Department of Pharmacology and Toxicology, Medical College of
Virginia Campus, Virginia Commonwealth University, Richmond, Virginia
(C.S.B., G.G., B.R.M.); and Istituto per la Chimica di Molecole di
Interesse Biologico, Consiglio Nazionale delle Ricerche, Arco Felice
(NA), Italy (V.D.M.)
The purpose of these studies was to support the hypothesis that an
undiscovered cannabinoid receptor exists in brain.
[35S]GTP
S binding was stimulated by anandamide and
WIN55212-2 in brain membranes from both CB1+/+
and CB1
/ mice. In contrast, a wide variety
of other compounds that are known to activate CB1
receptors, including CP55940, HU-210, and 
9-tetrahydrocannabinol, failed to stimulate
[35S]GTPS binding in CB1/
membranes. In CB1/ membranes, SR141716A
affected both basal and anandamide- or WIN55212-2-induced stimulation
of [35S]GTPS binding only at concentrations greater
than 1 µM. In CB1+/+ membranes, SR141716A
inhibited only 84% of anandamide and 67% of WIN55212-2 stimulated
[35S]GTPS binding with an affinity appropriate for
mediation by CB1 receptors
(KB 
0.5 nM). The remaining
stimulation seemed to be inhibited with lower potency
(IC50 5 µM) similar to that seen in
CB1/ membranes or in the absence of
agonist. Further experiments determined that the effects of anandamide
and WIN55212-2 were not additive, but that the effect of µ opioid,
adenosine A1, and cannabinoid ligands were additive. Finally, assays of
different central nervous system (CNS) regions demonstrated significant
activity of cannabinoids in CB1/ membranes
from brain stem, cortex, hippocampus, diencephalon, midbrain, and
spinal cord, but not basal ganglia or cerebellum. Moreover, some of
these same CNS regions also showed significant binding of
[3H]WIN55212-2, but not [3H]CP55940. Thus
anandamide and WIN55212-2 seemed to be active in
CB1/ mouse brain membranes via a common G
protein-coupled receptor with a distinct CNS distribution, implying the
existence of an unknown cannabinoid receptor subtype in brain.
 |
Introduction |
Although
the principal active constituent in marijuana,
9-tetrahydrocannabinol (THC), has been known
for more than 35 years (Gaoni and Mechoulam, 1964
), the first
cannabinoid receptor (CB1) was cloned only 10 years ago (Matsuda et al., 1990). The only other known cannabinoid
receptor is CB2 (Kaminski et al., 1992; Munro et
al., 1993), which is localized primarily on cells of the immune system.
Both CB1 and CB2 are G
protein-coupled receptors that seem to couple to inhibitory
Gi and/or Go proteins
(Childers and Breivogel, 1998). CB1 receptors are
known to affect adenylyl cyclase (Howlett, 1984), a variety of
potassium (Deadwyler et al., 1995; Mackie et al., 1995; Mu et al.,
1999) and calcium (Mackie and Hille, 1992; Mackie et al., 1995)
currents, and the mitogen-activated protein kinase pathway (Bouaboula
et al., 1995). CB1 receptors have been shown to
activate at least six subtypes of Gi and
Go proteins, supporting the reports that they
effect a wide variety of intracellular signaling systems (Prather et
al., 2000). CB2 receptors also inhibit adenylyl
cyclase and activate mitogen-activated protein kinase and Krox-24
pathways (Bouaboula et al., 1996), but do not seem to effect ion
currents directly (Felder et al., 1995). In vivo, cannabinoids elicit a
characteristic spectrum of behaviors in laboratory animals. These
include catalepsy, analgesia, and decreases in spontaneous activity and
body temperature (Adams and Martin, 1996) and a disruption of memory
(Heyser et al., 1993).
Receptor activation of G-proteins can be measured using
agonist-stimulated binding of the nonhydrolyzable GTP analog,
[35S]guanosine-5'-O-(3-thiotriphosphate)
([35S]GTPS) to membranes (Breivogel et al.,
1997b). Using this technique (Sim et al., 1998) or effects on
adenylyl cyclase (Howlett, 1984), it is possible to garner evidence for
an unknown receptor before the development of selective, high-affinity radioligands.
There are two brain-derived chemicals that have been widely studied as
candidate endogenous ligands for cannabinoid receptors. The first to be
discovered was arachidonyl ethanolamide, or anandamide (Devane et al.,
1992). The second was also an arachidonic acid derivative,
2-arachidonyl glycerol (Mechoulam et al., 1995; Sugiura et al., 1995).
Neither compound has high affinity or potency at CB1 or CB2, yet they seem
to conform to many of the criteria for candidate neuromodulator
compounds (Di Marzo et al., 1998).
It has been proposed that the CB1 receptor is the
mediator of all of the CNS actions of cannabinoids. Evidence of this
relationship is 3-fold: the structure-activity relationship for
behavior and receptor affinity is highly correlated (Compton et
al., 1993); the neuroanatomical localization of
CB1 receptors corresponds well with the
CNS-mediated effects of cannabinoids (Breivogel and Childers, 1998);
and most of the CNS-mediated actions of cannabinoids seem to be
reversible by the CB1-selective antagonist
SR141716A (Compton et al., 1996). However, a number of recent studies
have found support for the concept of additional sites of action for cannabinoids (Jarai et al., 1999). There is evidence that anandamide induces spinal antinociception via a different mechanism than THC or
CP55940 (Welch and Eads, 1999; Houser et al., 2000), and that the
centrally-mediated in vivo actions of anandamide are not reversed by
the CB1-selective antagonist, SR147161A (Adams et
al., 1998). Most recently, a study using transgenic C57BL/6 mice
lacking the CB1 receptor
(CB1/) (Zimmer et al., 1999)
revealed that anandamide still affected spontaneous activity,
catalepsy, and analgesia, even though THC was inactive in these mice
(Di Marzo et al., 2000). In the same study, anandamide was found to
stimulate [35S]GTPS binding to brain
membranes from both CB1/ and
CB1+/+ mice, but THC was active
only in CB1+/+ membranes. In the
current study, WIN 55212-2 was also found to stimulate
[35S]GTPS binding in
CB1/ membranes. The goals of
this study were to determine whether additional experiments confirmed
or refuted the existence of a novel G protein-coupled cannabinoid
receptor in brain. To accomplish this goal, the structure-activity
profiles for various cannabinoids were determined, it was established
whether anandamide and WIN55212-2 act at the same or separate sites,
and it was determined whether there is a correlation between activity
and specific binding of [3H] ligands across
membranes from various CNS regions and across different ligands.
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Materials and Methods |
Materials.
CB1-receptor
"knock-out" mutants were developed in C57BL/6 mice as described
earlier (Zimmer et al., 1999). Mice were maintained on a 14-h/10-h
light/dark cycle with free access to food and water. [35S]GTPS (1250 Ci/mmol) and
[3H]WIN55212-2 (55.0 Ci/mmol) were purchased
from PerkinElmer Life Science Products (Boston, MA).
[3H]CP55940 (180 Ci/mmol) was obtained from
Amersham Pharmacia Biotech (Piscataway, NJ). CP55940 and CP55244
were generously provided by Pfizer Inc. (Groton, CT). HU-210 and HU-211
were a generous gift from Prof. Raphael Mechoulam (Hebrew University,
Jerusalem, Israel). Cannabinol, cannabidiol,
9-THC, 8-THC,
SR144528, SR141716A, and [3H]SR141716A (53.0 Ci/mmol) were provided by the National Institute on Drug Abuse.
WIN55212-2, R-(+)-methanandamide, and
R-(
)N6-(2-phenylisopropyl)adenosine
(PIA) were purchased from Research Biochemicals International (Natick,
MA). Other compounds were synthesized by Raj Razdan (O-prefix;
Organix, Woburn, MA) or John W. Huffman (JWH-prefix; Clemson
University, Clemson, SC). GDP and GTPS were purchased from Roche
Molecular Biochemicals (New York, NY).
[D-Ala2,N-Me-Phe4,Gly5-ol]
enkephalin (DAMGO) and all other reagent grade chemicals and enzymes
were obtained from Sigma Chemical Co. (St. Louis, MO) or Fisher
Scientific (Pittsburgh, PA).
Agonist-Stimulated [35S]GTPS binding
assays.
Spinal cords were taken whole and brains were taken whole
or divided on ice into seven regions designated cerebellum,
hippocampus, cortex, basal ganglia (striatum and globus pallidus),
brain stem, midbrain, and diencephalon (thalamus and hypothalamus).
Each preparation was homogenized with a Tissumizer (Tekmar, Cincinnati,
OH) in cold membrane buffer (50 mM Tris-HCl, pH 7.4, 3 mM
MgCl2, 0.2 mM EGTA, 100 mM NaCl, pH 7.7) and
centrifuged at 48,000g for 10 min at 4°C. Pellets were
re-suspended in membrane buffer, then centrifuged again at
48,000g for 10 min at 4°C. Pellets from second centrifugation were homogenized in membrane buffer and stored at
80°C. Frozen membranes were thawed and diluted in membrane buffer,
homogenized, and preincubated for 10 min at 30°C in 0.004 U/ml
adenosine deaminase (240 U/mg protein; Sigma Chemical Co.) to remove
endogenous adenosine, then assayed for protein content before addition
to assay tubes. Assays were conducted at 30°C for 1 h in
membrane buffer including 5 µg of membrane protein with 0.1% (w/v)
bovine serum albumin (BSA), 30 µM GDP, and 0.10 nM
[35S]GTPS in a final volume of 0.5 ml.
Nonspecific binding was determined in the absence of agonists and the
presence of 30 µM unlabeled GTPS. Reactions were terminated by
rapid filtration under vacuum through Whatman GF/B glass fiber filters,
followed by three washes with cold Tris-HCl buffer, pH 7.4. Bound
radioactivity was determined by liquid scintillation spectrophotometry
at 95% efficiency for 35S after overnight
extraction of the filters in 4 ml of BudgetSolve scintillation fluid
(RPI Corp., Mount Prospect, IL).
[3H]Ligand binding.
The methods used for
radioligand binding were as described previously (Griffin et al.,
1998). In initial experiments, binding was initiated by the addition of
25 µg of membrane protein to siliconized tubes containing 10 nM
tritiated radioligand (WIN 55212-2, SR141716A, or CP55940), and a
sufficient volume of buffer to bring the total volume to 0.5 ml. In
subsequent experiments, 100 µg of cerebral cortex or cerebellar
membranes and 8 to 85 nM [3H]WIN55212-2 were
used to determine dose dependence of specific radioligand binding. The
addition of a 2-µM concentration of the corresponding unlabeled
ligand was used to assess nonspecific binding. The membranes were then
incubated at 30°C for 60 min. The reaction was terminated by addition
of ice-cold wash buffer (50 mM Tris HCl and 0.5% BSA, pH 7.4) followed
by rapid filtration under vacuum through Whatman GF/C glass-fiber
filters using a 12-well sampling manifold. The tubes were washed twice
with 2 ml of ice-cold wash buffer and the filters rinsed twice with 4 ml of wash buffer. Filters were placed into 7-ml plastic scintillation vials and 5 ml of BudgetSolve scintillation fluid was added. After shaking for 1 h, bound radioactivity was determined by liquid scintillation spectrophotometry at 45% efficiency for
3H.
Data Analysis.
Net agonist-stimulated
[35S]GTPS binding values were calculated by
subtracting basal binding values (obtained in the absence of agonist)
from agonist-stimulated values (obtained in the presence of agonist).
Data analyses (including agonist concentration-effect and competition
curves) were conducted by iterative nonlinear regression using Prism
for Windows (GraphPad Software, San Diego, CA) to obtain
EC50, Emax,
IC50, and Imax values.
KB values were calculated using the
equation: KB = IC50/[([a]/EC50) + 1],
where IC50 is the concentration of SR141716A that
inhibits half of the stimulation by agonist, [a] is the concentration
of agonist, and EC50 is the concentration of
agonists that produces half-maximal stimulation. Percent
additivity = Sab/(Sa + Sb) × 100%, where Sab is stimulation produced by the combination of
agonists, Sa is stimulation produced by the first
agonist alone (or combination of WIN55212-2 and anandamide), and
Sb is stimulation produced by second agonist
alone. Significant stimulation by multiple concentrations of each
ligand or in each brain region was determined by ANOVA followed by
Dunnett's test at the p < 0.05 level to compare each concentration of ligand to basal binding. Significant specific binding
of single concentrations of radioligand was determined by one-sample
t tests (single concentrations of each ligand) or by ANOVA
(for multiple concentrations) followed by Dunnett's test at the
p < 0.05 level to compare with zero specific binding.
All data presented are mean ± S.E. of experiments performed in
duplicate or triplicate in membranes from at least two different
animals of each genotype, except where noted.
| |
Results |
Stimulation of [35S]GTPS Binding by Various
Compounds.
Results published previously by this laboratory (Di
Marzo et al., 2000) have indicated that, although anandamide seems to be the endogenous ligand for cannabinoid receptors, it still exhibits activity for the stimulation of [35S]GTPS
binding in brain membranes from mice lacking the
CB1 receptor (CB1/). Moreover, anandamide
activity seemed to be equally potent in both
CB1/ and
CB1+/+ C57BL/6 mice, both for
the stimulation of [35S]GTPS binding and in
whole-animal behavioral assays for cannabinoid activity. This activity
proved to be insensitive to the CB1- and CB2-selective antagonists SR141716A and SR144528,
at concentrations sufficient to block activity via each receptor. In
contrast, 9-THC was active only in
CB1+/+ mice (Di Marzo et al.,
2000).
To follow-up the initial experiments, the present study uses
agonist-stimulated [35S]GTPS binding to
further characterize the activity and selectivity of this receptor in
mouse brain. A total of 24 compounds were assayed for stimulation of
[35S]GTPS binding in both
CB1/ and
CB1+/+ mouse whole-brain
membranes. These compounds included an array of commonly used ligands
for cannabinoid receptors and a number of analogs of both anandamide
and WIN55212-2. This included: anandamide and 2-arachidonyl glycerol,
9-THC, 8-THC,
cannabinol, cannabidiol, HU-210, HU-211, CP55940, CP55244, SR141716A,
SR144528, the anandamide analogs 1'-methyl-anandamide (O-610),
2-methyl-anandamide (O-680), 2-dimethyl-anandamide (O-687) (Adams et
al., 1995b), 2-methylarachidonyl-(2'-fluoroethyl)amide (O-689)
(Adams et al., 1995a), WIN55212-2 (and several analogs: JWH-030,
JWH-031, JWH-032, JWH-036, JWH-044, JWH-045, and JWH-073) (Wiley et
al., 1998). The only compounds that produced statistically significant
dose-dependent stimulation of [35S]GTPS
binding in CB1/ membranes
were anandamide and WIN55212-2 (Fig. 1).
All of the commonly used compounds (with the exception of SR144528, the
inactive isomer HU-211, and the inactive cannabinol and cannabidiol)
produced significant effects on [35S]GTPS
binding in CB1+/+ C57BL/6 whole
brain membranes (Table 1 and Fig. 1). The
first three anandamide analogs (O-610, O-680, and O-687) differed from anandamide by one or two methyl groups, yet each exhibited greater potency and lower or equal efficacy than anandamide in
CB1+/+ (Table 1), and each
failed to stimulate [35S]GTPS binding in
CB1/ membranes. Although
there was no indication of any activity by THC, HU-210, or CP55940,
there was a hint of activation by O-610, O-1812 (Fig. 1), and others.
However, the degree of stimulation was quite weak compared with
anandamide and WIN55212-2, and the results for these ligands failed to
achieve statistical significance. Among the analogs of WIN55212-2, only
those that had previously been reported to compete for binding of
[3H]CP55940 to rat brain membranes
(Ki values < 10,000 nM) were found to
stimulate [35S]GTPS binding in
CB1+/+ C57BL/6 whole brain
membranes (Table 1). As with the anandamide analogs, none of the
analogs of WIN55212-2 significantly stimulated [35S]GTPS binding in
CB1/ membranes.
8-THC (not shown) produced effects in
CB1+/+ membranes similar to
those of 9-THC (Table 1), but neither produced
significant effects in CB1/
membranes. The antagonist SR141716A produced significant inhibition of
[35S]GTPS binding that was equivalent in
CB1+/+
(IC50, 4.7 ± 0.4 µM;
Imax, 48 ± 9 fmol/mg) and
CB1/ membranes
(IC50, 5.7 ± 2.1 µM;
Imax, 50 ± 9 fmol/mg).
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Fig. 1.
Concentration-effect curves for stimulation of
[35S]GTPS binding by WIN55212-2, anandamide, O-610,
JWH-030, O-680, and THC in CB1+/+ and
CB1/ C57BL/6 mouse whole brain membranes.
O-610 and O-680 are monomethylated analogs of anandamide and JWH-030 is
an analog of WIN55212-2. Assays were performed using 5 µg of
membranes and included 30 µM GDP, 100 mM NaCl, and 0.1 nM
[35S]GTPS as described under Materials and
Methods. Net agonist-stimulated [35S]GTPS
binding was determined by subtracting basal binding values obtained in
the absence of agonist from values obtained for each concentration of
agonist. Curve fits are to a one-site model. Data are mean values ± S.E. from at least three assays performed in duplicate or
triplicate. Although stimulation by each ligand was statistically
significant in CB1+/+ membranes, only
WIN55212-2 and anandamide significantly stimulated
[35S]GTPS binding in CB1/
membranes.
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TABLE 1
Comparison of CB1 receptor affinity values from rat brain and
concentration-effect parameters for selected agonists in
CB1+/+ C57BL/6 mouse brain membranes
Ki values were determined in rat whole brain membranes via
displacement of [3H]CP55940. Concentrations-effect parameters
for activation of [35S]GTPS binding to
CB1+/+ C57BL/6 mouse brain membranes were determined as
described under Materials and Methods. The maximal
theoretical effect for stimulating [35S]GTPS binding
(Emax), and concentrations that produce a half-maximal response
(EC50) are given. Values are mean ± S.E. from experiments
conducted in triplicate.
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In the present study, results for anandamide were similar to those
previously published (see Table 2):
Emax of 41 fmol/mg (30 ± 2%
stimulation over basal binding) with an EC50
value of 3.6 µM in CB1/,
whereas the corresponding values in
CB1+/+ membranes were 150 fmol/mg (91 ± 4%) and 1.4 µM (Table 2). Similar to anandamide,
stimulation of [35S]GTPS binding by
WIN55212-2 exhibited lower efficacy in
CB1/ (64 fmol/mg; 48 ± 6%) than in CB1+/+ (180 fmol/mg; 110 ± 9%) membranes, but in contrast, WIN55212-2 also
exhibited approximately 10-fold lower potency with an
EC50 value of 1.8 µM in
CB1/ and 0.17 µM in
CB1+/+ (Table 2).
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TABLE 2
Characterization of anandamide and WIN55212-2-stimulated
[35S]GTPS binding and inhibition of agonist-stimulated
[35S]GTPS binding by SR141716A
The first two rows were data obtained using various concentrations of
either anandamide or WIN55212-2 alone. The next three rows are data
obtained with 10 µM anandamide or 10 µM WIN55212-2 in the presence
of various concentrations of SR141716A (see "SR + 10 µM WIN"
and "SR + 10 µM anand." in Fig. 2). "High
KB" and "Low IC50" refer to the two
sites observed for inhibition of agonist-stimulated
[35S]GTPS binding in CB1+/+ membranes.
"High KB" is the affinity of the high potency
site at which SR141716A inhibited agonist stimulation in
CB1+/+ membranes and was calculated from the
high-affinity IC50 value as described under Materials and
Methods. "Low IC50" is concentration of SR141716A that
seem to inhibit half of the remaining stimulation by agonist in
CB1+/+ and was the only site observed in
CB1/ membranes. "% High potency" is the
percentage of agonist-stimulated binding inhibited by SR141716A at the
high KB value. The last two rows were obtained by
subtracting the effects of SR141716A alone from the data obtained for
agonists plus SR141716A for each experiment before nonlinear fitting of
the data. "KB for SR" is the affinity value
exhibited by SR141716A, and "% Blocked SR" is the percentage of
agonist-stimulated binding inhibited by SR141716A (see "SR + 10 µM WIN SR alone" and "SR + 10 µM anand. SR alone"
in Fig. 2). Data are mean values ± S.E. obtained by nonlinear
fitting from at least four experiments.
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Once the active ligands were found, additional experiments were
conducted to optimize assay conditions for
[35S]GTPS binding by anandamide and
WIN55212-2 in CB1/ membranes
and to determine whether this receptor behaves similarly to previously
characterized G protein-coupled receptors for the stimulation of
[35S]GTPS binding. Incubation time and
concentrations of membrane protein, guanosine diphosphate (GDP), and
NaCl were varied (data not shown). All of the results were
qualitatively the same as those previously observed using other ligands
for other receptor systems (Breivogel et al., 1997b; Selley et
al., 1998) or WIN55212-2 in wild-type rat brain membranes (Breivogel et
al., 1998). Briefly, increasing incubation times between 5 and 60 min
increased the amount of [35S]GTPS binding
obtained in either the absence (basal) or presence of agonist (30 µM
anandamide or 10 µM WIN55212-2). There was little change in binding
between 45 and 60 min. Increasing the quantity of membrane protein
between 3 and 100 µg increased the amount of
[35S]GTPS binding obtained in either the
absence (0.47-30 fmol of specific [35S]GTPS
binding) or presence of 30 µM anandamide (0.62-32 fmol of specific
[35S]GTPS binding) or 10 µM WIN55212-2
(0.67-33 fmol of specific [35S]GTPS
binding). However, the increase in binding over basal by agonists (and
thus percent stimulation over basal binding) was greatest at the lowest
concentration of protein (52% by WIN55212-2) and decreased with
increasing protein (9.6% by WIN55212-2). Standard assay conditions in
this study included 30 µM GDP and 100 mM NaCl. Lowering either the
concentration of GDP or NaCl increased the amount of binding obtained
in either the absence (basal) or presence of agonist (30 µM
anandamide or 10 µM WIN55212-2). However, because basal binding
increased whereas net agonist-stimulated binding remained virtually the
same as the concentrations of GDP and NaCl were decreased, percentage
stimulation over basal binding decreased dramatically (from 62 to 5.2%
by WIN55212-2). Although maximal percentage of stimulation was obtained
at increased (100 µM) GDP and 100 mM NaCl, net agonist-stimulated
binding was greater, and thus experimental values were more consistent,
at standard assay conditions (30 µM GDP). These results indicated
that this receptor behaves similarly to previously characterized G
protein-coupled receptors with regard to the effects of time, amount of
membranes, and concentrations of GDP and NaCl on
[35S]GTPS binding. Moreover, the effects of
varying GDP and NaCl were similar between
CB1/ and
CB1+/+ C57BL/6 brain membranes.
It was previously shown that neither 200 nM SR141716A nor 30 nM
SR144528 was able to affect stimulation of
[35S]GTPS binding by anandamide in
CB1/ membranes (Di Marzo et
al., 2000). To further investigate the effects of SR141716A,
concentration-effect curves were generated for SR141716A alone and in
the presence of anandamide or WIN55212-2 in whole-brain membranes from
both CB1+/+ and
CB1/ mice (Table 2). At
concentrations greater than 1 µM, SR141716A decreased basal
[35S]GTPS binding in a
concentration-dependant manner in
CB1+/+ by 48 ± 9 fmol/mg
and CB1/ membranes by
50 ± 9 fmol/mg, yielding IC50 values of
4.7 ± 0.4 µM and 5.7 ± 2.1 µM, respectively (Fig.
2). When combined with either 10 µM
anandamide or 10 µM WIN55212-2, SR141716A concentration-effect curves
fit better (F test, p < 0.05) to a two-site than to a
one-site model in CB1+/+
membranes: there was a high-potency site with
IC50 values from 2 to 24 nM and a low-potency
site with IC50 values between 3 and 13 µM
(Table 2). The high-potency sites exhibited calculated KB values of 0.26 nM and 0.40 nM and made
up 84% of anandamide-stimulated and 66% of WIN55212-2-stimulated
[35S]GTPS binding sites, respectively (Table
2). In CB1/ membranes,
concentrations of SR141716A below 1 µM had no effect on 10 µM
anandamide or 10 µM WIN55212-2-stimulated
[35S]GTPS binding (Fig. 2), and seemed
to exhibit IC50 values of 3.3 µM and 11 µM,
respectively. To define the effect of SR141716A on anandamide
and WIN55212-2-stimulated [35S]GTPS binding,
in the absence of any unrelated effects of SR141716A alone, binding
values obtained in the presence of SR141716A alone were subtracted from
those obtained in the presence of each agonist, and the resulting data
were analyzed by nonlinear fitting (Fig. 2). These calculations showed
stimulation by anandamide and WIN55212-2 that was not completely
blocked by SR141716A in CB1+/+
membrane and was not affected at all in
CB1/ membranes. In
CB1+/+ membranes, SR141716A
inhibited 78% of anandamide-stimulated
[35S]GTPS binding with a
KB value of 0.42 nM, and 67% of
WIN55212-2-stimulated [35S]GTPS binding with
a KB value of 0.71 nM (Table 2).
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Fig. 2.
Comparison of the effects of SR141716A on basal and
WIN55212-2 (SR + 10 µM WIN) or anandamide (SR + 10 µM anand.)
stimulated [35S]GTPS binding in
CB1+/+ (A) and CB1/
brain membranes (B). Net agonist-stimulated [35S]GTPS
binding was determined by subtracting basal binding values obtained in
the absence of agonist from values obtained at each concentration of
agonist. "SR + 10 µM WIN SR alone" and "SR + 10 µM
anand. SR alone" curves were obtained by subtracting binding
values obtained in the presence of SR141716A alone from values obtained
using SR141716A + 10 µM WIN55212-2 or SR141716A + 10 µM anandamide.
In other words, the last two curves are the difference between the
effect of SR141716A alone and the effects of SR141716A on stimulation
of [35S]GTPS binding by each of these agonists, to
account for the effect of SR141716A on basal [35S]GTPS
binding. These manipulations were performed before nonlinear curve
fitting of each of the resulting data sets. Curve fits are to one- or
two-site models as described under Results and in Table
2. Data are means ± S.E. from three or four assays performed in
triplicate.
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Because only anandamide and WIN55212-2, but none of their analogs, were
active for the stimulation of [35S]GTPS
binding in CB1/ membranes,
it was important to determine whether these ligands were working
through the same receptor. Experiments were conducted using 10 µM
anandamide, 10 µM WIN55212-2, 10 µM DAMGO (Selley et al., 1998),
and 1 µM PIA (Breivogel and Childers, 2000), each alone or in
combination. Results indicated that combination of any two agonists
except anandamide and WIN55212-2 were almost completely additive
(between 89 and 104%) and were always higher than for either agonist
alone for the stimulation of [35S]GTPS
binding in both wild-type and
CB1/ membranes (Table
3). Also, combinations of anandamide plus
WIN55212-2 with either DAMGO or PIA were also additive compared with
anandamide plus WIN55212-2 and either DAMGO or PIA alone (Table 3). The values for combinations of anandamide and WIN55212-2 were higher than
for anandamide alone and almost exactly equal to those obtained for
WIN55212-2 alone in both CB1+/+
and CB1/ membranes.
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TABLE 3
Additivity of the effects of agonists for cannabinoid, µ-opioid, and
A1 adenosine receptors in wild-type and CB1/ brain
membranes
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Although the previous experiments in this study were carried out in
membranes prepared from whole mouse brain, a further goal of this study
was to determine the brain regional distribution of the activity of the
putative new cannabinoid receptor. Concentration-effect curves for the
stimulation of [35S]GTPS binding by both
anandamide and WIN55212-2 were performed in eight regions of
CB1/ mouse CNS, including
basal ganglia (caudate-putamen and globus pallidus), brain stem,
cerebellum, cerebral cortex, diencephalon, hippocampus, midbrain, and
spinal cord. Significant stimulation (ANOVA; p < 0.05)
was found for both ligands in all regions except basal ganglia and
cerebellum, where there was no significant activity of either ligand,
and in spinal cord, significant stimulation was observed only with
anandamide (Fig. 3A). In
CB1/ animals, the regions
exhibiting the greatest level of activity were cortex, midbrain, and
hippocampus, followed by diencephalon and brain stem. Spinal cord
contained the lowest level of activity among the regions that showed
significant stimulation.
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Fig. 3.
Emax values for stimulation of
[35S]GTPS binding by anandamide and WIN55212-2 (A) and
specific binding of 10 nM [3H]WIN55212-2 and 10 nM
[3H]SR141716A (B) to membranes from various regions of
CB1/ mouse CNS. Assays shown in A were
performed by incubating membranes with various concentrations of each
agonist and 0.1 nM [35S]GTPS as described under
Materials and Methods. Emax values were
determined by nonlinear fitting to a one-site model. For assays in B,
specific binding was calculated as total minus nonspecific binding,
where nonspecific binding was determined in the presence of a 10-µM
concentration of the respective unlabeled ligand. Values are mean ± S.E. from at least three assays performed in triplicate. All data
shown were significantly different from basal binding at
p < 0.05 by repeated measures ANOVA (A) or no
specific binding at the p < 0.05 level by
one-sample t-tests (B). BG, basal ganglia; BS, brain
stem; CB, cerebellum; CX, cortex; DE, diencephalon; HI, hippocampus;
MB, midbrain; SC, spinal cord.
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[3H] Ligand Binding.
To determine whether the
stimulation of [35S]GTPS binding observed in
the CB1/ CNS regions could
be correlated with specific receptor binding, three radioligands were
assayed: [3H]WIN55212-2,
[3H]SR141716A, and
[3H]CP55940. Initially, it was found that
specific [3H]WIN55212-2 binding displayed a
linear relationship (r2 = 0.963) and was
significantly correlated with protein concentration (p = 0.019) in CB1/ cerebral
cortex membranes between 10 and 100 µg/0.5 ml (Fig. 4). Percent specific binding reached a
plateau from 50 to 100 µg at a level of 20%. In subsequent
experiments, 25 µg of tissue was used to maximize specific binding
while conserving tissue, which was of limited availability.
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Fig. 4.
Relationship between protein concentration and
specific binding obtained with 10 nM [3H]WIN55212-2 in
membranes prepared from CB1/ C57BL/6 mouse
cortex. Specific binding was calculated as total minus nonspecific
binding at each concentration of protein, where nonspecific binding was
determined in the presence of 10 µM WIN55212-2. Data are presented as
means ± S.E. determined from two experiments performed in
triplicate.
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Radioligands, at concentrations of 10 nM, were then tested in each
region prepared from both CB1+/+
and CB1/ C57BL/6 mice to
determine whether there was significant specific binding. Statistically
significant specific binding of each ligand was detected in each region
of CB1+/+ membranes (not shown).
In contrast, total bound radioligand was very much reduced in
CB1/ compared with
CB1+/+ tissues, although
significant specific binding (p < 0.05 one-sample t test) of [3H]WIN55212-2 and
[3H]SR141716A were observed in some regions
(Fig. 5). The areas that demonstrated
significant levels of bound [3H]WIN55212-2 were
the brain stem, cortex, and hippocampus (Fig. 3B). No
statistically-significant binding of
[3H]WIN55212-2 was observed in the basal
ganglia, cerebellum, diencephalon, midbrain, or spinal cord (not
shown). Significant levels of [3H]SR141716A
binding (p < 0.05 one-sample t test) were
observed in the brain stem, cortex, midbrain, and spinal cord, but not in the basal ganglia, cerebellum, diencephalon, or hippocampus (Fig.
3B). No significant binding of [3H]CP55940 was
observed (p < 0.05 one-sample t test) in
any of the brain regions tested (not shown).
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Fig. 5.
Specific binding of various concentrations of
[3H]WIN55212-2 to membranes from
CB1/ mouse cortex and cerebellum. Specific
binding was calculated as total minus nonspecific binding, where
nonspecific binding was determined in the presence of 10 µM unlabeled
WIN55212-2. Data are presented as mean ± S.E. determined from
three experiments performed in triplicate. **p < 0.01 by Dunnett's test versus control (no specific binding).
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To further characterize the specific
[3H]WIN55212-2 binding observed in some regions
of CB1/ mouse brain
membranes, it was important to determine whether the binding was
concentration-dependent using higher concentrations of
[3H]WIN55212-2 used in the single-concentration
analysis described above (Fig. 5). Because analysis of the binding by
high concentrations of these lipophilic radioligands was technically
difficult, and use of such large amounts of radioligand is impractical,
only two regions were selected. Cortex was chosen because it displayed some of the highest levels of both WIN55212-2-stimulated
[35S]GTPS binding and specific
[3H]WIN55212-2 binding among the
CB1/ brain regions, and
cerebellum was chosen as a negative control, because it exhibited a
lack of activity in either assay. Results indicated significant,
concentration-dependent binding of
[3H]WIN55212-2 to
CB1/ cortex membranes
(ANOVA; p = 0.003) at 38 and 86 nM (p < 0.01 by Dunnett's test versus control (no specific binding). In
contrast, no significant specific binding was detected at any
concentration of [3H]WIN55212-2 in
CB1/ cerebellar membranes
(ANOVA; p = 0.06) (Fig. 5).
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Discussion |
A previous study found that anandamide, but not THC, produced
pharmacological effects in
CB1/ mice (Di Marzo et al.,
2000). In the present study, none of the classes of compounds
represented by THC, CP55940, HU-210 or SR141716A were active, but
anandamide and WIN55212-2 activated G proteins in brain membranes from
CB1/ mice. In contrast, none
of the analogs of anandamide or WIN55212-2 were active. However, in an
attempt to find ligands selective for the unknown receptor, analogs of
WIN55212-2 were chosen that exhibited behavioral activity in mice but
failed to displace [3H]CP55940 binding in
previous studies (see Table 1). In the present study, these analogs did
not activate G proteins in
CB1+/+ or
CB1/ mouse membranes. Among
the analogs active in CB1+/+
membranes, all but JWH-073 exhibited lower potency than WIN55212-2 (see
Table 1) and contained significant departures from the chemical structure of WIN55212-2. Moreover, each exhibited at least 33% lower
efficacy than WIN55212-2, which represents stimulation by WIN55212-2
contributed by the putative new receptor.
That analogs of anandamide that differed structurally by one or two
methyl groups did not activate G proteins in
CB1/ was somewhat
unexpected. These compounds were more potent and exhibited equal or
lower efficacy than anandamide in
CB1+/+ membranes. One
interpretation is that O-610 and O-687 activated only
CB1 receptors and did so with efficacy similar to
that of anandamide and that the additional efficacy observed for
anandamide came from the activation of the unknown receptor, for which
anandamide exhibited lower potency as well (yielding a higher
EC50 value). In contrast, O-680 exhibited similar
efficacy but higher potency than anandamide in
CB1+/+ membranes. The lack of
activity of O-680 in CB1/
membranes may indicate higher intrinsic efficacy than anandamide at
CB1. That each of these modifications to the
structure of anandamide yielded compounds that were more potent at
CB1 and were no longer substrates for fatty acid
amide hydrolase (Deutsch and Chin, 1993) argues that these alterations
are significant and produce changes in three-dimensional structure.
Perhaps the putative new receptor's binding pocket is more similar to
that of fatty acid amide hydrolase than CB1.
Alternatively, the lower potency of anandamide in
CB1+/+ membranes and the
activity of anandamide in
CB1/ membranes might be
attributed to degradation of anandamide. This seems unlikely, because
pretreatment of the membranes with phenylmethylsulfonyl fluoride in the
previous study (Di Marzo et al., 2000) had no effect on anandamide
efficacy or potency.
Experiments designed to optimize the
[35S]GTPS binding assay for anandamide and
WIN55212-2 in CB1/ membranes
found that optimal conditions were virtually the same as those for
activation in CB1+/+ membranes
(Breivogel et al., 1997b) or for other receptor systems in brain
(Sim et al., 1995; Selley et al., 1998) or cell (Breivogel et al.,
1997a) membranes. These observations support the concept that
the activities of anandamide and WIN55212-2 were due to activation of a
G protein-coupled receptor.
In a previous study (Di Marzo et al., 2000), neither SR141716A nor
SR144528 affected activation of G proteins by anandamide in
CB1/ membranes at
concentrations at least 50-fold greater than the KD values of the antagonists at
CB1 and CB2, respectively.
In CB1+/+ membranes in the
present study, SR141716A inhibited CB1
receptor-mediated activity with high potency, and remaining agonist
stimulation was inhibited at the same concentrations that produced
inhibition of basal [35S]GTPS binding. If
the effect of SR141716A and the agonists at the low potency sites were
mediated in a competitive manner, the concentrations of SR141716A
necessary to produce inhibition in the presence of agonist would be
higher than in the absence of agonist. Moreover, when the effect of
SR141716A on basal binding was subtracted from the effect on
agonist-stimulated binding, the inhibition of agonist-stimulated
[35S]GTPS binding at the higher
concentrations of SR141716A disappeared. Thus, it seems that the
low-potency inhibition of [35S]GTPS binding
by SR141716A in both CB1+/+ and
CB1/ membranes was caused
not by blockade of the site of anandamide and WIN55212-2 activity but
by additive effects at different sites. These data demonstrate that the
"inverse agonist" activity of SR14716A was mediated by neither
CB1 nor the putative new cannabinoid receptor.
Another finding was that the amount of activity of the unknown receptor
seemed to be equivalent in
CB1+/+ and
CB1/ membranes. Stimulation
produced by anandamide and WIN55212-2 in
CB1/ membranes, 41 fmol/mg
and 64 fmol/mg, respectively, was virtually the same as that refractory
to SR141716A in CB1+/+
membranes, 33 and 60 fmol/mg, respectively (calculated from Table 2).
This indicated that deletion of the CB1 gene did
not effect the expression or activity of the putative new receptor, an
important consideration because the genes for these two receptors may
be related. Additivity experiments implied that anandamide and
WIN55212-2 acted at the same site in
CB1/ membranes, and that
anandamide is a partial agonist at this site relative to WIN55212-2.
Moreover, it seemed that neither µ nor A1 activity was altered by
deletion of CB1.
These data show that anandamide and WIN55212-2 activity was localized
to discreet CNS areas of
CB1/ mice, which coincided
to some degree with areas exhibiting specific binding of
[3H]WIN55212-2. Both were found in cortex,
hippocampus, and brain stem; furthermore, specific binding exhibited
linear dependence on protein concentration and
[3H]WIN55212-2 concentration when examined in
cortex. Two regions, diencephalon and midbrain, exhibited significant
stimulation of [35S]GTPS binding without
specific binding of [3H]WIN55212-2. Basal
ganglia, cerebellum and spinal cord failed to exhibit significant
activity or binding of WIN55212-2, which was confirmed when cerebellum
was subsequently assayed using multiple concentrations of
[3H]WIN55212-2. In contrast,
[3H]CP55940 failed to exhibit specific binding
in any CNS region of CB1/
mice, in agreement with the lack of activity of CP55940 for
[35S]GTPS binding.
[3H]SR141716A exhibited significant binding in
some CNS regions of CB1/
mouse membranes, but unlike [3H]WIN55212-2,
these regions did not correlate with those exhibiting significant
stimulation of [35S]GTPS binding by
anandamide and WIN55212-2. Furthermore, SR141716A decreased basal
[35S]GTPS binding in both
CB1+/+ and
CB1/ whole brain membranes.
Thus, it seems that the inhibition of [35S]GTPS binding by SR141716A at higher
concentrations is unrelated to the site activated by anandamide and
WIN55212-2 in CB1/
membranes, but may be the same sites for which
[3H]SR141716A showed significant binding in
CB1/ membranes.
Interestingly, cerebellum and basal ganglia, regions in which
anandamide and WIN55212-2 failed to significantly stimulate [35S]GTPS binding in
CB1/ mouse brain membranes,
are regions that contain high levels of cannabinoid-induced
[35S]GTPS binding (Breivogel et al.,
1997b), radioligand binding (Breivogel et al., 1997b),
and CB1 mRNA in situ hybridization (Mailleux and
Vanderhaeghen, 1992) in wild-type (nontransgenic) rats. As a direct
comparison, WIN55212-2 significantly stimulated [35S]GTPS binding in C57BL/6
CB1+/+ mouse membranes from
cerebellum by 492 ± 44 fmol/mg, basal ganglia by 489 ± 23 fmol/mg, and hippocampus by 347 ±19 fmol/mg (D. E. Selley,
W. K. Rorrer, C. S. Breivogel, B. R. Martin, and L. J. Sim-Selley, unpublished observations). Thus, the putative new receptor is not localized in all of the same CNS areas as
CB1. That it is missing in basal ganglia implies
that this receptor is not involved in cannabinoid influences on
locomotion mediated by this brain region. It may still mediate effects
on memory via hippocampal receptors (Breivogel and Childers, 1998) or
the pyramidal control of locomotion or other cortical functions.
Moreover, the presence of these receptors in spinal cord may support
previous conclusions that differential binding of cannabinoid ligands
in wild-type rat spinal cord versus forebrain regions indicated the presence of multiple receptor subtypes (G. Griffin and B. R. Martin, submitted), and that spinal antinociceptive effects of
different cannabinoid ligands are mediated by different receptors
(Welch and Eads, 1999; Houser et al., 2000).
That WIN55212-2 has previously been shown to be the most efficacious
cannabinoid agonist (Sim et al., 1996; Breivogel et al., 1998;
Breivogel and Childers, 2000), may be explained by the existence of
this second G protein-coupled target for WIN55212-2. It was shown
previously that WIN55212-2 was as efficacious as levonantradol in rat
cerebellum but was 20 to 30% more efficacious in hippocampus and
hypothalamus membranes (Breivogel and Childers, 2000). This agrees with
the data in the present study that showed no activity of WIN55212-2 in
CB1/ cerebellum, but highly
significant activity in hippocampus and diencephalon (which included
hypothalamus). That anandamide is not more efficacious than other
cannabinoid ligands despite its activity at a second target is
explained by its lower intrinsic efficacy at both
CB1 (Breivogel and Childers, 2000) and this new receptor.
These data strongly support the existence of a previously
uncharacterized, G protein-coupled cannabinoid receptor in brain, and
cast light into its kinetic and ligand recognition properties, as well
as its CNS distribution. It seems to be related to
CB1 and CB2, because two
chemically unrelated compounds that produce cannabinoid pharmacological
effects activate this receptor. It is interesting to note that
compounds derived from cannabis, from which "cannabinoid"
nomenclature is derived, do not activate this receptor. This receptor
may provide a new target for therapeutics, because it seems to have
very different structural requirements from CB1
and may have different physiological effects. However, although these
data provide strong evidence for a novel receptor, proof must involve
cloning and expression of a novel gene.
This work was supported by National Istitute on Drug Abuse
Training Grant DA07027 (C.S.B.) and Grants DA09789 and DA03672 (B.R.M.). V.D.M. was the recipient of a Human Frontier Science Program
short-term fellowship.
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Abstract
Introduction
-subunits with different potencies.
Mol Pharmacol
57:
1000-1010
