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Am J Physiol Regul Integr Comp Physiol 277: R869-R877, 1999;
0363-6119/99 $5.00
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Vol. 277, Issue 3, R869-R877, September 1999

Defective ACTH response to stress in previously stressed rats: dependence on glucocorticoid status

Octavi Martí, Rosa Andrés, and Antonio Armario

Departament de Biologia Cel .lular, de Fisiologia, i d'Immunologia, Unitat de Fisiologia Animal, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of previous exposure to stress on the pituitary-adrenal response to a further stress was characterized in rats with different glucocorticoid status: sham-operated rats (Sham), adrenalectomized (ADX) rats, and ADX rats supplemented with a low corticosterone (B) dose in the drinking saline (ADX + B). Previous exposure of Sham rats to 1 h of immobilization (Imo) reduced, 2 h later, the ACTH response to a second severe stressor (Imo) but not to a less severe stressor (tail shock). In ADX rats, previous Imo totally suppressed the ACTH response to Imo or to shock. In ADX + B rats the response to shock was blocked and that to Imo tended to be lower. These changes were not explained by depletion of adenohypophysial ACTH stores. After previous Imo, reduced response to corticotropin-releasing factor was observed in Sham and ADX + B, but not in ADX, rats. Taken together, the present results suggest that the reduced ACTH response of previously stressed rats to a second severe stress is observed in the presence and absence of glucocorticoids, but the main site at which such inhibition occurs might be critically dependent on the glucocorticoid status.

corticosterone; feedback; adrenalectomy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION of the hypothalamic-pituitary-adrenal (HPA) axis has been known for years as one of the main stress indexes. Peripherally, this activation results in increased plasma ACTH and in the release of glucocorticoids by the adrenal gland. The initial activation of the HPA axis is no longer maintained, despite continuous exposure to the stressor, and this might result from a combination of three different mechanisms: 1) familiarization with the stimulus, resulting in a lower impact of the stressor, 2) triggering of biochemical changes in the regulation of the HPA axis, such as desensitization of the corticotrope response to stimulatory factors (35), and 3) negative feedback of glucocorticoids released during stress at different targets within the central nervous system and the pituitary gland (13). The contribution of each factor to the reduction of the initial response is difficult to establish, but the contribution of the first mechanisms could be theoretically assessed by changing the stressor, an approach our laboratory has been using in chronic stress paradigms (4, 5).

Irrespective of the existence of other additional mechanisms, stress-induced glucocorticoid release might be expected to dampen subsequent ACTH release to new demands in the short term. However, Dallman and Jones (11) demonstrated that exogenous administration of corticosterone (B) mimicking stress-induced B release reduced the B response to an acute stress challenge, whereas previous stress did not. The authors hypothesized that stress might provoke a hyperexcitability of the HPA axis that balances the negative feedback by glucocorticoids, so that the response to a superimposed stress would be roughly maintained. There is good agreement with regard to the existence of such a phenomenon, which has been usually termed stress-induced facilitation of the HPA axis (9). However, there are contradictory results about the influence of previous exposure to stress on the response of the HPA axis to a superimposed stress. Careful inspection of the data in the literature reveals that the HPA response is maintained (or even increased) when the stressors are mild (25) or when the exposure to them was brief and the interstressor interval was relatively long (8, 11, 15, 17, 27, 28, 42). After exposure to stronger stressors or with shorter interstressor intervals, a reduced response to the second stress has been observed (6, 18, 21, 26).

Taken together, all these data suggest that facilitation might be unmasked only when a glucocorticoid negative signal is reduced or eliminated. Whereas facilitation of the HPA response to stress was observed after three previous, short-term (1-min) exposures to immobilization (Imo) in adrenalectomized (ADX) rats supplemented with a low dose of B in their drinking saline (ADX + B) (2a), in this work no evidence of facilitation of the ACTH response to a further stress was found in ADX or ADX + B rats exposed previously to 1 h of Imo. We decided to characterize the reason for the lack of facilitation in these animals and obtained evidence of an apparently paradoxical desensitization of the ACTH response to acute repeated stress in all rats, irrespective of their glucocorticoid status, although the intensity of the effect and the mechanisms involved might be related to the glucocorticoid status.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Two-month-old male Sprague-Dawley rats obtained from the breeding center of the Universitat Autònoma de Barcelona were used. They were housed two per cage under controlled conditions of temperature (22 ± 1°C) and photoperiod (lights-on from 0730 to 1930), with free access to food pellets and water (or saline). Rats were maintained under these conditions for >= 1 wk before and during the whole experimental period. The experimental protocols were approved by the Committee of Ethics of the Universitat Autònoma de Barcelona.

Surgery and Stress Procedures

ADX was performed in ether-anesthetized rats by a bilateral dorsal approach. Sham-ADX (Sham) animals were treated the same way as ADX animals, but the adrenal glands were not excised. ADX rats received 0.9% saline as drinking solution. Circulating B levels were measured, and the possible presence of remnant adrenal tissue was controlled at autopsy. The animals not subjected to complete ADX were eliminated from the statistical analysis. In some experiments, some ADX animals were supplemented with 50 mg/l B in the drinking saline to presumably maintain basal levels of B and its circadian rhythmicity. The drug (Sigma Chemical) was dissolved first in ethanol (50 mg/4 ml) and then in 1 liter of saline. Fresh solutions were prepared every 2 days.

Two different stressors were used: Imo and tail shock. Imo consisted of restricting movements of the animal in a prone position by taping the four limbs to metal mounts attached to a wooden board; two metal loops around the neck area restricted head movements as well (23). Tail-shocked animals were placed in a Plexiglas cylinder provided with several holes, and shock (1.5 mA for 5 s with an intershock interval of 25 s) provided by a multiple-channel shocker was applied to their tails with the aid of a clip.

Sampling and Assays

Blood samples (250 µl) were obtained by tail nick into EDTA-coated capillary tubes and kept on ice. After centrifugation, plasma was stored at -30°C. In some experiments, animals were killed by decapitation, and the hypophysis was quickly removed and dissected on a cold plate into adenohypophysis and neurointermediate lobe. The adenohypophysis was further processed for determination of ACTH content as follows: each adenohypophysis was placed in 1 ml of 0.1 N acetic acid containing 10 mM 2-mercaptoethanol at 90°C for 15 min. The tissues were thereafter sonicated and centrifuged at 1,000 g for 20 min at 4°C, and the supernatant was lyophilized and stored at -80°C. On the day of the assay, samples were reconstituted and diluted, and the ACTH content was determined by RIA with human ACTH as the standard (Sigma Chemical). Plasma ACTH was assayed immunoradiometrically with a commercial kit (Nichols Institute). Plasma B was determined by RIA, as described previously (24). Fifty microliters of plasma were used in the ACTH immunoradiometric assay, with a limit of detection in this condition of 16 pg/ml, and 1-5 µl of plasma were used in the B RIA, with a limit of detection of 0.5 µg/dl in the assay with use of the maximum volume.

Experiments

All experiments were performed between 0900 and 1300. The details and schedule of the different experiments are shown in Fig. 1.


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  		Fig. 1.   Protocols and time schedule corresponding to experiments 1-5. Time of day and period of exposure to stressors [immobilization (Imo) or tail shock] or administration of corticotropin-releasing factor (CRF) are indicated. Sham, sham-operated rats; ADX, adrenalectomized rats; ADX + B, ADX rats treated with low-dose corticosterone in saline drinking water.

Experiment 1. One week after surgery, one-half of the Sham and ADX animals were subjected to Imo for 1 h; the remaining animals were left undisturbed. Two hours later all the animals were exposed to tail shock for 30 min, and blood samples were taken before tail shock and 15 and 30 min after the onset of shock (experiment 1A). One week later the same animals underwent the same treatments, except the time lag between the two stress exposures was 24 h (experiment 1B).

Experiment 2. One week after surgery all the animals (Sham, ADX, and ADX + B) were subjected to Imo for 1 h, and blood samples were taken before Imo and 30 min after the onset of this first stress. Two hours later rats were reexposed to Imo, and blood was sampled before Imo and 30 min after the onset of the second Imo.

Experiment 3. One week after surgery animals from the three groups (Sham, ADX, and ADX + B) were left undisturbed or subjected to 1 h of Imo. Two hours later all animals were subjected to 30 min of Imo and then killed. The adenohypophyses were collected and processed for ACTH content measurement.

Experiment 4. One week after surgery one-half of the Sham, ADX, and ADX + B animals were subjected to Imo for 1 h; the remaining animals were left undisturbed. Two hours later all the animals received ovine corticotropin-releasing factor (CRF, 50 µg/kg ip; Sigma Chemical) dissolved in saline containing 0.1% BSA, and blood was sampled before injection and 15 and 30 min after CRF administration. This dose was chosen to obtain a maximum blood ACTH response.

Experiment 5. One week after surgery ADX + B rats were left undisturbed or subjected to 1 h of Imo, and 2 h later all the animals were subjected to shock for 30 min, as in experiment 1. Blood samples were taken just before and 30 min after exposure to shock.

Statistical Analyses

When necessary, data were logarithmically transformed to achieve homogeneity of variances. Data were analyzed using two-way ANOVA or one-way ANOVA with repeated measures for the factor time (exposures to stress or response to secretagogue). Post hoc comparisons were done with the Student-Newman-Keuls (SNK) test (when >2 means were compared, P < 0.05). When only two means were compared, Student's t-test (or the Wilcoxon test, if variances were not homogeneous) was used.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1

Experiment 1 was designed to study the effect of glucocorticoid removal by ADX on the ACTH response to two consecutive stress exposures. Sham and ADX rats were left undisturbed or subjected to 1 h of Imo. In experiment 1A the rats were subjected to the second stressor (shock) 2 h after completion of exposure to the first one. One week later the same rats were used, but they were subjected to the second stressor 24 h after the first one (experiment 1B). With the highly different magnitude of basal and stress levels of ACTH in Sham and ADX rats taken into account, the statistical analysis of both groups was done separately.

The ACTH data corresponding to experiment 1A are shown in Fig. 2A. In Sham rats a significant effect of present exposure to shock (P < 0.001), but not of previous Imo, was found, in that neither basal nor shock levels of ACTH were altered by previous exposure to 1 h of Imo. In ADX rats an effect of present exposure to shock (P < 0.001) and a significant interaction between previous Imo and subsequent shock (P < 0.005) were found. This interaction was due to the fact that in ADX rats the ACTH response to shock observed in rats that had not been previously immobilized (P < 0.01 and P < 0.002 at 15 and 30 min, respectively) completely disappeared in previously immobilized animals. Plasma B levels were under the detection limit of the assay (0.5 µg/dl) in ADX rats. In Sham rats (Table 1), significant effects of previous Imo (P < 0.002), present exposure to shock (P < 0.001), and the interaction between the two factors (P < 0.05) were found for B. This interaction was due to the fact that previous exposure to Imo resulted in higher B levels before shock (P < 0.02 vs. basal levels in rats that had not been previously stressed), although the net increase in B levels was not affected.


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  		Fig. 2.   Effect of prior stress (Imo) on subsequent ACTH response to tail shock stress 2 h later (A) and 24 h later (B). Values are means ± SE (n = 5-7). Significantly different from respective basal group: * P < 0.05, ** P < 0.01. In B, 2-way ANOVA revealed significant effects of exposure to shock, but not of previous exposure to Imo, in Sham and ADX rats.


                              
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  		Table 1.   Effect of previous Imo on B response of sham rats to tail shock 2 or 24 h later

When the same animals were used 1 wk later (experiment 1B, Fig. 2B), no effect of previous exposure to Imo, but a significant ACTH response to shock (P < 0.001 in both cases), was found in Sham or ADX rats when the interval between exposure to Imo and exposure to shock was 24 h. Exactly the same pattern was observed in plasma B levels of Sham rats (Table 1).

Experiment 2

ADX rats are characterized by a high tonic activation of the hypothalamus-corticotrope axis and might well not be appropriate for revealing subtle stress-induced changes in the HPA axis. Thus, in the next experiment, Sham, ADX, and ADX + B rats were left undisturbed or subjected to 1 h of Imo. After 2 h all rats were again subjected to 30 min of Imo, so that the same rats served as their appropriate controls. Blood samples were taken just before the first and the second Imo and after 30 min of the first and second Imo. The data were analyzed separately for Sham, ADX + B, and ADX rats with use of one-way ANOVA with repeated measures for time (exposures to Imo).

The ACTH response of each group to Imo is shown in Fig. 3A. A significant effect of exposures to Imo was found in the three experimental groups (P <=  0.005). In Sham rats ACTH levels increased in response to the first (P < 0.001) and the second Imo (P < 0.005). In ADX rats, ACTH levels responded to the first Imo (P < 0.005), were high just before the second Imo (P < 0.02 vs. basal values before any stress), and did not respond to the second Imo. Finally, ADX + B rats showed a significant increase in response to the first Imo (P < 0.001), high basal levels just before exposure to the second Imo (P < 0.001 vs. basal values before any stress), and a marginally significant response to the second Imo (P = 0.09). Direct comparisons of the net ACTH responses to the first and the second Imo in the three experimental groups, when the appropriate basal levels were subtracted (Fig. 3B), revealed that the ACTH response to the second Imo was reduced in Sham (P < 0.02) and ADX rats (P < 0.01), but not in ADX + B rats.


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  		Fig. 3.   Effect of prior stress (1st Imo) on subsequent ACTH response to a 2nd Imo 2 h later. Values are means ± SE (n = 7 or 8). A: absolute ACTH values. Significantly different from respective basal group: * P < 0.005, ** P < 0.001. # Marginally significantly different from respective basal group. B: net increases in ACTH after acute stress. Significant difference between response to 1st Imo and response to 2nd Imo: * P < 0.02, ** P < 0.005.

B levels in ADX + B rats averaged 0.8-1.4 µg/dl (Table 2). The one-way ANOVA of B levels in Sham rats revealed a significant effect of exposures to Imo (P < 0.001; Table 2), in that they showed a significant response to the first Imo (P < 0.001), high B levels just before the second Imo (P < 0.005 vs. basal values before any stress), and a significant response to the second Imo (P < 0.02). Direct comparisons of the response to the first and the second Imo when the appropriate basal levels were considered revealed that the B response to the second Imo was reduced compared with the first one (Wilcoxon test, P < 0.05).

                              
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  		Table 2.   Effect of two sequential exposures to Imo on B levels in Sham and ADX + B rats

Experiment 3

The defective ACTH response to the second Imo observed in Sham and ADX rats might have been due to depletion of ACTH stores (33), so that adenohypophysial ACTH content was measured in rats treated similarly to those in experiment 2, except the rats were killed 2 h after completion of the first exposure to Imo. No significant effect of glucocorticoid status or previous exposure to Imo on adenohypophysial ACTH content was found (Table 3).

                              
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  		Table 3.   Effect of previous Imo on ACTH content of adenohypophysis as a function of glucocorticoid status

Experiment 4

Despite a normal adenohypophysial ACTH content, previous Imo might have impaired the releasable ACTH pool. To test this possibility, Sham, ADX, and ADX + B rats were left undisturbed or subjected to 1 h of Imo, and 2 h later all rats received 50 µg/kg of CRF. The one-way ANOVA (Fig. 4A) revealed a significant effect of glucocorticoid status on plasma ACTH levels just before CRF administration in rats that had not been previously immobilized (P < 0.001) and in those that had been previously immobilized (P < 0.001). In those that had not been previously immobilized, ACTH levels were higher in ADX than in ADX + B rats and higher in ADX + B than in Sham rats. In previously immobilized rats, ACTH levels of ADX + B rats approached those of ADX rats, and differences were observed between these two groups and Sham rats.


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  		Fig. 4.   Effect of prior stress (1st Imo) on subsequent ACTH response to CRF administration 2 h later. Values are means ± SE (n = 10 sham, 5 ADX, and 7-9 ADX + B). A: absolute ACTH values. Only significant differences in ACTH levels before CRF administration among experimental groups, within each stress condition, are indicated; groups labeled with different letters (a-c) are statistically different (Student-Newman-Keuls test). B: net ACTH response to CRF (with consideration of peak ACTH value achieved). In stress-naive rats, bars labeled with different letters (a-c) are statistically different (Student-Newman-Keuls test). Significant difference in ACTH response to CRF between previously immobilized and stress-naive rats under same glucocorticoid status: * P < 0.05, ** P < 0.001.

Peak ACTH response to CRF was observed at 15 min in some rats and at 30 min in the others. Because no differences between groups were observed in the number of animals reaching the peak at either time, the ACTH response to CRF and its statistical significance were evaluated by subtracting pre-CRF levels from the peak value at 15 or 30 min (Fig. 4B). In rats that had not been previously immobilized, the ACTH response to CRF differed among the three experimental groups (P < 0.001), the response being greatest in ADX + B rats, intermediate in ADX rats, and least in Sham rats (SNK test). In the rats that were previously subjected to Imo, the net ACTH increase was also dependent on glucocorticoid status (P < 0.001), the response being greater in ADX and ADX + B than in Sham rats, with no differences between the two former groups (SNK test). Previous Imo caused a significant decrease in the net ACTH increase after CRF in Sham (P < 0.001) and ADX + B (P < 0.05), but not in ADX, rats.

Experiment 5

Experiment 5 complemented experiment 1A, in that the influence of previous Imo on the response to tail shock 2 h later was studied in ADX + B rats. Exposure to 1 h of Imo resulted in substantially high preshock ACTH levels in those animals, with no further response to acute shock (data not shown), and therefore they behaved similarly to ADX rats of experiment 1A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We present evidence that previous exposure to a severe stressor such as Imo reduces the ACTH response to the same stressor in the presence or absence of glucocorticoids. The results suggest that changes at the level of the pituitary and also at levels above the pituitary, depending on the glucocorticoid status of the animals, are taking place as a consequence of exposure to Imo.

When previously exposed to 1 h of Imo, adrenal-intact animals showed normal resting ACTH levels and a normal ACTH response to a second stressor (acute tail shock) applied 2 h after exposure to Imo was completed. However, plasma B levels were elevated before initiation of exposure to shock, suggesting that a normal ACTH response to the second stressor persisted even in the presence of high prestress B levels. The results in Sham rats are in accordance with previous data in the literature reporting a normal ACTH response to stress in acute repeated stress paradigms (8, 11, 15, 17) and would also be consistent with the idea that, despite corticosterone release triggered by the first stress exposure, stress per se is capable of overcoming the previous negative influence of glucocorticoids on the HPA axis (9, 11).

ADX rats, if not stressed previously, showed a consistent response to tail shock, despite their very high basal ACTH levels. On the contrary, previous exposure of ADX rats to Imo resulted, 2 h later, in elevated ACTH levels just before exposure to shock (compared with ADX, stress-naive rats) and also in a blockade of the ACTH response to the second stressor. When the same animals were studied 1 wk later but the interval between Imo and shock was prolonged up to 24 h, previous exposure to Imo did not alter resting or shock levels of ACTH. Therefore, a 24-h lag period appears to be enough for the recovery of normal hypothalamus-corticotrope function in ADX rats. The inhibitory effect of previous Imo on the ACTH response of ADX rats to a subsequent stress applied 2 h later does not coincide with what might have been expected according to the hypothesis of the existence of a stress-induced facilitation that is counteracted by glucocorticoids, inasmuch as negative glucocorticoid feedback mechanisms were absent in ADX rats. It might be argued that ACTH release was already maximal in previously immobilized ADX rats before exposure to the second stressor, but our data after CRF administration clearly indicate that this was not the case (see below).

The absence of B results in very high ACTH levels in ADX rats and a strong disinhibition of the hypothalamus-corticotrope axis (1, 10, 16), and this might have affected the above results. We therefore included a group of ADX + B rats and decided to use the same stressor the first and the second time, each rat thus serving as its appropriate control. The higher basal ACTH levels of ADX rats were completely normalized by corticosterone replacement of ADX rats (ADX + B animals), giving proof that corticosterone replacement restrained tonic activation of corticotropes. In response to the first Imo, ADX rats showed, as expected, a greater response than Sham or ADX + B rats. In contrast to experiment 1, a reduction of the ACTH response to the second exposure to Imo was observed in Sham rats. Therefore, previous Imo appears to reduce the ACTH response to the second Imo but not to shock. The precise reason for this discrepancy is not clear, but several possibilities exist. First, the reduction of the response might have been caused by habituation due to the repetition of the same stressor, but it appears unlikely to us in view of the fact that repeated exposure to short-term Imo does not reduce the ACTH response (2a). Second, the two stimuli might differ in terms of the set of hypothalamic regulatory factors they activate. Third, the reduction of the response might be related to the intensity of the second stressor, in that a defective ACTH response is observed with a stronger demand to the hypothalamus-corticotrope axis, and Imo is a stronger stressor than shock. The latter hypothesis is supported by the results of Graessler et al. (18) showing a reduction of the ACTH response to 35% but not 25% hemorrhage in rats previously subjected to 60-90 min of Imo just before hemorrhage.

ACTH levels were higher in ADX and ADX + B rats just before the second than before the first exposure to Imo (levels in ADX + B rats were intermediate between Sham and ADX rats), supporting the hypothesis that stress-induced release of corticosterone greatly contributes to normalization of the HPA axis after its initial activation by stress (37, 38). Whereas the response of Sham rats to the second Imo was still significant, although reduced, it was only marginally significant in ADX + B rats and null in ADX rats. Direct comparison of the ACTH response to the first and the second Imo showed a reduced ACTH response to the second Imo in Sham and ADX rats, but not in ADX + B rats (the lack of significance was due to the high variability, inasmuch as a trend toward a decrease was observed). When we studied the influence of previous Imo on the subsequent response to shock in ADX + B rats (experiment 5), exposure to Imo for 1 h resulted in high preshock ACTH levels with no further response to shock. These data suggest that after previous exposure to Imo the response of ADX + B rats to a further stress is similar to that of ADX animals.

Provided that neither ADX nor ADX + B animals had negative glucocorticoid feedback caused by stress-induced B release, the null or weak ACTH response to the second stress observed in these animals might be related to the inability of the anterior pituitary of these animals to respond under a situation of high demand, as represented by the acute repeated stress protocol used in this work. A decrease in the ACTH content of the pituitary might have compromised subsequent pituitary output (35), but we found that it was not affected by the glucocorticoid status or the previous exposure to 1 h of Imo.

Despite the fact that ACTH stores in the adenohypophysis were not exhausted in our experimental paradigm, previous exposure to stress might have altered the responsiveness of corticotropes to CRF (35), the main ACTH secretagogue. Therefore, the ACTH response to CRF was tested in rats belonging to the three glucocorticoid status groups that had not been previously stressed and those that had been previously stressed. In stress-naive rats, ACTH release caused by CRF was greater in ADX than in Sham rats and was even greater in ADX + B than in ADX rats. These results could be explained by assuming that the response to CRF was constrained in Sham rats by negative glucocorticoid feedback at the pituitary and in ADX rats by ADX-induced downregulation of adenohypophysial CRF receptors (CRFr) (14, 20, 34, 44). ADX + B rats also responded more than Sham rats because of the lack of fast glucocorticoid negative feedback and even more than ADX rats because of the lack of CRFr downregulation, since ADX-induced CRFr downregulation was prevented by glucocorticoid administration (14).

Previous exposure to acute Imo modified the above pattern of response to CRF in a manner dependent on the glucocorticoid status of the animals. Thus previous Imo reduced the ACTH response to CRF in Sham and ADX + B, but not in ADX, rats. The present results in intact rats are compatible with the finding that acute stress causes downregulation of mRNA for CRFr in the adenohypophysis (29, 31) and reduced the ACTH response to CRF and other secretagogues (35). Acute stress-induced B release appears to be, at least in part, involved, inasmuch as it also causes adenohypophysial CRFr downregulation (29, 32, 36) and inhibition of ACTH synthesis and release (3, 22). The normal response of acutely stressed ADX rats to CRF in our conditions is in apparent contrast to the reduced response observed by Rivier and Vale (35). The discrepancy could be explained by the fact that our experimental paradigm involves CRF administration 2 h after the first exposure to Imo, during which the pituitary could have partially recovered. In fact, ACTH stores were partially depleted in ADX rats before CRF administration in their experiment but not in our conditions. The factors involved in the previous stress-induced reduction of the ACTH response to CRF in ADX + B rats are unclear but are compatible with the finding by Makino et al. (31) that acute Imo stress causes downregulation of mRNA for CRFr in the adenohypophysis of ADX rats maintained with low-dose corticosterone pellets. Although arginine vasopressin (AVP) and CRF have been reported to cause rapid downregulation of mRNA for CRFr in cultured adenohypophysial cells (32, 36), these factors are unlikely to be involved, inasmuch as ADX rats presumably released more CRF and AVP to the median eminence than ADX + B rats (2, 12, 19, 27, 43). The lack of reduction of ACTH response to CRF in ADX rats might have been due to the fact that activation of the HPA axis relies more on AVP than on CRF in these rats, and the response to AVP might not undergo desensitization. At least in intact rats, acute Imo-induced upregulation of adenohypophysial AVP receptors and AVP-induced inositol phosphate formation has been observed (33).

Previous Imo-induced changes in the ACTH response to CRF do not fully explain what occurs in response to the second stress: 1) ADX + B rats previously exposed to Imo showed a weak response to the second Imo and no response to shock, despite a good response to CRF (although it was lower than that observed in stress-naive rats), and 2) the null response of ADX rats to the second exposure to stress cannot be explained by a lack of response of corticotropes to CRF. At first glance this is not surprising, since regulation of the HPA axis is very complex and depends not only on negative-feedback mechanisms of glucocorticoids, but also on a proposed negative feedback of ACTH at the hypothalamus (7, 30, 40). If it is assumed that stress-induced negative glucocorticoid feedback, the most important to date, is absent in ADX and ADX + B rats, the blockade of the ACTH response to the second Imo in both groups might rely on the potency of negative feedback exerted by ACTH, which is greater in ADX rats, which showed higher ACTH levels. In accordance with this hypothesis, it has been recently reported that AVP from the parvocellular paraventricular nucleus of the hypothalamus is more sensitive than CRF to negative ACTH feedback in hypophysectomized-adrenalectomized rats (39). The stronger inhibition of subsequent ACTH response to stress in ADX than in ADX + B rats is also compatible with the recent evidence that ADX rats are unable to maintain paraventricular nucleus CRF gene transcription in response to a sustained stress and that this defect is corrected by implantation of low-dose corticosterone pellets (41). These data suggest a facilitatory role of corticosterone in stress-induced activation of the hypothalamus-corticotrope axis, likely through corticosteroid type I receptors.

In conclusion, the results shown in this work indicate that previous exposure to stress is able to reduce the HPA response to a further stress, particularly when the second stressor is severe. In addition, evidence for a defective ACTH response to acute repeated stress in ADX rats and, to a lesser degree, in ADX + B animals was found, which seems to be neither related to exhaustion of ACTH stores of the adenohypophysis nor fully explained by decreased pituitary response to CRF, the main hypothalamic ACTH secretagogue. Whether this phenomenon is related to possible short-loop feedback mechanisms exerted by ACTH, more evident in ADX animals, with high ACTH levels, remains to be elucidated. Finally, no facilitation of the HPA axis by previous stress was observed with use of these particular stress paradigms.

Perspectives

The influence of previous exposure to stress on the capability of the HPA axis to respond to a further stress is of great relevance considering the biological impact of the HPA axis activation. A review of data in the literature in this regard revealed that results are controversial. In addition, little is known about the physiological mechanisms underlying such an HPA response. In the present work the role of glucocorticoids in determining the influence of a severe stressor such as Imo on the ACTH response to a novel (electric tail shock) or the same stressor was studied using Sham, ADX, and ADX + B rats. The results showed that previous exposure of intact rats to Imo caused an impairment of the ACTH response to a subsequent stressor when it was severe (Imo) and that a defective ACTH response was evident in ADX and usually in ADX + B rats in response to both stressors. The level (adenohypophysis vs. hypothalamus) at which the first stressor acts to reduce the subsequent ACTH response appears to be critically dependent on the glucocorticoid status of the animals.


    ACKNOWLEDGEMENTS

The authors thank Dr. Mary F. Dallman for critical reading of the manuscript.


    FOOTNOTES

This work was partially supported by Dirección General de Investigación Científica y Técnica Grant PM95-201 and CIRIT Grants GRQ93-2096 and 1995SGR-499.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: O. Martí, Unitat de Fisiologia Animal, Departament de Biologia Cel.lular, de Fisiologia, i d'Immunologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain (E-mail: o.marti{at}cc.uab.es).

Received 25 August 1998; accepted in final form 6 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Compar Physiol 277(3):R869-R877
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R. Andres, O. Marti, and A. Armario
Direct evidence of acute stress-induced facilitation of ACTH response to subsequent stress in rats
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