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MOLECULAR
PHARMACOLOGY,
34:605-613
ACCELERA
TED
Therapeutics
and Characterization
WILLIAM
A. DEVANE,
ALLYN
C. HOWLETT
FRANCIS
of Pharmacology,
Groton, Connecticut
A.
DYSARZ
III, M.
St. Louis University
Medical
06340 (M.R.J. and L.S.M.)
August 4, 1988; Accepted
Received
Experimental
COMMUNICATION
Determination
Rat Brain
Department
Research,
and
August
ROSS
School,
of a Cannabinoid
JOHNSON,1
St. Louis,
LAWRENCE
Missouri
63104
Receptor
S. MELVIN,
(WAD.,
FAD.
in
and
and A.C.H.)
and Pfizer
Central
30, 1988
,
Various
preparations
traditionally
been
ical manifestations
(2)].
\9-THC
is the
have
effects
\9-THC
on the
include
to
and
memory,
humans
(1,
cannabinoid
a characteristic
work
FAD.
was
University
1 Present
was supported
supported
Medical
Address:
27709.
in extracts
alterations
in
of cannabis
include
ataxia
in part
by a Medical
School.
Glaxo,
Inc.,
altered
by National
Student
5 Moore
Institutes
Drive,
with
in monkeys,
of Health
Fellowship
Research
binoid
drugs
present
time,
ical
neurons
NC
in
models
the
(3).
have
the
poorly
been
actions
effects
Ref.
active
model
4 for
for
the
may
that
cannabinoid
in
of canna-
At the
neuroanatomdrugs,
that
this
obstacle
class
of drugs
evaluation).
interact
by
has
remained
Our
recent
demonstrating
cannabinoid
drugs inhibit
adenylate
neuronal
system
(5, 6). The ability
Gpp(NH)p,
studied
the
clas-
with
can-
drugs
have
CNS.
a thorough
this
change
Although
understood.
concerning
to cannabinoid
pathways
or the
a biphasic
rodents
(3),
remain
is known
responsive
cells,
in
One reason
for our lack of insight
Concerning
cannabinoid
drugs in the CNS is that a clearly
overcome
Park,
of cells
nabinoceptive
from
Triangle
brain
little
neurotransmitter
mechanism(s)
Louis
animal
in the
very
and
activity
relationships
in these
location
sical
immobility,
locomotor
and
ROlto A.C.H.
T32-NS07254.
St.
cannabinoid
structure-activity
humans
on
analGrant
Award
K04-NS00868
Training
Grant
Summer
a
in
hypothermia,
a typical
spontaneous
extensive
and
associated
behavior
in
responses
well as
performance
and
gesia,
to
in cognition
patterns
in dogs,
Career
Development
by a Neuropharmacology
CNS
as
psychomotor
behavioral
have
psychologand
Dewey
THC, tetrahydrocannabinol;
DALN, desacetyllevonantradol;
HEPES, 4-(2-hydroxyethyl)-1
-piperazineethanesulfonic
acid.
ABBREVIATIONS:
system;
drowsiness,
actions
static
DA03690
and
Research
W.A D. was supported
by
CNS (3). The predominant
analgesia
and antiemesis,
Animal
drug
(marihuana)
and for their
Hollister
(1)
compound
a decrement
2).
sativa
Cannabis
major
high,”
“psychological
This
of
used therapeutically
[see reviews
the kinetic constants
approximately
55 M and exceeded
200
pM, respectively.
The binding of the agonist ligand [3H]CP-55,940
was decreased
by the nonhydrolyzable
GTP analog guanylylimidodiphosphate.
The guanine
nucleotide
induced
a more rapid
dissociation
of the ligand from the binding site, consistent
with
an allostenc
regulation
of the putative
receptor
by a G protein.
The binding was also sensitive
to MgCI2 and CaCI2. Binding of
[3H]CP-55,940
was displaced
by cannabinoid
drugs in the following order of potency:
CP-55,940
desacetyllevonantradol
> 11OH-9-THC
=
9-THC
> cannabinol.
Cannabidiol
and cannabigerol displaced
[3H]CP-55,940
by less than 50% at 1 M concentrations.
The (-)-isomer
of CP-55,940
displaced
with 50-fold
greater potency than the (+)-isomer.
This pharmacology
is comparable to both the inhibition
of adenylate
cyclase in vitro and
the analgetic
activity of these compounds
in vivo. The criteria for
a high affinity,
stereoselective,
pharmacologically
distinct
cannabinoid receptor
in brain tissue have been fulfilled.
guanyl ($,-y)-imidodiphosphate;
that
the actions
of
defined
cellular
elusive
studies
the
[see
have
centrally
cyclase
activity
in a
of cannabinoid
drugs
CNS, central
nervous
605
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
SUMMARY
The determination
and characterization
of a cannabinoid
receptor
from brain are reported. A biologically
active bicyclic cannabinoid
analgetic
CP-55,940
was tntium-Iabeled
to high specific activity.
Conditions
for binding to rat brain P2 membranes
and synaptosomes were established.
The pH optimum
was between 7 and
8, and specific binding could be eliminated
by heating the membranes to 60#{176}.
Binding to the P2 membranes
was linear within
the range of 1 0 to 50 g of protein/mi.
Specific binding (defined
as total binding displaced
by 1 M 9-tetrahydrocannabinol
(9
THC) or 1 00 nM desacetyllevonantradol)
was saturable.
The Kd
determined
from Scatchard
analysis was 1 33 pM, and the Bmax
for rat cortical P2 membranes
was 1 .85 pmol/mg
of protein. The
Hill coefficient
for [3H]CP-55,940
approximated
1 indicating
that,
under the conditions
of assay, a single class of binding sites was
determined
that did not exhibit cooperativity.
The binding was
rapid (k00
2.6 x 1 0
pM
min)
and reversible
(K
0.016
min)
and (k06’ > 0.06 min).
The two Kd values estimated
from
606
to
Devane
regulate
et al.
adenylate
cyclase
determined
was
to be
to
National
effects
in
were
could
be
related
Institute
on Drug
Abuse.
DALN
and
the
isomers
of CP-55,940
was demonstrated
for the inhibition
of adenylate
cyclase
that
paralleled
the isomeric
selectivity
exhibited
in analgetic
tests
in animals
(8). The inhibition
of adenylate
cyclase
occurred
at Pfizer Central
Research.
Cannabinoid
drugs were
stored
as 10 mM stock
solutions
in absolute
ethanol
at -20.
Drugs
were initially
diluted to 20 M in 9.4 mg/ml
fatty
acid-deficient
bovine
serum
albumin
using
Regisil-treated
glassware.
All subsequent
dilutions
were
made
into
a vehicle
containing
5 mg/ml
bovine
serum
albumin.
[3H)CP-55,940
was radiolabeled
at DuPont
NEN by tritiuin
reduction, in the presence
of a Pd catalyst,
of a double bond between
carbons
2 and 3 of the A-ring
alkyl side chain
(Fig. 1). Labile
tritium
was
removed
by several
washes
with methanol.
Product
was purified
by
high performance
liquid
chromatography
on a 25-cm
Zorbax
ODS
only
on
column
as
(65:35). Purified
material
was judged by high performance
liquid chromatography
to be greater
than
97% chemically
pure. The specific
activity
was determined
to be 93.4 Ci/mmol,
using the UV absorbance
to quantitate
the yield of product.
Tritium
exchange
with labile hydro-
the
ability
of
humans
these
and
compounds
animal
models
to
produce
8).
The
(7,
CNS
response
produced
at submicromolar
concentrations
(7,
8) and
thus
would be consistent
with drug levels that might
be expected
to
be present
in the brain
during
peak activity
(9-11).
Using
a
series
of cannabinoid
Research
for
in
certain
adenylate
cell
G1 (6,
of
studies
that
effect
such
membrane
demonstrated
requirement
protein
receptors
suggested
fluidity
the
regulatory
hormone
activity.
strongly
the
to
The
evidence
the
presence
that
ultimately
accrued
of
from
pharma-
cologically
unique
cannabinoid
receptors
on the cultured
neuronal
cells.
Logically,
neurons
in the CNS should
also possess
cannabinoid
receptors.
The tools to search
for a cannabinoid
receptor
in the brain
were not available
until
recently.
The relatively
low potency
and tendency
to partition
into biological
membranes
suggest
that 9-THC
is a poor candidate
for a radiolabeled
ligand
for
the
detection
9-THC
nM
(7).
would
molar
characterization
of cannabinoid
adenylate
cyclase with a K10h of 430
It might
be expected
that radioactively
labeled
9-THC
have an affinity
for cannabinoid
receptors
in the nanorange and would bind to receptors
estimated
to be present
brain
Reports
in
of
the
the
range
of
fmoles
membrane/buffer
per
milligram
partition
of
coefficient
tissue.
for
i-
THC
have ranged
from 400 (15) to 12,500
(16).
It can be
calculated
that
the amount
of labeled
z9-THC
binding
to
receptors
could potentially
be 5 or 6 orders
ofmagnitude
smaller
than
the
amount
that
would
be
expected
to
partition
into
A collaborative
interaction
between
lowed the investigation
of cannabinoid
our laboratories
has alreceptors
in the brain
using
cannabinoid
a highly
potent
analgetic
bicyclic
compound,
CNS
receptor
functional
that
groups
mediates
for
analgesia
agonist-receptor
hydroxyl,
functional
groups
of
to
adenylate
cyclase
were
found
in vitro
cyclase
by CP-55,940
was
2)
side
found
important
were
the phenolic
chain.
These
to be required
(8).
The
interaction
be
1) the C-ring
hydroxyl,
and
3) the
A-ring
alkyl
posed
(12).
for
the
The
regulation
to exhibit
a K10h
accounts
25 nM,
for binding
studies
to characterize
receptor.
The studies
reported
for [3H]CP-55,940
and provide
binding
site
is the
elusive
the
cannabinoid
and
than
the
enantiouseful
cannabinoid
here describe
the binding
convincing
evidence
that
activity
55,940
was
Materials.
The
natural
cannabinoid
Procedures
were
provided
by
the
labeling
was
in
NaH2PO4,
excess
stored
at
of
pH
the
1 mCi/mi
4.3
theoretical
in ethanol
at
to
the
radioligand
inhibit
the
was
adenylate
also
monitored.
cyclase
[3H]CP-
activity
of N18TG2
membranes
in a dose-dependent
manner,
using the protocol
previously
described
(6) (data not shown).
Membrane
preparations.
Male Sprague-Dawley
rats weighing
250
to 370 g were decapitated,
and the brains
were
rapidly
removed
and
with
on
ice.
a washed
of two
Unless
or three
rats
were
5 mM MgCl2.
mm.
The
indicated,
P2 preparation
in 45 ml of a solution
and
all
consisting
with
of 320
homogenate
supernatant
was
reported
as follows.
homogenized
The
results
prepared
mM
were
The
and
the
cortices
a Dounce
glass
homogenizer
sucrose,
2 mM
Tris.EDTA,
was centrifuged
saved,
obtained
entire
at 1600
pellets
were
x g for 10
washed
twice
as
above. The combined
supernatant
fractions
were then centrifuged
at
39,000 x g for 15 mm. The pellet was resuspended
in 90 ml of buffer A
(50 mM Tris.HC1,
pH 7.0 at 30*, 2 mM Tris.EDTA,
5 mM MgCl2),
incubated
at
37*
The
membranes
mm,
and
site
in
for
10 mm,
were
centrifuged
and
at 11,000
to be important
equilibrium
centrifuged
resuspended
at
in buffer
x g for
15 mm.
for observing
studies
(see
23,000
These
a single
Results).
x g for
A, incubated
10 mm.
at 30* for 40
two
washing
steps
homogeneous
The
final
binding
pellet
was
resus-
pended
in buffer B (50 mM Tris. HC1, pH 7.4 at 30, 1 mM Tris . EDTA,
3 mM MgCl2) at a protein
concentration
of 4 to 5 mg/ml
and stored
at
Storage
Protein
bovine
for
up
to
using
modifications.
The
Dounce
glass
tubes
Ti50
at
rotor
no
noticeable
effect
on
Tris
.
Ligand
initial
by
for
from
and
x g for
the
10 mm.
was
performed
homogenate
was
The
were performed
The
final
binding.
(17) using
were also
the
hippo-
preparation
with
several
using
a 50-
centrifuged
in
differential
sedimentations
at 50,000
rpm in a Beckman
synaptosomal
pellet
was
resuspended
protein
in a buffer containing
25 mM Tris . HC1, pH 7.4, 1
EDTA,
and 16.6 mM sucrose
and was stored
at -80.
binding
assays.
Ligand
binding
assays
were performed
in
test
and
exception
derived
homogenization
homogenizer
12 mm.
Regisil-treated
started
preparation
cortex of the rat. The synaptosomal
the protocol
of Dodd et al. (18)
1600
for
to 5 mg/ml
mM
had
a synaptosomal
campus
plus prefrontal
was made
following
ml
6 weeks
values were determined
by the method
of Bradford
‘y-globulin
as the standard.
Some
of the studies
performed
30’
receptor.
drugs
of
able
radioligand,
site
this
the
mM
for long
Biological
ml
Experimental
for
CH3CN/25
[3H]CP-55,940
over 1.2M and 0.8M sucrose
of adenylate
of
system
term storage
and at 20*
for routine
usage.
Purity
of the
stored material
was monitored
by thin layer chromatography
on silica
gel GHLF
plates
using
the solvent
system
ether/isopropanol
(98:2).
-80’
50-ml
inhibition
the (-)-isomer
was found
to be 200-fold
more
potent
poorly
analgetic
(+)-isomer
(8). The high affinity
and
selectivity
exhibited
by CP-55,940
made
it a potentially
radioligand
pro-
A-ring
same
solvent
activity.
-80.
CP-55,940
(8, 12). This structure
is one ofa series of compounds
that conform
to a postulated
three-point
agonist-receptor
interaction
model proposed
for the cannabinoid
association
with
the
probably
specific
were found
membranes.
the
gens
dissected
receptors.
was able to inhibit
the
in
and
using
the
tubes
in
addition
50 mm.
After
mm
at
13,000
and
counted
The
microfuge
drugs
experiments,
incubation,
microfuge
x g. After
to
1 ml
containing
specified.
The
samples
and
centrifugation,
were
the
drained
concentration
on
were
were
With
carried
the
out at
to
centrifuged
supernatant
of
drying
pins
B,
was
transferred
immediately
the
buffer
incubation
protein.
all reactions
the
tubes
determine
tubes
of
as
of 20 to 50 .sg of membrane
of the kinetic
polypropylene
a volume
cannabinoid
was
free
1.5for
aspirated
[3H]CP-55,940.
for
30
mm,
after
9
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
these
cyclase
Central
phenomenon
nucleotide
of
Pfizer
enantioselectivity
arguing
cannabinoid-induced
responses
adenylate
14),
clearly
at
(12),
a universal
a guanine
the
decrease
(13,
not
studies
13),
mediates
activity
types
was
be expected
Further
changes.
developed
analgetic
cyclase
would
for
compounds,
their
synthesized
Cannabinoid
Receptors
607
in Rat Brain
)H
Ta
Fig. 1. The synthesis of [3H]
CP-55,940
by the tritium reduction of compound 1.
T
1
3H,
Results
Conditions
studies
for
addressed
radiolabeled
and
cannabinoid
the
separation
ligand.
Williams
In agreement
(16)
and
receptor
binding.
of unbound
Harris
ligand
with the
et al. (19)
Initial
using
THC
as
the
glass
cellulose
nitrate
excessive
added.
and
detergents,
not provide
the
free
with
of the
the
filters
unsuccessful
using
by adsorption
the
acetate
concentration
with
polyethyleneimine,
or
acceptable
conditions
[:3H]Cp55940
coal was also
were
achieved
above.
tubes
varied
Treatment
or cellulose
onto
19).
was
radioligand
organic
bovine
serum
for separation.
(see also Ref.
sedimentation
filters
of
various
solvents,
albumin
did
dextran-coated
Optimal
procedure
ware
55,940
Separating
charconditions
described
The incubations
were performed
in Regisil-treated
glass
in an effort
to minimize
the adsorption
of cannabinoid
surface
in the
the
(16).
the optimal
different
buffers
bound
to
the
for
incubation
tried
were
effectively
to the
during
the exact
the
amount
and adhering
each assay.
conditions
at
glass-
[3H]CP-
of
glassware
by determining
for
of bovine
also
concentration
ligand
in the supernatant
after
the sedimentation
To determine
presence
would
drug
free
adherence
accounted
of radiolabeled
microfuge
tube
the
mixture
in the
from
were
Similarly,
of cannabinoid
alterations
resulting
several
(20).
incubation
amount
Any
incubation
to the
for binding,
various
concentrations
pH values.
Of the buffers
tested,
including
K HEPES,
KW-tris[Hydroxyrnethyl]methyl-2-aminoethanesulfonate,
K
phosphate,
and imidazole
Ct,
none performed
any better than
50 mM
Tris
C1. Optimal
binding
was observed
between
pH 7
and 8, with specific
binding
of only 60% of optimal
at pH 6 or 9
(data not shown).
The phenolic
moiety
of [3H]CP-55,940
might
be expected
to have a pK in the vicinity
ofpH
10. The 40% loss
of specific
binding
observed
between
pH 8 and 9 cannot
be
entirely
accounted
for by a modification
of the ligand.
The pH
requirement
can be postulated
to aLso reflect the optimal
pH for
amino
acids
that would
interact
with the ligand
or be required
to maintain
the optimaiprotein
conformation.
Under
the experimental
conditions
used,
linear
binding
extended
from
10 to 50 j.tg/ml
of protein
for the P2 membrane
preparation
(data not shown).
Experiments
were routinely
conducted
using
20 to 40 g/ml
P2 protein,
and in this range specific
binding
typically
represented
85% of the total binding.
Specific
binding
was linear through
120 to 150 g/ml
of protein
for the
synaptosomalpreparation
(data not shown).
As the concentration
of membranes
was increased,
there was a decline in the percentage of the total binding
that could be described
as specific.
Thermolability
of the specific
binding
was
demonstrated.
Membrane
preparations
that were incubated
at 600 for 12 mm
and
before
of free [3H]CP-55,940
using
a filtralittle
success.
The binding
of ligand
to
and
decrease
shown).
separation
met with
to the
albumin
of Roth
labeled
ligand,
tion technique
fiber
serum
bound
from
experience
compounds
assay
failed
These
[3H]CP-55,940
is subject
to show
significant
specific
binding
ing
to thermal
nonspecific
denaturation.
of
therefrom
was
obtained
from
four
pmol/mg
the
by
Scatchard
sites
ofprotein
Hill
for
(four
transformation
(21),
and
the
Scatchard
error).
The
plot
of 133 ± 11 pM
(21) of the data
Kd
preparation
yielding
An ex-
inset).
± standard
experiments).
increas-
2A,
transformation
P2 cortical
with
(Fig.
in Fig. 2. A
(mean
the
binding
site.
was saturable,
to increase
isotherm
are depicted
experiments
of binding
continued
[3H]CP-55,940
binding
of a saturation
obtained
by
binding
concentrations
ample
not
findings
would be consistent
with the binding
of
to a protein
component
of the membranes
that
Characterization
of the [3HJCP-55,940
[3H]CP-55,940
binding
to cortical
membranes
whereas
(data
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
which the tips of the tubes were sliced, using a heated
spatula
and a
cutting
block designed
to ensure
that the cut tips were of identical
size.
The tips were then placed in scintillation
vials and submersed
in 2 ml
of a solubilizing
solution
(5% ethanol,
5% Triton
X100, 0.2 N NaOH).
The vials were then shaken
for at least 4 hr in order to dissolve
the
pelleted
membranes.
Scintillation
cocktail
(10 ml) was added to each
vial and the radioactivity
was determined
using a Beckman
LS1800
with an efficiency
for tritium
of 30%. Nonspecific
binding
to the
microfuge
tip was assessed
in control
tubes having
radioligand
but no
protein
(typically
2% to 3% of the total radioactivity
available
in the
incubation
medium).
Subtracting
this value from total
binding
gave
the total binding
in the pelleted
membranes.
Specific
binding
was
defined
as the difference
between
total binding
to the membranes
in
the absence
and presence
of either
100 nM DALN
or 1 M 9-THC.
Nonspecific
binding
to the membranes
was typically
15% to 30% of
the total binding
to the membranes,
dependent
upon the membrane
and ligand
concentrations
(see Fig. 2). Assays
were carried
out in
triplicate
with an average
coefficient
of variation
for the samples
of
2.5%, and experiments
were repeated
at least three times.
Metabolism
of [3HJCP-55,940.
To determine
whether
[3H]CP55,940 was metabolized
during the binding
assay, 40 g of membrane
protein
were incubated
with 70 pM [3H]CP-55,940
for 90 mm at 30* in
the standard
assay
buffer
described
above.
After centrifugation,
the
supernatant
and the pellet were separated
and the radioactivity
was
extracted
from each using ether. After drying with N2 gas, the samples
and an unincubated
[3H]CP-55,940
control
were resuspended
in absolute ethanol,
and thin layer chromatography
was performed
using the
procedure
described
above. The plate was sprayed
with EN3HANCE
(NEN,
Boston,
MA),
placed against
Kodak X-Omat
film and stored at
_80* for 6 days. Upon developing,
a single band was observed
for the
samples
which
comigrated
with the control.
These
studies
demonstrated
that neither
the free nor bound [3H]CP-55,940
was metabolized
or chemically
modified
during the assay procedure.
-CP-55,940
The
was
data
were
a straight
This
density
1.85
± 0.26
analyzed
line
(Fig.
One
IDI
IIIIIIIIIIIIIIIIIII10
IIIIIIIII
K9SR-X5R.-
EQAQ
608
Devane
et al.
60
A
0
.
-,
E
9-
/
40
D
C
3
0
I.
.
I
.0
0
20
4-
0
Fig.
2. Equilibrium
binding
of [3H]CP55,940.
Membranes
(43 /Lg of protein)
were incubated
with various
concentrations of [3H]CP-55,940.
A, The saturation
isotherm of specific binding. Inset; binding
a)
0.
U)
Fr..
(3OcP-664O
M
0
0
2000
1000
[3H]CP-55,940
‘+L/tItJ
transformation
of [3H]CP-55,940
binding
data from A with the bound ligand being
expressed
in terms of concentration
(pM).
This experiment
exhibited Kd and Bmax values of 1 39 M and 1 .3 pmol/mg of protein,
respectively.
Inset; The Hill transformation
of data from A. F; free drug concentration;
B; specifically
bound drug. The Hill coefficient (nH) was calculated to be 0.90 for this
experiment.
The lines drawn represent the
best fit as determined
by least squares
linear regression
analysis.
(pM)
0.60
a)
a)
0.40
9-
-o
C
3
0
0.20
0.00
0
10
20
30
40
[3H]CP-55,940
2B, inset).
and
the
The
was
flH
that
the n
binding
sites
conditions
0.88
Kinetic
than
at 30.
hr.
specific
time
of the
through
The
3A).
binding
(pM)
± 12 pM
that
class
of
the assay
cooperativity
is
specific
binding
plateau
consistent
incubation
binding
was
of maximal
with
being
of
exists
the
component
reached
and
with
rapidly,
binding
within
for at least
determination
that
steady
state
no
the
already
earliest
ment
[3H]CP-55,940-receptor
dissociation
more
rapid
earliest
One
change
the
by the
addition
These
rectly
studies
in the
were
performed
microfuge
tubes
before
of 100
nM DALN
is depicted
by establishing
rather
than
by
sedimentation.
Semilog
in Fig.
equilibrium
transferring
plots
(K_1
mechanism
or
interaction
with
free
affinities
dithe
ies.
In preliminary
was
observed
equilibrium
the
this
were
not
that
binding
for
k_1
may
the
studies
the
be
slower
the
component
having
that
these
of
transient
affinity
states
not
of
tightly
for
that
equilibrium
unwashed
kinetic
receptor.
interaction
Evidence
in the
two
bound
such
shown).
binding
be discerned
An
an
binding
P2 membranes,
could
A
at t,.
two
the
possessing
using
45 mm).
=
forms
It is of interest
(data
displaceAssuming
(t,.
nucleotides.
multiple
of
was
that
the
be discerned,
either
below.
studies
the
for complete
expect
experiments)
also
G proteins
discernible
time
represent
It is possible
of guanine
3B.
suggest
(21),
(three
is described
the
of manipulation.
interchangeable
for
receptor
When
addition
at equilibrium
would
may
period
min).
3B).
upon
would
have
sedimented
within
Thus,
the data
obtained
for
could
0.06
that
one
depicted
min1
represent
binding
be noted
the
dissociation
may
specific
However,
points
during
± 0.001
11 mm
in-
time
(Fig.
immediately
of membranes
of centrifugation.
first-order
states
complex
of the
was
GDP
of the
20%
It should
occurring
0.016
monophasic
centrifuged
is 10 mm.
major
fraction
the first
3 mm
the
not
were
displaced.
sedimentation
non-
at the
was
tubes
nM DALN,
The
3 hr.
dissociation
mixture
100
dur-
further
70
dissociation
altered
Procedures).
underwent
the
with
attained
stable
or chemically
Experimental
measurable
ligand
reached
remained
metabolized
(see
to P2 mem-
the
60
microfuge
observation
of [3H}CP-55,940
association
itiated
reaction
The
a single
under
no significant
Equilibrium
The
is not
point
experiments).
that
by [3H]CP-55,940
that
116
was
suggests
labeled
a rapid
90%
This
radioligand
analysis
bound
sites.
(Fig.
50 mm
such
(four
one
and
analysis
greater
the
± 0.08
is being
indicates
receptor
from
approaches
binding
branes
ing
derived
described
among
2
Kd
50
explanation
studit
in
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
Free
of [3H]CP-55,940
the absence
(0) or
presence (U) of 1 ;LMin DALN.
B, Scatchard
A E’sE\I
3000
Cannabinoid
0
50
100
Time
for
these
100
50
results
might
tightly
be that
the
GDP
could
bound
200
(mm)
Time
possessing
(mm)
population
be
150
of G proteins
greater
in
to
for
Kd
binding
dissociation
mated
pM’
the
for
rapidly
rate
experiments).
the
slowly
method
(21,
22).
Using
(and
k+1
ological
to
thus
been
of G proteins
reviewed
by
on
agonist-receptor
Birnbaumer
and
and Gilman
(24). To summarize
believed
to exist in a heterotrimer
form
bound
GDP in the presence
of
the
values
calculated
binding
methodology
has
Allosteric
been
Thus,
that
system
via
anisms
similar
to those
homologous
x
the
The
protein
intermediate
for
agonist.
methods
briefly,
the
receptor
a k0h, as
the
receptor
theoretical
and
of the
linear
transformation
an internal
the k+, may
and the Kd
of
consistency
One would
its signal
would
that
be
have
receptors.
regulated
been
Our
to the
by
the
current
hypothesize
cyclase
allosteric
demonstrated
unit.
Dissociation
mechfor
understanding
other
GTP
the
the
decrease
in
The
of the
G protein
into
a and
the
a
t/.
from
calculated
in
the
0.059
pM.
for
± 0.009
This
dissociating
dissociation
min
value
component
the
described
for
from
The
effector
a sub-
receptor
and
hy-
the
system
to
allow
of hormone.
were
45 mm
Kd
to
not
in
12 mm.
and
An
the
for
in-depth
Kd
the
the
addition
The
of Gpp(NH)p
estimated
above.
(data
analyzed
(Fig. 4). The
presence
experiments)
to
ligand
GTP-bound
the
nucleotide
(three
is similar
affinity
dissociates
of [3H]CP-55,940
dissociation
presence
or absence
ofguanine
of Gpp(NH)p
reduced
the
In
equilibrium
binding
studies
iM Gpp(NH)p
resulted
in a
binding
of
high
agonist
the
subunit
of 100
specific
dissociates.
3y subunits.
from
receptor,
a
suf-
a nonhydrolyzable
concentrations
presence
kinetics
or
the
to altered
cannabinoid
that
shown).
on
GDP
affinity
hormone
with
confers
a. relatively
with
are
tightly
receptor-hormone-G
and
interacts
of the
respond
indicated
40%
separates
the
the
the
GTP
and
interaction
change
that
of
the
cyclase)
of
For
for the
adenylate
and
perpetually
similar
binding
(23)
G proteins
possessing
Upon
exhibits
complex
complex,
adenylate
drolysis
such
is decreased,
(e.g.,
method-
are
Kd
to
(afl’y)
nucleotides,
Upon
this
analog
for estimating
estimates
the
system
interactions
colleagues
a conformational
of guanine
Kd
for
pM.
to the
absence
An
Kd
180
rate,
min’
complex,
energy
the
a pseudo-first-order
dissociation
10
pM’
of binding.
transmits
a G protein
3.4 ± 0.76
value,
demonstrated.
regulation
a receptor
functionally
using
data.
was
this
47 pM.
receptor-agonist
ficient
esti-
k+1
be to estimate
using
have
kinetic
appreciably
to exceed
of these
values)
Kd
the
equilibrium
that
the
limitations,
both
was
have
would
the slower
2.6 ± 0.2 x
was estirequired
3
The
(22)
site
would
data
association
contribute
Using
to equilibrium
be calculated
to be
would be 62 pM. Although
the
not
displacing
of the
proceeds
the
of incubation.
of binding
site
treatment
reaction
5 mm
(three
from
rate of association
of the membranes
would
after
dissociating
alternative
calculated
initial
min’
calculated
the
dissociation
until
from
10
that
the
reaction
be
The initial
pelleting
rates.
that
and
the
the
may
by assuming
mm
influence
has
Casey
The
mated
the
of
unwashed
membranes.
and
Fig. 3. Time course of association (A) and dissociation
(B)
of [3H]CP-55,940.
[3H]CP-55,940
(81 pM) was incubated
with 28 ;hg of P2 membranes
at 30#{176}.
A, The times indicated
are those that elapsed
between
the addition
of protein
(start of the reaction) and the start of centrifugation
of the
microfuge
tubes. Specific and nonspecific
binding were
determined
with 100 flM DALN as described in Expenmental Procedures.
B, After equilibrium
binding
of [3H]CP55,940 had been reached (70 mm), 100 n DALN was
added (t = 0) and dissociation
was monitored.
Data presented are a first-order
representation
with B/B0 denoting
the specific binding at the time indicated/specific
binding
at t = 0. The results are means of triplicate determinations
from a single representative
experiment,
which was performed three times.
k
was
was
176
rapidly
analysis
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
0
150
609
in Rat Brain
Receptors
610
Devane et al.
I
Fig. 4. Effect of Gpp(NH)p
on the dissociation
rate
of [3HJCP-55,940.
[3H]CP-55,940
(81 pM) was incubated
with 28 g of P2 preparation
membranes
for 70 mm at 30#{176}
and dissociation
was monitored
after addition
of 1 00 nM DALN (t = 0). Control,
addition of 1 00 nM DALN; +Gpp(NH)p,
simultaneous addition
of 1 00 nM DALN
plus 1 00 M
Gpp(NH)p.
The x axis indicates
the time that elapsed
between
the addition of the above compounds
and
2 mm after the start of centrifugation.
The y axis is
a log-scale
presentation
of the percentage
of specific binding at the indicated time/specific
binding at
t = 0. The data are the means
of triplicate determinations
from a single representative
experiment,
which was repeated three times.
I
-j
0
15
30
Time
interaction
proteins
most
facile
the
of the
in various
nucleotides
with
Divalent
for
possesses
at
divalent
presence
affinity
one
has
for
been
the
whether
G
The
at
than
50%
with CaCl2.
similar
Studies
of the
the
(see
26 and
receptors
opioid
affinity
associated
(data
not
which
manner,
(26, 27).
indicated
Na
has
In
as 1 mM
binding
response
to
does
for
the
an
effort
-
opioid
Na
act
Ca2
Na
of K
NaCl
80%.
The
appear
to be
great,
specific
had
and
by about
not
intra-
reduced
minimal
100
mM
selectivity
KC1
of this
suggesting
that
salt
concentrations
as
is altering
the
ability
of the
ligand
to bind
receptor.
the
of
of [3H]CP-55,940
ability
the
(Fig.
6).
determined
using
1987)
(28).
A one-site
for
an
pmol/mg
computer
data
using
CP-55,940
± 0.20
ment
the
both
CP-55,940
criterion
had
analysis
model
fit the
and
on
natural
a K
mem-
of 68 ± 6.2
(three
better
cannabinoid
residual
variances
for
cortical
pM
experiments)
(version
data
other
the
several
of homologous
program
speci-
[3H]CP-55,940
in the
protein
LIGAND
F test
sites
The
by determining
and
with
compete
binding
Unlabeled
receptor.
established
compounds
to
specific
by
was
synthetic
compounds
of
cannabinoid
binding
of related
Bmax of 1.75
model
receptor
mM,
mM
tested
inhibition
may not be the result
of a specific
interaction
a Na
site. It is possible
that a chaotropic
effect
of higher
branes
to determine
120
were
this
with
occupancy
as
to
cations
monochloride
to be present
concentrations
of
specific
cannabinoid
to adenylate
Low
20
by
of various
be expected
At
40%.
effects
5). Monovalent
might
inhibited
the
effects
were
binding
in
that
Na
may
been
demonstrated
KCI
Concentrations
and
is coupled
In
by about
of [3H]CP-55,940
the
extracellularly.
effects.
ficity
[3H]CP-55,940
similar
specific
(Fig.
Pharmacology
the
binding
observed,
that
or
to the
the
be
determined
binding
aden-
(25).
as low
ligand
ligands
therein).
to
shown).
receptor,
of agonist
in
higher
with
Maguire
agonist
this
to increase
of MgCl2
of the
references
a much
of Mgi
by
for
of GDP
in addition
having
(Fig. 5). Qualitatively
MnCl2
also stimulated
cyclase
in an inhibitory
an allosteric
regulator
decrease
for
discussed
to MgCl2
A role
complex,
effects
affinity
G protein
dissociation
a site
The
binding
24).
the
were
of the
could
concentrations
cellularly
of a
the
cations
salts
is that
interaction
regulation
valent
by guanine
that
(23,
the
concentrations
specific
a manner
for
with
ligands
have
Mgi
shown
24).
investigation,
Ref.
here
to influence
receptor-hormone
(23,
of agonist
by greater
observed
with
with
study.
presented
It is believed
associated
Mgi
stimulated
site
of this
be influenced
reported
site
been
of the
cyclase
present
been
receptors.
least
functions
for
results
can
consistent
have
their
cation
affinity
scope
a G protein.
cations
of agonists
ylate
site
binding
the
of the
binding
in a manner
receptor
other
is beyond
interpretation
[4H]CP-55,940
the
[3H]CP-55,940
states
(mm)
displace2.3.11;
than
May,
a two-site
drugs
tested
at the
level
MgC2
200
0
150
Fig. 5. The influence of cations on specific binding of [3H]
CP-55,940.
Control samples contained
50 m Tns . HCI, 1
C
0
0
0
mM
C
a)
0
a)
Tns.EDTA,
and 0.1
mM
MgCl2. Experimental
samples
contained the same buffer plus the indicated concentrations
of salts. The data are the means ± standard error of triplicate
determinations
from a single representative
experiment.
All
values were different from control at p < 0.05 except 20
mM NaCI and 5 mM KCl. Similar
results were observed
in
two other experiments.
100
50
0
20
120
5
100
1
Millimolar
3
added
10
1
3
10
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
the
of
60
45
Cannabinoid
Receptors
611
in Rat Brain
0)
c
-o
c
-O
Fig. 6. Competitive
inhibition of [3H]CP55,940 binding by various synthetic and natural cannabinoid
drugs. [3H]CP-55,940
(5070 pM) was incubated
with P2 membranes
(1 6-30 g) for 50 mm at 30#{176}
with either the
indicated
concentrations
of drug or vehicle
alone. The results were normalized
to 100%
of specific binding,
which was determined
with 1 00 nM DALN as described
in Experimental Procedures.
Data points represent
the averages
of triplicate
determinations
from single representative
experiments.
The
K1 values listed in the inset table were determined using the LIGAND program and represent the mean ± standard
error of three
0
4-
0
a)
0.
U)
9-
0
a)
0
a)
independent
12
11
10
9
-Log
of
p
=
values
analysis
8
[drug]
7
CP-55,940
was 50-fold
less potent
than the (-)-isomer,
having
a K, of 3.4 nM. DALN
was nearly
equipotent
with CP-55,940,
having
a K, of 123 pM. z9-THC
and
11-OH-9-THC
both
exhibited
high affinity
binding
with K1 values
of 1.6 nM. Cannabinol
was 8-fold
less potent
than
9-THC,
having
a K1 of 13
This
with
The
cyclase
selective
tor.
nabinoid
fail
compounds
in
solution.
These
concentrations
latter
two
of cancompounds
to exhibit
cannabinoid
activity
in humans
or animal
models
(1-3). One of several
explanations
for the binding
results
could
apply.
1) The two compounds
could
bind to the receptor
with
low affinity,
but the concentrations
required
to observe
a biological response
may not be possible
to achieve
in vivo. 2) The
cannabinoid
by
drug
response
to
these
two
compounds
may
be masked
concentrations,
such
as membrane
perturbation
in in vitro
studies
and CNS
depression
in in vivo
studies.
3) The observed
binding
displacement
may be the result
of a contaminant
in the drug preparation.
The latter
explanation
is a possible
artifact
that
must
be dealt
with
in future
investigations.
These
drugs
were isolated
from organic
extracts
of cannabis
that
originally
contained
a variety
of cannabinoid
compounds,
including
\9-THC.
effects
at
high
Discussion
Studies
the ability
suggestive
of a cannabinoid
receptor
were
based
on
of centrally
active
cannabinoid
drugs
to interact
guanine
for this
interaction
The
findings
argument
would
with
presented
The
rat
from
what
would
CNS
(30).
It may
for
be expected
investigations
to
drugs
have
system.
To
strengthen
binoid
have
receptor
evidence
duction
with
drugs
rat
(31,
brain
32).
in the
site
the
its cortical
was more
binding
site
than
2 orders
its Kh
regulation
of adenylate
the
model
of the
canna-
CNS,
Cyclic
we
AMP
now
pro-
in response
agonist
in the absence
of magnitude
cyclase
by cannacell
is decreased
affinity
[H]CPPrevious
of the
preparations.
regions
for
brain.
cyclase
in
The
receptor
association
55,940
for
nucleotides
for
studies.
with
putative
The
of [3H]CP-
is consistent
binding
in
in
CNS
remarkably
binding
ligand
cyclase
a
and
for
agrees
neuroblastoma
slice
rapid
reported
of adenylate
adenylate
brain
The
Kd for binding
the
via
consistent
Bmax determined
a cloned
the
using
site.
the
cyclase
inhibition
utilized
in several
cannabinoid
that
adenylate
of the
binoid
system
a neuromodulatory
be hypothesized
is linked
The
a receptor
are
constants
agonist
for
strongest
of a neuromodulator
equilibrium
this
the
with
values
The
kinetic
from
determined
and
with
(30).
the
Kd obtained
the
affinity
binding
toxin
receptor.
messenger
expected
consistent
receptors
derived
this
saturability
are
provide
thermolability
properties
binding
cortex
pertussis
consistent
a second
the
require-
(6, 13).
a cannabinoid
and
for
are
neuromodulator
55,940
with
the
are
cation
receptors
is entirely
structure
binding
receptor.
with
here
be associated
a protein
divalent
study
for
recep-
cyclase
demonstrated
this
The pH sensitivity
reversible
55,940
in
available
described
G protein.
the
enantiothat these
mediating
adenylate
of G1-linked
currently
site
with
and
and
and
of a receptor
nucleotide
characteristic
potent,
hypothesis
system.
adenylate
membrane-bound
in favor
drugs
messenger
inhibit
to
the
a biological
of cannabinoid
sensitivity
that
with
arguments
characteristic
ments
cellular
second
compounds
cell type-specific,
would
support
interact
interaction
the
higher
in a reversible,
manner
(5-8)
Additional
would
to maintain
characterized,
of cannabinoid
compounds
binding
for
be necessary
6
a well
ability
order
of potency
generally
parallels
the
order
of
both
CNS
activity
in vivo (1, 3, 12, 29) and inhibition of adenylate
cyclase
in vitro (7, 8).
Cannabidiol
and cannabigerol
were much
less potent,
binding
the [3H]CP-55,940
site with K1 values
estimated
to be greater
than
500 nM. Cannabidiol
and
cannabigerol
were
unable
to
fully displace
the specifically
bound
[3H]CP-55,940
at the highest concentrations
tested
(1 1uM). Concentrations
greater
than
this were not tested
due to the limited
solubility
of cannabinoid
drugs
above
1 M
and
the confounding
aspects
of increasing
the
solvent
or bovine
serum
albumin
concentration,
which
nM.
for each drug.
(M)
0.05. Using
68 pM as the Kd for [3H]CP-55,940,
the K1
of the various
compounds
were determined
by LIGAND
of heterologous
displacement
data.
The (+)-isomer
of
potency
experiments
in the
to
[3H]CP-
of guanine
greater
than
neuronal
cell
Downloaded from molpharm.aspetjournals.org at Univ of Washington on April 14, 2009
cL
612
Devane
model
of
(8).
eta!.
However,
Gpp(NH)p
adenylate
for ligand
this
site
are
inhibition
One
responses
designed
to possess
activity
plate,
for CP-55,940
phenylbenzylquinone
tests
to the
(-)agrees
reported
here
binoid
been
displace
rat
latter
was
not
dependent
scribed
by
Nye
thylammonium
to this
site
behavioral
the
analog
does
not
models
CNS
depression
with
our
(35).
taming
the hydrophobic
from the A-ring
(8).
The
to discern
34)
ligand
ity
of
the
stereoisomers
and
in vivo
effects
of
z9-THC
may
was
(33).
z8-THC
was
binding
of [3H]trimethylammonium
was 3 orders
of magnitude
lower
8-THC
than
the
that,
throughout
adsorption
Evidence
analysis
finding
of the
may
these
to
suggests
glass
equilibrium
in part
be
that
this
yielded
a K
determined
binding
data
the
aqueous
not
site
the
(34).
linked
a G
that
the
is definitely
described
here
suggested
reported
or
that
[3H]
different
using
[3H]CP
cannabinoid
neuromodulators
(36)
that
the
drugs
to their
receptors.
concentrations
11-OH-z9-THC
increased
additional
that
drugs
in
specific
studies
similarly
altered
from
high
fluidity
in excess
the
presence
binding
that
of a
of the
f3-adre-
for
opioid
receptors
cannabinoid
We have
previously
demonstrated
in neuroblastoma
agonists
duction
indicate
in the
cells
corthat
that
is
which
of
plasma
cannabi-
membranes
(37).
Vaysse
with certain
by addition
drugs
laboratory,
concentrations
of synaptic
by fluorescence
polarization
(38) reported
a similar
interference
of several
of high
as
and
colbinding
concentrations
presence
of 100 mM
that
site of cannabinoid
is not
the
related
to the
to the #{244}
opioid
receptor
or to subsequent
(14). The studies
reported
in the present
the presence
of a pharmacologically
ethanol.
binding
signal
work
selective,
of
transclearly
high
affinity
binding
site for cannabinoid
drugs.
However,
membrane
perturbation
may
be the
mechanism
by which
high
concentrations
of cannabinoid
drugs
may interfere
with binding
determinations
made
for a variety
of other
receptor
types.
The development
noid
class
of drugs
that
brain
have
that
of a ligand
will allow
previously
binding
assay
investigations
for the cannabiof cannabinoid
been
Pathways
in the
action
can
can
be
be more
not
may
be involved
in
The cellular
regulation
examined.
cannabinoid
and
can
drugs
its interaction
be assessed.
can
possible.
cannabinoid
of the receptor
with
Perhaps
be developed
alternative
second
an antagonist
for
now
that
a binding
site
has been
found
that
correlates
with
a cellular
function
(inhibition
of adenylate
cyclase).
Furthermore,
efforts
to search
for
putative
endogenous
of the
make
a major
ligand
can
characterization
impact
now
proceed.
of a cannabinoid
on research
in this
Thus,
the
receptor
field.
Acknowledgments
The
excellent
clerical
assistance
of Maggie
Klevorn
is gratefully
appreciated.
References
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This
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nergic
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[3H]dihydroalprenolol
in mouse
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The interpretation
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the cannabinoid
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such
the binding
behavior
was modified
(36).
This
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observation
considered
is not
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4.
and
for
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Hillard
and Bloom
of 3 tM
9-THC
for
2-fold,
Kd
were
binding
than
derived
by
ligands
site
z8-
by nonhydrolyzable
It is clear
binding
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will
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levonantradol
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less
for
constants
Scatchard
The
(34).
z8-THC
cannabinoid
importance
that
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[3H]trimethylammonium
decreased
as the
Other
a
of main-
binding
kinetic
unusual
of
is consistent
be questioned.
observed
the
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the
animal
exception
models
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of this binding
site
The
dextronantradol
the
8-THC
and
no stereoselectivity
for binding
8-THC
indicated
greatest
affinity
in
and cannabidiol)
are
[3Hjtrimethylammonium
from
of
than
nucleotides
fully characterized
messenger
systems
side chain
extending
profile
for displace-
agonists
nor antagonists
in in vivo animal
(33). Thus,
the pharmacological
relevance
to cannabinoid
used
importance
alkyl
de-
[3H]5’-trime-
activity
of the
(16).
was
binding
rather
of guanine
actions
which
in typical
with
biological
nature
ofthe
pharmacological
affinity
9-THC
the
activity
action,
ment
of [3H]5’-trimethylammonium
cannabinoid
compounds
having
brain
regions
(e.g., cannabigerol
to
however,
a high
using
The
poor
able
adsorption,
offree
biological
This
were
membranes
demonstration
and
synaptosomal
concentration
of 8-THC.
not
and
as
trimethylammonium
action
have
Roth
8-THC;
1 jM
membrane
of cannabinoid
previous
canna-
[3H]8-THC
group
in brain
analogs
assays
pharmacological
displacewas not performed
(19).
(33,
exhibit
and
receptor
to analgesia.
site
colleagues
and
a cannabinoid
of
unable
was enhanced
noid
including
typical
or purified
with
than
binding
and
of the
former
inasmuch
THC
demonstrated
Other
this
in addition
and
ligands
of
CP-55,940
Thus,
characterize
binding
upon
affinity
order
hypothermia,
certain
The
other
the
order
receptor.
for
binding
were
of binding
dem-
of drugs,
(29).
to crude
brains.
of the
This
The
or in vivo functions
colleagues
(19) and
and
investigators
A high
class
and
in vitro
of the
the
activity,
demonstrated
up to 10%
component
to
in animals
the binding
was not saturable
ment
by other
cannabinoid
The
binding
find
with
Harris
from
analgetic
site.
by
demonstrated
respectively,
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is mimicked
with
to
receptor
associated
met
with
success.
[3HJ9-THC,
(12).
in the tail flick,
hot
tail
clamp,
and
flinch
cannabinoid
observed
(16)
receptor
site
specifically
was
binding
locomotor
attempts
Williams
data
protein
55,940
activity
to be enantioselective
responses
of potencies
described
for
reported
[3H]CP-55,940
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50-fold
the
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