Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor

Article abstract of DOI:10.1124/mol.63.3.699

The cannabinoid analog abnormal cannabidiol [abn-cbd; (-)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)-olivetol] does not bind to CB₁ or CB₂ receptors, yet it acts as a full agonist in relaxing rat isolated mesenteric artery segments. Vasorelaxation by abn-cbd is endothelium-dependent, pertussis toxin-sensitive, and is inhibited by the BK_ca channel inhibitor charybdotoxin, but not by the nitric-oxide synthase inhibitor N^ω-nitro-L-arginine methyl ester or by the vanilloid VR1 receptor antagonist capsazepine. The cannabidiol analog O-1918 does not bind to CB₁ or CB₂ receptors and does not cause vasorelaxation at concentrations up to 30 μM, but it does cause concentration-dependent (1-30 μM) inhibition of the vasorelaxant effects of abn-cbd and anandamide. In anesthetized mice, O-1918 dose-dependently inhibits the hypotensive effect of abn-cbd but not the hypotensive effect of the CB₁ receptor agonist (-)-11-Δ⁹-tetrahydro-cannabinol dimethylheptyl. In human umbilical vein endothelial cells, abn-cbd induces phosphorylation of p42/44 mitogen-activated protein kinase and protein kinase B/Akt, which is inhibited by O-1918, by pertussis toxin or by phosphatidylinositol 3 (PI3) kinase inhibitors. These findings indicate that abn-cbd is a selective agonist and that O-1918 is a selective, silent antagonist of an endothelial anandamide receptor , which is distinct from CB₁ or CB₂ receptors and is coupled through G_i/G_o to the PI3 kinase/Akt signaling pathway.

Full text of DOI:10.1124/mol.63.3.699

0026-895X/03/6303-699–705
MOLECULAR PHARMACOLOGY
U.S. Government work not protected by U.S. copyright
Mol Pharmacol 63:699–705, 2003
Vol. 63, No. 3
2209/1047483
Printed in U.S.A.
Selective Ligands and Cellular Effectors of a G Protein-
Coupled Endothelial Cannabinoid Receptor
´
´
´
´
´
LASZLO OFFERTALER, FONG-MING MO, SANDOR BATKAI, JIE LIU, MALCOLM BEGG, RAJ K. RAZDAN,
BILLY R. MARTIN, RICHARD D. BUKOSKI, and GEORGE KUNOS
Laboratory of Physiologic Studies, National Institute on Alcoholism and Alcohol Abuse, National Institutes of Health, Bethesda, Maryland (L.O.,
S.B., J.L., M.B., G.K.); Organix, Inc., Woburn, Massachusetts (R.K.R.); Department of Pharmacology and Toxicology, Virginia Commonwealth
University, Richmond, Virginia (B.R.M.); and Cardiovascular Disease Research Program, North Carolina Central University,
Durham, North Carolina (R.D.B.)
Received October 10, 2002; accepted December 3, 2002
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
The cannabinoid analog abnormal cannabidiol [abn-cbd; (Ϫ)- damide. In anesthetized mice, O-1918 dose-dependently inhib-
4-(3–3,4-trans-p-menthadien-[1,8]-yl)-olivetol] does not bind to its the hypotensive effect of abn-cbd but not the hypotensive
CB1 or CB2 receptors, yet it acts as a full agonist in relaxing rat effect of the CB1 receptor agonist (Ϫ)-11-OH-⌬9-tetrahydro-
isolated mesenteric artery segments. Vasorelaxation by abn- cannabinol dimethylheptyl. In human umbilical vein endothelial
cbd is endothelium-dependent, pertussis toxin-sensitive, and is cells, abn-cbd induces phosphorylation of p42/44 mitogen-
inhibited by the BKCa channel inhibitor charybdotoxin, but not activated protein kinase and protein kinase B/Akt, which is
␻
by the nitric-oxide synthase inhibitor N -nitro-L-arginine methyl inhibited by O-1918, by pertussis toxin or by phosphatidylino-
ester or by the vanilloid VR1 receptor antagonist capsazepine. sitol 3 (PI3) kinase inhibitors. These findings indicate that abn-
The cannabidiol analog O-1918 does not bind to CB1 or CB2 cbd is a selective agonist and that O-1918 is a selective, silent
receptors and does not cause vasorelaxation at concentrations antagonist of an endothelial “anandamide receptor”, which is
up to 30 ␮M, but it does cause concentration-dependent (1–30 distinct from CB1 or CB2 receptors and is coupled through
␮M) inhibition of the vasorelaxant effects of abn-cbd and anan- Gi/Go to the PI3 kinase/Akt signaling pathway.
Endocannabinoids are recently identified lipid mediators receptors on postganglionic sympathetic nerves (Ishac et al.,
that act as natural ligands for cannabinoid receptors and
elicit biological effects similar to those of plant-derived can-
nabinoids (Mechoulam et al., 1998). In addition to their well-
known neurobehavioral effects, cannabinoids influence a
number of physiological functions, including cardiovascular
variables (Hillard, 2000; Kunos et al., 2000; Randall et al.,
2002). It has long been known that ⌬9-tetrahydrocannabinol
(THC), the main psychoactive ingredient of the marijuana
plant, can cause long-lasting hypotension in rodents (Vollmer
et al., 1974). The endocannabinoid anandamide also causes
hypotension in anesthetized rats and mice, which is suscep-
tible to inhibition by CB1 receptor antagonists (Varga et al.,
1995; Lake et al., 1997) and is absent in mice devoid of CB1
receptors (Jarai et al., 1999; Ledent et al., 1999), clearly
implicating CB1 receptors. Additional findings ruled out a
central mechanism for this effect (Varga et al., 1996). Can-
nabinoids inhibit norepinephrine release via presynaptic CB1
1996), which probably accounts for their bradycardic effect
(Kunos et al., 2000; Wagner et al., 2001). However, cannabi-
noids can decrease blood pressure to levels lower than that
achieved by elimination of sympathetic tone (Lake et al.,
1997), which points to a direct vasodilator mechanism. In-
deed, anandamide and its metabolically stable analog (R)-
methanandamide cause vasodilation in the coronary (Wag-
ner et al., 2001; Ford et al., 2002) and cerebral vasculatures
(Ellis et al., 1995; Gebremedhin et al., 1999; Wagner et al.,
2001), as tested in anesthetized animals or in isolated or-
gans.
Surprisingly, anandamide-induced mesenteric vasodila-
tion, although moderately sensitive to inhibition by
SR141716A, could be dissociated from CB1 receptors by its
presence in mice deficient in CB1 or in both CB1 and CB2
receptors (Jarai et al., 1999). Furthermore, THC or potent
synthetic CB1 receptor agonists do not cause mesenteric va-
sodilation (Wagner et al., 1999). Anandamide binds to va-
nilloid VR1 receptors with micromolar affinity, and this in-
L.O. and F.-M.M. contributed equally to this work.
ABBREVIATIONS: THC, ⌬9-tetrahydrocannabinol; abn-cbd, (Ϫ)-4-(3–3,4-trans-p-menthadien-[1,8]-yl)-olivetol (abnormal cannabidiol); HUVEC,
␻
human umbilical vein endothelial cells; L-NAME, N -nitro-L-arginine methyl ester; CGRP, calcitonin gene-related peptide; PI3, phosphatidylinositol
3; MAP, mitogen-activated protein; NO, nitric oxide; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
zole-3-carboxamide; BKCa, large-conductance calcium-activated potassium channel.
699

700
Offerta´ ler et al.
teraction was shown to result in the release from sensory 11-OH-⌬9-tetrahydrocannabinol dimethylheptyl (HU-210) were dis-
solved in ethanol. In the in vivo experiments, these drugs were
emulsified using ethanol/Alkamuls EL-620/phosphate-buffered sa-
line (1:1:8) to make a 30 mM stock solution stored at 4°C. All drugs
were purchased from Sigma Chemical (St. Louis, MO), except for
Alkamuls EL-620 (Rhodia Inc., Cranbury, NJ), pertussis toxin (List
nerve endings of the potent vasodilator peptide CGRP (Zyg-
munt et al., 1999). There is evidence that this mechanism
may account for the endothelium-independent, SR141716A-
insensitive component of the vasodilator effect of anandam-
ide in some (Zygmunt et al., 1999; Mukhopadhyay et al.,
2002) but not in other vascular beds (White et al., 2001;
Harris et al., 2002). The vasodilator response to anandamide
also has an endothelium-dependent component that is sensi-
tive to inhibition by SR141716A (White and Hiley, 1997;
␻
Biological Laboratories Inc., Campbell, CA), N -nitro-L-arginine
methyl ester (L-NAME; Alexis Corporation, San Diego, CA), HU-210
(Tocris Cookson Inc., Ballwin, MO), and charybdotoxin and apamin
(Bachem Bioscience, King of Prussia, PA). Antibodies against the
phosphorylated and unphosphorylated forms of p42/44 MAP kinase
Chaytor et al., 1999; Wagner et al., 1999; Mukhopadhyay et and Akt were from New England Biolabs (Beverly, MA).
Tissue Preparation. The mesenteric artery preparation used in
this study has been described recently (Bukoski et al., 2002). Briefly,
male Sprague-Dawley rats (200–300 g) obtained from Harlan (Indi-
anapolis, IN) were anesthetized with isoflurane, and the mesentery
was removed. Branch II or III segments were isolated under a
dissecting microscope, and 2-mm segments were mounted in a wire
myograph (Kent Scientific Co., Torrington, CT) using 0.001-inch
wires. The preparations were maintained in Krebs-Henseleit buffer
(150 mM NaCl, 5.4 mM KCl, 1.17 mM MgSO4, 1.18 mM NaH2PO4,
6.0 mM NaHCO3, 1.0 mM CaCl2, 20.0 mM HEPES, 5.5 mM glucose,
pH 7.4) equilibrated with 5% CO2 in O2. A resting tone of 1.0 g was
set, and vessels were precontracted by the presence of 5 ␮M phen-
ylephrine in the medium. Vasodilator responses were expressed as
the percentage of relaxation of the phenylephrine-induced tone. The
functional integrity of the endothelium was tested in all preparations
by Ͼ90% relaxation elicited by 10 ␮M acetylcholine. Endothelial
denudation was achieved by rubbing the inside of the vessel with a
mounting wire and was verified by the loss of the relaxing response
to 10 ␮M acetylcholine (Ͻ10% residual relaxation). Concentration-
response curves were generated by the cumulative addition of an
agonist. Antagonists were added 20 min before the agonist and
al., 2002), insensitive to VR1 receptor blockade (Grainger and
Boachie-Ansah, 2001; Mukhopadhyay et al., 2002), and may
(Chaytor et al., 1999) or may not be inhibited by gap junction
inhibitors (White et al., 2001; Harris et al., 2002; Mukho-
padhyay et al., 2002). The finding that (R)-methanandamide
elicits a similar effect in the rat mesenteric arterial bed rules
out the role of arachidonic acid metabolites, which have been
proposed to mediate the effect of anandamide in some other
vascular preparations (Pratt et al., 1998; Grainger and
Boachie-Ansah, 2001).
Abnormal cannabidiol [(Ϫ)-4-(3–3,4-trans-p-menthadien-
[1,8]-yl)-olivetol; abn-cbd], a structural analog of the behav-
iorally inactive marijuana constituent cannabidiol, has been
reported to lack behavioral effects but to cause profound
hypotension in dogs (Adams et al., 1977). More recently, we
reported that abn-cbd elicits endothelium-dependent,
SR141716A-sensitive mesenteric vasodilation that could be
inhibited by cannabidiol, and that this effect remains un-
changed in mice lacking both CB1 and CB2 receptors (Jarai et
al., 1999). This has led us to postulate the existence of a novel remained in the medium throughout the test period.
Blood Pressure Measurements. Mice were anesthetized with
pentobarbital sodium (50 mg/kg i.p.), and the femoral artery and vein
were cannulated for monitoring blood pressure and drug injections,
respectively. The arterial cannula was connected to a pressure trans-
ducer and computerized data acquisition system (IOX-Datanalyst,
EMKA Technologies, Arlington, VA).
Cell Culture. Human umbilical vein endothelial cells (HUVEC)
were purchased from American Type Culture Collection (Manassas,
VA) and were maintained in primary culture as described previously
(Liu et al., 2000, 2002). Briefly, cells were plated onto 60-mm2 wells
endothelial “anandamide” receptor for which abnormal can-
nabidiol is a selective agonist and cannabidiol is an antago-
nist (Jarai et al., 1999; Wagner et al., 1999). Evidence has
been presented that a similar anandamide receptor in the
rabbit aortic endothelium is coupled to Gi/Go and functions
via NO release (Mukhopadhyay et al., 2002). In an attempt to
further characterize this putative receptor, in the present
study we examined the mechanisms by which abn-cbd causes
vasorelaxation in small arteries isolated from the rat mesen-
tery, identified a chemically modified analog of cannabidiol in plastic culture dishes and maintained in EGM-2 BulletKit (Bio-
Whittaker, Walkersville, MD) containing 5% fetal calf serum at 37°C
under an atmosphere of 5% CO2 in air. Preconfluent cultures (two to
five passages) were incubated with vehicle or agonist in the presence
or absence of an antagonist, as described below.
as a selective, silent antagonist of this receptor, and identi-
fied some of its downstream effectors in cultured vascular
endothelial cells.
Western Blotting. The regular medium was replaced with se-
rum-free M199 medium, and cells were incubated for 16 h before the
addition of the agonist abn-cbd or vehicle for 30 min. O-1918 was
added to the medium 30 min before the agonist, whereas pretreat-
ment with pertussis toxin (400 ng/ml) was maintained for 30 min.
After drug treatment, the cells were lysed by the addition of 20 ␮l of
Materials and Methods
Reagents. Abnormal cannabidiol (Jarai et al., 1999) was synthe-
sized as described previously (Razdan et al., 1974). O-1918 (Fig. 1)
was synthesized by adding dry K2CO3 to a Wheaton-type pressure
tube, followed by a solution of (Ϫ)-2-(3–3,4-trans-p-menthadien-[1,8]-
yl)-orcinol in dry acetonitrile. Additional acetonitrile was used as
washings, which were transferred to the pressure tube. Subse-
quently, iodomethane was added, and the mixture was stirred and
heated to 70 to 80°C in an oil bath for 16 h. After cooling, the mixture
was filtered and washed with fresh acetonitrile. The filtrate was
concentrated on a rotary evaporator, and the residue was dissolved
in ether. The ether solution was washed with HCl and water, and
then it was dried and concentrated on a rotary evaporator. It was
purified by flash chromatography. The fractions containing the ma-
terial were monitored by thin layer chromatography, visualized by
phosphomolybdic acid, and concentrated. 1H NMR showed appropri-
ate peaks. For the in vitro experiments, abn-cbd, O-1918, and (Ϫ)-
Fig. 1. The chemical structure of O-1918.

Novel Endothelial Endocannabinoid Receptor
701
radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, BKCa channel inhibitor charybdotoxin caused a significant
150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxy-
cholate, 1 mM phenylmethylsulfonylfluoride, 1 ␮g/ml aprotinin, 1
mM activated Na3VO4, and 1 mM NaF). Aliquots of the cell lysate
containing 100 ␮g of protein were size-fractionated by 10% SDS-
polyacrylamide gel electrophoresis and transferred to a nitrocellu-
lose membrane. Phosphorylated and unphosphorylated forms of pro-
tein kinase B/Akt and p42/44 MAP kinase were visualized by
Western blotting using appropriate antibodies, as described previ-
ously (Liu et al., 2002).
right shift of the abn-cbd concentration-response curve,
which was slightly enhanced in the added presence of the
small-conductance calcium-activated potassium channel in-
hibitor apamin (Fig. 5B). Apamin alone did not significantly
affect the response to abn-cbd. The vanilloid VR1 receptor
antagonist capsazepine, which inhibits the endothelium-in-
dependent component of the vasodilator effect of anandamide
(Zygmunt et al., 1999; Mukhopadhyay et al., 2002), did not
affect abn-cbd–induced mesenteric vasorelaxation (Fig. 6). At
Ligand Binding. The ability of abn-cbd and O-1918 to bind to
CB1 or CB2 receptors was assessed in binding competition assays the concentration used (1 ␮M), capsazepine was an effective
using [3H]CP-55,940 as the labeled ligand and membranes from
antagonist of VR1 receptors, as indicated by its ability to
mouse cerebellum or from Chinese hamster ovary cells transfected
inhibit the vasorelaxant effect of capsaicin (Fig. 6, inset).
with the human CB2 receptor cDNA (Showalter et al., 1996), respec-
In anesthetized mice, the bolus intravenous injection of 10
tively. Details of the binding assays were described previously (Aung
mg/kg abn-cbd elicited a hypotensive response, which could
et al., 2000).
be dose-dependently inhibited by pretreatment of the animal
Statistical Analyses. pA2 values (Ϫlog EC50) were determined in
with 1 to 10 mg/kg O-1918. The higher dose of O-1918 did not
individual concentration-response curves by computerized curve-fit-
ting. Statistical comparisons of concentration-response curves were
made by a two-way analysis of variance of the whole data set,
followed by the Bonferroni/Dunn post hoc test for determining sig-
nificant differences (P Ͻ 0.05) between treatment groups.
Results
Abn-cbd elicits concentration-dependent relaxation of iso-
lated segments of phenylephrine-precontracted mesenteric
arteries with a pA2 of 5.67 Ϯ 0.07 and an Emax of 93 Ϯ 3%.
The vasorelaxation is gradual in onset and takes 2 to 3 min
to reach plateau at each concentration step. Endothelial de-
nudation results in a significant right shift of the abn-cbd
dose-response curve (pA2 ϭ 4.81 Ϯ 0.15; P Ͻ 0.01) (Fig. 2).
HU-210, a synthetic cannabinoid with very high affinity at
CB1 receptors (Lake et al., 1997), is ineffective in relaxing
mesenteric artery segments at concentrations up to 1 ␮M
(data not shown). Unexpectedly, cannabidiol, which antago-
nized abn-cbd–induced vasodilation without causing dilation
itself in the rat isolated, buffer-perfused mesenteric vascular
Fig. 2. The vasorelaxant effect of abn-cbd in intact (F) and endothelium-
denuded preparations (E). Endothelial denudation was achieved and
bed preparation (Jarai et al., 1999), caused concentration-
dependent near-maximal relaxation of isolated arterial seg-
ments with a pA2 value of 5.66 Ϯ 0.06 (data not shown).
Therefore, chemically modified analogs of cannabidiol were
tested as potential silent antagonists of abn-cbd induced
vasorelaxation. The analog O-1918 (Fig. 1) did not induce
relaxation at concentrations up to and including 30 ␮M, but
it caused a concentration-dependent right shift of the vasore-
laxant effect of abn-cbd (Fig. 3). O-1918 also inhibited vasore-
laxation by anandamide (pA2 ϭ 6.01 Ϯ 0.05 versus 4.59 Ϯ
0.49, P Ͻ 0.05, in the absence or presence of 10 ␮M O-1918,
respectively).
verified as described under Materials and Methods. Each pair of intact
and endothelium-denuded preparation was from the same animal. Points
and vertical bars represent the means Ϯ S.E. from six pairs of arteries.
Abn-cbd and O-1918 were tested for their ability to bind to
native CB1 receptors in mouse cerebellar membranes and
cloned mouse CB2 receptors transfected into Chinese ham-
ster ovary cells. Neither compound displaced the radiola-
beled agonist [3H]CP-55,940 from CB1 or CB2 receptors at
concentrations up to and including 30 ␮M.
Preincubation of isolated mesenteric arterial segments
with 400 ng/ml pertussis toxin for 90 min resulted in a
significant right shift of the abn-cbd concentration-response
curve in intact but not in endothelium-denuded preparations
(Fig. 4). The nitric-oxide synthase inhibitor L-NAME (100
␮M) did not affect abn-cbd–induced vasorelaxation (Fig. 5A),
Fig. 3. The vasorelaxant effect of abn-cbd is concentration-dependently
inhibited by O-1918 in endothelium-intact mesenteric arteries. The indi-
cated concentrations of abn-cbd were added cumulatively to the tissue
bath either alone (F, n ϭ 12), or in the presence of 1 ␮M (E, n ϭ 4), 10 ␮M
suggesting the lack of involvement of endothelial NO. The (ƒ, n ϭ 10), or 30 ␮M O-1918 (Ⅺ, n ϭ 5).

702
Offerta´ ler et al.
influence the hypotensive response to subsequently admin-
istered HU-210 (Fig. 7), which illustrates the selectivity of
the inhibitory effect of O-1918 and confirms its lack of inter-
action with CB1 receptors.
Incubation of cultured HUVEC with 10 ␮M abn-cbd in-
duced rapid phosphorylation of p42/44 MAP kinase and pro-
tein kinase B/Akt, which were blocked in the presence of 20
␮M O-1918 (Fig. 8). Preincubation of HUVEC with pertussis
toxin for 90 min (400 ng/ml) also significantly attenuated the
activation of MAP kinase and Akt phosphorylation by abn-
cbd (Fig. 8). Protein kinase B/Akt is a downstream effector of
PI3 kinase (Cantley, 2002); therefore, we tested whether
inhibitors of PI3 kinase can prevent Akt phosphorylation
induced by abn-cbd. Both 2-(4-morpholinyl)-8-phenyl-4H-1-
benzopyran-4-one (25 ␮M, Fig. 8) and wortmannin (100 nM;
data not shown) blocked Akt phosphorylation induced by
abn-cbd in HUVEC.
Fig. 4. Pertussis toxin inhibits abn-cbd–induced vasorelaxation in endo-
thelium-intact (F) but not in endothelium-denuded mesenteric arteries
(E). Pairs of endothelium-intact or -denuded arteries were preincubated
for 90 min with vehicle (solid line) or 400 ng/ml pertussis toxin (broken
line) before the cumulative addition of abn-cbd.
Discussion
The endogenous cannabinoid anandamide causes vasodila-
tion through both endothelium-dependent and -independent
mechanisms, as documented in the rat isolated buffer-per-
fused mesenteric bed (Jarai et al., 1999; Wagner et al., 1999)
and in rabbit isolated aortic rings (Mukhopadhyay et al.,
2002), by interacting with sites distinct from CB1 and CB2
receptors. The present findings indicate that the synthetic
cannabinoid ligands abn-cbd and O-1918 act as a selective
agonist and a selective silent antagonist, respectively, of a
vascular endothelial receptor that mediates mesenteric va-
sorelaxation and is coupled to a PI3 kinase/Akt-dependent
pathway through Gi/Go. The finding that O-1918 also inhibits
the mesenteric vasorelaxant effect of anandamide strongly
suggests that the same endothelial receptor is the site of
action of anandamide. That this site is distinct from CB1 and
CB2 receptors is further indicated by the finding that neither
abn-cbd nor O-1918 binds to CB1 or CB2 receptors. The
Fig. 5. A, the mesenteric vasorelaxant effect of abn-cbd is independent of
endothelial NO. Pairs of arteries were preincubated for 60 min with
vehicle or 100 ␮M L-NAME before the cumulative addition of abn-cbd. B,
charybdotoxin, but not apamin, inhibits abn-cbd–induced mesenteric va-
sorelaxation. Preparations were incubated for 30 min with vehicle (E),
100 nM apamin (Œ), 100 nM charybdotoxin (ꢀ), or apamin plus charyb-
dotoxin (ࡗ) before the cumulative addition of abn-cbd.
Fig. 6. VR1 receptors are not involved in the mesenteric vasorelaxant
effect of abn-cbd. Pairs of arteries were exposed to cumulative concentra-
tions of abn-cbd or capsaicin in the absence (F) or presence (E) of 1 ␮M
capsazepine.

Novel Endothelial Endocannabinoid Receptor
703
possibility that abn-cbd binds to CB1 or CB2 receptors but at
The ability of pertussis toxin to inhibit anandamide-in-
a site distinct from the binding sites of conventional antago- duced vasorelaxation was first documented in rat mesenteric
nists can be also ruled out by the earlier observation that arteries (White and Hiley, 1997). In the rabbit isolated aortic
abn-cbd causes mesenteric vasodilation in preparations from ring preparation, anandamide interacts with a non-CB1/CB2
CB1/CB2 double knockout mice (Jarai et al., 1999).
endothelial receptor coupled to Gi/Go and NO synthase, as
deduced from the inhibition of its vasorelaxant effect by
pertussis toxin and L-NAME, respectively (Mukhopadhyay et
al., 2002). In the present experiments, pertussis toxin inhib-
ited the abn-cbd–induced endothelium-dependent vasodila-
tion in rat mesenteric artery segments as well as the activa-
tion of p42/44 MAP kinase and Akt phosphorylation in
HUVEC, confirming the involvement of a putative Gi/Go-
coupled receptor. However, the NO synthase inhibitor L-
NAME did not affect abn-cbd–induced vasorelaxation in rat
mesenteric artery segments, which is similar to earlier find-
ings (White and Hiley, 1997; Jarai et al., 1999). In contrast,
this effect is inhibited by charybdotoxin, an inhibitor of BKCa
channels as well as delayed rectifier Kϩ channels (Garcia et
al., 1995). This is similar to previous findings that implicated
endothelial Kϩ channel activation in the vasorelaxant effect
of anandamide (Randall and Kendall, 1997; White and Hiley,
1997; Grainger and Boachie-Ansah, 2001; White et al., 2001),
which may be related to endothelium-derived hyperpolariz-
ing factor release (Edwards et al., 1998). It is well established
that the relative contribution of NO and endothelium-derived
hyperpolarizing factor to endothelially triggered vasodilation
varies with the size and location of blood vessels and with the
species involved (Hill et al., 2001), and the above discrepancy
may reflect such differences.
Fig. 7. O-1918 inhibits the hypotensive effect of abn-cbd, but not that of
HU-210, in anesthetized mice. Mice anesthetized with sodium pentobar-
bital were instrumented as described under Materials and Methods.
Columns and bars represent the means Ϯ S.E. for the peak hypotensive
response to 10 mg/kg abn-cbd or 100 ␮g/kg HU-210 in the absence or
presence of the indicated dose of O-1918, injected i.v. 10 min before
abn-cbd. Baseline mean blood pressure was 92 Ϯ 6 mm Hg.
In an earlier study using the rat isolated buffer-perfused
mesenteric bed preparation, the vasodilator action of abn-cbd
was fully endothelium-dependent, and cannabidiol acted as a
silent antagonist of this effect (Jarai et al., 1999). In the
present study, endothelial denudation revealed a residual
endothelium-independent component in the dilator action of
abn-cbd, and cannabidiol itself caused vasorelaxation, which
precluded its testing as an antagonist. It is possible that
cannabidiol acts as a silent antagonist in the segment of the
mesenteric vasculature that determines the resistance re-
sponse of the perfused vascular bed preparation, but it does
so as a partial agonist in the isolated conduit arteries used in
the myograph. Alternatively, intraluminal injection of drugs
in the perfused preparation may limit their access to the
endothelium, whereas in tissue-bath experiments, they
would have equal or even better access to additional sites on
smooth muscle and on periarterial nerve terminals. Unlike
cannabidiol, its structural analog O-1918 does not cause va-
sorelaxation but acts as a competitive antagonist of the en-
dothelium-dependent vasodilator effect of abn-cbd and anan-
damide without antagonizing the response to the muscarinic
agonist charbachol. The endothelial site of action of O-1918 is
further documented by its ability to antagonize the activation
of p42/44 MAP kinase activation and Akt phosphorylation by
abn-cbd in HUVEC. Thus, O-1918 is the first selective, silent
antagonist of the endothelial anandamide/abn-cbd receptor.
Anandamide binds to vanilloid VR1 receptors with micro-
molar affinity, and its mesenteric vasodilator effect has been
attributed to a VR1-mediated release of CGRP from sensory
nerve endings (Zygmunt et al., 1999). Indeed, there is evi-
dence that the endothelium-independent (but not the endo-
thelium-dependent) component of the vasorelaxant effect of
anandamide is inhibited by the VR1 antagonist capsazepine
Fig. 8. Abn-cbd induces phosphorylation of p42/44 MAP kinase and
protein kinase B/Akt through a PI3 kinase-dependent, Gi/Go-coupled
mechanism in HUVEC. Phosphorylation of Akt and p42/44 MAPK in-
duced by 10 ␮M abn-cbd is inhibited by 20 ␮M O-1918 (A) and by 400
ng/ml pertussis toxin (PTX) (B). Akt phosphorylation by 10 ␮M abn-cbd is
inhibited by 25 ␮M 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
(LY294002) (C). Western immunoblotting was done using antibodies
against pAkt and p42/44 MAPK, as described under Materials and Meth-
ods. Hybridization with an antibody against the unphosphorylated form
of Akt was used to verify equal loading.

704
Offerta´ ler et al.
References
(Mukhopadhyay et al., 2002). In the present experiments
using endothelium-intact mesenteric arteries, capsazepine
did not affect abn-cbd–induced vasodilation at a concentra-
tion that inhibited the effect of capsaicin. This rules out a role
for VR1 in the endothelium-dependent effect of abn-cbd and
also indicates that different mechanisms are involved in the
endothelium-independent effect of the abn-cbd and anand-
amide.
Adams MD, Earnhardt JT, Martin BR, Harris LS, Dewey WL, and Razdan RK (1977)
A cannabinoid with cardiovascular activity but no overt behavioral effects. Expe-
rientia 33:1204–1205.
Aung MM, Griffin G, Huffman JW, Wu MJ, Keel C, Yang B, Showalter V, Abood ME,
and Martin BR (2000) Influence of the N-1 alkyl chain length of aminoalkylindole
cannabinoids to CB1 and CB2 receptor binding. Drug Alcohol Depend 60:133–140.
Breivogel CS, Griffin G, Di Marzo V, and Martin BR (2001) Evidence for a new G
protein-coupled receptor in mouse brain. Mol Pharmacol 60:155–163.
Bukoski RD, Batkai S, Jarai Z, Wang Y, Offertaler L, Jackson WF, and Kunos G
(2002) CB1 receptor antagonist SR141716A inhibits Ca2ϩ-induced relaxation in
CB1 receptor-deficient mice. Hypertension 39:251–257.
Additional cannabinoid receptors distinct from CB1 and
CB2 may also be present in the central nervous system. In
preparations obtained from CB1 receptor-deficient mice,
anandamide and the synthetic cannabinoid agonist
WIN55,212–2 have been reported to stimulate guanosine
5Ј-O-(3-thio)triphosphate–labeling in brain plasma mem-
brane preparations (Breivogel et al., 2001) and to inhibit
glutamatergic synaptic transmission in hippocampal slices
(Hajos et al., 2001). However, these putative receptors are
distinct from the vascular endothelial receptor described in
the present and previous studies (Jarai et al., 1999; Wagner
et al., 1999; Mukhopadhyay et al., 2002). First, these neuro-
nal sites seem to be uniquely sensitive to activation by
WIN55,212–2 (Breivogel et al., 2001; Hajos et al., 2001),
which was found to be devoid of vasodilator activity in rat
mesenteric arteries (Wagner et al., 1999) and in rabbit aortic
rings (Mukhopadhyay et al., 2002). Second, our unpublished
observations indicate that abn-cbd does not inhibit glutama-
tergic excitatory postsynaptic potentials in rat hippocampal
slices. There is also recent evidence for a THC- or cannabinol-
induced, CB1/CB2 receptor-independent release of CGRP
from sensory nerve terminals (Zygmunt et al., 2002). The
inability of THC to induce mesenteric vasorelaxation (Wag-
ner et al., 1999) indicates that this site is also different from
the endothelial cannabinoid receptor. Thus, there may be
more than one, as-yet-undefined, cannabinoid receptor.
The present observations also indicate that the vasodilator
response to activation of abn-cbd–sensitive vascular recep-
tors is sufficient to elicit hypotension in intact animals, at
least in the anesthetized state, and that this effect is distinct
from CB1 receptor-mediated hypotension. The question
arises as to the possible physiological function of this novel
vasodilator mechanism. It has been recently proposed that
the mesenteric vasodilator response to increased extracellu-
lar Ca2ϩ, such as occurs in the postprandial absorptive
phase, is mediated by the release of a sensory nerve-derived
endocannabinoid that activates CB1 receptors, as deduced
from the dependence of this response on intact sensory
nerves and its susceptibility to inhibition by SR141716A
(Ishioka and Bukoski, 1999). Recent evidence that calcium-
induced vasorelaxation is maintained in CB1 receptor knock-
out mice and is inhibited by O-1918 (Bukoski et al., 2002)
suggests that calcium-induced vasodilation is mediated by
the receptors described in the present study rather than by
CB1 receptors.
Cantley LC (2002) The phosphoinositide 3-kinase pathway. Science (Wash DC)
296:1655–1657.
Chaytor AT, Martin PEM, Evans WH, Randall MD, and Griffith TM (1999) The
endothelial component of cannabinoid-induced relaxation in rabbit mesenteric
artery depends on gap junctional communication. J Physiol (Lond) 520:539–550.
Edwards G, Dora KA, Gardener MJ, Garland CJ, and Weston AH (1998) Kϩ is an
endothelium-derived hyperpolarizing factor in rat arteries. Nature (Lond) 396:
269–272.
Ellis EF, Moore SF, and Willoughby KA (1995) Anandamide and delta 9-THC
dilation of cerebral arterioles is blocked by indomethacin. Am J Physiol 269:
H1859–H1864.
Ford WR, Honan SA, White R, and Hiley CR (2002) Evidence of a novel site
mediating anandamide-induced negative inotropic and coronary vasodilator re-
sponses in rat isolated hearts. Br J Pharmacol 135:1191–1198.
Garcia ML, Knaus H-G, Munujos P, Slaughter RS, and Kaczorowski GJ (1995)
Charybdotoxin and its effects on potassium channels. Am J Physiol 269:C1–C10.
Gebremedhin D, Lange AR, Campbell WB, Hillard CJ, and Harder DR (1999)
Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit
L-type Ca2ϩ channel current. Am J Physiol 276:H2085–H2093.
Grainger J and Boachie-Ansah G (2001) Anandamide-induced relaxation of sheep
coronary arteries: the role of the vascular endothelium, arachidonic acid metabo-
lites and potassium channels. Br J Pharmacol 134:1003–1012.
Hajos N, Ledent C, and Freund TF (2001) Novel cannabinoid-sensitive receptor
mediates inhibition of glutamatergic synaptic transmission in the hippocampus.
Neuroscience 106:1–4.
Harris D, McCulloch AI, Kendall DA, and Randall MD (2002) Characterization of
vasorelaxant responses to anandamide in the rat mesenteric arterial bed.
J Physiol (Lond) 539:893–902.
Hill CE, Phillips JK, and Sandow SL (2001) Heterogeneous control of blood flow
amongst different vascular beds. Med Res Rev 21:1–60.
Hillard CJ (2000) Endocannabinoids and cardiovascular function. J Pharmacol Exp
Ther 294:27–32.
Ishac EJN, Jiang L, Lake KD, Varga K, Abood ME, and Kunos G (1996) Inhibition
of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on
peripheral sympathetic nerves. Br J Pharmacol 118:2023–2028.
Ishioka N and Bukoski RD (1999) A role for N-arachidonylethanolamine (anandam-
ide) as the mediator of sensory nerve-dependent Ca2ϩ-induced relaxation of iso-
lated mesenteric resistance arteries. J Pharmacol Exp Ther 289:245–250.
Jarai Z, Wagner JA, Varga K, Lake KD, Compton DR, Martin BR, Zimmer AM,
Bonner TI, Buckley NE, Mezey E, et al. (1999) Cannabinoid-induced mesenteric
vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc
Natl Acad Sci USA 96:14136–14141.
Kunos G, Jarai Z, Varga K, Liu J, Wang L, and Wagner JA (2000) Cardiovascular
effects of endocannabinoids–the plot thickens. Prostaglandins Other Lipid Mediat
61:71–84.
Lake KD, Compton DR, Varga K, Martin BR, and Kunos G (1997) Cannabinoid-
induced hypotension and bradycardia in rats is mediated by CB1-like cannabinoid
receptors. J Pharmacol Exp Ther 281:1030–1037.
Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, Beslot F, Bohme GA, Imperato
A, Pedrazzini T, Roques BP, et al. (1999) Unresponsiveness to cannabinoids and
reduced addictive effects of opiates in CB1 receptor knockout mice. Science (Wash
DC) 283:401–404.
Liu J, Gao B, Mirshahi F, Sanyal AJ, Khanolkar AD, Makriyannis A, and Kunos G
(2000) Functional CB1 cannabinoid receptors in human vascular endothelial cells.
Biochem J 346:835–840.
Liu J, Tian Z, Gao B, and Kunos G (2002) Dose-dependent activation of antiapoptotic
and proapoptotic pathways by ethanol treatment in human vascular endothelial
cells. J Biol Chem 277:20927–20933.
Mechoulam R, Fride E, and Di Marzo V (1998) Endocannabinoids. Eur J Pharmacol
359:1–18.
Mukhopadhyay S, Chapnick BM, and Howlett AC (2002) Anandamide-induced va-
sorelaxation in rabbit aortic rings has two components: G protein dependent and
independent. Am J Physiol 282:H2046–H2054.
Pratt PF, Hillard CJ, Edgemond WS, and Campbell WB (1998) N-arachidonyleth-
anolamide relaxation of bovine coronary artery is not mediated by CB1 cannabi-
noid receptor. Am J Physiol 43:H375–H381.
Randall MD, Harris D, Kendall DA, and Ralevic V (2002) Cardiovascular effects of
cannabinoids. Pharmacol Ther 95:191–202.
Earlier findings have implicated macrophage-derived en-
docannabinoids acting at SR141716-sensitive vascular can-
nabinoid receptors in endotoxic hypotension (Varga et al.,
1998). Our unpublished observations that O-1918 inhibits
lipopolysaccharide-induced hypotension in mice suggest that
the novel endothelial site described here also may be involved
in the hypotension associated with septic shock.
Randall MD and Kendall DA (1997) Involvement of a cannabinoid in endothelium-
derived hyperpolarizing factor-mediated coronary vasorelaxation. Eur J Pharma-
col 335:205–209.
Razdan RK, Dalzell HC, and Handrick GR (1974) Hashish. A simple one-step
synthesis of (Ϫ)-delta1-tetrahydrocannabinol (THC) from p-mentha-2, 8-dien-1-ol
and olivetol. J Am Chem Soc 96:5860–5865.
Showalter V, Compton DR, Martin BR, and Abood ME (1996) Evaluation of binding
in transfected cell line expressing a peripheral cannabinoid receptor (CB2): iden-
tification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther
278:989–999.

Novel Endothelial Endocannabinoid Receptor
705
Varga K, Lake K, Huangfu D, Guyenet PG, and Kunos G (1996) Mechanism of the
hypotensive action of anandamide in anesthetized rats. Hypertension 28:682–686.
Varga K, Lake K, Martin BR, and Kunos G (1995) Novel antagonist implicates the
CB1 cannabinoid receptor in the hypotensive action of anandamide. Eur J Phar-
macol 278:279–283.
Varga K, Wagner JA, Bridgen DT, and Kunos G (1998) Platelet- and macrophage-
derived cannabinoids are involved in endotoxin-induced hypotension. FASEB J
12:1035–1044.
Vollmer RR, Cavero I, Ertel RJ, Solomon TA, and Buckley JP (1974) Role of the
central nervous system in the hypotension and bradycardia induced by (Ϫ)-⌬9-
trans-tetrahydrocannabinol. J Pharm Pharmacol 26:186–192.
Wagner JA, Jarai Z, Batkai S, and Kunos G (2001) Hemodynamic effects of canna-
binoids: coronary and cerebral vasodilation mediated by cannabinoid CB1 recep-
tors. Eur J Pharmacol 423:203–210.
induced relaxations in the rat isolated mesenteric artery. Br J Pharmacol 122:
1573–1584.
White R, Venessa Ho W, Bottrill FE, Ford WR, and Hiley CR (2001) Mechanism of
anandamide-induced vasorelaxation in rat isolated coronary arteries. Br J Phar-
macol 134:921–929.
Zygmunt PM, Andersson DA, and Hogestatt ED (2002) ⌬9–Tetrahydrocannabinol
and cannabinol activate capsaicin-sensitive sensory nerves via a CB1 and CB2
cannabinoid receptor-independent mechanism. J Neurosci 22:4720–4727.
Zygmunt PM, Petersson J, Andersson DA, Chuang HH, Sorgard M, Di Marzo V,
Julius D, and Hogestatt ED (1999) Vanilloid receptors on sensory nerves mediate
the vasodilator action of anandamide. Nature (Lond) 400:452–457.
Address correspondence to: Dr. George Kunos, National Institute on Alco-
hol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn
Drive, MSC-8115, Bethesda, MD 20892-8115. E-mail: gkunos@mail.nih.gov
Wagner JA, Varga K, Jarai Z, and Kunos G (1999) Mesenteric vasodilation mediated
by endothelial anandamide receptors. Hypertension 33:4529–434.
White R and Hiley CR (1997) A comparison of EDHF-mediated and anandamide-