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Department of Psychology, University of Colorado, Boulder, Colorado 80309
Address all correspondence and requests for reprints to: Robert L. Spencer, Department of Psychology, Campus Box 345, University of Colorado, Boulder, Colorado 80309. E-mail: spencer{at}clipr.colorado.edu
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Introduction |
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Two separate groups of investigators have been especially instrumental in defining the relative roles of these two receptor subtypes in mediating corticosteroid feedback regulation of the HPA axis. Dallman and colleagues have focused on the role of glucocorticoid and mineralocorticoid receptors in regulating the basal and stress-induced activity of the HPA axis and how those roles change across the circadian cycle of HPA axis basal activity (1, 10, 11, 12, 13, 14). de Kloet and colleagues performed many of the initial studies characterizing the distribution and hormone binding properties of mineralocorticoid and glucocorticoid receptors in brain and pituitary. They have also examined the role of corticosterone receptor subtypes in mediating corticosterone regulation of basal and stress-induced HPA axis activity during the circadian trough of activity (2, 15, 16).
Based primarily on the work of these two groups of investigators, a basic model of corticosteroid receptor mediation of HPA axis regulation by corticosteroids has been put forth in which: 1) the low levels of corticosterone present during the circadian trough act via the high affinity mineralocorticoid receptors to maintain low basal activity of the HPA axis; 2) the higher levels of corticosterone present during the circadian peak or acute stress act via the lower affinity glucocorticoid receptors to constrain HPA axis activity (1, 2, 15). A recent study indicates that mineralocorticoid receptors contribute to the corticosterone maintenance of basal HPA axis activity during the circadian peak, perhaps by potentiating corticosterone effects at glucocorticoid receptors (13). Whether mineralocorticoid receptors also contribute to feedback inhibition of the HPA axis by corticosterone during acute stress, however, is not clear. Ratka et al. (16) found that treatment of rats with the mineralocorticoid receptor antagonist, RU28318, prolonged stress-induced elevations of corticosterone, but Weidenfeld and Feldman (17) observed no effect of RU28318 treatment on corticosterone or ACTH responses to acute stress. Neither study examined the effects of blocking both mineralocorticoid and glucocorticoid receptors at the same time. In addition, studies have yet to address whether there are circadian differences in mineralocorticoid and glucocorticoid receptor involvement in corticosteroid feedback inhibition of the HPA axis acute stress response. Two studies have suggested that the shut-off of the HPA axis response to acute stress may not be as dependent on active corticosterone negative feedback in the evening as in the morning (18, 19).
The experiments reported in this paper were designed to further test this emerging model of corticosteroid receptor subtype mediation of corticosteroid feedback regulation of the HPA axis. The basic strategy of these experiments was to acutely administer selective mineralocorticoid and/or glucocorticoid receptor antagonists to rats and examine their effect on basal and acute stress corticosterone levels during both the time of trough and peak basal HPA axis activity.
For these studies, we used the mineralocorticoid receptor antagonist, RU28318, and the glucocorticoid receptor antagonist, RU40555. RU40555 has been described by its supplier as having similar receptor selectivity as the more widely used RU486 (both progesterone receptor and glucocorticoid receptor antagonist), but not as much potency (unpublished correspondence from Roussel Uclaf). The choice of RU40555 as glucocorticoid receptor antagonist over RU486 was 2-fold: 1) in preliminary studies we didn’t see any evidence for a partial glucocorticoid receptor agonist effect of RU40555 (20), as has been described for RU486 (12, 16, 21); and 2) during this project RU486 was temporarily unavailable in the United States.
Finally, in our studies we estimated the proportion of corticosteroid receptors occupied in vivo by our two antagonists. Based on these estimates we were able to verify the in vivo receptor selectivity profile for each compound. In addition, we obtained evidence that the dose of RU40555 used in these studies occupied only 50% of glucocorticoid receptors. The poor solubility of this compound made testing of the acute effects of higher doses of RU40555 impractical. This submaximal blockade of glucocorticoid receptors, appears, however to have been fortuitous in that it allowed for evidence of a cooperative effect between both mineralocorticoid and glucocorticoid receptors in regulation of HPA axis activity.
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Materials and Methods |
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Corticosteroid receptor antagonist treatment
The mineralocorticoid receptor antagonist used for these studies
was RU28318
([7,17alpha]-17-hydroxy-3oxo-7-propyl-pregn-4-ene-21-carboxylic acid
potassium salt) and the glucocorticoid receptor antagonist used was
RU40555
(17-ß-hydroxy-11-ß-/4-/[methyl)-[1-methylethyl]aminophenyl/-17[E0]-[prop-1-ynyl]estra-4,9-dien-3-one).
Both compounds were donated by Roussel Uclaf (Romainville, France). The
dose of RU28318 (50 mg/kg) used in our studies was based on separate
studies in which this dose was found to produce near maximal decreases
in available mineralocorticoid receptor binding without affecting
glucocorticoid receptor binding (20). The dose of RU40555 (30 mg/kg)
used in our studies was chosen based on our experience from other
studies and manufacturer’s information. We have found that sc
injection of this dose of RU40555 completely blocked dexamethasone (50
µg/kg sc) suppression of stress-induced corticosterone secretion
(20). Unpublished correspondence from Roussel Uclaf states that this
dose of RU40555 when administered orally exhibited near total
antagonist activity against dexamethasone’s effects on a variety of
measures, including plasma corticotrophic activity. Due to the poor
solubility of RU40555, all drugs were dissolved in propylene glycol and
the injections were given sc (0.9 ml per rat). For combined antagonist
treatment a single injection containing RU28318 (50 mg/kg) and RU40555
(30 mg/kg) was given (0.9 ml per rat). Control rats were given an equal
volume sc injection of propylene glycol (vehicle).
Antagonists were injected 1 h before the onset of restraint. Experiments conducted during the morning began with injections at 0800 h. Experiments conducted in the evening began with injections at 1900 h.
Restraint stress
Restraint stress consisted of placing rats in a clear Plexiglas
tube (23.5 cm in length and 7 cm in diameter) that inhibited
forward/backward and lateral movement, but did not interfere with
breathing. The animals were placed into the restraint tubes for 1
h.
Blood sampling procedure
The tail clip method was used for serial blood sampling. For
experiments in which only plasma corticosterone was measured, blood
(approximately 100 µl) was collected into heparanized tubes, and
plasma was stored at -20 C. For ACTH measurement, blood samples
(approximately 300 µl) were collected into EDTA coated tubes and
plasma was stored at -70 C. All rats within a cohort were sampled
simultaneously and blood sampling was completed within 3–5 min of
entering the animal room. Blood samples were taken immediately upon
restraint (basal corticosterone determination), 30 and 60 min into
restraint (restraint corticosterone response), and 60 min after the
termination of restraint (recovery from restraint determination).
Plasma corticosterone and ACTH determination
Plasma corticosterone was measured by RIA. Plasma samples (20
µl) were diluted in 0.01 M PBS and heat inactivated for
1 h at 70 C. Samples and corticosterone standards (25–2000
pg/tube) were incubated overnight with antiserum (B21–42, Endocrine
Sciences, Agoura Hills, CA) and [3-H] corticosterone (20,000
cpm/tube). Antibody-bound steroid was separated from free steroid with
dextran-coated activated charcoal. The intra and interassay
coefficients of variability were 7.3% and 13.1%, respectively. Assay
sensitivity was approximately 0.5 µg/ml for a 20-µl plasma sample.
Plasma ACTH was measured with a commercial kit (IncStar, Stillwater,
MN). The ACTH assay had a sensitivity of 10 pg/ml for a 100-µl plasma
sample.
Estimates of in vivo corticosteroid receptor occupancy by
antagonists
The effect of a 1-h treatment with RU28318 (50 mg/kg sc),
RU40555 (30 mg/kg sc), or a combination of both RU28318 (50 mg/kg sc)
and RU40555 (30 mg/kg sc) on mineralocorticoid and glucocorticoid
receptor binding in brain tissue was assessed in adrenalectomized rats.
Because only the form of the corticosteroid receptor that has not been
occupied and activated by ligand can be measured in a cytosolic
receptor binding assay (22, 23), we refer to our receptor binding
values as available receptors. Rats were adrenalectomized (ketamine 50
mg/kg + xylazine 10 mg/kg anesthetic) 24 h before antagonist
treatment to remove endogenous corticosteroids which would compete with
the antagonists for in vivo receptor occupancy. Rats were
killed (rapid decapitation) 1 h after antagonist treatment. The
hippocampus was rapidly dissected, frozen, and stored at -70 C until
subsequent corticosteroid receptor binding determination.
Corticosteroid receptor binding
Mineralocorticoid and glucocorticoid receptors were measured in
the cytosolic fraction of tissue according to the procedure described
by Spencer et al. (7, 19). A homogenization/incubation
buffer was used that was comprised of 10 mM Tris, 1
mM EDTA, 20 mM molybdic acid, 5 mM
dithiothreitol, and 10% glycerin, pH 7.4, at 4 C. Frozen tissue was
thawed, homogenized, and then centrifuged for 30 min at 100,000 x
g, and the resulting supernatant was used as cytosol.
Cytosol (0.5–1.5 mg/ml) was incubated overnight (4 C) with
[3H]dexamethasone ± unlabeled steroids. Bound
steroid was separated from unbound steroid by gravity filtration over
Sephadex LH-20 (Pharmacia LKB Biotechnology, Piscataway, NJ)
minicolumns in triplicate. Glucocorticoid receptor binding was
determined by the amount of total [3H]dexamethasone (10
nM) binding that was displaced by the selective
glucocorticoid receptor ligand RU28362 (0.5 µM).
Mineralocorticoid receptor binding was determined by the amount of
[3H]dexamethasone (10 nM) in the presence of
RU28362 (0.5 µM) that was displaced by the
mineralocorticoid and glucocorticoid receptor ligand, corticosterone
(10 µM). We have found that
[3H]dexamethasone in vitro is as effective as
[3H]aldosterone for measuring mineralocorticoid receptors
(7) and has the advantage over [3H]aldosterone and
[3H]corticosterone in that there is no potential
cross-reaction of [3H]dexamethasone with corticosteroid
binding globulin. A single saturating concentration of
[3H]dexamethasone was used so that available
mineralocorticoid and glucocorticoid receptors could be measured in the
hippocampus from each individual animal. Specific binding was expressed
as femtomoles of steroid bound per mg cytosolic protein. Proteins were
measured by the method of Bradford (24), with use of BSA as protein
standard.
Data analysis and statistics
Each experiment was analyzed by the appropriate multiway ANOVA.
To determine differences between individual groups at a particular time
point a one-way ANOVA was conducted, and in each case was followed by
both a conservative (Tukey’s test) and a more powerful (Fisher’s
least significant difference test) posthoc test. Significant results
from the Fisher’s least significant difference test are indicated only
in the cases where there was not a significant difference as determined
by the Tukey’s test. Data presented in text and figures are the
means ± SEM.
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Results |
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). The vehicle treated group, as
expected, exhibited relatively low basal corticosterone levels in the
morning (5.7 ± 1.6 µg/100 ml) and high basal corticosterone
levels in the evening (23.2 ± 4.4 µg/100 ml). RU28318 treatment
produced a significant elevation of basal corticosterone levels
relative to vehicle treatment in the morning (15.2 ± 3.5), but
not in the evening (23.4 ± 4.2).
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The maximum corticosterone levels attained during stress (occurring 30 min after restraint) were higher for the vehicle rats in the evening than in the morning. However, the acute stress response in the morning for vehicle-treated rats was lower in this experiment than in Exp 2–5, and this is the only experiment of this study in which there was a diurnal difference in stress corticosterone levels for vehicle rats.
Exp 2: RU40555 effects on morning and evening plasma corticosterone
levels
The second experiment examined the effects of the glucocorticoid
receptor antagonist, RU40555, on plasma corticosterone levels under the
same diurnal and stress conditions as were used in the first experiment
(Fig. 2). As was the case in the first
experiment, vehicle-treated rats had low basal corticosterone levels in
the morning (4.6 ± 1.4 µg/100 ml) and high basal corticosterone
levels in the evening (19.0 ± 1.9 µg/100 ml). RU40555
treatment had no effect on basal corticosterone levels at either time
of day.
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Exp 3: combined RU28318 and RU40555 effects on morning and evening
plasma corticosterone levels
The third experiment examined the effects of combined treatment
with RU28318 and RU40555 on plasma corticosterone levels (Fig. 3). For this experiment, two separate
cohorts of rats were tested on separate occasions. Although there was
an overall statistically significant cohort effect due to higher
stress-induced corticosterone levels of all treatment groups in one
cohort compared with the other cohort, there was not a significant
interaction effect of cohort and time of day or cohort and drug on
plasma corticosterone levels. Consequently, for presentation purposes
we have pooled the data from the two cohorts.
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Treatment with a combination of both RU28318 and RU40555 produced an overall drug effect on plasma corticosterone levels, F(1,44) = 15.8, P < 0.001. Post hoc analysis indicates that the combined antagonist treatment produced an elevation of basal corticosterone levels in the evening. In addition, there was a significant elevation of corticosterone levels during stress both in the morning and evening. Finally, both in the morning and evening, corticosterone levels of the receptor antagonist treated group were significantly elevated compared with the vehicle group 1 h after the termination of restraint.
Exp 4: within experiment comparison of RU28318, RU40555, or
combined RU28318 and RU40555 effects on morning plasma corticosterone
levels
The fourth experiment was conducted to determine the
reproducibility of the effects observed in the first three experiments.
In addition, the fourth experiment provided for a within experiment
comparison of the three drug conditions examined in the first three
experiments. Because there were very few diurnal differences in the
drug effects observed in the first three experiments, the fourth
experiment was conducted only in the morning.
The vehicle-treated rats exhibited basal corticosterone levels
(3.1 ± 0.8 µg/100 ml) that were similar to morning basal levels
observed in the three previous experiments (Fig. 4). The stress response of the
vehicle-treated rats was also similar to that observed in the
previous experiments.
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Exp 5: combined RU28318 and RU40555 effects on morning plasma ACTH
and corticosterone levels
The fifth experiment examined the effects of the combined RU28318
and RU40555 treatment on both plasma ACTH levels and plasma
corticosterone levels in the morning. This experiment was conducted to
verify that the increased corticosterone response observed after
treatment with RU28318 and RU40555 reflects also increased ACTH levels.
Although it seems unlikely that acute treatment with corticosteroid
receptor antagonists could alter corticosterone levels independent of
alterations in ACTH secretion, there is evidence for neural alteration
of adrenal activity independent of changes in other components of the
HPA axis (25). Because plasma ACTH levels are capable of rising more
rapidly after stress onset than are plasma corticosterone levels, in
this experiment blood samples were taken at 15 min after restraint
onset in addition to the later time points used in the previous
experiments (30 and 60 min).
Restraint stress produced a small rise in plasma ACTH levels in the
vehicle-treated rats, which reached its maximum 30 min after restraint
onset. There was an overall drug treatment effect on plasma ACTH
levels, F(2, 21) = 10.6, P = 0.004 (Fig. 5). Posthoc analysis indicated that
treatment with the combined corticosteroid receptor antagonists
produced an enhanced ACTH response to restraint that reached its peak
at 15 min after stress onset and remained significantly elevated 30 and
60 min after stress onset. In this experiment, the combined RU28318 and
RU40555 treatment produced a significant elevation in plasma
corticosterone levels only at the 60 min restraint time point.
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Treatment with RU28318 produced a selective 85% decrease in available
mineralocorticoid receptors and no effect on available glucocorticoid
receptors relative to vehicle treatment. Treatment with RU40555
produced a selective 50% decrease in available glucocorticoid
receptors relative to vehicle treatment, and an unexpected increase in
available mineralocorticoid receptors. Treatment with the combination
of RU28318 and RU40555 produced decreases in both mineralocorticoid
(80%) and glucocorticoid (40%) receptors relative to vehicle
treatment.
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Estimates of corticosteroid receptor occupancy by
antagonists
Receptor binding studies indicate that RU28318 treatment produced
a selective 85% decrease in available mineralocorticoid receptors in
rat hippocampal tissue, whereas RU40555 treatment produced a selective
50% decrease in available glucocorticoid receptors. Several studies
have demonstrated that corticosteroid receptor antagonists, in addition
to agonists, lead to translocation of corticosteroid receptors from the
cytoplasm to the nucleus (26, 27, 28). Thus, the decrease in available
corticosteroid receptor binding in the cytosolic/soluble tissue
fraction seen in this study after acute antagonist treatment most
likely reflects the occupancy and translocation of receptors by the
antagonist before the rats were killed. We have found in previous
studies that changes in systemic steroid levels produce changes in
available corticosteroid receptor levels that are similar across each
brain area examined (7, 19). Consequently, we infer from this study
that our treatment with RU28318 produced approximately 85% occupancy
of mineralocorticoid receptors throughout the rat brain, and treatment
with RU40555 produced approximately 50% occupancy of glucocorticoid
receptors in rat brain. We unexpectedly found that acute treatment with
RU40555 treatment alone produced an increase in available
mineralocorticoid receptors relative to vehicle treated rats. We note,
however, that this result was not reproduced in other similar studies,
and therefore may be spurious (20).
Regulation of basal HPA axis activity
Our study is consistent with the proposed role of corticosteroid
receptors in regulation of HPA axis basal activity. We found that
mineralocorticoid receptor activation was necessary for maintaining the
low levels of corticosterone secretion normally seen in the morning,
whereas activation of both mineralocorticoid and glucocorticoid
receptors contributed to the normal evening basal corticosterone
levels. This result supports other studies in which treatment of
adrenalectomized rats with a low level of corticosterone, estimated to
occupy only mineralocorticoid receptors, was able to maintain basal
ACTH levels in the morning, whereas treatment with higher levels of
corticosterone, estimated to occupy both mineralocorticoid receptors
and some glucocorticoid receptors, was required to maintain basal ACTH
levels in the evening (11). The interactive role of mineralocorticoid
and glucocorticoid receptor regulation of ACTH levels in the evening
was further demonstrated in a study in which treatment with low levels
of corticosterone was able to potentiate the ability of dexamethasone
(a relatively selective glucocorticoid receptor agonist) to maintain
ACTH levels in the evening (13).
Regulation of acute-stress-induced HPA axis activity
Our study provides some refinement to the prior proposed exclusive
role of glucocorticoid receptors in mediating acute corticosteroid
negative feedback on HPA axis activity during and after stress (2).
Thus, in our study it appears that activation of both mineralocorticoid
and glucocorticoid receptors contributes to a corticosteroid feedback
inhibitory effect that limits the magnitude and duration of
stress-induced HPA axis activity. Moreover, this receptor contribution
appears to be similar in both the morning and the evening.
The inability of the mineralocorticoid receptor antagonist alone to affect stress- and post stress-induced corticosterone levels is consistent with the results of Weidenfeld and Feldman (17). Of importance in our study was the finding that treatment with the glucocorticoid receptor antagonist alone was also insufficient to affect corticosterone levels during or following stress. This contrasts with the results of Weidenfeld and Feldman (17) and Ratka et al. (16) in which treatment with another glucocorticoid receptor antagonist, RU486, produced either an increase in peak stress levels of corticosterone (17) or sustained elevations of corticosterone after stress (16). The discrepancy between the effectiveness of RU40555 vs. RU486 most likely pertains to their difference in potency (i.e. magnitude of glucocorticoid receptor blockade). As discussed above, we have evidence that the dose of RU40555 used in our studies occupied approximately 50% of glucocorticoid receptors. Consequently, it appears that blockade of only 50% of glucocorticoid receptors is not sufficient to increase HPA axis responses during and after stress. On the other hand, blockade of greater than 50% of glucocorticoid receptors, as apparently was the case in studies using the more potent RU486 compound, may result in increased HPA axis responses during and after stress.
Importantly, our results indicate that the combined blockade of 50% of glucocorticoid receptors and a majority of mineralocorticoid receptors is a condition sufficient to significantly increase HPA axis responses during and after acute stress. Based on this result, we suggest that mineralocorticoid receptors contribute to the corticosterone active inhibition of the HPA axis during acute stress. This contribution may be a result of a direct facilitatory effect of mineralocorticoid receptors on glucocorticoid receptor function within the same corticosteroid target cells. Precedence for such an effect has been provided by recent studies demonstrating in corticosteroid receptor transfected cell lines a potentiating effect between mineralocorticoid receptors and glucocorticoid receptors on activation of a reporter gene. In those in vitro studies, the potentiating effect appears to be a result of the formation of heterodimers between the two subtypes of corticosteroid receptors (29). Such a potentiating effect may be limited to enhancer-like function of corticosteroid receptors. Whether the negative feedback effects of corticosteroids on HPA axis activity includes enhancement of the transcription of certain target genes remains to be determined.
Alternatively, mineralocorticoid receptor facilitation of glucocorticoid receptor dependent corticosteroid negative feedback could be a result of an interaction between separate cells/pathways converging on the paraventricular nucleus of the hypothalamus (PVN). Thus, an indirect negative feedback facilitatory effect may result from a mineralocorticoid receptor sensitive input to the PVN (e.g. from the hippocampus) that adjusts the sensitivity of PVN neurons to the inhibitory effects of corticosterone mediated by glucocorticoid receptors. Although, there is no direct support for such a mechanism, it is worth noting that 1) the hippocampus expresses a high level of mineralocorticoid receptors, and 2) lesions of the hippocampus have been demonstrated to decrease the sensitivity of the HPA axis to the inhibitory effects of glucocorticoids (30).
Revised model of the dependence of HPA axis activity on
corticosteroid receptor mediated function
Based on this study, in conjunction with the results from previous
studies, we propose that mineralocorticoid receptor activation alone is
sufficient to maintain normal HPA axis basal activity at the circadian
trough. On the other hand, normal levels of HPA axis activity during
the circadian peak or during times of stress requires activation of
glucocorticoid receptors. However, mineralocorticoid receptor
activation at these other times may contribute to HPA axis regulation
by facilitating glucocorticoid receptor function, either directly, or
indirectly. Thus, mineralocorticoid receptor activation may decrease
the threshold of glucocorticoid receptor activation necessary to
restrain the HPA axis during these circadian or stress
circumstances.
This proposed potentiating effect of mineralocorticoid receptors on glucocorticoid receptor mediated function is essentially the same process proposed by Bradbury et al. (13) to be operative during the regulation of peak basal HPA axis activity by corticosterone. Our results extend this potentiating effect to corticosteroid regulation of the HPA axis during acute stress.
An implication of this proposal is that complete blockade of glucocorticoid receptors in the absence of mineralocorticoid receptor blockade would be expected to impair corticosteroid feedback inhibition of the HPA axis during acute stress or the circadian peak, but not during the circadian trough. Studies in both humans and rats found that treatment with RU486 produced elevated basal corticosteroid levels during the circadian peak but not circadian trough (31, 32, 33, 34). As already noted above, two studies have also found that RU486 treatment of rats produced either increased stress or poststress corticosterone levels (16, 17). On the other hand, partial blockade of glucocorticoid receptors in the absence of mineralocorticoid receptor blockade, as occurred in our study, may not affect HPA axis function due to the potentiating effect of mineralocorticoid receptors on the remaining available glucocorticoid receptors.
Another implication of this proposal is that complete blockade of mineralocorticoid receptors may also have effects on HPA axis activity during acute stress or the circadian peak, but only in cases where glucocorticoid receptor activation alone is not sufficient to maintain normal HPA axis levels of activity. In an example of mineralocorticoid receptor blockade having an effect on stress-related HPA axis activity, Ratka et al. (16) found that RU28318 treatment resulted in elevated poststress corticosterone secretion. Those authors suggested that RU28318 interfered with a normal mineralocorticoid receptor-mediated suppressive effect of corticosterone on limbic function and stress responsivity. But it is also possible that in the Ratka et al. study there was not enough glucocorticoid receptor activation during acute stress to act alone to inhibit poststress HPA axis activity.
An example of mineralocorticoid receptor blockade having an effect on basal HPA axis activity at a time other than the circadian trough is provided in a study in which men were treated with the relatively selective mineralocorticoid receptor antagonist, canrenoate (35). Canrenoate treatment produced elevated basal cortisol levels both during the night, when basal cortisol levels were at their circadian trough, and early in the morning (0700 h), as cortisol levels approached their circadian peak (35). Because later time points were not examined, it is unknown whether the canrenoate treatment also increased cortisol levels later in the morning at the height of the circadian peak.
Time domains for corticosteroid regulation of HPA axis
activity
The necessary time-relationship between corticosteroid receptor
activation and subsequent effects on HPA axis activity were not
systematically evaluated in this study. However, some inferences about
that relationship can be determined from our experimental paradigm.
Because mineralocorticoid and glucocorticoid receptors function as
hormone activated transcription factors, their effects on cell activity
are believed to depend on the alteration of cellular levels of specific
proteins. It is generally accepted that the production of new
functional proteins following initiation of gene transcription requires
a minimum of 20 min to several hours. In our study, rats were treated
with antagonists for 1 h before any blood samples were collected.
We have found that our antagonist treatment paradigm produces maximal
decreases in available hippocampal corticosteroid receptors
(i.e. receptor occupation) by 60 min after injection (20).
Thus, it is likely that we are measuring HPA axis activity at a time
point in which steady state levels of proteins that are regulated by
mineralocorticoid receptors and/or glucocorticoid receptors are just
beginning to be altered. An implication of our results is that basal
HPA axis activity is actively regulated by circulating corticosteroids
and blockade of those effects for as little as 30–60 min is capable of
disinhibiting HPA axis activity. Our treatment paradigm does not allow
us to determine whether the increased stress and poststress
corticosterone levels produced by corticosteroid receptor antagonism
was a result of receptor blockade during the hour before the onset of
stress or was a result of receptor blockade during stress. However,
there is support from another study for the surge in corticosterone
that accompanies a stress response to contribute a negative feedback
effect on the magnitude of ACTH secretion present during that stress
response (36).
Concluding remarks
In summary, this study further illustrates the dynamic dependence
of HPA axis function on corticosteroid negative feedback. Short-term
pharmacological blockade of corticosteroid receptors leads to increased
corticosterone levels during basal and acute-stress conditions. Both
mineralocorticoid receptors and glucocorticoid receptors contribute
importantly to corticosteroid feedback inhibition of the HPA axis.
Activation of mineralocorticoid receptors by the very low levels of
corticosteroid present at the trough of the circadian cycle appears to
be sufficient to maintain low basal HPA axis activity. On the other
hand, glucocorticoid receptor activation is necessary to maintain
normal levels of HPA axis activity at the peak of the circadian cycle
and during acute stress. However, even under these circumstances,
mineralocorticoid receptor activation appears to play an important role
in potentiating the glucocorticoid receptor mediated effects of
corticosterone on HPA axis function.
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Footnotes |
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Received September 19, 1997.
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C. Mattsson, M. Lai, J. Noble, E. McKinney, J. L. Yau, J. R. Seckl, and B. R. Walker Obese Zucker Rats Have Reduced Mineralocorticoid Receptor and 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Expression in Hippocampus--Implications for Dysregulation of the Hypothalamic-Pituitary-Adrenal Axis in Obesity Endocrinology, July 1, 2003; 144(7): 2997 - 3003. [Abstract] [Full Text] [PDF]
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D. Owen and S. G. Matthews Glucocorticoids and Sex-Dependent Development of Brain Glucocorticoid and Mineralocorticoid Receptors Endocrinology, July 1, 2003; 144(7): 2775 - 2784. [Abstract] [Full Text] [PDF]
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B. A. Kalman and R. L. Spencer Rapid Corticosteroid-Dependent Regulation of Mineralocorticoid Receptor Protein Expression in Rat Brain Endocrinology, November 1, 2002; 143(11): 4184 - 4195. [Abstract] [Full Text] [PDF]
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F. Gomez, H. Houshyar, and M. F. Dallman Marked Regulatory Shifts in Gonadal, Adrenal, and Metabolic System Responses to Repeated Restraint Stress Occur within a 3-Week Period in Pubertal Male Rats Endocrinology, August 1, 2002; 143(8): 2852 - 2862. [Abstract] [Full Text] [PDF]
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, Significant
difference from the vehicle group at the same time point and same time
of day, P < 0.05, Student’s t
test. a, Significant difference from the morning vehicle
group at the same time point, P < 0.05, Tukey’s
test.
