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Characterization of muscarinic receptors on guinea pig gallbladder smooth muscle
Characterization of Muscarinic Receptors on Guinea Pig Gallbladder Smooth Muscle TAMMO
VON SCHRENCK,
J6RG
SIEVERS,
SILVIA
MIRAU,
ANDREAS
RAEDLER,
and HEINER
GRETEN
Department of Medicine, University Hospital Eppendorf, Hamburg, Germany
Background: Cholinergic agonists are of major importance for the regulation of gallbladder motility. However, the gallbladder muscarinic receptors have not been localized or characterized directly using radioligands, and it has not been clearly established which subtype of muscarinic receptor mediates contraction. The aim of the present study was to characterize the gallbladder muscarinic receptors. Methods: Binding studies to guinea pig gallbladder sections were performed using l-[N-methyl-3H] scopolamine methyl chloride. Carbachol-induced contraction was measured using muscle strips. Results: Binding of l-[NmethyL3H] scopolamine methyl chloride was reversible, dependent on time, temperature, and pH. Autoradiography showed binding only over the smooth muscle. Binding and carbachol-induced contractions were inhibited by muscarinic receptor antagonists with the following potencies: atropine > N-methylscopolamine > silahexocyclium-methylsulfate > AF-DX 384 I(+)-51 1-dlhydro-l l-([(2-{2-[(dipropylamino)methyl]- 1-piperidinyl}ethyl)amino]carbonyi}-6H- pyrido (2,3b) (1,4)-benzodiazepine-6-one] > hexahydro-siladifenidol hydrochloride > AF-DX 116 [(+)- 1 l-( { 2[(diethylamino)methyI]- 1-piperidinyl}-acetyl)-5,11 -dihydro-6H-pyrido (2,3b)( 1,4)benzodiazepine-6-one] > pirenzepine. Carbachol inhibited binding to gallbladder sections over the same range of concentrations that caused contractions. The concentration-contraction curves for carbachoi were not altered by tetrodotoxin. Conc/usions: Gallbladder smooth muscle cells possess muscarinic receptors of the M3 type. These receptors mediate carbachol-induced contraction.
have
been
smooth
muscle
been studied
by their
ceptor
agonists
portance
allbladder
contraction
conditions,
an important
is,
under
factor
on
affinities
agonists
fect of CCK” indicating
the stimulation
muscarinic
receptors
or localized
subtype
in the guinea
muscarinic
of muscarinic
directly using
in
However, the musca-
radioligands.
receptor receptors
by
antagonists is mediating
study was therefore
directly
pig gallbladder
and by measurements subtype
the gallbladder, ceptors
contraction,
are involved
of the gallbladder.
tify and characterize
ceptor
also the ef-
it has not clearly been established
The aim of the present
ine which
the
contrac-
contraction.
in the gallbladder
the use of selective contraction
about
the effect of
species
receptors
of gallbladder
And furthermore, which
inhibited
and in certain
no studies characterized rinic
is known
and of SK” on gallbladder
that
the im-
gallbladder
atropine
re-
for the regulation
mediating
studies,
tachykinin
receptors.‘TiO Despite is2,16 little
receptors
tion. In several
have
pig and are charac-
transmitters
motility,
gallbladder
for tachykinins
for different
as NK-2
of gallbladder
cholinergic
single
in the guinea
of cholinergic
cholinergic
isolated
cells. l5 Receptors
in vitro
terized
antagonists
the muscarinic muscle
of contraction of muscarinic
the abilities
in the gallbladder
receptors
by binding
studies
in vitro. To examreceptor
of various
to interact
to iden-
is present
in
muscarinic
re-
with the muscarinic
re-
were tested.
Materials and Methods obtained
G
identified
Male Pirbright white guinea pigs (250-300 g) were from the Animal Section, University Hospital
physiological
in regulating
bile
flow in the digestive period’p2 and is considered to be necessary to prevent stasis of lithogenic bile in the gallbladder.3-5 Cholinergic ago nists, cholecystokinin (CCK)related peptides and tachykinins cause gallbladder contraction in various species in vitro and in viva.“” Receptors for CCK have been characterized in vitro also by radioligand studies in gallbladders from various species such as guinea pig, human, or bovine gallbladders,11-i4 and distinct receptors for gastrin and CCK
Abbreviations used in this paper: AF-DX 116, (+)-11-({2-[(diethylamino)methyl]-l-piperldinyl}-acetyl)-5,ll-dihydro-6H-pyrldo (2,3-b)( 1,4)benzodiazepine-6-one; AF-DX 384, (lt)-5,l l-dihydro1 l-{[(2-{2-[(dlpropylamino)methyl]-l-piperidlnyl}ethyl)amino]-carbonyl}-6H-pyrido(2,3-b)(l,4)benzodiazepine-6-one; BSA, bovine serum albumin; EC,, 50% effective concentration; EGTA, ethylene glycol tetraacetic acid; [3H]NMS, 1 -[N-methyL3H]scopolamine methyl chloride; HHSiD, hexahydro-sila-difenldol-hydrochloride; IC,, 50% inhibitory concentration; PTX, pertussis toxin; rCCiHP, rat calcitonin gene-related peptide; SIHC, silahexocyclium-methylsulfate. 0 1993 by the American Gastroenterologlcal Association 0016-5085/93/$3.00
1342
VON
SCHRENCK
ET AL.
Eppendorf, Hamburg, Germany. l-[N-methyl-3H]Scopolamine methyl chloride a3H]NMS) (sp act, 74 Ci/mmol) was obtained from Amersham Buchler (Braunschweig, Germany); atropine, tetrodotoxin, Tris, ethyleneglycol-bis(B-aminoethyl ether)Nfl,N’fl’-tetraacetic acid (EGTA), bacitracin, pirenzepine-dihydrochloride (pirenzepine), carbamylcholine chloride (carbachol), and unlabeled hJmethyl-scopolamine methyl chloride (NMS) were from Sigma Chemical Co. (Germany); bovine serum albumin fraction V (BSA), leupeptin hydrogen sulfate, and chymostatin were from Boehringer Mannheim (Mannheim, Germany). All other chemicals were obtained from Merck (Darmstadt, Germany). Substance P (SP), the sulfated octapeptide of cholecystokinin (26-33) (CCK-8), rat calcitonin gene-related peptide (rCGRP), gastrin, gastrin-releasing peptide (GRP), motilin, vasoactive intestinal polypeptide (VIP) were from Bachem Chemicals (Switzerland). Hexahydrosiladifenidol-hydrochloride (HHSiD) and silahexocyclium-methylsulfate (SiHC) were a gift from Professor Lambrecht, Department of Pharmacology, Johann-Wolfgang-Goethe-UniversitEt (Frankfurt-am-Main, Germany); (+)-ll-({2-[d’ ie th y 1amino)-methyl]-l-pipridinyl}-acetyl)5,1 I-dihydro-6H-pyrido(2,3-b)( 1,4) benzodiazepine-6-one (AF-DX 116) and (rt)-5,11-dihydro-1 l-{ [(2-{ 2-[(dipropylamino)methyl]-l-piperidinyl}-ethyl)amino]carbony1}bH-pyrido(2,3-b)-(l,4)benzodiazepine-6-one (AF-DX 384) were kind gifts from Dr. Karl Thomae, G.m.b.H. (Biberach, Germany).
Binding Studies Binding of [3H]NMS to tissue sections of the gallbladder was studied using the methods described previBriefly, guinea pigs were killed, and the gallbladously. 11*‘s,19 der was rapidly removed. Tissues were frozen at -7O’C. Tissue sections (15 pm) were cut on a cryostat microtome (Leica Instruments, Heidelberg, Germany) and mounted onto gelatine coated glass slides. Tissue sections were first incubated with 50 mmol/L Tris buffer containing 0.5% BSA. Subsequently the tissue sections were incubated with 5 nmol/L [3H]NMS in 50 mmol/L Tris buffer containing 0.025% bacitracin (wt/vol), 4 pg/mL leupeptin (wt/vol), 2 pg/rnL chymostatin (wt/vol), 130 mmol/L NaCl, 7.7 mmol/L KCl, 5 mmol/L MgCI, and 1 mmol/L EGTA at pH 7.4. Because increasing the pH of the incubation buffer above 7.4 or decreasing the pH below 7.4 decreased binding (data not shown), all experiments were performed at pH 7.4. The degree of nonsaturable binding of [3H]NMS was determined by incubating alternate slides with adjacent tissue sections with 5 nmol/‘L [3H]NMS alone or with 5 nmol/L [3H]NMS plus 1 l.tmol/L atropine. After the incubation, the tissue sections were washed in 50 mmol/L Tris containing 0.5% albumin at 4°C; subsequently, the tissue sections were wiped off the slides, transferred to a counting vial, and counted for bound radioactivity in a beta scintillation counter. Unless specified otherwise, the data in this paper
GASTROENTEROLOGY
Vol. 105,
represent saturable binding of [3H]NMS, nonsaturable ing was always <5% of total binding.
Autoradiography
No. 5
bind-
Studies
The tissue sections were preincubated, incubated, and washed as described above for the binding studies. After the washing the tissue sections were dried rapidly with a stream of cold air and dried completely in a dessicator for 24 hours. Subsequently the tissue sections were exposed to 3HUltrofilm in radiograph cassettes. Binding sites were localized by superimposing the developed film after exposure for 4 weeks with the stained histological section. Measurement of contraction of gallbladder muscle strips. Contraction was determined as previously described for the gallbladder. ‘I Guinea pigs we r e killed, and their gallbladders were carefully removed. Longitudinal muscle strips (0.2 cm X 1.0 cm) were prepared and attached in an organ bath using surgical silk. The muscle strips were placed in standard incubation solution containing 118 mmol/L NaCl, 25 mmol/L NaHCO,, 4.7 mmol/L KCl, 14 mmol/L glucose, 1 mmol/L MgSO,, 1.2 mmol/L NaH,PO,, 1.8 mmol/L CaCl, at pH 7.4 and were gassed with 95% 0, and 5% CO, at 37’C. The tissue was attached to isometrical transducers (Sachs, Germany) that were connected to amplifiers and to a recorder (Linseis, Germany). Before testing the effect of the various agents, the tension of the tissue was adjusted to the degree that gave the maximal response to 1 kmol/L carbachol (1.2 + 0.1 g, n = 30). Comparison of longitudinal, circular, and helical preparations of gallbladder muscle strips with respect to their dose-response to carbachol showed no significant differences between the different preparations; therefore, in all subsequent experiments, longitudinal muscle strips were used. Experiments described below for antagonists were also performed using longitudinal strips, but for each antagonist, control experiments were performed in which circular and helical strips were used; these experiments did not show any significant differences for the various preparations (data not shown). The effect of carbachol on gallbladder contraction was determined by exposing the tissue to the indicated concentration until the tension reached a plateau. Subsequently, the tissue bath was washed until the tension reached the level that was observed before testing the indicated concentration of carbachol. For testing the effect of the various antagonists, the tissue was exposed to the indicated contraction of the antagonist for 6 minutes and then to the concentration of carbachol that caused half-maximal concentration (1 pmol/L). The tissue bath was subsequently washed after the tension had reached a plateau. To examine the effect of tetrodotoxin on carbachol-induced contraction, gallbladder smooth muscle strips were preincubated with 1 l,tmol/L tetrodotoxin for 15 minutes and subsequently exposed to the various concentrations of carbachol (1 nmol/L-0. 1 mmol/L).
Results To determine the optimal ing of [3H]NMS to gallbladder
conditions for bindsections, the depen-
November 1993
GALLBLADDER MUSCARINIC RECEPTORS
dence on time and temperature was examined. Binding of [3H]NMS to gallbladder sections was maximal at 20°C with 410 f 20 cpm after 30 minutes; extending the incubation time to 90 minutes reduced binding to 340 f 45 cpm. Adding 1 pmol/L atropine to the incubation buffer reduced binding by 96% at 20°C after 30 minutes (Figure 1). At 4°C binding was significantly lower than at 2O”C, reaching maximally 200 k 45 cpm at 15 minutes (Figure 1). For all subsequent studies, gallbladder tissue sections were incubated at 20°C for 30 minutes. To examine the reversibility of binding of [3H]NMS to gallbladder tissue sections, the tissue sections were first incubated with [3H]NMS alone or with [3H]NMS plus 1 lmol/L atropine, washed, and subsequently incubated in wash buffer for the various time periods at the indicated temperature. The dissociation of specifically bound [3H]NMS from gallbladder sections was slow at 4°C with 15% of the bound radioactivity being dissociated after 120 minutes; at 2O”C, the dissociation was faster with 50% of the radioactivity being dissociated after 25 minutes (Figure 2).
1343
100
a0
60
40
TIME ( min ) Figure 2. Dissociation of specifically bound [‘H]NMS from gallbladder sections. Tissue sections were first incubated with 5 nmol/L [3H]NMS with or without 1 j.tmol/L atropine, washed and resuspended in wash buffer at the indicated temperatures. Results are expressed as means of 3 experiments with gallbladders from different guinea pigs.
To determine the specificity of binding of [3H]NMS to gallbladder sections, we measured the effect of CCK, gastrin, GRP, histamine, motilin, rCGRP, SP, and VIP on binding of [3H]NMS. Binding of [3H]NMS to gallbladder tissue sections was only inhibited by substances that are known to interact with cholinergic Table 1. Effect of Various Agents on Binding of [3H]NMS to
Gallbladder Sections
PLUS 1 pM ATROPINE L/, u 0
30
60
1 90
TIME (mh) Figure 1. Time and temperature depence of binding of [3H]NMSto tissue sections of gallbladder. Tissue sections were incubated with 5 nmol/L [3H]NMSalone (0, m or with 5 nmol/L [3H]NMSplus 1 pmol/L atropine (0, 0). Results are expressed as counts per minute bound per tissue section. In each experiment, each value was determined in duplicate. Results shown are means of 4 separate experiments with gallbladders from different guinea pigs, vertical bars represent SD.
Agent added
[3H]NMS bound (% control)
Alone Atropine (0.1 jfmo//L) rCGRP(1 pmo//L) GRP(1 pmo//L) CCK-8 (1 pmo//L) Gastrin (1 pmo//L) Histamine (1 pmo//L) Motilin (1 pmo//L) SP (1 pmol/L) VIP (1 pmo//L)
100 1 f la 10026 101 f 10 99 f 5 92 f 8 lOOk5 10328 99+9 99 * 5
NOTE.Tissue sections were incubated with 5 nmol/L [3H]NMSalone or with the indicated agents. Results are means + 1 SD of at least 4 separate experiments using gallbladders from different animals. The data are expressed as the percentage of saturably bound radioactivity with no additions. “Values are signlflcantly less (P < 0.01) than control value by Student’s paired t test.
1344
SCHRENCH
VON
receptors
(Table
to interact
receptors
To localize der sections, The
with
contraction:
over the muscle (Figure
the serosa,
to inhibit
tissue sections. of binding
binding
of [3H]NMS
inhibition
squares,
(Figure were
curve-fitting
The binding site model
atropine
3B) 3C).
to the incu-
was only
found
was observed
or over
over
the liver
tissue
nmol/L),
using
computer
than by a one-site
(dissociation binding
gallbladder
23 f
were performed.
Carbachol
at 1 pmol/L
The dose-response perimposable chol
50% effective duced
curves
concentration
alone were sucurves
for carba-
tetrodotoxin
and the
(EC,,)
for contraction
differby car-
muscarinic
affinities
can be differentiated
muscarinic
2’-30 we tested the abilities
receptor
NMS
receptors
for various
antagonists
to gallbladder
For
(n = 4,
of carbachol
to
sections of
experiments contrac-
half-maximal
con-
contraction
at 0.1
potent
nmol/L)
than
binding
of [3H]-
the
to gallbladder
and 12 times
0.8 pmol/L), AF-DX
which
384 (I&,,
potent
than
more
potent
20 pmol/L)
NMS binding tagonists centrations,
the
than
SiHC (I&,, than
and 5.2 times
more
AF-DX
less potent than
muscarinic were
133
potent
at mid-log data
2 times
(ICso,
than
of [3H]-
receptor
and full-log
analyzed
116
pirenzepine
5). The inhibition
by the various
was determined and
(50% inhibi-
(ICso, 4.2 pmol/L).
(Figure
of
sections,
being
more
was 2.4 times (I&o 9 10 pmol/L) HHSiD but 2 times more potent (IC,,,
tissue
antagonist
3.2 pmol/L)
HHSiD
inhibition
67 nmol/L)
was 4 times
an-
musca-
to inhibit
sections.
receptor
of different
iV-methylscopolamine
for carba-
in-
was not significantly
more
(I& 204 + 58
was
ent from the EC,, for the contraction induced bachol in presence of 1 pmol/L tetrodotoxin.
rinic
alone
alone
of tetrodotoxin.
for carbachol
of 1 pmol/L
by carbachol
by tetrodo-
f 1 SD, n = 8) for
in presence
with the dose-response
in presence
response
for carbachol
[I&,],
caused detectable
and maximal
contraction
was the most potent
to gallbladder
strips at 1 nmol/L,
for
atropine
to cause contraction contraction
of maximal
tory concentration
of [3H]NMS strips,
on
“Ligand.“20
5 pmol/L)
the ability
Te-
the effect of carbachol
fit by a two-
(n = 4, P < 0.01);
for carbachol
gallblad-
was tested.
of [3H]NMS
least
of carbachol
muscle
tion of muscle
better
to influence
was not altered
(0.1 mmol/L)
the
contrac-
2.2 + 0.1 g and 2.2 k 0.2 g (means carbachol
No. 5
characterize
by carbachol
the magnitude
Vol. 105,
on gallbladder
binding
site had a low affinity constant
at
induced
maximal
tagonists,
curves
a nonlinear,
program
model
f 1 SE). To compare the ability
inhibition
further
of tetrodotoxin
(0.1 mmol/L)
Because
half-maximal
and complete
toxin;
To
did not influence
carbachol
by their
inhibition
4B). The dose-inhibition analyzed
car-
to gallbladder
at 1 nmol/L,
data were significantly
the other
agonist
of [3H]NMS
one site had a high affinity
traction
(Figure
caused detectable
at 1.6 pmol/L
for carbachol
inhibit
(Figure
(Figure
of the cholinergic
Carbachol
0.3 mmol/L
with
per-
3B).
bachol
means
alone
Binding
no binding
We tested the ability
chol
sections
atropine
1 pmol/L 39
layer,
over
was
of adjacent
by the silver grains was mark-
(Figure
the mucosa,
the ability
4A).
of carbachol
der contraction
of [3H]NMS
with [3H]NMS
by adding
(Figure
studies were performed.
histological
that is indicated buffer
tion,
from
on gallblad-
the autoradiographs
3A) after incubation
bation
of action
trodotoxin
of binding
the stained
edly reduced
distinct
mode
for [3H]NMS
or with [3H]NMS plus 1 pmol/L Binding
mmol/L
1).
autoradiographic
by comparing
sections
with receptors
(Table
the receptors
localization
formed
by the agents
1) but was not inhibited
that are known cholinergic
GASTROENTEROLOGY
ET AL.
using
anconthe
Figure 3. Autoradiography of binding of [3H]NMS to gallbladder sections. (A) H&E-stained tissue section of the gallbladder. Arrows, the muscle layer; L, the liver. (6) Autoradiography of the tissue section shown in A after incubation with [3H]NMS alone. (C) Autoradiography of the tissue section adjacent to A after incubation with t3H]NMS plus 1 kmol/L atrOpine.
November 1993
GALLBLADDER MUSCARINIC RECEPTORS
Figure 4. Ability of carbachol to cause contraction of gallbladder muscle strips (A) and to inhibit [3H]NMS that was saturably bound to gallbladder tissue sections (8). Results given are means of at least 5 separate experiments with gallbladders from different guinea pigs, vertical bars represent 1 SD.
“Ligand”
program, **Othis
each antagonist binding cantly
A analysis
one single
demonstrated
class of binding
data for each antagonist better
CARBACHOL
fit by a two-site
for
sites: the
tested were not signifimodel
than by a one-site
To
maximally (IC,,,
further
characterize
various
muscarinic
ceptors
identified
abilities
of the muscarinic
chol-induced For inhibition
the
receptor
antagonists
on gallbladder
contraction
interaction smooth
antagonists
with
caused
the
the re-
muscle,
to inhibit
was determined
of the contraction
of
the
carba-
(Figure
6).
by the half-
CARBACHOL
effective
pmol/L),
was the
5.2 nmol/L) than
times HHSiD (I&,,
being
21 nmol/L), AF-DX
more
potent
4 times which
384 (I&,, than
was 2 times 0.4 pmol/L)
(Figure
receptor
carbachol-induced
pmol/L) than
than and
AF-DX
(I&,,
11 116
0.4 pmol/
contraction 6). To examine
antagonist
contraction
potent
(ICso, 0.24 Fmol/L).
potent
strips
muscle
more
and pirenzepine
bladder
(1
antagonist
was 8 times more po-
HHSiD
more
potent
0.16
carbachol-induced
muscarinic
of carbachol
most
L) at inhibiting the
( log M )
concentration
atropine
SiHC (I&,, tent
model.
B
( log M )
1345
SiHC
of gallwhether inhibited
by a competitive
an-
roe
-a %
EM
I
a
20
CONCENlRAllON ( 109 M )
0 . .
Figure 5. AbiRty of various muscarinic receptor antagonists to inhibit binding of [3H]NMS to gallbladder sections. Tissue sections were incubated with [3H]NMS and the indicated concentrations of the various antagonists. Data are expressed as percentage of saturably bound radioactivity. Results given are means of at least 5 separate experiments with gallbladders from different guinea pigs. In each experiment each value was determined in duplicate. Vertical bars represent 1 SD.
-1
-I
car-rnmKm
-0
-1
[IogU)
Figure 6. Ability of various muscarinic antagonists to inhibit carbachol-induced contraction of the gallbladder muscle strips. Results are expressed as percentage of the contraction caused by 1 pmol/L carbachol alone. Values given are means of at least 5 separate experiments with gallbladders from different guinea pigs, vertical bars represent 1 SD.
1346
VON
SCHRENCKET AL.
GASTROENTEROLOGY Vol. 105. No. 5
tagonism, the effect of carbachol on gallbladder contraction was determined in presence of various concentrations of SiHC (Figure 7). SiHC caused a parallel rightward shift of the dose-response curve for carbachol (Figure 7). The data shown in Figure 7B were calculated according to the method of Arunlakshana and Schild3i using a linear regression; the slope of the linear regression was -0.902, and this value was not significantly different from unity. The pA, value estimated for the antagonism of SiHC on carbachol-induced gallbladder contraction was 9.41 (Figure 7).
Discussion The present study shows that [3H]NMS binds to specific receptors on gallbladder tissue sections: binding of [3H]NMS to tissue sections of the gallbladder was dependent on time and temperature and was saturable and reversible. Binding was specific for agents that are known to interact with cholinergic receptors but was not inhibited by agents that are known to interact with receptors different from cholinergic receptors such as CGRP, CCK, gastrin, GRP, histamine, motilin, SP, or VIP receptors. Therefore, binding of [3H]NMS to gallbladder tissue sections fits all criteria required for binding to a specific receptor. Several findings indicate that the receptors identified on gallbladder tissue sections using [3H]NMS are the same receptors that mediate the contraction caused by carbachol: first, binding was localized over the smooth muscle layer of the gallbladder. However, light microscopic autoradiographic studies do not allow differentiation of binding of [3H]NMS to neural structures in the smooth muscle from binding directly to smooth mus-
-
_
A ALONE A+SIHClOIM A+SiHC3rlO=M ?? +SiHClWM ??+SiHC3xlOlM 0 + SiHC WM
CARBACHOL
cle cells. Therefore, we tested the ability of the neural blocking agent tetrodotoxin to influence the contraction of gallbladder smooth muscle strips caused by carbachol. Tetrodotoxin did not influence the dose-response curve or magnitude of response for carbacholinduced contraction of gallbladder muscle strips. These findings provide strong evidence for a direct effect of carbachol on the smooth muscle cells. The insensitivity to tetrodotoxin can not rule out the possibility that carbachol is acting on nerve terminals to release a contractile transmitter.32 However, so far no transmitter that is released by occupation of muscarinic receptors has been identified in the gallbladder; in contrast, it has been shown that other agents such as SK” or in one study also CCK17 are able to cause the release of acetylcholine in the guinea pig gallbladder. Second, carbachol caused inhibition of binding of [3H]NMS over the same range of concentrations that caused contraction of gallbladder muscle strips: the ECsO (1 pmol/L) for gallbladder contraction caused by carbachol correlated closely with the ICso (1.6 pmol/ L) for inhibition of binding of [3H]NMS to gallbladder sections. Third, there was a close ageement between the abilities of the various muscarinic receptor antagonists to cause inhibition of [3H]NMS binding to gallbladder tissue sections and their abilities to inhibit carbachol-induced contraction of gallbladder muscle strips; specifically, the rank order of potencies for inhibition of [3H]NMS binding to gallbladder sections and for inhibition of carbachol-induced contraction of gallbladder muscle strips were atropine > SiHC > AF-DX 384 > HHSiD > AF-DX 116 2 pirenzepine. In the present study, the ICso that was determined for each
SCHILD
( log M )
-log
PLOT
[SiHC]
Figure 7. Effect of various concentrations of SiHC on carbachol-induced contraction of gallbladder smooth muscle strips (A) and plot of the data (6) according to the method of Arunlakshana et Schild.20 Values given are means of at least 5 separate experiments with gallbladders from different guinea pigs, vertical bars represent 1 SD.
November
1993
antagonist
GALLBLADDER
for
mately
inhibition
lo-fold
higher
for inhibition bladder
strips;
traction.
Binding
measuring sections
under
mined
for
the agonist [3H]NMS
different
of potencies
identified muscle
in the present
for inhibition
contraction. Several muscarinic tified
on
the
studies
in
of l-30
was the same
gallbladder
smooth that me-
smooth
muscle
antagonists
study to characterize gallbladder
tissue
NMS. The fact that binding
were used
the receptors sections
using
rinic
strong
receptors.
ceptors
evidence
can be divided
proposed
for the presence into
firstly by Goyal
molecular
sequencing
ine the structure
M2 receptors29 high affinity to CHO-Kl been
and Rattan.27
and cloning
of the different
inhibition
subtypes
shown
(ml-m5)
glandular
for M2 receptors. antagonists similar
smooth
25 The affinity
identified
receptors expressed
when
using
muscarinic
receptor
a competitive
that the
method value
was not data
pepsin-
the
gallbladder m3
SiHC had a high receptor
as de-
in rat ileal smooth gallbladder
antagonism significantly
were
mus-
contraction
with a linear different
calculated
regres-
from
according
unity to
the
and Schild.3’ The high pA,
gallbladder
of SiHC
contraction
with
identified
SiHC for the muscarinic gallbladder receptors is also comparable with the high affinity of SiHC for the M3
named
by ml-m5.
molecular
biology
Pharmacologically
techniques
were
muscarinic
recep-
tors have been differentiated by receptor antagonists with relative selectivity for the different types of muscarinic receptors; for the pharmacological characterization, the receptor types were named Ml-M5. The muscarinic receptor antagonists pirenzepine,23 AF-DX 1 16,24 AF-DX 384,28,29 HHSiD,25 and SiHC26 have been shown to be the antagonists that are most useful to distinguish different muscarinic receptor subtypes. Previous studies showed that pirenzepine has a high affinity for Ml receptors, which are found in neural ganglia and cerebral cortex.23 AF-DX 116 has a high
receptors ceptors
value
on carba-
is comparable
subtypes showed the existence of five distinct types of muscarinic receptors 22,30,35-35;these five different types
receptors
pA,
re-
of the mode of antagonism
of Arunlakshana
the high
muscarinic
to the cloned
muscarinic
of 9.41 for the antagonism
chol-induced
pig
i.e., on
cells.22?30 Specifically,
antagonist
of SiHC on carbachol-induced
as
to exam-
selective
is
for the various
for
is also similar
for the M3 receptors
showed
receptors,
p rofile
on CHO-Kl
cle.26 The characterization
sion
muscle
for M3 recep-
cells from guinea
antagonists
scribed
strategies
described
affinity
[3H]-
re-
study for the
smooth
pig chief cells mediating
37 The
receptor
receptors
for M3 than
for the various
cells,36 on rat pancreatic
secretion.
muscarinic
cells and rat
muscarinic
ceptors26 or on guinea
are
affinity
profile
on smooth
which
cells26 and on
in the present
profile
26 for glandular
ogen
SiHC has
parietal
in gallbladder
to the affinity
ileum
muscarinic antagonist
muscle
has a higher
that was found
receptors
human
for M3 receptors,
cells such as rat gastric 26*36HHSiD
affinity
of [3H]NMS binding
to be a M3 receptor-selective
on gastrointestinal
pancreas.
to possess
was measured.%
a high affinity
of
possessing
shown
cloned
for the gallbladder
of musca-
Studies
when
affinity
subtypes21-2”
1347
binding
homogenates
has also been
the M3-receptor
that muscarinic
different
and
cells expressing
by having found
at inhibiting
for m2 and m4 and intermediate
for m3 receptors receptor
116
iden-
of E3H]NMS to gallbladder
It has been shown
AF-DX
384 to rat heart
muscarinic receptor
tissue sections could be observed and that binding of [3H]NMS was inhibited with high potency by atropine provides
than
[3H]AF-DX
gastril- parietal
that the receptors
in gallbladder
potent
tors, i.e., for receptors
and for inhibi-
are the same receptors
carbachol-induced
of
study, the rank order
indicating
studies
might than the
binding
of binding
tested
by binding
in the present
is higher
contraction
using [3H]NMS
diate
methods
was in the range
of carbachol-induced
for all antagonists
dose of
for inhibition
in most other
However,
was deter-
effective
of atropine
the IC,, for atropine
nmol/L.
to tissue
the IC,, of each
to the gallbladder
that was found
which
by
antago-
conditions
of contraction
These
of con-
performed
in contrast,
that the I&
binding
likely
were ap-
receptor
the half-maximally
carbachol.
also explain
tion
equilibration
inhibition
by using
were
of the muscarinic
of the gallbladder;
antagonist
methods
RECEPTORS
affinity for M2 receptors, which are found on guinea , AF-DX 384 has been shown to be more pig atria 24*
of gall-
is most
and for measurements
experiments
binding
nist [‘H]NMS
I&,
studies
approxi-
contraction
this discrepancy
by the fact that different
plied for binding
was
that was observed
of carbachol-induced
muscle
explained
of binding
than the I&,
MUSCARINIC
in rat ileal
on rat gastric expressed
on
of 8.78 found
muscle.26
parietal CHO-Kl
The
high
for the M3 affinity
of
cells36 and for m3 recells.22
Similarly,
HHSiD had an intermediate affinity for the gallbladder muscarinic receptors as described for M3 receptors in guinea pig ileum. 25 AF-DX 116 had a low affinity for the muscarinic receptors in gallbladder smooth muscle as described previously for the M3 receptors on rat gastric parietal cells36 or guinea pig chief cells.37 The study of Miller et a1.28 and the study of Entzeroth and Mayer 29 showed that AF-DX 384 is more potent antagonist than AF-DX 116; accordingly, AF-DX 384
1348
VON
GASTROENTEROLOGY Vol. 105, No. 5
SCHRENCK ET AL.
was also a more potent antagonist muscarinic
receptor than AF-DX
at the gallbladder 116. Similarly,
AF-
the receptors in the guinea pig gallbladder. In the present study, we used the cholinergic
DX 384 has been shown to be more potent muscarinic
stimulate
receptor antagonist than AF-DX 116 at the M3 receptor in the ileal muscle. 3s Based on these comparisons
strips; carbachol than acetylcholine
with other cell systems, we conclude that the gallblad-
esterases.40 In addition,
der smooth muscle muscarinic
receptors are of the M3
contraction
antagonists
agonist carbachol
of gallbladder
smooth
has been shown to be more stable with respect to the degradation
that have been shown to be able to distin-
guish the different M-receptor
muscarinic
which possesses high affinity for M3 receptors.
muscarinic
receptors30*33,34; it is possible
gallbladder
smooth
that
also
muscle possesses more than one
subtype of muscarinic
receptors.
In agreement
with
by
we tested several muscarinic
type. It has been shown that most tissues that possess receptors express more than one subtype of
to
muscle
data
on
types, especially SiHC,
thermore,
the
agreement
with the data on binding
contraction
were
Fur-
in close
of [3H]NMS to
gallbladder
tissue sections.
findings in other studies,36,37 analysis of the dose-inhi-
characterize
the muscarinic
bition curves for [3H]NMS and the various receptor
gallbladder as M3 receptors,
antagonists
a recent study in which we found that carbachol-in-
showed only one single class of binding
sites. In contrast,
the dose-inhibition
curves for [3H]-
NMS binding and the agonist carbachol
were broad,
duced contraction muscle
Our present
data, which
receptors in the guinea pig are strongly supported by
of guinea pig gallbladder
is accompanied
with an increase
extended over more than 3 logs, and were best fit by a
phosphate
two-site model. This pattern of interaction
of guinea pigs with pertussis toxin (PTX)
carinic receptor agonist carbachol
of the mus-
and of the receptor
metabolism.41
ADP-ribosylation
gallbladder membranes
than one muscarinic
of carbachol
of inositol
pretreatment
of a 40/41 -kilodalton
antagonists may be indicative for the existence of more receptor subtype also in gallblad-
Furthermore,
smooth
resulted in protein from
but did not influence the effect
on gallbladder contraction
in vitro.4’ Be-
der smooth muscle; however, binding studies and mea-
cause M3 and m3 receptors have been shown in other
surements
cell systems to be linked to the phosphoinositide
of contraction
using the receptor
antago-
me-
nists showed clearly receptors of the M3 type and did
tabolism by a PTX-insensitive
not detect or distinguish
findings4’ provide additional support for the view that
subtypes in gallbladder The conclusion rinic receptors
other
muscarinic
receptor
smooth muscle.
M3 receptors
of the present study that the musca-
mediating
gallbladder
contraction
of the M3 type contrasts with the conclusion
are
of a re-
are mediating
gall-
Based on the data from our present study, we conclude that guinea pig gallbladder sesses specific muscarinic
various muscarinic
ized by their
on acetycho-
carbachol-induced
bladder smooth muscle contraction.
cent study by Kurtel et al. 3g; they studied the effect of receptor antagonists
pathway,42,43 our recent
affinities
smooth muscle pos-
receptors that are characterfor the various
muscarinic
muscle strips and on acetycholine-induced
contraction
receptor antagonists as M3 receptors. These receptors mediate carbachol-induced contraction of gallbladder
of guinea pig ileal muscle and concluded
that the re-
smooth muscle.
line-induced
contraction
ceptors mediating
of guinea
gallbladder
pig gallbladder
contraction
are not of
the M3, but rather of the M4 or M5 type. Several points might explain the discrepancies between the present study and the study by Kurtel et al. First, in the study of Kurtel et al., gallbladder contraction was stimulated by acetylcholine, a substance that is highly susceptible for rapid degradation by esterases.40 The potential degradation of acetylcholine is of particular importance when receptor affinities are determined by measuring the effect of antagonists on acetycholineinduced contraction: in the case of rapid degradation of acetylcholine by esterases a higher potency for the antagonist would be determined. Second, the antagonists with selectivity and high affinity for M3 receptors were not used in the study by Kurtel et al., especially not the MS-selective receptor antagonist SiHC. Third, no binding studies were performed to examine directly
References 1. Pallin 6, Skoglund S. Neural and humoral control of gallbladder emptying mechanism in the cat. Acta Physiol Stand 1964; 60:358-362. 2. Ryan J, Cohen S. Interaction of luminal volume and gastrointestinal hormone function on gallbladder motor function. In: Vantrappen G, Agg H, ed. Fifth international symposium on gastrointestinal motility. Belgium: Tydoff Press, 1976:55-63. 3. Fridhandler TM, Davison JS, Shaffer EA. Defective gallbladder contractility in the ground squirrel and prarie dog during the early stages of cholesterol gallstone formation. Gastroenterology 1983;85:830-836. 4. Pomeranz IS, Shaffer EA. Abnormal gallbladder emptying in a subgroup of patients with gallstones. Gastroenterology 1985; 88:787-79 1. 5. Doty JE, Pitt HA, Kuchenbecker SL, DenBesten L. Impaired gallbladder emptying before gallstone formation in the prarie dog. Gastroenterology 1983;85: 168- 174. 6. Fisher RS, Rock E, Malmu LS. Cholinergic effects on gallbladder emptying in humans. Gastroenterology 1985;89:7 16-722. 7. Behar J, Biancini P. Effect of cholecystokinin and the octapeptide
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of cholecystokinin on the feline sphincter of Oddi and gallbladder. J Clin Invest 1980;66:1231-1239. 8. Zettler G. Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK8): Effects on isolated intestinal preparations and gallbladders of guinea pig and mice. Peptides 1984;5:729736.
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9. Shook JE, Burks TF. A novel bioassay for the NK-2 Neurokinin receptor: the guinea pig gallbladder. Life Sci 1986;39:25332539.
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10. Yau WM. Mode of stimulation of gallbladder contraction stance K. Am J Physiol 1990;259:G838-G84 1.
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11. von Schrenck T, Moran TH, Heinz-Erian P, Gardner JD, Jensen RT. Cholecystokinin receptors on gallbladder muscle and pancreatic acinar cells: a comparative study. Am J Physiol 1988; 255:G512-G521. 12. Schjoldager B, Shaw MJ, Powers SP, Schmalz PF, Szurszewski J, Miller I-J. Bovine gallbladder muscularis: source of myogenic receptor for cholecystokinin. Am J Physiol 1988;254:G294-G299. 13. Shaw MJ, Hadac EM, Miller LJ. Preparations of enriched plasma membranes from bovine gallbladder muscularis for characterization of cholecystokinin receptors. J Biol Chem 1987;262: 14313-14318. 14. Steigetwaldt RW, Goldfine ID, Williams JA. Characterization of cholecystokinin receptors on bovine gallbladder membranes. Am J Physiol 1984;247:G709-G7 14.
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15. Grider JR, Makhlouf GM. Distinct receptors for cholecystokinin and gastrin on muscle cells of stomach and gallbladder. Am J Physiol 1990;259:G 184-G 190. 16. Ryan JP. Motility of the gallbladder and biliary tree. In: Johnson LR, ed. Physiology of the gastrointestinal tract. Volume 1. 2nd ed. New York: Raven, 1987:695-722.
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17. Takahashi T, Yamamura T, Kusunoki M, Kantoh M, lshikawa Y. Differences between muscular receptors and neural receptors for cholecystokinin-octapeptide in the guinea-pig gallbladder. Eur J Pharmacol 1987; 136:255-258.
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18. von Schrenck T, Heinz-Erian P, Moran TH, Mantey SH, Gardner JD, Jensen RT. Neuromedin B-preferring receptor in esophagus: evidence for subtypes of bombesin receptors. Am J Physiol 1989;256:G747-G758.
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19. von Schrenck T, Wang L-H, Coy DH, Villanueva ML, Mantey S, Jensen RT. Potent bombesin receptor antagonists distinguish bombesin receptor subtypes. Am J Physiol 1990;259:G468G473.
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Munson PJ, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand binding systems. Anal Biothem 1980;45:530-556.
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Lambrecht G, Wess J, Tacke R, Mutschler E. Heterogeneity of muscarinic receptors: evidence from structure-activity relationships of antimuscarinic agents related to pridinol and sila-pridinol. In: van der Goot H, Domany G, Pallos L, Timmerman H, eds. Trends in medicinal chemistry 88. Amsterdam: Elsevier, 1989:265-282.
40.
Buckley NJ, Banner TI, Buckley CM, Brann MR. Antagonist binding properties of five clones muscarinic receptors expressed in CHO-K 1 cells. Mol Pharmacol 1989;35:469-476.
42.
Hammer R, Berrie CP, Birdsall NJM, Burgen ASV, Hulme EC. Pirenzepine distinguishes between subclasses of muscarinic receptors. Nature 1980;283:90-92. 24. Giachetti A, Micheletti R, Montagna E. Cardioselective profile of AF-DX 116, a muscarinic M2 receptor antagonist. Life Sci 1986; 38:1663-1672.
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Lambrecht G, Feifel R, Wagner-Roder M, Strohmann C, Zilch H, Tacke R, Waelbroeck M. Christophe J, Boddeke H, Mutschler E. Affinity profiles of hexahydro-sila-difenidol analogues at muscarinic receptor subtypes. Eur J Pharmacol 1989; 168:7 l-80. 26. Waelbroeck M, Tastenoy M, Camu J, Christophe J, Strohmann C,
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Linoh H, Zilch H, Tacke R, Mutschler E, Lambrecht G. Binding and functional properties of antimuscarinics of hexocyclium/sila-hexocyclium and hexahydro-diphenidol/hexahydro-siladiphenidol type to muscarinic receptor subtypes. Br J Pharmacol 1989; 98: 197-205. Goyal RK, Rattan S. Neurohumeral, hormonal, and drug receptors for the lower esophageal sphincter. Gastroenterology 1978; 74:598-6 19. Miller JH, Gibson VA, Mckinney. Binding of [3H]AF-DX 384 to cloned and native muscarinic receptors. J Pharmacol Exp Therap 199 1;259:60 l-607. Entzeroth M, Mayer N. Labeling of rat heart muscarinic receptors using the new M2 selective antagonist [3H]AF-DX 384. Biochem Pharmacol 1990;40: 1674- 1676. Dbrje F, Wess J, Lambrecht G, Tacke R, Mutschler E, Brann MR. Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Therap 199 1;256:727-733. Arunlakshana 0, Schild HO. Some quantitative uses of drug antagonists. Br J Pharmcol 1959; 14:48-58. Angel F, Go VLM, Szurszewski JH. Innervation of the muscularis mucosae of canine proximal colon. J Physiol 1984;357:93108. Bonner TI, Buckley NJ, Young AC, Brann MR. identification of a family of muscarinic receptor genes. Science 1987;237:527532. Kubo T, Fukuda K, Mikami A, Maeda A, Takahashi H, Mishina M, Haga A, lchiyama A, Kangawa K, Kojima M, Matsuo H, Hirose T, Numa S. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetycholine receptor. Nature 1986;323:4 1 l-4 16. Peralta EG, Winslow JW, Peterson GL, Smith DH, Ashkenazi A, Ramachandran J, Schimerlick MI, Capon DJ. Primary structure and biochemical properties of an m2 muscarinic receptor. Science 1987;236:600-605. Pfeiffer A, Rochlitz H, Noelke B, Tacke R, Moser U, Mutschler E, Lambrecht G. Muscarinic receptors mediating acid secretion in isolated rat gastric parietal cells are of M3 Type. Gastroenterology 1990;98:2 18-222. Sutliff VE, Rattan S, Gardner JD, Jensen RT. Characterization of cholinergic receptors mediating pepsinogen secretion from chief cells. Am J Physiol 1989;257:G226-G234. Eberlein WG, Engel W, Mihm G, Rudolf K, Wetzel B, Entzeroth M, Mayer N, Doods HN. Structure-activity relationships and pharmacological profile of selective tricyclic antimuscarinics. TIPS 1989; 1O(Suppl):50-59. Kurtel H, Yegen BC, Dedeoglu A, Ulusoy NB, Oktay S. Muscarinic receptor subtypes of guinea-pig gallbladder smooth muscle. Arch Int Pharmacodyn 1990;308:39-46. Reynolds EF. Martindale-the extra pharmacopoeia. 28th ed. London: Ther Pharmaceutical Press 1982: 1037- 1039. von Schrenck T, Mackensen B, Mende U, Schmitz W, Mirau S, Raedler A, Greten H. Carbachol induces contraction of gallbladder smooth muscle by stimulating inositol phosphate formation (abstr). Gastroenterology 1992; 102:A53 1. Lechleiter J, Girard S, Clapham D, Peralta E. Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors. Nature 199 1;350:505-508. Candell LM, Yun SH, Tran LLP, Ehlert FJ. Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. Mol Pharmacol 1990;38:689-697.
Received August 3 1, 1992. Accepted December 22, 1992. Address requests for reprints to: Tammo von Schrenck, M.D., Medizinische Kernklinik und Polikhik, Universttatskrankenhaus Eppendorf, MartinistraDe 52, 20246 Hamburg, Germany. Supported by a grant from the Deutsche Forschungsgemeinschaft.
VON SCHRENCK,
J6RG
SIEVERS,
SILVIA
MIRAU,
ANDREAS
RAEDLER,
and HEINER
GRETEN
Department of Medicine, University Hospital Eppendorf, Hamburg, Germany
Background: Cholinergic agonists are of major importance for the regulation of gallbladder motility. However, the gallbladder muscarinic receptors have not been localized or characterized directly using radioligands, and it has not been clearly established which subtype of muscarinic receptor mediates contraction. The aim of the present study was to characterize the gallbladder muscarinic receptors. Methods: Binding studies to guinea pig gallbladder sections were performed using l-[N-methyl-3H] scopolamine methyl chloride. Carbachol-induced contraction was measured using muscle strips. Results: Binding of l-[NmethyL3H] scopolamine methyl chloride was reversible, dependent on time, temperature, and pH. Autoradiography showed binding only over the smooth muscle. Binding and carbachol-induced contractions were inhibited by muscarinic receptor antagonists with the following potencies: atropine > N-methylscopolamine > silahexocyclium-methylsulfate > AF-DX 384 I(+)-51 1-dlhydro-l l-([(2-{2-[(dipropylamino)methyl]- 1-piperidinyl}ethyl)amino]carbonyi}-6H- pyrido (2,3b) (1,4)-benzodiazepine-6-one] > hexahydro-siladifenidol hydrochloride > AF-DX 116 [(+)- 1 l-( { 2[(diethylamino)methyI]- 1-piperidinyl}-acetyl)-5,11 -dihydro-6H-pyrido (2,3b)( 1,4)benzodiazepine-6-one] > pirenzepine. Carbachol inhibited binding to gallbladder sections over the same range of concentrations that caused contractions. The concentration-contraction curves for carbachoi were not altered by tetrodotoxin. Conc/usions: Gallbladder smooth muscle cells possess muscarinic receptors of the M3 type. These receptors mediate carbachol-induced contraction.
have
been
smooth
muscle
been studied
by their
ceptor
agonists
portance
allbladder
contraction
conditions,
an important
is,
under
factor
on
affinities
agonists
fect of CCK” indicating
the stimulation
muscarinic
receptors
or localized
subtype
in the guinea
muscarinic
of muscarinic
directly using
in
However, the musca-
radioligands.
receptor receptors
by
antagonists is mediating
study was therefore
directly
pig gallbladder
and by measurements subtype
the gallbladder, ceptors
contraction,
are involved
of the gallbladder.
tify and characterize
ceptor
also the ef-
it has not clearly been established
The aim of the present
ine which
the
contrac-
contraction.
in the gallbladder
the use of selective contraction
about
the effect of
species
receptors
of gallbladder
And furthermore, which
inhibited
and in certain
no studies characterized rinic
is known
and of SK” on gallbladder
that
the im-
gallbladder
atropine
re-
for the regulation
mediating
studies,
tachykinin
receptors.‘TiO Despite is2,16 little
receptors
tion. In several
have
pig and are charac-
transmitters
motility,
gallbladder
for tachykinins
for different
as NK-2
of gallbladder
cholinergic
single
in the guinea
of cholinergic
cholinergic
isolated
cells. l5 Receptors
in vitro
terized
antagonists
the muscarinic muscle
of contraction of muscarinic
the abilities
in the gallbladder
receptors
by binding
studies
in vitro. To examreceptor
of various
to interact
to iden-
is present
in
muscarinic
re-
with the muscarinic
re-
were tested.
Materials and Methods obtained
G
identified
Male Pirbright white guinea pigs (250-300 g) were from the Animal Section, University Hospital
physiological
in regulating
bile
flow in the digestive period’p2 and is considered to be necessary to prevent stasis of lithogenic bile in the gallbladder.3-5 Cholinergic ago nists, cholecystokinin (CCK)related peptides and tachykinins cause gallbladder contraction in various species in vitro and in viva.“” Receptors for CCK have been characterized in vitro also by radioligand studies in gallbladders from various species such as guinea pig, human, or bovine gallbladders,11-i4 and distinct receptors for gastrin and CCK
Abbreviations used in this paper: AF-DX 116, (+)-11-({2-[(diethylamino)methyl]-l-piperldinyl}-acetyl)-5,ll-dihydro-6H-pyrldo (2,3-b)( 1,4)benzodiazepine-6-one; AF-DX 384, (lt)-5,l l-dihydro1 l-{[(2-{2-[(dlpropylamino)methyl]-l-piperidlnyl}ethyl)amino]-carbonyl}-6H-pyrido(2,3-b)(l,4)benzodiazepine-6-one; BSA, bovine serum albumin; EC,, 50% effective concentration; EGTA, ethylene glycol tetraacetic acid; [3H]NMS, 1 -[N-methyL3H]scopolamine methyl chloride; HHSiD, hexahydro-sila-difenldol-hydrochloride; IC,, 50% inhibitory concentration; PTX, pertussis toxin; rCCiHP, rat calcitonin gene-related peptide; SIHC, silahexocyclium-methylsulfate. 0 1993 by the American Gastroenterologlcal Association 0016-5085/93/$3.00
1342
VON
SCHRENCK
ET AL.
Eppendorf, Hamburg, Germany. l-[N-methyl-3H]Scopolamine methyl chloride a3H]NMS) (sp act, 74 Ci/mmol) was obtained from Amersham Buchler (Braunschweig, Germany); atropine, tetrodotoxin, Tris, ethyleneglycol-bis(B-aminoethyl ether)Nfl,N’fl’-tetraacetic acid (EGTA), bacitracin, pirenzepine-dihydrochloride (pirenzepine), carbamylcholine chloride (carbachol), and unlabeled hJmethyl-scopolamine methyl chloride (NMS) were from Sigma Chemical Co. (Germany); bovine serum albumin fraction V (BSA), leupeptin hydrogen sulfate, and chymostatin were from Boehringer Mannheim (Mannheim, Germany). All other chemicals were obtained from Merck (Darmstadt, Germany). Substance P (SP), the sulfated octapeptide of cholecystokinin (26-33) (CCK-8), rat calcitonin gene-related peptide (rCGRP), gastrin, gastrin-releasing peptide (GRP), motilin, vasoactive intestinal polypeptide (VIP) were from Bachem Chemicals (Switzerland). Hexahydrosiladifenidol-hydrochloride (HHSiD) and silahexocyclium-methylsulfate (SiHC) were a gift from Professor Lambrecht, Department of Pharmacology, Johann-Wolfgang-Goethe-UniversitEt (Frankfurt-am-Main, Germany); (+)-ll-({2-[d’ ie th y 1amino)-methyl]-l-pipridinyl}-acetyl)5,1 I-dihydro-6H-pyrido(2,3-b)( 1,4) benzodiazepine-6-one (AF-DX 116) and (rt)-5,11-dihydro-1 l-{ [(2-{ 2-[(dipropylamino)methyl]-l-piperidinyl}-ethyl)amino]carbony1}bH-pyrido(2,3-b)-(l,4)benzodiazepine-6-one (AF-DX 384) were kind gifts from Dr. Karl Thomae, G.m.b.H. (Biberach, Germany).
Binding Studies Binding of [3H]NMS to tissue sections of the gallbladder was studied using the methods described previBriefly, guinea pigs were killed, and the gallbladously. 11*‘s,19 der was rapidly removed. Tissues were frozen at -7O’C. Tissue sections (15 pm) were cut on a cryostat microtome (Leica Instruments, Heidelberg, Germany) and mounted onto gelatine coated glass slides. Tissue sections were first incubated with 50 mmol/L Tris buffer containing 0.5% BSA. Subsequently the tissue sections were incubated with 5 nmol/L [3H]NMS in 50 mmol/L Tris buffer containing 0.025% bacitracin (wt/vol), 4 pg/mL leupeptin (wt/vol), 2 pg/rnL chymostatin (wt/vol), 130 mmol/L NaCl, 7.7 mmol/L KCl, 5 mmol/L MgCI, and 1 mmol/L EGTA at pH 7.4. Because increasing the pH of the incubation buffer above 7.4 or decreasing the pH below 7.4 decreased binding (data not shown), all experiments were performed at pH 7.4. The degree of nonsaturable binding of [3H]NMS was determined by incubating alternate slides with adjacent tissue sections with 5 nmol/‘L [3H]NMS alone or with 5 nmol/L [3H]NMS plus 1 l.tmol/L atropine. After the incubation, the tissue sections were washed in 50 mmol/L Tris containing 0.5% albumin at 4°C; subsequently, the tissue sections were wiped off the slides, transferred to a counting vial, and counted for bound radioactivity in a beta scintillation counter. Unless specified otherwise, the data in this paper
GASTROENTEROLOGY
Vol. 105,
represent saturable binding of [3H]NMS, nonsaturable ing was always <5% of total binding.
Autoradiography
No. 5
bind-
Studies
The tissue sections were preincubated, incubated, and washed as described above for the binding studies. After the washing the tissue sections were dried rapidly with a stream of cold air and dried completely in a dessicator for 24 hours. Subsequently the tissue sections were exposed to 3HUltrofilm in radiograph cassettes. Binding sites were localized by superimposing the developed film after exposure for 4 weeks with the stained histological section. Measurement of contraction of gallbladder muscle strips. Contraction was determined as previously described for the gallbladder. ‘I Guinea pigs we r e killed, and their gallbladders were carefully removed. Longitudinal muscle strips (0.2 cm X 1.0 cm) were prepared and attached in an organ bath using surgical silk. The muscle strips were placed in standard incubation solution containing 118 mmol/L NaCl, 25 mmol/L NaHCO,, 4.7 mmol/L KCl, 14 mmol/L glucose, 1 mmol/L MgSO,, 1.2 mmol/L NaH,PO,, 1.8 mmol/L CaCl, at pH 7.4 and were gassed with 95% 0, and 5% CO, at 37’C. The tissue was attached to isometrical transducers (Sachs, Germany) that were connected to amplifiers and to a recorder (Linseis, Germany). Before testing the effect of the various agents, the tension of the tissue was adjusted to the degree that gave the maximal response to 1 kmol/L carbachol (1.2 + 0.1 g, n = 30). Comparison of longitudinal, circular, and helical preparations of gallbladder muscle strips with respect to their dose-response to carbachol showed no significant differences between the different preparations; therefore, in all subsequent experiments, longitudinal muscle strips were used. Experiments described below for antagonists were also performed using longitudinal strips, but for each antagonist, control experiments were performed in which circular and helical strips were used; these experiments did not show any significant differences for the various preparations (data not shown). The effect of carbachol on gallbladder contraction was determined by exposing the tissue to the indicated concentration until the tension reached a plateau. Subsequently, the tissue bath was washed until the tension reached the level that was observed before testing the indicated concentration of carbachol. For testing the effect of the various antagonists, the tissue was exposed to the indicated contraction of the antagonist for 6 minutes and then to the concentration of carbachol that caused half-maximal concentration (1 pmol/L). The tissue bath was subsequently washed after the tension had reached a plateau. To examine the effect of tetrodotoxin on carbachol-induced contraction, gallbladder smooth muscle strips were preincubated with 1 l,tmol/L tetrodotoxin for 15 minutes and subsequently exposed to the various concentrations of carbachol (1 nmol/L-0. 1 mmol/L).
Results To determine the optimal ing of [3H]NMS to gallbladder
conditions for bindsections, the depen-
November 1993
GALLBLADDER MUSCARINIC RECEPTORS
dence on time and temperature was examined. Binding of [3H]NMS to gallbladder sections was maximal at 20°C with 410 f 20 cpm after 30 minutes; extending the incubation time to 90 minutes reduced binding to 340 f 45 cpm. Adding 1 pmol/L atropine to the incubation buffer reduced binding by 96% at 20°C after 30 minutes (Figure 1). At 4°C binding was significantly lower than at 2O”C, reaching maximally 200 k 45 cpm at 15 minutes (Figure 1). For all subsequent studies, gallbladder tissue sections were incubated at 20°C for 30 minutes. To examine the reversibility of binding of [3H]NMS to gallbladder tissue sections, the tissue sections were first incubated with [3H]NMS alone or with [3H]NMS plus 1 lmol/L atropine, washed, and subsequently incubated in wash buffer for the various time periods at the indicated temperature. The dissociation of specifically bound [3H]NMS from gallbladder sections was slow at 4°C with 15% of the bound radioactivity being dissociated after 120 minutes; at 2O”C, the dissociation was faster with 50% of the radioactivity being dissociated after 25 minutes (Figure 2).
1343
100
a0
60
40
TIME ( min ) Figure 2. Dissociation of specifically bound [‘H]NMS from gallbladder sections. Tissue sections were first incubated with 5 nmol/L [3H]NMS with or without 1 j.tmol/L atropine, washed and resuspended in wash buffer at the indicated temperatures. Results are expressed as means of 3 experiments with gallbladders from different guinea pigs.
To determine the specificity of binding of [3H]NMS to gallbladder sections, we measured the effect of CCK, gastrin, GRP, histamine, motilin, rCGRP, SP, and VIP on binding of [3H]NMS. Binding of [3H]NMS to gallbladder tissue sections was only inhibited by substances that are known to interact with cholinergic Table 1. Effect of Various Agents on Binding of [3H]NMS to
Gallbladder Sections
PLUS 1 pM ATROPINE L/, u 0
30
60
1 90
TIME (mh) Figure 1. Time and temperature depence of binding of [3H]NMSto tissue sections of gallbladder. Tissue sections were incubated with 5 nmol/L [3H]NMSalone (0, m or with 5 nmol/L [3H]NMSplus 1 pmol/L atropine (0, 0). Results are expressed as counts per minute bound per tissue section. In each experiment, each value was determined in duplicate. Results shown are means of 4 separate experiments with gallbladders from different guinea pigs, vertical bars represent SD.
Agent added
[3H]NMS bound (% control)
Alone Atropine (0.1 jfmo//L) rCGRP(1 pmo//L) GRP(1 pmo//L) CCK-8 (1 pmo//L) Gastrin (1 pmo//L) Histamine (1 pmo//L) Motilin (1 pmo//L) SP (1 pmol/L) VIP (1 pmo//L)
100 1 f la 10026 101 f 10 99 f 5 92 f 8 lOOk5 10328 99+9 99 * 5
NOTE.Tissue sections were incubated with 5 nmol/L [3H]NMSalone or with the indicated agents. Results are means + 1 SD of at least 4 separate experiments using gallbladders from different animals. The data are expressed as the percentage of saturably bound radioactivity with no additions. “Values are signlflcantly less (P < 0.01) than control value by Student’s paired t test.
1344
SCHRENCH
VON
receptors
(Table
to interact
receptors
To localize der sections, The
with
contraction:
over the muscle (Figure
the serosa,
to inhibit
tissue sections. of binding
binding
of [3H]NMS
inhibition
squares,
(Figure were
curve-fitting
The binding site model
atropine
3B) 3C).
to the incu-
was only
found
was observed
or over
over
the liver
tissue
nmol/L),
using
computer
than by a one-site
(dissociation binding
gallbladder
23 f
were performed.
Carbachol
at 1 pmol/L
The dose-response perimposable chol
50% effective duced
curves
concentration
alone were sucurves
for carba-
tetrodotoxin
and the
(EC,,)
for contraction
differby car-
muscarinic
affinities
can be differentiated
muscarinic
2’-30 we tested the abilities
receptor
NMS
receptors
for various
antagonists
to gallbladder
For
(n = 4,
of carbachol
to
sections of
experiments contrac-
half-maximal
con-
contraction
at 0.1
potent
nmol/L)
than
binding
of [3H]-
the
to gallbladder
and 12 times
0.8 pmol/L), AF-DX
which
384 (I&,,
potent
than
more
potent
20 pmol/L)
NMS binding tagonists centrations,
the
than
SiHC (I&,, than
and 5.2 times
more
AF-DX
less potent than
muscarinic were
133
potent
at mid-log data
2 times
(ICso,
than
of [3H]-
receptor
and full-log
analyzed
116
pirenzepine
5). The inhibition
by the various
was determined and
(50% inhibi-
(ICso, 4.2 pmol/L).
(Figure
of
sections,
being
more
was 2.4 times (I&o 9 10 pmol/L) HHSiD but 2 times more potent (IC,,,
tissue
antagonist
3.2 pmol/L)
HHSiD
inhibition
67 nmol/L)
was 4 times
an-
musca-
to inhibit
sections.
receptor
of different
iV-methylscopolamine
for carba-
in-
was not significantly
more
(I& 204 + 58
was
ent from the EC,, for the contraction induced bachol in presence of 1 pmol/L tetrodotoxin.
rinic
alone
alone
of tetrodotoxin.
for carbachol
of 1 pmol/L
by carbachol
by tetrodo-
f 1 SD, n = 8) for
in presence
with the dose-response
in presence
response
for carbachol
[I&,],
caused detectable
and maximal
contraction
was the most potent
to gallbladder
strips at 1 nmol/L,
for
atropine
to cause contraction contraction
of maximal
tory concentration
of [3H]NMS strips,
on
“Ligand.“20
5 pmol/L)
the ability
Te-
the effect of carbachol
fit by a two-
(n = 4, P < 0.01);
for carbachol
gallblad-
was tested.
of [3H]NMS
least
of carbachol
muscle
tion of muscle
better
to influence
was not altered
(0.1 mmol/L)
the
contrac-
2.2 + 0.1 g and 2.2 k 0.2 g (means carbachol
No. 5
characterize
by carbachol
the magnitude
Vol. 105,
on gallbladder
binding
site had a low affinity constant
at
induced
maximal
tagonists,
curves
a nonlinear,
program
model
f 1 SE). To compare the ability
inhibition
further
of tetrodotoxin
(0.1 mmol/L)
Because
half-maximal
and complete
toxin;
To
did not influence
carbachol
by their
inhibition
4B). The dose-inhibition analyzed
car-
to gallbladder
at 1 nmol/L,
data were significantly
the other
agonist
of [3H]NMS
one site had a high affinity
traction
(Figure
caused detectable
at 1.6 pmol/L
for carbachol
inhibit
(Figure
(Figure
of the cholinergic
Carbachol
0.3 mmol/L
with
per-
3B).
bachol
means
alone
Binding
no binding
We tested the ability
chol
sections
atropine
1 pmol/L 39
layer,
over
was
of adjacent
by the silver grains was mark-
(Figure
the mucosa,
the ability
4A).
of carbachol
der contraction
of [3H]NMS
with [3H]NMS
by adding
(Figure
studies were performed.
histological
that is indicated buffer
tion,
from
on gallblad-
the autoradiographs
3A) after incubation
bation
of action
trodotoxin
of binding
the stained
edly reduced
distinct
mode
for [3H]NMS
or with [3H]NMS plus 1 pmol/L Binding
mmol/L
1).
autoradiographic
by comparing
sections
with receptors
(Table
the receptors
localization
formed
by the agents
1) but was not inhibited
that are known cholinergic
GASTROENTEROLOGY
ET AL.
using
anconthe
Figure 3. Autoradiography of binding of [3H]NMS to gallbladder sections. (A) H&E-stained tissue section of the gallbladder. Arrows, the muscle layer; L, the liver. (6) Autoradiography of the tissue section shown in A after incubation with [3H]NMS alone. (C) Autoradiography of the tissue section adjacent to A after incubation with t3H]NMS plus 1 kmol/L atrOpine.
November 1993
GALLBLADDER MUSCARINIC RECEPTORS
Figure 4. Ability of carbachol to cause contraction of gallbladder muscle strips (A) and to inhibit [3H]NMS that was saturably bound to gallbladder tissue sections (8). Results given are means of at least 5 separate experiments with gallbladders from different guinea pigs, vertical bars represent 1 SD.
“Ligand”
program, **Othis
each antagonist binding cantly
A analysis
one single
demonstrated
class of binding
data for each antagonist better
CARBACHOL
fit by a two-site
for
sites: the
tested were not signifimodel
than by a one-site
To
maximally (IC,,,
further
characterize
various
muscarinic
ceptors
identified
abilities
of the muscarinic
chol-induced For inhibition
the
receptor
antagonists
on gallbladder
contraction
interaction smooth
antagonists
with
caused
the
the re-
muscle,
to inhibit
was determined
of the contraction
of
the
carba-
(Figure
6).
by the half-
CARBACHOL
effective
pmol/L),
was the
5.2 nmol/L) than
times HHSiD (I&,,
being
21 nmol/L), AF-DX
more
potent
4 times which
384 (I&,, than
was 2 times 0.4 pmol/L)
(Figure
receptor
carbachol-induced
pmol/L) than
than and
AF-DX
(I&,,
11 116
0.4 pmol/
contraction 6). To examine
antagonist
contraction
potent
(ICso, 0.24 Fmol/L).
potent
strips
muscle
more
and pirenzepine
bladder
(1
antagonist
was 8 times more po-
HHSiD
more
potent
0.16
carbachol-induced
muscarinic
of carbachol
most
L) at inhibiting the
( log M )
concentration
atropine
SiHC (I&,, tent
model.
B
( log M )
1345
SiHC
of gallwhether inhibited
by a competitive
an-
roe
-a %
EM
I
a
20
CONCENlRAllON ( 109 M )
0 . .
Figure 5. AbiRty of various muscarinic receptor antagonists to inhibit binding of [3H]NMS to gallbladder sections. Tissue sections were incubated with [3H]NMS and the indicated concentrations of the various antagonists. Data are expressed as percentage of saturably bound radioactivity. Results given are means of at least 5 separate experiments with gallbladders from different guinea pigs. In each experiment each value was determined in duplicate. Vertical bars represent 1 SD.
-1
-I
car-rnmKm
-0
-1
[IogU)
Figure 6. Ability of various muscarinic antagonists to inhibit carbachol-induced contraction of the gallbladder muscle strips. Results are expressed as percentage of the contraction caused by 1 pmol/L carbachol alone. Values given are means of at least 5 separate experiments with gallbladders from different guinea pigs, vertical bars represent 1 SD.
1346
VON
SCHRENCKET AL.
GASTROENTEROLOGY Vol. 105. No. 5
tagonism, the effect of carbachol on gallbladder contraction was determined in presence of various concentrations of SiHC (Figure 7). SiHC caused a parallel rightward shift of the dose-response curve for carbachol (Figure 7). The data shown in Figure 7B were calculated according to the method of Arunlakshana and Schild3i using a linear regression; the slope of the linear regression was -0.902, and this value was not significantly different from unity. The pA, value estimated for the antagonism of SiHC on carbachol-induced gallbladder contraction was 9.41 (Figure 7).
Discussion The present study shows that [3H]NMS binds to specific receptors on gallbladder tissue sections: binding of [3H]NMS to tissue sections of the gallbladder was dependent on time and temperature and was saturable and reversible. Binding was specific for agents that are known to interact with cholinergic receptors but was not inhibited by agents that are known to interact with receptors different from cholinergic receptors such as CGRP, CCK, gastrin, GRP, histamine, motilin, SP, or VIP receptors. Therefore, binding of [3H]NMS to gallbladder tissue sections fits all criteria required for binding to a specific receptor. Several findings indicate that the receptors identified on gallbladder tissue sections using [3H]NMS are the same receptors that mediate the contraction caused by carbachol: first, binding was localized over the smooth muscle layer of the gallbladder. However, light microscopic autoradiographic studies do not allow differentiation of binding of [3H]NMS to neural structures in the smooth muscle from binding directly to smooth mus-
-
_
A ALONE A+SIHClOIM A+SiHC3rlO=M ?? +SiHClWM ??+SiHC3xlOlM 0 + SiHC WM
CARBACHOL
cle cells. Therefore, we tested the ability of the neural blocking agent tetrodotoxin to influence the contraction of gallbladder smooth muscle strips caused by carbachol. Tetrodotoxin did not influence the dose-response curve or magnitude of response for carbacholinduced contraction of gallbladder muscle strips. These findings provide strong evidence for a direct effect of carbachol on the smooth muscle cells. The insensitivity to tetrodotoxin can not rule out the possibility that carbachol is acting on nerve terminals to release a contractile transmitter.32 However, so far no transmitter that is released by occupation of muscarinic receptors has been identified in the gallbladder; in contrast, it has been shown that other agents such as SK” or in one study also CCK17 are able to cause the release of acetylcholine in the guinea pig gallbladder. Second, carbachol caused inhibition of binding of [3H]NMS over the same range of concentrations that caused contraction of gallbladder muscle strips: the ECsO (1 pmol/L) for gallbladder contraction caused by carbachol correlated closely with the ICso (1.6 pmol/ L) for inhibition of binding of [3H]NMS to gallbladder sections. Third, there was a close ageement between the abilities of the various muscarinic receptor antagonists to cause inhibition of [3H]NMS binding to gallbladder tissue sections and their abilities to inhibit carbachol-induced contraction of gallbladder muscle strips; specifically, the rank order of potencies for inhibition of [3H]NMS binding to gallbladder sections and for inhibition of carbachol-induced contraction of gallbladder muscle strips were atropine > SiHC > AF-DX 384 > HHSiD > AF-DX 116 2 pirenzepine. In the present study, the ICso that was determined for each
SCHILD
( log M )
-log
PLOT
[SiHC]
Figure 7. Effect of various concentrations of SiHC on carbachol-induced contraction of gallbladder smooth muscle strips (A) and plot of the data (6) according to the method of Arunlakshana et Schild.20 Values given are means of at least 5 separate experiments with gallbladders from different guinea pigs, vertical bars represent 1 SD.
November
1993
antagonist
GALLBLADDER
for
mately
inhibition
lo-fold
higher
for inhibition bladder
strips;
traction.
Binding
measuring sections
under
mined
for
the agonist [3H]NMS
different
of potencies
identified muscle
in the present
for inhibition
contraction. Several muscarinic tified
on
the
studies
in
of l-30
was the same
gallbladder
smooth that me-
smooth
muscle
antagonists
study to characterize gallbladder
tissue
NMS. The fact that binding
were used
the receptors sections
using
rinic
strong
receptors.
ceptors
evidence
can be divided
proposed
for the presence into
firstly by Goyal
molecular
sequencing
ine the structure
M2 receptors29 high affinity to CHO-Kl been
and Rattan.27
and cloning
of the different
inhibition
subtypes
shown
(ml-m5)
glandular
for M2 receptors. antagonists similar
smooth
25 The affinity
identified
receptors expressed
when
using
muscarinic
receptor
a competitive
that the
method value
was not data
pepsin-
the
gallbladder m3
SiHC had a high receptor
as de-
in rat ileal smooth gallbladder
antagonism significantly
were
mus-
contraction
with a linear different
calculated
regres-
from
according
unity to
the
and Schild.3’ The high pA,
gallbladder
of SiHC
contraction
with
identified
SiHC for the muscarinic gallbladder receptors is also comparable with the high affinity of SiHC for the M3
named
by ml-m5.
molecular
biology
Pharmacologically
techniques
were
muscarinic
recep-
tors have been differentiated by receptor antagonists with relative selectivity for the different types of muscarinic receptors; for the pharmacological characterization, the receptor types were named Ml-M5. The muscarinic receptor antagonists pirenzepine,23 AF-DX 1 16,24 AF-DX 384,28,29 HHSiD,25 and SiHC26 have been shown to be the antagonists that are most useful to distinguish different muscarinic receptor subtypes. Previous studies showed that pirenzepine has a high affinity for Ml receptors, which are found in neural ganglia and cerebral cortex.23 AF-DX 116 has a high
receptors ceptors
value
on carba-
is comparable
subtypes showed the existence of five distinct types of muscarinic receptors 22,30,35-35;these five different types
receptors
pA,
re-
of the mode of antagonism
of Arunlakshana
the high
muscarinic
to the cloned
muscarinic
of 9.41 for the antagonism
chol-induced
pig
i.e., on
cells.22?30 Specifically,
antagonist
of SiHC on carbachol-induced
as
to exam-
selective
is
for the various
for
is also similar
for the M3 receptors
showed
receptors,
p rofile
on CHO-Kl
cle.26 The characterization
sion
muscle
for M3 recep-
cells from guinea
antagonists
scribed
strategies
described
affinity
[3H]-
re-
study for the
smooth
pig chief cells mediating
37 The
receptor
receptors
for M3 than
for the various
cells,36 on rat pancreatic
secretion.
muscarinic
cells and rat
muscarinic
ceptors26 or on guinea
are
affinity
profile
on smooth
which
cells26 and on
in the present
profile
26 for glandular
ogen
SiHC has
parietal
in gallbladder
to the affinity
ileum
muscarinic antagonist
muscle
has a higher
that was found
receptors
human
for M3 receptors,
cells such as rat gastric 26*36HHSiD
affinity
of [3H]NMS binding
to be a M3 receptor-selective
on gastrointestinal
pancreas.
to possess
was measured.%
a high affinity
of
possessing
shown
cloned
for the gallbladder
of musca-
Studies
when
affinity
subtypes21-2”
1347
binding
homogenates
has also been
the M3-receptor
that muscarinic
different
and
cells expressing
by having found
at inhibiting
for m2 and m4 and intermediate
for m3 receptors receptor
116
iden-
of E3H]NMS to gallbladder
It has been shown
AF-DX
384 to rat heart
muscarinic receptor
tissue sections could be observed and that binding of [3H]NMS was inhibited with high potency by atropine provides
than
[3H]AF-DX
gastril- parietal
that the receptors
in gallbladder
potent
tors, i.e., for receptors
and for inhibi-
are the same receptors
carbachol-induced
of
study, the rank order
indicating
studies
might than the
binding
of binding
tested
by binding
in the present
is higher
contraction
using [3H]NMS
diate
methods
was in the range
of carbachol-induced
for all antagonists
dose of
for inhibition
in most other
However,
was deter-
effective
of atropine
the IC,, for atropine
nmol/L.
to tissue
the IC,, of each
to the gallbladder
that was found
which
by
antago-
conditions
of contraction
These
of con-
performed
in contrast,
that the I&
binding
likely
were ap-
receptor
the half-maximally
carbachol.
also explain
tion
equilibration
inhibition
by using
were
of the muscarinic
of the gallbladder;
antagonist
methods
RECEPTORS
affinity for M2 receptors, which are found on guinea , AF-DX 384 has been shown to be more pig atria 24*
of gall-
is most
and for measurements
experiments
binding
nist [‘H]NMS
I&,
studies
approxi-
contraction
this discrepancy
by the fact that different
plied for binding
was
that was observed
of carbachol-induced
muscle
explained
of binding
than the I&,
MUSCARINIC
in rat ileal
on rat gastric expressed
on
of 8.78 found
muscle.26
parietal CHO-Kl
The
high
for the M3 affinity
of
cells36 and for m3 recells.22
Similarly,
HHSiD had an intermediate affinity for the gallbladder muscarinic receptors as described for M3 receptors in guinea pig ileum. 25 AF-DX 116 had a low affinity for the muscarinic receptors in gallbladder smooth muscle as described previously for the M3 receptors on rat gastric parietal cells36 or guinea pig chief cells.37 The study of Miller et a1.28 and the study of Entzeroth and Mayer 29 showed that AF-DX 384 is more potent antagonist than AF-DX 116; accordingly, AF-DX 384
1348
VON
GASTROENTEROLOGY Vol. 105, No. 5
SCHRENCK ET AL.
was also a more potent antagonist muscarinic
receptor than AF-DX
at the gallbladder 116. Similarly,
AF-
the receptors in the guinea pig gallbladder. In the present study, we used the cholinergic
DX 384 has been shown to be more potent muscarinic
stimulate
receptor antagonist than AF-DX 116 at the M3 receptor in the ileal muscle. 3s Based on these comparisons
strips; carbachol than acetylcholine
with other cell systems, we conclude that the gallblad-
esterases.40 In addition,
der smooth muscle muscarinic
receptors are of the M3
contraction
antagonists
agonist carbachol
of gallbladder
smooth
has been shown to be more stable with respect to the degradation
that have been shown to be able to distin-
guish the different M-receptor
muscarinic
which possesses high affinity for M3 receptors.
muscarinic
receptors30*33,34; it is possible
gallbladder
smooth
that
also
muscle possesses more than one
subtype of muscarinic
receptors.
In agreement
with
by
we tested several muscarinic
type. It has been shown that most tissues that possess receptors express more than one subtype of
to
muscle
data
on
types, especially SiHC,
thermore,
the
agreement
with the data on binding
contraction
were
Fur-
in close
of [3H]NMS to
gallbladder
tissue sections.
findings in other studies,36,37 analysis of the dose-inhi-
characterize
the muscarinic
bition curves for [3H]NMS and the various receptor
gallbladder as M3 receptors,
antagonists
a recent study in which we found that carbachol-in-
showed only one single class of binding
sites. In contrast,
the dose-inhibition
curves for [3H]-
NMS binding and the agonist carbachol
were broad,
duced contraction muscle
Our present
data, which
receptors in the guinea pig are strongly supported by
of guinea pig gallbladder
is accompanied
with an increase
extended over more than 3 logs, and were best fit by a
phosphate
two-site model. This pattern of interaction
of guinea pigs with pertussis toxin (PTX)
carinic receptor agonist carbachol
of the mus-
and of the receptor
metabolism.41
ADP-ribosylation
gallbladder membranes
than one muscarinic
of carbachol
of inositol
pretreatment
of a 40/41 -kilodalton
antagonists may be indicative for the existence of more receptor subtype also in gallblad-
Furthermore,
smooth
resulted in protein from
but did not influence the effect
on gallbladder contraction
in vitro.4’ Be-
der smooth muscle; however, binding studies and mea-
cause M3 and m3 receptors have been shown in other
surements
cell systems to be linked to the phosphoinositide
of contraction
using the receptor
antago-
me-
nists showed clearly receptors of the M3 type and did
tabolism by a PTX-insensitive
not detect or distinguish
findings4’ provide additional support for the view that
subtypes in gallbladder The conclusion rinic receptors
other
muscarinic
receptor
smooth muscle.
M3 receptors
of the present study that the musca-
mediating
gallbladder
contraction
of the M3 type contrasts with the conclusion
are
of a re-
are mediating
gall-
Based on the data from our present study, we conclude that guinea pig gallbladder sesses specific muscarinic
various muscarinic
ized by their
on acetycho-
carbachol-induced
bladder smooth muscle contraction.
cent study by Kurtel et al. 3g; they studied the effect of receptor antagonists
pathway,42,43 our recent
affinities
smooth muscle pos-
receptors that are characterfor the various
muscarinic
muscle strips and on acetycholine-induced
contraction
receptor antagonists as M3 receptors. These receptors mediate carbachol-induced contraction of gallbladder
of guinea pig ileal muscle and concluded
that the re-
smooth muscle.
line-induced
contraction
ceptors mediating
of guinea
gallbladder
pig gallbladder
contraction
are not of
the M3, but rather of the M4 or M5 type. Several points might explain the discrepancies between the present study and the study by Kurtel et al. First, in the study of Kurtel et al., gallbladder contraction was stimulated by acetylcholine, a substance that is highly susceptible for rapid degradation by esterases.40 The potential degradation of acetylcholine is of particular importance when receptor affinities are determined by measuring the effect of antagonists on acetycholineinduced contraction: in the case of rapid degradation of acetylcholine by esterases a higher potency for the antagonist would be determined. Second, the antagonists with selectivity and high affinity for M3 receptors were not used in the study by Kurtel et al., especially not the MS-selective receptor antagonist SiHC. Third, no binding studies were performed to examine directly
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Received August 3 1, 1992. Accepted December 22, 1992. Address requests for reprints to: Tammo von Schrenck, M.D., Medizinische Kernklinik und Polikhik, Universttatskrankenhaus Eppendorf, MartinistraDe 52, 20246 Hamburg, Germany. Supported by a grant from the Deutsche Forschungsgemeinschaft.