Original Paper
Pharmacology 2008;82:193–200
DOI: 10.1159/000156485
Effect of Cannabinoid Receptor Agonists
on Streptozotocin-Induced Hyperalgesia
in Diabetic Neuropathy
Magdalena Bujalska
Department of Pharmacodynamics, Medical University of Warsaw, Warsaw, Poland
Key Words
AM1241 ⴢ Cannabinoid system ⴢ CB-1 and CB-2 receptors ⴢ
Diabetes ⴢ Hyperalgesia ⴢ Indomethacin ⴢ L-NOArg ⴢ
Met-F-AEA ⴢ Streptozotocin ⴢ WIN 55,212-2
Abstract
The effect of CB-1 and CB-2 receptor agonists, as well as an in-
fluence of a non-selective inhibitor of nitric oxide synthase
(NOS), L-NOArg, and an inhibitor acting preferentially on cy-
clooxygenase-1 (COX-1), indomethacin, on the action of can-
nabinoid receptor agonists in a streptozotocin (STZ)-induced
neuropathic model was investigated. When administered
alone, a non-selective cannabinoid receptor agonist, WIN
55,212-2, a potentially selective CB-1 cannabinoid receptor ag-
onist, Met-F-AEA, and a selective CB-2 cannabinoid receptor
agonist, AM1241, dose-dependently reduced STZ-induced hy-
peralgesia. The results of the present study also demonstrated
that inhibitors of COX and NOS increase antihyperalgesic ac-
tivity of low doses of CB-1 and CB-2 receptor agonists. Hypo-
thetical consequences of this phenomenon are discussed.
Copyright © 2008 S. Karger AG, Basel
Introduction
Cannabinoids, the bioactive constituents of Cannabis
sativa extracts like hashish and marijuana, have been
used to treat pain for many centuries. However, only the
© 2008 S. Karger AG, Basel
0031–7012/08/0823–0193$24.50/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/pha
Received: October 30, 2007
Accepted after revision: March 21, 2008
Published online: September 23, 2008
development of new experimental methods during the
past several decades has made it possible to understand
the mechanisms of cannabinoid action [1, 2]. The exis-
tence of endogenous ligands for cannabinoid receptors,
e.g. arachidonylethanolamide (anandamide), 2-arachid-
onyl glycerol, noladin ether, virodhamine and N-arachi-
donoyl-dopamine, has been demonstrated [3].
CB-1 and CB-2 cannabinoid receptors are the primary
targets of endogenous and exogenous cannabinoids. The
CB-1 receptors are expressed predominantly by neurons
of the central and peripheral nervous system. They have
been demonstrated in laminae of the spinal cord dorsal
horn intimately concerned with the processing and mod-
ulation of nociceptive information. Expression of the CB-
1 receptors has also been identified in dorsal root ganglia
[4, 5]. The CB-2 receptors are present centrally and pe-
ripherally in certain non-neuronal tissues, particularly in
immune cells. It was observed that activation of CB-2 re-
ceptors down-modulates mast cell function [4]. There is
also evidence of the presence of CB-2 receptors in rat
brain microglia [6]. CB-2 receptors probably participate
in mediating the analgesic effects of endocannabinoids,
particularly during inflammation [4].
Neuropathies complicating chronic diabetes are ac-
companied by neuropathic pain. Therefore, it was of
interest to investigate the effect of the non-selective can-
nabinoid receptor agonist, WIN 55,212-2, a potentially
selective CB-1 cannabinoid receptor agonist, Met-F-AEA,
and a selective CB-2 cannabinoid receptor agonist,
Magdalena Bujalska, PhD
Department of Pharmacodynamics, Medical University of Warsaw
Krakowskie Przedmieście 26/28, PO Box 3
PL–00-927 Warsaw 64 (Poland)
Tel./Fax +48 22 826 1366 or +48 22 828 1088, E-Mail mbujalska@gmail.com
AM1241, in a streptozotocin (STZ)-induced diabetic
neuropathic model. The influence of L-NOArg, which is
the non-selective inhibitor of nitric oxide synthase (NOS),
and indomethacin (IND), an inhibitor acting preferen-
tially on cyclooxygenase-1 (COX-1), on the action of can-
nabinoid receptor agonists was also investigated.
Material and Methods
Laboratory Animals
The study was conducted according to the guidelines of the
Ethical Committee for Experiments on Small Animals, Medical
University of Warsaw. The aforementioned Committee approved
the experimental protocols. Male Wistar rats (270–320 g) were
housed in a room maintained at a temperature of 20 8 2 ° C, un-
der a 12–12 h light–dark cycle. The experimental groups consist-
ed of at least 6 rats. The animals had free access to food and water.
Food was removed 16 h before STZ administration. The individ-
ual animals were used in only one experiment (i.e. for administra-
tion of STZ + one agent).
Chemicals
AM1241 [(R)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piper-
idinylmethyl)-1H-indole], Met-F-AEA [(+/–)-2-methylarachi-
donoyl-2ⴕ-fluoroethylamide], STZ (N-[methylnitrosocarbamo-
yl]- ␣-D -glucosamine), WIN 55,212-2 [(R)-(+)-WIN 55,212-2 me-
sylate salt] were purchased from Sigma Chemical Co., USA; IND
[indomethacin] was from Polfa Kutno, and L-NOArg [NG-nitro-
L-arginine] from Research Biochemicals International.
STZ-Induced Diabetes
Diabetes was induced by intramuscular administration of
STZ at a dose of 40 mg/kg body weight, as described by Nakhoda
and Wong [7]. STZ was dissolved in citrate buffer at pH 4.5 and
administered in only one dose on the first day of the study into
the thigh muscles of rats. Before induction of diabetes the animals
were fasted over 16 h. Following the injection, food and water were
available ad libitum during the remaining 28 days of the experi-
ment. Control rats received an equal volume of buffer. Starting on
day 3 (72 h) after STZ administration, glucose levels were deter-
mined using a blood glucometer (Accu-Check Active, Roche Di-
agnostics Corp.). Blood samples for the glucose determinations
were drawn from the tail vein. Permanent hyperglycemia was ob-
served in all rats (1 400 mg/dl).
In the STZ-untreated animals, glucose levels amounted to
about 120 mg/dl and remained stable during the 28-day observa-
tion period. The STZ-induced hyperglycemia was accompanied
by the gradual decrease of body mass, the increase in food con-
sumption, as well as considerable increase in water intake.
Drug Administration
STZ was administered as described above. AM1241 and WIN
55,212-2 were dissolved in dimethyl sulfoxide (DMSO), Met-F-
AEA in 0.9% saline, L-NOArg and IND were suspended in 0.1%
solution of methylcellulose. AM1241 and Met-F-AEA in doses of
0.5 and 5 mg/kg, WIN 55,212-2 in a dose of 0.3 and 1.0 mg/kg, and
L-NOArg in a dose of 0.1 mg/kg were applied intraperitoneally
194 Pharmacology 2008;82:193–200
(i.p.), IND in a dose 0.1 mg/kg was injected subcutaneously (s.c.).
Control animals were injected i.p. with DMSO (control to AM1241,
WIN 55,212-2); i.p. with 0.9% saline (control to Met-F-AEA); i.p.
or s.c. with 0.1% solution of methylcellulose (control to L-NOArg
and IND, respectively) according to the same time schedule.
Time Schedule
All the drugs (except STZ given only on the first day) were ap-
plied on 5 consecutive days of the experiment (from 18 to 22 days
after STZ administration). L-NOArg was administered 5 min be-
fore and IND was applied 15 min before AM1241, Met-F-AEA
and WIN 55,212-2.
Measurement of the Nociceptive Threshold
The changes in pain thresholds were determined using me-
chanical stimuli – the modification of the classic paw withdrawal
test described by Randall and Selitto [8]. In order to perform me-
chanical stimulation, a progressively increased pressure was ap-
plied to the dorsal surface of the rat’s paw using an analgesimeter
(Ugo Basile Biological Research Apparatus). The instrument used
increased force on the paw at a rate of 32 g/s. The nociceptive
threshold was defined as force in grams, at which the rat attempt-
ed to withdraw its hind paw, and values of pressure were recorded
at this moment. Nociceptive threshold was measured in dupli-
cates and mean was drawn for further calculations. At least two
observers controlled the response.
Baseline nociceptive thresholds (average of two trials) were
measured for each animal immediately before STZ was estab-
lished (A). Measurements of prolonged activity of the drugs in-
vestigated were performed on 5 consecutive days (e.g., measure-
ment on the days following administration of drugs and before
consecutive drug administration) from 19 to 23 days (B) after STZ
administration and then, after cessation of drug administration,
to 28 day (B).
Measurements of withdrawal threshold to mechanical stimuli
for the STZ group of animals were performed the first day before
STZ administration (A) and then every day from day 2 to 28 of the
experiment (B). Nociceptive thresholds were also determined at 15,
30, 60, 90, 120 and 180 min after WIN 55,212-2, AM1241 and Met-
F-AEA (B) administered in single dose on 18 day of the experiment
(B).
In all experimental sessions the thresholds (B) obtained were
compared to baseline (A). Changes in pain threshold were calcu-
lated as percentage of baseline value according to the following
formula:
% of analgesia = (B/A) ⴢ 100% – 100%
where A = pressure (in g), baseline pain threshold, and B = pres-
sure (in g) in consecutive measurements.
Percents of analgesia values calculated as above for individual
animals were subsequently used to calculate average values in
particular experimental groups and for statistical analyses.
Statistical Analysis
The results are expressed as mean 8 SEM. The statistical sig-
nificance of differences between groups was evaluated by Stu-
dent’s t test and the Newman-Keuls multiple range test. p ^ 0.05
was accepted as statistically significant. All statistical calcula-
tions were performed using the computer software described by
Tallarida and Murray [9].
Bujalska
 20
10 ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **
0
% of analgesia
–10
–20
–30
–40
–50
–60
1 2 3 4 5 6 7 8
Fig. 1. Influence of STZ at a dose of 40 mg/kg i.m. on threshold to mechanical stimuli (days 1–28 of experiment).
Values are means 8 SEM. STZ vs. control; ** p ^ 0.01. i = STZ; ––_–– = control.
Results
Effect of STZ on Threshold to Mechanical Stimuli
As shown in figure 1, starting on day 2, a statistically
significant gradual decrease of the nociceptive threshold
was observed in STZ-treated animals. The decrease
reached its plateau phase on day 18 and remained fairly
stable for the next 10 days, i.e. until the end of the exper-
iment. No significant changes were observed when the
nociceptive threshold was measured up to 180 min on 18
day of the experiment (fig. 2).
Effect of WIN 55,212-2, AM1241 and Met-F-AEA on
STZ-Induced Hyperalgesia (Randall-Selitto Test)
As shown in figure 2a, single administration of WIN
55,212 at a dose of 0.3 or 1.0 mg/kg significantly reduced
STZ hyperalgesia. This effect was dose-dependent. It
reached its maximum in 15 min after drug administra-
tion and then slowly diminished. After a higher dose of 1
mg/kg in 15 min, even antinociception was seen. Similar
effects were also observed after Met-F-AEA or AM1241
at doses of 0.5 and 5.0 mg/kg. However, the higher dose
of Met-F-AEA (5.0 mg/kg) only abolished STZ hyperal-
gesia while the same dose of AM1241 produced antinoci-
ception from 15 to 60 min of the experiment (fig. 2b, c).
WIN 55,212-2 at a dose of 0.3 mg/kg i.p., AM1241 at a
dose of 0.5 mg/kg i.p. and Met-F-AEA at a dose of 0.5
mg/kg i.p. administered on 5 consecutive days dimin-
ished STZ hyperalgesia. This effect was markedly greater
Cannabinoid System Activity and
Hyperalgesia
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Time (days)
for AM1241 and Met-F-AEA than for WIN 55,212-2, and
it was gradually intensified for 4 consecutive days after
administration. After cessation of drug administration
this effect slowly decreased, however it never reached the
values of control animals; STZ-treated only (fig. 3–5).
Effect of IND on Activity of WIN 55,212-2,
Met-F-AEA and AM1241 in a Model of STZ-Induced
Hyperalgesia
IND given on 5 consecutive days reduced STZ-pro-
duced hyperalgesia. Daily pretreatment with IND pro-
gressively increased the antihyperalgesic action of low
doses of WIN 55,212-2 (0.3 mg/kg i.p.), AM1241 (0.5 mg/
kg i.p.) and Met-F-AEA (0.5 mg/kg i.p.) and on days 4 and
5 concomitant administration of IND and Met-F-AEA or
AM1241 (22 and 23 days after STZ administration) re-
sulted in abolishment of STZ hyperalgesia. After drug
withdrawal, a gradual decrease of the analgesic activity of
cannabinoids (especially for AM1241 and Met-F-AEA)
with IND pretreatment occurred (fig. 3–5).
Effect of L-NOArg on the Activity of WIN 55,212-2,
Met-F-AEA, and AM1241 in a Model of STZ-Induced
Hyperalgesia
L-NOArg administered on 5 consecutive days re-
duced STZ-produced hyperalgesia. Premedication L-
NOArg before low doses of WIN 55,212-2 and AM1241
not only progressively reduced (from the first or second
day of measurements, respectively) but in the last 2 days
Pharmacology 2008;82:193–200 195
 30 30
20
20 **
10 10
** ** ** **

% of analgesia
0
% of analgesia

0
–10
–10
** ** **
–20
** –20 ** **
–30
** ** –30
**
–40 ** ** **** ** –40 ** ** ** **
–50 –50
–60 –60
15 30 60 90 120 180 15 30 60 90 120 180
a Time (min) b Time (min)

30
20 ** ** **
10
0

% of analgesia
–10

Fig. 2. Influence of WIN 55,212-2 (a) administered at single dos-

–20
** ** ** **
es of 1.0 mg/kg (WIN 1) or 0.3 mg/kg (WIN 0.3), Met-F-AEA (b) –30 ** **
and AM1241 (c) at doses of 5.0 mg/kg (Met-F-AEA 5, AM 5) or
0.5 mg/kg (Met-F-AEA 0.5, AM 0.5) on STZ hyperalgesia. Mea-
–40
–50
** ****
surements on day 18 of experiment. Values are means 8 SEM. –60
STZ vs. WIN; STZ vs. Met-F-AEA, STZ vs. AM; ** p ^ 0.01. k = 15 30 60 90 120 180
WIN 0.3, Met-F-AEA 0.5, AM 0.5; i = WIN 1, Met-F-AEA 5, AM c Time (min)
5; ––o–– = STZ.

abolished STZ hyperalgesia (after 4 and 5 days of mea-
surements). Premedication of L-NOArg intensified the
antihyperalgesic activity administered in the low dose
of Met-F-AEA. On days 4–5 of measurements (days 22–
23 of the experiment) even a significant antinociception
was observed. After a cessation of drug administration,
hyperalgesia quickly returned and nociceptive thresh-
olds were comparable to those obtained in WIN 55,212
-2, AM1241 or Met-F-AEA alone treated groups (fig.
6–8).
Discussion
Diabetic neuropathy is typically accompanied by neu-
ropathic pain and hyperalgesia, and despite many stud-
ies, the mechanism of the development of diabetic hyper-
algesia has still not been satisfactorily explained. It is
commonly known that hyperglycemia (especially long-
lasting) is a basic factor determining the development of
diabetic neuropathy.
In the present investigations the diabetogenic action
of STZ was accompanied by development of irreversible
hyperalgesia. The considerable lowering of the withdraw-
al threshold to mechanical stimuli occurred on day 2 of
investigations and threshold values gradually decreased
until day 18, when hyperalgesia remained at an approxi-
mately similar level.
In diabetes, damage of small fiber C and A delta is re-
sponsible for the early symptoms of hyperalgesia. These
fibers have been identified to carry the capsaicin vanil-
loid receptor. It is known that cannabinoids suppress a
release of nociceptive neuropeptides such as substance P,
calcitonin gene-related peptide from central and periph-
eral terminals of capsaicin-sensitive primary afferent fi-
bers [10]. Other mechanisms through which some can-
nabinoids may act to induce antinociception include the
inhibition of inflammatory eicosanoid production [6,
11].
The results of previous studies suggest that analgesic
and antihyperalgesic effects of cannabinoids are medi-
ated not only by activation of CB-1 receptors [12–14] but

196 Pharmacology 2008;82:193–200 Bujalska

 20

10
** ** x** xx * xx * xx * xx * xx *
0

–10

% of analgesia
Fig. 3. Effect of IND at a dose of 0.1 mg/kg
s.c. on the antihyperalgesic activity of –20
WIN 55,212-2 (WIN) at a dose of 0.3 mg/
kg i.p. in STZ-treated rats. Days 19–23: –30
measurements of prolonged activity of in-
–40
vestigated drugs; days 24*–28*: after dis-
continuation of administration. Values
–50
are means 8 SEM. WIN vs. IND + WIN;
* p ^ 0.05, ** p ^ 0.01; WIN vs. control;
x –60
p ^ 0.05, xx p ^ 0.01. k = WIN; i = 19 20 21 22 23 24* 26* 28*
IND + WIN; g = IND; ––o–– = STZ; Time (days)
––_–– = control.

20

10

0
x ** **
xx xx ** **
xx xx ** xx xx **
xx
% of analgesia

–10
Fig. 4. Effect of IND at a dose of 0.1 mg/kg
s.c. on the antihyperalgesic activity of Met- –20
F-AEA at a dose of 0.5 mg/kg i.p. in STZ-
treated rats. Days 19–23: measurements of –30
prolonged activity of investigated drugs;
days 24*–28*: after discontinuation of ad- –40
ministration. Values are means 8 SEM.
–50
Met-F-AEA vs. IND + Met-F-AEA; ** p ^
0.01; Met-F-AEA vs. control; x p ^ 0.05, –60
xx
p ^ 0.01. k = Met-F-AEA; i = IND 19 20 21 22 23 24* 26* 28*
+ Met-F-AEA; g = IND; ––o–– = STZ; Time (days)
––_–– = control.

20

10

0
xx ** **
xx xx ** xx ** xx ** **
xx xx xx

–10
% of analgesia

Fig. 5. Effect of IND at a dose of 0.1 mg/kg –20

s.c. on the antihyperalgesic activity of
–30
AM1241 (AM) at a dose of 0.5 mg/kg i.p.
in STZ-treated rats. Days 19–23: measure-
–40
ments of prolonged activity of investigated
drugs; days 24*–28*: after discontinua-
–50
tion of administration. Values are means
8 SEM. AM vs. IND + AM; ** p ^ 0.01; –60
AM vs. control; xx p ^ 0.01. k = AM; i = 19 20 21 22 23 24* 26* 28*
IND + AM; g = IND; ––o–– = STZ; ––_–– = Time (days)
control.

Cannabinoid System Activity and Pharmacology 2008;82:193–200 197

Hyperalgesia
 20

10
** **
0
** ** **
–10

% of analgesia
Fig. 6. Effect of NG-nitro-L-arginine (L- –20
NOArg) at a dose of 0.1 mg/kg i.p. on the
–30
antihyperalgesic activity of WIN 55,212-2
(WIN) at a dose of 0.3 mg/kg i.p. in STZ-
–40
treated rats. Days 19–23: measurements of
prolonged activity of investigated drugs;
–50
days 24*–28*: after discontinuation of ad-
ministration. Values are means 8 SEM. –60
WIN vs. L-NOArg + WIN; ** p ^ 0.01. 19 20 21 22 23 24* 26* 28*
k = WIN; i = L-NOArg + WIN; g = Time (days)
L-NOArg; ––o–– = STZ; ––_–– = control.

20

**
10 **
0
** ** ** **
% of analgesia

–10
Fig. 7. Effect of NG-nitro-L-arginine (L-
NOArg) at a dose of 0.1 mg/kg i.p. on the –20
antihyperalgesic activity of Met-F-AEA at
a dose of 0.5 mg/kg i.p. in STZ-treated rats. –30
Days 19–23: measurements of prolonged
–40
activity of investigated drugs; days 24*–
28*: after discontinuation of administra- –50
tion. Values are means 8 SEM. Met-F-
AEA vs. L-NOArg + Met-F-AEA; ** p ^ –60
0.01. k = Met-F-AEA; i = L-NOArg + 19 20 21 22 23 24* 26* 28*
Met-F-AEA; g = L-NOArg; ––o–– = STZ; Time (days)
––_–– = control.

20

10

0
** ** ** **
–10
% of analgesia

Fig. 8. Effect of NG-nitro-L-arginine (L- –20

NOArg) at a dose of 0.1 mg/kg i.p. on the
antihyperalgesic activity of AM1241 (AM) –30
at a dose of 0.5 mg/kg i.p. in STZ-treated
–40
rats. Days 19–23: measurements of pro-
longed activity of investigated drugs; days –50
24*–28*: after discontinuation of adminis-
tration. Values are means 8 SEM. AM vs. –60
L-NOArg + AM; ** p ^ 0.01. k = AM; 19 20 21 22 23 24* 26* 28*
i = L-NOArg + AM; g = L-NOArg; Time (days)
––o–– = STZ; ––_–– = control.

198 Pharmacology 2008;82:193–200 Bujalska

also by activation of CB-2 receptors [15–18]. In the litera-
ture only limited data exist concerning participation of
cannabinoid receptors in modification of hyperalgesia-
evoked STZ administration. In an in vitro and ex vivo
study, Zhang et al. [19] demonstrated that high glucose
concentrations are associated with a decreased expres-
sion of CB-1 receptors in nerve cells, which may contrib-
ute to the pathogenesis of diabetic neuropathy. Studies
performed by Ulugöl et al. [20] showed that i.p. and local
administration of WIN 55,212-2 reduced mechanical al-
lodynia in diabetic animals in a model of STZ neuropa-
thy. Doğrul et al. [21] demonstrated that WIN 55,212-2
administered systemically (1.5 and 10 mg/kg) produced
a dose-dependent antinociception in radiant tail-flick
test both in diabetic and control mice. This compound
also blocked tactile allodynia.
In the current study it was shown that single or chron-
ic administration of non-selective cannabinoid agonist,
WIN 55,212-2, as well as potentially selective CB-1 can-
nabinoid receptor agonist, Met-F-AEA, and selective CB-
2 cannabinoid receptor agonist, AM1241, dose-depend-
ently alleviated STZ hyperalgesia.
Only one report in the literature has demonstrated
that (R)-AM1241 enantiomer, which was used also in this
study at a dose of 1 and 3 mg/kg i.v., reduced the second
phase of nociceptive behaviors in a mouse model of for-
malin intraplantar test. In rats, (R)-AM1241 (3 and 6 mg/
kg i.v.) reduced allodynia elicited by L5-L6 spinal nerve
ligation. (R)-AM1241 also dose-dependently reduced
capsaicin-induced calcitonin gene-related peptide release
[22]. It is of interest to note that in an in vitro study, Bing-
ham et al. [23] demonstrated that (R)-AM1241, as well as
(R,S)-AM1241, was found to be an agonist in human CB-
2 receptor, but an inverse agonist in rats and mouse CB-2
receptors. (R)-AM1241 bound with a more than 40-fold
higher affinity than (S)-AM1241 to all three CB-2 recep-
tors, what perhaps indicates that it is a specific ligand of
CB-2 receptors. This surprising and very intriguing phe-
nomenon requires further explanation.
In previous studies it was shown that COX as well as
nitric oxide (NO) systems contribute to transmission of
pain stimuli in STZ-induced diabetic neuropathy [24].
The results of the present study appear to indicate that the
interaction between endogenic cannabinoid, COX and
NOS(s) systems might exist. It was shown that both an
inhibitor of COX and a non-specific NOS inhibitor inten-
sified antihyperalgesic activity of WIN 55,212-2, Met-F-
AEA and AM1241. However, the mechanism of the inter-
action between cannabinoid, COX and NOS pathways
was not fully elucidated. It has been shown that arachi-
Cannabinoid System Activity and
Hyperalgesia
donoylethanolamide (anandamide) is a metabolite of ar-
achidonic acid (AA). However, it is not clear which AA
pathway contributes to the synthesis of endocannabi-
noids. AA mobilization is a condition favoring increased
anandamide or other endocannabinoids synthesis [25]. It
is also known that activity of enzyme responsible for the
metabolism of anandamide and other endocannabinoid
(fatty-acid amide hydrolase (FAAH)) is inhibited by non-
steroidal anti-inflammatory drugs such as ibuprofen, ke-
torolac and flurbiprophen inhibit an activity of FAAH
[26]. Moreover, COX and FAAH inhibition can intensify
endocannabinoid production [25].
There is also evidence that endocannabinoids regulate
the glutamate (NMDA)-mediated central sensitization [4].
In spinal cord, endocannabinoids released from the sec-
ond-order neurons act as retrograde neurotransmitters
lowering the release of excitatory amino acids on the first-
order neurons [25], what subsequently leads to inhibition
of e.g. the phospholipase A2 family or NOS activation.
It has been demonstrated that cannabinoids inhibited
NO production in cultured glia cells [27] as well as in cer-
ebellar granule cells in vitro [28]. Coffey et al. [29] showed
that ⌬9-tetrahydrocannabinol reduced NO production in
peritoneal macrophages. On the other hand, Costa et al.
[30] suggested that cannabidiol (isomer of ⌬9-tetrahydro-
cannabinol) reduced the overexpression of the endothe-
lial but not neuronal and inducible NOS. The lifespan of
endocannabinoids is limited by a rapid cellular uptake of
endocannabinoids via a membrane transporter with sub-
sequent intracellular degradation. Currently, it is com-
monly known that NO can activate the endocannabinoid
transporter with subsequent cellular uptake and degra-
dation of endocannabinoids. Interestingly, the antinoci-
ceptive activity of NOS inhibitor N-nitro-L-arginine
methyl ester (L-NAME) was reversed by AM251, whereas
the pronociceptive effect of an NO donor (RE 2047) was
abolished by co-administration of the endocannabinoid
transporter blocker AM404 [31]. In other study, repeated
treatment with WIN 55,212-2 lowered the increased level
of PGE2 in plasma. WIN 55,212-2 also abolished the in-
crease of NO levels in paw tissues as well as overexpres-
sion of neuronal NOS in injured sciatic nerve in a model
of chronic constriction of the sciatic nerve in rats. Costa
et al. [18] suggested that WIN 55,212-2 could modulate
the levels of NO and PGE2 by activating on CB-2 recep-
tors present in inflammatory cells, such as macrophages
and T lymphocytes.
It is interesting to note that in patients the treatment
of acute pain often requires relatively high doses of can-
nabinoids, which are associated with considerable side
Pharmacology 2008;82:193–200 199
effects such as dizziness and sedation. Nevertheless, pre-
clinical and clinical data suggest that lower doses of can-
nabinoids may be effective for the treatment of allodynia
and hyperalgesia in neuropathic pain. It seems that can-
nabinoids should be applied as low-dose co-analgesics to
inhibit neuroplasticity and central sensitization rather
than as analgesics in acute pain [1]. It is concluded that
the stimulation of both CB-1 and CB-2 receptors inhib-
ited transmission of pain stimuli in STZ-induced diabe-
tes. The results of the present study also demonstrated
that inhibitors of COX and NOS intensified antihyperal-
gesic activity of CB-1 and CB-2 receptor agonists. It is
possible that in future this observation may be relevant
in relief of diabetic neuropathic pain.

References

1 Azad SC, Rammes G: Cannabinoids in an-
aesthesia and pain therapy. Curr Opin An-
aesthesiol 2005;18:424–427.
2 Palmer SL, Thakur GA, Makriyannis A:
Cannabinergic ligands. Chem Phys Lipids
2002;121:3–19.
3 Goya P, Jagerovic N, Hernandez-Folgado L,
Martin MI: Cannabinoids and neuropathic
pain. Mini Rev Med Chem 2003;3:765–772.
4 Rice ASC, Farquhar-Smith WP, Nagy I: En-
docannabinoids and pain: spinal and pe-
ripheral analgesia in inflammation and neu-
ropathy. Prostagl Leukotr Essent Fatty Acids
2002;66:234–256.
5 Hohmann AG: Spinal and peripheral mech-
anisms of cannabinoid antinociception: be-
havioral, neurophysiological and neuroana-
tomical perspectives. Chem Phys Lipids
2002;121:173–190.
6 Pertwee RG: Cannabinoid receptors and
pain. Prog Neurobiol 2001; 63:569–611.
7 Nakhoda A, Wong HA: The induction of di-
abetes in rats by intramuscular administra-
tion of streptozotocin. Experientia 1979; 35:
1679–1980.
8 Randall LO, Selitto JJ: A method for mea-
surement of analgesic activity on inflamed
tissue. Arch Int Pharmacodyn Ther 1957;
111:409–419.
9 Tallarida RJ, Murray RB: Manual of Pharma-
cologic Calculation with Computer Pro-
grams, ed 2. New York, Springer, 1986, pp
113–117.
10 Richardson JD, Aanonsen L, Hargreaves KM:
Antihyperalgesic effects of spinal cannabi-
noids. Eur J Pharmacol 1998;345:145–153.
11 Seidel K, Hamza M, Ates M, Gühring H: Flur-
biprofen inhibits capsaicin-induced calcito-
nin gene-related peptide release from rat spi-
nal cord via an endocannabinoid-dependent
mechanism. Neurosci Lett 2003;338:99–102.
12 Hama A, Sagen J: Antinociceptive effect of
cannabinoid agonist WIN 55,212-2 in rats
with a spinal cord injury. Exp Neurol 2006;
204:454–457.
13 Fox A, Kesingland A, Gentry C, McNair K,
Patel S, Urban L, James I: The role of central
and peripheral cannabinoid1 receptors in the
antihyperalgesic activity of cannabinoids in
a model of neuropathic pain. Pain 2001; 92:
91–100.
14 Bridges D, Ahmad K, Rice ASC: The synthet-
ic cannabinoid WIN 55,212-2 attenuates hy-
peralgesia and allodynia in a rat model of
neuropathic pain. Br J Pharmacol 2001; 133:
586–594.
15 Ibrahim MM, Deng H, Zvonok A, Cockayne
DA, Kwan J, Mata HP, Vanderah TW, Lai J,
Porreca F, Makriyannis A, Malan TP Jr:
Activation of CB2 cannabinoid receptors by
AM1241 inhibits experimental neuropathic
pain: pain inhibition by receptors not pres-
ent in the CNS. Proc Natl Acad Sci USA 2003;
100:10529–10533.
16 Ibrahim MM, Porreca F, Lai J, Albrecht PJ,
Rice FL, Khodorova A, Davar G, Makriyan-
nis A, Vanderah TW, Mata HP, Malan TP Jr:
CB2 cannabinoid receptor activation pro-
duces antinociception by stimulating pe-
ripheral release of endogenous opioids. Proc
Natl Acad Sci USA 2005;102:3093–3098.
17 Scott DA, Wright CE, Angus JA: Evidence
that CB-1 and CB-2 cannabinoid receptors
mediate antinociception in neuropathic pain
in the rat. Pain 2004;109:124–131.
18 Costa B, Colleoni M, Conti S, Trovato AE,
Bianchi M, Sotgiu ML, Giagnoni G: Repeat-
ed treatment with the synthetic cannabinoid
WIN,212-2 reduces both hyperalgesia and
production of pronociceptive mediators in a
rat model of neuropathic pain. Br J Pharma-
col 2004;141:4–8.
19 Zhang F, Hong S, Stone V, Smith PJ: Expres-
sion of cannabinoid CB1 Receptors in mod-
els of diabetic neuropathy. J Pharmacol Exp
Ther 2007;323:508–515.
20 Ulugöl A, Karadag HC, Ipci Y, Tamer M,
Dokmeci I: The effect of WIN 55,212-2, a
cannabinoid agonist, on tactile allodynia in
diabetic rats. Neurosci Lett 2004; 371: 167–
170.
21 Doğrul A, Gűl H, Yildiz O, Biling F,
Gűzeldemir E: Cannabinoids blocks tactile
allodynia in diabetic mice without attenua-
tion of its antinociceptive effect. Neurosci
Lett 2004;368:82–86.
22 Beltramo M, Bernardini N, Bertorelli R,
Campanella M, Nicolussi E, Fredduzzi S,
Reggiani A: CB2 receptor-mediated antihy-
peralgesia: possible direct involvement of
neural mechanisms. Eur J Neurosci 2006;23:
1530–1538.
23 Bingham B, Jones PG, Uveges AJ, Kotnis S,
Lu P, Smith VA, Sun SC, Resnick L, Chlenov
M, He Y, Strassle BW, Cummons TA, Piesla
MJ, Harrison JE, Whiteside GT, Kennedy JD:
Species-specific in vitro pharmacological ef-
fects of the cannabinoid receptor 2 (CB2)-se-
lective ligand AM1241 and its resolved enan-
tiomers. Br J Pharmacol 2007;151:1061–1070.
Erratum in: Br J Pharmacol 2007;151:1137.
24 Bujalska M, Tatarkiewicz J, deCorde A,
Gumułka W: Effect of cyclooxygenase and
nitric oxide synthase inhibitors on strepto-
zotocin-induced hyperalgesia in rats. Phar-
macology 2008;81:151–157.
25 Ates M, Hamza M, Seidel K, Kotalla CE, Le-
dent C, Gühring H: Intrathecally applied
flurbiprofen produces an endocannabinoid-
dependent antinociception in the rat forma-
lin test. Eur J Neurosci 2003;17:597–604.
26 Guindon J, De Léan A, Beaulieu P: Local in-
teraction between anandamide, an endocan-
nabinoid, and ibuprofen, a nonsteroidal ant-
inflammatory drug, in acute and in-
flammatory pain. Pain 2006;121:85–93.
27 Molina-Holgado F, Pinteaux E, Moore JD,
Molina-Holgado E, Guaza C, Gibson RM,
Rothwell NJ: Endogenous interleukin-1 re-
ceptor antagonist mediates anti-inflamma-
tory and neuroprotective actions of cannabi-
noids in neurons and glia. J Neurosci 2003;
23:6470–6474.
28 Hillard CJ, Muthian S, Kearn CS: Effects of
CB(1) cannabinoid receptor activation on
cerebellar granule cell nitric oxide synthase
activity. FEBS Lett 1999;459:277–281.
29 Coffey RG, Snella KJ, Pross S: Inhibition of
macrophage nitric oxide production by tet-
rahydrocannabinol in vivo and in vitro. Int J
Immunopharmacol 1996;18:749–752.
30 Costa B, Trovato AE, Comelli F, Giagnoni G,
Colleoni M: The non-psychoactive cannabis
constituent cannabidiol is an orally effective
therapeutic agent in rat chronic inflamma-
tory and neuropathic pain. Eur J Pharmacol
2007;556:75–83.
31 Gühring H, Hamza M, Sergejeva M, Ates M,
Kotalla CE, Ledent C, Brune K: A role for
endocannabinoids in indomethacin-in-
duced spinal antinociception. Eur J Pharma-
col 2002;454:153–163.

200 Pharmacology 2008;82:193–200 Bujalska

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.