Exposure to restraint stress resulted in antagonism of the analgesic effect of administered morphine in adult male rats. This antagonism of morphine-induced analgesia by restraint stress was not affected by adrenalectomy one day prior to testing, suggesting that stress-induced secretion of corticosteroids is not critical to this antagonism. In addition, parenteral administration of exogenous adrenocorticotropin (ACTH) mimicked the effect of stress in antagonizing morphine's analgesic efficacy. The hypothesis that ACTH is an endogenous opiate antagonist involved in modulating pain sensitivity is supported

Heybach, J. P.

Shannon, L.

Vernikos, J.

English

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NASA Technical Memorandum 81282
Stress Antagonizes Morphine-
induced Analgesia in Rats
Joan Vernikos, Lynn Shannon, and John P. Heybach
(NASA-TM-81282) STRESS ANTAGONIZES
	 N81-21732
MURPH.INE-INDUCED .ANALGESIA IN RATS Final
Report (NASA) 18 p HC A02/,MF A01
	 CSCL 06C
Unclas
G3/51 42065
April 1981
n W VI IL
National Aeronautics and
Space Administration
N&Vk
National Aeronautics and
Space Administration
Ames Research Cuter
Moffett Field, California 94035
N
NASA Technical Memorandum 81282
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Stress Antagonizes Morohinem I 	 I
Induces Analgesia an Rats	 I I
Joan Vernikos
!..ynn Shannon
John P. Heybach, Ames Research Center, Moffett Field, California
J. YERNIKOS, L. SHANNON and J. P. HEXBACH, Stress antagonizes morphine-
E
.	 induced analgesia in rats, European J. Pharmacol. XX(1981)XX-XX.
Exposure to restraint stress resulted in antagonism of the analgesic
effect of administered morphine in adult male rats. This antagonism of
morphine-induced analgesia by restraint stress was not affected by adrenalectomy
one day prior to testing, suggesting that stress-induced secretion of cortico-
steroids is not critical to this antagonism. In addition, parenteral adminis-
tration of exogenous adrenocorta. cotropin (ACTH) mimicked the effect of stress
in antagonizing morphine's analGasic efficacy. These: results support the
hypothesis that ACTH is an endogenous opiate antagonist involved in modulating
pain sensitivity.
1	 Adrenocorticotropin
	 Morphine-induced analgesia.
	 Stress
1
1
1. Introduction.
We have shown previously, through classical endocrine manipulations, that
altering the endogenous circulating level of ACTH results in changes in sensi-
tivity to pain (Heybach and Vernikos, 1978). Hypophysectomy reduces pain
sensitivity In the rat and in man (Luf t and Olivecrona, 1955), and this effect
Is reversed in the rat by the intracerebroventricular (ICV) administration of
the synthetic opiate antagonist naloxone (Heybach and Vernikos, in press).
These observations promoted us to suggest that pituitary ACTH acts as an.
endogenous hyperalgesic factor and opiate antagonist (Heybach and Vernikos,
19118; 1981). Evidence in favor of this suggestion were the findings that the
ICV injection of crude ACTH, AOTHz- 2 4, or a-MSH resulted In increased sensi-
tivity to a painful thermal stimulus in the rat and that the peripheral admin-
istration of these peptides antagonized the analgesia in morphine pretreated
rats (Gispen et al., 1976; Paroli, 1967; Heybach and Vernikos, 1981). In the
course of these experiments we also observed that the stress of a saline injec-
t ,.(on in our control animals also reduced the analgesic effect of morphine pre-
treatment though to a much 'lesser extent than peptide treatment.
It is well known that the pituitary gland synthesizes and secretes ACTH
in response to stressful stimuli (Vernikos-Danellis, 1963). The present study
was therefore undertaken to determine the effect of stress-induced release of
endogenous ACTH on the analgesic action of morphine and on pain sensitivity in
general. The results indicate that restraint stress reduces the efficacy of
administered morphine in reducing the sensitivity to a painful thermal stimulus.
This antagonism occurs in adrettalectomized animals as well as in normal animals
and can be reproduced by the administration of exogenous ACTH.
2
c?
c
i
V	 2. Materials and methods
r. Male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, Calif.) weighing
i.
200 ±10 g were used throughout these experiments. They were housed 4-5 per
R
cake in plastic.-top loading cages with woodchp bedding and maintained on a
12--h light/dark cycle (lights on at 0700 h) in a vivarium with temperature
maintained at 23 ±3°C. Rats had ad libitum access to standard laboratory chow
and tap water. Groups of at least  rats were used in each experimental
condition.
Drugs used and their suppliers were: ACTH from Armour Labs, Kankakee,
Illinois; morphine sulphate from Endo Labs, Garden City, New York. Doses of
morphine were calculated as the free base.
Adrenalectomies were performed via the dorsal approach under ether
anesthesia. After surgery, adrenalectomized rats were provided with 0.9%
saline instead of tap water. Following all experimental procedures,
adrenalectomized rats were sacrificed and examined for completeness of
adrenalectomy.
TLe apparatus used to as6ess pain sensitivity has been described in detail
previously (Heybach and Vernikos, 1978). The procedure for estimating pain
sensitivity was standardized for all experiments. All animals were initially
allowed three 90-sec sessions, one session per day for 3 consecutive days, with
the temperature of the grid floor maintained at 23 ±1°C. This procedure
familiarized the rats withhandling and allowed for habituation to the novelty
of the apparatus. To initiate a testing session, the rat was placed on ther
grid floor of the apparatus with the temperature maintained at 55 ±1°C and a
stop watch was started simultaneously. Two responses were then recorded:
z
	
	
the latency to lift from the floor and. lick one paw (i.e., paw lick latency)
and, subsequently, the Latency to show a jump response consisting of
3
vigorously lifting both hind paws off the grid floor simultaneously (i.e., jump
latency). The paw lick and jump latencies were used as indices of the sensi-
tivity to the painful stimulus. The experimenter was not aware of the pre-
treatment of the rats during testing. If neither a paw lick nor jump response
occurred within 90 sec the test was terminated and a latency of 90 sec was
recorded for each response. All testing was carried out between 0800-1200 h.
Restraint stress consisted of placing the rat in a semicylindrical plexi-
glass device that comfortably restricted movement. Animals were injected
intraperitoneally (i.p.) with morphine immediately upon removal from the
restraint device and tested for pain sensitivity at 10, 20, and 30 min later.
In one experiment rats were 41so tested 60 min after the morphine injection to
determine the duration of its effect,
3. Results
Figure 1 shows the results of an experiment investigating the effect of
the duration of the restraint stress on the analgesic efficacy of morphine.
Three different dose levels of morphine (2.5, 5.0, and 10 mg/kg) were used.
They were injected using a Latin square design to minimize errors due to
experimental bias. Restraint stress alone for 15, 30, or 60 min had no effect
on the sensitivity to this painful thermal stimulus. The three doses of
morphine resulted in a dose-related degree of analgesia. Exposure to restraint
stress for 15 min completely prevented the analgesic effect of 2.5 mg/kg
morphine and significantly reduced that of 5.0 mg/kg, whereas it had no effect
on the 10 mg/kg dose. Thirty minutes of restraint also significantly reduced
the analgesic action of both the 2.5 and 5.0 mg/kg doses, having little effect
on the largest dose. On the other hand, 60 min of restraint had no effect on
<Fig.
g. 2
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<ig
<Fig.
f'.
the action of either of the lower doses of morphine, but significantly
potentiated the 10 mg /kg doge.
Figure 2 shows the time course of the analgesia produced by the three
doses of morphine as compared to saline-injected controls and the effect of
15 min of restraint stress on that response as measured by jump latencies.
The analgesic effect of morphine was evident as early as 10 min after its
injection and with the two higher doses contiinued to increase at 30 min.
Fifteen minutes of restraint stress effectively antagonized the 2.5 and
5.0 mg/kg doses at all time intervals tested. The data show that although
stress effectively antagonized the 10.0 mg /kg dose when the animals were tested
at 10 and 20 mitt, the effect was transient and at 30 min after the morphine
injection this large dose was as effective in the stressed as in the non-
stressed animals.
To determine whether stress-induced adrenocortical secretion was involved
in this antagonism . of the analgesic effect of morphine, animals that had been
adrenalectomized one day previously were exposed to 15 min of restraint or no
restraint and then given 5 . 0 mg/kg morphine. Figure 3 shows that the absence
of the adrenals did not affect the ability of stress to reduce the analgesic
action of this dose of morphine.
Figure 4 shows the comparison of the effect of 15 min of restraint or of
a single dose of ACTH (50 uU, i.p.) administered 15 min prior to the injection
of morphine (5.0 mg /kg). Animals were tested 30 min later. It can be seen
that this dose of ACTH is as effective as 15 min of restraint in reducing the
analgesic action of morphine.
w^
5
4. Discussion
__19
The antagonism of the analgesic effect of morphine by prior exposure of
an animal to 15 or 30 min of stress adds additional support to the hypothesis
that an endogenous opiate antagonist exists and that this substance may be
ACTH or an ACTH-like peptide from the pituitary. During restraint stress, as
with other stressful stimuli, ACTH is secreted very rapidly with maximal
levels occurring within 2.5 min after the stress begins; the level then falls
gradually for the next 60 min, but remains above control. values (Sakellaris
and Vernikos-Danellis, 1975).
Plasma corcicosterone reaches maximal levels within 10 min after restraint
begins and is sustained for the remaining 60 min. However, the persistence of
the stress-induced antagonism of morphine analgesia in the adrenalectomized
animals suggests that the secretions of the adrenal cortex are not involved
in this effect. However, the potentiation of the analgesia produced by the
largest dose of morphine by prior exposure to 60 min of restraint may be partly
attributable to the high circulating levels of corticosteroids. Holaday et al.
(1979) obtained evidence that corticosteroids may increase the parenteral
potency of morphine by increasing tts bioavailability through inhibition of its
metabolism. Even more likely is the possibility that endogenous opiate activ-
ity may be increased relative to ACTH at 60 min and may result is the poten-
tiation observed at that time of the effect of the largest dose of morphine.
Although it has been reported that pituitary endorphin is secreted concom-
itantly with ACTH in response to acute stress (Cuillemin et al., 1977), the
dynamics of its response to a more sustained stress, such as restraint, have
not been described. Such description has also been hampered by the problem of
specific,?.ty of the antibodies used in the radioimmunoassay of endorphins
6
in the early phases of this research and by the lack of correlation between
immunoreactivity and opiate activity (Li et al., 1977). Nevertheless, poten-
tiation of morphine analgesia by more long-lasting stress, as seen in this study,
may involve mechanisms that are akin to the stress-induced analgesia reported
by others (Akil et al., 1976). In our study we never observed an analgesic
effect of this form of restraint stress alone.
Fifteen minutes of restraint was as effective in reducing the analgesic
efficacy of morphine as 50 u U of ACTH (i.p.) administered 15 min earlier. Thus,
it would appear that the effect of short periods of stress in antagonizing
morphine-induced analgesia may be mediated by this pituitary peptide. ACTH is
effective in antagonizing morphine whether it is given before the opiate as in
this study, or after the opiate (Heybach and Vernikos, 1981). It is also
effective in this r;, spect°at doses that do not in themselves produce increased
sensitivity,, 
to. 
pain.
-'ACTH and ACTH-like peptides have been shown to antagonize morphine
(Terenius et al., 1975) and O-endorphin binding (Akil et al., 1980) to prepara-
tions of rat brain homogenates in vitro. Therefore, from this data and that of
our studies (Heybach and Vernikos, 1978; in press) it would appear that the
pituitary is the source of an endogenous opiate antagonist and hyperalgesic
factor, probably ACTH oran ACTH-like peptide secreted and regulated in
exactly the same way as ACTH, and that its action is via a direct effect on
central nervous system opiate receptors.
i
7
y
References
Akil, H., J. Fadden, R. L. Patrick and J. D. Barchas, 1976, Stress induced
increase in endogenous opiate peptides: Concurrent analgesia and its
partial reversal, by naloxone, in: Opiates and Endogenous Opioid Peptides,
ed. H. W. Kosterlitz (Amsterdam, Elsevier, North Holland), pp. 63-70.
Aki,l, H., W. A. Hewlett, J. D. Barchas and C. H. Li, 1980, Binding of
9H-0-endorphin to rat brain membranes: characterization of opiate
properties and interaction with ACTH, Eur. J. Pharmac. 64, 1-8.
Gispen, W. H., J. Buitelaar, V. M. Wiegant, L. Tereni,us and D. de Wied, 1976,
Interaction between ACTH fragments, brain opiate receptors, and morphine
induced analgesia, Eur. J. Pharmac. 39, 393-397.
Guillemin, R., T. Vargo, J. Rossi,er, Minck, N. Ling, C. Rivier, W. Vale and
F. Bloom, 1977, S-endophin and adrenocorticotrophin are secreted concom-
itantly by the pituitary gland, Science 197, 1367-1369.
Heybach, J. and J. Vernikos, 1.978, The effect of pituitary-adrenal function in
the modulation of pain sensitivity in the rat, J. Physiol. (London) 283,
331-340.
Heybach, J. P. and J. Vernikos, (in press), ACTH-Pike peptides produce hyper-
algesia and antagonize morphine induced analgesia, Eur. J. Pharmac.
Holaday, J. W., P. Y."Law, H. H. Loh and C. H. Li, 1979, Adrenal steroids
indirectly modulate morphine and R-endorphin effects, J. Pharmacol.
E. Therap. 208, 176-183-.
Li, C. H. J. Rao, B. A. Doneen and D. Yams.shiro, 1977, Lack of correlation
between opiate activity and immunoreactivity by radioimmunoassay,
Biochem. riophys. Res. Commun. 75, 576-580.
Luft, R. and H. Olivecrona, 1955, 1Hypophysectomy in man, Experience in metastic
cancer of the breast, Cancer Res. 8, 261-266.
1
8
Paroli, E., 1967, Indogini sul.l efetto antimorfi iem dell ACTH, I. Relazioni
con it corcitosurrene ed i livelli emotica 11-OH steroidi, Arch. Ital.
Sci. Farmacel. 13, 234-237.
Sakellaris, P. C. and J. Vernikos-Danellis, 1975, Increased rate of response of
the pituitary-adrenal system in rats adapted to chronic stress,
Endocrinology 97, 597-602.
Terenius, L., W. H. Gispen and D. de Weid, 1975, ACTH-like peptides and opiate
receptors in the rat brain: structure-activities studies, Eur. J.
Pharmac. 33, 395-399.
Vernikos-Danellis, J., 1963, Effect of acute stress on the pituitary glands
Changes in blood and pituitary ACTH concentrations, Endocrinology 72,
574-561,
.«q
w	 9
Fig. 1. The effect of restraint stress for 15, 30, or 60 min on the analgesic
action of various doses of morphine (2.5, 5, and 10 mg/kg; i.p.). Analgesia
was measured as the effect on paw lick (A) and jump (B) latencies, 30 min
after the administration of morphine.
Morphine 2.5 mg/kg
15 min stress vs no stress (NS)
Paw lick: F(1, 23) = 16.1, p < 0.005
,lump: F(I, 23) 36,8, p < 0.001
30 min stress vs no stress (NS)
Paw lick: F(1, 21) -	 6.27, p < 0.05
Jump: F(1, 21) = 12.2 0 p < 0.01
Morphine 5.0 mg/kg
15 min stress vs no stress (NS)
Paw Lick: F (I. , 25) = 13.2, p < 0.005
Jump: NS
30 min stress vs no stress (NS)
Paw Lick: F(1, 22) = 24.2, p < 0.001
Jump: NS
Fig. 2. Time course of the effect of three different doses of morphine
(2.5, 5.0, and 10.0 mg/kg) administered i.p. or saline in unstressed animals
or in animals subjected to 15 min restraint. Analgesia wan measured as the
effect on jump latencies.
Fig. 3. The lack of effects of adrenalectomy on the antagonism of morphine
analgesia by restraint stress.
10
Fig. 4. The effect of treatment with ACTH (Armour; 50 uU i.p. 15 min previously)
or restraint stress for 15 min on paw lick and Jump latencies 34 min after
morphine pretreatment (5 mg/kg; i.p.). Each bar represents the mean i S.E. of
the mean.
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15


Stress antagonizes morphine-induced analgesia in rats

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