Proc. Nat. Acad. Set. USA
Vol. 72, No. 2, pp. 590-594, February 1975

Morphine Receptors as Regulators of Adenylate Cyclase Activity

(neuroblastoma X glioma hybrid/prostaglandin E,/narcotic dependence)

SHAIL K. SHARMA*t, MARSHALL NIRENBERG*, AND WERNER A. KLEEt

* Laboratory of Biochemical Genetics, National Heart and Lung Institute, and the ¢ Laboratory of General and Compurative
Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014

Contributed by Marshall Nirenberg, November 11, 1974

ABSTRACT Morphine inhibits adenylate cyclase (EC
4.6.1.1) activity of neuroblastoma X glioma hybrid cells.
The inhibition is stereospecific and is reversed by the
antagonist, naloxone. The relative affinities of narcotics
for the opiate receptor agree well with their effectiveness
as inhibitors of adenylate cyclase. Morphine-sensitive and
-insensitive cell lines were found, and the degree of sensi-
tivity was shown to be dependent upon the abundance of
narcotic receptors. Thus, morphine receptors are func-
tionally coupled to adenylate cyclase. A molecular mech-
anism for narcotic addiction and tolerance is proposed.

 

A neuroblastoma xX glioma hybrid cell line with morphine
receptors has been described (1). The receptors are stereo-
specific with respect to narcotic binding, and receptor affinities
for narcotics closely resemble those of rat brain (2-4) and
correlate well with their pharmacologic potencies.

Collier and Roy have reported that prostaglandin E de-
pendent cAMP synthesis is inhibited by narcotics in rat
brain homogenates (5, 6). In this communication the effects
of morphine and other narcotics upon adenylate cyclase
(EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity
of neuroblastoma X glioma hybrids and parental cells are
examined. We find that morphine and other narcotics inhibit
adenylate cyclase activity and that the inhibition is dependent
upon morphine receptors.

MATERIALS AND METHODS

Materials. [G-?H]Naloxone (26.3 Ci/mmol) and [(G-3H]-
cAMP (22.1 Ci/mmol) were from New England Nuclear;
[a-PJATP (6-10 Ci/mmol) was from ICN; and {U-*4C]-
cAMP (0.295 Ci/mmol) was from Amersham/Searle. Mor-
phine sulfate was from Merck; naloxone-HCl was a gift of
Endo Labs; etorphine was from Dr. R. Willette, NIDA;
other narcotics were from Dr. Everette May; PGE, was from
Dr. John Pike, Upjohn Co.; Ro20-1724 [4-(3-butoxy-4-
methoxy benzy])-2-imidazolidinone was from Hoffman La-
Roche; IBMX (3-isobutyl-l-methylxanthine) was from
Aldrich; Gpp(NH)p (5’-guanylylimidodiphosphate) was from
ICN; cAMP, ereatine phosphate, and creatine kinase (155
U/mg of protein) were from Sigma.

Cell Lines. Neuroblastoma X glioma hybrid NG108-15 was
obtained§ by fusion of mouse neuroblastoma clone N18TG-2

 

Abbreviations: Ro20-1724, 4-(3-butoxy-4-methoxybenzy])-2-imid-
azolidinone; IBMX,  3-isobutyl-l-methylxanthine; Gpp-
(NH)p, 5’-guanylylimidodiphosphate; PGE,, prostaglandin Ey.
t Fogarty International Fellow, on leave from the Department
of Biochemistry, All India Institute of Medical Sciences, New
Delhi, India.

§ B. Hamprecht, T. Amano, and M. Nirenberg, in preparation.

590

(7) and rat glioma clone C6BU-1 (8), derived from C6 (9).
Cells were grown as described (1).

Assay of cAMP of Intact Cells. Confluent cells (8-4 my of
protein 60 min dish) were washed 3 times with 5 m] of medium
A [Dulbecco’s modified Eagle’s medium with 25 mM Hepes
(N-2-hydroxyethylpiperazine-N‘-2-ethanesulfonie acid) pH
7.4, instead of NaHCO, adjusted to 340 mosmol/liter with
1.1 g of NaCl/liter] and incubated with medium A plus 0.5
mM Ro20-1724 and 0.6 mM 13MX for 30 min at 37°.
Reactions were initiated by the addition of 30 ul of narcotic
in water, 30 ul of PGE, in ethanol, or solvent. Ethanol
(0.5% with Ro20-1724 present or 1% when Ro20-1724 and
PGE, were present) had no effect upon cAMP formation.
After incubation the medium was discarded and 3 ml] of 5%
trichloroacetic acid with 5 pmol of [4C]cAMP (3000 cpm) at
3° were added. The extract and 2 washes (each 1.5 ml of 5%
trichloroacetic acid) were combined and centrifuged and the
supernatant fluids applied to 0.8 * 8-cm columns of AG
50W-X4 resin, 200-400 mesh, H+ form (BioRad). The 3 ml of
eluate following a 6-ml water wash was assayed for cAMP by
the method of Gilman (10). Values reported are for duplicate
dishes and are corrected to 100% recovery of cAMP.

Adenylate Cyclase Assay. Cells that had been washed three
times were homogenized in 0.32 M sucrose, 10 mM Tris-HCl,
pH 7.4 (15 mg of protein per ml) with 10-15 strokes, by hand,
of a ground-glass homogenizer. Enzyme activity was <le-
termined by a modification of method C of Salomon et al.
(11). Each tube contained: 45 mM Tris-HCl, pH 7.4; 5 mM
MgCl; 160 mM sucrose; 20 mM creatine phosphate; 10 U of
creatine kinase; 1 mM cAMP; 0.5 mM Ro20-1724 (0.5%
ethanol, final concentration); 1 mM [a-@P]ATP (3 to 5 & 10°
cpm); and 100-300 ug of homogenate protein in a final volume
of 100 yl. The reaction was terminated by addition of 50 yl
of 15% trichloroacetic acid. For product characterization, the
32P product purified through the alumina column step (11)
was subjected to Dowex-1 formate column chromatography.
The 2 M formic acid eluate was lyophilized, and the radioac-
tivity was characterized by paper or thin-layer chromatog-
raphy in: (A) isopropanol-NH,OH-0.1 M boric acid (7:1:2);
(B) 1 M ammonium acetate-95% ethanol (3:7); (C) isobutyric
acid-2 M NH,OH (2:1); and (D) H,O-washed polyethylene-
imine-cellulose thin-layer plates developed sequentially with
HO and 0.25 M LiCl. Greater than 90% of the radioactivity
recovered from the alumina columns was cAMP.

Opiate Binding. The assay (1) was modified so that incuba-
tion mixtures contained the components of the adenylate
cyclase assay and 875 ug of homogenate protein; & X 10-§ M
(*J]I]naloxone (4150 cpm); and unlabeled narcotic in a final
volume of 200 ul. Under these conditions, narcotic affinities
Proc, Nat. Acad. Sci. USA 72 (1978)

 

A CELLS

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1200
1000
800
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Morphine Receptors and Adenylate Cyclase 591

 

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Fic. 1. Inhibition by morphine of the rate of cAMP accumulation in intact neuroblastoma X glioma hybrid NG108-15 cells and of
adenylate cyclase activity in homogenates. The effect of morphine on basal and PGE,-stimulated cAMP levels in intact cells is shown in
part A. In B, the rate of adenylate cyclase activity in NG108-15 homogenates is shown. Concentrations of PGE, and morphine were

10 «M.

for the receptor are lower than those as measured (1) by the
following factors: naloxone, 2; etorphine, 13; dextrorphan,
67; levorphanol, 667; morphine, 200; and 3-allylprodine, 100.

RESULTS

The effects of morphine upon basal and PGE,-stimulated
cAMP levels in intact NG108-15 hybrid cells and on adenylate
cyclase activity in cell-free homogenates are shown in Fig. 1A
and B, respectively. The addition of PGE, to intact cells
results in a 40-fold increase in intracellular cAMP levels.
The addition of morphine markedly reduces cAMP levels,
both in the presence and absence of PGE. As shown in panel
B, the addition of PGE; to homogenates similarly results in a
10-fold increase in adenylate cyclase activity (5- to 12-fold
increase in other experiments), Morphine inhibits basal and
PGEi-dependent adenylate cyclase activity about 45% (30-
60% inhibition in other experiments). The rate of cAMP
formation was lincar for about 8 min; therefore, in all sub-
sequent experiments reaction mixtures were incubated for
5 min.

The effect of morphine upon adenylate cyclase activity was
verified in the following ways: (1) the [#?P]product of the re-
action was shown to have the chromatographic mobility of
authentic cAMP in five systems (see Materials and Methods) ;
(ii) the concentration of substrate, ATP, did not change
appreciably during incubation by direct measurement with
the luciferase assay (12); and. (itt) the specific activity of ATP
changed less than 5% during the incubation.

The effect of naloxone, an antagonist that displaces mor-
phine from the opiate receptor but has no narcotic activity,
is shown in Table 1. Naloxone reverses the inhibitory effect
of morphine upon adenylate cyclase activity both in the
presence and absence of PGE,, but naloxone alone has little or
no effect upon basal or PGE;-stimulated adenylate cyclase
activity.

The relation between molarity of PGE, and the specific
activity of adenylate cyclase is shown in Fig. 2. Adenylate
cyclase is stimulated by concentrations of PGE, greater than
10-8 M, A discontinuity in the concentration curve is observed
at 5 X 10-77M PGE.

The effects of different concentrations of naloxone, in the
presence and absence of morphine, on adenylate cyclase are
shown in Fig. 3. The concentration of morphine was 10-5 M,
which inhibits adenylate cyclase activity maximally. Naloxone
completely reverses the inhibitory effect of morphine on
adenylate cyclase activity at 2 X 1075 M; naloxone at higher
concentrations stimulates adenylate cyclase activity 15-20%.
Stimulation has not been observed in all experiments, and
further experiments are needed to clarify this observation.

In Fig. 4 the decrease in binding of [*H]naloxone to the
narcotic receptor, as a function of narcotic concentration, is
compared with the effectiveness of the narcotics as inhibitors
of adenylate cyclase activity. Naloxone, a pure antagonist,
binds with high affinity to the opiate receptor but does not
inhibit adenylate cyclase activity (panel A). Etorphine,
levorphanol, morphine, and 3-allylprodine, each a potent
narcotic, inhibit adenylate cyclasc at essentially the same

TaB.e 1. Reversal of morphine inhibition of adenylate cyclase
by naloxone in cell-free preparations of NG108-16

 

Membranet

Homogenatet fraction

(pmol of cAMP formed/

 

Addition* min per mg of protein)

None 17 24
Morphine 8 17
Naloxone 17 22
Morphine + naloxone 17 23
PGE; 113 124
PGE, + morphine 68 | 101
PGE, + naloxone 110

PGE, + morphine + naloxone 100 122

 

* 10 uM, each component.

{ Unfractionated homogenate.

t The homogenate was centrifuged at 1500 X g for 10 min and
the supernatant fraction at 17,000 x g for 30 min. The 17,000 x
g pellet was washed and resuspended in the homogenizing me-
dium (membrane fraction).
592 Biochemistry: Sharma eé al.

 

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PGE, MOLARITY
Fic. 2. Activation of adenylate cyclase in homogenates of

NG108-15 by PGF, as a function of PGE, concentration.

concentrations as those needed to displace ["H ]naloxone from
the opiate receptor (panels B, C, E, and F). In contrast to
levorphanol, the analgesically inactive enantiomer dex-
trorphan is without effect on adenylate cyclase activity and
has a much lower affinity for the opiate receptor (panel D).

Apparent dissociation constants for [narcotic- opiate re-
ceptor] complexes are shown in Table 2, along with apparent
K, values for adenylate cyclase calculated from the data of
Fig. 4. A striking similarity is observed between the dissocia-
tion constants and the concentrations of narcotics required for
50% inhibition of adenylate cyclase activity.

Narcotic binding and enzyme inhibition curves shown in
Fig. 4 are not superimposable, even though the narcotic
concentrations for half-maximal inhibition of binding and
enzyme activity agree well. With each of the four narcotics,
enzyme activity changes over a relatively small range of
narcotic concentration compared to narcotic binding. This
relationship is seen more clearly in Fig. 5A and B, which shows
Hill plots of narcotic binding and adenylate cyclase inhibition,
respectively. Each slope for narcotic binding is approximately
1, whereas the maximum slopes for adenylate cyclase inhibi-
tion are 2.3. These results suggest that the binding of nar-
cotics to receptors is not a cooperative process, whereas the
effects of narcotics on adenylate cyclase activity exhibit a
cooperative character.

The relation between the molarity of Gpp(NH)p, a rela
tively stable analog of GTP, and adenylate cyclase activity,
in the presence and absence of morphine, is shown in Fig. 6.

TasBLe 2. Comparison of narcotic affinity for the opiate
receptor and ability to inhibit adenylate cyclase

 

 

Ka* Kit
Narcotic Adenylate
receptor cyclase
Narcotic nM nM
Etorphine 5 10
Levorphanol 200 200
Morphine 4,000 2,000
3-Allylprodine 10,000 50,000
Dextrorphan 10,000 _
Naloxone 20 _—

 

* Apparent dissociation constant of the narcotic-recéptor com-
plex.

t Narcotic concentration required for 50% of maximal in-
hibition of adenylate cyclase.

—, no inhibition.

Proc. Nat. Acad. Sei. USA 72 (1976)

- —

 

 

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|

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PMOL cAMP/MIN PER MG PROTEIN
°

L 10° 10° 107 10% 10° 10% 10°
NALOXONE MOLARITY
Fig. 3. The effect of naloxone upon adenylate cyclase activity
of an NG108-15 homogenate in the presence and absence of 10
uM morphine.

The GTP analog activates adenylate cyclase of NG108-15
homogenates, similar to the effects reported with other
systems (13). Complete reversal of morphine inhibition of
adenylate cyclase was observed in the presence of 1 X 10-5 M

 

 

 

 

 

    

 

 

 

  
   

 

 

 

 

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Fic. 4. The effectiveness of various narcotics as inhibitors

of adenylate cyclase activity of NG108-15 homogenates is com-

pared with their ability to displace [*H]naloxone from the opiate

receptor. Symbols represent the following: ©, [*H]naloxone

bound to receptor; 4, [#7P]cAMP formed per 5 min per tube (250
ug of protein per tube).
Proc. Nat, Acad. Set. USA 72 (1978)

 
 

 

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NARCOTIC MOLARITY

Fie. 5. (A) Hill plots of narcotic binding to receptor; and (B)
inhibition of adenylate cyclase activity by narcotics. The sym-
bols represent the following: O, etorphine; m, naloxone; @, levor-
phanol; 4, morphine; and 4, 3-allylprodine. In panel A the symbol
n in the ordinate represents the maximum amount of [*H]-
naloxone displaced at saturating concentrations of unlabeled
narcotic and » is the amount of [*H]naloxone displaced at non-
saturating concentrations of narcotic. In panel B, n represents
the maximum inhibition of adenylate cyclase observed at satu-
rating narcotic concentrations and » is the inhibition observed at
each nonsaturating concentration of narcotic.

Gpp(NH)p. In the absence of morphine, <1 X 10-° M Gpp-
(NH)p has little effect upon adenylate cyclase activity.
Similar results were obtained with partially purified mem-
brane preparations (not shown),

The effects of morphine upon adenylate cyclase activity of
intact neuroblastoma N18TG-2 and glioma C6BU-1 cells, the
parents of hybrid NG108-15, are shown in Fig. 7A and B,
respectively. Morphine reduces cAMP levels of neuro-
blastoma Ni8TG-2 cells slightly but does not affect cAMP
levels of glioma C6BU-1 cells.

The effects of morphine and naloxone upon adenylate
cyclase activity of homogenates prepared from parent
neuroblastoma N18TG-2 and gliorna C6BU-1 cells are shown
in Table 3. Morphine inhibits adenylate cyclase activity of
N18TG-2 slightly, and the inhibition is reversed by naloxone;
however, these compounds do not affect adenylate cyclase
activity of C6BU-1. Thus, the neuroblastoma parent, which
has few morphine receptors,‘ is slightly sensitive to morphine
whereas the glioma parent, which lacks these receptors, is
insensitive to morphine.

DISCUSSION

‘The results show that morphine reduces basal and PGE,-
stimulated cAMP levels of intact neuroblastoma x glioma
hybrid cells and inhibits adenylate cyclase activity of homoge-
nates. The effects of morphine are blocked by the narcotic

antagonist, naloxone, which competes with narcotics for sites”

on the opiate receptor. The inhibition of adenylate cyclase
activity by narcotics is specific for the levorotatory, pharma-
cologically active, stereoisomer. The relative affinities of
narcotics for the opiate receptor agree well with their effective-
ness as inhibitors of adenylate cyclase and with their pharma-
cologic potency. Morphine-sensitive and -insensitive cell lines

 

1 Neuroblastoma clone NI8TG-2 can be shown to have a rela-
tively small number of morphine receptors when binding experi-
ments are performed with {*H]naloxone in place of the [#H]-
dihydromorphine used previously (1). Glioma clone C6BU-1
shows no specific binding of either naloxone or dihydromorphine.

Morphine Receptors and Adenylate Cyclase 593

Taste 3. Effect of morphine on adenylate cyclase activity
of neuroblastoma and glioma parents

 

 

Homogenate
Neuro- .
blastoma Glioma
N18TG-2 C6BU-1
, (pmol of cAMP formed/
Addition * min per mg of protein)
None 6 20
Morphine 4 19
Naloxone 5 20
Morphine + naloxone 5 20
PGE, 75 24
PGE, + morphine 63 25
PGE, + naloxone 72 24
PGE, + morphine + naloxone 70 23

 

* 10 »M, each component.

were found, and the degree of sensitivity to morphine is
dependent upon the abundance of narcotic receptors.

Thus, two kinds of adenylate cyclase complex were de-
tected, one sensitive, the other insensitive, to narcotics.
Sensitivity to narcotics is dependent upon functional inter-
action of the {morphine-receptor] complex with adenylate
cyclase. The partial inhibition of adenylate cyclase observed
in the preseice of saturating concentrations of narcotics may
be due, at least in part, to the presence of narcotic-sensitive
and -insensitive adenylate cyclase molecules within cells and,
possibly, heterogeneity in the cell population with respect to
the number of narcotic receptors per cell.

Interactions that couple morphine receptors with adenylate
cyclase exhibit positive cooperativity; however, the inter-
action of narcotic with receptor is not a cooperative process.
The Hill coefficient of 2.3 suggests that multiple interactions
between [narcotic-receptor] arid the adenylate cyclase com-
plex may be required for inhibition. Alternate mechanisnis for’
apparent cooperativity may be envisaged involving threshold
effects and spare receptors. The cooperative activation of
adenylate cyclase has been reported for thyroid stimulating
hormone (14), but glucagon (15) and oxytocin (16) do not
exhibit cooperativity.

The available information suggests that one molecule of

 

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Fic. 6. Effect of Gpp(NH)p on adenylate cyclase activity of
an NG108-15 homogenate, in the presence and absence of 10
uM morphine.
594 Biochemistry: Sharma et al.

 

 

 

 

 

 

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MINUTES

Fig. 7. Cyclic AMP accumulation, as a function of time, in
intact parental cells. Cells used were neuroblastoma Ni8TG-2
in panel A and glioma C6BU-1 in panel B. Concentrations of mor-
phine and PGE, were 10 »M.

adenylate cyclase can respond to different kinds of hormones,
depending upon the species of receptors present, but that only
one kind of hormone is translated at a time. Usually, hor-
mones activate adenylate cyclase but enzyme inhibition
mediated by some a- or prostaglandin receptors has been
reported (17, 18). Morphine thus resembles an inhibitory
hormone.

In essence, three kinds of mechanism have been proposed
for the regulation of adenylate cyclase activity by hormones
and other effector molecules: (¢) Hormones and GTP are
allosteric effectors that activate the enzyme by reducing its
affinity for free ATP, a competitive inhibitor of the substrate
{[ATP:Mg] (19, 20). Gt) Mg++ or Ca++ bind to other sites
and thereby regulate enzyme activity (21, 22). (iz) Adenylate
cyclase is inactivated by phosphorylation, catalyzed by a
protein kinase, and activated by dephosphorylation, catalyzed
by a phosphatase (23).

Reactions that terminate reading of messages by adenylate
cyclase regulate the duration of response of the enzyme to
each message. GTP may be involved both in initiation and
termination of reading of hormonal messages by adenylate
cyclase-in ways that may be analogous to the roles of GTP
in aminoacyl-tRNA bindirig to ribosomes and translocation
reactions in protein synthesis.

The GTP analog, Gpp(NH)p, activates adenylate cyclase,
and morphine reduces the threshold for activation 10-fold.
Thus morphine and Gpp(NH)p act in a concerted fashion,
which suggests that both are physically coupled to the adeny-
late cyclase complex. The K,, for glucagon stimulation of
adenylate cyclase is reduced 10-fold by GTP (24). The possi-
bility that GTP is required for morphine action, as it is for
hormone action, should bé considered.

Collier and Roy have presented cogent arguments support-
ing the thesis that PGE-stimulated adenylate cyclase is the
primary site of morphine action (5, 6). Our results support
their view, although we find that morphine inhibits adenylate
cyclase both in the presence and absence of added PGE.
The inhibition of adenylate cyclase should have many secon-

Proc. Nat. Acad. Sci. USA 72 (1978)

dary consequences which might account for the varied pharma-
cologic effects of morphine.

We wish to propose the following hypothesis concerning
the molecular basis for narcotic addiction and tolerance.
Inhibition of adenylate cyclase by morphine reduces intra-
cellular levels of cAMP. This may lead to a compensatory
shift in enzyme synthesis, degradation, or activity which
restores the normal level of cAMP. The cell then is dependent
upon narcotic because the level of cAMP is normal in the
presence of the drug and abnormally high upon withdrawal.
Our working hypothesis is that the number of adenylate
cyclase molecules increases as cells become addicted to nar-
cotics. This also leads to tolerance since, at a given narcotic
concentration, the amount of uninhibited enzyme is greater
in addicted than in normal cells.

We thank Doyle Mullinex for growing cells and Richard
Streaty for his help.

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Pert, C. B. & Snyder, S. H. (1973) Sczence 179, 1011-1014.

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Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature New

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Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell

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