The adrenal actions were stereospecific since neither the positve stereoisomer of morphine, nor that of naloxone, had any effect on the adrenal response to exogenous adrenocorticotrophic hormone (ACTH). The administration of human beta endorphin to phyophysectomized rats had no effect on the adrenal corticosterone concentration nor did it alter the response of the adrenal gland to ACTH. These results indicate that morphine can potentiate the action of ACTH on the adrenal by a direct, stereospecific, dose dependent mechanism that is prevented by naloxone pretreatment and which may involve competition for ACTH receptors on the corticosterone secreting cells of the adrenal cortex

Heybach, J. P.

Vernikos, J.

English

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81253
Naloxone Inhibits and
Morphine Potentiates
The Adrenal Steroidogenic
Response to ACTH
John Hevbach and
Jean Vernikos, Ames Research Center, Moffett Field, California
Antes Rasearch Center
J. T.
	
and J_ YM	 . Nalamome inhibits and ao=phiee potentiates
the adrenal steroi4wale tesee!!E to ACM. European J. ldatmacol. U(1481 IM-1[_
The administration of morphine to hypophy taained rats potentiated the
steroidogenic response of the adrenal cortex to e,=gPmoa1 adrenocorticotrophic
hormone (ACT's) in a dose dependent fashion. Conversely, the opiate antagonist
naloxone inhibited the adrenal response to ACTS. Naloxone pretreatment Also
antagonized the potentiating effect of morphine on ACM-induced steroidogenesis
in a dose-dependent saner_ r6either morphine nor nalowne, administered to
by.pophysectomized rats, had any direct effect on adrenal steroidogenesis.
These adrenal acticas were stereospecific since neither the (+)-stereoisomer
of morphine, nor that of naloxone, had any effect on the adrenal response to
ACTH. The ad:in stration of husan d-endorphin to hypophysectomized rats had
no effect on the adrenal corticosterone concentration nor did it alter the
response of the adrenal gland to ACTH. These results indicate that morphine
can potentiate the action of ACTH on the adrenal by a direct, stereospecific,
dose-dependent mechanism that is prevented by naloxone pretreatment and which
may involve competition for ACTH receptors on the r_orticosterone-secreting
cells of the adrenal cortex.
1 .0'
1. Introduction
Adainistration of morphine has diverse effects on various endocrine
systems believed to reflect the action of this ding on hypothalamic endocrine
control mechanisms. For example, morphine administration results in
inhibition of ovulation in rats (Cicero et al., 1976; Packman and Rothchild,
1976) and reduces the proestrous increase of circulating luteinizing hormone
(Pang et al., 1977). Both of these effects are reversed by treatment with
the opiate antagonist naloxone.
Intrahvpothalamic (Lotti et al., 1969) and peripheral (Kokka and George,
1974) morphine injections stimulate the secretion of adrenocorticotrophic
hormone (ACTH) from the pituitary, as reflected by increased plasma levels of
adrenal corticosterone in the rat. This stimulation is also abolished by
naloxone pretreatment (Kokka and George, 1974). Morphine also stimulates the
release of growth hormone, but this effect is not alrered by naloxone
pretreatment (Kokka and George, 1974).
It has been demonstrated that ACTH can competitively inhibit opiate
binding in vitro showing mixed agonist-antagonist actions (Krivoy et al.,
1977; Terenius et al., 1975; Wiegant et al., 1977). In vivo studies have
shown that naloxone blocks the effect of chronic ACTH treatment in retarding
the development of reproductive function in mice (Yasukawa et al., 1978).
These observations suggest that compounds with an affinity for opiate receptors
might interact with ACTH receptors. We investigated this hypothesis by
determining in viva the action of acute morphine, 6-endorphin, and naloxone
pretreatment on the steriodogenic response of the adrenal cortex to ACTH.
2. Materials and methods
Yale Sprague-Lawley rats weighing 100 110 g (Simonsen Laboratories,
Gilroy, Calif.) were used. The rats were hotmed 5 per cage in large plastic
cages and maintained with free access to food and water in an animal colony
.&-
	 with controlled temperature (23%) on a regulated photoperiod of 12L:12D
(lights on at 0800 h).
Drugs used and their suppliers mere: Aj=—' l i _-= .'."'*ter !Abs, Kankakee,
Illinois; Human $-endorphin from Sachem, Inc., Torrance, California; both
(-)-morphine and (-)-naloxone from Endo Labs, Garden City, New York;
stereoisomers (+)-morphine and (+)-naloxone supplied by Dr. Kenner C. Rice,
Medicinal Chemistry Section, National Institutes of Health, Bethesda,
Maryland. The s-endorphin and ACTH were dissolved in 0.9% NaCl solution
containing 0.12 acetic acid. Morphine and naloxone were dissolved in 0.9%
NaCl. Doses of morphine and naloxone were calculated as the free base.
All rats used in these experiments were hyrophysectorized. At 18-20 h
prior to drug administration, the pituitary glads were removed under ether
anesthesia to eliminate the source of endogenous circulating ACTH. Pituitaries
were removed by a transaucicular approach in a stereotaxic instrument (Gay,
1967). Experimental procedures were carried out between 0800-1100 h on the
morning following hypophysectomy.
Initially, each drug was administered to different groups of rats to
determine any direct effect they might have on adrenal gland steroidogenesis.
ACTH (100 UU), S-endorphin (100 pg), or vehicle solution was injected into the
femoral vein of the anomals under ether anesthesia. They were sacrificed
5 min later and their adrenal glands removed, cleaned, and weighed. Various
doses of (-)-morphine (0, 5, 10, and 20 mg/kg) or (-)-naloxone (0, 12.5, 25,
and 50 mg/kg) were injected intraperitoneally and rats were sacrificed
3
20 min later. All compounds were injected in a total volume of 1 ml. Adrenal
glands were homogenized in 0.9: saline solution and frozen for later
determination of corticosterone content by a fluorometric method
(Vernikos-Danellis et al.. 1966).
To determine the time course of the effect of pretreatment with these
compounds on the adrenal steroidogenic response to ACTH, (-)-morphine
(10 mg/kg, i.p.) or (-)-naloxone (50 mg /kg, i.p.) were injected at various
times prior to ACTH (100 uU, i.v.). Rats Pere sacrificed 5 min following the
injection of ACTH and the corticosterone concentration in their adrenal glands
was determined. Dose-response relationships of the effect of morphine or
naloxone on the steroidogenic responses to ACTH (100 uU, i.v.) were also
determined 20 min following pretreatment with various doses of (-)-morphine
(0, 5, 10, and 20 mg/kg, i.p.) or (-)-naloxone (0, 12.5, 25, and 50 mg/kg,
i.p.) or 5 min following pretreatment with a-endorphin (100 vg, i.v.). Rats
were sacrificed 5 min following ACTH injection for measurement of adrenal
corticosterone concentrations.
Finally, the steroidogenic response to ACTH (100 pU, i.v.) was determined
20 min following pretreatment with either the (-)- or (+)-stereoisomeric forms
of morphine and naloxone (i.p.). Rats were sacrificed 5 min following the
ACTH injection and adrenal glands were removed and processed as described
above.
Results are expressed as ug of corticosterone per 100 mg of adrenal gland
tissue, fresh weight. The data were statistically analyzed by using overall
one-way analyses of variance followed by constructing 95% confidence limits
around the appropriate control group mean with the conservative post-hoc
procedure of Dunnett (Keppel, 1973).
4
3. Results
3.1. Effect of ACTH, morphine, and naloxone on adrenal gland content of
corticosterone
As shown in Table 1, ACTH (100 kU) stimulated steroidogenesis and led to
ff	 an almost threefold increase in the corticosterone content of the adrenal
glands. Neither morphine, naloxone, nor 8-endorphin, when administered alone,l
had any effect on the adrenal gland content of corticosterone at any of the
doses injected.
3.2. Time course cf the effect of morphine and naloxone on the steroidogenic
response to ACTH
Pretreatment with (-) -morphine (10 mg/kg) potentiated the steroidogenic
response of the adrenal gland to ACTH (100 uU), [F(5,41) - 12.1, p < 0.011
(fig. IA). This effect was most evident when the time interval between the
morphine pretreatment and ACTH injection was greater than 10 min (p < 0.05
at 20, 40, and 60 min). Although adrenal gland corticosterone levels were
elevated when ACTH was injected at 10 min following morphine pretreatment,
this elevation was not significantly different from that produced by ACTH
alone (p < 0.05).
Pretreatment with (-)-naloxone antagonized the steroidogenic response of
the adrenal gland to ACTH [F(5,40) - 9.7, p < 0.011, with a time course of
action similar to that seen for morphine's potentiation of the response
(fig. 1B).
3.3. Effect of increasing doses of morphine and naloxone on the steroidogenic
responses to ACTH
Figure 2A shows that morphine, injected 20 min before the ACTH, potentiate
the steroidogenic responses to ACTH at doses up to 10 mg/kg [F(6,44) - 10.7,
5
p < 0.011. However, at higher doses (20 mg/kg), morphine no longer potentiated
ACTH and may even have caused some inhibition of this response.
There was a dose-response relationship in the antagonism of the
steroidogenic response to ACTH by naloxone. This drug was particularly
effective at two higher doses (25 and 50 mglkg) [F(6,44) - 10.1, p < 0.011
(fig. _B).
3.4. Stereospecificity of the effects of morphine and naloxone on the
steroidogenic response to ACTH
Pretreatment with (-)-morphine (10 mg/kg) significantly (p < 0.05)
increased the adrenal steroidogenic response to ACTH (100 uU) injected 20 min
following morphine pretreatment (fig. 3). Pretreatment with (+)-morphine
(10 mg/kg) was without effect.
Similarly, (-)-naloxone (10 mg/kg) inhibited the response to ACTH
(p < 0.05) while (+)-naloxone (10 mg/kg) was without effect. When (-)-naloxone
(10 mg/kg) was injected prior to the (-)-morphine (10 mg/kg), the response of
the adrenal cortex to ACTH (100 pU) did not differ from the steroidogenic
response to the injection of ACTH alone. Pretreatment with 6-eudurphin
(100 ug) 5 min prior to ACTH injection (100 uU) had no effect on the
steroidogenic response of the adrenal.
4. Discussion
Morphine administration has been shown to stimulate corticosterone
secretion and adrenal gland function (Briggs and Munson, 1955). This response
has been presumed to be mediated solely by the hypothalamic-pituitary axis or
by a direct action at the pituitary (Kokka and George, 1974; Lotti et al.,
1969; Zimmerman and Critchlow, 1973). The present results indicate that
h
.0'
morphine can also influence the steroidogenic response to ACTH by a direct
action on the adrenal cortex.
At doses up to 10 mg/kg, the potentiation of ACTR-induced steroidogenesis
by morphine appears to be dose-dependent and stereospecific since (+)-morphine
was without effect. The opiate antagonist naloxone exerted effects on the
steroidogenic response to AC R that were also stereospecific and dose-dependent
but opposite to those of morphine. In addition, naloxone prevented the
potentiation by morphine of the ACTH-induced steroidogenic response. These
results suggest that morphine and naloxone interact with receptors on
corticosterone-secreting cells of the adrenal cortex.
It has been shown that addition of the opiate antagonists naloxone or
naltrexone to dispersed rat adrenal cell preparations inhibited the steroidogenic
response to ACTH, but had no effect on the steroidogenic response to the
addition of dibutyryl cyclic AMP (Ginsburg et al., 1978). This observation
and our results lend further support to the hypothesis that naloxone and,
presumably, morphine exert effects on the adrenocortical response to ACTH by
competition at ACTH adrenal receptor sites.
Alternately, mediation by an adrenal opiate receptor must also be
considered. However, this is unlikely since, in the present work, the
administration of 8-endorphin or of morphine alone, at doses up to 20 mg/kg,
did not stimulate steroidogenesis. Morphine's effect was only detectable in
the presence of ACTH. Furthermore, other work has failed to demonstrate the
presence of specific opiate receptor binding in adrenal-gland homogenates
(Simantov et al., 1978).
The evidence that ACTH can interact with opiate receptors in the central
nervous system and in the periphery (Krivoy et al., 1977; Gispen et al., 1976;
Terenius et al., 1975; Wiegant et al., 1977) suggests the possibility that
7
opiates may also interact with ACTH receptors. The block by naloxone of the
ACTH-induced delay of puberty and the reversal of ACTH- induced decreases in
reproductive organ weights of mice (Uasukaws et al., 1978), as well as the
antagonism by naloxone and potentiation by morphine of the steroidogenic
.&' 
	 effects of ACTH seen here, lend support to this possibility.
The present results strongly indicate that morphine and naloxone exert
opposite effects on the response of the adrenal cortex to ACTH by acting at
ACTH receptor sites. The results also demonstrate a pharmacological effect
of morphine most probably at the adrenal gland level that is dose-dependent,
stereospecific, and prevented by naloxone. The lack of effect of the endogenous
opiate S-endorphin on the response of the adrenal to ACTH might then be a
function of the dose or time course used here, or may indicate no significant
physiological effect of this particular endogenous opiate on the adrenal
cortex. On the other hand, the recent evidence that the adrenal medulla is
the primary source of circulating enkephalins (Tam and Yu, 1980) introduces the
possibility that this opiate, of adrenomedulary origin and in close proximity
to the adrenal cortex, may serve a physiological function in regulating
adrenal sensitivity to ACTH. Some evidence exists to suggest that 8-endorphin
and enkephalins may bind differently to the same receptor or exert their
opiate-like effects by binding to different receptors but with overlapping
specificity (Hazum et al., 1979).
8
References
Briggs, P. N. and P. L. Munson. 1955, Studies on the mechanism of stimulation
of ACTH secretion with the aid of morphine as a blocking agent,
Endocrinology 57, 205-219.
Cicero, T. J., E. R. Meyer, R. D. Bell and G. A. Koch, 1976, Effects of
morphine and methadone on serum testosterone and luteinizing hormone
1Fv_ls and in the secondary sex organs of the male rat, Endocrinology
98, 367-372.
Edwards, C. and V. L. Gay, 1967, A sterotaxic approach to transauricular
hypophysectomy in the rat, Endocrinology 81, 1177-1179.
Ginsburg, M., A. Gibson, M. Hall and S. L. Hart, 1978, Steroidogenesis in
adrenal cortex: efferents of Li+, opioid antagonists and Na+ dependence
of ACTH response, Proc. Int. U. Pharmac. 7, 1256.
Gispen, W. H., J. Buitelaar, V. M. Wiegant, L. Terenius and D. de Wied, 1976,
Interaction between ACTH fragments, brain opiate receptors, and morphine
induced analgesia, Eur. J. Pharmac. 39, 393-397.
Hazum, E., K. -J. Change and P. Cuatrecasas, 1979, Interaction of iodinated
human ID-Ala 2 ] S-endorphin with opiate receptors, J. Biol. Chem. 254,
1765 -1767.
Keppel, G., 1973, Design and Analysis (Prentice-Hall, Inc., Englewood Cliffs,
New Jersey), pp. 153-157.
Kokka, N. and R. George, 1974, Effects of narcotic analgesics, anesthetics,
and hypothalamic lesions on growth hormone and adrenocorticotrophic
hormone secretion in rats, in: Narcotics and the Hypothalamus, eds.
E. Zimmerman and R. George (Raven Press, New York), pp. 137-157.
Krivoy, W. A., D. C. Kroeger and E. Zimmerman, 1977, Neuropeptideinfluence
acute and chronic effects of opiates, Psychoneuroendocrinology 2, 43-51.
9
Lotti, V. J., N. Kokka and R. George, 1969. Pituitary-adrenal activation
following intrahypothalataic microinjection of morphine, Neuroendocrinology
4, 326-332.
Packman, P. M. and J. A. Rothchild, 1976, Morphine inhibition of ovulation:
reversal by naloxone. Endocrinology 99, 7-10.
Pang, C. N., E. Zimmerman and C. H. Sawyer, 1977, Morphine inhibition of the
preuvulatory surges of plasma luteinizing hormone and follicle stimulating
hormone in the rat, Endocrinology 101, 1726-1732.
Simantov, R., S. R. Childers and S. H. Snyder, 1978, The opiate receptor
binding interactions of 3H-methionine enkephalin, an opioid peptide,
Eur. J. Pharmac. 47, 319-331.
Tan, L. and P. H. Yu, 1980, Biosynthesis of enkephalins by chromaffin cells
of bovine adrenal meCi. Ia, Biochem. Biophys. Res. Communic. 95, 1901-1908.
Terenius, L., W. H. Gispen and D. de Wied, 1975, ACTH-like peptides and opiate
receptors in rat brain: structure-activity studies, Eur. J. Pharmac.
33, 395-399.
Vernikos-Danellis, J., E. Anderson and L. Trigg, 1966, Changes in adrenal
corticosterone concentration in rats: Method of bio-assay for ACTH,
Endocrinology 79, 624-630.
Wiegant, W. M., W. H. Gispen, L. Terenius and D. de Wied, 1977, ACTH-like
peptides and morphine. Interaction at the level of the CNS,
Psychoneuroendocrinology 2, 63-69.
Yasukawa, N., H. Monder, S. D. Michael and J. J. Christian, 1978, Opiate
antagonist counteracts reproductive inhibition by porcine ACTH extract,
Life Sci. 22, 1381-1-390.
Zimmerman, E. and V. Critchlow, 1973, Inhibition of morphine-induced
pituitary-adrenal activation by dexamethasone in the female rat, Proc.
Soc. Exp. Biol. Med. 143, 1224-1226.
10
W.4
TABLE 1
Effect of administration of ACTH, morphine, or naloxone
on the adrenal gland content of corticosterone.a
Adrenal gland corticosteroneb
Drug	 Dose
content, µg/100 mg ± S.E.
LLB
ACTH	 0	 1.23 ± 0.05
100
	
3.40 * 0.07e
S-endorphins	100 1.23 ± 0.05
mg/kg
(-)-morphined	0 1.30 ± 0.05
5 1.27 ± 0.10
10 1.24 t 0.06
20 1.05 t 0.03
(-)-naloxoned 	 0 1.25 ± 0.05
12.5 1.30 ± 0.08
25 1.41 *- 0.19
50 1.0 t 0.06
aAll rats were hypophysectomized 18-20 h prior :o
drug administration.
bN = 5 rats/group.
Administered intravenously.
dAdministered intraperitoneally.
e 	 < 0.01.
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ACTH MORPHINE NALOXONE
100µU 10 mg/Kg	 50 mg/Kg
ACTH 100µU ACTH 100uU
H-NALOXONE
+ 50 mg/Kg
H-MORPHINE
+ 10 mg/Kg
ACTH 000µU)
Figure 3.- The effect of pretreatment with the stereoisomeric forms of
morphine or naloxone on the adrenal island steroidogenic response to ACTH
and the adrenal res ponse to morphine following naloxone pretreatment.
Asterisks indicate significant differences from the group administered
ACTH alone (n = 10), at 1> ^ 0.05. N = 5 rats/group.
14


Naloxone inhibits and morphine potentiates. The adrenal steroidogenic response to ACTH

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