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The Journal of Neuroscience, January 1, 1998, 18(1):451-457
Hypoactivity of the Spinal Cannabinoid System Results in
NMDA-Dependent Hyperalgesia
Jennelle Durnett
Richardson1,
Lin
Aanonsen3, and
Kenneth M.
Hargreaves1, 2
Departments of 1 Pharmacology and
2 Restorative Sciences, University of Minnesota,
Minneapolis, Minnesota 55455, and 3 Department of Biology,
Macalester College, St. Paul, Minnesota 55105
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ABSTRACT |
Cannabinoids, such as  9-THC, are capable of
inhibiting nociception, i.e., pain transmission, at least in part, by
interacting with spinal Gi/Go-coupled
cannabinoid receptors. What is not known, however, is the
antinociceptive role of endogenous spinal cannabinoids. If endogenous cannabinoids modulate basal nociceptive thresholds, then
alterations in this system could be involved in the etiology of certain
pain states. In this report we provide evidence for tonic modulation of
basal thermal nociceptive thresholds by the spinal cannabinoid system.
Administration of oligonucleotides directed against CB1
cannabinoid receptor mRNA significantly reduced spinal cannabinoid
binding sites and produced significant hyperalgesia when compared with
a randomer oligonucleotide control. A second method used to reduce
activity of the spinal cannabinoid receptor was intrathecal
administration of the cannabinoid receptor antagonist SR 141716A. SR
141716A evoked thermal hyperalgesia with an ED50 of 0.0012 fmol. The SR 141716A-induced hyperalgesia was dose-dependently blocked
by the administration of D-AP-5 or MK-801, two antagonists to the NMDA receptor. These results indicate that there is tonic activation of the spinal cannabinoid system under normal conditions. Furthermore, hypoactivity of the spinal cannabinoid system results in
an NMDA-dependent hyperalgesia and thus may participate in the etiology
of certain chronic pain states.
Key words:
endogenous cannabinoid; SR 141716A; glutamate; NMDA; hyperalgesia; nociceptive threshold; tonic; spinal cord
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INTRODUCTION |
Antinociception induced by
administration of exogenous cannabinoids has been widely reported.
Available evidence supports the hypothesis that the antinociception
induced by intrathecal administration of cannabinoids is mediated, at
least in part, via activation of receptors located in the spinal cord.
Autoradiographic studies have identified cannabinoid receptors
localized within the dorsal horn of the spinal cord in regions known to
receive input from nociceptors (Herkenham et al., 1991 ). Cannabinoid
administration can prevent the expression of c-fos in the dorsal horn
(Tsou et al., 1996) and inhibit the firing of wide dynamic range
neurons (Hohmann et al., 1995) in response to noxious stimuli.
Additionally, antinociception induced by either intravenous or
intrathecal administration of the synthetic cannabinoid CP 55,940 persists after spinal transection (Lichtman and Martin, 1991),
indicating that descending control from supraspinal sites cannot
completely account for cannabinoid-mediated antinociception.
Collectively, these data indicate that cannabinoids have a spinal site
of action for at least some of their antinociceptive effects.
Although there is a large body of evidence that exogenously
applied cannabinoids in normal animals are capable of producing antinociception, the role of endogenous cannabinoids in
modulating basal nociceptive thresholds is not well understood. Many
spinal antinociceptive systems do not seem to modulate basal
nociceptive thresholds but are invoked only in response to nociceptive
stimuli. For example, intrathecal administration of naloxone does not
produce a consistent effect on nociceptive responses (Besson and
Chaouch, 1987). Additionally, animals that lack the µ opioid receptor
subtype show little, if any, change in basal thermal nociceptive
thresholds (Sora et al., 1997). These studies indicate that the spinal
opioid receptors have, at most, a limited role in modulating basal
thermal nociceptive thresholds. The question of whether a system
modulates basal nociceptive thresholds is important because
hypoactivity of such a system could be involved in the etiology of
hyperalgesic states. Accordingly, understanding the mechanism
responsible for the transition to hyperalgesia is important for the
development of novel therapeutic drugs.
The mechanism by which spinal cannabinoids produce
antinociception is not well understood but may stem from their ability to inhibit the release of neurotransmitters. Cannabinoids have been
reported to inhibit electrically evoked acetylcholine release from
hippocampal slices (Gifford and Ashby, 1996), norepinephrine release
from sympathetic nerves (Ishac et al., 1996), and glutamate release
from hippocampal cultures (Shen et al., 1996). In addition to the
hippocampus, glutamate is located in terminals in the spinal cord (De
Biasi and Rustioni, 1988), and its intrathecal application results in
hyperalgesia (Aanonsen and Wilcox, 1987). Thus, inhibition of glutamate
release into the spinal cord is one potential mechanism for the
antinociceptive effects of cannabinoids.
The purpose of the present study was to evaluate the hypothesis that
the spinal endogenous cannabinoid system modulates basal nociceptive
thresholds and that hypoactivity of this system results in
hyperalgesia. The mouse hot plate assay was used to measure hyperalgesia to thermal stimuli. First, the effects of the
administration of a selective CB1 cannabinoid receptor
antisense oligonucleotide were determined on both cannabinoid receptor
density and hot plate latencies. Next, we evaluated the effects of
administration of a selective CB1 cannabinoid receptor
antagonist on hot plate latencies. Finally, we determined whether the
observed hyperalgesia was mediated by the NMDA receptor.
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MATERIALS AND METHODS |
Animals. Male ND4 Swiss mice (20-25 gm; Harlan
Laboratories, Indianapolis, IN) were maintained on a 12 hr light/dark
cycle with free access to food and water. All procedures were approved by the University of Minnesota Animal Care and Use Committee.
Materials. Materials were obtained from the following
companies: oligonucleotides, Microchemical Facilities (University of Minnesota, Minneapolis, MN); [3H]CP 55,940, DuPont
NEN (Boston, MA); SR 141716A
[N-(piperidin-1-yl) 5-(4-chlorophenyl)1(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxyamide], a gift from Sanofi Recherche (Montpellier, France); [3H]SR 141716A, Amersham (Arlington
Heights, IL); MK-801, RBI (Natick, MA); AP-5, Tocris Cookson (Ballwin,
MO); sterile saline, Baxter (McGaw Park, IL); UltimaGold scintillation
cocktail, Packard (Meriden, CT). All other reagents were purchased from
Sigma (St. Louis, MO).
Intrathecal injections. Intrathecal injections were
performed as described by Hylden and Wilcox (1980). Briefly, a 30 gauge needle was inserted into the subarachnoid space approximately between
vertebrae L5 and L6. Successful placement was indicated by a prototypic
tail flick reflex. All drugs were injected in a volume of 5 µl in a
saline vehicle. SR 141716A was initially solubilized in ethanol and
then diluted in saline, with a final ethanol concentration of
<0.00001%.
Oligonucleotide treatment. Mice received a 12.5 µg
intrathecal injection once a day for 4 d of either an 18-mer
oligonucleotide complementary to bases 4-21 of the CB1
receptor mRNA ("antisense") or an 18-mer randomer control showing
identical G/C composition but not complementary to any known mRNA
(Edsall et al., 1996).
P2 membrane preparation. Animals were
decapitated, and their spinal cords were removed via hydraulic
extrusion. The lumbar and cervical enlargements were dissected and
immediately frozen on dry ice. P2 membranes were prepared
using the method described by Devane et al. (1988). Briefly, tissue was
pooled and homogenized in 30 ml of sucrose (320 mM), EDTA
(2 mM), and MgCl2 (5 mM), and then
centrifuged at 2000 × g for 10 min. The pellet was
washed twice. The supernatants were then centrifuged at 39,000 × g for 15 min. The pellet was resuspended in 30 ml buffer A
[Tris-HCl (50 mM, pH 7.0, at 30°C), EDTA (2 mM), and MgCl2 (5 mM)] at 37°C for 10 min. This was centrifuged at 23,000 × g for 10 min. The pellet was resuspended in 30 ml buffer A at 30°C for 40 min.
This was centrifuged at 11,000 × g for 15 min. The
pellet was resuspended in 1 ml Tris-HCl (50 mM, pH 7.4, at
30°C), EDTA (1 mM), and MgCl2 (3 mM), and stored at 80°C.
Radioreceptor binding. Homogenates [60 or 100 µg of
protein, determined by the Bradford assay using Sigma fatty acid-free bovine serum albumin (BSA) as the standard] were incubated for 1 hr at
room temperature in assay buffer [Tris-HCl (50 mM, pH 7.4), MgCl2 (3 mM), EDTA (1 mM),
and fatty acid-free BSA (1 mg/ml)] with various concentrations of the
cannabinoid receptor agonist [3H]CP-55940 or the
cannabinoid receptor antagonist [3H]SR 141716A.
Total volume per reaction tube was 200 µl. Nonspecific binding was
determined with unlabeled SR 141716A (1 µM). The reaction was terminated with rapid filtration over a Whatman GF/C filter (Hillsboro, OR) that had been soaked for 15 min in 0.5%
polyethylenimine. Tissue was then washed three times with 4 ml of
ice-cold assay buffer. After they were washed, filters were transferred
to a scintillation vial, and UltimaGold scintillation cocktail was added. Disintegrations per minute were determined by counting the vials
with a liquid scintillation counter for 1 min. Binding to tissue from
animals in the knockdown experiment consisted of two separate
experiments, each in triplicate, using homogenates of tissue from
10-15 animals. Binding to tissue from naive animals with
[3H]SR 141716A consisted of two separate
experiments, each in triplicate, using homogenates of tissue from
15-30 animals.
Hot plate latencies. Mice were placed on a Harvard Hot Plate
Analgesia Meter (Edinbridge, KY) set at 54-55°C and immediately removed when a response to the thermal noxious stimulus, either licking
of a hindpaw or jumping from the surface of the hot plate, was
observed. A 40 sec cutoff was used to prevent tissue damage. Hot plate
latencies were recorded in triplicate for each animal, with ~5 min
separating each trial. The average baseline latency is 17.6 ± 0.2 sec (mean ± SEM; n = 364). Average baseline
latencies were similar between the different treatment groups. After
baseline latencies were recorded, a 5 µl intrathecal injection of the
appropriate drug was administered to the animals. In the studies using
oligonucleotides, postinjection latencies were recorded on the day
after the final injection immediately before collection of the tissue
for radioreceptor binding studies. In the studies using SR 141716A,
postinjection latencies were recorded 5 min after drug administration.
Difference scores were determined for each mouse by subtracting its
average baseline latency from its average postinjection latency. When blocked results were analyzed across different days of testing, inter-day experimental variability was removed by subtracting the
vehicle control from the experimental group. The injector and hot plate
observer were blind to treatment allocations.
Statistics. Receptor binding results were analyzed with
GraphPad Prism software (San Diego, CA). Kd and
Bmax values were determined with nonlinear
regression, and one-site and two-site analyses were compared to
determine the better fit. Pharm/PCS software was used to calculate the
ED50 values. Other data were analyzed either with
Student's t test or with ANOVA followed by a post hoc test, as appropriate. Results were considered significant when
the probability that they occurred because of chance alone was <5%
(i.e., p < 0.05). Data are reported as mean ± SEM.
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RESULTS |
If the cannabinoid system is tonically active in modulating basal
thermal nociceptive thresholds, then a decrease in cannabinoid receptor
number should produce hypoactivity and thus hyperalgesia. To test this
hypothesis, we used the "receptor knockdown" technique. Animals
received an intrathecal injection of either the antisense or randomer
oligonucleotide once a day for four consecutive days. Hot plate
latencies were recorded before injection and on the day after
termination of the injections. After the animals were tested, the
lumbar and cervical enlargements of the spinal cord were collected and
processed for receptor binding. There were no differences in the
Kd values, regardless of treatment. However, as
indicated in Figure 1 and Table
1, application of the antisense oligonucleotide produced a significant decrease in the amount of
cannabinoid receptor binding in the lumbar enlargement when compared
with the randomer oligonucleotide control (0.62 ± 0.15 vs
1.58 ± 0.19 pmol/mg protein). Application of the randomer
oligonucleotide produced no change in cannabinoid receptor levels in
the lumbar enlargement when compared with animals receiving the same
regimen of saline injections (Table 1). There were also no differences in cannabinoid receptor levels in the cervical enlargement regardless of treatment (Table 1), indicating that the CB1 antisense
treatment selectively reduced cannabinoid receptors localized in the
lumbar enlargement of the spinal cord.
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Figure 1.
Effects of intrathecal administration of an
antisense oligonucleotide complementary to the CB1 receptor
mRNA on cannabinoid receptor binding (A) and
hyperalgesia (B). A, Binding of
the cannabinoid receptor agonist [3H]CP 55,940 to
tissue from animals receiving the randomer control is represented by
the open squares (Bmax = 1.6 ± 0.2 pmol/mg protein). Binding of the cannabinoid receptor
agonist [3H]CP 55,940 to tissue from animals
receiving the antisense oligonucleotide is represented by the
filled circles (Bmax = 0.6 ± 0.2 pmol/mg protein). Error bars are SEM
(B). Hot plate latencies were recorded before
injection on the first day of oligonucleotide treatment and before
tissue collection 24 hr after the last oligonucleotide injection.
Observers were blind to treatment allocations. n = 12-15. Error bars are SEM. *p < 0.05 (Student's
t test).
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After 4 d of receiving intrathecal oligonucleotide injections,
animals receiving the CB1 antisense oligonucleotide
demonstrated significant hyperalgesia when compared with the random
controls (3.5 ± 1.2 vs 0.3 ± 0.7 sec) (Fig. 1). This
effect corresponds with the decrease in cannabinoid receptor binding,
which is consistent with the hypothesis that a decrease in
CB1 receptors in the lumbar spinal cord results in the
development of thermal hyperalgesia.
An additional series of studies independently tested the hypothesis
that endogenous cannabinoids modulate basal nociceptive thresholds. We
first determined that the CB1 receptor antagonist [3H]SR 141716A bound to the mouse lumbar spinal
cord with a Kd of 600 ± 200 pM
and a Bmax of 0.8 ± 0.1 pmol/mg protein
(Fig. 2). We then determined whether an
intrathecal injection of SR 141716A could evoke thermal hyperalgesia.
If the cannabinoid receptor is tonically active, then administration of
a cannabinoid receptor antagonist would be expected to block basal
cannabinoid activity, resulting in hyperalgesia. The results are
presented in Figure 3. Animals injected
with SR 141716A demonstrated a dose-dependent hyperalgesia 5 min after
injection with an ED50 of 0.0012 fmol/5 µl (0.24 pM) and a 95% confidence interval of 0.0018-0.0073 fmol/5 µl (0.36-1.46 pM). This effect was transient, with
latencies returning to preinjection values by 20 min postinjection
(Fig. 4) [p < 0.05; ANOVA (F(2, 28) = 5.13)]. It should be
noted that although hyperalgesia was consistently observed at these
concentrations, preliminary data suggest that at higher concentrations
SR 141716A is not as effective at producing hyperalgesia. The
production of hyperalgesia by intrathecal administration of the
cannabinoid receptor antagonist in otherwise naive animals is
consistent with the hypothesis that spinal cannabinoid receptors act
tonically to modulate basal nociceptive thresholds in intact
animals.
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Figure 2.
Binding of [3H]SR 141716A to
tissue from normal animals. Receptor binding was performed in
P2 membranes from mouse lumbar spinal cord using
[3H]SR 141716A. A shows a
saturation curve. Error bars are SEM. Error bars that are not visible
are contained within the symbol. B shows a Rosenthal
(Scatchard) plot of the same data.
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Figure 3.
Effect of SR 141716A on hot plate latencies in
normal animals. Mice received a 5 µl intrathecal injection of either
the saline vehicle (n = 49) or SR 141716A
(0.0006-0.01 fmol; n = 10-29). Hot plate
latencies were measured before and 5 min after injection. ED50 = 0.0012 fmol (95% confidence interval = 0.00018-0.0073 fmol). Observers were blind to treatment allocations.
Error bars represent SEM.
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Figure 4.
Time course of SR 141716A-induced hyperalgesia.
After baseline hot plate latencies were recorded, mice received an
intrathecal injection of either saline (n = 10;
open squares) or 0.005 fmol SR 141716A
(n = 10; closed circles). Hot plate
latencies were again recorded at 5, 20, and 40 min postinjection.
Observers were blind to treatment allocation. Error bars are SEM.
p < 0.05; ANOVA with Duncan's multiple range
test.
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In addition to the cannabinoid receptor, glutamate is located on
certain terminals of the spinal cord (De Biasi and Rustioni, 1988). The
release of glutamate from these terminals results in hyperalgesia, in
part by interacting with the NMDA glutamate receptor subtype (Aanonsen
and Wilcox, 1987). Interestingly, cannabinoids have been reported to
presynaptically inhibit the release of glutamate from cultured neurons
(Shen et al., 1996). Thus, one potential mechanism for the tonic
modulation of basal nociceptive thresholds by cannabinoids is the
presynaptic inhibition of basal glutamate release. One prediction of
this hypothesis is that the hyperalgesic action of SR 141716A is caused
by the disinhibition of glutamate release into the dorsal
spinal cord. The possibility that the hyperalgesia mediated by SR
141716A has an NMDA component was evaluated by intrathecal
co-administration of NMDA receptor antagonists with SR 141716A. The
results are presented in Figures 5 and
6. The intrathecal administration of SR
141716A (0.005 fmol/5 µl; 1 pM) produced a similar degree
of thermal hyperalgesia versus that of vehicle-treated animals in four
independent experiments [6.19 ± 0.81 vs 0.01 ± 0.68 sec
(Fig. 5); p < 0.001; ANOVA (F(5,
76) = 12.41); 5.35 ± 1.03 vs 0.00 ± 0.85 sec (Fig.
6); p < 0.01; ANOVA (F(5, 67) = 5.0); and previous data presented in Figs. 3 and 4]. Co-administration
of SR 141716A with the competitive NMDA receptor antagonist D-AP-5
(0.5-5.0 pmol/5 µl) resulted in a dose-dependent inhibition of SR
141716A-induced hyperalgesia [(Fig. 5) p < 0.001; ANOVA/linear regression (F(1, 29) = 12.87)].
The maximal inhibition was observed at 5 pmol D-AP-5, at which
concentration latencies had returned to preinjection values
(1.14 ± 0.79 vs 0.01 ± 0.68 sec). Application of D-AP-5
in the absence of SR 141716A had no effect on hot plate latencies when
compared with controls (0.52 ± 0.76 vs 0.01 ± 0.68 sec).
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Figure 5.
Inhibition of SR 141716A-induced hyperalgesia by
the NMDA antagonist D-AP-5. Hot plate latencies were recorded in mice
before and 5 min after a 5 µl injection of one of the following:
saline vehicle (n = 20; open
triangle), 5.0 pmol D-AP-5 (n = 11;
open square), or 0.005 fmol SR 141716A in the absence
(n = 20; filled circle) or presence
of 0.5 pmol (n = 10), 2.5 pmol
(n = 10), or 5.0 pmol (n = 11)
D-AP-5 (filled squares). Data are normalized to
the vehicle control. Observers were blind to treatment allocations. Error bars are SEM. **p < 0.01 versus vehicle
(F(5, 76) = 12.41); p < 0.001; ANOVA with Duncan's multiple range test.
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Figure 6.
Inhibition of SR 141716A-induced hyperalgesia by
the NMDA antagonist MK-801. Hot plate latencies were recorded in mice
before and 5 min after a 5 µl intrathecal injection of one of the
following: saline vehicle (n = 20; open
triangle), 2.5 pmol MK-801 (n = 9; open square), or 0.005 fmol SR 141716A in the absence
(n = 20; filled circle) or presence
of 0.025 pmol (n = 9), 0.25 pmol
(n = 7), or 2.5 pmol (n = 8)
MK-801 (filled squares). Data are normalized to
the vehicle control. Observers were blind to treatment allocations. Error bars are SEM. **p < 0.01 versus vehicle
(F(5, 67) = 4.95); p < 0.001; ANOVA with Duncan's multiple range test.
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To eliminate the possibility of a nonselective interaction between SR
141716A and D-AP-5, the effect of the noncompetitive NMDA receptor
antagonist MK-801 was also evaluated (Fig. 6). At concentrations of
0.025-2.5 pmol, co-administration of MK-801 with SR 141716A also
dose-dependently inhibited SR 141716A-induced hyperalgesia
[p < 0.05; ANOVA/linear regression
(F(1, 22) = 7.5)]. Maximal inhibition was
observed at 2.5 pmol MK-801; at this concentration there was no
difference from the vehicle control (0.40 ± 1.5 vs 0.00 ± 0.85 sec). Additionally, at its highest concentration, MK-801 showed no
effect on its own (1.37 ± 1.45 vs 0.00 ± 0.85 sec) when
compared with the vehicle control. Together, these results comprise
four independent replicates of SR 141716A-induced hyperalgesia and
indicate that it is mediated by an NMDA receptor mechanism.
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DISCUSSION |
In the present studies we have evaluated the hypothesis that the
spinal cannabinoid system modulates basal thermal nociceptive thresholds. Our first study indicates that decreasing cannabinoid receptor density in the lumbar but not cervical spinal cord results in
hyperalgesia. After 4 d of administration of an oligonucleotide complementary to the CB1 receptor mRNA, there was a
decrease in cannabinoid receptor binding, as determined with
[3H]CP 55,940, when compared with binding from the
tissue of animals receiving the randomer control. The antisense
oligonucleotide is not complementary to any known mRNA (Edsall et al.,
1996). Because of the specificity, it is reasonable to assume that the decrease in binding observed after treatment with the antisense oligonucleotide was caused by a decrease in the CB1 subtype
of cannabinoid receptors. However, the magnitude of reduction in CB1 binding sites cannot be determined because
[3H]CP 55,940 is not selective for the
CB1 receptor over the CB2 receptor. The binding
sites remaining after treatment with the antisense oligonucleotide may
therefore be either CB1 or CB2 receptors. However, the presence of CB2 receptors in spinal cord has
not been reported in the literature. Our results indicate that there is
little difference in the amount of binding determined with [3H]CP 55,940 or the selective CB1
receptor antagonist [3H]SR 141716A, suggesting
that if CB2 receptors are located in rat spinal cord, they
are expressed at a very low density. Thus, assuming that only
CB1 receptors were labeled with [3H]CP
55,940, the reduction in CB1 receptors was 61%.
Animals receiving the antisense oligonucleotide treatment displayed
significant hyperalgesia on the day after termination of treatment, the
same day at which the decrease in cannabinoid receptor binding was
measured. A similar treatment regimen with the same oligonucleotide
sequences administered intracerebroventricularly had no effect on
basal hot plate latencies but did result in a shift to the right in the
dose-response curve for intracerebroventricular cannabinoid-induced
antinociception (Edsall et al., 1996). In that study, cannabinoid
receptor levels were not determined, and thus there is no way of
knowing at which regions and to what extent the oligonucleotides were
effective in reducing cannabinoid receptor densities. Our results
indicate that administration of the oligonucleotide to the lumbar
enlargement produced a very localized effect, i.e., not even extending
to the cervical enlargement. Taken together, these studies suggest that
tonic modulation of nociceptive thresholds by cannabinoid receptors is
not a ubiquitous event but rather may be localized to the lumbar
enlargement of the spinal cord.
Our subsequent studies independently evaluated the hypothesis of
regulation of basal thermal nociceptive thresholds by cannabinoid receptors by intrathecal administration of SR 141716A. SR 141716A is a
selective, high-affinity antagonist at the CB1 receptor
that demonstrates a Ki for the transfected
CB1 receptor of 12 ± 2 nM but only
973 ± 280 nM for the transfected CB2
receptor (Felder et al., 1995). We found that the
Kd of [3H]SR 141716A in
mouse lumbar spinal cord was 600 ± 200 pM, which is
similar to what has been reported for rat whole brain synaptosomes (610 ± 60 pM) (Rinaldi-Carmona et al., 1996) and rat
cerebellum (610 ± 120 pM) (Hirst et al., 1996). The
IC50 of SR 141716A at 35 noncannabinoid receptors,
including the NMDA receptor, is >1 µM (Rinaldi-Carmona
et al., 1994). Together, available data indicate that SR 141716A is
selective for the CB1 receptor. Thus it was used to
evaluate the involvement of CB1 receptors in modulation of
basal nociceptive thresholds. Our results indicate that SR 141716A was
very potent in producing hyperalgesia. In addition, its ability to
produce hyperaglesia was short-lived, lasting <20 min. It is
interesting that this time course is similar to that observed after
intrathecal administration of NMDA (Aanonsen and Wilcox, 1987). The
short time course of SR 141716A hyperalgesia may reflect the diffusion
from the active receptors, which appear to be located discretely in the
lumbar enlargement, or redistribution of the highly lipophilic SR
141716A. Alternatively, it may be secondary to depletion of releasable
glutamate or a short time course of glutamate activity at NMDA
receptors. The transience of the effect argues against neurotoxicity of
the drug and supports the hypothesis that SR 141716A inhibits tonic
activity of the cannabinoid receptor and in this way produces
hyperalgesia.
It is curious that the ED50 for SR 141716A (0.24 pM) is well below the determined Kd
for SR 141716A in mouse lumbar spinal cord (600 pM). The
reason for this difference is currently unclear. One possibility is
that SR 141716A is interacting with high affinity at a noncannabinoid
binding site. However, if this were the case, we would not have
expected hyperalgesia after treatment with the CB1
antisense oligonucleotide. Another possibility is that a low-density, high-affinity CB1 receptor subtype is present in spinal
cord and its detection is difficult with current ligands. The
selectivity of SR 141716A for the CB1 receptor together
with the hyperalgesia obtained after the CB1 receptor
knockdown support the involvement of the CB1 receptor in
modulation of basal nociceptive thresholds.
An alternative explanation must be considered for the effects of the
CB1 receptor knockdown and SR 141716A on hot plate
latencies. Along with antinociception, two classic behavioral responses
to cannabinoid agonist administration are catalepsy and hypomotility. Thus, it is possible that the shorter latencies observed after SR
141716A and CB1 antisense oligonucleotide treatment may be caused by increased locomotor activity rather than hyperalgesia. There
are two arguments against this possibility. First, Yaksh (1981)
demonstrated that at concentrations necessary to produce antinociception, intrathecally administered cannabinoids did not produce any detectable changes in motor activity. Catalepsy was observed only at concentrations four times the ED50 for
producing antinociception and only after a delay, suggesting that
nociceptive thresholds are more sensitive to intrathecal administration
of cannabinoids than are the locomotor effects. Indeed, in the present study, blinded observers were unable to detect differences in gross
locomotor activity among the treatment groups. Second, Compton and
colleagues (1996) report that intravenous SR 141716A is capable of
stimulating locomotor activity. However, these effects are seen
only at concentrations >3 mg/kg (~0.137 µmol). This
dose is 20-fold greater than dosages required to block
cannabinoid-induced antinociception in the same studies. Together,
these support the hypothesis that the decreased latencies reflect
changes in nociceptive transmission rather than locomotion.
Collectively, these studies support the hypothesis that tonic
activity at the spinal CB1 receptor maintains thermal
nociceptive thresholds. Tonic activity of the cannabinoid receptor has
been reported previously in a different model: SR 141716A enhances short-term memory in rats and mice, suggesting that the cannabinoid system tonically modulates memory (Terranova et al., 1996). Tonic activity of receptors can be explained either by the activation of the
receptor by an endogenous ligand or by the spontaneous coupling between
the receptor and G-protein in the absence of ligand (Costa et al.,
1992). Although the latter occurs in cell expression systems, it has
not been reported in vivo, suggesting that there may be
tonic release of endogenous cannabinoids in the spinal cord.
In the present study, tonic activity of the spinal cannabinoid receptor
under basal conditions was demonstrated by the hyperalgesia induced by
a decrease in cannabinoid receptor density and by administration of the
cannabinoid receptor antagonist. Thus, tonic spinal cannabinoid receptor activation results in the modulation of basal thermal nociceptive thresholds. The mechanism for such modulation may be
similar to the mechanism of antinociception produced by the exogenous
administration of cannabinoids. Although the mechanism for spinal
cannabinoid-induced antinociception has not been determined, several
lines of evidence are consistent with it involving the inhibition of
neurotransmitter release. Activation of the CB1 receptor
inhibits adenylyl cyclase activity (Howlett, 1984), which has been
implicated in the regulation of exocytosis (Chavez-Noriega and Stevens,
1994). Additionally, CB1 activation can close certain calcium channels whose activity is necessary for neurotransmitter release (Mackie and Hille, 1992; Caulfield and Brown, 1992). Finally, CB1 activation can enhance potassium currents that can lead
to hyperpolarization of the membrane (Deadwyler et al., 1993; Henry and
Chavkin, 1995). Thus, the mechanism for cannabinoid antinociception may
be the inhibition of the release of neurotransmitters involved in
nociception, such as glutamate. Inhibition of such cannabinoid activity
would result in glutamate release and potentially NMDA receptor
activation and hyperalgesia. In support of this hypothesis, SR
141716A-induced hyperalgesia could be inhibited by both the competitive
NMDA antagonist D-AP-5 and the noncompetitive NMDA antagonist MK-801.
These results support the hypothesis that SR 141716A-induced
hyperalgesia is attributable to disinhibition of glutamate release.
Collectively, these results are consistent with the hypothesis that the
endogenous spinal cannabinoid system modulates basal thermal
nociceptive thresholds. These studies demonstrate that a decrease in
cannabinoid receptor number in the lumbar spinal cord is correlated
with hyperalgesia and that inhibition of cannabinoid activation by
administration of a cannabinoid receptor antagonist results in an
NMDA-dependent hyperalgesia. A recent report demonstrates that
intrathecal administration of pertussis toxin, which inactivates Gi and Go proteins via ADP ribosylation, will
produce hyperalgesia in mice (Womer et al., 1997). The results from the
present study suggest that one potential mechanism by which pertussis
toxin may act to produce hyperalgesia is by inactivating spinal
cannabinoid receptors. Taken together, these results provide a strong
rationale for the hypothesis that hypoactivity of the cannabinoid
system may be involved in the etiology of certain chronic pain states. Because opioids are not thought to be involved in modulation of basal
nociceptive thresholds, these findings provide a major difference between these two endogenous analgesic systems. Thus, there may be pain
states that are unresponsive to opioids but are relieved by
administration of cannabinoids. In animals, this is the case with
neuropathic pain that responds poorly to opioids but has recently been
demonstrated to be sensitive to cannabinoids (Herzberg et al., 1997).
Accordingly, drugs that activate cannabinoid receptors or gene therapy
directed at increasing activity of the cannabinoid system may have
therapeutic use in treating certain types of chronic pain.
| |
FOOTNOTES |
Received July 25, 1997; revised Oct. 3, 1997; accepted Oct. 8, 1997.
This work was supported by a predoctoral grant from the Howard Hughes
Medical Institute (J.D.R.) and by National Institutes of Health Grant
DE9860 (K.M.H.). SR 141716A was a gift from Sanofi Recherche
(Montpellier, France). We thank M. A. Sabino for technical assistance.
Correspondence should be addressed to Dr. Ken Hargreaves, Department of
Endodontics, Dental School, University of Texas Health Science Center,
7703 Floyd Curl Drive, San Antonio, TX 78284.
Dr. Richardson's present address: Department of Neurobiology, Harvard
Medical School, Boston, MA 02115.
| |
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Top
Abstract
Introduction
