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Vol. 59, Issue 5, 1029-1036, May 2001
Research Center, Montreal Heart Institute, Montreal, Quebec, Canada (H.W., H.H., L.Z., H.S., G.S., S.N., Z.W.); Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada (S.N., Z.W.); and Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada (S.N.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified by molecular cloning and designated m1-m5, of which four correspond to the functional subtypes M1, M2, M3, and M4 in primary tissues. The presence of M5 receptors in tissues remains uncertain. The present study was designed to explore the diversity and cellular distribution of various mAChR subtypes in human hearts. Competition binding of [N-methyl-3H]-scopolamine methyl chloride with various mAChR antagonists yielded data consistent with the presence of multiple subtypes (M1/M2/M3/M5) of mAChRs in both human atrial (HA) and ventricular (HV) tissues. Expression of mRNAs encoding all five subtypes was readily detected by reverse transcription-polymerase chain reaction in both HA and HV samples. Immunoblotting with subtype-specific antibodies confirmed the presence of M1, M2, M3, and M5, but not M4, proteins in membrane preparations from both HA and HV. The protein levels of M1 and M2 were comparable between HA and HV. Although the density of M3 appeared ~10-fold higher in HV than HA, that of M5 was ~5 times lower in HV than in HA. Positive immunostaining of single ventricular myocytes by M1, M2, M3, and M5 antibodies, respectively, was consistently detected. Under confocal microscopy, M5 showed characteristic localization to the intercalated discs, whereas other subtypes were more evenly distributed throughout the surface membrane. Our results provide the first molecular evidence for the presence of multiple subtypes of mAChR, including endogenous M5 receptors, in human hearts and suggest that different subtypes have different tissue distributions and cellular localization.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Not
until the 1990s were multiple subtypes (M1,
M2, M3, and
M4) of mAChRs functionally defined in primary
tissues with the development of pharmacological probes (Doods et al.,
1987
; Mitchelson, 1988; Goyal, 1989; Mutschler et al., 1989; Hulme et
al., 1990; van Zwieten and Doods, 1995; Eglen and Watson, 1996). cDNAs
representing five different isoforms of mAChR subunits,
m1 through m5, have been
cloned from a variety of mammals, including humans (Bonner et al.,
1987; Peralta et al., 1987; Goyal, 1989; Brann et al., 1993).
M1-M4 receptors seem to
correspond to the cloned
m1-m4 isoforms, whereas
the physiological counterpart of m5 is yet to be established.
Muscarinic receptor stimulation by acetylcholine plays an important
role in mediating parasympathetic control of cardiac functions such
because heart rate, conduction, and contractility. In contrast to most
peripheral tissues, the myocardium has generally been considered to
possess a single mAChR subtype: M2 receptors have long been believed to be the only functional mAChR subtype in the heart
(Bonner et al., 1987; Peralta et al., 1987; Dörje et al., 1991;
Pappano, 1991). In fact, the heart is commonly used because a model
illustrating the exclusive presence of M2
receptors. However, the concept that the heart possesses a single
M2 subtype of mAChR is now being challenged. Many
physiological responses to mAChR stimulation cannot be explained on the
basis of a single M2 subtype in the heart.
Accumulating evidence suggests the existence of mAChR subtypes other
than M2 in cardiac tissues of chickens, rats,
guinea pigs, rabbits, and dogs (Jaiswal et al., 1989; Akahane et al.,
1990; Tietje and Nathanson, 1991; Ford, 1992; Yang et al., 1992; Gadbut
and Galper, 1994; Kan et al., 1996; Sharma et al., 1996, 1997; Sun et
al., 1996), with large variations of subtype expression among species.
The cDNA of the m5 muscarinic receptor was first
cloned over a decade ago. In artificial expression systems,
m5 receptors have been shown to couple to
multiple signaling mechanisms, including stimulation of phospholipase
C-protein kinase C, phospholipase A2-arachidonic
acid, and mitogen-activated protein kinase; inhibition of cAMP
production; activation of nitric-oxide synthase; and intracellular calcium mobilization (Kohn et al., 1996; Reever et al., 1997; Kukkonen
et al., 1998; Wotta et al., 1998). The physiological significance of
the m5 subtype remains a mystery because of a lack of m5-selective ligands and a lack of
evidence that primary tissues express the M5
receptor. Only recently has the presence of native
M5 receptors in the brain been suggested by
pharmacological approaches (Reever et al., 1997).
We recently reported functional and molecular evidence for the presence
of M3 and M4 receptors in
canine (Shi et al., 1999a,b; Wang et al., 1999b) and
M3 receptors in guinea pig atrial myocytes (Shi
et al., 1999b; Wang et al., 1999b). We found these receptors to be
functionally coupled to two novel and distinct K+
channels. Activation of M3 receptors in guinea
pig atrial preparations promotes membrane repolarization and slows
sinus rate (Shi et al., 1999b; Wang et al., 1999b). Whether the human
heart possesses mAChRs other than M2 remains
unknown. To date, no systematic studies have examined mAChR subtypes in
the human heart. We therefore examined the expression of various mAChR
subtypes in human hearts by analyzing pharmacological properties
(radioligand binding displacement), mRNA expression (RT-PCR), tissue
protein concentrations (Western blot), and cellular protein
distribution (immunostaining/confocal microscopy).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Membrane Protein Preparation. Specimens of human right atrial appendage (HA) were obtained from the hearts of four patients undergoing aortocoronary bypass surgery. The atria were from patients without heart failure, atrial arrhythmias, or electrocardiographic P-wave abnormalities and were grossly normal in appearance. Left ventricular tissues (HV) were dissected from the explanted hearts of four patients (aged 57-71) receiving heart transplantations. The procedure for obtaining the tissue was approved by the Ethics Committee of the Montreal Heart Institute.
The procedures for membrane protein preparation have been described previously (Shi et al., 1999a,b; Wang et al., 1999b). The preparations
were minced and washed with ice-cold phosphate-buffered saline (PBS)
buffer. The tissues were then homogenized with a Polytron in 15 ml of
ice-cold lysis buffer containing 5 mM Tris-HCl, 2 mM EDTA, pH 7.4, and
a protease inhibitor cocktail consisting of 5 mg/ml
phenylmethylsulfonyl fluoride, 10 mg/ml benzamidine, and 5 mg/ml
soybean trypsin inhibitor. The homogenate was centrifuged at
500g for 15 min at 4°C. The pellets were then homogenized
as before, spun again, and the supernatants pooled. The supernatants were centrifuged at 45,000g for 15 min and the pellets
washed twice in the same buffer. The membrane fractions were
resuspended in a buffer containing 75 mM Tris-HCl, pH 7.4, 12. 5 mM
MgCl2, and 5 mM EDTA. The protein content was
determined with Bio-Rad Protein Assay kit (Bio-Rad, Mississauga, ON,
Canada) using bovine serum albumin as the standard. Chemicals were
purchased from Sigma Chemical Co. (St. Louis, MO).
Membrane Receptor Binding Assay.
Saturation binding assays
were performed using eight concentrations of
[N-methyl-3H]scopolamine methyl
chloride ([3H]NMS, 82 Ci/mmol) ranging from 2 to 2500 pM. Binding measurements were obtained in triplicate for each
experiment with total of eight individual preparations (from four atria
and four ventricles). Incubations at a volume of 1 ml (90 min at room
temperature) were terminated by rapid filtration with Whatman GF/C
filters (Xymotech, Montreal, PQ, Canada), and radioactivity was counted
with an LS6500 Scintillation Counter (Beckman, Fullerton, CA).
Nonspecific binding was defined as that measured in the presence of 1 µM atropine. Specific binding (averaging ~90% of total binding)
was determined by subtracting nonspecific from total binding (Shi et
al., 1999a,b; Wang et al., 1999).
,b; Wang et al., 1999b): Homogenates were incubated with 400 pM
[3H]NMS and pirenzepine (0.3 nM-100 µM),
methoctramine (0.3 nM-100 mM),
4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 0.1 nM-100 µM), or tropicamide (1 nM-1 µM), respectively. In
addition, choline chloride (10 nM-10 mM) and
tetramethylammonium (30 nM-1 mM), which have been shown to
activate M3 receptors in canine myocardium (Shi
et al., 1999a,b), were also studied in competition binding experiments.
A fixed amount of membrane protein (100 µg) was used for each binding
study. Reagents for binding experiments were purchased from
RBI/Sigma (Natick, MA).
RT-PCR.
RNA was isolated as described previously (Wang et
al., 1998, 1999a; Shi et al., 1999; Yue et al., 1999). RNA
samples were isolated from adjacent tissues of the same hearts used for
membrane protein extraction. Total RNA samples extracted from human
atrial and ventricular tissues were incubated with DNase I (0.1 units/ml) at 37°C for 15 min, and this was followed by
phenol/chloroform extraction to remove genomic DNA.
; Bonner
et al., 1988), and unique oligonucleotide sequences were chosen from
cDNA regions with minimal homology among different mAChR isoforms. The
specificity of primer pairs was verified by comparison with the entire
GenBank database using BLAST and by amplification of a PCR product of
the predicted size visible as a single discrete band with ethidium
bromide staining on an agarose gel.
Western Blot Analysis.
Membrane proteins (60 µg) were
fractionated by SDS-polyacrylamide gel electrophoresis (10%
polyacrylamide gels) and transferred to nitrocellulose membranes
(Immobilon-P, pore size 0.45 mm; Millipore Corporation, Bedford, MA) in
a transfer buffer (25 mM Tris-base, 192 mM glycine, 20% methanol, and
0.01% SDS) (Wang et al., 1999a; Yue et al., 1999). After
transfer, the membrane was washed in Tris-buffered saline (Tris-HCl,
NaCl, distilled H2O, pH 7.5) with 0.05% Tween 20 for 10 min and then incubated in a blocking buffer containing 5%
nonfat dry milk in 0.1% Tris-buffer saline/Tween 20 (Tris-buffered + 0.1% Tween 20) for 2 h, followed by overnight incubation at 4°C
with the primary antibody. The M1,
M2, M3, and M5 antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA) and the M4
antibody from Chemicon International (Temecula, CA). The next day, the
membrane was washed three times in 0.1% Tris-buffered saline/Tween 20 (10 min/wash) and incubated for 2 h with the secondary antibody
(avidin-horseradish peroxidase-conjugated goat anti-mouse IgG for
M4 and goat anti-rabbit IgG for other subtypes)
in the blocking buffer. The membrane was then washed in the blocking
buffer for 15 min. Bound antibodies were detected using
chemiluminescent substrate (Western Blot Chemiluminescence Reagent
Plus; PerkinElmer Life Science Products, Boston, MA). A rat brain
protein sample (20 µg) was used as a positive control for mAChRs and
negative controls were performed by preincubating the antibodies with
the respective peptides against which they are generated. The presence
of a given subtype of mAChR was verified by the presence of a prominent
band with molecular mass in the range of previous reports and by
elimination of the band in preparations preincubated with the antigenic
peptide. Potential cross-reaction of antibodies was excluded using
purified M1, M2,
M3, and M5 receptors obtained from Santa Cruz Biotechnology. Coomassie staining was performed to verify the amount of protein inputs by incubate the membranes in Coomassie Brilliant Blue (prepared as a working solution in 50% methanol and 10% acetic acid solution) for 2 min and then washed to remove the background. Experiments were discarded if any
visible differences between samples for comparison (HA versus HV) were found.
Immunocytochemistry. Human ventricular myocytes were isolated from the explanted hearts of three patients receiving heart transplantation, using methods described previously in detail (Li et al., 1995). A segment of the left ventricular free wall containing a coronary artery was perfused via a Langendorff-type system with Ca2+-containing Tyrode's solution at 37°C until the effluent was clear of blood. The perfusate was then changed to Ca2+-free Tyrode's solution for 20 min at 12 ml/min, followed by perfusion with the same solution containing collagenase (110 U/ml CLS-II collagenase; Worthington Biochemical, Freehold, NJ) and 0.1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO). The Tyrode's solution contained 136 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4, 5 mM HEPES, 10 mM glucose, and 1 mM CaCl2; pH was adjusted to 7.4 with NaOH.
The dispersed cells were washed twice with fresh PBS and plated on laminin (15 µg/ml)-coated coverslips in the wells of culture dishes containing Dulbecco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (10 mg/ml penicillin and 1 mg/ml streptomycin). Cells were incubated at 37°C for 1 h. The culture medium was then discarded and the cells were incubated with 1 ml of paraformaldehyde (1%, pH 7.4) for 30 min. After two washes with PBS, the cells were treated with Triton (1%) for 5 min to permeabilize cell membranes and then incubated overnight with 1% bovine serum albumin at 4°C. The following day, the cells were exposed to primary anti-mAChR antibodies for 2 h. After a PBS wash, the cells were incubated in secondary antibody for 1 h. Coverslips were then placed onto glass slides and sealed with mounting medium for confocal microscopy.Data Analysis.
Group data are expressed as the mean ± standard error. Binding data were analyzed using curve-fitting
functions in GraphPad Prism software (GraphPad Software, San Diego,
CA). Linear regression was performed on the percentage of bound versus
the ratio of bound over free ligand, and only data with a regression
coefficient of 
0.9 were used for analyses. The F test was
used to compare fits for the competition binding data, and the best fit
(one-site binding versus two-site binding) was determined by the
probability value for the F test and by the change in the
residual sum of squares for the two different fits. One- and
two-site models were tested for all data sets, and the model yielding
the least residual sum of squares was taken to describe the data.
Densities of the immunoblot bands were quantified with the use of a
Molecular Imager System (GS-505; Bio-Rad).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pharmacological Identification of Multiple Subtypes of mAChR in
Human Hearts.
[3H]NMS binding to membrane
homogenates from human atrial and ventricular tissues was saturable
over the concentration range (2-2500 pM) examined. As illustrated in
Fig. 1, the experimental data were well
fitted by a one-site binding model. Mean Kd
values were 220 ± 20 pM for HA and 259 ± 29 pM for HV, and
the Bmax value was 280 ± 40 fmol/mg
protein for HA and 259 ± 51 fmol/mg protein for HV
(n = 4/group). A Scatchard analysis of saturation
binding data (Fig. 1A, inset) was linear, consistent with binding to a single class of receptor sites.
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; van Zwieten and Doods, 1995), methoctramine (M2-selective) (Michel and Whiting, 1988; van
Zwieten and Doods, 1995), 4-DAMP (M3-selective)
(Barlow and Shepherd, 1986; Michel et al., 1989; Araujo et al., 1991;
van Zwieten and Doods, 1995), and tropicamide
(M4-selective) (Lazareno et al., 1990; Lazareno and Birdsall, 1993). The data from each curve gave the best fit to a
two-site binding model (Fig. 1). Table
1 provides
pKi values, together with the percentage of
total binding represented by the high-affinity site for
each antagonist. Comparison with previously published values of the
relative affinities for these antagonists (Doods et al., 1987;
Mitchelson, 1988; Goyal, 1989; Mutschler et al., 1989; Hulme et al.,
1990; Lazareno et al., 1990; Lazareno and Birdsall, 1993; van Zwieten
and Doods, 1995; Eglen and Watson, 1996) suggests the presence of
multiple mAChR subtypes
(M1/M2/M3/M5).
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Expression of mRNAs Encoding Different mAChR Isoforms in Human
Hearts.
RT-PCR showed the presence of mRNA corresponding to all
five mAChR isoforms
(m1-m5) in HA (Fig.
2A). Note bands at the expected molecular
weights for m1 [275 base pairs (bp), lane 1],
m2 (297 bp, lane 3), m3
(432 bp, lane 5), m4 (257 bp, lane 7), and
m5 (391 bp, lane 9). Similar results were
obtained from the other three mRNA samples from HA and from a total of
three HV samples. Note also the absence of bands in RT-negative
controls (laness 2, 4, 6, 8, and 10).
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4 subunit (for
neural contamination) and the maxi-K channel
(Ca2+-activated K+ channel,
for vascular contamination) (Wang et al., 1998, 1999a). Primers
for 4 and maxi-K detected strong signals in
RNA samples extracted from rat brain and rat vascular smooth muscle,
respectively (Fig. 2B). The absence of corresponding signals with
cardiac RNA samples excludes significant neural and vascular tissue
contamination of cardiac samples (Dixon and McKinnon, 1994; Wang et
al., 1998).
Immunoblotting of mAChR Proteins in Human Hearts.
To clarify
whether multiple mAChR subtypes also express at the protein level in
human hearts, immunoblotting analysis was performed with fractionated
HA and HV membrane proteins. Western blots with the
anti-m1, m2,
m3, or m5 antibody revealed
prominent protein bands at 
100, 84, 113, and 80 kDa, respectively
(Fig. 3A). The bands were eliminated when
the antibodies were preincubated with the respective peptides against
which they were generated. Similar results were consistently obtained
with all four preparations from different patients. The presence of
proteins representing M1,
M2, M3, and
M5 receptors were also consistently revealed in all three HV samples with band sizes identical to the respective subtypes found in the HA preparations. M4
receptors were detected neither in HA nor in HV, even with increased
quantities of both antibody and membrane preparation (up to 150 µg/reaction). In contrast, the anti-m4 antibody
labeled a clear band representing M4 receptors in
the rat brain preparation, the positive control. For
M1-3 and M5 receptors, the
band sizes of the positive (rat brain) controls were in the same range
as for the corresponding subtypes in the human heart.
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Immunostaining of mAChRs in Isolated Human Ventricular
Myocytes.
To investigate further the specific cellular
localization of various mAChR subtypes in human heart cells, we
performed immunocytochemistry with isolated human ventricular myocytes.
Figure 4 presents typical examples of
such experiments, showing the green fluorescence staining of various
subtypes of mAChR under confocal microscopy. Consistent with Western
blot studies, cells exposed to antibodies against M1, M2,
M3, and M5 receptors showed
clear sarcolemmal staining; whereas antibody against
M4 receptors failed to produce any positive signals. M5 receptor staining seemed to be
largely restricted to the intercalated discs, whereas other receptor
subtypes were evenly distributed along the surface membrane.
M3 receptors also demonstrated stronger staining
on the intercalated discs relative to other regions of the plasma
membrane. Nonspecific and subtype cross-reactivities were excluded by
the elimination of staining after pretreatment of the antibodies with
their respective antigenic peptides.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present study, we obtained several lines of evidence suggesting that multiple subtypes (M1/M2/M3/M5) of mAChR coexist in human hearts. Our results provide the first evidence for the presence of the endogenous M5 receptor in mammalian hearts and suggest that different mAChR subtypes have distinct distributions in HA versus HV and characteristic cellular localizations.
Evidence for the Presence of Multiple Subtypes of mAChR in Human
Hearts.
The results from our competition binding experiments
suggest that besides the long-recognized M2
receptor, M1 and M3
subtypes are also expressed in the membrane of human cardiac cells. At present, there is no selective M5 receptor
antagonist. Thus, the data from binding experiments neither confirm nor
exclude the presence of M5 receptors. The
displacement of [3H]NMS binding by pirenzepine
in HA yielded pKi values of 8.4 and 6.3 for
high- and low-affinity binding, respectively (Table 2). The
high-affinity pKi (8.4) is consistent with
the previously reported binding affinity of pirenzepine for
M1 receptors (8.0) (Brann et al., 1993; van
Zwieten and Doods, 1995; Eglen and Watson, 1996) and the fractional
binding with this affinity is 28.7%, indicting a significant
participation of M1 receptors (Watson et al.,
1983; van Zwieten and Doods, 1995). On the other hand, the low-affinity
binding (pKi = 6.5) does not discriminate
among subtypes. The high-affinity binding of methoctramine apparently confirms the existence of M2 receptors (van
Zwieten and Doods, 1995), and the low-affinity binding
(pKi = 6.9) identifies but does not
distinguish M1 and M4
subpopulations. Similarly, 4-DAMP binding also revealed two groups of
mAChRs, with high-affinity binding (pKi = 9.2) consistent with 4-DAMP affinity to M3 and M1 receptors (van Zwieten and Doods, 1995) and
low-affinity binding (pKi = 8.0) typical of
4-DAMP binding to M2 receptors. Competition binding of tropicamide showed a high-affinity binding site
(pKi = 7.4), which fits well with the
existence of M3 and M2
receptors (Lazareno et al., 1990; Lazareno and Birdsall, 1993). The
low-affinity binding of tropicamide (pKi = 6.2) does not fit with previously reported binding to a specific
subtype. Taken together, the results from our binding experiments point
to the existence of M1, M2, and M3 receptors in HA. Similar results were
obtained from HV preparations. One important weakness of using receptor
antagonists is a lack of perfect specificity toward different subtypes,
although the compounds used in our study represent the best choices
available. Therefore, caution must be taken when interpreting the
binding data.
Previous Studies Related to mAChR Subtypes in Hearts.
Expression of multiple isoforms of mRNA encoding different subtypes of
mAChRs
(M1/M2/M3/M4)
in chick hearts has been reported by two groups (Tietje and Nathanson,
1991; Gadbut and Galper, 1994). Sun et al. (1996) studied the
antagonism of carbachol-induced chronotropy and inositol
monophosphate accumulation in neonatal rat ventricular myocytes.
They found that HHSiD, an M3-selective antagonist, blocked carbachol effects, whereas pirenzepine and AF-DX
116 (M1 and M2 antagonists)
had no effect. They speculated that neonatal ventricular myocytes have
a heterogeneous population of muscarinic receptors including
M2 and M3 subtypes. Sharma
et al. (1996, 1997) used single-cell PCR, subtype-specific antibodies, and the measurement of Ca2+ transients to provide
convincing molecular and functional evidence for the presence of
M1 receptors in rat ventricular myocytes. Ford et
al. (1992) analyzed mAChR-mediated phosphoinositide hydrolysis in
guinea pig atria and ventricles. The inhibition of the response to
agonist by several antagonists, including HHSiD and
p-F-HHSiD, generated an affinity profile dissimilar to the
pure M2 response, suggesting "a second
population of muscarinic sites". In the isolated rabbit heart,
acetylcholine increased prostaglandin synthesis and the effects were
inhibited by low concentration of 4-DAMP (10 nM) (Jaiswal et al.,
1989). Although the investigators considered 4-DAMP an
M2 antagonist, the concentration they studied
would probably block M3 receptors, with minimal
effects on M2 receptors. The same group (Kan et
al., 1996) has recently reevaluated the mAChR subtypes involved in
prostacyclin synthesis and the authors now believe that acetylcholine
can function via M3 receptors in ventricular
myocytes. In isolated blood-perfused dog atria, Akahane et al. (1990)
compared the inhibitory potency of 4-DAMP, AF-DX 116, and pirenzepine
for carbachol-induced negative chronotropic and inotropic responses.
They found that the potency of 4-DAMP (an
M3-preferring antagonist) was equal to that of
atropine but greater than AF-DX 116 (an
M2-preferring antagonist) and much greater than
pirenzepine (an M1-selelctive inhibitor),
suggesting a role of the M3 subtype. Yang et al.
(1992) performed a detailed pharmacological characterization of mAChR
subtypes in membrane homogenates from dog left ventricular tissues.
Their data favored the existence of M2 and
M3 subtypes, and argued against the presence of
M1 receptors. Because of the imperfect
selectivity of available pharmacological probes, none of these studies
provided clear and unequivocal definitions of mAChR subtype distribution.
suggested, based on binding data, that there is a single subtype of
mAChR (M2) in HA and HV. However, they used only
two subtype-selective antagonists, pirenzepine
(M1) and AF-DX 116 (for
M2).
Potential Significance.
One of the major innovations in the
field of the cholinergic nervous system was the discovery of multiple
subclasses of muscarinic receptors. Although many cellular responses to
mAChR stimulation are mediated by the various subtypes of mAChR,
M2 receptors are commonly believed to be the only
functional mAChR subtype in the heart (Dörje et al., 1991; Gadbut
and Galper, 1994; Mizushima et al., 1987; Pappano, 1991; Tietje and
Nathanson, 1991). In many studies, the heart is often taken as a model
for the exclusive presence of M2 receptors. The
present study, in conjunction with previous findings in other species,
strongly suggests that multiple subtypes, and not just
M2 receptors, coexist in mammalian hearts. It
seems that the one-receptor (M2) concept needs to
be revised and the potential participation of other mAChR subtypes in
the cholinergic control of heart function must be considered. Prior work indicates that there are large species variations in terms of the
subtypes of mAChR expressed in the hearts. M1 and
M2 subtypes exist in rat hearts (Sharma et al.,
1996). M2, M3, and
M4, but not M1 and
M5, receptors are present in dog hearts (H. Shi,
H. Wang, and Z. Wang, unpublished data; Shi et al., 1999a) and
there are M2, M3, and
M5 receptors in guinea pig hearts (H. Shi, H. Wang, and Z. Wang, unpublished data; Shi et al., 1999b). Thus, extrapolation of data from animal species to humans must be done with caution.
;
Kukkonen et al., 1998; Reever et al., 1997). The present study provides
the first evidence for the presence of an endogenous M5 receptor subtype in a peripheral non-neuronal
tissue (Reever et al., 1997). Although the function of the
M5 receptor is still unknown, our findings point
to a potential functional role in the human heart.
We previously reported that stimulation of M3
receptors by choline (Shi et al., 1999b), pilocarpine (Wang et al.,
1999b), or tetramethylammonium (Shi et val., 1999a) in dog and guinea pig atrial myocytes activates a novel K+ current
with both delayed and inward rectifier properties. Activation of
M3 receptors caused significant slowing of the
heart rate and shortening of the action potential duration. Stimulation
of M4 receptors by 4-aminopyridine also
induces a distinct K+ current in dog cells (Shi
et al., 1999a). Sharma et al. (1996) found that activation of
M1 receptors stimulates
Ca2+ transients in rat heart. The present study
suggests potential relevance of the above-mentioned physiological
observations to humans. Nevertheless, further direct experimentation is
necessary to establish the function of M1,
M3, and M5 receptors in the
human heart.
Our immunoblotting analysis indicated that M3 and
M5 distributions are quantitatively different
between HA and HV, with M3 density higher in HA
than in HV and M5 higher in HV than in HA. In
addition, immunostaining also revealed a characteristic localization of
M5 receptors to the intercalated discs. Other
receptor subtypes were more evenly distributed throughout the surface
membrane, although M3 receptors also demonstrated
a stronger staining on the intercalated discs relative to other regions
of the plasma membrane. The data indicate that different subtypes of
mAChR might have different patterns of cellular localization. Whether
this heterogeneous distribution has functional implications awaits future studies.
In summary, we have obtained evidence with receptor binding, mRNA
expression, and protein detection in support of the presence of
multiple mAChR subtypes in human hearts. The tissue distribution (HV
versus HA) and cellular localization vary among subtypes. These
findings may open up opportunities for improved understanding of the
subtype specificity of mAChR modulation of heart function and for the
development of new drugs selectively targeting different subtypes of mAChRs.
| |
Acknowledgments |
|---|
We thank XiaoFan Yang for excellent technical assistance.
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Footnotes |
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Received August 22, 2000; Accepted January 18, 2001
This work was supported in part by the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, an Establishment Grant for young investigators from the Fonds de Recherche en Santé du Québec awarded to Dr. Wang, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. Z.W. is a Research Scholar of the Heart and Stroke Foundation of Canada. H.W. and H.S. are Research Fellows of the Medical Research Council of Canada. H.H. is a Research Fellow of the Heart and Stroke Foundation of Canada.
Send reprint requests to: Zhiguo Wang, Ph.D, Research Center, Montreal Heart Institute, 5000 Bélanger St. East, Montreal, Quebec, Canada H1T 1C8. E-mail: wangz{at}icm.umontreal.ca
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Abbreviations |
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mAChR, muscarinic acetylcholine receptor; RT, reverse transcription; PCR, polymerase chain reaction; HA, human atrium; HV, human ventricle; PBS, phosphate-buffered saline; [3H]NMS, [N-methyl-3H]scopolamine methyl chloride; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; bp, base pair(s); HHSiD, hexahydro-siladifenidol.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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H. Wang, B. Yang, Y. |
,
specific binding; 
, nonspecific binding obtained in the presence of
atropine (1 µM). Shown in the inset of A is the Scatchard plot of the
atrial data, with the regression line to the data points shown [the
title for the y-axis, Bound/Free, and for the
x-axis, Bound (fmol)]. Data shown are averaged from
four individual preparations carried out in triplicate for each
experiment. B, competition binding of [3H]NMS with
various compounds. Each data point represents mean ± S.E. from
six experiments (six hearts) assayed in duplicate. Curves are best fits
of the experimental data to a two-site binding model.
indicates omission of reverse
transcriptase from the reaction to exclude the possible contamination
by genomic DNAs. B, test for contamination by neuronal and vascular
tissues. Lane 1, 
