CB1 Receptor
Antagonist Prolongs the Survival in Severe AP
Tohoku J. Exp. Med., 2005,
207, 99-107
99
The Cannabinoid 1 Receptor Antagonist, AM251,
Prolongs the Survival of Rats with Severe Acute
Pancreatitis
KAZUHISA MATSUDA, YUKIO MIKAMI, KAZUNORI TAKEDA, SHOJI FUKUYAMA,
SHINICHI EGAWA, MAKOTO SUNAMURA, IKUROU MARUYAMA1 and SEIKI MATSUNO
Division of Gastroenterological Surgery, Tohoku University Graduate School of
Medicine, Sendai, Japan, and
1
Department of Laboratory and Molecular Medicine, Faculty of Medicine,
Kagoshima University, Kagoshima, Japan
MATSUDA, K., MIKAMI, Y., TAKEDA, K., FUKUYAMA, S., EGAWA, S., SUNAMURA, M.,
MARUYAMA, I. and MATSUNO, S. The Cannabinoid 1 Receptor Antagonist, AM251, Prolongs the Survival of Rats with Severe Acute Pancreatitis. Tohoku J. Exp. Med., 2005,
207 (2), 99-107 ── It has recently been recognized that anandamide (arachidonylethanolamide), which is an endogeneous-cannabinoid (endocannabinoid), mediates septic shock.
Cannabinoid means a mind-active material in cannabis (marijuana). Anandamide is mainly produced by macrophages. Cannabinoid 1 (CB1) receptor, which is one of the cannabiniod receptors, is also known to mediate hypotensive shock. The role of endocannabinoids
in the progression of acute pancreatitis is unclear. The aims of this study are to clarify
their relationship and to find a new therapeutic strategy by regulating the endocannabinoid
signaling in acute pancreatitis. Male Wistar rats were injected with caerulein intravenously
to induce mild edematous pancreatitis or injected with 5% sodium taurocholate to the
bilio-pancreatic duct to induce severe necrotizing pancreatitis. The animals in the latter
group were also injected with a CB1 receptor antagonist, AM251, or vehicle solution to
see if the inhibition of endocannabinoids improves their survival. Plasma anandamide level was measured by the liquid chromatography/tandem mass spectrometry method. In both
models of acute pancreatitis, the plasma anandamide levels were increased, and the levels
were significantly higher in rats with severe necrotizing pancreatitis than those in rats with
mild edematous pancreatitis. The mean arterial pressure and survival rate were significantly improved by the treatment with AM251, despite that the local inflammatory changes in
the pancreas and various parameters (white blood cells, hematocrit, serum amylase, and
serum interleukin-6) were similar. This is the first report to show that endocannabinoids
are involved in the deterioration of acute pancreatitis and that the down-regulation of
endocannabinoid signaling may be a new therapeutic strategy for severe acute pancreatitis.
──── severe acute pancreatitis; septic shock; anandamide; endocannabinoids; AM251
© 2005 Tohoku University Medical Press
Received September 6, 2004; revision accepted for publication July 9, 2005.
Correspondence: Kazuhisa Matsuda, M.D., Ph.D., Division of Gastroenterological Surgery, Tohoku University
Graduate School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980-8574, Japan.
e-mail: [email protected]
99
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K. Matsuda et al.
Severe acute pancreatitis is one of the intractable pancreatic diseases, in which the mortality
rate extends to 20%, yet there are still many unclear points about the deterioration mechanism.
However, it recently became clear that several
mediators such as cytokines, reactive oxygen species, and platelet-activating factor (PAF), participate in the deterioration of acute pancreatitis
(Nonaka et al. 1992; Yotsumoto et al. 1994;
Rongione et al. 1997) and new therapeutic strategies for these have been developed. Despite the
evidence, it is difficult to conclude that adequate
results are provided (Kingsnorth et al. 1995;
Dumot et al. 2001; Schulz et al. 2001). Therefore,
discovery of a new factor participating in the deterioration of acute pancreatitis and development
of a new therapy are desired.
In 1964, tetrahydrocannabinol (THC) was
isolated and identified as a mind-active material
in cannabis (marijuana) (Mechoulam and Gaoni
1965), which has many similar structures, termed
cannabinoids. The cDNA of a cannabionid 1
(CB1) receptor for cannabinoids was cloned from
the brain (Matsuda et al. 1990). Subsequently arachidonic acid derivatives, anandamide (arachidonylethanolamide, AEA) (Devane et al. 1992)
and 2-arachidonoylglycerol (2-AG) (Mechoulam
et al. 1995; Sugiura et al. 1995), were discovered
as candidates of endogenous ligands of the receptor. Anandamide is mainly produced by macrophages, and 2-AG is by platelets.
The CB1 receptor, which is one of the cannabinoid receptors, is expressed in the central
nervous system, endothelial cells and the smooth
muscle cells of blood vessels. The stimulation of
CB1 receptor brings about hypotension, tachycardia, and disturbance of consciousness (Lake et al.
1997). It has also been established that endocannabinoids are one of the mediators in septic shock
(Varga et al. 1998). In addition, it was reported
that the endotoxin-absorption therapy (PMX-DHP,
direct hemoperfusion with polymixin-B immobilized fiber) absorbs endocannabinoids as well as
endotoxins and that it is effective even in some
cases of septic shock when endotoxins are not
high level. This suggests that absorption/removal
of endocannabinoids may improve shock (Wang
et al. 2000, 2001).
Severe acute pancreatitis frequently causes
circulatory insufficiency and disturbance of consciousness. Although endocannabinoids may participate in the deterioration of acute pancreatitis,
the relationship between acute pancreatitis and
endocannabinoids is not clarified. The primary
aim of this study is to clarify the role of endocannabinoids in the deterioration of acute pancreatitis
through animal experiments.
MATERIALS AND METHODS
Animals
Male Wistar rats weighing 200-250 g were maintained at 23°C in a 12 h: 12 h light-dark cycle and allowed free access to water and standard laboratory chow.
Twelve hours prior to the start of the experiments, the
animals were deprived of food but allowed access to water. This study was conducted with the consent of the
Ethics Committee for the Use of Experimental Animals
of Tohoku University School of Medicine.
Plasma anandamide level and immunohistochemical examination of the CB1 receptor in acute pancreatitis
Study design. Rats were randomly divided into
three groups: (a) Normal rat control (Normal) (n = 4); (b)
Caerulein-induced pancreatitis (CE-AP) (n = 16); and (c)
5% sodium taurocholate induced pancreatitis (TCA-AP)
(n = 16).
Caerulein pancreatitis rats were used as the mild
edematous acute pancreatitis model and 5% sodium taurocholate pancreatitis rats were used as the severe necrotizing pancreatitis model.
Under diethylether (Wako Pure Chemical, Osaka)
anesthesia, CE-AP was induced by a bolus injection of
caerulein (Sigma, St. Louis, MO, USA, 100 μ g/kg body
weight) into the right jugular vein. TCA-AP was induced
by the method of Aho et al. (1980). In brief, under diethylether anesthesia and laparotomy, 5% sodium taurocholate (TCA; Sigma, 1 ml/kg body weight) was injected
into the biliopancreatic duct at a rate of 0.2 ml/min with
a microinfusion pump (MINIPULS 3; Gilson Medical
Electronics, Villiers le Bel, France).
Under diethylether anesthesia, blood was collected
after 1, 6, 12, and 24 hours, and the plasma anandamide
was measured in each group. The plasma was separated
by 3,000 rpm, 10 min centrifusion, and stored at −80°C.
In all rats, the pancreas and kidney were resected at 12
CB1 Receptor Antagonist Prolongs the Survival in Severe AP
hours after the induction of pancreatitis.
Histological examination. The pancreas and the
kidney were fixed by immersion in 5% paraformaldehyde, dehydrated and then embedded in paraffin wax.
Sections of 3 μ m thickness were cut, dewaxed, and
stained with haematoxylin and eosin for histological examination. A pathologist blind to the experimental design performed histological assessment.
Plasma anandamide level. The plasma anandamide
level was measured by liquid chromatography/tandem
mass spectrometry (LC/MS/MS). Internal standard material was added in the measurement samples, and the
samples were stuck to a cartridge (OASIS HLB, Waters,
Milford, AR, USA). After washing with distilled water
and hexan, the samples were extracted with ethyl acetate.
Then, the samples were concentrated and dried at 40°C
under nitrogen current, and then dissolved in 80% methanol again. Finally, the fraction containing anandamide
was collected using ultrafiltration (ULTRAFREE-MC,
Millipore, Billerica, MA, USA, 10,000 rpm, 3 min, 4°C),
and measured on API3000 (Applyed Biosystems/MDS,
Ontario, Canada).
Immunohistochemical localization of CB1 receptors. The organs were embedded in OCT compound
(Tissue-Tek) (Sakura Finetek, Torrance, CA, USA) at
−80°C. Sections of 5 μ m thickness were cut using cryostat (CM3000, LEICA, Wetzlar, Germany) and were
fixed in acetone.
1) Enzyme antibody method: The sections were pretreated in phosphate buffered saline (PBS), 0.1% sodium
azide and 0.3% H2O2/PBS and blocked in 10% bovine
serum albumin (BSA) (Wako Pure Chemical). Anti-CB1
receptor antibody (rabbit, Cheyman chemical, Ann
Arbor, USA) or CD31 antibody (mouse, BD Biosciences,
Franklin Lakes, USA) were added as a primary antibody,
and peroxidase-conjugated second antibody (Jackson
ImmunoResearch, West Grove, USA) was used.
Coloring reaction was made with DAB (3, 3-diaminobenzidine, Wako Pure Chemical) with haematoxylin nucleus counter staining.
2) Fluorescence antibody method (double staining):
Some of the sections were blocked in 10% BSA and prepared with anti-CB1 receptor rabbit antibody and antiCD31 mouse antibody mixture. After washing in PBS,
FITC-conjugated anti-rabbit antibody and Texas
Red-conjugated anti-mouse antibody (Jackson Immuno
Research) mixture were added as the second antibodies.
CB1 receptor positive cells (Green) and CD31 positive
101
cells (Red) were observed.
Treatment with a CB1 receptor antagonist
Study design. In the severe pancreatitis model
(TCA-AP), the effects of CB1 receptor antagonist were
analyzed. Just after inducing severe pancreatitis by TCA
injection and 6 hours later, a CB1 receptor antagonist,
AM251 (Tocris Cookson Inc., Ballwin, USA, 3 mg/kg
per once) (Gatlay et al. 1996), dissolved in the mixture of
ethanol, vegetable oil, and saline (1 : 1 : 18), was injected
into the right jugular vein (AM251 [+], n = 6). As a control, animals group were induced severe pancreatitis but
were injected only vehicle (AM251 [−], n = 6).
Histological examination of pancreas. The pancreas was resected in all rats at 12 hours after the induction
of TCA-AP for histological examination.
Measurement of white blood cells, hematocrit, serum amylase, serum interleukin (IL)-6, and mean arterial
pressure. Blood samples were drawn at 12 hours after
the induction of TCA-AP. The number of white blood
cells, hematocrit, serum amylase, and serum IL-6 were
measured. The concentration of serum IL-6 was measured with Rat IL-6 ELISA kits (TFB, Tokyo). Each assay was performed in duplicate with 100 μ l of supernatant using 96-well plates. The detection limit of these
assays was 10 pg/ml. For measurement of mean arterial
pressure (MAP), 24G catheter was inserted into the right
femoral artery after anesthesia by Nembutal (Dainippon
Pharmaceutical, Osaka, Japan; 1 ml/kg body weight,
subcutaneous injection), and was connected to the
DMS-760 (Nishiyama Production, Osaka).
Survival rate. We analyzed the AM251 (+) group (n
= 10) and the AM251 (−) group (n = 10), treated as described above. After the second injection of AM251, rats
were allowed free access to water and food, and the survival time was observed for 72 hours after the induction
of pancreatitis.
Statistical analysis
Results are expressed as means ± S.E.M. One-way
Analysis of variance (ANOVA) and Fisher protected
least significant difference post hoc tests were used to
determine the significance for plasma anandamide and
serum IL-6 levels. Differences for other studies were
evaluated with a two-tailed unpaired Student’s t-test.
Survival rate was evaluated by the log rank method. A p
value of less than 0.05 was accepted as significant.
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K. Matsuda et al.
RESULTS
Plasma anandamide level and immunohistochemical examination of the CB1 receptor in
acute pancreatitis
At 12 hours after induction of pancreatitis, the
remarkable edematous swelling and slight inflam-
Fig. 1. Histological examination of pancreas; H-E
(Hematoxylin and eosin) staining.
The remarkable edematous swelling and slight
inflammatory cell permeation in the CE-AP rat,
and broad necrosis of acinar cells in the TCAAP rat were recognized.
matory cell permeation were recognized in the
CE-AP rats, while there was broad necrosis of acinar cells in the TCA-AP rats (Fig. 1).
In CE-AP rats, there was a slight increase of
plasma anandamide but the difference was not
statistically significant from that of normal rats.
On the other hand, significant increase was observed in TCA-AP rats after the induction of pancreatitis (Fig. 2).
The enzyme antibody method was used in
the renal artery of the normal rat and the TCA-AP
rat. CB1 receptors were expressed in the endothelium and the smooth muscle of the blood vessel and CD31 was strongly stained only in the endothelium of the blood vessel. There was no
recognizable difference between the CB1 staining
in normal rats and in the TCA-AP rats (Fig. 3).
The fluorescence antibody method revealed that
the CB1 receptor was localized in the smooth
muscle of the renal artery and the pulmonary artery of the TCA-AP rats. Co-localization of CB1
Fig. 2. Plasma anandamide level; Mean ± S.E.M.
(n = 4), * p < 0.05 vs CE-AP group.
There was no significant difference between
normal rats and CE-AP rats, but there was significant increase in TCA-AP rats at 6, 12, and
24 hours after the induction of pancreatitis.
CB1 Receptor Antagonist Prolongs the Survival in Severe AP
103
B
A
D
C
Fig. 3. Immunohistochemical localization of CB1 receptors (Enzyme antibody method). [Normal] A:
CB1 receptor, B: CD31; [TCA-AP] C: CB1 receptor, D: CD31.
In the kidney of the normal and the TCA-AP rats, CB1 receptor was stained in the endothelium and
the smooth muscle of the blood vessel and CD31 was strongly stained in the endothelium of the
blood vessel. There seems to be no remarkable differences between the staining of normal rat and
that of the TCA-AP rat.
A
B
Fig. 4. Immunohistochemical localization of CB1 receptors in TCA-AP rats (Fluorescence double staining); Green: CB1 receptor, Red: CD31; A: kidney, B: lung.
CB1 receptor was strongly expressed in the smooth muscle of the arteries in the kidney and the lung
of the TCA-AP rats. Similarly, CB1 receptors were also partially recognized in the endothelium of
the blood vessel. Co-localization of CB1 receptor and CD31 were stained in yellow.
104
K. Matsuda et al.
receptors and CD31 were stained in yellow, suggesting that CB1 receptors are also partially recognized in the endothelium of the blood vessel
(Fig. 4).
Effects of a CB1 receptor antagonist
In order to clarify the role of endocannabinoids in the deterioration of acute pancreatitis, a
CB1 receptor antagonist, AM251, was administered in TCA-AP rats. Massive areas of necrosis
were observed in both the AM251 (+) group and
the AM251 (−) group, accompanied by infiltration
with inflammatory cells and hemorrhages compatible with severe acute pancreatitis. The histological findings of local inflammation in the pancreas
were not different regardless of the injection of
AM251 (Fig. 5).
Serum amylase, white blood cells, hematocrit and serum IL-6 were measured. All parameters showed remarkable increase, but neither recognized significant difference between AM251 (+)
group and AM251 (−) group (Table 1).
Although the blood examination and local
inflammatory changes in the pancreas were not
different regardless of the injection of AM251, the
MAP and the survival rate was significantly improved by the injection of AM251. At six hours
after the induction of pancreatitis, the MAP of the
AM251 (+) group was significantly higher than
that of the AM251 (−) group. After the second injection, the MAP elevated immediately in both
groups, but this was thought to be a volume effect. Briefly, in the AM251 (+) group, the MAP
was improved significantly compared with the
Fig. 5. Histological examination of pancreas; H-E
(Hematoxylin and eosin) staining.
Massive areas of necrosis were observed in both
the AM251 (+) group and the AM251 (−)
group, accompanied by infiltration with inflammatory cells and hemorrhages compatible with
severe acute pancreatitis. The inflammatory
changes in the pancreas were not different regardless of the injection of AM251.
TABLE 1. Comparison of parameters in AM251 (+) group and AM251 (−) group; Mean ±
S.E.M. (n = 6)
Normal
Serum amylase (IU/l)
WBC (/μ l)
Ht. (%)
IL-6 (pg/ml)
5,710 ± 560
9,900 ± 3,010
39.9 ± 3.49
81.0 ± 22.0
TCA-AP
AM251 (−)
AM251 (+)
9,945 ± 419
11,833 ± 2,246
57.8 ± 7.2
177.1 ± 174.2
9,942 ± 448
8,375 ± 1,644
57.6 ± 8.3
197.9 ± 160.2
Serum amylase, white blood cells, hematocrit, and serum IL-6 were measured. Both
parameters showed high value, but neither recognized significant difference.
CB1 Receptor Antagonist Prolongs the Survival in Severe AP
Fig. 6. The change of mean arterial pressure (MAP)
(n = 4). * p < 0.05 vs AM251 (−) group.
At six hours after the induction of TCA-AP, the
MAP of the AM251 (+) group was higher than
that of the AM251 (−) group. In the AM251 (+)
group, the MAP was improved significantly
compared with the AM251 (−) group.
AM251 (−) group (Fig. 6).
The survival was observed for 72 hours after
the induction of pancreatitis. The survival of rats
in AM251 (+) group was significantly higher than
that of AM251 (−) group (AM251 [+]: 60% [6/10],
AM251 [−]: 10% [1/10]; p < 0.05) (Fig. 7).
DISCUSSION
Cannabinoid is the principal ingredient of
cannabis (marijuana). The CB1 receptor is mainly expressed in the central nervous system, the
endothelial cells and the smooth muscle cells of
the blood vessel. Cannabinoid also shows mind
nervous system action and vasodilatation (Lake et
al. 1997). Also, the CB2 receptor is located in
blood cells and immunity system cells, which are
used in the immune system control (Facci et al.
1995). Cannabinoids stimulate/excite the nervous
system, promote coagulation, and activate immune system whereas they create hypotension
and have inhibitory effects on the nervous system,
coagulation system, and immune system (Wang et
al. 2000). In addition, anandamide, one of the endocannabinoids, is produced by macrophages and
105
Fig. 7. Survival rate.
The survival rate was observed for 72 hours after the induction of pancreatitis (n = 10). * p <
0.05 vs AM251 (−) group.
The survival rate was significantly improved
by the injection of AM251 (AM251 [+]: 60%
[6/10], AM251 [−]: 10% [1/10]; p < 0.05).
is a mediator of septic shock (Varga et al. 1998).
Even though severe acute pancreatitis can
produce circulatory insufficiency and disturbance
of consciousness, a role of endocannabinoids in
the pathogenesis of acute pancreatitis is not clarified. We measured the plasma anandamide level
by LC/MS/MS method in normal rats, the CE-AP
rats (mild pancreatitis model), and the TCA-AP
rats (severe pancreatitis model).
Anandamide is an endogenous neurotransmitter which binds to the cannabinoid receptors
and is proven to have a pharmacological action
similar to THC, which is an active principle of
cannabis (Felder et al. 1993). In this study, the
plasma anandamide level was significantly elevated after the induction of severe acute pancreatitis.
Severe pancreatitis model showed significantly
high level when compared with the mild pancreatitis model. In the cases of sepsis, it has been
proven that macrophages produce anandamide
and participate in shock by LPS stimulation (Varga
et al. 1998). In severe acute pancreatitis it bacteremia occurs by bacterial translocation (Runkel et
al. 1991). However it is rare that complication of
bacteremia is observed by 6 hours after the induc-
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K. Matsuda et al.
tion of acute pancreatitis, as reported in our previous study (Iwasaki et al. 1994). Since the plasma
anandamide level was elevated six hours after the
induction of acute pancreatitis in this study, anandamide production mechanism other than the production by endotoxin stimulation from macrophages should be further investigated.
The CB1 receptor antagonist AM251 is an
analog of SR141716A (Gatlay et al. 1996).
Injection of AM251 showed remarkable improvement in MAP and survival rate, but there was no
clear change in the blood exam and histological
findings of the pancreas. Endocannabinoids are
known as materials inducing hypotension. It is
thought that there are three pathways for this
mechanism. First, nitrogen oxide (NO) is released by activating the CB1 receptor in the blood
vessel. Second, endocannabinoids are taken into
the cells through transporter to be used for PGI2
production. Third, calcitonin-gene related peptide
(CGRP), which is known to cause hypotension, is
produced by activating capsaicin receptor on
nerve cells (Hogestatt and Zygmunt 2002). In a
rat ischemic model, it was reported that injection
of SR141716A, which is one of the CB1 receptor
antagonists, brought about improvement of blood
pressure and extension of survival time (Wagner
et al. 1997). The mechanism of improvement in
survival rate in this study is considered to be the
inhibition of hypotension by blocking the CB1 receptor.
In summary, the rat experimental model of
severe acute pancreatitis showed a significantly
elevated level of plasma anandamide compared to
the mild pancreatitis. The blood examination and
local inflammatory changes in the pancreas were
not different regardless of the injection of the
CB1 receptor antagonist, AM251, after the induction of severe acute pancreatitis, but improvement
of mean arterial pressure and survival rate were
observed.
CONCLUSION
This is the first report to provide evidence
that endocannabinoids are involved in the deterioration of acute pancreatitis. Using plasma anandamide level as an index of severity of acute pan-
creatitis a new therapeutic strategy of severe acute
pancreatitis by down-regulation of endocannabinoid signaling is expected in the future.
References
Aho, H.J., Koskensalo, S.M. & Nevalainen, T.J. (1980) Experimental pancreatitis in the rat: Sodium-taurocholate induced
acute hemorrhagic pancreatitis. Scand. J. Gastroenterol.,
15, 411-416.
Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson,
L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A.
& Mechoulam, R. (1992) Isolation and structure of a brain
constituent that binds to the cannabinoid receptor. Science,
258, 1946-1949.
Dumot, J.A., Conwell, D.L., Zuccaro, G., Jr., Vargo, J.J., Shay,
S.S., Easley, K.A. & Ponsky, J.L. (2001) A randomized,
double blind study of interleukin 10 for the prevention of
ERCP-induced pancreatitis. Am. J. Gastroenterol., 96,
2098-2102.
Facci, L., Dal Toso, R., Romanello, S., Buriani, A., Skaper, S.D.
& Leon, A. (1995) Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide
and palmitoylethanolamide. Proc. Natl. Acad. Sci. USA,
92, 3376-3380.
Felder, C.C., Briley, E.M., Axelrod, J., Simpson, J.T., Mackie, K.
& Devane, W.A. (1993) Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal
transduction. Proc. Natl. Acad. Sci. USA, 90, 7656-7660.
Gatlay, S.J., Gifford, A.N., Volkow, N.D., Lan, R. &
Makriyannis, A. (1996) 123I-labeled AM251: a radioiodinated ligand which binds in vivo to mouse brain cannabinoid
CB1 receptors. Eur. J. Pharmacol., 307, 331-338.
Hogestatt, E.D. & Zygmunt, P.M. (2002) Cardiovascular pharmacology of anandamide. Prostaglandins Leukot. Essent.
Fatty Acids, 66, 343-351.
Iwasaki, G., Takeda, K., Sunamura, M., Kobari, M. & Matsuno,
S. (1994) The role of intestinal flora in the pathogenesis of
infection and aggravation of experimental acute pancreatitis in rats. Nippon Geka Gakkai Zasshi, 95, 669-677.
Kingsnorth, A.N., Galloway, S.W. & Formela, L.J. (1995) Randomized, double-blind phase II trial of Lexipafant, a platelet-activating factor antagonist, in human acute pancreatitis.
Br. J. Surg., 82, 1414-1420.
Lake, K.D., Compton, D.R., Varga, K., Martin, B.R. & Kunos, G.
(1997) Cannabinoid-induced hypotension and bradycardia
in rats mediated by CB1-like cannabinoid receptors. J.
Pharmacol. Exp. Ther., 281, 1030-1037.
Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C. &
Bonner, T.I. (1990) Structure of a cannabinoid receptor
and functional expression of the cloned cDNA. Nature,
346, 561-564.
Mechoulam, R. & Gaoni, Y. (1965) A total synthesis of dl-delta-1-tetrahydrocannabinol, the active constituent of hashish.
J. Am. Chem. Soc., 87, 3273-3275.
Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M.,
Kaminski, N.E., Schatz, A.R., Gopher, A., Almog, S.,
Martin, B.R. & Compton, D.R. (1995) Identification of an
endogenous 2-monoglyceride, present in canine gut, that
binds to cannabinoid receptors. Biochem. Pharmacol., 50,
83-90.
Nonaka, A., Manabe, T., Kyogoku, T., Tamura, K. & Tobe, T.
CB1 Receptor Antagonist Prolongs the Survival in Severe AP
(1992) Evidence for a role of free radicals by synthesized
scavenger, 2-octadecylascorbic acid, in cerulein-induced
mouse acute pancreatitis. Dig. Dis. Sci., 37, 274-279.
Rongione, A.J., Kusske, A.M., Kwan, K., Ashley, S.W., Reber,
H.A. & McFadden, D.W. (1997) Interleukin 10 reduces the
severity of acute pancreatitis in rats. Gastroenterology,
112, 960-967.
Runkel, N.S., Moody, F.G., Smith, G.S., Rodriguez, L.F.,
LaRocco, M.T. & Miller, T.A. (1991) The role of the gut in
the development of sepsis in acute pancreatitis. J. Surg.
Res., 51, 18-23.
Schulz, H.U., Hoenl, H., Schrader, T., Kropf, S., Halangk, W.,
Ochmann, C., Matthias, R., Letko, G., Roessner, A.,
Lippert, H. & Niederau, C. (2001) Randomized, placebocontrolled trial of lazaroid effects on severe acute pancreatitis in rats. Crit. Care. Med., 29, 861-869.
Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A.,
Itoh, K., Yamashita, A. & Waku, K. (1995) 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor
ligand in brain. Biochem. Biophys. Res. Commun., 215,
89-97.
Varga, K., Wagner, J.A., Bridgen, D.T. & Kunos, G. (1998)
Platelet- and macrophage-derived endogenous cannabi-
107
noids are involved in endotoxin-induced hypotension.
FASEB J., 12, 1035-1044.
Wagner, J.A., Varga, K., Ellis, E.F., Rzigalinski, B.A., Martin,
B.R. & Kunos, G. (1997) Activation of peripheral CB1
cannabinoid receptors in haemorrhagic shock. Nature, 390,
518-521.
Wang, Y., Liu, Y., Sarker, K.P., Nakashima, M., Serizawa, T.,
Kishida, A., Akashi, M., Nakata, M., Kitajima, I. &
Maruyama, I. (2000) Polymyxin B binds to anandamide
and inhibits its cytotoxic effect. FEBS Lett., 470, 151-155.
Wang, Y., Liu, Y., Ito, Y., Hashiguchi, T., Kitajima, I.,
Yamakuchi, M., Shimizu, H., Matsuo, S., Imaizumi, H. &
Maruyama, I. (2001) Simultaneous measurement of anandamide and 2-arachidonoylglycerol by polymyxin B-selective adsorption and subsequent high-performance liquid
chromatography analysis: increase in endogenous cannabinoids in the sera of patients with endotoxic shock. Anal.
Biochem., 294, 73-82.
Yotsumoto, F., Manabe, T., Kyogoku, T., Hirano, T., Ohshio, G.,
Yamamoto, M., Imamura, T. & Yoshitomi, S. (1994) Platelet-activating factor involvement in the aggravation of
acute pancreatitis in rabbits. Digestion, 55, 260-267.