1993; Karaalp et al., 1999; Akici et al., 2000). The possibility that M4 receptors may be involved in gallbladder contraction deserves careful consideration because it markedly
contrasts with the lack of M4 receptor involvement in the
contraction of other smooth muscle preparations (Stengel et
al., 2000).
The availability of muscarinic M2, M3, and M4 receptor
knockout mice coupled to the use of pharmacological tools
selective for these receptors has prompted the present study
to evaluate definitively the role of each of these receptors in
gallbladder contractility. Furthermore, unlike the guinea pig
gallbladder that possesses an extensive intramural neuronal
network (Sutherland, 1967; Yoshida and Tsuruta, 1988;
Parkman et al., 1999), gallbladder from the mouse does not
possess such an extensive neuronal network (Yoshida and
Koeda, 1991, 1992). Thus, use of the M2, M3, and M4 muscarinic knockout mice can permit a detailed evaluation of the
ABBREVIATIONS: M1 to M5 receptors, muscarinic acetylcholine receptors; AF-DX 116, 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one.
643
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
ABSTRACT
Muscarinic receptors play a major role in gallbladder function,
although the muscarinic receptor(s) mediating smooth muscle
contractility is unclear. This study compared smooth muscle
contractile responses to carbamylcholine (10⫺7-10⫺3 M) in isolated gallbladder from wild-type and M2, M3, and M4 receptor
knockout mice. Carbamylcholine-induced contraction in gallbladder was associated with tachyphylaxis and the release of a
cyclooxygenase product because indomethacin (10⫺6 M) inhibited carbamylcholine-induced contraction. The M3 receptor was
the major muscarinic receptor involved in contraction because
carbamylcholine-induced contractility was inhibited in gallbladder
from M3 receptor knockout mice. Furthermore, the muscarinic
receptor antagonists 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6one (AF-DX 116) and pirenzepine dextrally shifted contraction to
carbamylcholine in gallbladder from wild-type, M2, and M4 recep-
644
Stengel and Cohen
role of these muscarinic receptors in gallbladder smooth muscle contraction.
Materials and Methods
Results
Carbamylcholine-Induced Contractility in Gallbladder from Wild-Type, M2, M3, and M4 Receptor Knockout Mice. Carbamylcholine produced marked contraction in
gallbladder from wild-type mice with a pEC50 of 5.77 ⫾ 0.06
(n ⫽ 25) (Fig. 1). In fact, the maximum contractile force to
carbamylcholine (10⫺5 M) was approximately 2.5-fold
greater than the response to 67 mM KCl, using a concentration of KCl that produced a near maximal contractile response in smooth muscle.
In the M2 receptor knockout mice, low concentrations of
carbamylcholine (ⱕ3.0 ⫻ 10⫺6 M) produced a contractile
concentration response similar to the response in gallbladder
from wild-type mice. However, as the concentration of carbamylcholine increased (10⫺5-10⫺4 M), the maximal contractile force to carbamylcholine tended to be lower in gallbladder
from M2 receptor knockout mice compared with the response
in wild-type mice, an effect that reached significance only at
3.0 ⫻ 10⫺5 M carbamylcholine (Fig. 1, top).
Carbamylcholine-induced contraction of the mouse gallbladder was most affected in the M3 receptor knockout mice
(Fig. 1, middle), where the maximal response to carbamylcholine was dramatically reduced relative to the maximal
response in gallbladder from wild-type mice. Nevertheless,
even in the M3 receptor knockout mice, a small contraction to
high concentrations of carbamylcholine remained.
In contrast to these changes in the contractile response to
carbamylcholine in gallbladder from the M2 and M3 receptor
knockout mice, the maximal response to carbamylcholine in
gallbladder from M4 receptor knockout mice was similar to
the response in gallbladder from wild-type mice (Fig. 1, bottom). However, the EC50 for carbamylcholine-induced contraction was higher in gallbladder from M4 receptor knockout
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Animals. The generation of M2, M3, and M4 muscarinic receptor
knockout mice has been described previously (Gomeza et al.,
1999a,b; Matsui et al., 2000; Yamada et al., 2001). M2 receptor
knockout mice (129J1/CF-1 hybrids) and M3 and M4 receptor knockout mice (129SvEv/CF-1 hybrids) were obtained from either the
National Institute of Diabetes and Digestive and Kidney Diseases
(Bethesda, MD) or Taconic (Germantown, NY). Wild-type mice of the
same genetic background as M2, M3, and M4 receptor knockout mice
served as controls. 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 (6:00 AM– 6:00 PM
light). Food (Laboratory Rodent Diet 5001; PMI Feeds, St. Louis,
MO) and water were supplied ad libitum. Mice (33–58 g) were killed
by cervical dislocation, and the gallbladder was 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䡠2H2O, and 24.8 mM
NaHCO3. Experimental protocols and procedures were approved by
the Eli Lilly and Company Animal Care and Use Committee.
Smooth Muscle Preparation. The gallbladder body (cut from
the cystic duct to the neck) was prepared for in vitro examination.
One end of the gallbladder was attached with thread to a stationary
glass rod, whereas the other end was tied with thread to the transducer. In some experiments, urinary bladder from wild-type mice
was prepared as previously described (Stengel et al., 2000). Tissues
were placed in organ baths containing 10 ml of Krebs-bicarbonate
buffer (see the above-mentioned description for composition). The
organ bath solution was maintained at 37°C and aerated with a
95:5% mixture of O2/CO2. Gallbladders and urinary bladders were
placed under an initial optimal force of 1.0 and 4.0g, respectively,
and equilibrated for 1 h during which time the tissues were washed
at 15-min intervals. Isometric force in grams was measured with
transducers (Sensotec, Columbus, OH). Tissues 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, M3, or M4 receptor knockout and
wild-type mice. Cumulative contractile concentration-response
curves to carbamylcholine (10⫺7-10⫺4 M) were generated and expressed as a percentage of the 67 mM KCl maximum contraction
determined for each tissue. For experiments comparing responses of
gallbladder from muscarinic receptor knockout and wild-type mice,
tissues from muscarinic receptor knockout and wild-type mice were
used on each day to avoid the possibility of any daily systematic
effect.
In some experiments, gallbladder from wild-type mice was exposed to an initial contractile concentration-response curve to carbamylcholine. Tissues were washed, returned to baseline, and 45
min later, the contractile concentration-response curve to carbamylcholine was repeated. Because tachyphylaxis was observed (see Results), subsequent studies used only a single concentration-response
curve to carbamylcholine per tissue.
In other experiments, tissues were incubated with 3.0 ⫻ 10⫺7 M
neostigmine, 10⫺6 M indomethacin, 10⫺6 M atropine, 3.0 ⫻ 10⫺6 M
AF-DX 116, 10⫺7 or 10⫺6 M pirenzepine, or vehicle for 20 min, and
contractile concentration-response curves to 10⫺7 to 10⫺3 M carbamylcholine were generated. Only one antagonist or vehicle was examined in each tissue.
The equilibrium dissociation constants (KB) for antagonists versus
carbamylcholine were 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.
In some experiments where maximal carbamylcholine-induced
contraction was markedly reduced in the presence of antagonists,
antagonist equilibrium dissociation constants were determined as
described for noncompetitive receptor antagonists according to the
following equation: KB ⫽ [B]/[slope ⫺ 1], where [B] equals the antagonist concentration and slope is determined from a double reciprocal plot of 1/x1 versus 1/x, where x1 and x are the equieffective
concentrations of carbamylcholine in the presence (x1) and absence
(x) of inhibitor (Kenakin, 1993; Cohen et al., 1998). The antagonist
equilibrium dissociation constant was expressed as the negative
logarithm of the KB (i.e., pKB).
Statistical Analyses. Results were expressed as the mean ⫾ S.E.
Agonist concentration-response curves were analyzed by a threeparameter 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 using
SAS (SAS Institute, Cary, NC) on a Deskpro 5133 personal computer
(Compaq Computer Corporation, Houston, TX). Unpaired Student’s
t test was used to compare mean KCl contractile responses and
pEC50 (the negative logarithm of the EC50) values between two
groups. Analyses were run using SigmaStat for Windows (version
2.03; SPSS, Inc., Chicago, IL) on a Compaq personal computer
(Deskpro 5133). Comparisons were considered significant for P values of 0.05 or less.
Drugs. Carbamylcholine chloride, pirenzepine dihydrochloride,
neostigmine bromide, indomethacin, and atropine sulfate were purchased from Sigma-Aldrich (St. Louis, MO). AF-DX 116 was provided
by the Lilly Research Laboratories.
Muscarinic Receptor Knockout Mice: Gallbladder Contraction
645
Fig. 1. Effect of carbamylcholine to contract gallbladder from wild-type
mice and M2 (top), M3 (middle), and M4 (bottom) receptor knockout mice.
Values are mean ⫾ S.E.M. of the number of tissues in parentheses.
Asterisks denote a significant difference between the contraction observed in gallbladder from muscarinic receptor knockout and wild-type
mice. Absence of error bars indicates that the magnitude of error was less
than the symbol size.
than from wild-type mice. Thus, removal of the M4 receptor
did not affect maximal contraction to carbamylcholine, but
did reduce the sensitivity of the gallbladder to carbamylcholine.
Carbamylcholine-Induced Gallbladder Contraction
after Repeated Administration. Before initiating studies
with muscarinic receptor knockout mice and pharmacological
antagonists, we examined the ability of carbamylcholine to
Fig. 2. Contractile concentration-response curves in gallbladder from
wild-type mice to exposures to carbamylcholine separated by 45 min.
Values are mean ⫾ S.E.M. of the number of tissues in parentheses.
Asterisks denote a significant difference between the values of the second
generated curve and the values obtained on initial exposure to carbamylcholine. Absence of error bars indicates that the magnitude of error was
less than the symbol size.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
produce a response in the gallbladder from wild-type mice
after previous exposure to a contractile concentration response to carbamylcholine. Surprisingly, carbamylcholineinduced contraction in the gallbladder was markedly reduced
after previous exposure to carbamylcholine (Fig. 2). Both the
potency of carbamylcholine and its maximal response were
reduced in each tissue after previous exposure to carbamylcholine (10⫺6-10⫺4 M), indicating the rapid development of
tachyphylaxis. For this reason, only a single carbamylcholine
concentration-response curve was generated in all subsequent tissues studied.
Effect of Neostigmine on Carbamylcholine-Induced
Contraction in Gallbladder from Wild-Type Mice. Because tachyphylaxis to the contractile response to carbamylcholine occurred, we considered the possibility that carbamylcholine might, in part, activate presynaptic cholinergic
receptors to release acetylcholine, which might contribute to
the contractile response. We reasoned that if carbamylcholine were exerting a modulating effect on acetylcholine release from presynaptic cholinergic nerves then neostigmine,
an inhibitor of acetylcholinesterase, should alter carbamylcholine-induced contraction in gallbladder from wild-type
mice. However, neostigmine (3.0 ⫻ 10⫺7 M) pretreatment did
not significantly affect carbamylcholine-induced contraction
in gallbladder from wild-type mice (Fig. 3), suggesting that
carbamylcholine did not exert an effect on neuronal acetylcholine release.
Effect of Indomethacin on Carbamylcholine-Induced Contraction in Gallbladder from Wild-Type
Mice. In an effort to understand further the tachyphylaxis to
the contractile response to carbamylcholine, we considered
the possibility that carbamylcholine was activating the release of an endogenous cyclooxygenase product that could be
depleted by excessive stimulation. For this reason, we evaluated the effect of indomethacin (10⫺6 M), a cyclooxygenase
646
Stengel and Cohen
inhibitor, on the contractile response to carbamylcholine in
gallbladder from wild-type mice. Indeed, indomethacin (10⫺6
M) produced a dramatic inhibition of the contractile response
to carbamylcholine (Fig. 4, top).
To determine whether the effect of indomethacin on carbamylcholine-induced contractility was unique to the gallbladder, indomethacin was also examined for its effect in
mouse urinary bladder, a tissue possessing a cholinergic
receptor profile similar to the gallbladder (Stengel et al.,
2002). Indomethacin (10⫺6 M) did not alter carbamylcholineinduced contraction in mouse urinary bladder (Fig. 4, bottom). These results indicate that 1) indomethacin was not a
cholinergic receptor antagonist at 10⫺6 M and 2) the effect of
indomethacin was restricted to the gallbladder and did not
generalize to all M3 receptor-mediated contractile responses.
These data were consistent with the contention that carbamylcholine was activating release of a cyclooxygenase product
that mediated contraction in the gallbladder.
Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from
Wild-Type Mice. AF-DX 116 is a muscarinic receptor antagonist relatively selective for the M2 receptor, in contrast to
pirenzepine, which possesses approximately 3-fold lower affinity than AF-DX 116 at the M2 receptor and has highest
affinity at M1 and M4 receptors (Table 1). AF-DX 116 (3 ⫻
10⫺6 M) dextrally shifted the contractile response to carbamylcholine in gallbladder from wild-type mice (Fig. 5), resulting in a pKB value of 6.03 ⫾ 0.16 (n ⫽ 4) consistent with the
affinity of AF-DX 116 for M3 receptors (Table 1). Pirenzepine
(10⫺7 M) modestly inhibited the contractile response to carbamylcholine (Fig. 5) with a pKB value of 7.05 ⫾ 0.16 (n ⫽ 5),
also consistent with the affinity of pirenzepine at M3 receptors (Table 1).
Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M2
Receptor Knockout Mice. As in wild-type mice, 3 ⫻ 10⫺6
M AF-DX 116 produced a marked dextral shift in the con-
Fig. 4. Contractile concentration-response curves to carbamylcholine after either vehicle or 10⫺6 M indomethacin in gallbladder (top) and urinary bladder (bottom) from wild-type mice. Values are mean ⫾ S.E.M. of
the number of tissues in parentheses. Asterisks indicate those values that
significantly differ from vehicle. Absence of error bars indicates that the
magnitude of error was less than the symbol size.
tractile response to carbamylcholine in gallbladder from M2
receptor knockout mice (Fig. 6) with a resulting pKB value of
5.88 ⫾ 0.08 (n ⫽ 4), consistent again with the affinity of
AF-DX 116 for M3 receptors. Furthermore, 10⫺7 M pirenzepine produced a small dextral shift of the contractile response to carbamylcholine in gallbladder from the M2 receptor knockout mice (Fig. 6) with a resultant pKB value of
6.96 ⫾ 0.19 (n ⫽ 4), again consistent with the affinity of
pirenzepine for M3 receptors (Table 1).
Effect of Atropine, AF-DX 116, and Pirenzepine on
Carbamylcholine-Induced Contraction in Gallbladder
from M3 Receptor Knockout Mice. Because the residual
contractile response to carbamylcholine in M3 receptor
knockout mice was small, we first established whether this
effect was mediated by activation of muscarinic receptors.
For this reason, we evaluated the effect of the nonselective
muscarinic receptor antagonist atropine (10⫺6 M). Atropine
(10⫺6 M) abolished the contractile response to carbamylcholine in gallbladder from M3 receptor knockout mice (Fig. 7).
In addition, 3.0 ⫻ 10⫺6 M AF-DX 116 dextrally shifted the
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 3. Contractile concentration-response curves to carbamylcholine after either 3.0 ⫻ 10⫺7 M vehicle or neostigmine in gallbladder from
wild-type mice. Values are mean ⫾ S.E.M. of the number of tissues in
parentheses. Absence of error bars indicates that the magnitude of error
was less than the symbol size.
Muscarinic Receptor Knockout Mice: Gallbladder Contraction
647
TABLE 1
Radioligand binding profile of AF-DX 116 and pirenzipine at cloned human muscarinic receptor subtypes
Muscarinic Receptor Subtype
Ligand
Reference
M1
M2
M3
M4
M5
pKi (M)
5.89
6.37
6.24
5.72
N.A.
6.06
6.73
7.26
7.27
6.91
7.17
7.07
6.08
6.16
6.10
5.67
N.A.
6.00
N.D.
6.79
6.96
6.52
N.A.
6.76
5.55
5.52
5.29
N.A.
N.A.
5.45
Buckley et al. (1989)
Dörje et al. (1991)
Esqueda et al. (1996)
Loudon et al. (1997)
Kovacs et al. (1998)
7.80
8.20
7.77
8.04
7.80
N.A.
8.15
7.96
6.04
6.65
5.96
6.28
6.69
6.43
6.52
6.37
6.74
6.96
6.59
6.80
7.12
N.A.
7.12
6.89
N.A.
7.43
7.23
6.98
7.66
N.A.
7.77
7.41
N.A.
7.05
6.55
6.90
N.A.
N.A.
7.18
6.92
Buckley et al. (1989)
Dörje et al. (1991)
Esqueda et al. (1996)
Hegde et al. (1997)
Loudon et al. (1997)
Kovacs et al. (1998)
Moriya et al. (1999)
N.A., data not available; NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate; MQNB, N-methyl-3-QNB.
a
Information from Stengel et al. (2000).
Fig. 5. Effect of 3.0 ⫻ 10⫺6 M AF-DX 116 and 10⫺7 M pirenzepine on the
contractile response to carbamylcholine in gallbladder from wild-type
mice. Values are mean ⫾ S.E.M. of the number of tissues in parentheses.
Absence of error bars indicates that the magnitude of error was less than
the symbol size.
Fig. 6. Effect of 3.0 ⫻ 10⫺6 M AF-DX 116 and 10⫺7 M pirenzepine on
contractile response to carbamylcholine in gallbladder from M2 receptor
knockout mice Values are mean ⫾ S.E.M. of the number of tissues in
parentheses. Absence of error bars indicates that the magnitude of error
was less than the symbol size.
contractile response to carbamylcholine in gallbladder from
M3 receptor knockout mice, resulting in a pKB value of 6.83 ⫾
0.10 (n ⫽ 6), a value consistent with the affinity of AF-DX 116
for M2 receptors (Table 1). Furthermore, 10⫺7 M pirenzepine
did not alter the contraction to carbamylcholine in gallbladder from M3 receptor knockout mice (Fig. 7). However, 10⫺6
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
AF-DX 116a
[3H]-NMS
[3H]-NMS
[3H]-NMS
[3H]-QNB
[3H]-MQNB
Mean
Pirenzepine
[3H]-NMS
[3H]-NMS
[3H]-NMS
[3H]-NMS
[3H]-QNB
[3H]-MQNB
[3H]-NMS
Mean
648
Stengel and Cohen
(Fig. 8), with a pKB value of 5.83 ⫾ 0.10 (n ⫽ 5), consistent
again with the affinity of AF-DX 116 for M3 receptors (Table
1). Pirenzepine (10⫺7 M) did not alter the contractile response to carbamylcholine in gallbladder from the M4 receptor knockout mice (Fig. 8), consistent with the contention
that M3 receptors are mediating the contractile response to
carbamylcholine in the gallbladder from M4 receptor knockout mice.
Discussion
Fig. 7. Effect of 10⫺6 M atropine, 3.0 ⫻ 10⫺6 M AF-DX 116, and 10⫺7 and
10⫺6 M pirenzepine on the contractile response to carbamylcholine in
gallbladder from the M3 receptor knockout mice. Values are mean ⫾
S.E.M. of the number of tissues in parentheses. Absence of error bars
indicates that the magnitude of error was less than the symbol size.
M pirenzepine produced a dramatic inhibition of the contractile response to carbamylcholine, inhibiting both the EC50
and maximal response to carbamylcholine. Calculation of a
noncompetitive antagonist dissociation constant for pirenzepine resulted in a pKB value of 6.58 ⫾ 0.13 (n ⫽ 4), consistent with affinity of pirenzepine at M2 receptors (Table 1).
Effect of AF-DX 116 and Pirenzepine on Carbamylcholine-Induced Contraction in Gallbladder from M4
Receptor Knockout Mice. AF-DX 116 (3 ⫻ 10⫺6 M) produced a dextral shift in the contractile response to carbamylcholine in gallbladder from the M4 receptor knockout mice
Fig. 8. Effect of 3.0 ⫻ 10⫺6 M AF-DX 116 and 10⫺7 M pirenzepine on the
contractile response to carbamylcholine in gallbladder from M4 receptor
knockout mice. Values are mean ⫾ S.E.M. of the number of tissues in
parentheses. Absence of error bars indicates that the magnitude of error
was less than the symbol size.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
The receptors responsible for muscarinic-induced contraction of gallbladder have been the subject of intense experimentation and debate. M1, M2, M3, and M4 muscarinic receptors have been implicated in the contractile response to
muscarinic agonists in gallbladder (Parkman et al., 1999). In
addition, M1, M2, M3, and M4 receptor mRNA exists in human gallbladder, again raising the possibility that each of
these receptors may be involved in the cholinergically mediated contractile response of gallbladder (Heinig et al., 1997).
Previous studies examining the role of multiple muscarinic
receptors in the contractile response of gallbladder smooth
muscle relied heavily upon the use of relatively nonselective
pharmacological tools. With the development of muscarinic
Muscarinic Receptor Knockout Mice: Gallbladder Contraction
lease and formation of cyclooxygenase contractile products.
In addition, carbamylcholine stimulated arachidonic acid
and eicosanoid release from brain synaptosomes (Strosznajder and Samochocki, 1992). Consistent with these reports,
10⫺6 M indomethacin markedly inhibited the contractile response to carbamylcholine in the mouse gallbladder, an effect
that did not occur in urinary bladder, a tissue with a muscarinic receptor profile (Stengel et al., 2002) similar to the
gallbladder. Thus, unlike other M3 receptor-mediated responses, the cholinergic signaling mechanism in mouse gallbladder appears to involve release of a cyclooxygenase product for contraction to carbamylcholine.
A marked inhibition of the contractile response to carbamylcholine occurred in gallbladder from the M3 receptor knockout mice, clearly indicating that M3 receptors play a predominant role in the contraction to carbamylcholine. This
conclusion was further supported by pharmacological observations demonstrating that AF-DX 116 inhibited carbamylcholine-induced contraction in the wild-type mouse with an
antagonist dissociation constant similar to the antagonist
dissociation constant reported for AF-DX 116 at M3 receptors
(Table 1). Furthermore, the antagonist dissociation constant
for AF-DX 116 was similar in gallbladder from wild-type
(pKB ⫽ 6.03), M2 (pKB ⫽ 5.88), and M4 (pKB ⫽ 5.87) knockout
mice, again consistent with M3 receptors mediating the contractile response to carbamylcholine in gallbladder from M2
and M4 receptor knockout mice. Also, 10⫺7 M pirenzepine
produced only a modest inhibition of the contractile response
to carbamylcholine in gallbladder from the wild-type mice,
ruling out a role for M1 receptors. The antagonist dissociation
constant (pKB ⫽ 7.05 ⫾ 0.46) for pirenzepine in gallbladder
was similar to the antagonist dissociation constant for pirenzepine at M3 rather than M1, M2, or M4 receptors (Table 1).
Thus, M3 receptors are the predominant receptors mediating
the contractile response to carbamylcholine in mouse gallbladder.
However, M2 receptors have been demonstrated in gallbladder (Oktay et al., 1998) and our data along with others
(Chen et al., 1995) suggest that M2 receptors also play a
modest role in carbamylcholine-induced contraction of mouse
gallbladder. This conclusion is based on 1) a modest inhibition of the maximum contractile response to carbamylcholine
in M2 receptor knockout mice compared with wild-type mice
(Fig. 1); 2) a small residual contraction to carbamylcholine in
tissues from M3 receptor knockout mice; and 3) the demonstration that AF-DX 116 and pirenzepine both inhibited the
residual contractile response to carbamylcholine in gallbladder from M3 receptor knockout mice with affinities similar to
that for M2 receptors (Table 1). The future availability of M2/3
double receptor knockout mice will be important to confirm
the lack of involvement of other cholinergic receptors in carbamylcholine-induced gallbladder contraction.
The gallbladder was a uniquely interesting smooth muscle
based on the proposed presence of M4 receptors and their role
in mediating smooth muscle contractility (Ozkutlu et al.,
1993; Karaalp et al., 1999; Akici et al., 2000). Carbamylcholine-induced contraction in gallbladder from M4 receptor
knockout mice was dextrally shifted compared with the response in gallbladder from wild-type mice, suggesting that
the M4 receptor participated in the contraction to carbamylcholine. However, antagonism of carbamylcholine-induced
contraction by AF-DX 116 or pirenzepine was not consistent
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
knockout mice, it became possible to examine definitively the
role of M2, M3, and M4 receptors in carbamylcholine-induced
gallbladder contraction.
Carbamylcholine produced a contractile concentration-response curve in gallbladder from wild-type mice with a pEC50
(5.77 ⫾ 0.06, n ⫽ 25) close to that described for carbamylcholine-induced contraction in guinea pig gallbladder (von
Schrenck et al., 1993; Takahashi et al., 1994; Eltze et al.,
1997). However, the contractile potency of carbamylcholine
was approximately 5- to 10-fold lower in gallbladder compared with other smooth muscle preparations (Stengel et al.,
2000). Furthermore, like the stomach fundus (Stengel et al.,
2000), the maximal gallbladder response to carbamylcholine
was 3-fold greater than the maximal response to 67 mM KCl.
Maximal carbamylcholine contraction in gallbladder was
greater than the maximal contractile response in trachea or
urinary bladder (Stengel et al., 2000). These differences in
potency and maximal contractility might reflect a lack of
receptor reserve in this tissue or more likely a difference in
signal transduction mechanisms.
The possibility of a unique signaling mechanism for carbamylcholine in gallbladder is consistent with the observation that contraction was associated with tachyphylaxis upon
a second exposure to carbamylcholine, an effect that did not
occur in other smooth muscle preparations (Stengel et al.,
2000). Several possible explanations could account for the
tachyphylaxis observed in mouse gallbladder. First, carbamylcholine could be increasing presynaptic neuronal release of
acetylcholine, a possibility supported by the fact that certain
peptides such as cholecystokinin can release acetylcholine in
gallbladder (Garrigues et al., 1992), that nicotinic receptor
activation can alter acetylcholine release in gallbladder
(Parkman et al., 1998), and that presynaptic M1 inhibitory
autoreceptors and M2 excitatory autoreceptors have been
reported in guinea pig gallbladder (Parkman et al., 1999).
However, neostigmine, an inhibitor of acetylcholinesterase,
in concentrations that dramatically potentiated smooth muscle responses to acetylcholine (unpublished observations), did
not affect carbamylcholine-induced gallbladder contraction.
Thus, carbamylcholine did not exert a functional presynaptic
effect to alter acetylcholine release in mouse gallbladder.
Second, carbamylcholine could be releasing inhibitory
transmitters that may impact subsequent contraction to
cause tachyphylaxis. Nicotinic receptor activation can both
activate (Parkman et al., 1998) and inhibit gallbladder contractility (Pozo et al., 1989; Parkman et al., 1998) and nicotinic receptor-induced smooth muscle responses have been
associated with tachyphylaxis (Rand and Li, 1992). The fact
that neostigmine did not alter carbamylcholine-induced contraction suggests that carbamylcholine was not interacting
with nicotinic receptors to alter acetylcholine release, but
does not rule out the possibility that carbamylcholine could
be interacting directly with nicotinic receptors to alter release of other neurotransmitters.
Last, tachyphylaxis to carbamylcholine may be occurring
because carbamylcholine released other contractile mediators in the mouse gallbladder, which become depleted. Although gallbladder M3 receptors have been linked to phosphoinositide hydrolysis and adenylate cyclase inhibition
(Takahashi et al., 1994), M1, M3, and M5 receptor activation
has also been linked to stimulation of phospholipase A2
(Felder, 1995), which could stimulate arachidonic acid re-
649
650
Stengel and Cohen
Acknowledgments
We are grateful for the expert administrative assistance of
Priscilla Kirsch. We also acknowledge Drs. Jesus Gomeza, Masahisa
Yamada, and Jürgen Wess for the initial generation of the muscarinic receptor knockout mice and global collaboration in understanding muscarinic receptor responses.
References
Akici A, Karaalp A, Iskender E, Christopoulos A, El-Fakahany EE, and Oktay S
(2000) Further evidence for the heterogeneity of functional muscarinic receptors in
guinea-pig gallbladder. Eur J Pharmacol 388:115–123.
Akici A, Karaalp A, Iskender E, El-Fakahany EE, and Oktay S (1999) Muscarinic M2
receptors are not primarily involved in the contraction of guinea-pig gallbladder
smooth muscle. Pharmacol Res 40:443– 449.
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.
Chen Q, Yu P, De Petris G, Biancani P, and Behar J (1995) Distinct muscarinic
receptors and signal transduction pathways in gallbladder muscle. J Pharmacol
Exp Ther 273:650 – 655.
Cohen ML, Bloomquist W, Calligaro D, and Swanson S (1998) Comparative 5-HT4
receptor antagonist activity of LY353433 and its active hydroxylated metabolites.
Drug Dev Res 43:193–199.
Conklin BR, Brann MR, Buckley NJ, Ma AL, Bonner TI, and Axelrod J (1988)
Stimulation of arachidonic acid release and inhibition of mitogenesis by cloned
genes for muscainic receptor subtypes stably expressed in A9 L cells. Proc Natl
Acad Sci USA 85:8698 – 8702.
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.
Dörje F, Wess J, Lambrecht G, Tacke R, Mutschler E, and Brann MR (1991)
Antagonist binding profiles of five cloned human muscarinic receptor subtypes.
J Pharmacol Exp Ther 256:727–733.
Eltze M, König H, Ullrich B, and Grebe T (1997) Contraction of guinea-pig gallbladder: muscarinic M3 or M4 receptors? Eur J Pharmacol 332:77– 87.
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.
Felder CC (1995) Muscarinic acetylcholine receptors: signal transduction through
multiple effectors. FASEB J 9:619 – 625.
Furchgott RF (1972) The classification of adrenoceptors (adrenergic receptors). An
evaluation from the standpoint of receptor theory, in Handbook of Experimental
Pharmacology, Catecholamines (Blaschko H and Muscholl E eds) vol 33, pp 285–
335, Springer Verlag, Berlin.
Garrigues V, Ponce J, Cano C, Sopena R, Hoyos M, Del Val A, and Berenguer J (1992)
Effect of selective and nonselective muscarinic blockade on cholecystokinininduced gallbladder emptying in man. Dig Dis Sci 37:101–104.
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.
Hegde SS, Choppin A, Bonhaus D, Briaud S, Loeb M, Moy TM, Loury D, and Eglen
RM (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.
Heinig T, von Schrenck T, De Weerth A, Kolsch K, Schulz M, Izbicki JR, and Greten
H (1997) Characterization of gallbladder muscarinic receptor subtypes: signal
transduction and RT-PCR studies. Gastroenterology 112:A746.
Karaalp A, Akici A, Akbulut H, Ulusoy NB, and Oktay S (1999) Distinct functional
muscarinic receptors in guinea-pig gallbladder, ileum and atria. Pharmacol Res
39:389 –395.
Kenakin T (1993) Allotopic, noncompetitive and irreversible antagonism, in Pharmacologic Analysis of Drug Receptor Interaction, pp 323–343, Raven Press, New
York.
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.
Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, Takahashi S,
and Taketo MM (2000) Multiple functional defects in peripheral autonomic organs
in mice lacking muscarinic acetylcholine receptor gene for the M3 receptor subtype. Proc Natl Acad Sci USA 97:9579 –9584.
Moriya H, Takagi Y, Nakanishi T, Hayashi M, Tani T, and Hirotsu I (1999) Affinity
profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular
gland. Life Sci 64:2351–2358.
Oktay S, Cabadak H, Iskender E, Gören Z, Caliskan E, Orun O, Aslan N, Karaalp A,
Tolun A, Ulusoy NB, et al. (1998) Evidence for the presence of muscarinic M2 and
M4 receptors in guinea-pig gallbladder smooth muscle. J Autonomic Pharmacol
18:195–204.
Ozkutlu U, Alican I, Karahan F, Onat F, Yegen BC, Ulusoy NB, and Oktay S (1993)
Are m-cholinoreceptors of guinea pig gallbladder smooth muscle of m4 subtype?
Pharmacology 46:308 –314.
Parkman HP, Pagano AP, and Ryan JP (1998) Investigation of endogenous neurotransmitters of guinea pig gallbladder using nicotinic agonist stimulation. Dig Dis
Sci 43:2237–2243.
Parkman HP, Pagano AP, and Ryan JP (1999) Subtypes of muscarinic receptors
regulating gallbladder cholinergic contractions. Am J Physiol 276:G1243–G1250.
Pozo MJ, Salido GM, and Madrid JA (1989) Cholecystokinin induced gallbladder
contraction is influenced by nicotinic and muscarinic receptors. Arch Int Physiol
Biochim 97:403– 408.
Rand MJ and Li CG (1992) Activation of noradrenergic and nitrergic mechanism in
the rat anococcygeus muscle by nicotine. Clin Exp Pharmacol Physiol 19:103–111.
Stengel PW, Gomeza J, Wess J, and Cohen ML (2000) M2 and M4 receptor knockout
mice: muscarinic receptor function in cardiac and smooth muscle in vitro. J Pharmacol Exp Ther 292:877– 885.
Stengel PW, Yamada M, Wess J, and Cohen ML (2002) M3-receptor knockout mice:
muscarinic receptor function in atria, stomach fundus, urinary bladder and trachea. Am J Physiol Regul Integr Comp Physiol, in press.
Strosznajder J and Samochocki M (1992) Carbachol-stimulated release of arachidonic acid and eicosanoids from brain cortex synaptoneurosome lipids of adult and
aged rats. Adv Exp Med Biol 318:251–258.
Sutherland SD (1967) The neurons of the gallbladder and gut. J Anat 101:701–709.
Takahashi T, Kurosawa S, and Owyang C (1994) Regulation of PI hydrolysis and
cAMP formation by muscarinic M3 receptor in guinea pig gallbladder. Am J
Physiol 267:G523–G528.
von Schrenck T, Sievers J, Mirau S, Raedler A, and Greten H (1993) Characterization of muscarinic receptors on guinea pig gallbladder smooth muscle. Gastroenterology 105:1341–1349.
Wess J (2000) Physiological roles of G-protein-coupled receptor kinases revealed by
gene-targeting technology. Trends Pharmacol Sci 21:364 –367.
Yamada M, Miyakawa T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa
M, Chou CJ, Xia B, Crawley JN, et al. (2001) Mice lacking the M3 muscarinic
acetylcholine receptor are hypophagic and lean. Nature (Lond) 410:207–212.
Yoshida M and Koeda T (1991) Studies on the intramural nerves of the hamster and
mouse gallbladders. J Smooth Muscle Res 27:23–34.
Yoshida M and Koeda T (1992) Studies on the electrical stimulation-induced contractile responses of hamster and mouse gallbladders. J Smooth Muscle Res
28:111–120.
Yoshida M and Tsuruta Y (1988) Observation on the distribution of ganglia in the
ganglionated plexus of guinea-pig gallbladder. Jpn J Smooth Muscle Res 24:113–
125.
Address correspondence to: Dr. Peter W. Stengel, Eli Lilly and Company,
Lilly Research Laboratories, Neuroscience Research, Lilly Corporate Center,
Indianapolis, IN 46285. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
with an interaction with M4 receptors. It is possible that an
intact M4 receptor is required for optimal functioning of M2
and M3 receptor-mediated responses to carbamylcholine as
proposed for isolated atria (Stengel et al., 2000). We speculate that activation of the M4 receptor may be permissive to
the intracellular events necessary for optimal efficacy of M2
and/or M3 receptor activation. For example, activation of
phospholipases and protein kinases (Chen et al., 1995) along
with activation of G protein-coupled receptor kinases (Wess,
2000) has been linked to M2 and M3 receptor activation and
their possible desensitization. We speculate that M4 receptor
activation may synergize with these G protein-coupled transduction events. Further studies will be required to understand more fully the precise role of M4 receptors in permitting optimal expression of functional responsiveness to
muscarinic responses.
In conclusion, these data using gallbladder from M2, M3,
and M4 receptor knockout mice have unequivocally demonstrated that both M2 and M3 receptors are involved in carbamylcholine-induced contractility of gallbladder smooth
muscle. In addition, gallbladder contractility to carbamylcholine is dependent for optimal efficacy on the presence of the
M4 receptor. Last, carbamylcholine-induced gallbladder contraction is associated with the release of a cyclooxygenase
product that mediates gallbladder contractility consistent
with previous observations that M3 receptors can couple to
phospholipase A2 activation (Conklin et al., 1988). Because
cholinergic innervation and muscarinic receptor activation
regulate gallbladder contractility, these studies may provide
information useful to our understanding of the postprandial
responses that regulate biliary excretion and dynamics.