0022-3565/97/2811-0470$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 281:470 –477, 1997
Vol. 281, No. 1
Printed in U.S.A.
M2, M3 and M4, but not M1, Muscarinic Receptor Subtypes
are Present in Rat Spinal Cord1
A. U. HÖGLUND AND H. A. BAGHDOYAN
Department of Anesthesia, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania
Accepted for publication December 16, 1996
Activation of muscarinic ACh receptors in spinal cord of
animals produces antinociception (Abram and O’Connor,
1995; Abram and Winne, 1995; Detweiler et al., 1993;
Iwamoto and Marion, 1993; Naguib and Yaksh, 1994) and
hypertension (Buccafusco, 1996; Carp et al., 1994; Feldman
et al., 1996). Similar responses have been noted in human
subjects (Hood et al., 1995; Lauretti et al., 1996). It has been
proposed that spinal antinociception is mediated by M1
and/or M2 muscarinic receptors (Iwamoto and Marion, 1993)
and that neostigmine-induced hypertension is mediated, in
part, by spinal M2 receptors (Lothe et al., 1994).
Muscarinic receptors previously have been demonstrated
to be present in spinal cord of human, rat and cat using in
vitro autoradiography with a variety of tritiated ligands,
including [3H]-QNB (Gillberg et al., 1984; Kayaalp and Neff,
1980; Scatton et al., 1984; Seybold and Elde, 1984; Villiger
and Faull, 1985; Yamamura et al., 1983), [3H]-PZ (Villiger
Received for publication September 16, 1996.
1
This work was supported by the Departments of Anesthesia and Comparative Medicine at The Pennsylvania State University College of Medicine, the
Medical Faculty at Uppsala University, U.S. P.H.S. grants MH-45321 (H.A.B.)
and HL-47749 and grant B96-99Z-11159-02 from the Swedish Medical Research Council (A.U.H.).
receptor assays using [3H]-N-methylscopolamine and the unlabeled antagonists pirenzepine, 11-2[(-[(diethylamino)methyl]1-piperidinyl)-acetyl]-5, 11-dihydro 6H-pyrido(2, 3-b)(1, 4) benzodiazepine-one, methoctramine, and methoctramine in
combination with atropine corroborated the autoradiographic
findings and also revealed the presence of M4 binding sites.
The finding that M2 and M3 binding sites were localized to the
superficial laminae of the dorsal horn where nociceptive Ad and
C fibers terminate suggests the possibility that either or both of
these muscarinic receptor subtypes modulate antinociception.
The present demonstration of M4 binding sites in spinal cord is
consistent with the possibility that M2 and/or M4 receptors are
involved in the regulation of blood pressure at the spinal level.
and Faull, 1985), [3H]-ACh (Gillberg et al., 1988) and [3H]NMS (Wamsley et al., 1984). Wamsley and colleagues (1984)
showed that high- and low-affinity muscarinic binding sites
are present in spinal cord. Villiger and Faull (1985) named
these receptors M1 and M2, respectively.
Since the foregoing autoradiographic studies were performed, additional muscarinic receptor subtypes have been
identified by molecular cloning, and there are now known to
be five muscarinic receptor subtypes (Bonner et al., 1987;
Kubo et al., 1986). By convention, molecularly identified subtypes are referred to as m1 to m5, and pharmacologically
identified subtypes are designated M1 to M4 (Birdsall et al.,
1989). The M1 to M4 subtypes generally correspond to the m1
to m4 subtypes (Caulfield, 1993; Waelbroeck et al., 1990).
However, it is important to note that muscarinic antagonists
are only relatively selective, not exclusively specific, for individual subtypes. The relative subtype selectivity of many
muscarinic antagonists has been evaluated (Caulfield, 1993),
and it is now clear that [3H]-NMS (Dörje et al., 1990) and
[3H]-QNB (Bolden et al., 1991; Jakubik et al., 1995) label m1
to m5 receptors with equal and high affinity and that PZ has
a high affinity for m1/M1 receptors, a relatively lower affinity
for m3/M3 and m4/M4 receptors and a low affinity for m2/M2
ABBREVIATIONS: AF-DX 116, 11-2[(-[(diethylamino)methyl]-1-piperidinyl)-acetyl]-5, 11-dihydro-6H-pyrido(2, 3-b)(1, 4)-benzodiazepine-one;
ANOVA, analysis of variance; CV, cresyl violet; 3H, tritium; IML, intermediolateral nucleus; Kd, dissociation constant; LFB, luxol fast blue; METH,
methoctramine; nH, Hill number; NMS, N-methylscopolamine; PZ, pirenzepine; QNB, quinuclidinyl benzilate; RT-PCR, reverse transcriptasepolymerase chain reaction; T, thoracic.
470
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ABSTRACT
Muscarinic receptors in the spinal cord have been shown to
mediate antinociception and alter blood pressure. Currently,
there is much interest in identifying which muscarinic receptor
subtypes regulate these functions. Toward that end, this study
aimed to identify and localize the muscarinic receptor subtypes
present in spinal cord using in vitro receptor autoradiography
with [3H]-pirenzepine and [3H]-N-methylscopolamine. The results showed that M2 binding sites were distributed throughout
the dorsal and ventral horns, whereas M3 binding sites were
localized to laminae I to III of the dorsal horn. Only background
levels of M1 binding sites were detected. Saturation binding
assays using [3H]-pirenzepine in spinal cord homogenates confirmed the absence of M1 receptors. Competition membrane
1997
Muscarinic Receptors in Spinal Cord
receptors (Waelbroeck et al., 1990). Thus previous in vitro
autoradiographic studies using [3H]-NMS, [3H]-QNB and
[3H]-PZ may have erroneously classified the muscarinic receptor subtypes present in spinal cord.
Increased knowledge about the multitude of muscarinic
receptor subtypes (Caulfield, 1993), the ability to selectively
visualize these subtypes (Flynn and Mash, 1993) and the
rising interest in muscarinic pharmacology for use in clinical
anesthesia (Hood et al., 1995; Lauretti et al., 1996) encouraged us to undertake the present study, which tested the
hypothesis that spinal cord contains M1 to M4 muscarinic
receptors.
Materials and Methods
The NIH Image program was used to digitize and analyze autoradiographic images and histological sections, as described previously (Baghdoyan et al., 1994; Mallios et al., 1995). For every sheet of
film, the autoradiographic image made from one set of [3H] microscales was digitized to make possible the calibration of optical density to fmol of binding per mg tissue equivalent (Geary et al., 1985).
Autoradiograms of spinal cord sections were magnified four times
using a microscope and then were digitized and written to disk. All
slide-mounted, LFB/CV-stained spinal cord sections used to generate
autoradiograms were also magnified, digitized, and written to disk.
Because of the low resolution of film autoradiography, it was not
possible to distinguish between laminae I and II on the computerized
autoradiographic images. Therefore, laminae I and II are discussed
hereafter as one entity called laminae I/II. The digitized LFB/CV
images were used as templates to identify the boundaries of laminae
I/II and lamina III. Each of these boundaries was traced on the
digitized image of the LFB/CV section using the outlining feature of
NIH Image. The outline then was copied to the digitized autoradiogram of the same section. Binding density was measured within the
outlined region on the digitized autoradiographic image, and binding
density values were pasted to a spreadsheet for subsequent statistical analyses.
Means and standard errors of the means were calculated on 16 to
20 samples from each segmental level of the thoracic spinal cord.
Two-way ANOVA was used to test the hypothesis that M2 and M3
binding sites were differently distributed across all 12 segments of
the thoracic spinal cord and between laminae I/II and lamina III. A
one-way ANOVA with a Tukey’s post-hoc test identified the thoracic
cord segments that contributed to any significant differences in
muscarinic receptor density. To evaluate quantitatively which laminae contributed to any significant differences in muscarinic receptor
distribution, further analyses applied multiple t tests with Bonferroni correction factors (aactual 5 0.05/number of comparisons).
Membrane receptor assays. Eleven rats were decapitated by
guillotine, and the thoracic spinal cord and hippocampus were extracted rapidly and placed in a glass petri dish on ice. The remaining
meninges and blood vessels were removed, and the tissue was
weighed and diluted to 50 times its wet weight in ice-cold 20 mM
Tris-HCl 1 1 mM MnCl2 buffer, pH 7.4 (for [3H]-PZ saturation
binding) or 50 mM phosphate buffer 1 1 mM MgCl2, pH 7.4 (for
[3H]-NMS saturation and competition binding). The tissue was homogenized, centrifuged and resuspended three times. After the second centrifugation, the pellet was resuspended in distilled water,
and a 0.2-ml sample was taken to determine the protein content of
the homogenate using the BCA Protein Assay Reagent (PIERCE,
Rockford, IL).
Saturation binding assays were performed according to the methods of Waelbroeck and colleagues (1986), using six concentrations of
[3H]-PZ ranging from 0.2 to 20 nM or six concentrations of [3H]-NMS
ranging from 35 to 3500 pM. Competition binding assays also were
performed according to procedures described by Waelbroeck et al.
(1986; 1990). Homogenates of spinal cord were incubated with 240
pM [3H]-NMS and with PZ alone, AF-DX 116 alone, METH alone or
METH in combination with atropine (METH 1 atropine) as competitors. PZ (0.4 nM–100 mM) was used in order to distinguish between
M1 and M3 binding sites. AF-DX 116 alone (0.4 nM–100 mM) and
METH alone (0.4 nM–100 mM) were used to distinguish between M2
and M3 binding sites. As described by Waelbroeck et al. (1990),
METH (0.4 nM–100 mM) in combination with atropine (1 mM) was
used to distinguish between M3 and M4 binding sites. The rationale
for using METH 1 atropine to identify M3 and M4 binding sites is
based on the previously demonstrated finding that [3H]-NMS dissociates more slowly from m3/M3 and m4/M4 receptors than from
m1/M1 and m2/M2 receptors (Flynn and Mash, 1993; Waelbroeck et
al., 1990). Therefore, the addition of 1 mM atropine for the final 35
min of incubation should differentially displace [3H]-NMS from the
four muscarinic receptor subtypes, leaving residual [3H]-NMS bind-
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Animals and chemicals. This study was approved by the Institutional Animal Care and Use Committee of The Pennsylvania State
University, College of Medicine. Male LBNF1 rats (Harlan SPD,
Pottsville, AL), 10 weeks of age, were housed in stainless steel cages
and had access to food (Harlan Teklad rodent diet (W) 8604, Madison, WI) and water ad libitum. Standardized environmental conditions included lights on between 0700 and 1900, lights off between
1900 and 0700, temperature 21°C to 23°C, humidity 40% to 60%, and
11 to 15 air changes per hour. Animals were given at least 1 week of
acclimatization in home cages before use.
Radiolabeled ligands [3H]-PZ (77.9 Ci/mmol) and [3H]-NMS (84
Ci/mmol) were obtained from Amersham (Arlington Heights, IL). PZ
and atropine sulfate were obtained from Sigma Chemical Co. (St.
Louis, MO). METH was purchased from Research Biochemicals International (Natick, MA). The antagonist AF-DX 116 was provided
as a gift by Boeringer-Ingelheim (Ridgefield, CT).
In vitro receptor autoradiography. Five rats were decapitated
by guillotine, and the thoracic spinal cords were extracted rapidly
and frozen on dry ice. Spinal cords were sectioned serially using a
Hacker-Bright cryostat (Bright Instrument Company LTD, Huntingdon, England), and 25-mm transverse sections were thaw-mounted
on chrome alum-coated glass slides. Slide-mounted tissue sections
were dried under reduced pressure at 4°C for at least 2 h and kept at
270°C until processed according to standard autoradiographic techniques (Kuhar and Unnerstall, 1990).
Consecutive slides were used to label M1, M2 and M3 muscarinic
receptors at each level of the thoracic cord in accordance with the
binding assays described by Flynn and Mash (1993). These assays
take advantage of the selectivity of PZ binding and the kinetic
properties of NMS binding and have been shown to be useful for
localizing muscarinic receptor subtypes throughout monkey brain
(Flynn and Mash, 1993) and in cat brain stem (Baghdoyan et al.,
1994; Mallios et al., 1995). Briefly, to visualize M1 binding sites,
tissue sections were labeled with 3 nM [3H]-PZ for 60 min. Because
[3H]-NMS associates most rapidly to the m2 subtype (Flynn and
Mash, 1993; Waelbroeck et al., 1990), M2 binding sites were labeled
with a 2-min incubation in 0.5 nM [3H]-NMS, which followed a
60-min preincubation in 0.3 mM PZ to occlude m1, m3 and m4 sites.
[3H]-NMS dissociates most slowly from the m3 subtype (Flynn and
Mash, 1993; Waelbroeck et al., 1990). Thus M3 binding sites were
labeled by preincubating the sections for 3 min in 0.5 nM NMS to
occlude m1, m2 and m4 sites and then incubating them in 0.25 nM
[3H]-NMS for 2 h. This labeling was followed by a 75-min dissociation
of the radioligand by the addition of 1 mM atropine. For all three
muscarinic receptor subtypes, nonspecific binding was determined in
selected adjacent sections by adding 10 mM atropine to the assay
buffer. Radiolabeled sections and [3H] micro-scales (Amersham Corporation, Arlington Heights, IL) were apposed to [3H]-Hyperfilm
(Amersham) for 6 weeks at 4°C. Films were developed in Kodak
D-19; then tissue sections were fixed in paraformaldehyde vapors
(Herkenham and Pert, 1982) before staining with luxol fast blue
(LFB; 0.2%) and cresyl violet (CV; 0.1%).
471
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Höglund and Baghdoyan
ing to 3% of M1, 0% of M2, 66% of M3, and 50% of M4 receptors
(Waelbroeck et al., 1990).
Nonspecific binding was measured in the presence of 1 mM atropine. Incubations were terminated by rapid filtration using GF/C
filters (Biomedical Research and Development Laboratories, Inc.,
Gaithersburg, MD) that had been soaked in 0.1% polyethyleneimidine (Sigma Chemical Co., St. Louis, MO), and radioactivity was
counted with an average efficiency of 58%. Saturation and competition binding data were analyzed using the LIGAND program (Munson and Rodbard, 1980).
Results
Fig. 1. Digitized autoradiographic images showing the distribution of
M1, M2 and M3 binding sites in the left half of the gray matter at the first
thoracic segmental level (T1). These images are typical of the spinal
cord sections from which quantitative measures of receptor density
were obtained. Rectangular boxes under each image show film background. Roman numerals indicate laminae of Rexed (Rexed, 1952). This
figure clearly shows the absence of M1 binding sites in all laminae and
the background levels of M3 binding in all laminae except I/II and III. In
contrast, M2 binding sites are distributed throughout the dorsal and
ventral horn. IML, intermediolateral nucleus.
first was verified using tissue from hippocampus, where a
high density of M1 receptors has been demonstrated (Waelbroeck et al., 1986). Assays performed using hippocampal
homogenates showed saturable binding with a dissociation
constant (Kd) of 5.5 nM and a maximal binding (Bmax) of
100.8 fmol/mg tissue. These results agree with previously
published values (Kd 5 2–8 nM, Bmax 5 94.3 fmol/mg tissue,
Watson et al., 1983; Kd 5 4–6 nM, Waelbroeck et al., 1986).
Having thus demonstrated that the saturation binding protocol was working, the assay then was applied to tissue from
spinal cord. Specific binding of [3H]-PZ to homogenates of
spinal cord was negligible, which strongly indicates that M1
receptors are not present in this tissue.
Competition binding assays provide evidence for
M2, M3 and M4 binding sites in spinal cord. The presence of M2, M3 and M4 binding sites in spinal cord was
determined by competition binding measured with [3H]-NMS
and PZ, AF-DX 116, METH and atropine as unlabeled competitors. Figure 5 shows the competition curves obtained
from these experiments, and table 1 reports the Kd and Bmax
values calculated for the competing ligands. Table 1 Kd values were compared with previously published dissociation
constants for PZ, AF-DX 116 and METH (table 2) in order to
identify which muscarinic receptor subtypes were present in
spinal cord homogenates.
Competition of [3H]-NMS binding with PZ (fig. 5) showed a
normal competition curve with a Hill coefficient (nH) of
0.95 6 0.03, which indicates that PZ detected one binding
site. Comparison of the Kd value obtained for PZ in the
present study (table 1, 197.1 6 29.9 nM) with Kd values for
PZ obtained using known muscarinic receptor subtypes (table 2) suggests that PZ labeled M2 and/or M3 receptors in the
spinal cord. Table 2 indicates that the M2 and M3 Kd values
for PZ are not sufficiently different to distinguish between
these two subtypes.
Competition of [3H]-NMS binding with METH alone (fig. 5)
produced a steep competition curve with a nH of 1.21 6 0.06.
This nH was significantly greater than unity (t 5 3.4; df 5 8;
P , .001), which indicates positive cooperativity. This positive cooperativity probably resulted from the METH-induced
allosteric inhibition of [3H]-NMS displacement (Waelbroeck
et al., 1990). To minimize the effects of this allosteric inhibition, only data obtained with concentrations of METH that
were below 1 mM were used to calculate Kd and Bmax. Comparison of the METH Kd value (table 1, 24.5 6 1.8 nM) with
previously reported Kd values for METH (table 2) suggests
that the spinal cord Kd value might represent both M2 and
M4 receptors. The relatively high density of receptors obtained from the fit of the present METH data (Bmax 5
391.7 6 61.0 fmol/mg protein) suggests the possibility that
the Kd value derived from the METH competition represents
M2 and M4 receptors in spinal cord.
Competition with AF-DX 116 alone and METH in combination with atropine (fig. 5) produced shallow competition
curves, a result that indicates the presence of more than one
binding site. A two-binding-site model provided a significantly (P , .001) better fit than a one-site model when these
competition curves were analyzed. For AF-DX 116, the fitted
Kd values (table 1) and the shallow competition curve with nH
of 0.77 6 0.04 (significantly different from unity, t 5 6.3; df 5
8; P , .001) indicate that AF-DX 116 recognized M2 and M3
receptors (compare table 1 Kd values with table 2 Kd values).
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In vitro receptor autoradiography reveals M2 and
M3 binding sites in dorsal horn. The main finding to
emerge from the autoradiographic data was that the thoracic
spinal cord contains M2 and M3, but no M1, binding sites.
The distribution of binding sites is shown in figures 1 and 2.
Only background levels of M1 binding (fig. 1) were observed,
whereas M2 sites (figs. 1 and 2) were distributed throughout
the gray matter of the thoracic spinal cord. Relatively high
M2 densities were found in the superficial layers of the dorsal
horn corresponding to laminae I/II and III of Rexed (Rexed,
1952), the intermediolateral nucleus (fig. 1, IML), laminae VI
and VII, lamina IX in the ventral horn and lamina X. M3
binding sites (figs. 1 and 2) were localized mainly to laminae
I/II, with a lower M3 density in lamina III. The ventral horn
was devoid of M3 binding sites.
Figure 3 plots the density of M2 binding sites in laminae
I/II and lamina III for thoracic (T) segments T1 through T12.
These data show that there were no major differences in the
density of M2 sites across thoracic segments and between
laminae I/II and lamina III. Figure 4 shows the distribution
of M3 binding sites in laminae I/II and lamina III across the
12 segments of the thoracic cord. In contrast to M2 distribution, M3 density was significantly (P , .05) higher in laminae
I/II than in lamina III for all segments of thoracic cord.
Saturation binding assays confirm the absence of M1
binding sites in spinal cord. Saturation binding with [3H]PZ was used to confirm the autoradiographic finding of no M1
binding sites in spinal cord. The saturation binding protocol
Vol. 281
1997
Muscarinic Receptors in Spinal Cord
473
After addition of 1 mM atropine to the METH incubation,
[3H]-NMS was still bound to the homogenate (fig. 5), which
indicates the presence of slowly dissociating (non-M1, nonM2) binding sites. Taken together, the nH of 0.60 6 0.03
(significantly less than unity, t 5 12.5; df 5 6; P , .001) and
the affinity constants fitted to the METH 1 atropine competition curve (table 1) indicate the presence of both M3 and M4
muscarinic receptors (table 2) in spinal cord homogenates.
Discussion
These results provide new information about the distribution of muscarinic receptor subtypes in spinal cord. No M1
binding sites were found in the present study. Instead, the
data offer evidence for the presence of M3 receptors localized
to the same areas where M1 receptors previously had been
suggested to be present (Gillberg et al., 1988; Villiger and
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Fig. 2. Autoradiograms showing
the distribution of M2 and M3
binding sites in thoracic spinal
cord. M2 (left column) and M3
(right column) sites are shown at
thoracic segmental levels T3, T6,
T9 and T12. The color scale at the
right provides a quantitative index
of total binding in fmol/mg tissue
equivalent. Calibration bar at bottom equals 1 mm.
474
Höglund and Baghdoyan
Vol. 281
Fig. 3. M2 muscarinic receptor density (total
binding, mean 6 S.E.M.) in the thoracic spinal
cord measured in laminae I/II and lamina III.
Each mean represents four measurements per
rat obtained from four or five rats (N 5 16 –20
measurements). Nonspecific binding was ,3
fmol/mg. Note that M2 binding sites in laminae
I/II were distributed homogeneously across the
thoracic cord. In lamina III, there were fewer
(P , .05) M2 sites at the T1 level than at most
all other thoracic segments. With the exception
of segment T1, there was no significant difference in the density of M2 binding sites between
laminae I/II and lamina III.
Faull, 1985). The distribution of M2 binding sites corresponds well to previous results (Villiger and Faull, 1985). The
present study also provides evidence for the existence of M4
receptors in spinal cord.
Absence of M1 muscarinic receptors in spinal cord.
Spinal cord M1 receptors were reported by earlier studies,
yet the present study found only background levels of M1
binding (fig. 1). How can this apparent discrepancy be explained? Previous autoradiographic studies (Villiger and
Faull, 1985; Yamamura et al., 1983) used a 20 nM concentration of [3H]-PZ to visualize M1 receptors, whereas the
present study used 3 nM [3H]-PZ. According to the formula
% occupancy 5 100 3 [PZ]/([PZ] 1 Kd)
where [PZ] is 3 nM or 20 nM and Kd for PZ is 6 nM or 85 nM,
respectively (Kd values from Flynn and Mash, 1993), it can be
shown that 3 nM PZ would occupy 33% of M1 sites but only
3.4% of M3 sites. Likewise, 20 nM PZ would occupy 77% of
M1 sites and 19% of available M3 sites. Although these
percentages are calculated under the assumption that M1
and M3 receptors are available in equal concentrations, these
calculations help explain the differences between the present
results and previous findings. At a concentration of 20 nM
(Villiger and Faull, 1985; Yamamura et al., 1983), PZ would
label enough M3 sites to be visible by autoradiography. Because only M1 and M2 muscarinic receptors were known to
exist in the early 1980s, the [3H]-PZ binding sites originally
found in spinal cord were identified as M1.
Autoradiographic data revealing an absence of spinal cord
M1 binding sites were confirmed in spinal cord homogenates.
A recent study using RT-PCR and HPLC demonstrated that
only background levels of m1 mRNA are present in spinal
cord (Wei et al., 1994). Most recently, m1-toxin only minimally displaced [3H]-NMS binding in the lumbar region of
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Fig. 4. M3 muscarinic receptor density (total
binding, mean 6 S.E.M.) in the thoracic spinal
cord measured in laminae I/II and lamina III.
Each mean represents four measurements per
rat obtained from four or five rats (N 5 16 –20
measurements). Nonspecific binding was ,3
fmol/mg. The distribution of M3 binding sites
across segments T1 through T12 was relatively
homogeneous for laminae I/II and lamina III,
with the exception of lower (P , .05) densities
in segments T4 and T5. Note that for each of
the 12 thoracic segments, M3 densities in lamina III were significantly lower (P , .001) than
M3 densities in laminae I/II (multiple t test comparisons, Bonferroni correction applied; depending on sample size, degrees of freedom
varied between 30 and 38; t was always .5.6).
1997
Muscarinic Receptors in Spinal Cord
475
TABLE 2
Dissociation constants (Kd, nM) for selected muscarinic antagonists
TABLE 1
Dissociation constants (Kd) and receptor densities (Bmax)
obtained from fitted competition binding data
Kd (nM)
METH
PZ
AF-DX 116
METH 1 atropine
Bmax (fmol/mg protein)
METH
PZ
AF-DX 116
METH 1 atropine
M2
M3
M4
24.5 6 1.8
197.1 6 29.9
64.0 6 6.2
—
—
—
1642.6 6 344.9
875.2 6 99.5
—
—
—
16.9 6 4.0
391.7 6 61.0
204.3 6 24.3
318.1 6 44.7
—
—
—
22.6 6 4.0
9.0 6 1.4
—
—
—
8.4 6 0.9
Means 6 S.E.M. were calculated from four or five individual experiments
performed in duplicate. These Kd values were used to identify the muscarinic
receptor subtypes in spinal cord. The competition curves for METH and PZ were
fit to a one-binding-site model, and the calculated Kd values indicate the presence of M2 receptors. A two-binding-site model provided a significantly (P ,
.001) better fit than a one-site model when analyzing the competition curves for
AF-DX 116 and METH 1 atropine. Competition binding with AF-DX 116 demonstrated the presence of M2 and M3 receptors in spinal cord homogenates. The
METH 1 atropine competition binding protocol provided evidence for the existence of M3 and M4 receptors in spinal cord. Kd values reported in this table are
within the published range (table 2). Thus the spinal cord muscarinic receptor
subtypes identified in the present study are pharmacologically similar to known
muscarinic subtypes found in other tissues (Waelbroeck et al., 1990) and similar
to cloned muscarinic receptor subtypes expressed in vitro (Caulfield, 1993).
human spinal cord (Borenstein et al., 1996). Thus multiple
techniques now support the conclusion that the spinal cord
does not contain M1 receptors.
Evidence for the presence of M2, M3 and M4 receptors in spinal cord. The present data provide autoradiographic evidence for M2 and M3 receptors in thoracic cord
(figs. 1 and 2). In spinal cord homogenates, competition of
[3H]-NMS binding with AF-DX 116 revealed two binding
sites (fig. 5; table 1), and the Kd values corresponded to M2
and M3 receptors (table 2). Kd values obtained from competition binding with METH and with PZ (table 1) also are
consistent with the presence of M2 and M3 receptors. The
existence of m2 and m3 mRNA in spinal cord has been
demonstrated by RT-PCR (Wei et al., 1994). Taken together,
Antagonist
m1/M1
m2/M2
m3/M3
m4/M4
PZ
AF-DX 116
METH
5–13
200–400
25–80
200–500
50–80
5–15
80–200
1260–1600
125–1000
25–80
250–300
8–23
These values, compiled from the literature (Caulfield, 1993; Waelbroeck et al.,
1990), were obtained in radioligand-binding displacement studies using either
tissue expressing a single muscarinic receptor subtype or cloned muscarinic
receptors expressed in cell lines. The Kd values for PZ, AF-DX 116 and METH
obtained in the present study (table 1) were comparable to the values in this table
and demonstrated that spinal cord contains M2, M3 and M4 receptors.
these findings confirm the presence of M2 and M3 receptors
in thoracic spinal cord.
The autoradiography protocol used in the present study
(Flynn and Mash, 1993) did not permit localization of M4
receptors. The existence of M4 receptors in the spinal cord,
however, can be suggested based on the reinterpretation of
previous autoradiographic data using 20 nM [3H]-PZ to label
muscarinic receptors (Villiger and Faull, 1985; Yamamura et
al., 1983). According to the % occupancy formula described
above, 20 nM PZ (Villiger and Faull, 1985; Yamamura et al.,
1983) may have labeled a substantial proportion of M4 sites
(31%, assuming a Kd value of 44 nM; Flynn and Mash, 1993).
In the present study, the 3 nM [3H]-PZ concentration used for
autoradiography would be expected to label only 6% of M4
sites, which evidently is too low an occupancy to be detected.
Thus competition binding in spinal cord homogenates was
performed to determine whether M4 receptors were present.
METH 1 atropine discriminated between two slowly dissociating (non-M1, non-M2) receptors (fig. 5; table 1). [3H]NMS dissociates slowly from genetically defined m3 and m4
muscarinic receptors (Flynn and Mash, 1993; Jakubik et al.,
1995), and METH has a higher affinity for m4 than for m3
receptors (Dörje et al., 1990) (table 2). The METH 1 atropine
competition data revealed Kd values (table 1) consistent with
the assumption that METH 1 atropine distinguished between M3 and M4 receptors (table 2). The competition bind-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 5. Competition binding performed
using homogenates from spinal cord.
[3H]-NMS binding was displaced by
METH (ç), AF-DX 116 (F), PZ (É) or
METH 1 atropine (E). Each point represents the mean 6 S.E.M. from three experiments assayed in duplicate. The dissociation constants (Kd) and maximal
numbers of binding sites (Bmax) calculated from these competition binding
data are summarized in table 1. Competition of [3H]-NMS binding by METH
alone is consistent with the presence of
M2 and/or M4 receptors in spinal cord
homogenates. Competition with PZ
alone yielded data consistent with the
presence of M2 and/or M3 receptors.
AF-DX 116 competition revealed M2 and
M3 receptors, and METH 1 atropine revealed that spinal cord contains M3 and
M4 receptors.
476
Höglund and Baghdoyan
m4 sites, respectively. It is the unique kinetic binding properties of [3H]-NMS to m1 to m4 muscarinic receptors that
permit this minimal amount of autoradiographic cross-labeling.
The above values for percent occupancy of muscarinic receptors were derived using cloned muscarinic receptor subtypes expressed in A9L cells and were verified for native
muscarinic receptors using primate brain and rabbit peripheral tissue (Flynn and Mash, 1993). It is possible that the
dissociation rates and occupancy values for muscarinic receptor subtypes expressed in rat spinal cord may differ from
those published by Flynn and Mash (1993). The pharmacological characteristics of muscarinic receptors, however, have
been noted to be quite similar between species (Caulfield,
1993). In addition, differential kinetics of [3H]-NMS binding
to muscarinic receptors expressed in rat brain have been
demonstrated (Waelbroeck et al., 1986; 1990). Therefore, it is
likely that the kinetic parameters of [3H]-NMS binding that
were applied in the present study are relevant for muscarinic
receptors expressed in rat spinal cord. Characterizing the
kinetics of [3H]-NMS binding in rat spinal cord will be of
interest for future studies.
The present data revealed that M2 and M3 receptors are
localized to laminae I to III. The limitations of film autoradiography made it impossible to distinguish between laminae
I and II. The present results encourage the use of muscarinic
receptor subtype-specific antibodies to identify which cell
types in the spinal cord express muscarinic receptors and to
determine the pre- and/or postsynaptic localization of spinal
cord muscarinic receptor subtypes (Levey et al., 1995).
In conclusion, this study provides new insights into the
distribution of muscarinic receptor subtypes in spinal cord.
Until now, the only muscarinic receptors thought to be
present were M1 and M2. The data presented here show that
M2, M3 and M4, but not M1, receptors exist in spinal cord.
Given that intrathecal injections of cholinergic agonists and
acetylcholinesterase inhibitors produce antinociception, an
exciting opportunity for future research is to determine
which spinal muscarinic receptor subtypes modify nociceptive input.
Acknowledgments
The authors thank Boeringer-Ingelheim for providing AF-DX 116
and thank J. L. DiVittore and P. P. Myers for expert technical and
secretarial assistance.
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