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Department of Pharmacology and Toxicology, Medical College of Virginia/Virginia Commonwealth University. Richmond,
Virginia
Accepted for publication August 19, 1994
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ABSTRACT
Dose-effect
Administration to
Mice: Possible Mechanisms for Interaction with Morphine’SANDRA P. WELCH, CHARITY THOMAS and GRAY S. PATRICK
THC and
tracerebroventricularly (icv.)] and compared with those previ-
ously generated after administration intrathecally
The ED,,
values after administration of
56,667 was significantly more potent after icv. administration
than
administration, and was nearly 10 times more potent
than CP 55,940 (icv.j. CP 55,940 and
for 7 days
totally abolished the antinociception produced by the cannabi-
ABBREVIATIONS:
sulfoxide.
310
manner nearly 1 O-fold
after icv. administration. The
or Cl-CAMP (10
the cannabinoids.
Recent work has shown that the cannabinoids produce
potent antinociceptive effects in mice and rats through both
spinal and supraspinal mechanisms (Welch and Stevens,
1992; Welch, 1993; Smith and Martin, 1992; Lichtman and
Martin,
the
salt; DMSO,
Modulation of Cannabinoid-Induced Antinociception After
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56,667, which did not
produce greater than additive effects in combination with
shifted phine when the drugs were administered
the mor-
phine (icv.) dose-effect curve in a
nabinoids (icv.) were not blocked by ICI 174,864 (20
mouse),
significantly attenuated the antinociception produced
by the
and CP 55,940. However, when administered
icv., forskolin and Cl-CAMP produced antinociception, but did
not block or produce greater than additive effects with the
possible effect; THC, tetrahydrocannabinol;
monosodium salt; 4-AP, 4-aminopyridine; TEA, tetraethylammonium;
or naloxone (20 kg/mouse or
10
Received for publication September
This
CL, confidence limit; AD,,, dose producing 50% antagonism; Cl-c-AMP,
antinociception in the rat has been proposed to
(icv. and i.t.). Pretreatment of the mice with forskolin
the mechanism by which cannabinoids produce
supported by the Nationai
Pertussis toxin pretreatment
Although the spinal mechanism of cannabi-
(79
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were generated for the canrrabinoids
. VS
icv. did not differ significantly.
intrathecally; icv, intracerebroventricularly;
and
receptor
i.t.), which produced no
1993.
of Drug Abuse Grants
Versus
and Martin,
55,940,
of the
intravenously;
tration of the cannabinoids. Conversely.
the antinociceptive effects of the cannabinoids. The AZ.
ues
antinociception produced by the cannabinoids
failed to alter the antinociception produced by the
which did not alter the antinociceptive effects of
Thapsigargin (icv.) blocked the antinociception
icv. The
CP 55,940 were 215
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nmollmouse, respectively.
effect curves of
ATP- and apamin-sensitive potassium channels, these
intracellular systems may be common points of
nificantly reversed the calcium-induced blockade of
sium channels, produced a parallel rightward shift in the
apamin (5
nabinoids. Because acute administration of
tinociception has not been
tion of a specific cannabinoid receptor has been the to]
intense investigation leading to the cloning of a
have been shown to interact with
In receptor
addition. an endoge
ligand for the cannabinoid receptor has been identified
ane et al.,
tion-What are the transduction mechanisms
with the activation of the cannabinoid receptor’?
modulation of adenylate
sible transduction mechanisms have been proposed
calcium. prostaglandin
opioids. The role of adenylate cyclase, calcium
and potassium channel modulation, and to a lesser
decrease calcium entry to and content of neurons.
levels and produce hyperpolarization of neurons
dibutyryl cyclic
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and CP 55,940. Apamin, blocker
for calcium-induced
nor-binaitorphimine; ICI 174,864.
administration of calcium and calcium
subcutaneously:
These reports led to the next logical
icv.) failed to block icv.
176 (122-253) and
and
determined. The
monophosphate
percentage of the
of
protein-coupled
55,940
(1
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1995
role of
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of the cannabinoids is the focus of this paper. These
mechanisms will be discussed individually, although the in-
terplay of these various systems in the transduction
the
effects of the cannabinoids is clearly a possibility.
CAMP. The cloning of the cannabinoid receptor was per-
formed in
modula-
tion after receptor activation
Numerous studies have shown the involvement of the
cannabinoids in the modulation of
amide, the endogenous cannabinoid ligand, has also been
shown to inhibit
levels in cells of
various types in culture as well as in homogenates of brain
regions (Kelly and Butcher. 1979; Zimmerman et al..
et
al., 1986). The potency of numerous cannabinoids to inhibit
and Ng, 1984; Howlett and Fleming, 1984; Howlett, 1984;
Howlett,
The interaction of the cannabinoids with a
membrane protein
bosylated protein was identified as the
formation in the neuroblastoma cells was found to
correlate to the antinociceptive effects of the drugs in
The effects of the cannabinoids on
spinal
accumulation have not been examined. This
study utilizes modulators of adenylate cyclase or
levels
in
to evaluate the effects of such treatments on theantinociceptive effects of the cannabinoids in the brain and in
the spinal cord.
transmission and an-
tinociception. Previous work has indicated that
An alteration of intracellular cal-
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cium by the cannabinoids could be the trigger for a
events leading to decreased
de-
creases the release of acetylcholine presynaptically by a de-
crease in the influx of calcium into presynaptic nerve
terminals
studies of the involvement
of calcium in the antinociceptive effects of the cannabinoids
have not been reported previously. In addition, potassium
channel opening and the resultant hyperpolarization of
19821 found that cannabinoids decrease calcium uptake to
several brain regions, an effect which did not correlate to the
psychoactivity of the drugs. Direct measurement of the ef-
fects of cannabinoids on free intracellular calcium
has shown that depolarization-in-
duced rises in intracellular calcium are attenuated by
vitro studies indicate a role for calcium in the
effects of the cannabinoids, in
membrane could account for the antinociceptive
effects of cannabinoids. The modulation of potassium chan-
nels by the cannabinoids may differ in the brain and in the
spinal cord. The involvement of potassium channels in
nabinoid-induced effects has been reported in
although the nature of the interaction of the
drugs with potassium channels has not been determined.
et al., 1973; Chesher and
Jackson, 1985; Sanders et al., 1979; Martin,
fails to block the effects of various parenterally admin-
istered cannabinoids
also failed to block the antinociception in-
duced by a variety of
al.,
is unclear. Many investigators have shown that
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icv. or
in concentrations of 1
Welch,
using
et
The role of opioids in cannabinoid antinocicep-
as possible mechanisms in the antinoeiceptive
1981; Gilbert, 1981; Welch and Stevens,
It is intriguing that, despite the data
with an evaluation of
and
ADP-ribosylation was shown. The
production in
icv. or spinally administered
or higher
et al.,
protein
et
Naloxone
Stokes
1988
brain
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311
suggesting that cannabinoids and opiates have distinct
of action, the effects of morphine have been
found to be enhanced by crude cannabis extract
and
Bhattacharya,
et al., 1984). Intrathecal administration
of several cannabinoids leads to synergism with
istered morphine in the production of antinociception in mice
binoids
intraventricular injections. Intrathecal in-
jections were performed according to the protocol of Hylden and
Wilcox
antinociception, but not
catalepsy,
or hypoactivity has been also re-
ported
Although we have evaluated the
effects of opioid antagonists on the antinociceptive effects of
(Welch and Stevens, 1992;
Welch,
with icv. administered opioids and opioid antagonists
has not been reported previously.
Thus, the research presented in this manuscript was de-
signed to evaluate the involvement of the cannabinoids
routes both
of administration) on the modulation
of
in the
production ofantinociception. In addition, we wanted to com-
pare and contrast the interaction of the carmabinoids with
morphine in the brain and in the spinal cord. Finally, from
our results we hoped to generate a hypothesis as to the possible
Mice were lightly anesthetized with ether and an incision was made
in the scalp such that the bregma was exposed. Injections were
performed using a 26-gauge needle with a sleeve of PE
produced significant antinociceptive effects
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alone in the tail flick test and were not used as the cannabinoid
vehicle when performing i.t. injections. Preliminary time course
studies of the various drugs (at the highest dose which exhibited no
toxicity) or their respective controls were initially performed in com-
bination with the ED,, dose of each cannabinoid. Once the time of
the peak effect of those drugs which altered cannabinoid
were administered. lntraventricular injec-
tions were performed according to the method of Pedigo et al.
tubing to
control the depth of the injection, An injection volume of 5
DMSO. Nor-BNI, ICI 174.864, naloxone hydrochloride, morphine
sulfate, apamin, pertussis toxin, calcium chloride, magnesium chlo-
ride.
and verapamil were dissolved in distilled
water. The cannabinoids or DMSO vehicle
min before determination of the response latency of the mice in the
tail flick test. This time point represents the peak
DMSO vehicle produced scratching behavior in mice
which lasted 2 min after injection. Other vehicles have been tested
previously in our laboratory.
needle.
and L6 area of the spinal cord with a
Injection volumes of 5
at a site
2-mm rostra1 and 2-mm
to the bregma at a depth of 2 mm was
administered to the mice. The cannabinoids,
charybdotoxin. TEA.
of the drugs
as determined in previous studies in our laboratory (Welch and
Stevens, 1992
spinal and supraspinal sites.
Methods
Intrathecal
administered cannabinoids, and the interaction of
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by
K 8644, ryanodine and nimodipine were prepared in
was determined. all further testing was performed at that
and Stevens, 1992; Smith and Martin, 1992; Smith et
and
of the A”-THC-induced
Recently, the blockade of cannabinoid antinocicep-
kappa opioid antagonist,
with morphine
Unanesthetized mice were injected between the
been reported
et
the interaction of icv. administered
Cannabinoid-Induced
1:
interaction of the opiates and the
and potassium channels in
and by orally administered
Cl-CAMP, forskolin.
1993
were administered
The blockade by
and emulphor!
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thapsigargin,
1975
and
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Welch et al.
time point. With few exceptions, the time of the peak effects of the
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drugs was 5 to 10
before the administration of the cannabinoids.
The exceptions include glyburide
pretreatment; thapsigargin, which produced peak
blockade after a 1-hr pretreatment and pertussis toxin, which was
administered i. t. (0.5
potas-
sium channel openers and morphine (Welch and
days before testing in combination
with the cannabinoids. Thapsigargin, Bay K 8644 and calcium pro-
duced antinociception when administered i,t. Forskolin,
1993)
using a 15
and Cl-CAMP and Bay K 8644 produced antinociception when
administered
To test for greater than additive effects of the
intrinsically antinociceptive drugs
in combination with
the cannabinoids, doses of the drugs were chosen which produced no
significant antinociception. These doses were then tested in combi-
nation with a wide range of doses of the cannabinoids. For the
studies of the enhancement of the effects of morphine
nabinoids
before
morphine. The mice were tested 10 min after the administration of
morphine using the tail flick test.
To study the effect of
on calcium-induced blockade of
the cannabinoids, the
was administered icv. at 5 min
before the calcium
which was administered at 10 min before the
cannabinoid
The mice were tested 15 min later using the tail
flick test.
The tail flick test. The tail flick procedure used was that of
and
a cut-off time of 10
were empioyed. Antinociception was quanti-
fied as the %MPE as developed by Harris and Pierson
using
the following formula:
Percent MPE was calculated for each mouse using at least 12 mice
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per dose. Using the
modification of the
were
determined using the method of Litchfield and
as
well as the
1.Using at least three doses of blocker,
for each mouse, the mean effect and
standard error of the mean
was calculated for each dose.
Dose-response curves were generated using at least three doses of
test drug.
method omitting doses pro-
ducing 100% or 0% MPE) and 95% confidence limits
et al., 1977).
The AD,, for blockade the cannabinoid-induced antinociception
was determined by calculation of the percent antagonism of
ciception (using a dose of the cannabinoid that resulted in at least
80% MPE) by the blockers according to the following formula:
% antagonism = 100
values were determined
by log-probit analysis (a modification of the
method omitting doses producing 100% or 0%
1949).
were
At least 12 mice per dose were used for all determinations.
Statistical analysis. Significant differences between treatment
and control groups were determined using analysis of variance andthe Dunnett’s
19551. The dose-response curves were
evaluated for the parallelism of shifts using the method of Tallarida
and Murray
Drugs. All of the cannabinoids were obtained from the National
Institute on Drug Abuse with the exception of CP 55.940, CP 56,667,
levonantradol and dextronantradol which were obtained from Dr.
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Lawrence Melvin, Pfizer Central Research. Nor-BNI, Bay K 8644,
MA). chemicals, Inc.
ICI 174,864 was purchased from
Cambridge Research
and Smith
and forskolin were purchased from Research
test
the cannabinoids were administered 10
values were determined by log-probit analysis
control\%MPE = 100 x
using the method of Litchfield and
program
as well as the
1
Control reaction times of
(test
(Valley Stream, NY). Calcium
of antagonist + cannabinoid)
of vehicle + cannabinoid)
which blocks both
or
control)
program
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and 95%
to 4
by
et
monosodium
was provided by Miles, Inc. Pharmaceutical
Division (West Haven, CN). Thapsigargin was purchased from LC
GVIA was purchased from Pen-
insula (Belmont, CA). Ryanodine and pertussis toxin were purchased
from Calbiochem (San Diego, CA).
Results
values for a series of cannabinoids are listed in
table 1. The ED,, values of the cannabinoids after
admin-
istration were taken from Welch and Stevens
and
were compared with the ED,, values generated for the
icv. administration. The dose-effect curves
used to generate the
of the cannabinoids were full
agonists in the tail flick test
precluded but the solubility of the
the use of higher
doses. The
did not differ significantly after adminis-
tration
after icv. administration. Conversely,
the
us. and CP
icv. were nearly identical. The ED,,
values for
or icv.. although a trend toward higher potency of
the drug was
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did not differ significantly after
administration
or icv., although a trend toward higher
potency of the drug was observed after i.t. administration. CP
56,667 was significantly more potent after icv. administra-
tion than
administration, and was nearly 10 times more potent than CP 55,940
and CP 56,667 are
stereoisomers and CP 55,940 has been described to be the
active isomer in many tests (Johnson and Melvin, 1986;
Little
icv.
administration. However, 25 &mouse produced 44% MPE
in the tail flick test. Dextronantradol was inactive at 25
and icv. administration.
Interaction with opioids. Table 2 lists the ED,, valuesfor morphine
(taken from Welch and Stevens, 1992) and the ED,, values
for morphine (icv.)
and levonantradol
produced greater than additive effects in combination with
TABLE 1
ED, values
and 95% confidence limits for various
cannabinoids in the tail flick test in miceThe cannabinoids were administered erther
using the tail flick test. The ED,, values and 95%
2.89 (1.6-5.1)
CP 56,667 4.17
Levonantradol 0.04
Dextronantradol Inactive at 25
11
shown in figure 1.
55,940 2.28
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IN).
Drug
which produced only an average of 68% MPE at
under “Methods.”
magnesium chloride, verapamil. glyburide.
al.,
to complete a dose-effect curve of the drug
and apamin were purchased from Sigma
values for
after both
We tried to administer higher doses of
was
MA).
As-THC,
values after administration of
obtained from Boehringer Mannheim
after pretreatment with cannabinoids
We were unable to obtain enough
44.97 (22.96-88.09) 16.4 (1 l-24.8)
72.07 (36.06-l 44) 125.8
14.67
pretreatment with cannabinoids
values for the cannabinoids
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CP
both
or
and icv.
at
the exception of
0.02
0.18
were determined
25
min before
= 44%
4-U
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icv.)
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Fig. 1. Dose-effect
icv. administration.
Cannabinoids were injected icv. and tested for the production of
tinociception using the tail flick test and the ED,, values were deter-
mined as described under “Methods.” The following cannabinoids were
tested: A = levonantradol; B
At least 12 mice were used per dose.
morphine when the drugs were administered
nantradol) failed to produce greater than additive effects
with morphine
CP 55,940,
CP 56,667 and dextronantradol
and CP 56,667 produced greater than additive effects with morphine when
the drugs were administered icv. The
values for mor-
phine were shifted from 2.4
produced greater than additive effects with morphine when
the drugs were administered either i.t. or icv. The shifts in
the morphine dose-effect curve in combination with the
mouse by CP
nabinoids were parallel.
Various opioid antagonists were administered
before
icv. administered cannabinoids and did not alter
noid-induced antinociceptive effects. These drugs are listed
in Table 3 and include the delta opioid antagonist. ICI
174,864 (20
the kappa
opioid antagonist, nor-BNI (70
(Takemori et al.,
1988) and naloxone (20
Interaction with
C
for 7 days, which ribosylates
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doses used blocks mu, delta and kappa receptors
et al., 1987
E =
0.001 0.01 0.1
10 100 1000
Dose
AMP. Pertussis toxin pretreatment
Conversely, CP
for the cannabinoids
and CP 56,667, respectively.
CP 56,667: C = CP 55,940; D =
or 10
et al.,
and/or
inactive isomer of
to 0.28 and 0.65
proteins (for a
which at
313TABLE 2
ED, values
for morphine various cannabinoids in the tail
in combination with
and icv.
administration
Mice were injected with cannabinoids either
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binoid pretreatment or vehicle pretreatment. Cannabinoids were administered 10
min before morphine and the mice were tested using the tail flick test at 10 min
after morphine administration. The
were determined as
described under “Methods.”
PretreatmentED,, of morphine
review see Brown and Birnbaumer, 19901, totally abolished
the antinociception produced by the cannabinoids
and significantly attenuated or blocked the antinocicep-
tion produced by the cannabinoids
administered i.t. was least affected by the pertussis toxin
pretreatment. Pretreatment with pertussis toxin
did not
alter the baseline latencies for the mice in the tail flick test
and did not produce any overt toxicity in the mice. However,
pretreatment with pertussis toxin icv. (0.5
for 7
days) led to loss in body weight, and lethality in greater than
30% of the animals. Pretreatment of the mice with 5 and 25
&mouse forskolin
which produced no antinociception,
significantly attenuated the antinociception produced by the
A similar effect was ob-
served using CP 55,940. Forskolin (25 &mouse
pre-treatment significantly reduced the antinociceptive effects of
CP 55,940
forskolin MPE. However, when administered
produced
antinociception. The ED,, of forskolin
which produced
no antinociceptive effects, did not block or produce greater
than additive effects with the antinociception produced by
the cannabinoids administered icv. Administration of
partially, but significantly,
blocked the antinociceptive effects produced by the
ad-
ministration of ED,, doses the three cannabinoids tested (fig.
failed to alter the antinocicep-
tion produced by the cannabinoids administered i.t. How-
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ever,
produced antinociceptive effects when ad-
ministered icv. [ED,, values = 0.5
was
obtained using higher doses of Cl-CAMP. Both Cl-CAMP and
dibutyryl
and
2.3)
dibutyryl
respectively) did
not block or produce greater than additive effects with the
antinociception produced by the cannabinoids administered
icv.
Interaction with calcium. Various calcium modulators
were tested in combination with the cannabinoids (table
The
1.6)
None 1 (0.6-l
2.7 (1.7-4.3)
DMSO 0.61 (0.26-I
Levonantradol 0.06 (0.015.24) 0.66
N.T.
11
Dextronantradoi 0.51
morphine. The dose-effect curve for morphine was determined after
55.940 0.3
However, no greater blockade of the
(3.13
(6.25
(25
administration of calcium
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administered
(10
Forskolin (0.1
Cannabinoid-Induced Antinociception
respectively]. Inactive doses of
(0.02 and 0.05
0.15
0.05
0.05
0.08
0.26
(fig.
from 83
values and 95%
test in mice after
before
morphine or icv. before
MPE to 16
(fig.
2.4 (1.7-3.3)
0.65
and 0.3
was 0.5
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8
N.T.
(fig.
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itself did not alter the effects of the cannabinoids, it did
significantly block calcium-induced blockade of the cannabinoids.
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3
gargin (0.1 &mouse), which increases intracellular calcium
314 Welch et al.
TABLE 3
cannabinoids after
before
cannabinoids
and
the time
pools
of the endoplasmic reticulum by blocking
activity
(Bian et al., 1991; Koshiyama and Tashjian, 1991); Bay K
8644 (0.3 &mouse), which predominantly increases calciumentry through dihydropyridine-sensitive calcium channels;
verapamil (30
&mouse),
which block voltage-gated
which alters calcium flux from
the intracellular caffeine-sensitive (but not
sensitive)
calcium pool (Thayer et al., 1988;
cal-
toxin (1 &mouse) which blocks both
cium channels
and ryanodine
et al., 1989) failed to
produce antinociception or to alter the antinociception
administration of the cannabinoids. The
doses listed represent the highest non-antinociceptive dose
for each of the drugs. At higher doses, calcium, thapsigarginand BAY K 8644, which increase intracellular calcium, pro-
duced antinociception after
blocked the antinociceptive effects of the
cannabinoids (fig. 4, panel A). The AD,, values
for
calcium-induced block of
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mouse, respectively. Higher doses of calcium (greater than
450
because we could not use higher doses of calcium. This diffi-
culty is reflected in the large confidence limits for the AD,, of
calcium us.
which didnot alter the antinociceptive effects of
and CP 55,940
were 215
produced hyperactivity in the mice. It
was difficult to generate an
icv.
Opioid antagonists
Naloxone No block No block
ICI 174,864 No block No block
nor-BNI
Blocked No block
Calcium modulators
Calcium No block Blocked
Verapamil No block No block
No block No block
Nimodipine No block No block
Thapsigargin No block Blocked
BAY-K No block No block
Ryanodine No block No block
Modulators of
Blocked No block
Forskolin Blocked No block
Magnesium Chloride No block No block
Modulators of potassium channels
No block No block
Apamin Blocked No block
l
administration.
Calcium
No block No block
TEA No block No block
Glyburide No block No block
blockade of entry of calcium into the
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by the
et al.
were administered
of blockade of the antinociceptive effects of
toxin Blocked Blocked
as described under “Methods”
1986;
Antinociception was determined using the tail flick
Drugs
and icv. administration
176 (122-253) and 123
et al., 1987; Reynolds et al.,
et al., 1987; Zemig,
and nimodipine
before
(1 &mouse icv.
calcium channels (God-
for calcium us.
No block No block
and
significantly
or
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Fig. 2. The effect of pertussis toxin pretreatment (i.t.) on the antinoci
(panel A) c
icv. (panel
7 vehicle was administered
days before the administration of th
cannabinoids
or icv.) in distilled water-pretreated mice (the vehicle for the
At least 12 mice per treatment group were used.
l
vehicle. Th
mice were tested for antinociceptive effects after either
administration of the cannabinoids or DMSO. The clear bar denote
6% (icv.), respectively. Bars denoted
represent the antinociceptive effects of those cannabinoids in mic
pretreated for 7 days i.t. with distilled water vehicle. The bars denote
PANEL B
100
80
group.
respective distilled
reversed the calcium-induced blockade of
blocked the antinociception produced by
gargin us.
panel B!.
Pretreatment of the mice icv. with thapsigargin for 1
55,940, data not shown) administered icv.
Other calcium modulators were tested including:
or tion after either
icv. administration. All of these dru:
were tested at the highest nontoxic doses in combination
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the cannabinoids administered either i.t. or icv. and failed
alter cannabinoid-induced antinociception (table 3).
PANEL A
represents the antinociception produced by the administration
40
vehicle in PT-treated mice. Not shown is the effect of DMSO
indicate the effects of the cannabinoids in PT-pretreated
generated in those treatment groups were 5
effects of the cannabinoids administered either
0
and nimodipine
omega conotoxin
None of these drugs produced
= 0.003
toxin
or CP 55,940) or
(0.5 &mouse) or distilled
and magnesiu:
and “CP
verapamil
3%
P
55940
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for
0.01
(fig.
and 8
(fig.
or
Th
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PANEL A
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Fig. 3. The effect of forskolin pretreatment (i.t.) (panel A) or Cl-c-AMP
[panel B) on the antinociceptive effects of the cannabinoids adminis-
tered
or distilled water vehicle
was administered
or DMSO
vehicle. The mice were tested for antinociceptive effects after
represents the antinociception produced by the admin-
istration of distilled water
in distilled water-pretreated mice (the vehicle for the forskolin).
The
55,940” represent the antinociceptive effects of those
Cannabinoids in mice pretreated
which block voltage-gated potassium
channels (North, 1992;
mouse), which blocks ATP-gated potassium channels
mouse), which blocks the large
with distilled water vehicle. The
bars denoted
with potassium channels. Various potas-
sium channel blockers were tested: TEA
sett et al., 1988; Gaines et al., 1988;
at these doses, which are the highest nontoxic doses of
the drugs, failed to alter cannabinoid-induced
mice per treatment group were used.
were used for all treatment groups. Panel
and
However. apamin. blocker of
Panel A, Forskolin
potassium channels (Smith et al., 1986;
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et al., 1988). These drugs administered either
effects of distilled water administration before DMSO. At least
and Lazdunski,
generated in that treatment group was
of the cannabinoids or
i.t.)-pretreated mice. “VEH” denotes the
before the administration of
indicate the effects of the cannabinoids in
before
1 5 25
@mouse,
and charybdotoxin
fast) conductance
1992); glyburide
P
Not shown is the effect of
The dark bar denoted
0.01.
Bars denoted
et al., 1989; and
I
4%. At
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conductance
and
12
or
Fig. 4. The effect of calcium pretreatment (icv.) (panel A) or w-conotoxin
CP 55,940 or
DMSO vehicle. The mice were tested for antinociceptive effects after
icv. administration of the cannabinoids or DMSO. The bar denoted
represents the antinociception produced by the administration
of distilled water icv. before DMSO icv. The AD,, values for calcium
blockade of the cannabinoids were determined as described under
“Methods.” At least 12 mice per treatment group were used.
Mice were pretreated
with dual injections icv. of vehicle (v), calcium (Ca, 300
binoids were shifted from
or distilled water vehicle was administered
icv. before the icv. administration of
et al., 1982; Cas-
tle et
as described
under “Methods.” The mice were tested 15 min after the injection of
At least 12 mice per treatment group were used. w-Conotoxin
significantly reversed the calcium-induced blockade of
calcium-gated potassium channels
produced a parallel rightward
shift in the dose-effect curves of
was shifted
from 6
failed respectively. However, apamin (5
to
block icv. administered cannabinoids.
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and CP
55,940 (fig. 6, panels A-C). The ED,, values for the
to 33-fold by administration of
apamin
P
120, 300
315
PANEL A
(OC) icv. before calcium pretreatment (panel B) on the
- -Calcium Calcium Calcium
PANEL
100
80
0.01 from
60
40
20
effects of the cannabinoids administered icv. Panel A. Calcium
antinociception.
and CP 55,940 were shifted from 21
.
1989; Strong,
&mouse to 123
to 82
Cannabinoid-Induced
P
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The ED,, of
and/or
0.01 from
Panel
and 20
or
The ED,, values for
P
0.05 from
P
and 0.6
0.05;
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to
greater than lo-fold by the pretreatment with cannabinoids
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at doses which did not have any antinociceptive effects. W’hen
we compared the enhancement of morphine-induced
administration ciception by the cannabinoids
with
those data previously generated after i.t. administration, we
found that some cannabinoids enhanced morphine in the
1981; Gilbert, 1981; Lichtman and Martin, 1991, a and b;
Welch and Stevens, 1992). Intrathecally
nabinoids seem to act at predominantly spinal sites in the
production of antinociception (Smith and Martin, 1992). It is
not clear whether the icv. administration of the cannabinoids
activates both spinal and supraspinal sites in the production
of antinociception, Due to the lypophilicity of the drugs, pas-
sage to the spinal cord is very likely.
In summary, the cannabinoids seem to produce potent
antinociceptive effects at both spinal and supraspinal sites,
However, the mechanism by which the cannabinoids produce
antinociceptive effects at these two sites seemed to differ and
is the focus of the remainder of this manuscript.
Interaction with opiates. We have
previously
that the ED,, values for morphine were shifted from
316 Welch et al.
The mice were tested 15 min later in the tail flick
test. The clear bar denoted
represents the antinociceptive ef-
fects of DMSO icv. administered 1 hr before
binoids produced antinociception after both i.t. and icv. ad-
ministration and are approximately
at either site
with the exception of CP 56,667. These results are consistent
with previous studies which indicate that the cannabinoids
are active as antinociceptive drugs when injected
icv.
Fig. 5. Blockade of
gargin (icv.). Thapsigargin, at the doses listed, or vehicle was injected at
1 hr before
The remain-
ing bars represent thapsigargin administration before
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At least
12 mice were used per dose.
l
P
represents
20
VEH 0.001 0.005 0.01
THAPSIGARGIN
vehicle
Discussion
(icv.)-induced antinociception by
of the cannabinoids.
at 1 hr before
0.05 from
icv. The bar labeled
P
0.01 from
been shown to be dense in the striatum and the
which are associated with dense binding of the opiatt
et al., 1988; Gamse et al., 1979). Thus, neuroanatom
cal location of the receptors does not seem to account for
differences observed between the effects of CP 55,940
were determined 15 min later using the tail flick test. The ED
values and the parallelism of the shifts in the dose-effect curves we
determined as described under “Methods” using 12 mice per dose.
brain whereas others enhanced the effects of morphine in
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bility that subtypes of cannabinoid receptors may exist
brain and spinal cord and differ in their interactions
opiate-sensitive pathways either via their neuroanatomic:
proximity to opiate receptors or differ in the convergence
second messenger systems with those of the opiates. Hot;
ever, in the case of one cannabinoid, CP 55,940, binding
gelatinosa of the spinal cord
are administered
spinal cord. Our data are suggestive of the intriguing
Fig. 6. Blockade of the antinociceptive effects of
min before the cannabinoids. The dose-effect curves of the
PANEL A
i.t.)
PANEL C
(panel
B
and CP 55,940 (panel C) by apamin when all
10
Apamin (5
1000
A8
1 10
doses
doses
doses
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was administered at
et
A9 +
apamin
ED50 = 82
(panel
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opioid or the direct interaction with opiate receptors.
Thus, the enhancement of opiate antinociception by
binoids icv. may be due to interactions with common intra-
cellular second messenger systems. Some possible points of
interaction include the modulation of calcium,
sensitive, high-voltage-activated calcium channel, an effect
that is blocked by the administration of pertussis toxin and
independent of the formation of
C
AMP. Because the L-type
calcium channel blocker, nitrendipine, fails to alter such an
effect, the cannabinoids were hypothesized to interact with
an N-type calcium channel (Mackie and Hille, 1992). A sim-
ilar study produced similar results. The cannabinoids were
found to inhibit I,, current in newoblastoma cells, an effect
which was not. dose related, but was pertussis toxin- and
administration of calcium
to mice
administration of the cannabinoids is not sensitive to
calcium modulation and thus may not directly involve cal-
cium modulation.
Conversely, the icv. administered cannabinoids were sen-
sitive to blockade by calcium icv. and the AD,, values for
calcium icv. us. the cannabinoids
of the cannabinoids at supraspinal sites are not me-
diated
ciceptive effects of icv. administered cannabinoids. These
data suggest that the icv. administration of the cannabinoids
does not result in the release of an antinociceptive
and
potassium flux.
Modulation of calcium and
enhance or fail
blastoma cells indicate cannabinoids inhibit an
1986) or by
modulators of intracellular pools of calcium such as
et al., 1988; Welch et al., 1992; Smith and
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Dewey, 19923, as well as Bay K 8644 and thapsigargin
(Damaj et
before the cannabinoids did not
produce greater than additive effects with the cannabinoids.
These data indicate that the antinociception produced by the
did not differ signifi-
cantly from that previously shown for calcium blockade of
calcium entry to
a variety of tissues. Electrophysiological studies in
All such
previous studies were performed in
Based on the re-
sults of such studies we hypothesized that the antinocicep-
produced by the cannabinoids might be sensitive to
calcium, or modulators of calcium.
administration of calcium chloride or a
series of modulators of calcium flux across voltage-gated
calcium channels
ciceptive effects result from the
1993). Administration of non-antinociceptive
doses of these drugs
effects of the cannabinoids administered i.t. were not
nor-BNI administered icv. failed to block the
have previously reported that the kappa antagonist,
and ICI 174,864
data support the hypothesis that the antinociceptive
1990;
by the
produced by the cannabinoids administered
interaction with nor-BNI-sensitive receptors.
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Thayer et al., 1988;
et al., 1991; Koshiyama and Tashjian, 1991
(Godfraind et
but not other opioid antagonists, blocks
alter
antinociception in the brain
et
et
(Caulfield and Brown
antinociception (Welch, 1993).
1988; Harris and Stokes,
1987; Reynolds et
also failed to block the
et al.,
nimodipine, Bay K 8644 or
1986;
Cannabinoids either
the antinocicep-
1989
et al., 1987;
or
morphine icv. (Harris et al., 1975; Chapman and Way, 1980;
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Guerrero-Munoz and
and stimulates calcium influx into cells
(for a review see Irvine,
also blocked the antinocicep-
tion produced by
which increases intracellular calcium (Koshiyama
and Tashjian,
which increases calcium
entry to cells via the voltage-gated
calcium channel,
as well as via nonspecific mechanisms (Bouchelouche et
nabinoids is due to calcium entry through voltage-gated cal-
cium channels because
induced blockade (fig.
alone did not
alter cannabinoid-induced antinociception. Thus, it appears
that the entry of calcium to neurons must be stimulated by
the administration of calcium to observe effects of omega
calcium chan-
nels, and found no reversal of the effects of icv. calcium (data
not shown). Thus, the calcium-induced blockade of
a similar logic we attempted to reverse the
calcium-induced blockade of cannabinoids with both
pamil and nimodipine, which block
calcium noid-induced antinociception seems to be due
en-
try through the
tinociception by thapsigargin, which raises intracellular cal-
cium levels, indicates that the cannabinoids may decrease
and Stokes (1982) and Martin et al. (1988). Inaddition, the stimulation of calcium entry to neurons after
thapsigargin treatment would further increase calcium con-
centrations intsacellularly, an effect insensitive to blockade
by classic calcium channel blockers (for review, see Irvine?
1992; Jackson et al., 1988; Takemura et al., 1991) and con-
sistent with our data. Our data therefore suggest that,
formation follows a structure-activity relationship and
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stereoselectivity similar to that observed for behavioral mea-
sures (Howlett, 1985; Dill and Howlett, 1988; Howlett et al.,
1986; Howlett and Fleming, 1984; Howlett, 1987; Howlett et
al., 1990; Devane et
1989
calcium channel. These results are
consistent with reported effects observed in vitro (Mackie
and Hille, 1992). The blockade of cannabinoid-induced
entry to neurons which is consistent with the in vitro
work
nabinoids produce antinociception at supraspinal, but not
spinal, sites by decreasing intracellular calcium levels via
blockade of calcium entry through
calcium chan-
nels.
Consistent with the report of Mackie and Hills
antinociception resulting from supraspinal administra-
tion of the cannabinoids seems to involve interaction with a
due
to the blockade of antinociception by pertussis toxin pretreat-
ment which inactivates
lation (for a review, see Brown and
proteins, nabinoids with
but indicating a lack of involve-
ment of
induced antinociception or other behavioral effects of the
cannabinoids (Hillard and Bloom, 1983; Little and Martin,
1991; Dolby and Kleinsmith, 1974; Dolby and Kleinsmith,
1977; Askew and Ho, 1974). In
studies indicate a some-
what different profile of cannabinoid action. Studies in
1986; Bidaut-Russell et al., 1991;
Howlett et al.,
Our
data agree with studies indicating an interaction of the
did not alter cannabinoid antinociception.
It is likely that the calcium-induced blockade of the
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or
doses of BAY K 8644
protein and be independent of changes in
cells show that cannabinoid-induced inhibition of
in the brain in the modulation of
Cannabinoid-induced Antinociception
and is mediated via coupling to the
although
and CP 55,940.
and
1982;
proteins via.
reversed the
et al.,
in
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al.
protein because pertussis toxin attenuates binding of CP
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55,940
These studies, along with supporting work evaluating
the stereoselectivity and the Hill coefficient for binding of the
synthetic
ylation of calcium channels
nabinoid receptor linked through a
our studies indicate that modula-
tion of
tion by alteration of calcium entry to neurons
protein to the modu-
lation of
C
AMP.
is not directly involved in the production of
antinociception, we can not rule out the possibility that
and Birnbaumer, 1990;
Schultz et
cannabinoids
could decrease calcium entry to neurons.
In the spinal cord, the cannabinoids seem to produce
protein tinociception
in conjunction with the
modulation of
because the antinociceptive effects ofi.t.
cannabinoids are blocked by pertussis toxin, forskolin and
Cl-CAMP, The lack of blockade of antinociception by
may be due to the lack of penetration of the
Gimenez-Gallego
et
Morphine has been shown to modulate
ratios, High ATP levels close these channels leading to a less
negative membrane potential and depolarization. Opening-of
these channels leads to hyperpolarization. Pharmacologi-
cally, these channels are blocked by the oral hypoglycemic
agents, such as glyburide, which stimulate the entry of
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to the
site of action. The effects of
on antinociception are the
opposite in the brain us. in the spinal cord, which is similar to
the opposite effects of calcium observed in brain
administration may reflect differences in the function
of
in the brain us. in the spinal cord in the- modulation
of antinociception.
Modulation of potassium.
basic classes of potas-
sium channels exist in the central nervous system (for a
review, see Aronsen, 1992). This classification is clearly a
generalization and the drugs discussed often interact with
more than one
of
the channels. In general these potassiumchannels are either voltage-activated, calcium-gated,
gated or ligand-activated. The
nels are either opened or closed in response to membrane
electrical activity and are blocked pharmacologically by TEA
and
proteins or other second messenger systems,
Calcium-gated potassium channels have been studied using
the bee venomapamin, which blocks the small (low)
conductance (SK or
et al., 1982;
Castle et al., 1989; Strong, 1990). An endogenous ligand for
the apamin receptor has been found in brain
tive calcium-gated potassium channels in peripheral sensory
neurons
channels are gated by changes in intracellular
et al.,
1988: Howlett et al..
spinal
cord
The calcium-gated potassium channels are activated
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et al.,
channel
potassium
et al.,
channel in
of
et
Anatomically, these channels exist in areas where high levels
of opiate binding occur
and Lazdunski, 1990). ATP-gated potassium channels have
been found in the brain, with particularly high levels in the
substantia nigra, hippocampus, basal ganglia and thalamus.
and to a lesser degree in the spinal cord, although the func-
tion of such channels remains unknown
et al., 1988). Opiates also
interact with l&and-activated potassium channels, a diverse
collection of channels which are modulated by a variety of
neurotransmitters and drug classes. These channels are
most
effects produced by the cannabinoids, although the
is blocked by potassium channel blockers and con-
versely, that potassium channel openers administered
produce antinociception that is blocked by opiate antagonists
(Welch and
The administration of apamin. a
blocker of calcium-gated potassium channels, produced a
complete blockade of morphine-induced antinociception.
Evaluation of several potassium channel blockers
blocked the antinociceptive ef-
fects of the cannabinoids
producing parallel rightward
shifts in the dose-effect curves of the cannabinoids.
the parallel shifts could be coincidental and the
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could be nonspecific, this is unlikely because
parallel shifts were observed with three cannabinoids.
it is likely that cannabinoids interact spinally with a
gated, apamin-sensitive potassium channel in the production
of antinociception. Cannabinoids administered icv. appear to
produce antinociception which does not involve
on cannabinoid-induced antinociception can not
be attributed to lack of binding of the apamin.
Summary. Cannabinoids produce antinociception after
administration to both spinal and supraspinal sites; how-
ever, modulation of antinociception by calcium,
and
potassium seems to differ in the brain
in the spinal cord.
Additionally, the cannabinoid-induced modulation of opiate
antinociception seems to differ in the brain and the spinalcord. Based on our data, we hypothesize that in the brain,
cannabinoids produce antinociception
decreasing calcium
levels in neurons through receptor linkage to
proteins
involved in the modulation of the function of the
calcium channel. Cyclic
seem to not be involved in the production of antinociceptior
induced by icv. administered cannabinoids. In the
ing in a decreased
calcium-gated potassium channels. The involvement
of
tion with G proteins has the potential to alter both
and potassium channel function.
Because acutely administered
have beer shown to interact with
of
apamin in
sitive potassium channels. Since apamin binding has been
shown in the brain
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the lack of effects of
apamin
or to play an indirect role. In addition, potassium
cord, our data indicate that the cannabinoids produce
ciceptive
1978; Childers and Snyder,
We have shown previously that opiate-induced
in combination with the cannabinoids
into the ceils of both the pancreas and the brain
appears to not play a direct role in the
1988; Gaines
that only apamin
coupled to a G protein (Brown and Birnbaumer.
via the interaction with G,, proteins
1988;
protein-coupled receptors
production and interaction
et
t.
seems to either not be
decrease calcium entry
et al.,
and
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et
and
cal
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and content of neurons (Harris et al., 1976; Cardenas and
R
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OSS
,
1976; Welch and Olson,
levels
(Collier and Roy,
1974;1978) and
produce hyperpolarization of neurons
these three intracellular systems
may be common points of interaction with the cannabinoids.
For example, it has been hypothesized that the end result of
bination may be the phosphorylation of similar proteins
which are proposed to be synapsins I and II which are
volved in the release of
(Childers et al.,
1992). Finally, the data suggest that cannabinoids interact
spinally with nor-BNI-sensitive receptors, This could indi-
cate some interaction of the cannabinoids directly with opi-
ate-sensitive pathways. Kappa receptor-selective opioids
have not been shown to be displaced by the cannabinoids.
Thus, although we can hypothesize as
be done to
delineate conclusively the mechanisms underlying thegreater than additive effects observed using cannabinoids
and opiates in combination.
Acknowledgments
The author wishes to thank Dr. Bill R. Martin, Department of
the points of inter-
action ofcannabinoids and opiates based on the in
mech-
anisms of action of the drugs indicated by this work and that
and Toxicology, Medical College of Virginia for his
assistance and collaboration on this project.References
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