Section on Biochemical Pharmacology, Departments of Neurosciences
(M.C.O., P.O.) and
Medical Sciences (A.I., C.M.), University of
Cagliari, Italy; and
Department of Clinical Neuroscience, Geriatric
Section, Karolinska Institute, Stockholm, Sweden (A.A., E.K.)
 |
Introduction |
In
the central nervous system and in peripheral tissues, many actions of
acetylcholine (ACh) occur through the activation of muscarinic
receptors. Molecular biology studies have led to the identification of
five distinct molecular forms of the muscarinic receptors, named
m1-m5, and the artificial expression of the cloned receptor subtypes
in host cells has allowed the characterization of their signal
transduction pathways (Bonner et al., 1987
; Peralta et al., 1987; Hulme
et al., 1990). Thus, it has been shown that m1, m3, and m5 receptors
are predominantly coupled to phospholipase C through G proteins of
Gq/11 type, whereas m2 and m4 receptors preferentially regulate adenylyl cyclase and ion channel activities by
coupling to Gi/Go proteins
(Peralta et al., 1988). However, a great limitation to the study of the
physiological role played by each receptor subtype in the different
tissues is the lack of highly selective ligands. Indeed, the muscarinic
receptor antagonists currently available display an affinity for one
receptor subtype that is <10-fold greater than that for the other
subtypes (Caufield, 1993). The limited selectivity of the drugs
complicates the receptor characterization in tissues expressing a
heterogeneous muscarinic receptor population. In addition, none of the
classic antimuscarinic drugs bind with high selectivity to the m4 or m5
receptor subtype (Caufield, 1993). The need of m4-selective ligands is
particularly critical because the m4 receptor is almost exclusively
expressed in neurons (Wood et al., 1996; Mieda et al., 1997) and is
preferentially localized in central neuronal pathways regulating motor
and cognitive functions (Levey et al., 1991; Ferrari-Dileo et al.,
1994).
Recently, Karlsson and collaborators (1994) reported the isolation of a
new peptide toxin, named MT3, from green mamba venom. In radioligand
binding studies using Chinese hamster ovary (CHO) cells separately
expressing the five cloned muscarinic receptors, muscarinic toxin 3 (MT3) showed a high affinity for the m4
(pKi = 8.70), a lower affinity for the m1
(pKi = 7.11), and a very low affinity for
the m2, m3, and m5 subtypes (pKi < 6.0)
(Jolkkonen et al., 1994). The distribution of m4 receptors in the rat
brain has been studied by using radiolabeled
[125I]MT3 (Adem et al., 1995; Adem and
Karlsson, 1997). MT3 was found to be a potent antagonist of muscarinic
inhibition of rat striatal adenylyl cyclase activity, a putative
m4-mediated response (Olianas et al., 1996). Thus, MT3 appeared to be a
potent and selective antagonist of m4 receptors and a unique tool with
which to investigate m4 receptor function.
In the present study, we examined the receptor subtype selectivity of
MT3 in functional assays of the cloned human m1-m4 receptors and of
the native m4 receptor expressed in two cell lines: the N1E-115
neuroblastoma and the NG108-15 neuroblastoma × glioma hybrid.
| |
Experimental Procedures |
Materials.
[
-32P]ATP (30-40 Ci/mmol),
[2.8-H3]cAMP (25 Ci/mmol), and
[35S]guanosine-5'-O-(3-thio)triphosphate
([35S]GTP
S) (1306 Ci/mmol) were obtained from New
England Nuclear-Du Pont (Bad Homburg, Germany).
[3H]N-methylscopolamine (NMS) (83 Ci/mmol)
was from Amersham (U.K.). MT3 was purified from the venom of
Dendroaspis angusticeps as described previously
(Jolkkonen et al., 1994). Forskolin and GTPS were from Calbiochem
(La Jolla, CA). Himbacine was a generous gift of Prof. W. C. Taylor (Department of Organic Chemistry, University of Sydney,
Australia). Pituitary adenylate cyclase activating polypeptide (PACAP)
38 was purchased from Peninsula Laboratories (Merseyside, U.K.). ACh,
carbachol chloride (CCh), physostigmine hemisulfate, and the other
reagents used were from Sigma Chemical (St. Louis, MO).
Cell Culture and Membrane Preparation.
CHO cells stably
expressing the cloned human m1-m4 receptors were kindly provided by
Prof. A. D. Strosberg (Institut Cochin de Genetique Moleculaire,
Paris, France). The cells were grown as a monolayer culture in Ham's
F-12 medium (GIBCO BRL) supplemented with 10% fetal calf serum (GIBCO
BRL) in a humidified atmosphere (5% CO2) at 37°C. Cells
were grown to ~80% confluency in plastic Petri dishes (Falcon), the
medium was removed, and the cells were washed with ice-cold
phosphate-buffered saline. The cells were then scraped into an ice-cold
buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/NaOH and 1 mM EDTA (pH 7.4) and lysed with a Dounce tissue
grinder. The cell lysate was centrifuged at 1000g for 2 min at 4°C. The supernatant was collected and centrifuged at
32,500g for 30 min at 4°C. The pellet was resuspended
in homogenization buffer to a protein concentration of ~3.0 mg/ml.
The membrane preparations were either used immediately or stored at
70°C.
N1E-115 neuroblastoma and NG108-15 neuroblastoma × glioma cell
lines were obtained from European Collection of Cell Cultures (U.K.).
N1E-115 cells were grown in Dulbecco's modified Eagle's medium
containing 2 mM glutamine and 10% fetal calf serum, whereas NG108-15
cells were grown in the same medium supplemented with 10% HAT (100 µM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine). Cells
were grown in 75-cm2 flasks (Falcon) in the
presence of 20 to 30 ml of medium, which was changed on day 2 of
subculture and every subsequent day. When cells reached confluency
(6-8 days), the medium was removed and the cells were washed with
ice-cold phosphate-buffered saline. The cells were then scraped into an
ice-cold buffer containing 10 mM HEPES/NaOH, 1 mM ethylene glycol
bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, and 1 mM MgCl2 (pH 7.4) and lysed with a
Dounce tissue grinder. The cell lysate was centrifuged at
1000g for 2 min at 4°C, and the supernatant was collected
and centrifuged at 32,500g for 30 min at 4°C. The pellet
was resuspended in homogenization buffer to a protein concentration of
2.0 to 3.0 mg/ml. The membrane preparations were either used
immediately or stored at 70°C.
Assay of [35S]GTPS Binding.
CHO cell
membranes were diluted 10-fold in 10 mM HEPES/NaOH, 1 mM EDTA, and
0.1% bovine serum albumin (BSA) (pH 7.4); centrifuged; and resuspended
in the same buffer. The binding of [35S]GTPS was
assayed in a reaction mixture (final volume, 100 µl) containing 25 mM
HEPES/NaOH (pH 7.4), 10 mM MgCl2, 1 mM EDTA, GDP (0.1 µM
for m1 and m3 and 1 µM for m2 and m4 receptor activities), 100 mM
NaCl, 10 kallikrein inhibitor units (KIU) of aprotinin, and 1.0 to 1.5 nM [35S]GTPS. The incubation was started by adding the
membrane suspension (1.5-2.0 µg of protein) and was carried out at
30°C for 60 min. The incubation was terminated by adding 5 ml of
ice-cold buffer containing 10 mM HEPES/NaOH (pH 7.4) and 1 mM
MgCl2, immediately followed by rapid filtration through
glass-fiber filters (Whatman GF/C) presoaked in the same buffer. The
filters were washed twice with 5 ml of buffer, and the radioactivity
trapped was determined by liquid scintillation spectrometry.
Nonspecific binding was determined in the presence of 100 µM GTPS.
Assays were performed in duplicate.
Assay of Adenylyl Cyclase Activity.
The enzyme activity was
assayed in a reaction mixture (final volume, 100 µl) containing 50 mM
HEPES/NaOH (pH 7.4), 2.3 mM MgCl2, 0.3 mM ethylene glycol
bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 0.05 mM [-32P]ATP (150-200 cpm/pmol), 0.5 mM
[3H]cAMP (160 cpm/nmol), 100 µM GTP, 1 mM
3-isobutyl-1-methylxanthine, 5 mM phosphocreatine, 50 units/ml creatine
kinase, 50 µg of BSA, 10 µg of bacitracin, 10 KIU of aprotinin, and
10 µM physostigmine. The incubation was started by adding the tissue
preparation (30-40 µg of protein) and carried out at 25°C for 20 min. [32P]cAMP was isolated according to Salomon et al.
(1974).
Assay of [3H]NMS.
The binding of
[3H]NMS to NG108-15 cell membranes was performed in an
incubation mixture (final volume, 0.5 ml) containing 50 mM Tris·HCl,
0.5 mM EDTA, 100 KIU/ml aprotinin, and 0.1% BSA (pH 7.4). Membrane
protein concentration was 100 to 150 µg/ml. In saturation binding
assay, the [3H]NMS concentration ranged from 10 pM to 3 nM, whereas in competition experiments, the radioligand concentration
was 0.5 nM. Binding assays were performed at 32°C for 120 min.
Nonspecific binding was determined in the presence of 1 µM atropine.
The binding of [3H]NMS to CHO/m4 cell membranes
was assayed in a buffer containing 25 mM sodium phosphate buffer (pH
7.4), 5 mM MgCl2, 100 KIU/ml aprotinin, 0.1%
BSA, and 8 to 10 µg of membrane protein. The concentration of
[3H]NMS was 0.5 nM, and the incubation volume
was 0.5 ml. The incubation was performed at 30°C for 90 min.
Nonspecific binding was determined in the presence of 1 µM atropine.
The incubation was stopped by adding 4 ml of ice-cold buffer (without
aprotinin and BSA) to each sample followed by immediate filtration
through GF/C filters presoaked in 0.1% polyethylenimine for 
18 h.
The filters were washed twice with the same buffer and dried, and the
bound radioactivity was counted by liquid scintillation. Binding data
were analyzed by the computer program LIGAND (Munson and Rodbard,
1980).
Reverse Transcription-Polymerase Chain Reaction Analysis of
Muscarinic Receptor Subtypes.
Total RNA was extracted from
NG108-15 and N1E-115 cells by using TRIzol (GIBCO BRL) according to the
manufacturer's protocol. The RNAs were first treated with 5 units of
RNase-free DNase (Boehringer-Mannheim) for 30 min at 37°C in
the presence of RNase inhibitor (GIBCO BRL). First-strand cDNA was
synthesized by using 2 µg of total RNA and SuperScript II reverse
transcriptase (GIBCO BRL) in a final volume of 20 µl containing 0.5 µg of oligo(dT)12-18 primers, 10 mM dithiothreitol, 0.5 mM deoxynucleotide triphosphates, 50 mM Tris·HCl (pH 8.3), 3 mM
MgCl2, and 75 mM KCl. The amplification reaction mixture
(final volume, 50 µl) contained 20 mM Tris·HCl (pH 8.4), 50 mM KCl,
2 mM MgCl2, 0.2 mM concentration of deoxynucleotide triphosphates, 20 pmol of each primer, 2 µl of cDNA, and 1.25 units
of Taq DNA polymerase (GIBCO BRL). The primers used to
identify the m2 and m4 receptor subtypes were taken from Drescher et
al. (1992) and corresponded to sequences 633 to 653 and 1184 to 1164 of
the rat m2 receptor DNA and to sequences 543 to 566 and 1052 to 1029 of
the rat m4 receptor cDNA. Polymerase chain reaction (PCR) was performed
by an initial denaturation at 94°C for 2 min, followed by 35 cycles
at 94°C for 45 s, 55°C for 45 s, 72°C for 60 s,
and a final extension at 72°C for 10 min. PCR products (10 µl) were
resolved on 1.6% agarose gels and visualized by UV illumination after
ethidium bromide staining.
Protein content was determined by the method of Bradford (1976), with
BSA as a standard.
Statistical Analysis.
Results are given as mean ± S.E.
Concentration-response curves were analyzed by a least-squares
curve-fitting computer program (GraphPAD Prism, San Diego, CA).
Antagonist effects of MT3 were examined according to
Arunlakshana-Schild analysis (Arunlakshana and Schild, 1959), and the
potencies were determined from the ratios between the EC50
values of the agonist in the absence and in the presence of different
concentrations of the antagonist. The pA2 values were
calculated by using the PHARM/PCS program of Tallarida and Murray
(1987). In other experiments where the effect of a single concentration
of antagonist was examined, the inhibition constant
(Ki) was calculated from the equation:
|
(1)
|
where EC50a and
EC50b are the concentrations of the agonist
producing half-maximal effect in the absence and in the presence of the
antagonist, respectively, and I is the antagonist
concentration. MT3 was also examined for its ability to completely
reverse the agonist effect. Experiments were therefore performed in
which the effects of multiple concentrations of MT3 on the response elicited by a fixed concentration of the agonist were determined. The
data were analyzed as competition curves by nonlinear regression analysis for models of one or two noninteracting sites. The MT3 inhibition constants were calculated according to the equation (Cheng
and Prusoff, 1973):
|
(2)
|
where IC50 is the concentration of
antagonist producing half-maximal inhibition, A is the
agonist concentration, and EC50 is the
concentration of the agonist producing half-maximal effect. For
comparison with pA2 values, the
Ki values were converted to negative
logarithmic form (pKi). Statistical
significance of the difference between means was determined by
Student's t test.
| |
Results |
Effects of MT3 on [35S]GTPS Binding to CHO/m1-m4
Cell Membranes.
In membranes of CHO/m1, m2, m3, and m4 cells, ACh
elicited a concentration-dependent increase of
[35S]GTPS binding with EC50 values of
8.5 ± 0.8, 0.28 ± 0.03, 9.0 ± 0.6, and 1.0 ± 0.08 µM, respectively (Fig. 1). The
maximal stimulatory effects corresponded to 71 ± 8%, 205 ± 15%, 25 ± 1.2%, and 248 ± 18% increase in basal value,
respectively. In CHO/m1 cells, MT3 (0.1-2.0 µM) antagonized the ACh
response with a pA2 value of 6.78 ± 0.08 and a slope
of 0.97 ± 0.05. In CHO/m2 cells, the toxin failed to produce a
significant shift in the agonist curve at concentrations up to 0.5 µM. Conversely, the M2 antagonist himbacine (100 nM)
increased the EC50 of ACh by 9-fold, a shift yielding a
pKi of 7.91 ± 0.1. In CHO/m3 cells,
MT3 (0.1-2.0 µM) was a weak antagonist of the ACh effect with a
pA2 value of 6.30 ± 0.03 and a slope of 0.96 ± 0.08. In contrast, in CHO/m4 cells, the toxin, tested at concentrations
ranging from 15 to 500 nM, potently antagonized the ACh stimulation
with a pA2 of 8.33 ± 0.05 and a slope value of
1.01 ± 0.08. At each muscarinic receptor subtype investigated,
MT3 per se failed to affect [35S]GTPS binding.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of MT3 on ACh stimulation of
[35S]GTPS binding to membranes of CHO cells expressing
the cloned human m1 to m4 receptors. [35S]GTPS binding
was determined at the indicated concentrations of ACh in the absence
( ) and in the presence of: MT3 100 ( ), 500 ( ), and 2000 ( )
nM for the m1; MT3 100 () and 500 () nM and himbacine 100 nM
() for the m2; MT3 100 (), 500 (), and 1500 () nM for the
m3; and MT3 15 (), 100 (), and 500 () nM for the m4 receptor.
Data are the mean ± S.E. of four experiments for m1 and m3 and
three experiments for m2 and m4 receptors.
|
|
Effects of MT3 on Native Muscarinic Receptors Coupled to Adenylyl
Cyclase in N1E-115 and NG108-15 Cells.
In N1E-115 cells, 10 nM
PACAP 38 stimulated basal adenylyl cyclase activity by 10-fold. CCh
inhibited the PACAP-stimulated enzyme activity in a
concentrationdependent manner with an EC50 value of
1.6 ± 0.02 µM. The maximal inhibitory effect corresponded to a
25.0 ± 3.5% reduction of control activity (P < .001, n = 8). The addition of 3, 30, and 100 nM MT3 shifted to
the right the CCh curve by 6.2-, 40.4-, and 130-fold, respectively,
without affecting the maximal inhibitory effect (Fig.
2A). Arunlakshana-Schild analysis of the
MT3 antagonism yielded a pA2 value of 8.81 ± 0.1 with
a slope of 0.94 ± 0.04. Increasing concentrations of MT3 completely antagonized the inhibition of PACAP-stimulated cAMP formation elicited by 30 µM CCh (Fig. 2B). The competition curve was
monophasic with a Hill coefficient of 0.96 ± 0.07. The
pKi value of MT3 calculated according to eq.
2 was 8.33 ± 0.08.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
MT3 antagonism of muscarinic inhibition of PACAP
38-stimulated adenylyl cyclase activity in N1E-115 cells. A, the enzyme
activity stimulated by 10 nM PACAP 38 was assayed at the indicated
concentrations of carbachol (CCh) in the absence () and in the
presence of 3 (), 30 (), and 100 () nM MT3. Data are expressed
as percentage of the maximal enzyme inhibition elicited by CCh and
represent the mean ± S.E. of three experiments. B, the enzyme
activity stimulated by 10 nM PACAP 38 was assayed at the indicated
concentrations of MT3 in the absence and in the presence of 30 µM
CCh. Data are expressed as percentage of the CCh inhibitory effect
observed in the absence of MT3 and represent the mean ± S.E. of
three experiments. At the concentrations used, MT3 per se failed to
affect the enzyme activity.
|
|
In NG108-15 cells, CCh inhibited the forskolin-stimulated adenylyl
cyclase activity with an EC50 value of 12.5 ± 1.2 µM and a maximal effect corresponding to a 20.2 ± 1.2%
reduction in control activity (P < .001, n = 8).
MT3 antagonized the muscarinic inhibition of cAMP formation less
potently than that occurring in N1E-115 cells. Thus, the addition of 3 nM MT3 failed to affect the CCh inhibitory curve, whereas at 30, 100, and 300 nM the toxin increased the agonist EC50
value by 2.6-, 4.8-, and 28-fold, respectively (Fig.
3A). The Schild plot yielded a
pA2 value of 7.60 ± 0.09 and a slope of
1.10 ± 0.08. Moreover, in NG108-15 cells, MT3 failed to
completely reverse the CCh inhibition of adenylyl cyclase at concentrations as high as 3 µM (Fig. 3B). The MT3 inhibition curve was biphasic, with a high-affinity (pKi = 8.46 ± 0.09) and a low-affinity component
(pKi < 6.0). The high-affinity component
comprised ~60% of the CCh inhibitory effect.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
MT3 antagonism of muscarinic inhibition of
forskolin-stimulated adenylyl cyclase activity in NG108-15 cells. A,
the enzyme activity stimulated by 10 µM forskolin was assayed at the
indicated concentrations of carbachol (CCh) in the absence () and in
the presence of 3 (), 30 (), 100 (), and 300 ( ) nM MT3.
Data are expressed as percentage of the maximal enzyme inhibition
elicited by CCh and represent the mean ± S.E. of three
experiments. B, the enzyme activity stimulated by 10 µM forskolin was
assayed at the indicated concentrations of MT3 in the absence and in
the presence of 30 µM CCh. Data are expressed as percentage of the
CCh inhibitory effect observed in the absence of MT3 and represent the
mean ± S.E. of three experiments. At the concentrations used, MT3
per se failed to affect the enzyme activity.
|
|
Effects of MT3 on [3H]NMS Binding.
Saturation
experiments indicated that [3H]NMS bound to CHO/m4 cell
membranes with a KD of 0.20 ± 0.04 nM.
Increasing concentrations of MT3 completely displaced the binding of
0.5 nM [3H]NMS with a pKi
value of 8.68 ± 0.09 and a Hill slope of 1.0 (Fig.
4A). In NG-108-15 cell membranes,
[3H]NMS binding displayed a KD
of 0.17 ± 0.02 nM and a Bmax of
169 ± 25 fmol/mg protein. MT3 displaced 85% of the specific
[3H]NMS binding with a pKi of
8.4 ± 0.2 (Fig. 4B). The remaining fraction of
[3H]NMS binding sites was unaffected by toxin
concentrations up to 1 µM.
View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4.
Concentration-dependent inhibition of
[3H]NMS binding. A, the binding of [3H]NMS
to CHO/m4 cell membranes was determined as described in Experimental
Procedures at the indicated concentrations of MT3. Data are the
mean ± S.E of three experiments. B, the binding of
[3H]NMS to NG108-15 cell homogenates was determined as
described in Experimental Procedures at the indicated
concentrations of MT3. Data are the mean ± S.E. of three
experiments.
|
|
Reverse Transcription-PCR Analysis of m2 and m4 Receptor
Expression.
Amplification of cDNA transcribed from total RNA of
NG108-15 cells using primers specific for the m2 subtype yielded a band of the expected size of 552 bp (Fig. 5,
lane 2). This product was not detected in N1E-115 cells (lane 4). On
the other hand, reverse transcription (RT)-PCR analysis using primers
specific for the m4 subtype yielded a band of the expected size of 510 bp in both NG108-15 (lane 3) and in N1E-115 cell samples (lane 5). No
amplification product was obtained in samples of both cell lines when
the reverse transcriptase step was omitted (lanes 6 and 7 for NG108-15
and N1E-115, respectively).
View larger version (48K):
[in this window]
[in a new window]
|
Fig. 5.
Agarose gel electrophoresis with ethidium bromide
staining of PCR products amplified from NG108-15 (lanes 2 and 3) and
N1E-115 (lanes 4 and 5) cells, using primers specific for either the m2
(lanes 2 and 4) or the m4 (lanes 3 and 5) receptor. The expected size
of the amplification product is 552 bp for the m2 and 510 bp for the m4
receptor. Lane 1, DNA standard; lanes 6 and 7, control samples of
NG108-15 and N1E-115 cells, respectively, without reverse transcriptase
addition.
|
|
| |
Discussion |
In the present study, the muscarinic receptor selectivity of MT3
has been evaluated in functional assays of cloned and native receptors.
The agonist-induced stimulation of [35S]GTPS
binding to CHO cell membranes expressing the cloned muscarinic receptor
subtypes was used to characterize the pharmacological activity of the
toxin. This assay has previously been shown to constitute a valid
method for the analysis of the receptor response to a variety of
muscarinic agonists and antagonists (Lazareno and Birdsall, 1993;
Olianas and Onali, 1996). At each receptor subtype, MT3 failed to
stimulate [35S]GTPS binding, indicating a
lack of agonist activity. However, the toxin was able to antagonize the
receptor activation induced by ACh with a marked subtype selectivity.
In fact, MT3 blocked the m4-mediated
[35S]GTPS binding with a potency
(pA2 = 8.33) that was 38-fold higher than that
displayed at the m1 subtype (pA2 = 6.78). The
toxin failed to block the m2 receptor subtype at concentrations up to 500 nM and displayed a quite low potency (pA2 = 6.3) in counteracting the activation of the m3 subtype. Both in terms
of absolute values and affinity differences, the selectivity profile
determined in the functional studies agrees well with that previously
observed in radioligand binding studies (Jolkkonen et al., 1994). At
the concentrations used, the inhibitory activity of MT3 was
characterized by a progressive displacement to the right of the agonist
concentration-response curve without depression of the maximal
response. Moreover, the slopes of the Schild plots were not
significantly different from unity. These data indicate that the toxin
behaved as a competitive antagonist.
As the m4 selectivity of MT3 was assessed in radioligand binding
(Jolkkonen et al., 1994) and functional (present work) studies using
the cloned receptor subtypes overexpressed in host cells, it was of
interest to investigate whether the toxin was capable of recognizing
the m4 receptor with high affinity in cells expressing the receptor in
a native membrane environment. We have therefore considered two cell
lines, the N1E-115 and NG108-15 cells, which have previously been shown
to express the mRNA encoding the m4 receptor (Fukuda et al., 1988;
Peralta et al., 1988; McKinney et al., 1991). In addition, in both cell
lines, the activation of the m4 receptor has been demonstrated to cause
inhibition of cAMP accumulation (Baumgold and White, 1989; McKinney et
al., 1991). Accordingly, we found that CCh elicited a significant
inhibition of PACAP 38- and forskolin-stimulated adenylyl cyclase
activities in membranes of N1E-115 and NG108-15 cells, respectively.
MT3 antagonized the CCh inhibitory effects in the two cell lines with a
different pattern. In N1E-115 cells, the toxin counteracted the
muscarinic response with high potency (pA2 = 8.81) and generated monophasic inhibitory curves with a complete
reversal of the CCh effect. These data are consistent with the
involvement of a homogeneous population of m4 receptors. Conversely, in
NG108-15 cells, the Schild plot of the MT3 antagonism yielded a
pA2 of 7.6, which appeared too low for a
selective action on m4 receptors. The analysis of the toxin inhibitory
curve revealed the presence of two components in the CCh effect: one
blocked by the toxin with a potency (pKi = 8.46) comparable to the affinity for the m4 subtype, and another not
reversed by MT3 at a concentration as high as 3 µM. A possible explanation of these findings is that in NG108-15 cells, the muscarinic inhibition of cAMP is mediated not only by m4 but also by another receptor subtype, possibly the m2. The presence of a receptor population relatively insensitive to MT3 was also demonstrated by the
data obtained in radioligand binding studies showing that MT3 was
unable to completely displace [3H]NMS bound
with high affinity. To investigate the possibility that NG108-15 cells
express the m2 in addition to the m4 receptor, RT-PCR analysis was
conducted using primers specific for the two receptor subtypes. The
results indicate that the NG108-15 cells contain the mRNA for both m2
and m4 subtypes, whereas the N1E-115 cells lack the mRNA for the m2
subtype. The failure of previous studies (Peralta et al., 1987; Fukuda
et al., 1988) to detect the expression of m2 mRNA in NG108-15 cells by
Northern blot analysis may be attributed to the lower sensitivity of
the latter assay compared with RT-PCR. It is noteworthy that
immunological studies using subtype-selective antisera have previously
demonstrated the expression of both m4 and m2 receptors in NG108-15
cells (Yasuda et al., 1993). In addition, Akiyama et al. (1984)
reported that pirenzepine, a drug with higher affinity for
M1 and M4 than for M2 receptors (Caufield, 1993), inhibited
[3H]quinuclidinyl benzilate binding to NG108-15
cell lysate according to a two-site model, with 72% of the labeled
sites displaying a high affinity for pirenzepine. This heterogeneity of
binding sites was not observed in other studies (Evans et al.,
1984; Baumgold and White, 1989; Michel et al., 1989). However,
the presence in NG108-15 cells of M2 sites was
postulated by Lazareno et al. (1990) on the basis of kinetic and
equilibrium radioligand binding data.
In conclusion, the present study shows that in functional studies, MT3
is capable of recognizing both the cloned and the native m4 receptors
with high affinity and selectivity. Moreover, the data demonstrate that
in cells where both m4 and m2 receptors may regulate a common response,
the toxin constitutes a powerful tool for determining the relative
contribution by each receptor subtype.
The authors thank Prof. A. D. Strosberg (Institut Cochin de
Genetique Moleculaire, Paris, France) for the gift of CHO cells transfected with the human muscarinic receptor genes.
ACh, acetylcholine chloride;
CCh, carbachol
chloride;
CHO, Chinese hamster ovary;
BSA, bovine serum albumin;
EGTA, ethylene glycol bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid;
GTPS, guanosine-5'-O-(3-thio)triphosphate;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
MT3, muscarinic toxin 3;
NMS, N-methylscopolamine;
PACAP, pituitary adenylate cyclase activating polypeptide;
RT, reverse
transcription;
PCR, polymerase chain reaction.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
