Endocrinology Vol. 140, No. 12 5642-5650
Copyright © 1999 by The Endocrine Society
Differential Regulation of Corticotropin-Releasing Hormone and Vasopressin Transcription by Glucocorticoids
Xin-Ming Ma1 and
Greti Aguilera
Section on Endocrine Physiology, Developmental Endocrinology
Branch, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Greti Aguilera, M.D., Section on Endocrine Physiology, DEB, NICHD, NIH Building 10, Room 10N 262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862. E-mail: greti{at}helix.nih.gov
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Abstract
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CRH and vasopressin (VP), the main regulators of pituitary ACTH
secretion, co-exist in parvocellular cells of the PVN, but their levels
of expression are regulated differentially during manipulations of the
hypothalamic pituitary adrenal (HPA) axis. The effects of
glucocorticoids on this system was studied using in situ
hybridization with intronic and exonic probes to measure changes in CRH
and VP messenger RNA (mRNA) and heteronuclear (hn) RNA in 48-h
adrenalectomized (ADX) rats receiving injections of corticosterone (2.8
mg/100 g, ip) or vehicle. We also determined the time course of changes
in VP expression following the first 72 h of ADX. Levels of VP
heteronuclear (hn) RNA and the number of parvocellular cells containing
VP hnRNA remained very low in sham operated rats, whereas biphasic
changes were observed after ADX. Grain density levels increased
11.5-fold over sham-operated controls by 6 h, declined to 2-fold
by 18 h, to increase again to 10- and 20-fold by 48 and 72 h,
respectively. In 48-h ADX rats, vehicle injection increased CRH hnRNA
levels transiently (11-fold the basal by 15 and 30 min), returning to
basal at 60 min, whereas VP hnRNA levels increased progressively up to
28-fold the basal by 2 h. Corticosterone injection had no
significant effect on vehicle-induced increases in CRH hnRNA, in spite
of marked elevations in circulating corticosterone. In contrast to CRH,
VP hnRNA levels increased only transiently by 15 min, and then
decreased below basal (near sham-ADX levels) by 2 h. The data show
that in normal conditions the responsiveness of parvocellular neurons
to stress is under marked inhibition by the low resting levels of
glucocorticoids, and that the sensitivity of CRH and VP transcription
to glucocorticoid feedback is markedly different.
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Introduction
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CRH AND VASOPRESSIN (VP) produced by
parvocellular neurons of the hypothalamic paraventricular nucleus (PVN)
are the major regulators of ACTH secretion during stress (1, 2). While
CRH is a potent ACTH secretagogue and is essential for the stress
response (3, 4, 5), VP is a weak stimulus on its own but it markedly
potentiates the effect of CRH, both in vivo and in
vitro (2, 6, 7). A number of immunohistochemical and in
situ hybridization studies have shown that basal levels of
expression of both peptides are under feedback inhibition by
glucocorticoids. Immunoreactive (ir) CRH and CRH messenger RNA (mRNA)
levels in the PVN decrease after glucocorticoid administration or
implantation of dexamethasone in the PVN (8, 9, 10), but increase markedly
following ADX, the latter effect being prevented by glucocorticoid
replacement (11, 12, 13, 14). The increase in CRH mRNA after ADX is relatively
slow, requiring 48 h to become detectable and reaching stable
levels of about 3-fold the controls after 72 h (9, 12, 15). The
study of VP expression in parvocellular neurons has been more difficult
because the low levels of expression in these cells is masked by the
much higher expression levels in magnocellular neurons intermingled in
the parvocellular PVN. Recent studies using intronic probes to measure
changes in newly transcribed mRNA, or heteronuclear (hn) RNA, have
shown that the kinetics of VP transcription in parvocellular neurons
are different from those of CRH following acute stress paradigms, such
as ether exposure or ip hypertonic saline injection (16, 17). Another
study shows that levels of VP hnRNA, but not of CRH hnRNA levels, are
elevated in 6-day ADX rats, and that the increased VP transcription is
rapidly inhibited by corticosterone administration (18). However, in
the latter study it was not possible to study the effect of
glucocorticoids on CRH transcription because CRH hnRNA levels were
found to be very low in 6-day ADX rats (18, 19). Although it is clear
that both CRH and VP are under glucocorticoid feedback inhibition, the
role of glucocorticoids on the different patterns of expression, or in
the transcriptional regulation of these peptides remains to be
elucidated.
The purpose of this study was to investigate the effects of
glucocorticoids on CRH and VP transcription in parvocellular PVN
neurons in rats subjected to adrenalectomy and corticosterone
administration using sensitive intronic in situ
hybridization techniques. Because in recent studies we show that CRH
hnRNA levels increase between 12 and 72 h after ADX (20), the
effect of glucocorticoids on CRH transcription was studied 48 h
after ADX, a time at which the increase in CRH transcription reached a
plateau. Because the stress of vehicle injection was sufficient to
induce marked increases in CRH and VP hnRNA levels, the experiments
also allowed determination of the effect of glucocorticoids on
stress-induced transcription. In addition, the time course of the
changes in VP hnRNA and mRNA during early ADX were compared with those
previously observed for CRH.
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Materials and Methods
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Animals and in vivo procedures
Adult male Sprague Dawley rats (Harlan Sprague Dawley, Frederick, MD) weighing 250–300 g were housed 3 per
cage with a 14-h light, 10-h dark cycle with food and water available
ad libitum, for at least 5 days before study. All
procedures were performed according to the NIH guidelines, and the
experimental protocols were approved by the NICHD Animal Care and Use
Committee.
Rats were adrenalectomized bilaterally via dorsal approach or sham
operated under ketamine/xylazine anesthesia. After recovery from
anesthesia, rats were returned to their home cages, and ADX rats were
given access to 0.9% NaCl in the drinking water. To determine the time
course of changes in VP transcription following ADX, groups of 6 ADX or
sham-operated rats were killed by decapitation at the times indicated
in the figures, between 3 and 72 h after surgery. The effect of
glucocorticoids was studied after injection of corticosterone (2.8
mg/100 g body weight in 100 µl of saline containing 50% ethanol, ip.
Injection was performed 48 h after ADX, a time at which CRH hnRNA
levels had reached maximal levels. Control rats received injections of
the same volume of vehicle. Rats were killed by decapitation at the
time point indicated in results and figures. Injections were performed
between 0800 and 0900 h, and rats were killed between 0900 and
1200 h. Trunk blood was collected into ice-cold tubes containing
EDTA for corticosterone determination by RIA using 50 µl of plasma
and the rat corticosterone kit (DPC, Los Angeles, CA). The sensitivity
of the assay was 7.5 ng/ml. Brains were removed, frozen on dry ice, and
stored at -80 C until processed.
In situ hybridization
Serial 12-µm coronal brain sections were cut though the medial
parvocellular subdivision of the PVN (8 sections per rat) in a cryostat
at -20 C, thaw-mounted on poly-L-lysine
(Sigma, St. Louis, MO) coated slides, and stored at -80 C
until used for in situ hybridization.
The rat CRH intronic (CRHin) probe (kindly supplied by Dr. Robert
Thompson, University of Michigan, Ann Arbor, MI), was a 530 bp pvu II
fragment of the CRH gene subcloned into pGEM-3 (Promega Corp., Madison, WI) and linearized with XbaI. The rat
CRH (CRHex 2) complementary DNA (cDNA) (Dr. Robert Thompson) was a 770
bp BamHI fragment subcloned in pGEM-3Z (Promega Corp.), linearized by HindIII. The VP exonic
probe was an EcoRI-BamHI 200 bp fragment of the
rat AVP cDNA (AVPex) kindly provided by Drs. Susan Wray and Harold
Gainer (NINDS, NIH, Bethesda, MD), linerarized with BamHI.
The AVP intron probe (kindly supplied by Dr. Thomas G. Sherman,
Georgetown University, Washington, DC) was generated from a 735 bp
PvuII fragment of AVP intron I subcloned into pGEM-3 and
linearized by HindIII. High specific activity antisense
complementary RNA (cRNA) probes for CRH and VP intronic and
exonic probes were produced using 35S-ATP and
35S-UTP (21). In situ hybridization was
performed as previously described (21). Briefly, before hybridization,
stored sections were air-dried at room temperature, fixed with 4%
formaldehyde for 5 min at room temperature, washed 3 times with PBS,
and acetylated using 0.25% acetic anhydride in 0.1 M
triethanolamine/0.9% NaCl (pH 8.0) for 10 min at room temperature.
Slides used for mRNA stability evaluation followed the same
prehybridization and hybridization procedures after fixation. Sections
were dehydrated in serial ethanol solutions, defatted in chloroform,
and dried before hybridization with the radiolabeled probes. Sections
were hybridized overnight at 55 C with 2 x 106 cpm
labeled CRHin or CRHex probe per slide containing four sections each.
Nonhybridized probe was removed by washing with 50% formamide/250
mM NaCl at 60 C for 10–15 min, ribonuclease A treatment
for 30 min at 37 C, followed by 3 washes with 0.1 x SSC.
Semiquantification of the CRH and VP transcripts was performed for
films autoradiograms and emulsion dipped slides, respectively, as
previously described (17, 22). While the sensitivity of both evaluation
methods is similar under appropriate exposure conditions, it is
necessary to use emulsion dipped slides to distinguish parvocellular
neurons hybridized for VP from intermingled magnocellular neurons that
are larger and express high levels of VP (17, 22). For analysis of CRH
hnRNA and CRH mRNA in the PVN, sections were exposed to
Kodak BIOMAX film (Kodak, Rochester, NY)
together with 14C-labeled standards (American
Radiochemical, St. Louis, MO), for 10 h (CRHex) or 15 days for
(CRHin). For cellular localization of CRH mRNA or CRH hnRNA, slides
were subsequently dipped in nuclear emulsion diluted 1:1 in distilled
water (NTB2, Kodak), exposed for appropriate times (CRHin,
40 days; CRHex, 4 days) and counterstained with cresyl violet acetate
(Sigma). The optical density of film autoradiographic
images of parvocellular CRH mRNA and CRH hnRNA was measured in a
computerized image analysis system (Imaging Research, Inc., St. Catherine, Ontario, Canada), using the public domain
NIH Image Program (developed at the National Institutes of Health, and
available on the Internet at: http://rsb.info.nih.gov/nih-image).
Optical densities from two matched sections per rat were averaged after
subtracting the background and used to calculate group means. The
results are presented as mean and SEM of the percent of
change from basal levels in sham-operated rats.
Analysis of grain density levels of VP hnRNA and mRNA and number of
cells containing VP hnRNA and VP mRNA in the medial parvocellular of
the PVN was carried out in the cresyl violet counterstained sections
using a x40 objective with bright field condenser as described (17, 22). Medial parvocellular VP neurons in the PVN were differentiated
histologically from magnocellular neurons on the basis of their overall
size, their relatively low level of VP expression, and their small,
dense-staining nuclei. The relative grain density levels of VP hnRNA
and VP mRNA was quantified in the medial parvocellular subdivision of
PVN using computerized densitometry as described above after
subtracting the background measured in the immediate proximity of the
cells. The grain density measurements for parvocellular VP hnRNA and VP
mRNA were made on individual cells identified as parvocellular after
excluding scattered magnocellular cells in the medial parvocellular
subdivision of the PVN. For each animal, at least two sections were
measured bilaterally, and the average value for each rat was used to
calculate group means. The results of grain density measurement are
presented as mean and SE of the percent change from the
basal level in naive rats.
Statistical analyses were performed by one-way ANOVA followed by
Fisher’s least significant difference procedure test to assess
statistical significance between control and experimental groups at
each time point. P < 0.05 was considered to be
statistically significant.
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Results
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Plasma corticosterone
Plasma levels of corticosterone decreased rapidly after
adrenalectomy and had reached levels below the detection limit of the
assay by 3 h. As shown in Fig. 1
, injection of corticosterone increased plasma levels rapidly to 3,479
ng/ml at 15 min, followed by a gradual decrease to levels still above
those in sham operated rats by 2 h (861 ng/ml, P
< 0.01 vs. sham). Vehicle injection had no effect on plasma
corticosterone in ADX rats but increased them to 307, 282, 282, and 129
ng/ml at 15 (P < 0.01), 30 (P <
0.05), 60 (P < 0.05) and 120 min, respectively, in
sham-operated rats.
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Figure 1. Time course of the changes in plasma
corticosterone levels following injection of corticosterone (2.8 mg/100
g BW, ip) in 48-h adrenalectomized or sham-operated rats. Data points
represent the mean and SE of values obtained in six rats
per experimental group. **, P < 0.01
vs. ADX basal 0 min; ##, P < 0.01
vs. sham basal 0 min.
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VP hnRNA and mRNA levels after adrenalectomy
VP hnRNA expression in parvocellular neurons of the PVN was very
low in sham-operated rats and showed no significant changes at any time
point measured from 3 to 72 h after surgery (Fig. 2A). In ADX rats, VP hnRNA grain density
was significantly higher than that in sham-operated controls 3 h
after ADX, and further increased by 6 h (Table 1, Figs. 2A and 3B and D). Unexpectedly, VP hnRNA started
to decrease at 12 h, and after reaching the lowest level at
18 h, increased again progressively from 24 to 72 h (Table 1;
Figs. 2A and 3, E and F). This biphasic pattern was different from the
changes in CRH hnRNA, which started to increase by 12 h from near
undetectable levels and reaching maximum levels by 48 h (Table 1).
As observed for VP hnRNA grain density, the changes in the number of
parvocellular neurons containing VP hnRNA were also biphasic. Values
were significantly higher than in sham-operated rats at 3 h and
6 h after ADX (403% and 320% of sham values, P
< 0.01, respectively), decreased at 18 h (200% of sham,
P < 0.05; 53% lower than ADX at 6 h,
P < 0.01 or 39% lower than ADX at 12 h,
P < 0.01), and increased again by 24 h (Fig. 2B).
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Figure 2. Changes in VP hnRNA grain density levels (A) and
the number of cells expressing VP hnRNA (B) after adrenalectomy,
measured in emulsion dipped slides hybridized with a
35S-labeled VP intronic cRNA probe. Rats were
adrenalectomized or sham-operated and killed at the indicated time
points. Data points are the mean and SE of values obtained
in six rats per group. *, P < 0.05 sham; **,
P < 0.01 vs. sham; ##,
P < 0.01 vs. ADX 6 h or ADX
48 h.
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Table 1. Time course of the changes in VP hnRNA and CRH hnRNA
in parvocellular neurons of the PVN following adrenalectomy
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Figure 3. Representative dark field photographs of PVN
sections from adrenalectomized (ADX) rats hybridized with VP intronic
cRNA probes. Rats were killed at the times indicated after
sham-operation (A and C) or ADX (B, D, E and F). PM, Posterior
magnocellular subdivision of the PVN; MP, medial parvocellular
subdivision of the PVN.
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There were no changes in VP mRNA levels or the number of cells
containing VP mRNA between 3 and 72 h after sham surgery. In
adrenalectomized rats, VP mRNA grain density levels were similar to
sham operated rats at 3 h, and in contrast to the biphasic changes
in VP hnRNA increased progressively between 6 h (530%,
P < 0.01 vs. sham) and 72 h (1,635%,
P < 0.01 vs. sham 72 h, Fig. 4A). The number of cells containing VP
mRNA increased slightly but significantly 3 h after ADX (133%,
P < 0.05 vs. sham at 3h), and remained at
values about 150% higher than sham-operated rats between 6 and 72
h (P < 0.01 vs. sham at all time points,
Fig. 4B).
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Figure 4. Changes in VP mRNA grain density levels (A) and
the number of cells expressing VP mRNA (B) after adrenalectomy,
measured in emulsion-dipped slides hybridized with exonic cRNA probes.
Rats were adrenalectomized or sham-operated, and killed at the
indicated time points. Data points are the mean and SE of
values obtained in six rats per group. *, P < 0.05
vs. sham 3 h; **, P < 0.01
vs. sham.
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Effect of corticosterone on CRH hnRNA and CRH mRNA
Vehicle injection had no effect on CRH hnRNA or CRH mRNA in
sham-operated rats at any time point. In 48-h adrenalectomized rats,
both CRH hnRNA and CRH mRNA levels were significantly higher than those
in 48-h sham-operated controls. In contrast to sham-operated rats,
vehicle injection caused a striking increase in CRH hnRNA in ADX rats
indicating a much higher sensitivity of the parvocellular neuron to
stress. CRH hnRNA levels increased by about 3.5-fold from 15 to 30 min,
P < 0.01, and returned to levels observed before
vehicle injection by 60 min. Corticosterone injection had no
significant inhibitory effect on basal CRH hnRNA levels or on the
elevations induced by vehicle injection. Although CRH hnRNA levels
tended to decrease at 30 and 60 min, the difference between vehicle and
corticosterone injected rats was not significantly different. (Fig. 5A, Fig. 6B). CRH mRNA levels had a tendency to
increase in both vehicle and corticosterone injected rats but the
changes were not significant (Fig. 5B).
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Figure 5. Time course of the changes in CRH hnRNA (A) and
CRH mRNA (B) after injection of corticosterone (2.8 mg/100 g BW, ip) or
vehicle in 48-h adrenalectomized or sham operated rats. Data points are
the mean and SE of the optical density values obtained from
film autoradiograms in six rats per experimental group. **,
P < 0.01 vs. sham basal 0 min; ##,
P < 0.01 vs. ADX basal 0 min.
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Figure 6. Representative dark field photographs of PVN
sections hybridized with CRH intronic probes, showing the increase in
CRH hnRNA after 48 h ADX (A-1 and 2), and the effect of an ip
injection of vehicle (B-1 and 3) or corticosterone, 2.8 mg/100 g BW,
(B-2 and 4) in 48-h ADX rats.
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Effect of corticosterone on VP hnRNA and VP mRNA
Vehicle injection had no effect on VP hnRNA or mRNA levels
in parvocellular neurons of the PVN in sham-operated rats (Fig. 7, A and B). In 48-h ADX rats, levels of
VP hnRNA were 7.9-fold higher than in sham-operated rats
(P < 0.01, Fig. 8A), and
levels increased progressively after vehicle injection (19-, 22-, 24-,
and 28-fold at 15, 30, 60, and 120 min, respectively, Fig. 7A, Fig. 8B). In corticosterone injected rats, VP hnRNA increased significantly
at 15 min but declined markedly to levels below basal by 30 min, and
reaching levels not significantly different from sham-operated controls
by 120 min (Fig. 7A, Fig. 8B). In contrast to the minor effects of
corticosterone on CRH hnRNA, levels of VP hnRNA were significantly
lower in corticosterone injected rats compared with vehicle injected
rats at all time points.
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Figure 7. Time course of the changes in VP hnRNA (A) and VP
mRNA (B) after injection of corticosterone (2.8 mg/100 g BW, ip) or
vehicle, in 48-h adrenalectomized or sham operated rats. Data points
are the mean and SE of the optical density values obtained
from film autoradiograms in six rats per experimental group. #,
P < 0.05 vs. ADX basal 0 min; ##,
P < 0.01 vs. ADX basal 0 min; **,
P < 0.01 vs. sham.
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Figure 8. Representative dark field photographs of PVN
sections hybridized with VP intronic probes, showing the increase in VP
hnRNA after 48 h ADX (A-1 and 2), and the effect of an ip
injection of vehicle (B-1 and 3) or corticosterone, 2.8 mg/100 g BW,
(B-2 and 4) in 48 h ADX rats.
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VP mRNA levels were 13-fold higher in 48-h adrenalectomized rats than
in sham-operated controls. Levels of VP mRNA showed a further increase
after vehicle injection reaching significant levels only 2 h after
injection, and this effect was prevented by the injection of
corticosterone (Fig. 7B
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Discussion
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This study shows that the responsiveness of parvocellular neurons
to minor stressful stimuli is normally under inhibition by basal levels
of circulating glucocorticoids. In addition, it provides further
evidence that CRH and VP expression in stress responsive parvocellular
neurons is differentially regulated by glucocorticoids. The
demonstration of marked increases in both CRH and VP hnRNA in response
to the minor stress of vehicle injection in adrenalectomized rats
indicates that the parvocellular neuron becomes much more sensitive to
stress in the absence of glucocorticoids. The same injection caused
only minor increases in plasma corticosterone and had no detectable
effect on CRH or VP hnRNA levels in sham-operated rats, suggesting that
in normal conditions low prevailing levels of glucocorticoids restrain
the responsiveness of the parvocellular neuron to stress. This is in
agreement with previous studies showing enhanced CRH and VP mRNA
responses to stress in ADX compared with intact rats (23). This effect
of low levels of glucocorticoids may act as a protective mechanism to
prevent inappropriate activation of the HPA axis in response to
exposure to minor stimuli that occur frequently in physiological
conditions.
While glucocorticoid withdrawal sensitized the responses of both CRH
and VP to stress, exogenous glucocorticoids had differential effects on
these genes, markedly inhibiting VP but not CRH transcription.
Corticosterone injection up to 2 h had no inhibitory effect on
either stress-stimulated CRH transcription or adrenalectomy-induced
basal levels of CRH hnRNA in spite of the rapid and massive increase in
plasma corticosterone levels. The similar kinetics of CRH hnRNA in
vehicle and corticosterone-injected rats suggests that glucocorticoids
are not responsible for the usually self-limiting CRH hnRNA also
observed in response to ether exposure, ip hypertonic saline injection,
or restraint (16, 17, 22). While the present experiments show that
stress induced-increases in CRH transcription are relatively refractory
to glucocorticoid inhibition, it is not possible to rule out that
prolonged exposure to glucocorticoids (for more than 2 h) may
inhibit basal CRH transcription. Such a delayed effect of
glucocorticoids would not explain the rapid decline of CRH
transcription during stress, but it could contribute to the reduction
in CRH mRNA known to occur following local or systemic administration
of glucocorticoids (9, 12). Alternatively, or in addition, a decrease
in CRH mRNA stability by glucocorticoids is likely to play a role on
the inhibitory effect of glucocorticoids on CRH mRNA levels (20).
In striking contrast to the lack of effect of corticosterone on CRH
expression, there was a rapid and marked inhibition the VP hnRNA
responses to the stress of vehicle injection. Corticosterone not only
reduced the effect of stress but also decreased the elevated basal VP
hnRNA levels observed after 48-h ADX to near levels observed in sham
operated animals 2 h after injection. This is consistent with
previous observations in 6-day adrenalectomized rats (18) and supports
the hypothesis that VP mRNA levels in the parvocellular PVN are largely
controlled at the level of transcription.
The increase in VP hnRNA, 3 to 6 h after ADX, preceded the
increases in CRH hnRNA that occur 12 h after removal of the
adrenals (20). This is consistent with the view that VP transcription
is more sensitive to glucocorticoid feedback than CRH transcription.
However, the biphasic pattern of change in VP transcription, with a
marked decrease 18 h before a progressive rise between 24 and
72 h, suggests the involvement of more than one mechanism. Thus,
the early rise in VP hnRNA could be the result of an exacerbated
response to the surgical stress in the absence of glucocorticoids
rather than to glucocorticoid withdrawal per se. The
kinetics of the changes in VP hnRNA during the first 18 h are
consistent with an effect of stress and are similar to the long-lasting
VP hnRNA responses observed in other stress paradigms such as ip
hypertonic saline injection (17). Although the effect of stress could
mask the onset of the VP response to glucocorticoid withdrawal, the low
VP hnRNA levels at 18 h suggests that, similar to CRH, release of
VP transcription from glucocorticoid inhibition is not immediate
following removal of glucocorticoids.
It has been postulated that differential sensitivity of CRH and VP to
glucocorticoid feedback can explain the differential regulation in
expression of both peptides during stress. This hypothesis is based on
the demonstration that the declining phase of CRH hnRNA responses to
ether exposure are preceded by the peak corticosterone response,
whereas the VP hnRNA responses coincide with the decrease in plasma
corticosterone (16). The present demonstration in 48-h adrenalectomized
rats that CRH hnRNA responses to the stress of vehicle injection had
declined to basal levels by 1 h in the absence of the
stress-induced glucocorticoid surge indicates that this self limiting
pattern of response is independent of glucocorticoid feedback. The fact
that at the time of the plasma corticosterone peak VP hnRNA reaches
levels similar to vehicle injected rats, is against the possibility
that the stress-induced glucocorticoid surge causes the delay in VP
transcription response described during ether exposure and ip
hypertonic saline injection (16, 17). In stress models such as
restraint and ip hypertonic saline injection, the onset of the VP
transcription responses also occurs at the time of maximal increases in
plasma corticosterone (17, 22), and VP hnRNA can be sustained for up to
4 h, despite equally sustained elevations in plasma corticosterone
(17). While it is clear from these experiments and previous reports
(18) that VP transcription is highly sensitive to glucocorticoid
inhibition, stress can overcome this inhibition to different degrees,
probably depending on the glucocorticoid levels and intensity of the
stress.
The mechanism responsible for the modulatory effect of stress on
glucocorticoid inhibition may involve interaction of glucocorticoids
with neurotransmitters and neuropeptides activated by stress (24, 25).
At the molecular level, second messenger-induced expression of
intermediate early genes or transcription factors could modify
glucocorticoid receptor activity by heterodimerization with the
receptor or by binding to regulatory elements and by changing the
conformation of the promoter (26, 27, 28, 29). In the parvicellular cell,
increases in VP hnRNA induced by ether exposure are closely preceded by
the induction of c-fos mRNA and fos protein (16), which has
been shown to interact with the glucocorticoid receptor modifying its
activity at the DNA responsive element (30).
In conclusion, the marked increases in CRH and VP hnRNA in response to
the minor stress of vehicle injection in ADX rats, indicate that low
prevailing levels of circulating glucocorticoids in normal intact rats
reduce the responsiveness of parvicellular neurons. This effect of
resting levels of glucocorticoids would prevent the onset of CRH and VP
transcription and inappropriate activation of the HPA axis in response
to minor stresses. While it is clear that glucocorticoids have distinct
effects on CRH and VP transcription, the data show that increases in
glucocorticoid levels alone cannot explain the differential regulation
of CRH and VP transcription during stress.
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Footnotes
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1 Present address: Johns Hopkins University, Department of
Neuroscience, Baltimore, Maryland. 
Received May 20, 1999.
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Abstract
Introduction
