OPIATE BECEPTGES AS REGULATGBS OF ADENYLATE CYCLASE

Werner A. Klee, Shail K. Sharma* and Marshall Nirenberg
Laboratory of General and Comparative Biochemistry, NIMB,
and the Laboratory of Biochemical Genetics, IBILI, NIH,
Bethesda, Maryland 20014

(Received in final form day 24, 1975)

  We have reported the presence of opiate receptors in some
neuroblastoma derived cell lines cultured in vitro and
that a neuroblastoma x glioma hybrid cell line, NG108-15, contains
a particularly large number of morphine receptors (1). Table 1
shows that whereas the hybrid cell line has opiate receptors

     II-dihydromorphine         H naloxone
HYBRIDS
-15       fmoleslmg protein
            17                   37     (19)
PARENTS
-2      0                   11     (6)
c6BU-1       1                    1     (1)

The concentration of radioactive narcotic was 1 n&l in
each case.  In neither case is this close to a saturating amount,
naloxone one has twice the affinity of dihydromorphine and so to
be comparable the ,naloxone data should be divided by 2 (numbers in
parenthesis);, NG108-15 (also called lOSCCl5) was obtained by B.
Siamprecht, T. Amano and M. Nirenberg (in preparation), NlOTG-2
by Minna et al. (2), CLBU-1 by Amano et al. (3).

which are rea
  dilydemonstrntod gands those
of N18TG2 were only detected with=II-naloxone binding whereas the
C6BU-1 line does not have a detectable number of opiate receptors
by either assay method.  This group of cell lines with no, few
and an abundance of opiate receptors has provided us with
material with which to study the biochemical consequences of
the interaction of morphine with its receptor.

  Collier and Boy reported that morphine and related drugs
inhibit the PGE. stimulated conversion of3B ATP into CAMP
by rat brain homogenates in a way that correlates with agonist
potency and receptor affinity (4.5). These exneriments promoted us
to examine the effect of morphine on adenylate cyclase activity
and on the CAMP levels of 1?G108-15 hvbrid cells and their oarental
cell lines (6).  We found that morphine inhibits the adenyiate
cvclase activity of NG108-15 cells and lowers cellular cAMP levels
in the presence and in the absence of added PGFJ (fig 1).
'*Fogarty InternationalFellow, on leave from the Department of
Diochemistry, All India Institute of Medical Sciences, Dew Delhi,
India.

117


Figure 1. Inhibition by morphine (lOdM) of the rate of CAMP
accumulation in intact NG108-15 hybrid cells(part A) and of adeny-
late cyclase activity in homogenates (part B). Basal and PGQ
(lO-SM) stimulated results are shown (6).
There is also a dramatic reduction in the adenosine stimulated
rise in cellular cAMP levels in the presence of morphine (17).
Thus, in this cell, morphine inhibits both stimulated ,-
and unstimulated adonylate cyclase.                     \-

  Morphine inhibits the adenylate cyclase of the neuro-
blastoma parent somewhat, but does not affect the activity
the enzyme found in the glioma parent (Table II). Thus, the de-

Tablemxcx of morphine on adenylate cyclase activity of

      neuroblastoma and glioma parents
                         N18'IG-2      C6BU-1
    Addition*            pmole/min/mg protein
None                             6           20
Morphine                          4           19
Naloxone                                     20
Morphine + naloxone       55           20
PGC,                                       24
PGQ + morphine        67:
PGQ + naloxone                  72     ii
PGQ morphine + naloxone           70           23
o lQrm of each component

gree of inhibition or adenylate cyclase by morphine is correlated
with the number of opiate receptors.  There are a number of other
properties of the enzyme of NG108-15 cells which show that the
inhibition by opiates is mediated by their receptors.  Thus,
naloxone, an apparently pure antagonist of narcotic drugs reverses
morphine inhibition of adenylate cyclase (6).  Furthermore,
the inhibition is sterospecific in that levorphanol, but not its
inactive isomer, dextrorphan, inhibits adenylate cyclase (6).
Traber et al. (7-9) have also reported that morphine reduces
PGE .clevation of cAt!P levels and Dlosser et al. (10) have reported
that morphine inhibits the PGI$ dependent activation of adenylate
cylase in neuroblastoma or hybrid cell lines derived from neuro-
blastoma cells.

   There is a good correlation between the concentrations at
which opiate agonists displace JIl-naloxone from the receptors and
and those required for inhibition of adenylate cyclase.  This
agreement is readily apparent in the data presented in Table III.
                                118


Table III Comparison of narcotic affinity for the opiate

      receptor and ability to inhibit adenylate cyclase
                       Kd              Ki
         Narcotic receptor     Adenylate cyclase
Narcotic                   nM              nM

Btorphine                  5                10
Levorphanol              200                200
Morphine            4,000           2,000
3-Nlylprodine       10,000           50,000
Dextrorphan         10,000                  --
Naloxone                 20                  --

However, we found th  the opiate binding and enzyme inhibition
curves not superimpo:ible (6)   Enzyme inhibition takes place
over a nmch narrower range of-drug concentrations than does dis-
placement of %I-naloxone from the receptors. This behavior im-
plies cooperativity among liganded receptors in their interaction
with the adenylate cyclase complex.  Analysis of the data by means
of Hill plots shaws that the slope for narcotic binding (reaction1
of scheme 1) is close to 1 indicating little or no cooperativity

                a              ?
???   ?? receptor4 receptor*M o ?o
                      i enzyme*receptor.M 1
in the formation of Garcotic.receptod complex but that the
maximum slopes of the cumes for adenylate cyclase inhibition by
narcotics (reaction 2) are between 2 and 3, indicat'  g strong
positiv  cooperativity in the reactions that couple K arcotic-
recepto 3 wmplexes with adenylate cyclase.

   Coupling of opiate-receptor complexes to adenylate cyclase

may occur by any of 3 general mechanisms:
1) Direct interaction of receptor and enzyme, by analogy with
enzyme systems composed of catalytic and regulatory subunits,or
linkage via a modulator (15).
2) Opiate-receptor complexes may elicit the production of chem-
ical messages, suggested as a rather unlikely possibility by 1I.O.J.
Collier (personal wmmunication).
3) Indirect coupling mediated by conformational transitions of
the membrane.  The membrane conformation may reflect either the
proportions of receptors in states A and B or may change in re-
ponse to the process of transition of the conformation of the
receptors betweenhtates A and B induced by the association and
dissociation'of agonists (but not of antagonists, since Na', main-
tains receptors in the B state). The indirect coupling mechanisms,
which involve membrane chanqes , may allow opiate receptors to
wpress their interaction with narcotics in more than one way.
Thus, the inhibition of adenylate cyclase and the electro-
physiologocal effects of narcotics on NG108-15 cells found by
Traber et al. (9), Myers and Livengood (16) and in our own
laboratories can be different manifestations of the same
fundamental effects on membrane structure.

   An important property of many of the narcotic analgesics
is that of mixed agonist-antagonist behavior.  Perhaps the best
studied example of such a compound is nalorphine which is a
potent antagonist of morphine, but also is a goad analgesic in
its cwn right (11).  fIow may this dualism of action be understood

119


in the context of opiate action as an inhibitor of adenylate
cyclase?  Figure 2 shows the effects of nalorphine upon adenylate
              l-l-

Figure 2.  Effects of nalorphine on the adenylate cyclase activity
of homogenates of NG108-15 cells.  The curves, reading from top
to bottom, represent experiments performed at the following
wncent ations
   5     of morphine:  none, 2 x 10 MI lo-*M, and
2 x 10' M.  The data have been normalized so that uninhibited
adenylate cyclase activity is wnstant.

cyclase activity at several wncentrations of morphine.  In the

absence of morphine, nalorphine inhibits the enzyme but only
partially when compared with the degree of inhibition produced
by morphine.  In the presence of morphine, on the other hand,
the effect of nalorphine is to reverse the inhibition produced
by morphine.  !rhe reversal of morphine inhibition by nalorphine
is also not complete but only restores enzyme activity to the
level seen in the presence of nalorphine alone.

   There is evidence that opiate receptors exist in two wnfor-
mational states (12, 13) as shown below:

                  Na+
                       A .d Receptor B

inhibition of adenylate cyclase            uncoupled

Receptor form A has a high affinity for agonists and form B for
antagonists as shown.  Binding of agonists to the receptor will
shi   the equilibrium  o the left and convert most receptors to
the fL onisteReceptor A wmplex which will result in inhibition of
aden late cyclase.  Ii
i              Conv  sely, when receptors are in the form of
the Antagonist*Receptor B complex,  as the result of interaction
with a pure antagonist, adenylate cyclase is not inhibited. Mixed
agonist-antagonists, such as nalorphine, may have a comparable af-
finity for receptors in both states.  The interaction of opiate
receptors with agonist-antmonist narcotics will then result in
the receptor complexes being partitioned between states A and B in
comparable amounts.  Inhibition of adenylate cyclase will thus be
only partial as is observed.

   When NGlO8-15 cells are cultured in the preselre of morphine
for a number of days, the level of adenylate cyclase activity in-
creases by approximately 50-1008.  An experiment which dcmon-
strate, this phenomenon is shown in fig 3.  The cells after 2 or
more days of exposure to morphine are tolerant in the sense that
adenylate cyclase activity is nearly normal when assayed in the
                             120


adenylate cyclase activity is nearly normal when assayed in the

              34YS CELLS TREATED WITH MORPHINE
Figure 3.
activity of nasal and PGQ stinulatcd adenylate cyclase
       homogenates of 16108-15 cells cultured in the
presence of 10 bl morphine for the tines shown (17).

presence of morphine.  They are dependent upon it in the sense
that adenylate cyclasa activity measured in its absence is
abnormally high.  The dependence phenomenon is dranatically seen
when cNIP levels of cells cultured in the presence of morphine
for 48 hours are measured after a brief exposure to naloxone.

Table IV CAMP levels of nornal and addicted cells (11)

                               CELLS
Conditions of Assay

basal          pnolcs cNJP/ng protein
                       20          23
naloxone                 21          37

PGC                    264          81
PGE + naloxqne         241        1183
adenoske '             103          65
adenosine + naloxone      72         217
naloxone

e of IlQxlal

  113
  175
4::
3::

Addicted cells show as much as a 4 to 5 fold increase in ci%P
levels Over the control, in the presence but not in the absence
of naloxonc precipitated withdra%lal.  There ia no change in the
number of opiate receptors in tolerant cells (17).

Pigure 4 summarizes the general conclusions which we have
reached wncerning tolerance and dependence on the basis of
these and other (17), related, experiments. Ne find that
morphine inhibits adenylate cyclase activity and thus de-
creases cAflP levels.  On continued exposure tc morphine
the cells adapt by an increase in adonylate cyclase activity
which results in tolerance and dependence.  The fully toler-
ant cells have CAMP levels close to normal in the presence
of morphine.  When the opiate is withdratm on addition of an
antagonist, CAM? levels rise to abnormally high values. This
abrupt increase in cA!lP indicates that the cells are depen-

121


         ADD             WITHDRAW
        MORPHINE            MORPHINE
ii
~200-  1      f
J
0.
5 ISO-
0

              0
Figure 4.                    TIME

dent upon morphine and is the biochemical counterpart of the
abstinence syndrome.  Recovery of the cells from the'addictcd
state requires the return of adenylate cyclase activity to
its normal levels.  These results support the suqgestions of
Caldstein and Goldstein (14) and Shuster (19), made many
years ago, that drugs may act as enzyme inducers. Increases
in CAMP levels in the abstinence syndrome of animals
has recently been demonstrated by Collier and Francis (18).

1.

2.

3.

4.
5.
6.

7.

8.

9.

10.
11.

12.

13.
E:

16.
17.
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