(Received for publication, March 1, 1996)
From the
(-)--Tetrahydrocannabinol
((-)-
-THC) is the major active psychotropic
component of the marijuana plant, Cannabis sativa. The
membrane proteins that have been found to bind this material or its
derivatives have been called the cannabinoid receptors. Two GTP-binding
protein-coupled cannabinoid receptors have been cloned. CB1 or the
neuronal cannabinoid receptor is found mostly in neuronal cells and
tissues while CB2 or the peripheral cannabinoid receptor has been
detected in spleen and in several cells of the immune system. It has
previously been shown that activation of CB1 or CB2 receptors by
cannabinoid agonists inhibits adenylyl cyclase activity. Utilizing
Chinese hamster ovary cells and COS cells transfected with the
cannabinoid receptors we report that(-)-
-THC
binds to both receptors with similar affinity. However, in contrast to
its capacity to serve as an agonist for the CB1 receptor,
(-)-
-THC was only able to induce a very slight
inhibition of adenylyl cyclase at the CB2 receptor. Morever,
(-)-
-THC antagonizes the agonist-induced
inhibition of adenylyl cyclase mediated by CB2. Therefore, we conclude
that (-)-
-THC constitutes a weak antagonist for
the CB2 receptor.
Most of the original cannabinoid receptor research was devoted
to the analysis of the function of the cannabinoid receptor designated
CB1 due to its earlier cloning and the large amount of work performed
on neuronal cells and tissues, which contain this
receptor(1, 2, 3, 4, 5, 6) .
Activation of CB1 leads to inhibition of adenylyl cyclase (AC) ()(1) in various brain tissues and neuronal cells as
well as to the inhibition of N-type voltage-dependent calcium channels
in a number of in vitro systems(3, 4, 5, 6) .(-)-
-THC,
the active cannabinoid compound from Cannabis sativa, has been
shown to be a potent agonist for this receptor and to initiate various
receptor-mediated biochemical and behavioral
responses(5, 6, 7, 8, 9) .
Although the original focus of cannabinoid function was on the
nervous system, it has been noted that there are specific binding sites
for cannabinoid ligands in non-neuronal tissues and
cells(1, 10) . Cannabinoid binding sites have been
localized to regions of the mouse and rat spleen, and it has been
proposed that these sites are located on myeloid cells, B-cells, or
mast cells(10, 11, 12) . Indeed, a second
cannabinoid receptor, designated CB2, has recently been cloned from the
HL-60 promyelocytic leukemia cell line(13) . Like CB1, it
belongs to the seven-transmembrane GTP-binding protein (G
protein)-coupled receptor family and was shown to be able to bind
(-)--THC as well as
various(-)-
-THC
derivatives(13, 14, 15) .
Since(-)--THC is the active ingredient of
marijuana, which is popularly used as a mood-altering drug by many
human subjects, we and others have investigated the effect
of(-)-
-THC on the signal transduction of the CB1
receptor(2, 3, 5, 7, 8) .
It was shown that(-)-
-THC inhibits AC in
neuronal cells (e.g. NG108-15 neuroblastoma
glioma and N
TG
neuroblastoma cells) as well as
in CB1-transfected cell
lines(5, 7, 8, 16, 17) .
Since cannabinoids also have effects on immunological
functions(18, 19, 20, 21) , which
may be mediated by the CB2 receptor, it was of interest to define the
activity of (-)-
-THC on this receptor. Here we
show that although many cannabinoid agonists inhibit AC activity
through the activation of
CB2(14, 15, 22) ,(-)-
-THC
showed a very weak agonistic activity.
Moreover,(-)-
-THC reversed the effects obtained
with other cannabinoid agonists of the CB2 receptor.
Figure 1:
(-)--THC
inhibits the FSK-stimulated AC activity in N
TG
neuroblastoma cells. 100% cAMP accumulation represents AC activity in
the absence of(-)-
-THC and is equivalent to 4540
± 315 cpm. Data are means ± S.E. of two independent
experiments performed in duplicate.
Figure 2:
Effect of(-)--THC
on cAMP accumulation and [
H]HU-243 binding in COS
cells transfected with CB1 or CB2 cDNA. A shows the effect of
increasing concentrations of(-)-
-THC on
FSK-stimulated AC activity in COS cells transfected with AC type V and
CB1 or CB2 cDNAs. 100% represents cAMP accumulation in the absence
of(-)-
-THC (about 5000 cpm for both receptors). B shows the competition for binding of
[
H]HU-243 by various concentrations of
(-)-
-THC. 100% binding represents 302 ± 8
and 305 ± 18 fmol of [
H]HU-243 bound per
mg of protein to COS-CB1 and COS-CB2, respectively. Data are means
± S.E. of two independent experiments performed in
duplicate.
Figure 3:
(-)--THC is an
efficient agonist of the CB1 but not of the CB2 receptor. COS cells
were transiently cotransfected with AC type V and CB1 or CB2 cDNAs, and
the FSK-stimulated AC activity was determined in the presence of 0.1
µM of the indicated cannabinoids. The difference in AC
activity observed between HU-293a and HU-293a together with
(-)-
-THC was significant according to
Student's t test (*, p < 0.005). Data are
means ± S.E. of four independent experiments performed in
triplicate.
The difference between the results obtained with CB1- and
CB2-transfected COS cells was not due to variations in the efficiency
of AC or cannabinoid receptor transfection, since both groups of
transfected cells were equivalently stimulated by FSK (demonstrating
equivalent transfection by AC type V) and both showed similar levels of
receptor expression, as determined by specific binding of
[H]HU-243 (see Fig. 2B legend).
Moreover, as shown in Fig. 2B,(-)-
-THC binds to both
receptors on COS cells and competes with
[
H]HU-243 binding with similar affinities. The K
values of (-)-
-THC
calculated from these data are 39.5 ± 3 and 40 ± 6 nM for the CB1 and CB2 receptors, respectively. These values are
comparable with those recently reported by
others(14, 22) . The (+) isomer
of(-)-
-THC, known to be relatively inactive on
CB1(7) , showed very weak affinity for both CB1 and CB2 (data
not shown) and did not inhibit AC through either of the two receptors (Fig. 3).
This phenomenon
was observed not only with transiently transfected COS cells but also
with CHO cells stably transfected with the CB2 receptor. Fig. 4shows the dose-response curves for the inhibition of AC by
two potent cannabinoid receptor agonists in the presence or absence
of(-)--THC. It shows that HU-293a and HU-210
inhibit AC with EC
values of 8.2 ± 3 nM and 105 ± 1.2 pM, respectively. The EC
of HU-293a in the presence of 0.1 µM (-)-
-THC was shifted by 15-fold (to 125
± 2 nM) and that of HU-210 in the presence of 1
µM(-)-
-THC was shifted by 40-fold
(to 4.2 ± 2 nM). This result shows that as with the COS
7-transfected cells, the activation of the CB2 receptor in CHO cells is
antagonized by(-)-
-THC.
Figure 4:
(-)--THC
antagonizes the capacity of the cannabinoid agonists HU-293a and HU-210
to inhibit the FSK-stimulated AC activity in CHO-CB2 cells. The
cannabinoids (-)-
-THC and HU-293a (A)
or HU-210 (B) were added at the indicated concentrations. 100%
represents the amount of cAMP in the absence of cannabinoids and ranged
between 1500 and 2200 cpm. Data are means ± S.E. of two to three
independent experiments performed in
triplicate.
The two members of the cannabinoid receptor family, CB1 and
CB2, have been shown to share approximately 68% homology in their amino
acid sequence(13) . Until today, no selective agonists have
been found, and no marked differences between the two receptors have
been reported for agonist binding
parameters(10, 13, 14, 15, 22) .
(-)--THC is the most active psychotropic
compound in C. sativa. Here, we demonstrate that this agonist
is functionally selective in activating the CB1 but not the CB2
receptor.
(-)--THC inhibits the
FSK-stimulated AC activity in N
TG
neuroblastoma as well as in CB1-transfected CHO or COS
cells(1, 2, 7, 8, 15, 31) .
We and others have recently demonstrated that various cannabinoids
(including HU-243, HU-210, HU-293, HU-293a, WIN 55, 212-2,
and(-)CP55,940) serve as agonists of the peripheral cannabinoid
receptor and inhibit AC activity (14, 15, 22) . On the other
hand,(-)-
-THC did not significantly inhibit the
FSK-stimulated AC activity via this receptor (15) .
However,(-)-
-THC binds to the CB2 receptor with
the same affinity as it binds to the CB1 receptor. Therefore, it should
inhibit the action of an agonist. Here we show that
(-)-
-THC blocks the agonistic activity of other
cannabinoid ligands, such as HU-293a and HU-210, on the CB2 receptor,
shifting the AC inhibition curves to the right by more than 1 order of
magnitude, thus serving as a partial agonist/antagonist of the CB2
receptor. It has been shown that(-)-
-THC is
chemically stable when applied to cells in culture(5) . It thus
seems that the antagonistic effect is due
to(-)-
-THC itself and does not result from the
formation of a possible degradation product. The effect
of(-)-
-THC antagonism was dependent on the
affinity of the agonist, and as agonist affinity for the receptor
increased, higher concentrations of (-)-
-THC
were required to antagonize the agonist efficiently (see Fig. 4). It is of interest to note that anandamide also did not
efficiently inhibit AC activity in B6C3F1 mouse splenocytes (20) or in CHO transfected with CB2 receptor (15) and
that at low concentrations, it partially blocked CB1-mediated agonistic
activity of(-)-
-THC both in vivo and in vitro(32) .
The difference in the response
to(-)--THC leads to the conclusion that it is
related to intrinsic differences between the CB1 and CB2 receptors
themselves. The two cannabinoid receptors share high homology in their
transmembrane domains, whereas the intracellular loops, which are known
to mediate the signaling of G protein-coupled receptors(33) ,
are significantly different(13) . Line up of the two sequences
demonstrates that the CB2 receptor lacks a group of 13 amino acids in
its IC
loop as compared with CB1(13) . The
difference of 13 amino acids in the IC
between the CB1 and
CB2 receptors may affect the efficacy of receptor-G-protein coupling.
In cases when the efficacy of coupling of the G-protein to the receptor
is low, a weaker agonist may not be able to activate the receptor
efficiently even though the ligand binds to the receptor. We previously
observed that(-)-
-THC is a weaker agonist than
HU-210 or HU-293a(15) . Consequently, this could serve as the
basis for the functional selectivity observed
for(-)-
-THC. Similar situations have recently
been reported for partial agonists of the muscarinic
receptor(34, 35) . Additional differences between the
sequences of the cannabinoid receptors are present in the transmembrane
and extracellular domains and may also be involved in the
(-)-
-THC partial agonism/antagonism phenomenon.
According to the above data,(-)--THC does not
mediate a strong signal through the CB2 receptor. It has been shown
that cannabinoids affect cAMP levels and other modulatory factors in
immune system cells, but the concentrations
of(-)-
-THC used in these experiments exceed
those required to mediate effects in the central nervous
system(19, 20) . Moreover, the exact repertoire of
cannabinoid binding sites in these cells is not completely clear.
Cannabinoids have been shown to modulate proliferation of B-cells, and
the CB2 receptor was implicated in this activity; however,
(-)-
-THC was much less potent than CP55,940 and
WIN 55,212-2(18) . The exact nature of the signaling
process of the cannabinoid receptors in these cells remains to be
elucidated. Only with the development of specific potent antagonists
for CB2, like the one that has been developed for CB1(36) ,
will it be possible to observe the effects of blocking the activities
of the CB2 receptor in both in vivo and in vitro systems. The results presented above indicate
that(-)-
-THC is a weak antagonist for the CB2
receptor. Its structure can therefore serve as a model for the chemical
synthesis of such an antagonist.