opiates and Endogenous Opioid Peptides
> 1976, Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands 153

DUAL REGULATION OF ADENYLATE CYCLASE BY
ENDOGENOUS OPIATE PEPTIDES

Werner A. Klee, Arthur Lampert and Marshall Nirenberg
Laboratory of General and Comparative Biochemistry, NIMH
and Laboratory of Biochemical Genetics, NHLI
National Institutes of Health
Bethesda, Maryland 20014

Morphine and related narcotics affect adenylate cyclase in two ways,
both mediated by the opiate receptor. The first process is the in-
hibition of adenylate cyclase activity (1-3). The second phenomenon
is a delayed increase in the specific activity of the enzyme, which
compensates for the inhibitory action of the narcotics, but requires
hours of exposure to the drugs before being expressed (4,5). This
dual regulation of adenylate cyclase has been proposed to account for
oniate tolerance and dependence, as well as for their acute effects
(4).

The discovery and characterization of endogenous opiate peptides
(6) prompted us to ask whether these substances also inhibit adenylate
cyclase and evoke a delayed increase in enzyme activity. Our studies
have shown that methionine enkephalin (Tyr-Gly-Gly-Phe-Met) , and other
endogenous opiate peptides, are potent. receptor mediated, inhibitors
or adenylate cyclase activity in homogenates of neuroblastoma x glioma
NG108-15 hybrid cells. Furthermore, upon prolonged incubation of
NG108-15 cells with Met-enkephalin, the specific activity of adenylate
cyclase is increased. Thus, Met-enkephalin causes both the immediate
inhibition and the delayed increase in adenylate cyclase activity
characteristic of dual regulation.

The high potency of Met-enkephalin as an inhibitor of basal and
PGE] stimulated adenylate cyclase in homogenates of NG108-15 cells is
shown in Fig. 1. The concentrations of Met-enkephalin required to

inhibit both basal and PGE, stimulated adenylate cyclase half-
154

 

 

 

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: = 30 3s
of a
iz eo <
& = 2
3 z
a 4 20 =
wi =
a a
=z uw
= ao 8
io Ly-— ; .

0 9 8 7 6
—LOG MET—ENKEPHALIN MOLARITY

Fig. 1. Inhibition of basal —"O° —:; and PGE, (10 uM) stimulated
—e —, adenylate cyclase activity of homogenates (97 pe protein/
tube of neuroblastoma x glioma NG108-15 cells by Met-enkephalin.
Assays were performed as described by Sharma et al (2) except that
incubations were for three minutes. Materials used in this work are

as described elsewhere (8).

maximally, along with similar data for Leu-enkephalin, morphine, and
etorphine (2), are shown in Table 1. Clearly, the enkephalins are of
very high potency in this assay, as they also are in the mouse vas
deferens (6). Indeed, in the adenylate cyclase assay, Met-enkephalin
is comparable to etorphine in activity.

Inhibition of adenylate cyclase by Met-enkephalin is receptor
mediated since it is reversed by naloxone, as shown in Fig. 2. As
expected for a competitive interaction at the opiate receptor, the
concentration of naloxone required to reverse the inhibitory action
of Met-enkephalin increases with increasing peptide concentration.
The dissociation constant for naloxone, Ke, calculated from these
data by the dose-ratio method (7), is 30 nM, in ,good agreement with
the value of 20 nM calculated from similar’ experiments with morphine
(8), instead of enkephalin, or obtained by direct measurement of

fialoxone binding affinity for the opiate receptor (2).

 

155

TABLE 1

ACTIVITY OF PEPTID.

E AND OTHER N.

OF AD ARCOTICS A

ENYLATE CYCLASE IN NG108-15 HOMOGENATES (2).

 

 

 

Compound x, °” (nM)
Basal (
PGE, 6°)
Tyr-Gly-Gly~Phe-Met 1
(met-enkephalin) 12 20
Tyr-Gly-Gly-Phe-Leu

(leu-enkephalin) 40, 100
Morphine 150
0

Etorphine 1500
10

 

(a) Assays
(ey ys performed as described previously (2).

Concentration of
i
{c) 10 uM in assays. nhibitor required for 50% of maximal effect

CAMP (pmoles/mg/min)

 

 

L I l
10° 10-4 1023

|
to or" 107 10-*

NALOXONE (Molarity}

Fig. 2. The relati
betwe
of aden on en naloxone con
ylate cyclase inhibition in homogenates (90 ug protes Weakest
ein/tube)

of NG108-1

mney oes AS ak SeheMGBDMann tb mt {SUUPTUME concentrations
The inhibitory action of Met-enkephalin on NG1O8-15 adenylate

“yetase is short lived, typically only 15 minutes or so (Fig. 3)

Note that morphine continues to inhibit the activity of the enzyne

for at least 90 minutes. The short duration of action of Met-
Lao

 

 

 

 

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Zz f T | a | T
9 2.0
a
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0 io 20 30 60 90
MINUTES

Fi 3 Adenylate cyclase activity of a homogenate (111 e protein/
tube) of NG108-15 cells, as a function of time. Additions to the
standard assay mixture were as follows: none —o—;
0.16 nM —A—; morphine, 20 uM —O—.
enkephalin is due to its destruction during the incubation rather
hl
than to desensitization since the enzyme is inhibited by a freshly
added portion of methionine enkephalin after 25 minutes of pre-
incubation with the peptide (Table 2). The relative lability of
ee-
Met-enkephalin activity compared with that of morphine is in agr
t
ment with results of analgesic assays of these materials in intac
animals (9-11), and with tests performed with the guinea pig ileum
preparation (6). -
Incubation of cultures of NG1O8-15 cells for 12 or more hours Ww
fic
methionine enkephalin results in a marked increase in the speci
f{ the
activity of adenylate cyclase as shown in Table 3. In view o
d
lability of Met-enkephalin, the experiments shown were performe
h
with a 100 fold excess of peptide over that required to saturate the
dz . Control experi-
receptors, and the medium was changed twice. aily
n
ments showed that approximately 90% of the enkephalin activity

he
such cultures is destroyed in 12 hours of incubation, and so t

 

157

TABLE 2

ADENYLATE CYCLASE ACTIVITY OF HOMOGENATES OF NG108-15
CELLS PREINCUBATED WITH AND WITHOUT MET-~ENKEPHALIN

 

Adenylate cyclase activity
- pmoles cAMP/min/mg protein

 

Preincubation
Additions to assay
Control Enkephalin
Water 16.4 16.5
6
Naloxone (10 1M) 16.4 17.2
Met-enkephalin 11.5 11.5
(0.16 uM)

 

Homogenates of NG1O8-15 cells (111 ug protein/tube) were pre-

incubated at 37° in the standard adenylate cyclase assay mixture,

omitting only radioactive ATP, for 25 min, with and without 0.16 uM

Met-enkephalin. Control experiments showed that adenylate cyclase

was inhibitgg by enkephalin for less than 20 minutes. After 25 min,
P

1 uCi of [a ] ATP was added to each tube along with other additions
as shown in column 1 of the table.

for an additional 5 min and [2p] ca
was determined in triplicate.

Incubations at 37° were continued
MP formed during this interval

amount of enkephalin present is in excess throughout the experiment

(12). Note that the extent of the increase in adenylate cyclase

activity elicited by enkephalin is similar to that found with etor-

phine; the time dependence is also similar.
The experiments described here demonstrate that an endogenous

opiate peptide acts as a dual regulator of adenylate cyclase exactly

as morphine and other conventional narcotics. Thus, these neuro-

hormones, or transmitters, can be expected to inhibit adenylate

cyclase activity of neurons with opiate receptors and thereby
suppress the effects of other neurohormones or transmitters which
activate adenylate cyclase. As a result of the delayed increase in

adenylate cyclase activity caused by the endogenous opiates, some

neurons may be rendered supersensitive to the effects of agents

which stimulate the enzyme. Shifts in the levels of endogenous

oplate peptides may increase or decrease the flow of information
TABLE 3

ADENYLATE CYCLASE ACTIVITY IN HOMOGENATES OF NG108-15
CELLS CULTURED FOR THE TIMES SHOWN WITH MET-ENKEPHALIN
OR ETORPHINE

 

Adenylate cyclase activity

Ho.vs of treatment
: pmoles cAMP/min/mg protein

 

Control Met-enkephalin Etorphine
0 7 - -
12 9 17 19
25 11 19 22
48 15 23 31
97 17 38 43

 

Logarithmically growing cultures of NG108-15 cells were maintained
with additions to the culture media as follows: Met~enkephalin,

10 uM, and etorphine, 1 uM. Media were changed twice daily, and
cells were harvested, homogenized, and assayed for basal adenylate
cyclase activity in the presence of 100 1M naloxone. PGE stimulated
adenylate cyclase activities also were increased, although to a
smaller extent. (12).

through specific neural circuits, and also regulate the responses

of adenylate cyclase to other kinds of receptor mediated reactions.
Cellular tolerance and dependence induced by the endogenous opiate
peptides may play a normal role in switching certain neural pathways

on or off.

REFERENCES

1. Collier, H. 0. J. and Roy, A. C. (1974) Nature 248, 24-27.

2. Sharma, S. K., Nirenberg, M. and Klee, W. A. (1975) Proc. Natl.
Acad. Sci. U.S. 72, 590-594.

3. Traber, J., Fischer, K., Latzin, S. and Hamprecht, B. (1975)
Nature 253, 120-122.

4. Sharma, S. K., Klee, W. A. and Nirenberg, M. (1975) Proc. Natl.

Acad. Sci. U.S. 72, 3092-3096.

 

10.

ll.

12,

Loy

Traber, J., Gullis, R. and Hamprecht, B. (1975) Life Sci. 16,
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Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A.,
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Klee, W. A. and Nirenberg, M., in preparation.

Belluzzi, J. D., Grant, N., Garsky, V., Sagantakis, D., Wise,

C. D. and Stein, L. (1976) Nature 260, 625-626.

Buscher, H. H., Hill, R. C., Romer, D., Cardinaux, F., Closse, A.,
Hauser, D. and Pless, J. (1976) Nature 261, 423-425.

Pert, A., Simantov, R. and Snyder, 8. H. (1976) Proc. Natl. Acad.
Sci. U.S., in press.

Lampert, A., Klee, W. A. and Nirenberg, M., in preparation.