Neuropharmacology Vol. 26, No. 5, pp. 507-512, 1987 0028-3908/87$3.00+ 0.

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Printed in Great Britain. All rights reserved Copyright 0 1987Pergamon Journals Ltd

CANNABINOID INHIBITION OF ADENYLATE CYCLASE:

RELATIVE ACTIVITY OF CONSTITUENTS AND
METABOLITES OF MARIHUANA

A. C. HOWLETT
Department of Pharmacology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis,
MO 63104, U.S.A.

(Accepted 29 May 1986)

Summary-A9Tetrahydrocannabinol (THC) has been shown to inhibit the activity of adenylate cyclase
in the N18TG2 clone of murine neuroblastoma cells. The concentration of A9THC exhibiting half-
maximal inhibition was SOOnM. A*Tetrahydrocannabinol was less active, and cannabinol was only
partially active. Cannabidiol, cannabigerol, cannabichromene, olivetol and compounds having a reduced
length of the C3 alkyl side chain were inactive. The metabolites of A8THC and A9THC hydroxylated at
the Cl 1 position were more potent than the parent drugs. However, hydroxylation at the C8 position of
the terpenoid ring resulted in loss of activity. Compounds hydroxylated along the C3 alkyl side chain were
equally efficacious but less potent than A9THC. These findings are compared to the pharmacology of
cannabinoids reported for psychological effects in humans and behavioral effects in a variety of animal
models.

Key words: A9tetrahydrocannabinol, neuroblastoma cells, adenylate cyclase.

It has been demonstrated in this laboratory that METHODS

A*tetrahydrocannabinol (THC) and A9THC inhibit
basal or hormone-stimulated activity of adenylate
cyclase in N18TG2 neuroblastoma cell membranes
(Howlett, 1985; Howlett and Fleming, 1984). This
inhibition was not competitive with respect to the
prostanoid or the peptide hormone (secretin) stimu-
lators (Howlett and Fleming, 1984). A number of
hormones and neuromodulators inhibit adenylate
cyclase via pharmacologically distinct receptors
that interact with a guanosine triphosphate (GTP)-
binding protein complex, referred to as Gi (Gilman,
1984). The response to A9THC also appears to be
mediated by the G, protein complex (Howlett, 1985;
Howlett, Qualy and Khachatrian, 1986). In the
N18TG2 cell, muscarinic cholinergic, a,-adrenergic
and d-opioid receptors are known to inhibit the
activity of adenylate cyclase. However, none of these
receptors is responsible for the inhibitory effects of
cannabinoids (Devane, Spain, Coscia and Howlett,
1986; Howlett and Fleming, 1984). Thus, the
response to cannabinoids may be the result of
stimulation of a “cannabinoid” receptor.
The present communication attempts to define the
pharmacological profile of the inhibition of adenylate
cyclase by cannabinoids by examining the activities of
several compounds extracted from the marihuana
plant. The study further characterizes the activity of
a variety of hydroxylated metabolites produced in
humans and experimental animals. The results are
compared to the relative activities of these com-
pounds, reported by others for effects of cannabinoid
drugs in humans and in a variety of experimental
animal models.
Determination of adenylate cyclase in plasma
membranes
Neuroblastoma cells (N18TG2) were grown
in Dulbecco’s modified Eagle’s: Ham’s F12 (1: 1)
medium containing 10% heat-inactivated calf serum.
A plasma membrane fraction was isolated by differ-
ential and sucrose gradient centrifugations and stored
in small aliquots at -80°C as previously described
(Howlett, 1985).
Adenylate cyclase was determined by addition of
1Opg of rapidly thawed membranes to a reaction
mixture containing (in 100 ~1): 50 mM Na 4-(2-
hydroxyethyl)-1-piperazine ethane sulfonate (Na
HEPES), pH 8.0; 0.1 mg/ml fatty acid-deficient bo-
vine serum albumin; 1 mM ethylene diaminetetra
acetic acid (EDTA); 5 mM MgCl,; 100pM GTP;
0.5 PM secretin; 0.1 mM 4-(3-butoxy-4-methoxy-
benzyl)-2-imidazolidinone (R020-1724); 0.5 mM
(cz-~~P)-ATP (0.5 PCi); 10 pg/ml pyruvate kinase;
3 mM phosphoenolpyruvate; 0.1 mM (3H)cyclic
AMP (10 nCi); and the indicated cannabinoid drugs.
Cannabinoid drugs were stored as 10 mM stock
solutions in absolute ethanol at -20°C. Serial dilu-
tions were made in a buffer containing 20 mM Na
HEPES, pH 8.0, 1 mM ethylene diamine tetra acetic
acid (EDTA), 2 mM MgCl, and 0.1 mg/ml fatty
acid-deficient bovine serum albumin using silylated
glassware as previously described (Howlett, 1985).
All assay points were determined in triplicate. Reac-
tions were stopped after 20 min at 30°C and
(3zP)-cyclic AMP was isolated by the procedure of
Salomon, Londos and Rodbell (1974).

507
508 A. C. HOWLETT
Four different membrane preparations were used
for these experiments, and the secretin-stimulated
activity of adenylate cyclase ranged from 180
to 260pmol/min/mg protein (5 to &fold above
GTP-basal).
The data points in each Figure are the mean of the
percentage inhibition of secretin-stimulated activity
of adenylate cyclase from three individual experi-
ments (except Fig. l(B) where nine experiments were
performed for A9’6HC and four for A’THC). The
error bars represent the standard error. The Kin,,
values (the concentrations causing half-maximal in-
hibition) were determined by linear regression analy-
sis of data transformed using the Hill equation
(Bennet and Yamamura, 1985). Slope factors for all
active cannabinoid drugs approximated 1.O, ranging
between 0.8 and 1.25. The curves are computer-
generated plots having the slope factors equal to 1.
Materials
Radioactive compounds were purchased from
New England Nuclear. Secretin was from Bachem
and other reagents were from standard sources.
R020-1724 was generously donated by Hoffmann-
LaRoche. Cannabinoid drugs were provided by the
National Institute on Drug Abuse.
RESULTS
Previous studies demonstrated a dose-dependency
for the ability of ABTHC and A9THC to inhibit
adenylate cyclase in preparations of membranes from
neuroblastoma cells (Howlett and Fleming, 1984).
These studies have been expanded to include
other constituents commonly found in extracts of
30
20
‘0 10
‘Z
:g 0
f 30
20
10
a
0.6
- log DmLbinoiclGdrugl &I
Fig. 1. Inhibition of adenylate cyclase by constituents and
analogs of marihuana. (A) Cannabinoid compounds (CBN,
cannabinol; CBD, cannabidiol; CBG, cannabigerol; CBC,
cannabichromene; OLVTL, olivetol) were at a final concen-
tration of 10 PM. Data are the mean + standard error for
n = 3 experiments. (B) Cannabinoid drugs were tested at the
concentrations indicated. Maximum inhibition produced by
A9THC was defined as 1.Ofor comparative purposes. Drugs:
-.- AQ-LC, Kiti0.53pM; ---O--- A*THC, Kin,, Fig. 2. Chemical structure of A9THC and sites of possible
0.56j1M; ...a... CBN, Kinh 1.4bM.
marihuana. In Figure l(A) cannabinoid drugs were
compared for their ability to inhibit the secretin-
stimulated activity of adenylate cyclase. A’Tetra-
hydrocannabinol inhibited the enzyme by about 30%
and A*THC was nearly as active. Cannabinol was
partially effective and cannabidiol, cannabigerol and
cannabichromene were not effective in inhibiting the
activity of adenylate cyclase. In studies not shown,
neither cannabinol nor cannabidiol antagonized the
response to A9THC.
The complete log dose-response curves for the
active constituents of marihuana are shown in Figure
l(B). The I&, determined for A*THC was similiar
to that for A9THC, however A*THC appeared to
produce a slightly smaller maximal inhibition than
A9THC. The response at large concentrations of drug
(> 3 PM) deviated from that expected, based on the
majority of points along the log dose-response curve.
This phenomenon might be related to the insolubility
in aqueous solution of these compounds at concen-
trations exceeding 5 PM under the experimental con-
ditions of the assay (Garrett and Hunt, 1974). The
response to cannabinol faiied to reach the same
maximal value as for A’THC, however the response
to cannabinol also diminished at concentrations
greater than 10pM.
Certain structural features are required for in-
hibition of adenylate cyclase activity by cannabinoids
[Fig. l(A)]. Olivetol, comprising the phenolic ring
plus the C3 alkyl side chain, was not sufficient for
inhibition of adenylate cyclase. A9,“Tetrahydro-
cannabinol retained significant activity, but the maxi-
mal inhibition (at 10 PM) never exceeded 60% of the
inhibition in response to A9THC (data not shown).
The poor activity observed for A9*“THC and canna-
binol suggest that the conformation of the terpenoid
ring is important for activity. 1’,2’,3’,4’,5’-
Pentanor-A9THC-3-carboxylic acid and 3’,4’,5’-
trinor-A9THC-2’-carboxylic acid were also tested
(data not shown). These experiments showed that
reducing the length of the C3 alkyl side chain and
adding carboxylic acid residues resulted in total loss
of activity.
Enzymatic hydroxylation is a prominent form of
metabolism of the cannabinoid drugs. Shown in
Figure 2 are possible sites of hydroxylation reported
oHa*
7
metabolic hydroxylation.
 Cannabinoid inhibition of adenylate cyclase
4 B
Fig. 3. Inhibition of adenylate cyclase by hydroxylated
metabolites of A9THC (A) and A8THC (B). (A) Drugs:
-O-- A9THC, Ki,,,, 360nM; ...6... IlOH A9THC, Kinh
100 nM. + 8aOH A9THC; A @OH A9THC; n 8,l I-diOH
A9THC. (B) Drugs: ---O--- A*THC, Kinh 560nM;
.CJ. 110H A’THC, Kin,, 260 nM.
for man (Lemberger, Silberstein, Axelrod and Kopin,
1970) monkeys (Ho, Estevez, Englert and McIsaac,
1972) and rodents (Ben-Zvi, Burstein and Zikopoulos,
1974; Christensen, Freudenthal, Gidley, Rosenfeld,
Boegli, Testino, Brine, Pitt and Wall, 1971; Gill,
Jones and Lawrence, 1973). As shown in Figure 3,
1lOH-A9THC and 1lOH-A*THC exhibited a greater
potency than their respective parent drugs to inhibit
the activity of adenylate cyclase. Hydroxylation at
the Cl 1 of cannabinol resulted in a product more
active than cannabinol itself [Fig. l(A)]. A complete
log dose-response analysis for 1lOH-cannabinol
(n = 3) resulted in a curve exhibiting an efficacy
similar to A9THC and having a 4nh of 320 nM (data
not shown). As for the other cannabinoid com-
pounds, the inhibition observed at concentrations of
1IOH-cannabinol greater than 3 PM were less than
maximal.
Hydroxylation at the C8 position yielded con-
trasting results. Both c1and B isomers of 80H-A9THC
were inactive compounds [Fig. 3(A)]. Hydroxylation
at the C&z-position also rendered the 1IOH-A9THC
inactive. In some experiments, these inactive com-
pounds increased the activity of adenylate cyclase by
up to 15% at concentrations exceeding 3 PM (data
not shown).
Hydroxylation along the C3 alkyl side chain re-
sulted in compounds having lesser potency but
efficacy similar to A9THC (Fig. 4). In data not
shown, the activity of 5’OH-A9THC was similar to
4’OH-A9THC. The position of the hydroxyl along the
length of the chain was not directly related to the
potency change.
DISCUSSION
The above studies were performed in order to
compare the pharmacology of the cannabinoid drugs,
509
observed in the neuroblastoma adenylate cyclase
system, to the pharmacology demonstrated for the
human experience and for various animal models.
These comparisons must be semiquantitative at best.
Uptake, distribution, metabolism and elimination of
drug are factors that modify the determinations of
potency in human and animal investigations. These
factors are not influential in the in oitro studies
reported here.
In contrast, a problem in many in vitro studies is
that concentrations of drug are difficult to assess
accurately because of the poor aqueous solubility and
the tight binding to glass and plastic of the canna-
binoid compounds. Further, many published experi-
ments have employed organic solvents or detergent
surfactants as solubilizing agents; these agents can
modify the biochemical response. The present studies
were performed using glass test-tubes and pipets that
had been treated with bis(trimethylsilyl)trifluoro-
acetamide in order to minimize surface adherence of
the cannabinoid drugs (Garrett and Hunt, 1974). The
solubility problems have been minimized by making
initial dilutions of drugs in fatty acid-deficient
bovine serum albumin in the absence of detergents or
organic solvents (Perez-Reyes, Timmons, Lipton,
Davis, and Wall, 1972), thereby creating a micro-
suspension that can be further diluted to the soluble
range [5-10pM for A9THC (Garrett and Hunt,
1974)]. Although these precautions inspire confidence
in the data at concentrations below this range, it is
apparent, from experiment to experiment, that data
points at 10 or 20 p M may deviate below what would
be expected for a sigmoidal log dose-response re-
lationship. For this reason, the compounds found to
be inactive at 10pM have not been tested at larger
concentrations.
The studies with A9THC in humans have moni-
tored either cardioacceleration or psychological high
ratings reported by the subjects (Table 1). Estimates
of potency have suggested that the psychological high
experience is greater for A9THC than for A’THC
(Hollister, 1974; Hollister and Gillespie, 1973) but
that the cardioacceleration effects may be greater
for AsTHC (Hollister and Gillespie, 1973). Neither
cannabinol nor cannabidiol produced responses in
human subjects at the oral doses tested (Hollister,
D,“D Kl”ki#M)
-*- #THS 0.30
,30- . -+-
- o-
P’O”l 7°C 2.8
9’0”2 THC0.8. *,: 7
,,. ..“,
gzo_ -o-. 4’0”A0T”C I.0 , .
, .,. $..” I.
E /...j/ .d
4-/’
=10- ,,’ ,..p’
de . . .:“:
0 __ /; +_& .-.“‘.
$1, P
I
0 8 7 6 5
- log [Cannabinold drug1 (M)
Fig. 4. Effect of C3 alkyl chain hydroxylation on inhibition
of adenylate cyclase.
510 A. C. HOWLETT

Table 1. Relative ootencv estimates for constituents and metabolites of marihuana in human and animal studies
Relative potency (percentage of A9THC)
Ps” Ca DAb Mn Ht MI An MA Dis RS NbC’
Constituents of marihuann
A9THC 100 100 100 100 100 100 100 100 100 100 100
AsTHC 75-90 140 3&50 50 50 - 65-l IO 30-90 50 65-95
Cannabidiol 0 0 0 - 0 0 3 0 0
Cannabinol O-IO &lo - 0 - - - 3 0.3 37d
Cannabichromene - - - 0 - 0.1 0
Metabolic products of THC
I I -OH A*THC 90 - - - - - 500 200 I2&200 250 I40
II-OH A9THC 100-220 w-220 500 100 - 700 500-800 IO&200 25&350 375 360
SE-OH A’THC a-25 0 25 - - 40 15 0 0 0
8b-OH A9THC 20-30 I5 - 25 - - 0 30 <2 I 0
8a,l I-diOH A9THC 0 - ---- 20 <2 0 0 0
3’ /Y-OH A9THC - - II&200 - II&350 90 - 11&400 - 250 46
‘Activity in man-Ps: psychological high; Ca: cardioacceleration (Hollister, 1973, 1974; Hollister and Gillespie, 1973; Lemberger etal., 1973;
Perez-Reyes et al., 1972; 1973a; 1973b). bAnimal models-DA: ataxia in the dog (Handrick et al., 1982; Martin et al., 1981; Wilson et
al., 1976); Mn: behavior in the monkey (Ben-Zvi et al., 1971; Edery and Grunfeld, 1971); Ht: hypothermia in the mouse (Handrick et
al., 1982; Martin et al., 1981); MI: immobility in the mouse (Gill et al., 1973; Pertwee, 1972); An: analgesia (mouse hot plate) (Uliss
et al., 1975; Wilson and May, 1975; Wilson et al., 1976); MA: motor activity in the mouse (Christensen et al., 1971; Handrick et al.,
1982; Perez-Reyes et al., 1973); Dis: drug discrimination (pigeons, rats) (Jarbe and McMillan, 1980); RS: THC-seizure prone rabbits
(Consroe and Fish, 1981). eNeuroblastoma inhibition of adenylate cyclase (data presented here). “Cannabinol was a partial agonist,
exhibiting 50% the efficacy of A9THC.

1973; Hollister, 1974). However, when infused intra-
venously, cannabinol (but not cannabidiol) produced
a psychological high and cardioacceleration at
lo-fold the dose of A9THC (Hollister, 1973; Perez-
Reyes, Timmons, Davis and Wall, 1973a). The
effects of these compounds to inhibit the activity
of adenylate cyclase, paralleled these responses.
A9Tetrahydrocannabinol was somewhat more potent
and efficacious than A’THC and considerably more
potent and efficacious than cannabinol. Cannabidiol
was inactive.
Similar parallels occurred for the hydroxylated
metabolites. In oiuo studies suggest that llOH-
A9THC was equally potent (Perez-Reyes, Timmons,
Lipton, Christensen, Davis and Wall, 1973b; Perez-
Reyes ef al., 1972) or up to twice as active (Hollister,
1974; Lemberger, Martz, Rodda, Forney and Rowe,
1973) as the parent compound in producing a psycho-
logical high. Heart rate was increased to the same
or greater extent by the hydroxylated metabolite
(Hollister, 1974; Lemberger et al., 1973; Perez-Reyes
et al., 1972). Similarly, 1lOH-A*THC was about 20%
more potent than the parent drug (Hollister, 1974).
When the 8a- and &!I-hydroxylated metabohtes were
administered intravenously to three human subjects,
4- and S-times the dose of A9THC, respectively, was
required to produce a comparable psychological high
Hollister, 1974). In another study, the ,!3isomer pro-
duced a psychological high and cardioacceleration at
2.5- and 6-times the dose of A’THC, respectively, but
the a isomer was inactive at up to IO-times the dose
of A’THC (Perez-Reyes et al., 1973b). For inhibition
of adenylate cyclase, the 1I-hydroxylated metabolites
of A’THC and A9THC were 3-fold more potent than
the parent drugs. In contrast, neither &OH-A’THC
nor 8/3OH-A9THC inhibited adenylate cyclase at
concentrations up to 50-fold greater than the &,, for
A9THC.
Several animal models have been investigated as
indicators of the activity of cannabinoid drugs (Table
1). Typically, A8THC was less potent than A9THC
in such tests as ataxia in the dog (Martin, Balster,
Razdan, Harris and Dewey, 1981; Wilson, May,
Martin and Dewey, 1976) behavior of monkeys
(Edery and Grunfeld, 1971), hypothermia in the
mouse (Martin et al., 1981), hot plate analgesia
(Uliss, Dalzell, Handrick, Howes and Razdan, 1975;
Wilson and May, 1975), spontaneous activity in the
mouse (Christensen et al., 1971) and in genetically
THC-seizure prone rabbits (Consroe and Fish, 1981).
Cannabidiol (Consroe and Fish, 1981; Edery and
Grunfeld, 1971; Pertwee, 1972; Uliss et al., 1975)
cannabinol (Edery and Grunfeld, 1971) and canna-
bichromene (Edery and Grunfeld, 1971) were found
to be inactive in a number of these models. The
THC-seizure prone rabbits responded to cannabinol
in large doses (Consroe and Fish, 1981). Studies of
A9.“THC found it to be at least 100 times less potent
than A9THC in rhesus monkeys, but only seven times
less potent in drug-discrimination tests, performed
using rats (Semjonow and Binder, 1985). The data
reported here for inhibition of the activity of adenyl-
ate cyclase are consistent with this pharmacological
pattern. For the inhibition of adenylate cyclase,
cannabinol and A9,“THC appeared to be partial
agonists. It is possible that the discrepancies in their
activity in various animal models may be a reflection
of this property.
The anticonvulsant property of A9THC and other
cannabinoid drugs in the maximal electroshock test
does not exhibit the same pharmacology as reported
for the human psychological high or for a variety of
animal models (Karler, Cely and Turkanis, 1975).
This difference is mainly due to the anticonvulsant
activities of cannabidiol and cannabinol. The anti-
convulsant property of cannabinoid compounds may
 Cannabinoid inhibition of adenylate cyclase
not be due to the same m~hanism as that for the
other behavioral effects. Since cannabinol was a
poor inhibitor of adenylate cyclase and cannabidiol
was inactive, it is not likely that the anticonvulsant
property of cannabinoid compounds is related to a
decrease in the production of cyclic AMP.
The II-hydroxylated metabolites of A8THC and
A’THC were more potent than the parent com-
pounds in producing ataxia in the dog (Wilson et al.,
1976), immobility in the mouse (Gill et al., 1973) hot
plate analgesia (Wilson and May, 1975) spontaneous
activity in the mouse (Christensen et ai., 1971) and
TX-seizure prone rabbit model (Consroe and
Fish, 1981) as well as in drug-discrimination studies,
using rats or pigeons (Jarbe and McMillan, 1980).
The ability of 1IOH-A’THC and 1lOH-A8THC to
inhibit adenylate cyclase with a greater potency than
the non-hydroxylated compounds paralleled these
findings. I 1-Hydroxylation of cannabinoi increased
the activity to half that of A9THC in the static ataxia
test in the dog (Martin, Harris and Dewey, 1984).
This metabolite of cannabinol also exhibited a greater
ability to inhibit adenylate cyclase than the parent
compound.
Animal studies of the hydroxylated metabolites at
the 8-position produced inconsistent results, although
the responses to these compounds were poor com-
pared with those to A9THC (Table 1). Both &OH-
and XfiOH-A9THC affected behavior in the monkey
at the same dose (Ben-Zvi, Mechoulam, Edery and
Porath, 1971). The 8a isomer exhibited analgesic
activity, whereas the 86 isomer did not (Wilson and
May, 1975). In contrast, the S/J isomer was more
effective in decreasing motor activity in the mouse
(Perez-Reyes et al., 197313).Only the SD isomer was
recognized in drug-disc~mination studies (Jarbe and
McMillan, 1980) and produced seizures in the gen-
etically susceptible rabbits (Consroe and Fish, 1981),
but the doses required were excessive.
811 -Dihydroxy-A’THC was nearly inactive in the
spontaneous activity test in the mouse (Christensen et
al., 1971). The inhibition of adenylate cyclase by the
8hydroxylated metabolites was most consistent with
these latter three models.
The in vitro activity of the C3-side chain hydroxy-
lated metabolite (3’) was not consistent with findings
in animal models. This compound was more potent
than A9THC in producing ataxia in the dog, hypo-
thermia in the mouse and motor activity in the mouse
(Handrick, Duffley, Lambcrt, Murphy, Dalzell,
Howes, Razdan, Martin, Harris and Dewey, 1982;
Martin et al., 1984) yet was less potent than A9THC
in inhibiting adenylate cyclase. One possible expla-
nation for the discrepancy is that the pure /J isomer
was used for the animal studies (Handrick et al.,
1982) and the racemic mixture was used for the
present investigation. Alternatively, 3’OH-A9THC
might be further metabolized to a more active prod-
uct in animals (Harvey, Martin and Paton, 1977).
The similar pharmacology of cannabinoids existing
511
for the inhibition of adenylate cyclase in neuronal
cells and the human experience or behavior in a
variety of animal models is intriguing. One possible
mechanism of action for the cannabimimetic com-
pounds at the cellular level may involve regulation of
the synthesis of cyclic AMP in certain populations of
neurons associated with modification of such behav-
ior. It will be of interest in the future to locate cells
sensitive to A9THC in the brain.
Aeknowied~e~e~ts-The donation of the cannabinoid
drugs by NIDA is greatly appreciated. Technical assistance
by Richard Fleming and Gerald Wilken, and clerical
assistance by Linda Russell were vital to the completion of
this work. This study was supported by research grant
DA03690 and by RCDA NS00868.
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