Cannabinoid CB1 receptor of cat cerebral arterial
muscle functions to inhibit L-type Ca2⫹ channel current
DEBEBE GEBREMEDHIN,1 ANDREW R. LANGE,1 WILLIAM B. CAMPBELL,2
CECILIA J. HILLARD,2 AND DAVID R. HARDER1
1Departments of 1Physiology and 2Pharmacology and Toxicology and Cardiovascular Research
Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Gebremedhin, Debebe, Andrew R. Lange, William B.
Campbell, Cecilia J. Hillard, and David R. Harder.
Cannabinoid CB1 receptor of cat cerebral arterial muscle
functions to inhibit L-type Ca2⫹ channel current. Am. J.
Physiol. 276 (Heart Circ. Physiol. 45): H2085–H2093, 1999.—
The CB1 subtype of the cannabinoid receptor is present on
neurons in the brain and mediates the perceptual effects of
⌬9-tetrahydrocannabinol and other cannabinoids. We found
that cat cerebral arterial smooth muscle cells (VSMC) contain
the protein for the CB1 receptor and express a cDNA that has
⬎98% amino acid homology to the CB1 cDNA expressed in rat
and human neurons. Activation of the CB1 cannabinoid
receptor has been shown to decrease the opening of N-type
voltage-gated Ca2⫹ channels in neurons through a pertussis
toxin-sensitive GTP-binding protein. In the present study we
tested the hypothesis that activation of the cannabinoid CB1
receptor in cerebral VSMC inhibits voltage-gated Ca2⫹ chan-
nels and results in cerebral vasodilation. The predominant
Ca2⫹ current identified in cat cerebral VSMC is a voltage-
gated, dihydropyridine-sensitive, L-type Ca2⫹ current. The
cannabimimetic drug WIN-55,212-2 (10–100 nM) induced
concentration-dependent inhibition of peak L-type Ca2⫹ cur-
rent, which reached a maximum of 82 ⫾ 4% at 100 nM
(n ⫽ 14). This effect was mimicked by the putative endog-
enous CB1-receptor agonist anandamide, which produced a
concentration-related reduction of peak L-type Ca2⫹ current
with a maximum inhibition (at 300 nM) of 39 ⫾ 4% (n ⫽ 12).
The inhibitory effects of both ligands on peak L-type Ca2⫹
currents were abolished by pertussis toxin pretreatment and
application of the CB1-receptor antagonist SR-141716A (100
nM, n ⫽ 5). Both WIN-55,212-2 and anandamide produced
concentration-dependent relaxation of preconstricted cere-
bral arterial segments that was abolished by SR-141716A.
These results indicate that the CB1 receptor is expressed in
cat cerebral VSMC and that the cerebral vasculature is one of
the targets for endogenous cannabinoids. These findings
suggest that the CB1 receptor and its endogenous ligand may
play a fundamental role in the regulation of cerebral arterial
tone and reactivity by modulating the influx of Ca2⫹ through
L-type Ca2⫹ channels.
anandamide; WIN-55,2121–2; SR-141716A; whole cell; patch
clamp; vasodilation
THE MAJOR PSYCHOACTIVE CONSTITUENT of Cannabis sa-
tiva, ⌬9-tetrahydrocannabinol (THC) (10), exerts its
cellular effects by binding and activating a cell-surface,
cannabinoid-selective receptor (26). Two major sub-
types of the cannabinoid receptor have been cloned and
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/99 $5.00 Copyright r 1999 the American Physiological Society
Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.
characterized; the first subtype is found primarily in
the brain (CB1) (26), and the second is found primarily
in cells of myeloid lineage (CB2) (27). Both receptor
subtypes have the structural features typical of G
protein-coupled receptors, including seven-transmem-
brane domains (26, 27). The cellular effects of CB1-
receptor activation in neural cells, including inhibition
of adenylyl cyclase (18), inhibition of voltage-operated
N-type Ca2⫹ channels (3, 23), and activation of inward
rectifier K⫹ current and K⫹ A-current (5, 16, 24) are
inhibited by pertussis toxin, which ribosylates and
inhibits G proteins of the Gi/Go subtype. The CB1
receptor is also activated by a novel eicosanoid, N-
arachidonylethanolamine (anandamide), which has
been isolated from the brain and has been suggested to
be an endogenous CB1 agonist (7).
There is evidence in the literature that marijuana
and THC have a direct vasodilator effect on cerebral
blood vessels. Marijuana smoking produces a dose-
related increase in global cerebral blood flow (CBF) in
humans that is not due to changes in sympathetic
cerebrovascular regulation or PCO2 and that is consis-
tent with cerebral vasodilation (25). The increase in
CBF is accompanied by a parallel increase in the
velocity of flow through the middle cerebral artery (25).
In addition, marijuana-intoxicated individuals show
evidence of impaired cerebral autoregulation in re-
sponse to a change in posture from supine to standing
(25). The mechanism for this effect is not known;
however, it is not accompanied by impaired peripheral
sympathetic activity and suggests that cerebral arteri-
oles are maximally dilated by THC and, therefore, are
unable to respond to reduced perfusion pressure. A
recent study has demonstrated that CB1 agonists
dilate cerebral arterioles in an in situ perfusion prepa-
ration (9), supporting the hypothesis that the effects of
marijuana on CBF are due to direct effects of the drugs
on vascular tone. However, the mechanism(s) and the
cell type in the vessel wall expressing the CB1 receptor
responsible for the cerebrovascular effects of CB1 ago-
nists are not known. The present studies were under-
taken to determine whether cannabinoid receptors are
expressed in cerebral vascular smooth muscle cells
(VSMC), to investigate the electrophysiological bases of
cannabinoid-induced cerebral vasodilation by determin-
ing the effect of agonists on voltage-activated L-type
Ca2⫹ channel in cat cerebral VSMC, and to determine
whether the CB1 receptor mediates the actions of
cannabinoids on the cerebral vascular tone. The results
of these studies demonstrate that cat cerebral VSMC
express cDNA encoding the CB1 receptor, which, when
stimulated by WIN-55,212-2 or anandamide, functions
H2085
H2086 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL
to dilate cat cerebral microvessels by inhibiting Ca2⫹
influx through a dihydropyridine-sensitive L-type Ca2⫹
channel in cerebral VSMC.
METHODS
Vascular muscle cell dispersion. Adult mongrel cats of
either sex were deeply anesthetized with Telazol (10 mg/kg
im; Aveco, Fort Dodge, IA) and their brains removed. Cerebral
microvessels [200–230 µm outer diameter (OD)] were dis-
sected free of arachnoid, cut into small pieces, and placed in
2-ml vials containing an enzyme solution of the following
composition (in mM): 134 NaCl, 5.2 KCl, 1.2 MgSO4, 0.33
KH2PO4, 0.05 CaCl2, 11 glucose, and 10 HEPES, with 100
U/ml collagenase (class II, Worthington), 0.5 mg/ml bovine
serum albumin, and 1 mM trypsin inhibitor (Sigma, St.
Louis, MO). The enzyme solution containing the arterial
pieces was maintained at 37°C and pH 7.4 and was continu-
ally stirred at 10 rpm for 45 min. Supernatant fractions were
collected every 5 min, and the dispersion of VSMC was
examined under a microscope. The vascular smooth muscle
origin of the cells was confirmed by indirect immunocytochemi-
cal staining. The freshly isolated vascular muscle cells did not
show specific staining with fluorescein isothiocyanate-
conjugated rabbit anti-human von Willebrand’s factor (vWF),
a marker for endothelial cells that cross-reacts with cat vWF
(Zymed Laboratories, San Francisco, CA), whereas the cells
exhibited high-intensity staining with anti-␣-actin-Cy3-
conjugated antibody (Sigma).
RT-PCR, cloning, and sequencing. Total RNA was extracted
from freshly dispersed cat cerebral VSMC using silica-based
column chromatography (RNAeasy, Qiagen, Chatsworth, CA).
RNA (2 µg) was reverse transcribed using commercial kits
(Pharmacia First Strand cDNA Synthesis Kit) in a volume of
33 µl. Aliquots of RT reactions (5 µl) were amplified with
KlenTaq DNA polymerase mix (Clontech Laboratories, Palo
Alto, CA) using primers specific to the rat CB1 receptor
(GenBank accession no. X55812): forward, 58-ATGAAGTC-
GATCCTAGATGGCC-38; reverse, 58-TCACAGAGCCTCGGC-
GGACGTG-38 (26). Components of the reactions were mixed,
overlaid with mineral oil, and heated to 95°C for 3 min; cycled
for 10 cycles of 94°C for 30 s, 66°C for 30 s, and 68°C for 3 min;
followed by 30 cycles of 94°C for 30 s, 61°C for 30 s, and 68°C 3
min; and ending with a 30-min extension at 72°C. Fifty
microliters of each reaction were purified by electrophoresis
on a 1.5% agarose gel (SeaKem GTG, FMC BioProducts),
stained with ethidium bromide, and scanned using a Molecu-
lar Dynamics FluorImager. Bands of expected size were
excised by using the scan printout as a guide, solubilized in
NaI at 50°C for 10 min, and purified using a GlassMax
column (GIBCO). Purified product (150 ng) was ligated into
pCR2.1 using the T/A Cloning Kit (Invitrogen, Carlsbad, CA),
transformed into Escherichia coli INV␣F8, and plated on
LB-kanamycin (50 µg/ml) plates containing X-Gal. Positive
colonies were grown in LB-kanamycin overnight, and plas-
mid DNA was purified using the Qiaprep Spin Miniprep
plasmid purification kit (Qiagen). Plasmid DNA (1 µg) was
sequenced using dye-primer (M13 forward and reverse prim-
ers) Texas Red cycle sequencing (Amersham) on a Vistra
model 725 automated DNA sequencer to confirm the identity
of inserts. Four independent clones were identified that
contained sequences with high homology to human, rat, and
mouse CB1 receptors. Both strands of these four independent
clones were sequenced repetitively using dye-terminator cycle
sequencing on an ABI system (Perkin Elmer).
Western blot analysis. Cat and rat cerebral cortex micro-
somal fractions were prepared separately using standard
Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.
differential centrifugation methods. Freshly dispersed cat or
rat cerebral VSMC were pelleted by centrifugation and
resuspended in buffer (150 mM NaCl, 1.0% NP-40, 0.5%
deoxycholate, 0.1% SDS, 1 mM EDTA, and 50 mM Tris, pH
8.0) containing a protease inhibitor cocktail (PharMingen,
San Diego, CA) consisting of 16 µg/ml benzamidine hydrochlo-
ride, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 10 µg/ml pepstatin A, and 50 mM phenylmethylsul-
fonyl fluoride. Cells were solubilized by vigorous vortexing
and sonication. Insoluble material was pelleted by centrifuga-
tion for 5 min at 14,000 g. Cerebellar microsomes (50 µg) and
VSMC lysates (10 µg) were mixed separately with 2⫻ Laemmli
SDS sample buffer, boiled for 5 min, and loaded onto SDS-
PAGE gels (4% stacking, 10% resolving; Bio-Rad Ready-gels).
After fractionation, proteins were electrophoretically trans-
ferred to nitrocellulose membranes (Bio-Rad) and probed
with the primary antibody anti-rat CB1 antibody (1–77)
(1:1,000; kindly provided by Dr. Ken Mackie, University of
Washington, Seattle, WA) and the secondary antibody goat
anti-rabbit horseradish peroxidase (1:1,000; Bio-Rad).
Prestained molecular mass marker proteins (Bio-Rad) were
used to estimate sizes of immunoreactive protein bands.
Electrophysiology. Macroscopic Ca2⫹ currents were re-
corded at room temperature (22°C) using a conventional
whole cell voltage-clamp technique (13) with Ba2⫹ as a charge
carrier. The VSMC were dialyzed with the intracellular
solution containing (in mM) 135 CsCl, 5 Mg-ATP, 5 HEPES,
and 1 ethylene glycol-bis(␤-aminoethyl ether)-N,N,N8,N8-
tetraacetic acid, with pH adjusted to 7.2 with CsOH. The
extracellular solution bathing the cells was composed of (in
mM) 135 tetraethylammonium (TEA) chloride, 5 BaCl2, 1
MgCl2, 11 glucose, 10 HEPES, 5,4-aminopyridine, and 0.001
tetrodotoxin, with pH adjusted to 7.4 with TEA hydroxide.
Inward Ca2⫹ currents were elicited every 1 s by 200-ms
depolarizing pulses from a holding potential of ⫺70 mV to test
potentials between ⫺50 and ⫹50 mV in 10-mV increments.
Whole cell Ca2⫹ currents were recorded through a List EPC-7
patch-clamp amplifier (Darmstadt, Germany) before and
after addition to the bath of CB1 agonists in the presence or
absence of the various blockers. Cell capacitance was deter-
mined from the current amplitude in response to a hyperpolar-
izing voltage pulse of 1 mV for 25 ms from a holding potential
of 0 mV. Average cell membrane capacitance was 26 ⫾ 3 pF
(range 15–28 pF, n ⫽ 22). The inward Ca2⫹ current was
normalized by dividing its amplitude by the cell capacitance
(expressed in pA/pF). The signal was low-pass filtered at 1
kHz (⫺3 dB frequency) by an eight-pole low-pass Bessel filter
and digitized at a sampling frequency of 2.5 kHz. Data were
analyzed using a pCLAMP software package (Axon Instru-
ments).
Isolated vessel studies. Isolated cat cerebral microvascular
segments (8–10 mm in length, 0.20- to 0.30-mm OD) were
placed in a perfusion chamber, cannulated at both ends with
glass micropipettes, and secured in place with 8-0 polyethyl-
ene sutures (Ethicon, Somerville, NJ) using a stereomicro-
scope. Side branches of the arteries were tied off with 10-0
polyethylene sutures. The arterial segments were superfused
with physiological saline solution that was aerated with a
95% O2-5% CO2 gas mixture and maintained at 37°C and pH
7.35. The inflow cannula was connected in series with a
volume reservoir and a pressure transducer (Gould, Cleve-
land, OH) to allow continuous monitoring of transmural
pressure. Internal diameter of the arteries was measured
using a video microscopy system composed of a television
camera and a video micrometer, as previously described (11).
After an equilibration period of 15 min, the cannulated
arteries were pressurized to 90 mmHg and then maintained
 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL
at this pressure throughout the course of the experiment.
After an additional 15-min equilibration, the functional integ-
rity of the vessels was verified by the contractile response to
60 mM KCl and the vasodilator response to the direct
vasorelaxant sodium nitroprusside (1 µM) after preconstric-
tion with serotonin (5 µM). After three successive washes, the
arterial segments were allowed to equilibrate for an addi-
tional 15 min. The concentration-dependent relaxation re-
sponses of the cerebral arterial preparations to anandamide
(10, 100, and 300 nM) or WIN-55,212-2 (10, 30, and 100 nM)
were then determined in the absence and presence of the CB1
cannabinoid receptor antagonist SR-141716A (100 nM) after
preconstriction of the vascular segments with 5 µM serotonin.
Statistics. Data are presented as means ⫾ SE. Differences
between groups were assessed using ANOVA or Student’s
t-test with a Bonferroni correction for multiple comparisons.
Drugs and chemicals. All chemicals were analytic grade.
SR-141716A was the kind gift of Sanofi Researche (Montpe-
lier, France). ATP (magnesium salt), sodium nitroprusside,
BaCl2, nifedipine, CdCl2, TEA chloride, TEA hydroxide, 4-ami-
nopyridine, and tetrodotoxin were all purchased from Sigma
Chemical (St. Louis, MO). Anandamide was purchased from
Biomol (Plymouth Meeting, PA), and R-(⫹)-WIN-55,212-2
was purchased from RBI (Natick, MA).
RESULTS
Identification of cannabinoid CB1 receptor mRNA in
cat cerebral VSMC. To determine whether cat cerebral
VSMC express CB1 mRNA, we amplified cDNA encod-
ing the CB1 receptor with the use of RT-PCR with
oligonucleotide primers derived from the published
sequence of rat CB1 (see RT, cloning, and sequencing)
(26). The sequence of the amplified and cloned product
showed ⬎98% homology to the rat (26) and human (12)
CB1 cannabinoid receptor (Fig. 1A). This finding pro-
vides evidence for the expression of the CB1 cannabi-
noid receptor mRNA in cat cerebral VSMC (GenBank
accession no. U94342). The existence of the protein for
the CB1 receptor in cerebral VSMC and the brain
cortex was determined by Western blot analysis. Re-
sults of the Western immunoblot studies (Fig. 1B)
revealed the expression of an immunoreactive band
with an apparent molecular mass of 56 kDa in VSMC
lysate and brain cortex microsomes obtained from cats
or rats when probed with a polyclonal anti-rat CB1(1–
77) antibody raised in rabbits. An additional band with
a molecular mass of 61 kDa was also detected in the cat
VSMC lysate. This 61-kDa band was not detected in
either the VSMC lysate or cortical microsomes of the
rat. It is possible that the CB1 receptor of cat cerebral
VSMC has a different pattern of glycosylation and,
therefore, a different molecular mass. It is likely that
the 56-kDa protein is the nonglycosylated receptor.
These results indicate that the protein for the CB1
receptor is expressed in cerebral arterial muscle cells
and the cerebral cortex of the cat and rat.
Characterization of macroscopic Ca2⫹ currents in cat
cerebral VSMC. Inward Ca2⫹ currents carried by Ba2⫹
were recorded from freshly dispersed cat cerebral arte-
rial muscle cells with high-Cs⫹ solution in the pipette
and with bath solution containing TEA and tetrodo-
toxin (1 µM) to block outward K⫹ currents and inward
Na⫹ currents, respectively. During step depolarization
Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.
H2087
from a holding potential of ⫺70 mV to test potentials
from ⫺50 to ⫹50 mV, an inward Ca2⫹ current began to
activate at ⫺40 mV and peaked at ⫹10 mV. The peak
current-voltage relation over the range of more positive
potentials started to reverse between ⫹40 and ⫹50 mV
(Fig. 2A, c) and remained stable for ⬎20 min. As shown
in Fig. 2A, a and c, the inward Ca2⫹ current, elicited by
a 200-ms depolarizing pulse from a holding potential of
⫺70 mV to a test potential from ⫺50 to ⫹10 mV, was
inhibited ⬎96% by nifedipine (2 µM). This inward
current was also completely blocked by 50 µM Cd2⫹
(data not shown). The high voltage threshold of channel
activation and the sensitivity to blockade by either
nifedipine or Cd2⫹ indicated that the inward current
recorded from freshly dissociated cat cerebral arterial
VSMC passes through a high voltage-activated, dihy-
dropyridine-sensitive, L-type Ca2⫹ channel. We have
previously shown (11) that the magnitude of this in-
ward macroscopic current is not associated with sub-
stantial rundown or inactivation during alteration of
the frequency of depolarization from slower to higher
rates under identical experimental conditions.
Effects of the CB1 agonists anandamide and WIN-
55,212-2 on peak macroscopic L-type Ca2⫹ channel
current in isolated cerebral VSMC. As depicted in Fig.
2, A and B, the application of 300 nM anandamide to
the bath induced a 37 ⫾ 4% (n ⫽ 12) maximum
reduction in the amplitude of peak Ca2⫹ current elicited
by 200-ms depolarizing pulses from a holding potential
of ⫺70 mV to test potentials from ⫺50 to ⫹10 mV in
10-mV increments. The inhibitory action of anan-
damide on Ca2⫹ current was not readily reversible on
washout (data not shown); therefore, each cell was
exposed to only one concentration of anandamide. The
inhibitory action of anandamide on normalized peak
Ca2⫹ current is concentration dependent; the peak Ca2⫹
current was reduced by 12 ⫾ 3% at 10 nM (n ⫽ 5) and
by 23 ⫾ 5% at 100 nM (n ⫽ 5; Fig. 2B). Higher
concentrations of anandamide (1 µM) did not cause
further inhibition of the peak L-type Ca2⫹ current (data
not shown).
The concentration-related effects of the synthetic
cannabimimetic drug WIN-55,212-2 on the peak Ca2⫹
current recorded from cat cerebral VSMC was also
examined. WIN-55,212-2 was both a more potent and a
more efficacious inhibitor of peak Ca2⫹ current than
anandamide (Figs. 2A and 4A). At concentrations of 10,
30, and 100 nM, WIN-55,212-2 reduced peak Ca2⫹
current by 24 ⫾ 2% (n ⫽ 4), 48 ⫾ 3% (n ⫽ 4), and 82 ⫾
4% (n ⫽ 14), respectively. Similarly to anandamide,
WIN-55,212-2 did not shift the position of the peak
current-voltage curve, which indicates that the canna-
binoid agonists inhibit Ca2⫹ current by decreasing
either the number of active channels or the probability
that a channel remains open.
The time course of the reduction in peak L-type Ca2⫹
current induced by either anandamide (300 nM) or
WIN-55,212-2 (100 nM) is depicted in Fig. 3. Both
agonists produced a rapid decrease in Ca2⫹ current.
Control peak L-type Ca2⫹ current as well as the maxi-
mum inhibitory action of both anandamide and WIN-
H2088 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL

Fig. 1. A: deduced amino acid se-

quences of cat cerebral vascular smooth
muscle cell (VSMC) cannabinoid CB1
receptor cDNA. Amino acids are indi-
cated in a single-letter code. Compari-
sons show amino acid sequences of
product amplified from reverse-tran-
scribed mRNA extracted from cat cere-
bral VSMC (GenBank accession no.
U94342) with previously reported rat
and human CB1 receptor sequences.
Identical amino acid residues are shown
together in black boxes; sequences
share ⬎98% overall homology. B: West-
ern blot analysis showing expression of
immunoreactive bands in both cerebel-
lar microsomes and VSMC lysate of cat
and rat at ⬃56 kDa when probed with
polyclonal anti-CB1(1–77) antibody.

55,212-2 on the peak L-type Ca2⫹ current remained
unchanged over a period of 5 min (n ⫽ 10–14).
SR-141716A is a selective antagonist of the CB1
receptor at a concentration of 100 nM (33). At that
concentration, SR-141716A completely antagonized the
inhibition of peak Ca2⫹ current produced by 300 nM
anandamide (n ⫽ 5) or 100 nM WIN-55,212-2 (n ⫽ 5;
Fig. 4). Pretreatment of the VSMC for 3 min with
SR-141716A alone did not affect the peak Ca2⫹ current
(Fig. 4) or change the shape of the peak current-voltage
relation of this whole cell Ca2⫹ current. These findings
support a role for the CB1 receptor in the inhibitory
actions of anandamide and WIN-55,212-2 on L-type
Ca2⫹ current recorded from cat cerebral VSMC.
To determine whether the effects of anandamide and
WIN-55,212-2 on the L-type Ca2⫹ channel current were
mediated by a pertussis toxin-sensitive G protein, the
freshly isolated cerebral VSMC were treated with 1
µg/ml pertussis toxin for 5–6 h and the effects of both
anandamide and WIN-55,212-2 on peak Ca2⫹ current

Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.

 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL H2089

Fig. 2. A: inhibition of L-type Ca2⫹ current by CB1

agonists anandamide (AEA) and WIN-55,212-2 (WIN)
in cerebral VSMC. Bath application of either 300 nM
AEA (a) or 100 nM WIN (b) markedly inhibited whole
cell Ca2⫹ current elicited by depolarizing steps from
⫺50 to ⫹10 mV from a holding potential of ⫺70 mV
within 1–3 min. Nifed., nifedipine. Broken lines indi-
cate zero current level. c: Effects of AEA (300 nM) and
WIN (100 nM) on normalized mean peak current-
voltage relation of Ca2⫹ currents in cerebral VSMC.
WIN was more efficient in inhibiting mean peak Ca2⫹
current than AEA. Both AEA and WIN depressed mean
peak current without shifting current-voltage curve. B:
comparison of averaged percent maximum peak Ca2⫹
current inhibition evoked by increasing concentrations
of either WIN (10, 30, and 100 nM) or AEA (10, 100, and
300 nM) in cat cerebral VSMC at a test potential of ⫹10
mV. AEA was less potent in inhibiting L-type Ca2⫹
current at all concentrations studied. Data are means ⫾
SE; nos. in parentheses represent no. of cells studied.

were determined (Fig. 4). Pertussis toxin pretreatment

Fig. 3. Time course of inhibitory effects of AEA and WIN on peak
L-type Ca2⫹ current. Normalized peak L-type Ca2⫹ current (ICa ) is
plotted as a function of time. Inhibitory effects of both AEA (300 nM)
and WIN (100 nM) remained stable over a period of 5 min. There is no
apparent rundown or inactivation associated with normalized peak
L-type current recorded from cerebral VSMC. Data points are means,
and bars are SE.
abolished the inhibitory effects of both anandamide and
WIN-55,212-2 and had no effect on the magnitude of
control peak L-type Ca2⫹ current elicited with a test
potential from ⫺50 to ⫹10 mV from a holding potential
of ⫺70 mV (Fig. 4). Thus 300 nM anandamide produced
a 38 ⫾ 3% (n ⫽ 12) reduction of the Ca2⫹ current in
control cells compared with a 5 ⫾ 2% (n ⫽ 6, P ⬍ 0.05)
decrease in cells preincubated with 1 µg/ml pertussis
toxin. Similarly, the effect of 100 nM WIN-55,212-2 was
to reduce peak Ca2⫹ current by 83 ⫾ 5% (n ⫽ 14) in
control cells and 5 ⫾ 1% (n ⫽ 5, P ⬍ 0.05) in cells
pretreated with pertussis toxin. These results demon-
strate that an intact pertussis toxin-sensitive G protein
is required for coupling of CB1 receptors to L-type
channel inhibition in cat cerebral VSMC.
Effects of anandamide and WIN-55,212-2 on cerebral
arterial tone. The vasodilator effects of the endogenous
CB1 ligand anandamide and the cannabimimetic drug
WIN-55,212-2 were determined in either intact or
endothelium-denuded cat cerebral microvascular seg-
ments pressurized to 90 mmHg and preconstricted with
5 µM serotonin. Under control conditions, the basal
diameter of the cerebral microvascular segments aver-

Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.

H2090 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL

Fig. 4. Blockade of WIN- and AEA-induced inhibition of peak L-type Ca2⫹ current by cannabinoid (CB1)-receptor
antagonist SR-141716A (SR) and pertussis toxin (PTX). Application of CB1 antagonist SR (100 nM, 3 min) had no
effect on basal peak L-type Ca2⫹ current but completely prevented WIN (100 nM)- and AEA (300 nM)-induced
inhibition of L-type Ca2⫹ current in cerebral VSMC (A and B, top). VSMC were pretreated with PTX (1 µg/ml) for
5–6 h before experiment (A and B, bottom). PTX pretreatment eliminated both WIN- and AEA-evoked inhibition of
peak L-type Ca2⫹ current compared with control peak L-type Ca2⫹ current recorded from VSMC treated with
vehicle.

aged 214 ⫾ 25 µm (n ⫽ 6) and decreased to 160 ⫾ 10 µm
when stimulated with 5 µM serotonin. Cumulative
addition of either anandamide (10, 100, and 300 nM) or
WIN-55,212-2 (10, 30, and 100 nM) to the bath induced
concentration-related relaxation of the arterial seg-
ments (Fig. 5, A and B). Similar to the electrophysiologi-
cal findings, anandamide was less potent than WIN-
55,212-2 in inducing relaxation of the arterial segments.
The vasodilator effects of anandamide and WIN-
55,212-2 were greatly attenuated after pretreatment of
the cerebral arterial preparations with the CB1-
receptor antagonist SR-141716A (100 nM) (Fig. 5, A
and B; n ⫽ 6, P ⬍ 0.05). Anandamide induced a similar
degree of relaxation in cerebral arterial preparations
contracted with high extracellular KCl (60 mM) (data
not shown).
DISCUSSION
In these studies we have demonstrated that isolated
cerebral VSMC of the cat express the protein and the
mRNA for CB1 cannabinoid receptor. Furthermore,
external application of the CB1 agonist WIN-55,212-2
produced potent and profound inhibition of Ca2⫹ cur-
rent recorded in cat cerebral VSMC. We have identified
the Ca2⫹ current carriers as L-type Ca2⫹ channels on
the basis of a characteristically high threshold of
activation, slow activation and inactivation kinetics,
and sensitivity to blockade by nifedipine. The proper-
ties of this L-type Ca2⫹ channel current are similar to
those reported in a variety of tissues (2, 32). The effect
of WIN-55,212-2 was mimicked by the putative endog-
enous cannabinoid ligand anandamide (7) and reversed
by the CB1-receptor antagonist SR-141716A (33). Inhi-
bition of the L-type Ca2⫹ current by either anandamide
or WIN-55,212-2 was abolished by pretreatment of the
cells with pertussis toxin, which is consistent with the
findings of others (3, 19, 23) and supports the involve-
ment of a G protein-mediated signaling pathway. We
have also demonstrated that activation of the CB1
receptor results in dilation of isolated pressurized
cerebral vessels preconstricted with serotonin or high
KCl. These findings suggest that CB1 agonists mediate
the inhibition of Ca2⫹ influx through a G protein-
mediated pathway, resulting in a decrease in intracellu-
lar Ca2⫹ available to sustain smooth muscle cell contrac-
tion (1) and decreased vessel tone. Because the extent
of cerebral microvascular tone is one of the mechanisms
by which CBF is regulated (39), these results are
consistent with the hypothesis that the endogenous
cannabinoid agonist plays a potential role in the regula-
tion of regional CBF.
Several other investigators have demonstrated that
activation of the CB1 receptor results in decreased Ca2⫹
transients in hippocampal neurons (40) and neuroblas-

Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.

 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL
Fig. 5. Effect of CB1-receptor antagonist SR (100 nM) on AEA (10,
100, and 300 nM)- and WIN (10, 30, and 100 nM)-induced relaxations
(means ⫾ SE, n ⫽ 6) of cannulated and pressurized (90 mmHg) cat
cerebral arterial segments preconstricted with serotonin (5 µM).
Graphs show concentration-dependent relaxation of cerebral arterial
segments induced by AEA (A) and WIN (B) in absence or presence of
CB1 antagonist SR. * CB1 antagonist significantly prevented (P ⬍
0.05, n ⫽ 6) relaxations induced by either AEA or WIN at all
concentrations.
toma cells expressing the endogenous CB1 receptor (3,
23), in AtT20 cells (24), and in superior cervical gan-
glion cells (28) transfected with the CB1 receptor. In
these studies, CB1-receptor agonists produce inhibition
of Ca2⫹ current through N-type (23, 28, 40) or P/Q-type
Ca2⫹ channels (24, 40) but not through L-type Ca2⫹
channels (23, 24). CB1 receptor-mediated inhibition of
Ca2⫹ channel opening required an intact G protein
signaling cascade because pretreatment of the cells
with pertussis toxin (3, 23, 28), N-ethylmaleimide (22),
or guanosine 58-O-(2-thiodiphosphate) (28) abolished
the cannabinoid agonist response. The precise signal-
ing mechanism, i.e., whether CB1 receptor-mediated
inhibition is due to a direct interaction of a component
of the G protein with the channel or occurs as a result of
an as yet undefined change in a second messenger, is an
open question. One current hypothesis is that G sub-
units bind directly to a domain with the sequence
QXXER in the 1-2 linker region of N-, P-, and Q-type
Ca2⫹ channels (4).
Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.
H2091
The inhibition of L-type Ca2⫹ currents by CB1-
receptor agonists in cat cerebral microvessel VSMC has
many biochemical features in common with CB1 recep-
tor-mediated inhibition of Ca2⫹ current in cell lines and
neurons. In particular, the CB1 agonist WIN-55,212-2
is a very potent inhibitor of Ca2⫹ current, with an IC50
in the range of 10⫺8 M, and the effect is pertussis toxin
sensitive. However, the Ca2⫹ current in the cat cerebral
VSMC is carried predominantly through L-type Ca2⫹
channels, and our data demonstrate that activation of
the CB1 receptor in cerebral VSMC inhibits Ca2⫹ influx
through these L-type Ca2⫹ channels. This finding is
somewhat surprising because there are only a few
examples of coupling of heptahelical receptors to L-type
Ca2⫹channels via pertussis toxin-sensitive G proteins
and because the CB1 receptor itself does not affect
L-type Ca2⫹ currents in other cells (23, 24). However, Go
proteins are involved in the coupling of muscarinic
receptors to L-type Ca2⫹ channels in GH4C1 pituitary
cells, in which L-type rather than N-type Ca2⫹ channels
subserve stimulus-secretion coupling (15, 17, 37). Sev-
eral reports (8, 15) have suggested that the L-type Ca2⫹
channel is also subject to direct inhibition by a pertus-
sis toxin-sensitive G protein in neuronal cells. To our
knowledge, this is the first demonstration of the inhibi-
tion of cerebral VSMC L-type Ca2⫹ channels by the
activation of heptahelical receptors acting through a
pertussis toxin-sensitive G protein. However, it is not
clear from the present studies whether the mechanisms
by which activation of the CB1 receptor inhibits N- and
P/Q-type Ca2⫹ channels in neurons and L-type Ca2⫹
channels in cerebral VSMC are the same.
Although no single study has investigated the struc-
ture-activity relationships among more than two CB1-
receptor agonists, previous studies of the effect of
CB1-receptor activation on Ca2⫹ channel currents re-
vealed that WIN-55,212-2 is the most efficacious of the
CB1-receptor agonists investigated (22–24, 28, 40).
The intrinsic activity of anandamide is less than that of
WIN-55,212-2 in N18 cells (22), superior cervical gan-
glion cells transfected with CB1 cDNA (28), and cere-
bral VSMC (present study) but equal to that of WIN-
55,212-2 in hippocampal neurons expressing
endogenous CB1 receptor (40). In other studies, both
THC (3) and its synthetic congener, CP-55940 (28, 35),
also have intrinsic efficacies lower than that of WIN-
55,212-2. The most likely explanation for this discrep-
ancy, as suggested by Mackie and colleagues (40), is
that anandamide and the THC analogs have a lower
absolute intrinsic activity but can produce full agonist
effect in cellular systems in which the expression levels
of the CB1 receptor and/or G protein effectors are high.
There are several reports in the literature (9, 41) that
cannabinoids vasodilate cerebral and other vascular
beds and elicit hypotensive action in anesthetized
animals. Ellis and colleagues (9) have reported that low
concentrations of THC and anandamide produce dila-
tion of rabbit cerebral arterioles in situ. Whereas these
results are consistent with our finding that cerebral
VSMC Ca2⫹ channels are inhibited by CB1 agonists,
indomethacin inhibited the vasodilator effects of both
H2092 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL
anandamide and THC in this model. This supports the
alternative hypothesis that CB1-receptor activation
increases the production of vasodilator prostanoids.
Consistent with this hypothesis is the previous finding
(36) that anandamide and THC increase the release of
arachidonic acid from rat cortical astrocytes and that
this release is inhibited by both the CB1-receptor
antagonist SR-141716A and pretreatment with pertus-
sis toxin, indicative of the involvement of the CB1
receptor in the effect in a G protein-mediated pathway.
In light of the multiple cell types that express the CB1
receptor, it is entirely possible that multiple mecha-
nisms are involved in the regulation of cerebrovascular
tone by the CB1 receptor.
A number of studies (6, 21, 29–31, 43) in other
vascular beds have demonstrated that anandamide
acts as a vasorelaxant through a variety of diverse
mechanisms. Administration of anandamide to anesthe-
tized rats produces a triphasic effect on blood pressure
(21, 41, 42): first, a brief period of hypotension accompa-
nied by a profound reduction in heart rate; second, a
brief period of hypertension; and third, a period of
hypotension. The third period is consistent with inhibi-
tion of transmitter release from sympathetic terminals
and appears to be mediated by a cannabinoid receptor
because it is reversed by SR-141716A. Interestingly,
the third period of hypotension is not apparent in
conscious rats, possibly because sympathetic outflow is
diminished and the pressor effect predominates (21,
38). A recent study (43) has provided evidence that
activation of the CB1 receptor by an endogenous ligand
produced during hemorrhagic shock contributes to the
profound hypotension that occurs. This evidence sup-
ports the hypothesis that the endogenous ligand is
anandamide and that the cellular source is the macro-
phage. Although the mechanism for the vasodilation
was not explored in depth, changes in nitric oxide
synthase activity are not likely to be involved.
Anandamide produces vasorelaxation in isolated ar-
terial segments through a mechanism(s) that is not
clear and that may vary depending on the vessel bed. In
rat mesenteric artery, anandamide-induced relaxation
is not consistent with an effect on the CB1 receptor
because its effect is not mimicked by the high-affinity
CB1-receptor agonists HU-210 or WIN-55212-2 (29)
and is either partially inhibited by relatively high (i.e.,
⬎1 µM) concentrations of SR-141716A (31, 44, 45) or
not inhibited by SR-141716A at all (29). The mecha-
nism for this effect is not clear, although there is
evidence that anandamide inhibits the release of Ca2⫹
from caffeine-sensitive stores (45) and opens large-
conductance K⫹ channels in vascular smooth muscle
cells (29). In bovine coronary arteries, relaxation in
response to anandamide is abolished by diazomethylara-
chidonyl ketone, an inhibitor of anandamide amidohy-
drolase, suggesting that anandamide serves as an
arachidonic acid donor in this vessel (30). In the kidney,
low concentrations of anandamide stimulate the re-
lease of nitric oxide from endothelial cells, and anan-
damide vasorelaxation is sensitive to both SR-141716A
and the nitric oxide synthase inhibitor nitro-L-arginine
Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.
methyl ester, suggesting a role for CB1 receptor in the
regulation of nitric oxide synthase activity in this
tissue (6). In summary, there are several mechanistic
paradigms that explain anandamide-mediated vasodi-
lation in various tissues and under various physiologi-
cal and pathological conditions. It is remarkable that,
despite the variety of mechanistic explanations, the
physiological effect that is consistently observed is
vasorelaxation.
The physiological role of CB1-receptor regulation of
cerebral vascular tone remains to be determined. How-
ever, the studies reported here suggest that one func-
tion of the endogenous ligand for the CB1 receptor is to
increase regional CBF. Anandamide has been isolated
from the brain and has the functional characteristics of
a CB1-receptor agonist (7, 22), which has led to the
suggestion that it is the endogenous ligand of the CB1
receptor (7). Although the regulation of anandamide
production in the brain is not completely understood,
there is strong evidence that brain concentrations of
anandamide increase during severe brain ischemia (20,
34). Hansen and co-workers (14) have reported that
neurons exposed to an azide produce anandamide,
which suggests that dying neurons synthesize anan-
damide. In light of the actions of anandamide on the
regulation of Ca2⫹ influx into cerebral VSMC, one could
speculate that anandamide released from ischemic
cells acts via CB1 receptors of VSMC to increase blood
flow to ischemic regions of the brain. Furthermore,
these findings suggest that the role of the CB1 receptor
in the normal brain is more fundamental than previ-
ously appreciated. It is likely that the endogenous
ligand of the CB1 receptor targets multiple cell types in
the brain and plays a role in fundamental processes
including the regulation of regional CBF.
We thank Jayashree Narayanan, Adam D. Harder, and Mike R.
Aebly for excellent technical assistance.
This study was supported by National Institutes of Health Grants
DA-09155 (to C. J. Hillard and W. B. Campbell), R37-HL-33883-13 (to
D. R. Harder), and DA08098 (to C. J. Hillard), and by Veterans Affairs
Grant 3440-06-P (to D. R. Harder).
Address for reprint requests and other correspondence: C. J.
Hillard, Dept. of Pharmacology and Toxicology, 8701 Watertown
Plank Rd., Milwaukee, WI 53226 (E-mail: chillard@mcw.edu).
Received 30 July 1998; accepted in final form 3 February 1999.
REFERENCES
1. Alborch, E., J. B. Salom, and G. Torregrosa. Calcium chan-
nels in cerebral arteries. Pharmacol. Ther. 68: 1–34, 1995.
2. Catterall, W. Functional subunit structure of voltage-gated
calcium channels. Science 242: 50–61, 1988.
3. Caulfield, M. P., and D. A. Brown. Cannabinoid receptor
agonists inhibit Ca2⫹ current in NG108-15 neuroblastoma cells
via a pertussis toxin-sensitive mechanism. Br. J. Pharmacol.
106: 231–232, 1992.
4. Clapham, D. E., and E. J. Neer. G protein beta subunits. Annu.
Rev. Pharmacol. Toxicol. 37: 167–203, 1997.
5. Deadwyler, S. A., R. E. Hampson, J. Mu, A. Whyte, and S.
Childers. Cannabinoids modulate voltage sensitive potassium
A-current in hippocampal neurones via cAMP-dependent pro-
cess. J. Pharmacol. Exp. Ther. 273: 734–743, 1995.
6. Deutsch, D. G., M. S. Goligorsky, P. C. Schmid, R. J.
Krebsbach, H. H. O. Schmid, S. K. Das, S. K. Dey, G.
Arreaza, C. Thorup, G. Stefano, and L. C. Moore. Production
and physiological actions of anandamide in the vasculature of the
rat kidney. J. Clin. Invest. 100: 1538–1546, 1997.
 CANNABINOID RECEPTOR INHIBITS L-TYPE CA2⫹ CHANNEL
7. Devane, W. A., L. Hanus, A. Breuer, R. G. Pertwee, L. A.
Stevenson, G. Griffin, D. Gibson, A. Mandelbaum, A.
Etinger, and R. Mechoulam. Isolation and structure of a brain
constituent that binds to the cannabinoid receptor. Science 258:
1946–1949, 1992.
8. Dolphin, A. C., and R. H. Scott. Interaction between calcium
channel ligands and guanine nucleotides in cultured rat sensory
and sympathetic neurones. J. Physiol. (Lond.) 413: 271–288,
1989.
9. Ellis, E. F., S. F. Moore, and K. A. Willoughby. Anandamide
and ⌬9-THC dilation of cerebral arterioles is blocked by indo-
methacin. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1859–
H1864, 1995.
10. Gaoni, Y., R., and R. Mechoulam. Isolation, structure and
partial synthesis of an active constituent of hashish. J. Am.
Chem. Soc. 86: 1646–164, 1964.
11. Gebremedhin, D., A. R. Lange, J. Narayanan, M. R. Aebly,
E. R. Jacobs, and D. R. Harder. Cat cerebral arterial smooth
muscle cells express cytochrome P450 4A2 enzyme and produce
the vasoconstrictor 20-HETE which enhances L-type Ca2⫹ cur-
rent. J. Physiol. (Lond.) 507: 771–781, 1998.
12. Gerard, C. M., C. Mollereau, G. Vassart, and M. Par-
mentier. Molecular cloning of a human cannabinoid receptor
which is also expressed in testis. Biochem. J. 279: 129–134, 1991.
13. Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J.
Sigworth. Improved patch-clamp techniques for high-resolution
current recording from cells and cell-free membrane patches.
Pflügers Arch. 391: 85–100, 1981.
14. Hansen, H. S., L. Lauritzen, A. M. Strand, A. M. Vinggaard,
A. Frandsen, and A. Schousboe. Characterization of glutamate-
induced formation of N- acylphosphatidylethanolamine and
N-acylethanolamine in cultured neocortical neurons. J. Neuro-
chem. 69: 753–761, 1997.
15. Haws, C. M., P. A. Slesinger, and J. B. Lansman. Dihydropyri-
dine- and omega-conotoxin-sensitive Ca2⫹ currents in cerebral
neurons: persistent block of L-type channels by a pertussis
toxin-sensitive G-protein. J. Neurosci. 13: 1148–1156, 1993.
16. Henry, D. J., and C. Chavkin. Activation of inwardly rectifying
potassium channels (GIRK 1) by co-expressed rat brain cannabi-
noid receptors in Xenopus oocytes. Neurosci. Lett. 186: 91–94,
1995.
17. Hescheler, J., and G. Schultz. Heterotrimeric G proteins
involved in the modulation of voltage-dependent calcium chan-
nels of neuroendocrine cells. Ann. NY Acad. Sci. 733: 306–312,
1994.
18. Howlett, A. C. Cannabinoid inhibition of adenylate cyclase.
Biochemistry of the response in neuroblastoma cell membranes.
Mol. Pharmacol. 27: 429–436, 1985.
19. Howlett, A. C., J. M. Qualy, and L. L. Khachatrian. Involve-
ment of Gi in the inhibition of adenylate cyclase by cannabimi-
metic drugs. Mol. Pharmacol. 29: 307–313, 1986.
20. Kempe, K., F. F. Hsu, A. Bohrer, and J. Turk. Isotope dilution
mass spectrometric measurements indicate that arachidonyletha-
nolamide, the proposed endogenous ligand of the cannabinoid
receptor, accumulates in rat brain tissue post mortem but is
contained at low levels in or is absent from fresh tissue. J. Biol.
Chem. 271: 17287–17295, 1996.
21. Lake, K. D., B. R. Martin, G. Kunos, and K. Varga. Cardiovas-
cular effects of anandamide in anesthetized and conscious normo-
tensive and hypertensive rats. Hypertension 29: 1204–1210,
1997.
22. Mackie, K., W. A. Devane, and B. Hille. Anandamide, an
endogenous cannabinoid, inhibits calcium currents as a partial
agonist in N18 neuroblastoma cells. Mol. Pharmacol. 44: 498–
503, 1993.
23. Mackie, K., and B. Hille. Cannabinoids inhibit N-type calcium
channels in neuroblastoma-glioma cells. Proc. Natl. Acad. Sci.
USA 89: 3825–3829, 1992.
24. Mackie, K., Y. Lai, R. Westenbroek, and R. Mitchell. Canna-
binoids activate an inwardly rectifying potassium conductance
and inhibit Q-type calcium currents in AtT20 cells transfected
with rat brain cannabinoid receptor. J. Neurosci. 15: 6552–6561,
1995.
25. Mathew, R. J., and W. H. Wilson. Acute changes in cerebral
blood flow after smoking marijuana. Life Sci. 52: 757–767, 1993.
Downloaded from journals.physiology.org/journal/ajpheart (186.086.033.023) on August 8, 2021.
H2093
26. Matsuda, L. A., S. J. Lolait, M. J. Brownstein, A. C. Young,
and T. I. Bonner. Structure of a cannabinoid receptor and
functional expression of the cloned cDNA. Nature 346: 561–564,
1990.
27. Munro, S., K. L. Thomas, and M. Abu-Shaar. Molecular
characterization of a peripheral receptor for cannabinoids. Na-
ture 365: 61–65, 1993.
28. Pan, X., S. R. Ikeda, and D. L. Lewis. Rat brain cannabinoid
receptor modulates N-type Ca2⫹ channels in a neuronal expres-
sion system. Mol. Pharmacol. 49: 707–714, 1996.
29. Plane, F., M. Holland, G. J. Waldron, C. J. Garland, and J. P.
Boyle. Evidence that anandamide and EDHF act via different
mechanism in rat isolated mesenteric arteries. Br. J. Pharmacol.
121: 1509–1511, 1997.
30. Pratt, P. F., C. J. Hillard, W. S. Edgemond, and W. B.
Campbell. N-arachidonylethanolamide relaxation of bovine coro-
nary artery is not mediated by CB1 cannabinoid receptor. Am. J.
Physiol. 274 (Heart Circ. Physiol. 43): H375–H381, 1998.
31. Randall, M. D., S. P. H. Alexander, T. Bennett, E. A. Boyd,
J. R. Fry, S. M. Gardiner, P. A. Kemp, A. I. McCulloch, and
D. A. Kendall. An endogenous cannabinoid as an endothelium-
derived vasorelaxant. Biochem. Biophys. Res. Commun. 229:
114–120, 1996.
32. Reuter, H. Calcium channel modulation by neurotransmitter,
enzymes, and drugs. Nature 301: 569–574, 1993.
33. Rinaldi-Carmona, M., F. Barth, M. Heaulme, D. Shire, B.
Calandra, C. Congy, S. Martinex, J. Maruani, G. Neliat, D.
Caput, P. Ferrara, P. Soubrie, J. C. Breliere, and G. LeFur.
SR141716A, a potent and selective antagonist of the brain
cannabinoid receptor. FEBS Lett. 350: 240–244, 1994.
34. Schmid, P. C., R. J. Krebsbach, S. R. Perry, T. M. Dettmer,
J. L. Maasson, and H. H. O. Schmid. Occurrence and postmor-
tem generation of anandamide and other long-chain N-acyletha-
nolamines in mammalian brain. FEBS Lett. 375: 117–120, 1995.
35. Shen, M. X., T. M. Piser, V. S. Seybold, and S. A. Thayer.
Cannabinoid receptor agonists inhibit glutamatergic synaptic
transmission in rat hippocampal cultures. J. Neurosci. 16: 4322–
4334, 1996.
36. Shivachar, A. C., B. R. Martin, and E. F. Ellis. Anandamide
and ⌬9-tetrahydrocannabinol-evoked arachidonic acid mobiliza-
tion and blockade by SR141716A [N-(piperidin-1-yl)-5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxi-
mide hydrochloride]. Biochem. Pharmacol. 51: 669–676, 1996.
37. Somlyo, A. P., and A. V. Somlyo. Signal transduction and
regulation in smooth muscle. Nature 372: 231–236, 1994.
38. Stein, E. A., S. A. Fuller, W. S. Edgemond, and W. B.
Campbell. Physiological and behavioural effects of the endog-
enous cannabinoid, arachidonylethanolamide (anandamide), in
the rat. Br. J. Pharmacol. 119: 107–114, 1996.
39. Strandgaard, S., and O. B. Paulson. Regulation of cerebral
blood flow in health and disease. J. Cardiovasc. Pharmacol. 19,
Suppl. 6: S89–S93, 1992.
40. Twitchell, W., S. Brown, and K. Mackie. Cannabinoids inhibit
N- and P/Q-type calcium channels in cultured rat hippocampal
neurons. J. Neurophysiol. 78: 43–50, 1997.
41. Varga, K., K. D. Lake, D. Huangfu, P. G. Guyenet, and G.
Kunos. Mechanism of the hypotensive action of anandamide in
anesthetized rats. Hypertension 28: 682–686, 1996.
42. Varga, K., K. Lake, B. R. Martin, and G. Kunos. Novel
antagonist implicates the CB1 cannabinoid receptor in the
hypotensive action of anandamide. Eur. J. Pharmacol. 278:
279–283, 1995.
43. Wagner, J. A., K. Varga, E. F. Ellis, B. A. Rzigalinski, B. R.
Martin, and G. Kunos. Activation of peripheral CB1 cannabi-
noid receptors in haemorrhagic shock. Nature 390: 518–521,
1997.
44. White, R., and C. R. Hiley. A comparison of EDHF-mediated
and anandamide-induced relaxations in the rat isolated mesen-
teric artery. Br. J. Pharmacol. 122: 1573–1584, 1997.
45. Zygmunt, P. M., E. D. Hogestatt, K. Waldeck, G. Edwards,
A. J. Kirkup, and A. H. Weston. Studies on the effects of
anandamide in rat hepatic artery. Br. J. Pharmacol. 122: 1679–
1686, 1997.