The cannabinoid agonist WIN55,212-2 increases
intracellular calcium via CB1 receptor coupling
to Gq/11 G proteins
Jane E. Lauckner†‡, Bertil Hille‡§, and Ken Mackie†¶
†Department of Physiology and Biophysics, ‡Neurobiology and Behavior Graduate Program, and ¶Department of Anesthesiology, University of Washington,
Seattle, WA 98195
Contributed by Bertil Hille, November 3, 2005
Central nervous system responses to cannabis are primarily mediated by CB1 receptors, which couple preferentially to Gi/o G proteins. Here, we used calcium photometry to monitor the effect of
CB1 activation on intracellular calcium concentration. Perfusion
with 5 ␮M CB1 aminoalkylindole agonist, WIN55,212-2 (WIN),
increased intracellular calcium by several hundred nanomolar in
human embryonic kidney 293 cells stably expressing CB1 and in
cultured hippocampal neurons. The increase was blocked by coincubation with the CB1 antagonist, SR141716A, and was absent in
nontransfected human embryonic kidney 293 cells. The calcium rise
was WIN-specific, being essentially absent in cells treated with
other classes of cannabinoid agonists, including ⌬9-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was
independent of Gi/o, because it was present in pertussis toxintreated cells. Indeed, pertussis toxin pretreatment enhanced the
potency and efficacy of WIN to increase intracellular calcium. The
calcium increases appeared to be mediated by Gq G proteins and
phospholipase C, because they were markedly attenuated in cells
expressing dominant-negative Gq or treated with the phospholipase C inhibitors U73122 and ET-18-OCH3 and were accompanied
by an increase in inositol phosphates. The calcium increase was
blocked by the sarco兾endoplasmic reticulum Ca2ⴙ pump inhibitor
thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and
1,1ⴕ-diheptyl-4,4ⴕ-bipyridinium dibromide, but not by removal of
extracellular calcium, showing that WIN releases calcium from
intracellular stores. In summary, these results suggest that WIN
stabilizes CB1 receptors in a conformation that enables Gq signaling, thus shifting the G protein specificity of the receptor.
aminoalkylindole 兩 inositol phosphate 兩 neurons 兩 phospholipase C
T
he CB1 cannabinoid receptor (CB1) mediates the majority of
the psychotropic and behavioral effects of cannabis (1, 2).
CB1 is a member of the heptahelical G protein-coupled receptor
(GPCR) superfamily (1). It couples via pertussis toxin (PTX)sensitive Gi/o G proteins to inhibit adenylyl cyclase and L-, N-,
and P兾Q-type calcium channels and to activate potassium channels and mitogen-activated protein kinase (2). Rarely, CB1 has
also been found to couple to phospholipase C (PLC) in a
PTX-sensitive manner involving the ␤␥ subunits from Gi/o (3, 4).
Several distinct agonists activate CB1. The classical cannabinoids are tricyclic dibenzopyran compounds. The prototype of
this group is ⌬9-tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabis. THC is a low-affinity partial
agonist for CB1 (2), whereas other synthetic classical compounds
such as HU-210 are both more potent and efficacious (2).
Nonclassical cannabinoids lack the dihydropyran ring yet maintain some similarity to THC’s stereochemistry. The best known,
CP55,940 (CP), is a potent and efficacious CB1 agonist. The third
group, the eicosanoids, includes endogenous ligands for CB1,
such as anandamide and 2-arachidonoylglycerol (2-AG), and
their analogs (5, 6). The aminoalkylindoles (AAIs) were syn19144 –19149 兩 PNAS 兩 December 27, 2005 兩 vol. 102 兩 no. 52
thesized as antiinflammatory drugs, with the goal to reduce
gastrointestinal side effects. When tested, they were first found
to have potent antinociceptive activity and weak antiinflammatory properties (7) and later discovered to bind to cannabinoid
receptors (8). AAIs, and in particular WIN55,212-2 (WIN),
produce the full spectrum of in vivo effects seen with THC and
other cannabimimetic agonists (9).
Here, we are concerned with the G protein specificity of CB1.
Initial dogma was that each GPCR interacts with a certain class
of G protein. Subsequently, more promiscuous GPCR–G protein
coupling was recognized. Multiplicity in G protein coupling
activates multiple second-messenger cascades, each with distinct
effects. Such promiscuity should allow a cell greater range in
response to activation of a single GPCR. Some examples include
D1 dopamine and ␣2-adrenergic receptors, which couple to both
Gs and Gi, and the PACAP type I and H2 histamine receptors,
which activate both Gs and Gq/11 (10–13). CB1 has been shown
to activate both Gi/o and Gs (14). This multiplicity in G protein
coupling can be agonist-regulated. The prevailing idea here is
that receptors exist in different activated states capable of
coupling to different G proteins. Binding of a specific agonist
stabilizes a distinct active state, favoring coupling to a certain G
protein (15, 16). Examples of this have been reported for A1
adenosine receptors and the Ca2⫹-sensing receptor (17, 18). In
this study, we report the previously undescribed observation that
CB1 couples to Gq/11 G proteins in an agonist-specific manner to
increase the concentration of intracellular calcium.
Materials and Methods
Cells. Human embryonic kidney (HEK) 293 cells stably express-
ing rat CB1 cannabinoid receptor (CB1-HEK293) were generated by using standard techniques (19). Most experiments were
done with cells expressing CB1 at a density of 2.2 pmol兾mg
protein. Before an experiment, cells were plated onto poly
D-lysine-coated coverslips and, when required, transfected the
next day. cDNA for additional genes (M1R and dominantnegative G␣q) were transiently transfected with Lipofectamine
2000. DsRed (0.5 ␮g) was cotransfected as a transfection marker.
Cells were used for photometry experiments 24–36 h after
transfection. Hippocampal neurons were cultured according to
the method of Brewer (20).
Dye Loading and Photometry. Cytosolic Ca2⫹ was monitored with
the ratiometric calcium indicator fura-2. Cells were loaded at
Conflict of interest statement: No conflicts declared.
Abbreviations: CB1, CB1 cannabinoid receptor; AAI, aminoalkylindole; THC, ⌬9-tetrahydrocannabinol; WIN, WIN55,212-2; CP, CP55,940; 2-AG, 2-arachidonoylglycerol; HEK, human
embryonic kidney; PTX, pertussis toxin; PLC, phospholipase C; IP, inositol phosphate; IP3,
inositol 1,4,5-trisphosphate; GPCR, G protein-coupled receptor; [Ca2⫹]i, intracellular calcium concentration; ER, endoplasmic reticulum; SERCA, sarco兾ER Ca2⫹-ATPase; M1R, M1
muscarinic receptor; TG, thapsigargin; XeD, xestospongin D; SR, SR141716A; Oxo-M,
oxotremorine-M; RyR, ryanodine receptor.
§To
whom correspondence should be addressed. E-mail: [email protected].
© 2005 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0509588102
Receptor Expression Studies and Measurement of [3H]Inositol Phosphate (IP) Formation. To determine the level of CB1 expression,
CB1-HEK293 cells were grown in 10-cm plates and membrane
binding performed with [3H]CP, as described (22). [3H]Inositol
phosphate production was measured in cells loaded with myo[2-3H]inositol by using established techniques (23).
Solutions and Materials. Fura-2-AM and pluronic 127 were from
Molecular Probes, and thapsigargin (TG), xestospongin D
(XeD), and ET-18-OCH3 were from Calbiochem. PTX was from
List Biological Laboratories (Campbell, CA); and HU210,
U73122, and 1,1⬘-diheptyl-4,4⬘-bipyridinium dibromide were
from Tocris Cookson (Ellisville, MO). SR141716A (SR), THC,
and CP were supplied by the National Institute on Drug Abuse
Research Resources Drug Supply System. Zeocin, DMEM, FBS,
Lipofectamine 2000, and penicillin兾streptomycin were from
Invitrogen. The dominant-negative G␣q (Q209L兾D277N) construct was obtained from the Guthrie Research Institute (Sayre,
PA). AM356 was a gift of A. Makriyannis (Northeastern University, Boston). Cannabidiol, [3H]CP, and myo-[2-3H]inositol
were gifts from N. Stella (University of Washington, Seattle). All
other chemicals were from Sigma.
The control mammalian Ringer’s bath solution contained 160
mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
Hepes, and 8 mM glucose adjusted to pH 7.4 with NaOH.
Ca2⫹-free Ringer’s solution was the same as control solution
without CaCl2 and with 0.5 mM EGTA. PTX and oxotremorine-M (Oxo-M) were dissolved in water. All other compounds
were dissolved in DMSO. Final dilutions were made with
Ringer’s bath solution (DMSO ⬍0.05%). One milligram per
milliliter BSA was added as a carrier to all cannabinoidcontaining solutions except those used in the receptor expression
and IP measurements. After experiments, perfusion lines and tip
were flushed with 100% ethanol followed by dH2O. For the
receptor expression and IP formation assays, siliconized tubes
and pipette tips were used for drug preparation and delivery.
Results
WIN Evokes a Large Slow CB1-Specific Increase in Intracellular Calcium
Concentration Perfusion of 5 ␮M of the cannabimimetic AAI
WIN raised intracellular calcium in CB1-HEK293 cells by ⬎500
Lauckner et al.
Fig. 1. Activation of CB1 by WIN increased [Ca2⫹]i to a variable extent and
with varied kinetics. (A) The time course of changes in [Ca2⫹]i in HEK293 cells
loaded with fura-2 and perfused with 5 ␮M WIN. The lines above indicate drug
application. The solid line indicates the WIN-induced response in CB1-HEK293
(n ⫽ 8). A subset of CB1-HEK293 were coperfused with 1 ␮M of the CB1
antagonist SR (dashed line, n ⫽ 5). The change in [Ca2⫹]i in response to WIN in
nontransfected HEK293 cells is also shown (dotted line, n ⫽ 8). (B) Calcium
elevations with WIN recorded in four different CB1-HEK293 cells, tested on 4
different days. (C) CB1-HEK293 were perfused with 5 ␮M WIN (solid line), and
HEK293 cells transiently expressing M1R were perfused with 10 ␮M Oxo-M
(dashed line). These traces show the change in calcium in representative cells.
(D) The black bar shows the 10 –90% rise time for the calcium caused by 5 ␮M
WIN in CB1-HEK293 (n ⫽ 6). Open bar indicates the calcium rise time caused by
a 40-s application of 10 ␮M Oxo-M in HEK293 cells transiently expressing M1R
(n ⫽ 7). The rise times were significantly different (P ⬍ 0.01, t test).
nM (Fig. 1A). Perfusion of 5 ␮M WIN also increased intracellular calcium in HEK293 cells transiently expressing rCB1 (790 ⫾
120 nM, n ⫽ 9). The CB1 specificity of this response was shown
in three ways. First, coapplication of 1 ␮M of the CB1 antagonist
SR with WIN markedly attenuated the calcium increase (Fig.
1 A). Second, perfusion of 5 ␮M WIN55,212-3, the inactive
enantiomer of WIN (2), did not increase intracellular calcium
(data not shown). Third, the transient was absent in nontransfected HEK293 cells perfused with WIN (Fig. 1 A). WIN caused
a calcium transient in 100% of cells expressing CB1 (121兾121),
although the amplitude and speed of the response varied among
cells (Fig. 1B). Overall, the WIN-induced calcium increase was
markedly slower than that caused by 10 ␮M of the muscarinic
receptor agonist, Oxo-M, in HEK293 cells expressing M1R. The
M1R-mediated calcium increase had a 10–90% rise time of 4 ⫾
1 s, significantly faster than the WIN-induced calcium rise time
of 40 ⫾ 8 s (P ⬍ 0.01) (Fig. 1 C and D).
The effect of WIN is not restricted to expression systems. Five
micromolar WIN caused a calcium rise in cultured mouse
hippocampal neurons like that in CB1-HEK293 (the F340/380 ratio
increased to 0.9, n ⫽ 9). This response also depended on CB1,
because 1 ␮M of the antagonist SR attenuated WIN-induced
F340兾380 increase by 76% (P ⬍ 0.001). WIN had no effect on
calcium levels in astrocytes, cells generally regarded to not
express CB1 (data not shown).
WIN Is the Only CB1 Agonist to Increase Intracellular Calcium Robustly.
We tested several classes of CB1 agonists at concentrations at
least 200 times the CB1-binding Kd value [with the exception of
PNAS 兩 December 27, 2005 兩 vol. 102 兩 no. 52 兩 19145
NEUROSCIENCE
room temperature for 15–20 min with fura-2 acetoxymethyl ester
(AM) dissolved in DMSO, dispersed in 20% pluronic 127, and
diluted to 8 ␮M in 0.5 ml of Ringer’s solution. Test solutions were
applied to the incubation chamber with a solenoid-controlled
gravity-fed multibarreled local perfusion device.
During [Ca2⫹]i measurements, the dye was excited by 340 and
380 nm light and a photodiode collected emission above 520 nm
(Polychrome IV TILL Photonics, Planegg, Germany). The standard calibration parameters (21), Rmin (0.35), Rmax (2.5), and K*
(613 nM), were determined from HEK293 cells equilibrated in
KCl-based internal solutions containing ionomycin (10 ␮M) and
either 20 mM EGTA, 15 mM CaCl2, or 20 mM EGTA ⫹ 15 mM
CaCl2 (149 nM free Ca2⫹). For experiments with hippocampal
neurons, the relative [Ca2⫹]i is given as the fluorescence ratio
F340兾F380. Photometric measurements were analyzed in Igor
(WaveMetrics, Lake Oswego, OR) and statistical analyses performed in EXCEL (Microsoft), except ANOVAs, which were
performed on the statistical methods web site, developed by Tom
Kirkman at the College of St. Benedict兾St. John’s University (St.
Joseph and Collegeville, MN). All results are presented as
mean ⫾ SEM. Each drug was tested on at least 2 different days,
with concurrent interleaved controls. Each calcium measurement represents a single cell from an individual coverslip. The
measurement ‘‘rise in [Ca2⫹]i’’ was calculated as: maximum
[Ca2⫹]i during drug application ⫺ basal [Ca2⫹]i, where basal
[Ca2⫹]i was the mean [Ca2⫹]i for 47 s before drug application.
Fig. 2. Only the AAI agonist WIN caused a large increase in [Ca2⫹]i in
CB1-HEK293. The WIN-induced change in [Ca2⫹]i (black bar, n ⫽ 5) is significantly greater than that of other agonists (P ⬍ 0.05, ANOVA). Agonists tested:
the classical cannabinoids, THC (10 ␮M, n ⫽ 9), HU-210 (1 ␮M, n ⫽ 4), the
phytocannabinoid, cannabidiol (CBD, 3 ␮M, n ⫽ 4), the nonclassical cannabinoid, CP (n ⫽ 6), the endocannabinoid, 2-AG (n ⫽ 5), and the anandamide
analog, AM356 (n ⫽ 9). All agonists were perfused for 150 s.
2-AG, which was used at 20 times its Kd (2)]. THC (10 ␮M) raised
calcium by only 40 ⫾ 15 nM (Fig. 2), which is well within the
range seen for resting cells and was only 5% of the WIN-induced
increase. Agonists structurally similar to THC, such as cannabidiol (CBD, 3 ␮M), also showed no significant effects, with the
exception of 1 ␮M HU-210, which increased calcium slightly in
CB1-HEK293 cells (Fig. 2), but not in hippocampal neurons.
Neither the nonclassical cannabinoid, CP55,940 (CP, 1 ␮M), nor
the endogenous cannabinoid, 2-AG (1 ␮M), nor the metabolically stable anandamide analog, methanandamide (AM356, 10
␮M), caused a significant calcium increase (Fig. 2). The increase
in intracellular calcium by WIN was significantly more than for
other agonists (ANOVA, 95% confidence interval).
WIN Increases Intracellular Calcium via PTX-Insensitive G Proteins
from the Gq/11 Family. To determine whether the WIN-evoked
calcium increase was mediated by members of the Gi/o protein
family, cells were pretreated for 16 h with 500 ng兾ml PTX. Local
perfusion of these cells with increasing concentrations of WIN
revealed a concentration-dependent increase in intracellular
calcium. Unexpectedly, the increase was augmented by PTX at
all WIN concentrations (Fig. 3A). This augmentation was particularly dramatic for 1 ␮M WIN, where PTX increased the
calcium rise by 1,200%. PTX increased the 3 ␮M WIN-evoked
calcium rise by 220%, and the 5 ␮M WIN-evoked calcium rise
by 120% (Fig. 3A). PTX pretreatment did not affect the 10–90%
rise times for the WIN-evoked calcium increase (Fig. 3B). The
calcium increase in PTX-treated cells was mediated by CB1
receptors, because it was blocked by 1 ␮M SR (data not shown).
Fig. 3. PTX pretreatment augmented the WIN-induced [Ca2⫹]i increase in
CB1-HEK293. Black bars indicate the response in untreated cells, and open bars
indicate overnight treatment with 500 ng兾ml PTX. (A) Pretreatment with PTX
enhanced the rise in [Ca2⫹]i at all WIN concentrations. n ⫽ 5 for all conditions.
(B) PTX pretreatment had no effect on the 10 –90% rise time for the calcium
rise following perfusion of 3 or 5 ␮M WIN.
19146 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0509588102
Fig. 4. Gq/11 is involved in the WIN-induced increase in [Ca2⫹]i. (A) The rise in
[Ca2⫹]i during perfusion of either 5 ␮M WIN (left three bars) or 10 ␮M Oxo-M
(right two bars) is plotted. Control cells were not treated with PTX and did not
express dominant negative G␣q (black bars, n ⫽ 10 for WIN control, n ⫽ 4 for
Oxo-M control). The rise in [Ca2⫹]i in HEK293 cells expressing DN G␣q was less
than control (P ⬍ 0.001 for WIN-treated and P ⬍ 0.05 for Oxo-M treated, t test).
PTX pretreatment (open bar, n ⫽ 5) augments the rise in calcium (P ⬍ 0.05, t
test). (B) The change in calcium upon WIN application in a representative cell
for each treatment. Solid line indicates WIN only, dashed line is WIN⫹DN G␣q,
and dotted line is WIN⫹PTX.
In cells pretreated with heat-inactivated PTX, the WINincreased calcium was like that in untreated cells (data not
shown). The WIN-induced calcium rise in cultured hippocampal
neurons was also PTX-insensitive. WIN increased the F340/380
ratio to 0.5 ⫾ 0.1 (n ⫽ 4). After PTX treatment, WIN increased
the F340/380 ratio to 0.4 ⫾ 0.1 (n ⫽ 4). Activity of the PTX toward
Gi/o was confirmed by its ability to block CP-stimulated activation of mitogen-activated protein kinase (data not shown).
Together, these data show that the pathway we describe here is
not inhibited by PTX, so it does not involve Gi/o G proteins.
PTX pretreatment enhanced the calcium rise in response to 1
␮M CP to a much lesser extent. CP alone caused a negligible
calcium increase of 10 ⫾ 1 nM (n ⫽ 6). Overnight treatment with
PTX increased the calcium rise in response to CP to 28 ⫾ 5 nM
(n ⫽ 5, P ⬍ 0.05). Unlike PTX, heat-inactivated PTX did not
augment the response to CP (8 ⫾ 3 nM, n ⫽ 5). PTX pretreatment had small and nonsignificant effects on the calcium rise
following perfusion of 10 ␮M THC and 10 ␮M AM356 (data not
shown).
Because inhibition of Gi/o G proteins did not reduce the
WIN-induced calcium increase, we investigated the importance
of the PTX-resistant Gq/11 family of G proteins. Transient
expression of a dominant-negative G␣q (DN G␣q, Q209L兾
D277N) (24) suppressed the WIN-induced transient by 70%
(P ⬍ 0.001, Fig. 4A). M1R activation increases intracellular
calcium by Gq G proteins (25). Thus, as a positive control for DN
G␣q, we checked its action on the M1R-mediated calcium
transient. In cells transiently expressing M1R, overexpression of
DN G␣q reduced the 10 ␮M Oxo-M calcium increase by 40%
(P ⬍ 0.05, Fig. 4A). Thus, Gq/11 G proteins are involved in the
WIN-induced calcium increase.
The WIN-Induced Calcium Rise Depends on PLC Activation. The major
downstream effector for G␣q is PLC␤ (26). PLC␤ is a likely
candidate for the pathway identified in this study, because it
Lauckner et al.
cleaves the membrane lipid phosphatidylinositiol-4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol, both of which modulate intracellular
calcium. To test for the involvement of PLC, CB1-HEK293 cells
were pretreated for 160 s with 3 ␮M of the PLC inhibitor U73122
followed immediately by 5 ␮M WIN. This concentration of
U73122 has been shown to inhibit PLC (27) yet not increase
intracellular calcium on its own (Fig. 5B). The latter is an
important point, because U73122 at concentrations ⬎3 ␮M can
increase intracellular calcium concentration by releasing calcium
from intracellular stores (28). U73122 (3 ␮M) suppressed the 5
␮M WIN-evoked calcium transient by 80% (P ⬍ 0.01, Fig. 5A).
The same concentration of its inactive analog, U73343, affected
neither the calcium rise in response to 5 ␮M WIN (n ⫽ 4, Fig.
5A) nor intracellular calcium by itself (Fig. 5B). As a positive
control for U73122, we verified that it inhibited the M1Rinduced calcium rise, which requires PLC␤ (29). In cells transiently expressing M1R, 3 ␮M U73122 decreased the calcium rise
in response to 5 ␮M Oxo-M by 70%. The phosphatidylinositolspecific PLC inhibitor, ET-18-OCH3 (30), also attenuated the
WIN-induced calcium rise. A 20-min pretreatment with 15 ␮M
ET-18-OCH3 reduced the calcium increase by 70% (180 ⫾ 60
nM; n ⫽ 6) compared with that in control cells treated with 5 ␮M
WIN (650 ⫾ 145 nM; n ⫽ 12; P ⬍ 0.001; t test).
Accumulation of IP derivatives is a measure of PLC activity.
In CB1-HEK293 cells, 5 ␮M WIN (n ⫽ 6) increased IP levels by
90% over basal amounts (measured in untreated cells, n ⫽ 9, P ⬍
0.01; t test), as well as 80% over amounts measured in nontransfected cells (n ⫽ 6, P ⬍ 0.01; t test) stimulated with the same
concentration of WIN (Fig. 6). Incubation with 3 ␮M WIN also
increased IP levels (Fig. 6). As a positive control, 10 ␮M Oxo-M
increased IP accumulation by 210% over basal amounts in
HEK293 cells transiently expressing M1R (n ⫽ 9, P ⬍ 0.002; t
test), and by 150% over levels in nontransfected cells also treated
with 10 ␮M Oxo-M (n ⫽ 6, P ⫽ 0.006; t test, Fig. 6). IP production
was PLC-specific: 3 ␮M U73122 suppressed both the 3 ␮M and
5 ␮M WIN-mediated increases in IP accumulation by 70%, and
the 10 ␮M Oxo-M-mediated increase by 80% (data not shown).
The WIN-Evoked Calcium Rise Comes from IP3- and Ryanodine-Sensitive Intracellular Calcium Stores Two main candidate calcium
sources could contribute to the intracellular calcium increase.
First, extracellular calcium might enter through plasmalemmal
ion channels. Second, calcium might be released from the
endoplasmic reticulum (ER) or another intracellular pool. A
role for extracellular calcium was assessed by using a calciumLauckner et al.
free medium (0 Ca Ringer’s). External calcium entry did not
appear to be important, because the calcium transient was
unaffected when WIN was delivered in 0 Ca Ringer’s (Fig. 7).
The latency to onset and the time course of the calcium increase
were unchanged (Fig. 7 Inset).
Because CB1 activation did not promote calcium entry, it was
likely that calcium was being released from intracellular stores.
The sarco兾endoplasmic reticulum Ca2⫹ (SERCA) pump is an
ATP-dependent pump located on the ER surface that maintains
and replenishes agonist-sensitive calcium stores. If the SERCA
pump is blocked, the stores empty as ER calcium leaks out and
is not replaced. CB1-HEK cells transiently transfected with M1R
were incubated with 1 ␮M of the SERCA pump inhibitor
thapsigargin (TG) in 0 Ca Ringer’s for 20 min before the
experiment, a treatment sufficient to empty TG-sensitive intracellular stores (31). TG pretreatment reduced the 5 ␮M WIN
calcium transient by 90% (P ⬍ 0.05 by ANOVA, Fig. 7). As a
positive control, the same TG treatment almost abolished the
calcium increase from M1R activation, known to be mediated by
TG- and IP3-sensitive stores (29) (data not shown). These
Fig. 7. WIN releases calcium from IP3- and Ry-sensitive intracellular stores. In
CB1-HEK293, the 5 ␮M WIN-induced rise in [Ca2⫹]i did not require extracellular
calcium (‘‘0 Ca,’’ n ⫽ 12 vs. ‘‘Control,’’ black bar, n ⫽ 16). One micromolar TG
(n ⫽ 13), the IP3R inhibitor XeD, 1 ␮M, n ⫽ 10), the RyR inhibitors dantrolene
(Dan, 10 ␮M, n ⫽ 11) and 1,1⬘-diheptyl-4,4⬘-bipyridinium dibromide (DHBP)
(50 ␮M, n ⫽ 6) all reduced calcium rises in response to WIN (P ⬍ 0.05, ANOVA),
as did coapplied XeD and Dan (n ⫽ 4). The reversible SERCA pump inhibitor
2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) in the absence of WIN, modestly
increased calcium (100 ␮M, n ⫽ 5, P ⬍ 0.05, ANOVA). (Inset) Removal of
extracellular calcium (‘‘0 Ca,’’ open bar, n ⫽ 12) affected neither the latency
nor rise time of the calcium transient (‘‘Control,’’ black bar, n ⫽ 16). Latency
was calculated as the time from start of drug application until the [Ca2⫹]i rose
to 10% of the maximum value.
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Fig. 5. PLC mediates the increase in [Ca2⫹]i by WIN. (A) CB1-HEK293s were
either untreated (black bar, n ⫽ 12) or pretreated for 160 s with either 3 ␮M
of the PLC inhibitor U73122 (n ⫽ 5) or 3 ␮M of its inactive analog U73343 (n ⫽
4) followed immediately by perfusion of 5 ␮M WIN for 150 s. U73122 strongly
suppressed the calcium rise (P ⬍ 0.01, t test), whereas U73343 had no significant effect. (B) Neither 3 ␮M U73122 (solid line) nor 3 ␮M U73343 (dashed
line) increased [Ca2⫹]i on its own.
Fig. 6. WIN increases IP levels. CB1-HEK293 transiently transfected with M1R
(solid bars, n ⫽ 9) and nontransfected HEK293 cells (open bars, n ⫽ 6) were
stimulated with agonist for 10 min and then assayed for IP production. IP levels
increased in CB1-HEK293 treated with either 3 or 5 ␮M WIN compared with
basal levels or levels in nontransfected controls. In cells expressing M1R, 10 ␮M
Oxo-M robustly increased IP accumulation. *, comparison with basal control;
†, comparison with nontransfected HEK293 cells treated with the same agonist concentration. * and †, P ⬍ 0.05; ** and ††, P ⬍ 0.01.
observations suggest that either the calcium is released from
TG-sensitive intracellular calcium stores or that WIN inhibits the
SERCA pump. In the latter case, the rise in intracellular calcium
concentration would be due to leakage of calcium from SERCA
pump-dependent stores. To test this possibility, we applied 100
␮M 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), a fast and
reversible SERCA pump inhibitor, and measured the change in
intracellular calcium. We reasoned that if BHQ perfusion increased calcium to a lesser extent than WIN, we could discount
the possibility that the actions of WIN were solely due to SERCA
pump inhibition. BHQ treatment elicited a calcium increase only
10% of that seen with WIN (P ⬍ 0.05 by ANOVA, Fig. 7). This
suggests that inhibition of SERCA pump activity is not the major
cause of the WIN-evoked calcium transient.
IP3-sensitive ER calcium stores were a likely source contributing to our calcium increase because IP3 is produced by PLC␤,
which is central in the pathway (Figs. 5 and 6). A 30-s pretreatment with 1 ␮M of the IP3R inhibitor xestospongin D (XeD) in
0 Ca Ringer’s immediately followed by perfusion of 5 ␮M WIN
reduced the calcium rise by 75% (P ⬍ 0.05 by ANOVA, Fig. 7).
The ryanodine receptor (RyR) also plays a role in calcium
release, because pretreatment for 20–25 min with 10 ␮M of the
RyR inhibitor dantrolene in 0 Ca Ringer’s reduced the calcium
rise by 50% (P ⬍ 0.05 by ANOVA, Fig. 7). As further evidence
for RyR involvement, treatment with its inhibitor, 1,1⬘-diheptyl4,4⬘-bipyridinium dibromide (DHBP, 50 ␮M) attenuated the
WIN-evoked calcium increase by 70% (P ⬍ 0.05 by ANOVA,
Fig. 7). Pretreatment with both XeD and dantrolene decreased
calcium transients to 15% of that seen in untreated cells perfused
with WIN (P ⬍ 0.05 by ANOVA, Fig. 7). These data suggest that
the calcium transient is the result of calcium release from IP3and Ry- sensitive intracellular stores.
Discussion
We have found that WIN, a cannabinoid AAI agonist, can
increase intracellular calcium in CB1-HEK293 cells, as well as in
cultured hippocampal neurons. Because the WIN-induced calcium increase was absent in untransfected HEK293 cells and was
blocked by a CB1 antagonist, it is specific. The large slow
WIN-induced rise in intracellular calcium was PTX-insensitive
and required Gq/11 G proteins and PLC. WIN increased phosphatidyl inositide turnover, as measured by IP production, and
the IP3 released calcium from IP3-sensitive ER stores. RyRs also
contributed to the calcium release.
Other groups have reported instances where CB1 activation
increases intracellular calcium (3, 4, 32–35). Generally, the
reported calcium rises were more modest than those we report
here. Unlike our results, the calcium rise in the earlier studies
was mediated by PTX-sensitive Gi/o ␤␥ acting via PLC to release
calcium from TG-sensitive intracellular stores (3, 4, 32, 33, 35).
The one exception was a report that the nonclassical cannabinoid
agonist, DALN, increases intracellular calcium by enhancing the
entry of calcium ions through L-type calcium channels by a
PTX-insensitive mechanism (34). Because HEK293 cells do not
have L-type voltage-sensitive calcium channels, this could not be
the mechanism for the calcium increase we report here. Thus,
our study provides evidence that CB1 functionally couples to G
proteins from the Gq/11 family to increase intracellular calcium.
The WIN-induced calcium increase identified here is agonistspecific. For example, WIN was more potent and efficacious in
increasing intracellular calcium than CP. In contrast, WIN is less
potent than CP at inhibiting cAMP production, a Gi-coupled
pathway (36). As a working hypothesis, we propose that CB1
exists in several active states; the relative ratio of each is a
function of the ligand. WIN likely stabilizes a conformation of
CB1 that couples more readily to Gq/11. The reversal of agonist
potency in the inhibition of cAMP production (36) supports this
hypothesis. Similarly, accumulating evidence suggests that the
19148 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0509588102
binding site for WIN only partially overlaps that for other CB1
agonists. Although WIN displaces CP from CB1 in radioligandbinding assays (2), mutagenesis studies uncovered an amino acid
residue (K192) in helix three of CB1 that, when mutated to
alanine, abolished CP binding with little effect on binding or
receptor activation by WIN (37). A second mutation, where
V282 in helix five was mutated to phenylalanine, increased the
affinity of CB1 for WIN with no effect on affinity for CP (38).
Favored interactions with distinct residues may result in WIN
preferentially stabilizing a specific active conformation of CB1
that couples not only to Gi/o but also to Gq/11. Other structurally
related AAIs may also enhance CB1 coupling to Gq/11.
A relatively high concentration of WIN (5 ␮M) was required
for a substantial calcium increase (⬎400 nM); however, many
published studies use concentrations in this range or higher.
WIN-activated CB1 appears to couple preferentially to Gi/o, and
only when receptor occupancy is very high or when the ratio of
G␣q to functional Gi/o increases will coupling to less-favored G
proteins, such as Gq/11, be evident. This could explain how PTX
pretreatment enhanced the potency of WIN. By inhibiting the
Gi/o ␣ subunit, PTX prevents Gi/o heterotrimeric G proteins from
interacting with the receptor (39). This increases the likelihood
of CB1 coupling to Gq/11, leading to an enhanced calcium
response at the same WIN concentration. PTX pretreatment
also unmasked modest coupling between Gq/11 and CB1 activated
by CP and THC, but even after PTX, CP and THC were still far
less efficacious than WIN.
Our study is not the first example of agonist-driven coupling
of CB1 to a less-favored G protein. Bonhaus et al. (40) reported
a difference in the ability of cannabinoid agonists to activate
Gs-coupled pathways. They showed that WIN was more potent
and efficacious than CP in increasing forskolin-stimulated
cAMP accumulation, yet CP was more potent than WIN in
inhibiting adenylyl cyclase (40).
There are other examples of promiscuous receptors activating
different signal transduction pathways in an agonist-dependent
manner that may be explained by different receptor conformations coupling to different G proteins. Rey et al. (18) have
studied agonist-specific signal trafficking of the Ca2⫹-sensing
receptor (CaR) (18). They find that the CaR activated by
external calcium stimulates Gq兾PLC, increasing [Ca2⫹]i in an
oscillatory fashion. However, if the aromatic amino acid Lphenylalanine activates CaR, the receptor no longer couples to
Gq and instead transiently increases [Ca2⫹]i through the activation of the small GTPase Rho and G proteins of the G12
subfamily. A second example is from Cordeaux et al. (41), who
show that the A1 adenosine receptor, traditionally described as
Gi-coupled, can also couple to Gs and Gq. The efficacy of this
coupling depends on the agonist. The two agonists N(6)cyclopentyladenosine (CPA) and 5⬘-(N-ethylcarboxamido)adenosine (NECA) are equally efficacious in Gi coupling. After
PTX treatment in cells expressing a low level of A1R, NECA, but
not CPA, promoted A1R–Gs interactions. After PTX treatment
in cells expressing a high level of A1R, receptors activated by
NECA coupled to Gq more efficaciously than CPA-activated
receptors.
CB1 generally signals more slowly then other GPCRs, even
when modulating the same response. For example, Mackie and
Hille (42) found that WIN-activated CB1 inhibited N-type
calcium channels 15-fold more slowly than the norepinephrineactivated ␣2-adrenergic receptor, even at supramaximal agonist
concentrations. In the current study, the difference was just as
pronounced. The WIN-induced calcium increase was 10-fold
slower than the Oxo-M-induced calcium increase (Fig. 1D). The
reason for this marked kinetic difference is unknown. It may be
that CB1 assumes its activated conformation relatively slowly.
Another possibility is that CB1 activates G proteins less efficiently compared with other GPCRs (43). In this case, it would
Lauckner et al.
take longer to reach the threshold concentration of downstream
effectors necessary to initiate a response.
Our data indicate that unique among CB1 agonists, WIN
releases calcium from intracellular stores. WIN does so by
coupling to Gq/11 and enhancing PLC activity. Because PLC␤
initiates the production of the endocannabinoid 2-AG, this
suggests the intriguing possibility that WIN activation of CB1
might increase 2-AG levels. These results also suggest that
caution should be taken when extending observations made with
high levels of WIN to other drugs activating CB1. Although WIN
exhibits the full range of pharmacological and behavioral effects
seen with THC, there are some differences. Among these, WIN
is more potent in drug discrimination trials and in producing
We thank Dr. C. Kearn (University of Washington) for assistance in the
preparation of cultured neurons and the mitogen-activated protein
kinase assay; Dr. A. Makriyannis (Northeastern University, Boston) for
AM356; and Dr. N. Stella (University of Washington) for cannabidiol,
[3H]CP, myo-[2-3H]inositol, and, along with E. Cudaback and F. Reyes,
for help with the [3H]CP binding and [3H]inositol assays. This work was
supported by National Institutes of Health Grants DA00286, DA11322,
DA09150, DA14486, AR17803, and NS08174.
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NEUROSCIENCE
antinociception and less potent in the production of hypothermia
(9). The origin of these differences is unknown, but they may be
due to high concentrations of WIN increasing intracellular
calcium.
Lauckner et al.
PNAS 兩 December 27, 2005 兩 vol. 102 兩 no. 52 兩 19149