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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 280:416 –421, 1997
Vol. 280, No. 1
Printed in U.S.A.
Dynorphin A1–13 Stimulates Ovine Fetal Pituitary-Adrenal
Function through a Novel Nonopioid Mechanism1
CINDY C. TAYLOR, DUNLI WU, YI SOONG, JENNY S. YEE and HAZEL H. SZETO
Department of Pharmacology, Cornell University Medical College, New York, New York
Accepted for publication September 13, 1996
Dynorphin A is a 17-amino acid peptide initially isolated
from porcine pituitary tissue and postulated to be an endogenous k opioid receptor ligand (Goldstein et al., 1979; Goldstein et al., 1981; Huidoro-Toro et al., 1981; Chavkin et al.,
1982). Dynorphin A1–13 is the N-terminal tridecapeptide of
dynorphin A1–17 and has been found to be as potent as the
natural peptide in opioid-binding assays and in the guinea
pig ileum (Goldstein et al., 1979). The high density of k-opioid
receptors in the hypothalamus (Mansour et al., 1994) and the
high concentration of dynorphin peptides in both hypothalamus and pituitary (Hollt et al., 1980) suggest a possible
involvement of dynorphin in the regulation of release of anterior pituitary hormones. In fact, dynorphin A1–13 significantly increased plasma prolactin levels when administered
i.v. to adult male rhesus monkeys (Gilbeau et al., 1987), and
both dynorphin and U50488H produced a significant increase in plasma levels of ir-ACTH and ir-corticosterone in
adult rats (Pfeiffer et al., 1985; Iyenger et al., 1986).
The sites and mechanisms by which dynorphin alters
ACTH release have not been studied. The effects of opioids on
Received for publication May 20, 1996.
1
This research was supported by Grant DA02475-16. C.C.T. is supported by
training Grant from NIDA (DA07274-04).
levels of 383.3 6 43.8 pg/ml and 32.8 6 9.0 ng/ml, respectively,
at 15 min after administration. A similar increase in plasma
immunoreactive-ACTH was seen after the same dose of dynorphin A1–17 (P 5 .02) but not dynorphin A2–17. This ACTH response to dynorphin A1–13 was shown to be insensitive to the
opioid antagonist, naloxone (12 mg/hr), as well as antagonists
of corticotropin releasing factor and arginine vasopressin.
These data suggest that dynorphin A1–13 in the ovine fetus may
be acting through a mechanism distinct from the k-opioid system and that the dynorphins may serve as secretagogues of
ACTH directly at the anterior pituitary through nonopioid receptors.
the pituitary-adrenal axis are thought to occur via a hypothalamic mechanism of action, most likely through the modulation of CRF or AVP, the two most potent secretagogues of
ACTH (Pechnick, 1993). There is good evidence to support a
role for CRF in the release of ACTH resulting from k-opioid
agonists (Buckingham and Cooper, 1986; Nikolarakis et al.,
1988). The prospect of opioid modulation of ACTH secretion
via AVP has not previously been addressed. Studies investigating the interaction of the k-opioid system with AVP have
been limited to the actions of k-opioid agonists on posterior
pituitary function. It has been shown that administration of
U50488H or dynorphin A1–13 causes diuresis via inhibiting
AVP release most likely through a direct action of these
agonists at posterior pituitary k receptors (Oiso et al., 1988).
It is possible that dynorphin peptides may evoke ACTH release through a mechanism that is independent from AVP
altogether and may be dependent solely on regulation of
CRF. Alternatively, the combined hypothalamic secretion of
AVP, CRF and dynorphin peptides (Roth et al., 1982; Watson
et al., 1982) may implicate the dynorphin peptides as additional secretagogues of ACTH acting directly at the anterior
pituitary independent of either CRF or AVP. Supporters of
the view that dynorphin peptides act exclusively at opioid
receptors would argue that the flaw in this hypothesis is that
ABBREVIATIONS: HPA, hypothalamic-pituitary-adrenal; ACTH, adrenocorticotropin; AVP, arginine vasopressin; CRF, corticotropin releasing
factor; ir-ACTH, immunoreactive adrenocorticotropin; ir-cortisol, immunoreactive cortisol; U50,488H, trans-(6)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide; icv, intracerebroventricular; ANOVA, analysis of variance.
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ABSTRACT
We previously reported that U50488H, a k-selective opioid
agonist, stimulates the release of adrenocorticotropin (ACTH) in
the ovine fetus via the release of hypothalamic arginine vasopressin and corticotropin releasing factor. In this study we
examined the effects of the endogenous k-preferring opioid
peptide, dynorphin A1–13, on fetal ACTH release using the
unanesthetized, chronically catheterized fetal lamb model. Fetal plasma samples were collected at timed intervals after fetal
administration of dynorphin A1–13 (0.5 mg/kg, i.v.) and subsequently analyzed by radioimmunoassay for immunoreactiveACTH and immunoreactive-cortisol. Dynorphin A1–13 produced
a highly significant and rapid increase in immunoreactive-ACTH
(P 5 .002) and immunoreactive-cortisol (P 5 .002) with peak
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Fetal HPA Activation by Dynorphin
Materials and Methods
Surgical procedure. Surgery was performed on 22 fetal sheep
(gestational age 5 115–120 days) to insert chronic indwelling polyvinyl catheters as described in detail by Szeto et al. (1990). One
catheter was inserted into the femoral artery and advanced to the
distal aorta for blood sampling and another into the inferior vena
cava via the femoral vein for drug infusion. An additional catheter
was placed in the lateral cerebral ventricle for infusion of the CRF
antagonist. Guidelines approved by the Institution for the Care and
Use of Animals at Cornell University Medical College were followed
for all surgical procedures and experimental protocols.
Experimental protocol. Experiments were performed a minimum of 5 days after surgery to ensure complete recovery from
surgical stress. The ewe was placed in a mobile cart with free access
to food and water. All studies commenced at 0800 hr with a minimum of 2.5 hr to allow for acclimation of the animal to the study
conditions. The study environment was free of external stressful
stimuli to the best of our ability. As plasma hormone levels and
responsiveness of the HPA axis varies diurnally, drugs were administered at approximately the same time for each study (1100 hr 6 30
min). A sample of fetal blood (2 ml) was collected for determination
of basal arterial blood gases and pH (Radiometer ABL30, Cleveland,
OH) as well as basal ir-ACTH and ir-cortisol levels 30 min before
drug administration. An incomplete randomized cross-over design
was used in the administration of the various drug treatments with
each fetus receiving no more than three different treatments with a
minimum of 2 days between studies. Gestational age on the day of
the study ranged from 125 to 142 days. An estimation of fetal weight
was made from physical observation at the time of surgery allowing
for calculation of fetal weight on the day of the study factoring
growth of 2% per day (Joubert, 1956). A more accurate estimation of
the dose administered could then be determined after birth by measuring the actual birth weight and recalculating the study weight
based on the 2% per day principle.
Dynorphin A1–13 (Chiron Mimotopes Peptide Systems, San Diego,
CA), dynorphin A1–17 (Multiple Peptide System, San Diego, CA),
dynorphin A2–17 (Multiple Peptide System) or saline was administered to the fetus as an iv bolus (0.5 mg/kg) with n 5 5 in all groups
except for dynorphin A1–17 (n 5 3). This dose of dynorphin peptides
was chosen based on preliminary studies with doses ranging from
0.05 to 0.5 mg/kg. Blood samples (2 ml) were collected from the fetus
15, 30 and 60 min after drug administration. The opioid antagonist,
naloxone (gift from Du Pont-Merck, Wilmington, DE; 12 mg/hr, i.v.),
was administered to the fetus 1 hr before, during and 1 hr after the
dynorphin A1–13 bolus (n 5 5) to test whether it is acting through
classical opioid receptors.
To determine whether dynorphin A1–13 is acting via the hypothalamic secretagogues, CRF or AVP, some fetuses were pretreated with
antagonists of CRF or AVP before dynorphin A1–13 administration.
a-Helical CRF [Sigma Chemical Co,, St. Louis, MO; 20 mg icv followed by 10 mg/hr icv infusion (Bennet et al., 1990)], was given 15
min before the dynorphin A1–13 bolus (n 5 3). The AVP antagonist
[(b-mercapto-b, b-cyclopentamethylene-propionyl, O-Me2-Tyr, Arg8]vasopressin; Sigma)] was administered as an i.v. bolus (60 mg/kg) 10
min before dynorphin A1–13 (n 5 4) (Dunlap and Valego, 1989). It was
necessary to administer a-helical CRF via the icv route because
impractically large amounts would have had to be administered via
the i.v. route (2.3 mg/kg) (Rivier et al., 1984). The dose of AVP
antagonist was chosen because it has been shown to block an AVPinduced rise in ovine fetal ir-ACTH similar in magnitude to that
which is produced by dynorphin A1–13 administration (Apostolakis et
al., 1991). For the a-helical CRF, the dose used was shown to completely reverse the stimulation of ovine fetal breathing movements
associated with CRF infusion (Bennet et al., 1990). Furthermore,
previous studies in our laboratory have shown that these doses of
antagonists resulted in a significant attenuation of a comparable
increase in ovine fetal plasma ir-ACTH by U50488H (Taylor et al.,
1996). In all antagonist studies, an additional control sample was
collected after the administration of the antagonist and before the
administration of dynorphin A1–13.
Radioimmunoassay. Blood samples were centrifuged at 1500
rpm for 10 min at 4°C and subsequently frozen at -70°C until assayed. [125I]ACTH and [125I]cortisol radioimmunoassay kits (INCSTAR, Stillwater, MN) were used to measure fetal plasma ir-ACTH
and ir-cortisol concentrations. These assays have been standardized
in our laboratory using ovine fetal plasma (Taylor et al., 1996).
Statistical analysis. All data are reported as mean 6 S.E.M. A
single-factor ANOVA with repeated measures (factor 5 time) was
used to determine drug effects on fetal plasma ir-ACTH and ircortisol levels. Friedmans repeated measures ANOVA on ranks was
used if the normality test failed. The statistical significance of the
change from control for each time point was assessed by Dunnett’s
post hoc comparison. A multifactor ANOVA (factor 5 time, drug) was
used to analyze the influence of antagonists on the dynorphin A1–13
response. To compare plasma levels of ir-ACTH after dynorphin
A1–13 with different antagonists 15 min after drug administration, a
single-factor ANOVA (factor 5 drug) followed by Dunnett’s post hoc
test was used.
Results
Effects of dynorphin A1–13 on fetal plasma ir-ACTH
and ir-cortisol. Basal levels of ir-ACTH and ir-cortisol were
46.2 6 7.2 pg/ml and 9.4 6 2.5 ng/ml, respectively. Fifteen
minutes after the administration of dynorphin A1–13, there
was a highly significant increase from control values in
plasma ir-ACTH (x2 5 15.0, P 5 .002; see fig. 1) with peak
ir-ACTH level of 383.3 6 43.8 pg/ml. Although ir-ACTH
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opioid receptors have not been shown to be present in the
anterior pituitary (Mansour et al., 1995). However, the finding that dynorphin A1–17 is rapidly degraded to its nonopioid
analog, dynorphin A2–17 (Muller and Hochhaus, 1995), and
that many of the actions of dynorphin peptides are naloxoneinsensitive (Walker et al., 1982; Smith and Lee, 1988; Hooke
et al., 1995), indicate that they are capable of acting via a
nonopioid mechanism of action.
Our study was an attempt to understand the regulation of
the HPA axis by dynorphin peptides during fetal development. During fetal life the HPA axis plays a crucial role in
proper development of organ systems, initiation of parturition and homeostasis. The development of an appropriate
fetal stress response is essential to maintaining the wellbeing of the fetus and helps to ensure proper physiological
controls through birth and into adulthood. Further, it may
elucidate a mechanism by which exogenous administration of
opioids during pregnancy can have detrimental effects on the
fetus. We have administered dynorphin A1–13 and dynorphin
A1–17 to the fetal lamb in utero and found a potent increase in
fetal plasma ir-ACTH and ir-cortisol which was surprisingly
naloxone-insensitive, not mimicked by dynorphin A2–17, and
did not involve the release of AVP or CRF. This is in contrast
to recent studies in our laboratory which have shown
U50488H to stimulate fetal pituitary-adrenal function via a
naloxone-sensitive mechanism involving hypothalamic AVP
and CRF (Taylor et al., 1996). These data suggest that dynorphin A1–13 and dynorphin A1–17 in the fetus may be acting
through a mechanism distinct from the k-opioid system and
that the dynorphins may serve as secretagogues of ACTH
directly at the anterior pituitary through nonopioid receptors.
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Taylor et al.
Vol. 280
Fig. 1. Effects of dynorphin A1–13 (f) alone and in the presence of
naloxone (M) on fetal plasma (A) ir-ACTH and (B) ir-cortisol. Data are
represented as absolute change from predrug control levels. Dynorphin
A1–13 (0.5 mg/kg) was administered at time 0 min. Naloxone was given
as a constant rate iv infusion (12 mg/h, iv) starting 60 min before, and
continuing for 60 min after dynorphin A1–13 administration. The effects
of naloxone alone (F) are also shown. *P , .05 as compared to predrug
control levels.
levels subsequently declined toward to control levels, plasma
levels were still significantly elevated at 60 min after dynorphin A1–13 administration. There was a corresponding rise in
ir-cortisol (F3,16 5 9.1, P 5 .002) at 15 and 30 min after
dynorphin A1–13 administration. There was no effect of saline
vehicle administration on plasma ir-ACTH or ir-cortisol (n 5
5) (see fig. 2).
The effect of naloxone on the dynorphin A1–13 response. Administration of naloxone alone (n 5 6) had no
effect on either ir-ACTH or ir-cortisol levels (fig. 1). Dynorphin A1–13 in the presence of naloxone (n 5 5) resulted in a
significant rise in ir-ACTH levels (F3,16 5 20.1, P , .001)
with plasma ir-ACTH reaching a peak of 291.9 6 42.7 pg/ml
compared to 383.3 6 43.8 pg/ml with dynorphin A1–13 alone
(see fig. 1). There was also a significant increase in ir-cortisol
(F3,16 5 14.6, P , .001) with peak levels of 53.5 6 12.7 ng/ml
compared to 32.8 6 9.0 ng/ml after dynorphin A1–13 alone.
Neither the ir-ACTH nor the ir-cortisol response to dynorphin A1–13 was blocked by concurrent administration of naloxone (ir-ACTH: F1,32 5 1.5, P 5 .3, ir-cortisol: F1,32 5 1.1,
P 5 .3).
Effects of dynorphin A1–17 and dynorphin A2–17 on
plasma ir-ACTH and ir-cortisol. Administration of the
same dose of dynorphin A1–17 to the ovine fetus (n 5 3) also
resulted in a significant increase in fetal plasma ir-ACTH
(F 5 5.4, P 5 .02), although the magnitude of increase was
significantly less than with dynorphin A1–13 (see fig. 2). In
contrast, the same dose of dynorphin A2–17 had no effect on
either plasma ir-ACTH (x2 5 7.1, P 5 .1) or ir-cortisol (x2 5
4.6, P 5 .2) (n 5 5; see fig. 2).
The effects of AVP and CRF antagonists on the
dynorphin A1–13 response. Administration of the AVP antagonist or CRF antagonist alone had no effect on plasma
ir-ACTH or ir-cortisol. In the presence of the AVP antagonist,
dynorphin A1–13 resulted in a highly significant increase in
ir-ACTH (F3,12 5 80.0, P , .001) and ir-cortisol (F3,12 5 31.3,
P , .001) (n 5 4). There was also a highly significant increase
in ir-ACTH (F3,8 5 119.3, P , .001; n 5 3) and ir-cortisol (F3,8
5 12.6, P 5 .005; n 5 3) after dynorphin A1–13 in the presence
of the CRF antagonist. Figure 3 illustrates the fetal plasma
ir-ACTH levels after the administration of dynorphin A1–13
alone, and in the presence of the AVP antagonist or CRF
antagonist. No significant difference was observed between
administration of dynorphin A1–13 alone and in the presence
of the AVP antagonist or CRF antagonist.
Discussion
Dynorphin-related peptides are known to have actions on
the neuroendocrine system that are reversible by naloxone,
including the inhibition of AVP and oxytocin release from the
posterior pituitary (Pechnick, 1993). However, the role of
dynorphin-related peptides in the anterior pituitary is not as
well understood. Some evidence exists that the dynorphins
can stimulate the release of ACTH, however, there has been
conflicting data in regards to naloxone sensitivity. Iyengar et
al. (1987) reported a significant rise in rat plasma ir-corticosterone after a 10-mg icv dose of dynorphin A1–13 that was
blocked by naloxone (5 mg/kg, i.p.). However, Gunion et al.
(1991) showed a similar rise in rat plasma ir-corticosterone
after having administered icv dynorphin A1–17 (0.3–10 nmol/
rat) that was not blocked by naloxone (1 mg/kg, s.c.). Further,
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Fig. 2. Effects of saline (F), dynorphin A1–13 (f), dynorphin A1–17 (å)
and dynorphin A2–17 (ç) on fetal plasma ir-ACTH. Data are represented
as absolute change from predrug control levels. The dynorphin A1–13
data (f) are the same as shown in figure 1. All drugs were administered
at time 0 min. *P , .05 as compared to predrug controls.
1997
the sustained actions of dynorphin A1–17 with mixed dynorphin-naloxone intraventricular injections confirmed the conclusion that the observed effects of dynorphin A1–17 were, in
fact, not opioid mediated. Thus, a nonopioid component of
HPA stimulation by the dynorphins is implied.
In the present study, dynorphin A1–13 was capable of stimulating a highly significant and rapid rise in plasma ir-ACTH
and ir-cortisol in the ovine fetus. The later peak in cortisol
increase (30 min vs. 15 min) is consistent with the primary
action of dynorphin A1–13 being at the level of the hypothalamus or pituitary rather than at the adrenal. This magnitude
of ACTH release (7- to 10-fold) is comparable to that observed
after 1 mg/kg of U50488H to the ovine fetus (Taylor et al.,
1996), and is in the same range as those reported after high
doses of CRF (Kerr et al., 1992) and AVP (Apostolakis et al.,
1991), or a combination of CRF and AVP (Norman and Challis, 1987). Although higher doses of dynorphin A1–13 were not
tested in this study, it is likely that the ACTH response is at
near-maximal level. However, the time-action of dynorphin
A1–13 on ir-ACTH was very different from that of U50488H
(Taylor et al., 1996), with a much earlier peak and shorter
duration of action. After an i.v. dose of U50488H, ir-ACTH
increased gradually over a 60-min period, and plasma levels
at 3 hr were still significantly elevated compared to control.
The rapid increase in ir-ACTH after dynorphin A1–13 was
unexpected given that the diffusion of a peptide drug should
have been slower than that of the more lipid soluble benzeneacetamide. The magnitude of change in ir-ACTH was comparable for dynorphin A1–13 and U50488H even though the
molar dose of dynorphin A1–13 was 7-fold lower than that of
U50488H. The shorter duration of action of dynorphin A1–13
is consistent with the rapid degradation of dynorphin peptides in vivo (Muller and Hochhaus, 1995).
The increase in ir-ACTH by dynorphin A1–13 was not antagonized by naloxone pretreatment, suggesting that it is not
mediated by classical opioid receptors. The k-opioid antagonist, nor-binaltorphimine, was not practical for use in this
419
study because of its prolonged antagonism of 28 days (Horan
et al., 1992). It is highly unlikely that the dose of naloxone
administered was insufficient to block k-opioid receptors because it was three times the dose used to successfully block a
comparable release of ir-ACTH by U50488H (Taylor et al.,
1996). We therefore propose that dynorphin A1–13 is acting
via a nonopioid mechanism to increase the release of ACTH
from the ovine fetal pituitary. The apparent lack of action of
dynorphin A1–13 on k-opioid receptors was surprising given
that the affinity of dynorphin A1–13 for the recently cloned k
receptor is actually 10-fold more than that of U50488H
(Meng et al., 1993). This discrepancy may be explained by the
poor distribution of dynorphin peptides across the bloodbrain barrier to the hypothalamus after i.v. administration.
The N-terminal tyrosine of the endogenous opioid peptides
is known to be required for binding and pharmacological
activity at the opioid receptor (Walker et al., 1982) and the
naloxone-insensitive actions of the dynorphins have been
thought to be due to degradative fragments resulting from
the loss of this crucial amino acid. Dynorphin A2–12 accounts
for 15% of the dynorphin peptide fragments found in human
blood within minutes of dynorphin A1–13 administration
(Muller and Hochhaus, 1995). It has also been demonstrated
that most of the naloxone-insensitive actions of dynorphin
A1–17 can be mimicked by the des-[Tyr]1-dynorphin analogues (Shukla and Lemaire, 1994). Surprisingly, in this
study dynorphin A2–17 had no effect on fetal plasma levels of
ir-ACTH or ir-cortisol. It has been reported that b-endorphin
can increase thyrotropin stimulating hormone secretion from
superfused anterior pituitary tissue by a mechanism that is
naloxone insensitive (Judd and Hedge, 1983). However, the
nonopioid des-[Tyr]1-g-endorphin was unable to elicit a response. Thus, it appears as though the stimulatory effects of
the dynorphins and other opioid peptides on the fetal anterior pituitary may be via a yet unreported or “novel” nonopioid mechanism in which the N-terminal tyrosine is necessary.
The action of opioids on HPA function is generally thought
to occur at the level of the hypothalamus via modulation of
AVP and CRF (Pechnick, 1993). We recently reported that
the effects of U504488H on ACTH release in the ovine fetus
were naloxone-sensitive and significantly attenuated by both
AVP and CRF antagonists (Taylor et al., 1996). In contrast,
the stimulation of ACTH release by dynorphin A1–13 was
found to be both nonopioid and independent of AVP or CRF
modulation (see fig. 4). These results suggest a distinctly
different mechanism, and possibly site of action, of dynorphin
peptides and k-opioid agonists on ACTH release. Because
dynorphin peptides can have both opioid and nonopioid actions, the possibility exists for dual regulation of ACTH release via k-opioid receptor-mediated release of AVP and CRF
from the hypothalamus or directly at the anterior pituitary
via nonopioid receptors. The possibility that dynorphin peptides may serve as additional ACTH secretagogues is supported by the finding that the dynorphins are known to be
colocalized and coreleased with AVP and CRF (Roth et al.,
1982; Watson et al., 1982). Further, it has been reported that
fetal hypothalamic extracts contain a substantial ACTH-releasing capability that does not correspond with AVP or CRF
(Currie and Brooks, 1992). In fact, we have found that there
is a component of U50488H-induced ACTH release that is not
blocked by either AVP or CRF antagonists (Taylor et al.,
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Fig. 3. Effects of AVP antagonist (F) and CRF antagonist (å) on the
stimulation of fetal plasma ir-ACTH by dynorphin A1–13. Data are represented as the change from predrug control levels. The dynorphin
A1–13 data (F) are the same as shown in figure 1. The dynorphin A1–13
was administered at time 0 min with the AVP antagonist (60 mg/kg, iv)
given 10 min before dynorphin A1–13, and the CRF antagonist (20 mg,
icv 1 10 mg/hr infusion, icv) given 15 min before dynorphin A1–13. The
change in ir-ACTH was significant (P , .05) for all three treatment
groups at all time points.
Fetal HPA Activation by Dynorphin
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Taylor et al.
Vol. 280
Although this hypothesis remains to be explored further, it is
without question that the k-opioid and dynorphin systems
play a prominent neuroendocrine role that may include
maintenance of an appropriate fetal stress response and development of a functional HPA axis.
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release of dynorphin together with CRF and AVP. In our
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Fig. 4. Summary of all drug treatments on fetal plasma ir-ACTH. These
data represent the magnitude of plasma ir-ACTH levels 15 min after
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Send reprint requests to: Dr. Hazel H. Szeto, Department of Pharmacology,
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