Administration to
Mice: Possible Mechanisms for Interaction with Morphine’
SANDRA P. WELCH, CHARITY THOMAS and GRAY S. PATRICK
THC and
for 7 days
totally abolished the antinociception produced by the cannabi-
ABBREVIATIONS:
sulfoxide.
310
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,
mouse),
This
(79
were generated for the canrrabinoids
. VS
icv. did not differ significantly.
and
receptor
1993.
Versus
and Martin,
55,940,
of the
intravenously;
icv. The
effect curves of
apamin (5
In receptor
addition. an endoge
ligand for the cannabinoid receptor has been identified
ane et al.,
modulation of adenylate
calcium. prostaglandin
opioids. The role of adenylate cyclase, calcium
dibutyryl cyclic
and CP 55,940. Apamin, blocker
for calcium-induced
subcutaneously:
and
determined. The
monophosphate
percentage of the
of
protein-coupled
55,940
(1
1995
role of
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.
C
AMP. The cloning of the cannabinoid receptor was per-
formed in
modula-
tion after receptor activation
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
de-
creases the release of acetylcholine presynaptically by a de-
crease in the influx of calcium into presynaptic nerve
terminals
al.,
in concentrations of 1
Welch,
using
et
with an evaluation of
and
production in
or higher
et al.,
protein
et
Naloxone
Stokes
1988
brain
311
suggesting that cannabinoids and opiates have distinct
binoids
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
tubing to
control the depth of the injection, An injection volume of 5
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
and
of the A”-THC-induced
with morphine
been reported
et
Cannabinoid-Induced
1:
Cl-CAMP, forskolin.
1993
were administered
The blockade by
and emulphor!
thapsigargin,
1975
and
Welch et al.
time point. With few exceptions, the time of the peak effects of the
drugs was 5 to 10
before the administration of the cannabinoids.
The exceptions include glyburide
potas-
sium channel openers and morphine (Welch and
1993)
using a 15
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
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,
et al., 1977).
The AD,, for blockade the cannabinoid-induced antinociception
was determined by calculation of the percent antagonism of
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 and
the Dunnett’s
19551. The dose-response curves were
evaluated for the parallelism of shifts using the method of Tallarida
and Murray
and Smith
test
control\
%MPE = 100 x
program
as well as the
(test
of antagonist + cannabinoid)
of vehicle + cannabinoid)
or
control)
program
and 95%
to 4
by
et
monosodium
us. and CP
icv. were nearly identical. The ED,,
values for
icv.
administration. However, 25 &mouse produced 44% MPE
in the tail flick test. Dextronantradol was inactive at 25
(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
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
IN).
Drug
under “Methods.”
al.,
values for
after both
was
MA).
As-THC,
14.67
both
or
and icv.
at
the exception of
0.02
0.18
were determined
25
min before
= 44%
4-U
icv.)
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
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
mouse by CP
(Takemori et al.,
1988) and naloxone (20
Interaction with
C
et al., 1987
E=
10 100 1000
Dose
Conversely, CP
CP 56,667: C = CP 55,940; D =
or 10
et al.,
and/or
inactive isomer of
proteins (for a
which at
313
TABLE 2
ED, values
for morphine various cannabinoids in the tail
in combination with
and icv.
administration
Mice were injected with cannabinoids either
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.”
Pretreatment
ED,, of morphine
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
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
N.T.
11
Dextronantradoi 0.51
55.940 0.3
(3.13
(6.25
(25
administration of calcium
administered
(10
Forskolin (0.1
Cannabinoid-Induced Antinociception
0.15
0.05
0.05
0.08
0.26
(fig.
from 83
before
MPE to 16
(fig.
2.4 (1.7-3.3)
0.65
and 0.3
was 0.5
8
N.T.
(fig.
itself did not alter the effects of the cannabinoids, it did
significantly block calcium-induced blockade of the cannabinoids.
3
gargin (0.1 &mouse), which increases intracellular calcium
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 calcium
entry through dihydropyridine-sensitive calcium channels;
verapamil (30
&mouse),
which block voltage-gated
cal-
toxin (1 &mouse) which blocks both
cium channels
and ryanodine
and CP 55,940
were 215
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
et al.
were administered
1986;
Drugs
and nimodipine
before
(1 &mouse icv.
No block No block
and
significantly
or
Fig. 2. The effect of pertussis toxin pretreatment (i.t.) on the antinoci
(panel A) c
icv. (panel
vehicle. Th
mice were tested for antinociceptive effects after either
PANEL B
100
80
group.
respective distilled
reversed the calcium-induced blockade of
gargin us.
panel B!.
Pretreatment of the mice icv. with thapsigargin for 1
PANEL A
40
and nimodipine
omega conotoxin
= 0.003
toxin
or CP 55,940) or
and magnesiu:
and “CP
verapamil
3%
55940
for
0.01
(fig.
and 8
(fig.
or
Th
PANEL A
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
and
Panel A, Forskolin
and Lazdunski,
of the cannabinoids or
before
1 5 25
@mouse,
and charybdotoxin
fast) conductance
1992); glyburide
0.01.
Bars denoted
4%. At
conductance
and
12
or
CP 55,940 or
DMSO vehicle. The mice were tested for antinociceptive effects after
icv. administration of the cannabinoids or DMSO. The bar denoted
as described
under “Methods.” The mice were tested 15 min after the injection of
was shifted
from 6
to 33-fold by administration of
apamin
120, 300
315
PANEL A
--
Calcium Calcium Calcium
PANEL
100
80
0.01 from
60
40
20
antinociception.
1989; Strong,
&mouse to 123
to 82
Cannabinoid-Induced
P
The ED,, of
and/or
0.01 from
Panel
and 20
or
0.05 from
and 0.6
0.05;
to
greater than lo-fold by the pretreatment with cannabinoids
at doses which did not have any antinociceptive effects. W’hen
we compared the enhancement of morphine-induced
icv.
Fig. 5. Blockade of
The remain-
ing bars represent thapsigargin administration before
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
0.01 from
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
bility that subtypes of cannabinoid receptors may exist
are administered
PANEL A
i.t.)
PANEL C
(panel
10
Apamin (5
1000
A8
1 10
doses
doses
doses
was administered at
et
A9 +
apamin
ED50 = 82
(panel
in the
spinal cord.
opioid or the direct interaction with opiate receptors.
Thus, the enhancement of opiate antinociception by
administration of calcium
to mice
and
potassium flux.
Modulation of calcium and
enhance or fail
1986) or by
modulators of intracellular pools of calcium such as
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-
1990;
by the
(Godfraind et
alter
et
et
1987; Reynolds et
et al.,
1986;
Cannabinoids either
the antinocicep-
1989
et al., 1987;
or
calcium chan-
nels, and found no reversal of the effects of icv. calcium (data
not shown). Thus, the calcium-induced blockade of
1989
due
to the blockade of antinociception by pertussis toxin pretreat-
ment which inactivates
Our
data agree with studies indicating an interaction of the
Cannabinoid-induced Antinociception
although
and CP 55,940.
and
1982;
proteins via.
reversed the
et al.,
in
al.
protein because pertussis toxin attenuates binding of CP
55,940
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
Gimenez-Gallego
et
et al.,
1988: Howlett et al..
spinal
cord
et al.,
1987). Charybdotoxin, a scorpion venom, blocks predomi-
nantly the large
preparations
19901. By decreasing
et
cannabinoid. CP 55,940
et
fast) conductance
or
channel
potassium
et al.,
channel in
of
et
ing in a decreased
of
apamin in
ciceptive
1988; Gaines
1988;
protein-coupled receptors
et
t.
et al.,
and
et
and
cal
and content of neurons (Harris et al., 1976; Cardenas and
R
OSS
,
1976; Welch and Olson,
levels
(Collier and Roy,
1974;
1978) and
produce hyperpolarization of neurons
(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
be done to
delineate conclusively the mechanisms underlying the
greater than additive effects observed using cannabinoids
and opiates in combination.
Acknowledgments
The author wishes to thank Dr. Bill R. Martin, Department of
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