Brain Research, 532 (1990) 34-40 Elsevier

34

BRES 15986

CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress*'** Richard L. Hauger 1, Marge Lorang 1, Michael Irwin I and Greti Aguilera 2 1Veterans Administration Medical Center and Department of Psychiatry, University of California, San Diego, CA (U.S.A.) and eEndocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, MD (U.S.A.) (Accepted 8 May 1990)

Key words: Corticotropin releasing factor; Adrenocorticotropic hormone; Corticosteroid; Chronic stress

The relationship between corticotropin releasing factor (CRF) receptors and pituitary-adrenal responses was determined after chronic intermittent immobilization (2.5 h restraint/day) to examine the hypothesis that CRF receptor regulation is involved in the sensitization of the pituitary-adrenocortical axis to novel stimuli during repeated stress. Following the ll-fold stimulation of ACTH secretion on the first day of restraint stress, a desensitization of the pituitary ACTH response to immobilization was observed over the next 9 days of chronic intermittent stress. In contrast, the magnitude of the restraint-stimulated release of corticosterone on the 2nd and 4th day of stress was similar to the day 1 adrenocortical response. Furthermore, the significant stimulation of corticosterone secretion by restraint stress persisted to the 16th day of immobilization (P < 0.001), even though significant increases in plasma ACTH were absent. The concentration of anterior pituitary CRF receptors was unchanged after a single period of restraint; however, a down-regulation of anterior pituitary CRF receptors was observed following 4 days (P < 0.001) and 10 days (P < 0.005) of repeated immobilization stress. CRF receptors in the olfactory bulb were unchanged following acute or chronic restraint stress, consistent with previous observations that brain CRF receptors are neither changed by adrenalectomy, glucocorticoid administration, nor 18-48 h of continuous restraint stress. The concentration of CRF receptors in the intermediate lobe of the pituitary also was not influenced by immobilization stress. Despite the desensitization of the ACTH response during repeated immobilization, a highly significant potentiation (P < 0.001) of ACTH release following 5 min of ether vapor was observed 24 h following 9 or 15 days of repeated restraint stress. Consequently, the loss of anterior pituitary CRF receptors cannot account for the sensitization of the ACTH response to acute ether exposure following chronic immobilization stress. The data support the hypothesis that the increased pituitary corticotroph responses to novel stressful stimuli during chronic stress involves the 'facilitation' of stimulatory inputs to CRF-releasing neurons in the hypothalamus and the integrative actions of CRF and other ACTH regulators at the post-CRF receptor level.

INTRODUCTION Biologic stress elicits a biphasic response of the hypothalamic-pituitary-adrenal ( H P A ) axis 2"9"21-23'28-31. Acute stress increases the secretion of CRF, A C T H and other P O M C - d e r i v e d peptides, and adrenal corticosteroids. This large stimulation of H P A axis h o r m o n e release during the initial period of stress is usually transient followed by a decline in the concentration of circulating stress hormones to near basal levels during the adaptation phase of the response to continuous chronic stress. A similar desensitization of H P A axis responses is observed during intermittent exposure to chronic stress. The regulation of H P A axis h o r m o n e secretion during chronic stress could occur at the following levels of the brain-pituitary-adrenal system: (1) extrahypothalamic centers modulating PVN C R F neurons, (2) hypothalamic sites releasing C R F and other A C T H secretagogues, (3)

ACTH-secreting cells in the anterior pituitary, and (4) the adrenal cortex. It is well-known, however, that the magnitude of the acute stress-stimulated increases in H P A axis hormonal secretion and the temporal pattern of A C T H , fl-endorphin, and corticosteroid responses are influenced by the type of stressful stimulus and its intensity1'zl-24'31. For example, the acute hypersecretion of A C T H provoked by a single exposure of laboratory animals to ether, leg fracture, or tourniquet stress persists during continuous or repeated exposure to these stimuli in contrast to the loss of A C T H responses to chronic footshock or immobilization stress 5"14'17'21-23'28-31. H P A axis responses to novel stimuli can also vary with the type of prior chronic stress exposure. 'Systemic' forms of stress such as hypoglycemia can inhibit the release of A C T H provoked by a novel, superimposed stressor, while 'neurogenic' stress (e.g., skin incision, laparotomy, immobilization, forced swim)

* The work described in 'CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress' was done as part of our employment with the federal government and is therefore in the public domain. ** Portions of these data were presented in abstract form at the American Federation for Clinical Research, San Diego, CA, April, 1987. Correspondence: R.L. Hauger, (present address:) Oregon Health Sciences University, 3181 S.W. Sam Talleron Park Road, Portland, OR 97201-3098, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

35 can facilitate the A C T H r e s p o n s e to a s u b s e q u e n t stress 14"21-23. I n a d d i t i o n , n e u r o g e n i c stress causes a large s t i m u l a t i o n of A C T H s e c r e t i o n f r o m the n e u r o i n t e r m e diate l o b e w h i l e h u m o r a l stressors m a i n l y A C T H - r e l e a s i n g cells in the a n t e r i o r l o b e z2'23.

activate

W e h a v e p r e v i o u s l y d e m o n s t r a t e d that a large r e d u c tion in a n t e r i o r pituitary C R F r e c e p t o r s occurs in rats e x p o s e d to the s e v e r e and c o n t i n u o u s stress of 18-48 h of immobilization

TM.

In the p r e s e n t study, we h a v e e x a m -

ined the relationship between CRF receptor regulation and t h e h o r m o n a l r e s p o n s e s of the p i t u i t a r y - a d r e n a l axis d u r i n g r e p e a t e d restraint (2.5 h/day) which r e p r e s e n t s a stressor of m o d e r a t e intensity and b r i e f duration. T h e c h r o n i c i n t e r m i t t e n t i m m o b i l i z a t i o n stress p a r a d i g m allows for a d a p t a t i o n a l r e s p o n s e s of the H P A axis during the

period

each

day

when

the

animals

are

not

i m m o b i l i z e d 23. W e n o w r e p o r t that a d o w n - r e g u l a t i o n of C R F r e c e p t o r s in the a n t e r i o r pituitary, but not in the i n t e r m e d i a t e l o b e o r central n e r v o u s system, also occurs f o l l o w i n g 4 - 1 0 days of chronic i n t e r m i t t e n t i m m o b i l i z a tion stress and that the d i r e c t i o n of this C R F r e c e p t o r c h a n g e is consistent with the loss of the pituitary A C T H response

to restraint.

However,

a highly significant

p o t e n t i a t i o n o f t h e A C T H r e s p o n s e to acute e t h e r stress occurs f o l l o w i n g c h r o n i c i m m o b i l i z a t i o n stress, despite the d e c r e m e n t in C R F r e c e p t o r c o n c e n t r a t i o n , suggesting that the sensitization of the pituitary c o r t i c o t r o p h by c h r o n i c stress results f r o m a c o m p l e x i n t e r a c t i o n b e t w e e n post-CRF afferent

receptor pathways

mechanisms in the

brain

and

a facilitation of

which

stimulate

the

s e c r e t i o n of h y p o t h a l a m i c c o r t i c o t r o p i n releasing factor.

MATERIALS AND METHODS

Animals In all experiments adult male Sprague-Dawley rats weighing 300-350 g (Charles River, Boston, MA) were housed in groups of 3 animals per cage and maintained in a controlled temperature (24 °C) and light environment (12L;12D; lights on 06.00). The animals had free access to food pellets and water before and after being subjected to stress or while being housed in their home cage for 14 days before starting the stress experiments. Stress procedure After the initial period of environmental acclimation, groups of rats (n = 6-16/group) were randomly selected for the immobilization stress paradigms or served as non-restrained controls (e.g., left undisturbed in their home cages throughout the experiment). The body weight of animals was determined at the beginning of the experiment, before the first stress exposure, and 24 h prior to decapitation. Daily restraint stress consisted of exposing rats to 2.5 h/day of immobilization (from 08.00 to 10.30) for periods ranging from 1 to 16 days using a modification of the restraint method of Stone et ai. 32. Following each stress session, rats were returned to their home cage and were able to eat and drink ad libitum for the remainder of the day. CRF receptors and plasma hormone patterns were measured in chronically stressed rats immediately prior-to or following the last immobilization period on days 4, 10 or 16, and compared to animals acutely restrained for 2.5 h or controls. In

addition, the hormonal responses were measured in a subgroup of rats exposed to repeated immobilization for 3, 9, or 15 days and to 5 min of acute ether stress on the last stress day (days 4, 10, or 16). In experiments involving CRF binding in the intermediate lobe and brain, CRF receptors were also measured after 18-48 h of continuous immobilization as previously described TM.

Hormone analysis Immediately following the final stress period, all animals underwent decapitation in a separate room within 5 s of being removed from their home cage, released from 2.5 h of immobilization, or exposed to 5 min of ether vapors. Approximately 5 ml of truncal blood was collected in plastic conical centrifuge tubes containing 200 /~1 of a solution of 50 mg/ml EDTA and 500 klU of aprotinin (Sigma Co., St. Louis, MO). Plasma ACTH was measured by radioimmunoassay (RIA) in lyophilized eluates after extraction onto C18 Sep-Pak cartridges (Waters Associates, Inc., Milford, MA) and elution with 60% acetonitrile in triethylamine formate buffer, pH 3.2 (refs. 13, 14). Plasma corticosterone was measured by RIA as previously described 13A4'34. CRF receptor assay Pituitary glands were rapidly removed and separated into anterior and neural-intermediate lobes. Brains were promptly dissected on ice and brain regions collected in ice-cold PBS for measurement of CRF binding. CRF receptors were measured in brain and pituitary membrane-rich particles prepared as previously described 13"14'36. The CRF receptor assay consisted of incubating in 1.5-ml polystyrene microfuge tubes 100-/A aliquots of the membrane suspension with 100000 cpm (0.1 nM) of [125I]Tyr-ovine CRF (oCRF) (Dupont-NEN, Boston, MA) in a total volume of 300/~1 of buffer 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCI2, 2 mM EGTA, 0.1% BSA, 100 kIU/ml of aprotinin, and 1 mM DTT. Non-specific binding was measured as CRF binding in the presence o f 10 -6 M oCRE After incubation for 60 rain at 22 °C, tubes were centrifuged twice at 10,000 g for 3 min following the addition of 1 ml of 7.5% polyethylene glycol in 50 mM Tris-HCl, pH 7.4. The resultant pellets were analyzed for bound radioactivity in a gamma-spectrometer. Protein concentrations were measured by the Pierce BCA assay. Statistical analysis Calculation of receptor affinities and concentrations were performed by computer analysis of the equilibrium binding data using the non-linear least squares curve-fitting program LIGAND 25. Hormonal and binding data are presented as arithmetic mean _+ S.E.M. Statistical evaluations were made with paired and unpaired Student's t-test and with one-way analyses of variance (ANOVA) using Student-Newman-Keuls multiple-range tests to compare individual groups.

RESULTS

Pituitary-adrenocortical responses to chronic intermittent immobilization stress Fig. 1 depicts H P A axis h o r m o n a l r e s p o n s e s to r e p e a t e d r e s t r a i n t and a c u t e e t h e r stress. I m m o b i l i z a t i o n stress for 2.5 h on day 1 r e s u l t e d in l l - f o l d increases in the p l a s m a c o n c e n t r a t i o n s o f A C T H and c o r t i c o s t e r o n e which w e r e highly significant ( P < 0.001) c o m p a r e d to basal h o r m o n e levels in n o n - s t r e s s e d controls. H o w e v e r , t h e r e was a dissociation b e t w e e n the s e c r e t o r y r e s p o n s e s of the p i t u i t a r y c o r t i c o t r o p h and t h e a d r e n a l c o r t e x during c h r o n i c i n t e r m i t t e n t r e s t r a i n t stress. T h e i n c r e a s e in p l a s m a A C T H levels after i m m o b i l i z a t i o n o n day 4 was

36 significantly smaller than the A C T H response on day 1 (P < 0.001). Significant decreases in the A C T H responses to restraint occurred on days 10 ( P < 0.001) and 16 ( P < 0.05) c o m p a r e d to the day 4 A C T H response. Plasma A C T H levels on the 10th and 16th days of immobilization stress were not significantly different ( P > 0.05 by N e w m a n - K e u l s a posteriori test) from A C T H concentrations in non-stressed controls due to this progressive reduction in restraint-stimulated A C T H secretion. These d a t a suggest that a desensitization occurs in the plasma A C T H responses to restraint during chronic intermittent immobilization stress. In contrast to the p a t t e r n for A C T H responses, there were no statistically significant changes in the plasma

A.

800 •

[] []

Basalipre-Rastraint) RestraintStress(2,5h/day) AcuteEtherStress(5 rain)

a,b ,,

600

N ~

.~

400

a ~200

Day 1

B.

30

Day 4

Day 10

Day 16

e e

== 20 2m

•

Basal ~re-Restralnt)

i

RestraintStress(2.5 h/day) AcuteEtherStress(5 rain)

e, b

8

~ ~

"1~

~-

Day 1

Day 4

,

Day 10

,

Day 16

Fig. 1. Plasma concentrations of ACTH and corticosterone following chronic intermittent immobilization stress and/or acute ether stress. Values are the mean + S.E.M. of data obtained in 5-60 rats per time point. In a previous publication, a small subset of the plasma ACTH values after repeated restraint (but not pre-restraint or ether stress ACTH levels) have been correlated to splenic natural killer (NK) cytotoxicity in the same immobilized animals 17. The adaptation of pituitary ACTH responses to chronic restraint stress occurs earlier than the adaptational changes in splenic NK activity 17. A: ACTH values across the treatment groups were significantly different (F = 80.1, df 11,221, P < 0.001 by one-way ANOVA); B: corticosterone values across the treatment groups were significantly different (F = 41.0, df 11,228, P < 0.001 by one-way ANOVA). When the unpaired Student t-test or Newman-Keuls a posteriori test were used to compare individual groups, statistically significant differences were as follows: ap < 0.001 vs. non-stressed control; bp < 0.001 vs. day 1 restraint; cp < 0.05 vs. non-stressed control.

corticosterone responses to restraint on day 2 (21.1 + 2.3 /~g/dl) and day 4 (Fig. 1). M o r e o v e r , p l a s m a corticosterone responses on day 10 and d a y 16 continued to be significantly greater than basal levels in non-stressed cont-rols ( P < 0.001), although the magnitudes of the restraint-stimulated corticosterone responses were statistically lower than the responses on day 4 ( P < 0.001). This persistence of adrenocortical responses to chronic intermittent immobilization was associated with significant increases ( P < 0.05) in a d r e n a l weight on days 4, 10, and 16 of r e p e a t e d restraint stress (Table I).

Effect of chronic intermittent immobilization stress on pituitary-adrenocortical responses to acute ether stress The secretory responses of the H P A axis to 5 min of ether vapor after various periods of chronic intermittent immobilization is depicted in Fig. 1. A l t h o u g h the magnitude of the plasma A C T H response to acute ether stress was similar to that for acute restraint on day 1, a progressive sensitization of the e t h e r response was observed during chronic intermittent immobilization. A highly significant potentiation ( P < 0.001) of the A C T H response to e t h e r was m e a s u r e d on day 10 and day 16 of r e p e a t e d restraint stress c o m p a r e d to the day 1 A C T H response to e t h e r in non-restrained controls (Fig. 1). In contrast, a decline in the ether-stimulated increases in plasma corticosterone levels resulted after exposure to chronic immobilization, which was not statistically significant. T h e r e f o r e , no sensitization of adrenocortical responses to acute ether stress was o b s e r v e d following r e p e a t e d restraint stress (Fig. 1). The ratio of plasma A C T H : c o r t i c o s t e r o n e responses to ether in rats immobilized for 9 or 15 days is significantly increased ( P < 0.05). Since these changes in the A C T H : c o r t i c o s t e r o n e ratio indicate that the secretion of adrenal corticosteroids in response to the very large ether-induced increases in plasma A C T H levels is r e d u c e d on days 10 and 16, the adrenal cortex appears to be desensitized to A C T H (Table I). H o w e v e r , the significant decreases in the ratio of A C T H : c o r t i c o s t e r o n e responses to r e p e a t e d restraint stress alone on those days provides evidence that very low levels of A C T H can stimulate substantial amounts of corticosterone release (Table I). Therefore, adrenocortical responsivity to A C T H r e l e a s e d by immobilization vs. e t h e r is differentially regulated.

CRF receptor regulation during chronic immobilization stress H a b i t u a t i o n of rats to handling has been r e p o r t e d to lower p l a s m a corticosterone concentrations in truncal blood samples 35. H o w e v e r , in two e x p e r i m e n t s the C R F receptor content in the anterior pituitary (440 + 53 fmol/mg) of rats h a b i t u a t e d to 2 min of daily handling

37 TABLE I The effect o f chronic intermittent immobilization stress on body weight and adrenal weight, and A CTH: corticosterone response ratios

Values are the mean + S.E.M. Days o f immobilization stress 0

ANOVA

1

4

10

16

Final body weight (g)

317 + 9

305 + 5

279 + 4*

271 + 3*

254 + 3*

F = 1 9 . 7 ; d f = 4 , 5 2 ; P