Regulation of the hypothalamic pituitary adrenal axis during chronic stress: responses to repeated intraperitoneal hypertonic saline injection

262

Brain Research, 630 (1993) 262-270 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19503

Regulation of the hypothalamic pituitary adrenal axis during chronic stress: responses to repeated intraperitoneal hypertonic saline injection Alexander Kiss, Greti Aguilera

*

Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bldg 10 Rm10N262, Bethesda, MD 20892, USA (Accepted 20 July 1993)

Key words: Repeated stress; Corticotropin releasing hormone; Vasopressin; Paraventricular nucleus; Adrenocorticotropin; Glucocorticoid

Chronic osmotic stress inhibits, while repeated physical stress can increase pituitary ACTH responsiveness to a novel stress. The interaction between these effects was studied in rats subjected to repeated i.p. injection of hypertonic saline, a strong aversive stimulus with osmotic and painful and psychological stress components, for 14 days. Hypertonic saline injection caused marked drinking responses, transient increases in

plasma vasopressin (VP), and marked increases in VP mRNA and irVP in magnocellular cell bodies in the hypothalamus~ Parvicellular activity was also enhanced as indicated by increases in VP immunostaining in the external zone of the median eminence and CRH m R N A and irCRH in the PVN. Plasma ACTH levels increased 10-fold after 30 min hypertonic saline injection, returning to basal levels in 4 h, and there was no desensitization of the ACTH responses after repeated injections (from basal values of 76+ 10 to 782+ 57, 788_+83 and 779 +31 pg/ml 30 min after the first, 4th and 14th injection, respectively). Basal ACTH levels were normal 24 h after the last injection, but pituitary POMC mRNA levels were increased by 95%, and ACTH responses to a novel stress (15 min immobilization) were significantly larger than in controls (P < 0.01) despite increases in morning plasma corticosterone levels (1.5 -+0.4 and 9.2_+3.1 ~tg/dl in controls and stressed rats, respectively). Enhancing the osmotic component of the stress by daily withdrawal of the drinking water for 8 h following the injection, or by administration of 0.9% saline as drinking fluid, did not prevent the increases in CRH mRNA and hypersensitivity of the ACTH response to the novel stress. The data show that physical and psychological components of the stress overcome the inhibitory effect of chronic osmotic stimulation on ACTH secretion, and emphasizes the relationship between parvicellular activation and HPA hypersensitivity during chronic stress.

INTRODUCTION

effect of C R H on A C T H a n d c A M P p r o d u c t i o n in t h e p i t u i t a r y c o r t i c o t r o p h 2'5'19. I m m u n o h i s t o c h e m i c a l stud-

T h e c h a r a c t e r i s t i c s o f t h e r e s p o n s e s o f the h y p o t h a lamic p i t u i t a r y a d r e n a l axis d u r i n g stress vary a c c o r d i n g to t h e type of stress. Several r e p o r t s i n d i c a t e that in chronic stress p a r a d i g m s , such as r e p e a t e d i m m o b i l i z a tion a n d cold exposure, t h e r e is d e s e n s i t i z a t i o n o f the p i t u i t a r y A C T H r e s p o n s e s to t h e p r i m a r y stimulus, b u t h y p e r r e s p o n s i v e n e s s to a novel s u p e r i m p o s e d stress 3'2°-22'38. Similar r e s p o n s e p a t t e r n s a r e o b s e r v e d w h e t h e r t h e chronic stress is a p p l i e d c o n t i n u o u s l y 20,22,38 or i n t e r m i t t e n t l y 21. T h e m e c h a n i s m s r e s p o n s i b l e for the i n c r e a s e d r e s p o n s i v e n e s s a r e n o t fully e l u c i d a t e d , b u t m a y involve t h e c o o p e r a t i v e effect o f C R H and V P from p a r v i c e l l u l a r n e u r o n s o f t h e p a r a v e n t r i c u l a r nucleus of t h e h y p o t h a l a m u s 2'5'19'33'34'36. In rats a n d pri-

ies have shown activation o f t h e p a r v i c e l l u l a r vasop r e s s i n e r g i c system d u r i n g chronic i n t e r m i t t e n t stress, as i n d i c a t e d by progressive i n c r e a s e s in V P c o n t e n t in nerve e n d i n g s o f t h e e x t e r n a l z o n e of the m e d i a n e m i n e n c e 7'12't3. V P is c o l o c a l i z e d with C R H in parvicel-

m a t e s V P is a w e a k s t i m u l a t o r o f A C T H s e c r e t i o n on its own, b u t it m a r k e d l y p o t e n t i a t e s t h e s t i m u l a t o r y

* Corresponding author.

l u l a r n e u r o n s a n d r e c e n t studies have shown t h a t enh a n c e d V P i m m u n o r e a c t i v i t y in the m e d i a n e m i n e n c e o f chronically s t r e s s e d rats is d u e to an i n c r e a s e in t h e n u m b e r of V P positive C R H t e r m i n a l s 6'13'39. In c o n t r a s t to r e p e a t e d i m m o b i l i z a t i o n , chronic osm o t i c s t i m u l a t i o n results in activation o f t h e m a g n o c e l lular, b u t n o t t h e p a r v i c e l l u l a r v a s o p r e s s i n e r g i c system 8'16'17'27'31. D e s p i t e m a r k e d elevations in circulating VP, chronic o s m o t i c s t i m u l a t i o n inhibits, r a t h e r t h a n s t i m u l a t e s t h e h y p o t h a l a m i c p i t u i t a r y axis, with d e c r e a s e s in p i t u i t a r y A C T H r e s p o n s e s to s t i m u l a t i o n by a novel stress o r C R H injection 4'1°'25. T h e s e unexp e c t e d findings have suggested, e i t h e r t h a t V P o f m a g n o c e l l u l a r origin has little i n t e r a c t i o n with t h e a n t e r i o r

263 pituitary, or that osmotic stimulation activates the pro. duction of an inhibitory factor 9. Intrape'ritoneal injection of hypertonic saline, used as a modal of physical and psychological stress, is also a marked osmotic stimulus and has been shown to increase CRH mRNA levels in the paraventricular nucleus of the hypothalamus 23. Therefore, in these studies it was sought to use this aversive stimulus, with physical, psychological and osmotic stress components to study the relative importance of the parvicellular and magnocellular systems on pituitary function by comparing the activity of these systems with the changes in pituitary responsiveness. The results show that repeated i.p. hypertonic saline injection causes activation of the parvicellular system and increased responsiveness of the HPA axis despite the marked osmotic component of the stress. The data is in support of a strong relationship between parvicellular activation and the hypersensitivity of ACTH responses during chronic stress. MATERIAL AND METHODS

Animal procedures and sample collection Male Sprague-Dawley rats (Zivic Miller, Zelienople, PA) weighing 320-350 g were housed 3 per cage in a controlled environment (14 h light; 10 h dark) with free access to food and water. Rats were handled daily for 3 to 7 days prior to the experiment to minimize stress of experimental manipulation and then randomly assigned to control and chronically stressed groups. Rats were subjected to chronic intermittent stress by daily i.p. injections of 5 ml 1.5 M NaCI for 4 to 14 days (7.5 mEq Na/day). Twenty-four hours after the last injection, animals were tested for their HPA axis responses to acute stress and killed by decapitation for blood and tissue collection. To measure the response to acute stress, between 9.00 and 10.00 h, rats were subjected to 15 rain immobilization stress by placing them into a 2.5 x 6 inches plastic restrainers (Harvard Apparatus, South Natick, MA) for 15 min. Trunk blood was collected into ice-cold plastic tubes containing EDTA, centrifuged and plasma stored at - 20°C for ACTH, VP, and corticosterone radioimmunoassaysz°. ACTH and VP were measured in lyophilized eluates after extraction onto C-18 Sep-Pak cartridges (Waters Associates, Inc., Milford, MA), and elution with 60% acetonitrile in triethylamine formate buffer, pH 3.2. RIA for ACTH and VP were performed using a specific Nterminal antibody (IgG-ACTH-1, IgG Corp., Nashville, TN) and a VP antiserum kindly provided by Dr. G. Rougon (LCB, NIMH,Bethesda, MD), respectively. The IzsI-tracers were prepared by iodogen radioiodination of ACTH 1-39 and AVP and purified by HPLC. Immediately after death, brains were removed and fixed in paraformaldehyde for VP and CRH immunohistochemistry, or frozen in isopentane at -40°C for VP and CRH in situ hybridization. Pituitaries were individually frozen in dry ice for POMC mRNA, or collected in ice-cold PBS for CRH receptor determination.

When required for immunohistochemical study, rats were injected i.c.v, with 20 /zg colchicine under kethamine/xylazyne anesthesia, 6 h before sacrifice.

Immunohistochemistry Immediately after sacrifice, brains were removed and fixed for 1 h in 4% paraformaldehyde in 0.1 M acetate buffer, pH 6.5, at 4°C and then for an additional 20 h in 4% paraformaldehyde in 0.1 M borate buffer, pH 9.5. Immunohistochemical detection of CRH and VP was performed on 40 /zm thick free floating sections of the hypothalamic region using specific antibodies raised in rabbits (GA13 for CRH and a VP antibody kindly provided by Dr G. Rougon, Laboratory of Cell Biology, NIMH, NIH) and avidin-biotin peroxidase reagents from Vectostain ABC kits (Vector, Burlingame, CA) as previously describedz6. After staining, sections were mounted on glass slides and the density of the immunostaining was compared by visual examination using a Zeiss light microscopy, and semiquantitated using a computerized image analysis system (Image Research Inc., Ontario, Canada).

In situ hybridization Coronal 12 tzm sections of the hypothalamic region of the brain were obtained in a cryostate at -18°C, thaw-mounted in double coated gelatin slides, fixed with formaldehyde and acetylated. Hybridization was performed as previously described23, using [35S]deoxy-ATP labelled 48-mer oligonucleotides. Oligonucleotides complementary to the bases of amino aci~ls 22 to 37 rat/human proCRH, and 14 carboxyterminal amino acids of rat AVP-neurophysin synthesized by Synthecell, Rockville, MD. The specific activity of probes ranged from 1-3 x 10/12 dpm//zM. In each experiment, all control and experimental sections were processed in the same hybridization reaction and exposed to Hyperfilm-3H (Amersham), together with 3sS-labelled brain paste standards. The optical density of the autoradiographs were quantitated using a computerized image analysis system (Imaging Research Inc., Ontario, Canada), and the results expressed as cpm/mg, after subtracting the background values. Comparison between the different groups was performed from the values at least 4 sections per animal in a minimum of 5 rats per group• Selected sections for the detailed localization of silver grains were processed for emulsion dipping and evaluation under the Zeiss light microscope.

Statistic analysis Data are presented as the"tnean and standard error of the values in the number of observati6ns indicated in results. Unless otherwise indicated, statistical significance of the differences between the experimental groups were determined by analysis of variance by Scheffe's F test for multiple groups comparisons or unpaired t-test. RESULTS

General effects of acute and repeated i.p. hypertonic saline injection Administration of i.p. hypertonic saline was followed by a rapid drinking response, initiated 2 to 5 min

TABLE I

Effect of a single i.p. hypertonic saline injection on plasma osmolarity and levels of VP, ACTH and corticosterone Time after i.p. HS injection Plasma osmolarity (mOsm/kg) Vasopressin (pg/ml) ACTH (pg/ml) Corticosterone (/zg/dl)

0 (basal)

30 min

304.8+ 1.0 0.8+0.1 67.6 5:4.0 1.5 + 2.8

339.15:2.8 39.75:4.1 681.9 5:77.8 39.0 5:2.8

4h * * * *

305.25:3.1 # 1.05:0.4 ~ 251.0 + 17.9 * '# 10.8 5:4.0 * "# e

* P < 0.01 vs. basal; ~ P < 0.01 vs. 30 min. ,

264 1500 A

] ]

BASAL

i.p.HS

lOOO

500

o

~,

60 m

..=o,

40

o

20

*

o

0

1 day 4 day 14 day Fig. 1. Plasma ACTH (upper panel) and corticosterone (lower panel) levels 30 min after a single (1 day) or repeated (4 and 14 days) i.p. hypertonic saline injection. Bars are the mean and SE of values obtained from 6 rats per group. * P < 0.001 vs. basal; *P < 0.01 vs. 1 day

after the injection, with ingestion of 3.9 + 0.92 m l / 1 0 0 g BW vs. 0.4 + 0.12 m l / 1 0 0 g BW in controls, during the first 2 h after injection. Plasma osmolarity and VP levels were increased after 30 min but returned to basal levels 4 h after the injection (Table I). Similarly, plasma A C T H and corticosterone levels were markedly elevated 30 min after injection and returned to basal at 4 h (Table I). The plasma A C T H and corticosterone responses to repeated i.p. hypertonic saline injection for 4 and 14 days are shown in Fig. 1. Basal plasma A C T H levels

CONTROL

after 3 (day 4) and 13 (day 14) days injection were not significantly different from non-injected controls (93.7 + 12.9, 85.9 + 6.0 p g / m l and 51.5 + 1.3, respectively), but basal plasma corticosterone was significantly elevated in the morning of days 4 and 14 (5.0 + 0.8 and 9.2 + 3.1 / x g / d l compared with 1.47 + 0.4 ~ g / d l in controls). The increases in plasma A C T H levels 30 min after injection on days 4 and 14 were similar to those in naive rats (782 + 57, 788 + 83 and 779 + 31 p g / m l after the first, 4th and 14th injection, respectively). Plasma corticosterone also increased to similar levels 30 min after the first, 4th or 14th injection (44.2 + 3.2, 48.6 + 1.3 and 53.1 + 1.1 /xg/dl, respectively). Body weight decreased by 22% after 14 days hypertonic saline injection (402 + 11 and 312 + 6 g, in controls and stressed rats, respectively). Adrerial weight increased from 13.1 + 0.2 to 21.0 + 0.7 m g / 1 0 0 g body weight. Thymus weight was reduced from 702 + 21 to 523 + 41 mg, however when expressed per body weight the change was not significant. In additional groups of rats the osmotic component of the stress was prolonged by replacing the drinking water with normal saline solution or by restricting water intake for 8 h following the injection. In these conditions, plasma osmolarity remained significantly elevated 4 h after i.p. injection (from a control value of 303 + 2.7 to 316 + 2.9 and 320 + 3.9 m O s m / k g in saline drinking and water restricted rats, respectively). In the 8 h water-restricted group, plasma VP levels increased from 0.8 + 0.1 p g / m l to 4.4 + 0.7 and 3.9 + 0.3 p g / m l , 4 and 8 h after i.p. injection, but returned to normal after 24 h. In the saline drinking group, plasma VP was increased to 3.4 + 0.4 p g / m l 4 h after i.p. injection, but not different from controls after 24 h.

i.p. HYPERTONIC SALINE

Fig. 2. In situ hybridization of CRH mRNA and VP mRNA in the PVN of controls and rats injected for 14 days with hypertonic saline i.p. The figure is representative of the results obtained in 4 sections of 5 rats per group.

265

Expression of VP and CRH in the hypothalamus Repeated once daily i.p. administration of hypertonic saline for 14 days resulted in a 25 + 3.3% increase in VP mRNA (P < 0.01) in the PVN of the hypothalamus (Figs. 2 and 3). The SON was not consistently included in all sections, but in the sections analyzed showed higher VP mRNA levels in rats injected with hypertonic saline (not shown). As shown in Fig. 3, the increase in VP mRNA in the PVN after 14 days hypertonic saline injection was significantly enhanced in the rats in which the osmotic stimulation was prolonged by replacing the drinking water by normal saline solution (37 + 3.5%,), but not by 8 h water restriction (11 5: 2.6%). The effects of repeated hypertonic saline injection on VP immunostaining in the PVN, SON and median eminence without colchicine treatment are shown in Fig. 4. Consistent with the increases in VP mRNA, after fourteen days i.p. hypertonic saline there was an apparent increase in VP immunostaining in the PVN and SON of the hypothalamus. This was evident in the absence (not shown) or in the presence of colchicine treatment (Fig. 4A, D), from the increases in the number of stained magnocellular cell bodies and the intensity of the immunostaining. The intensity of the staining in the internal zone of the median eminence was not different from controls. However, in hypertonic

200

~FO

100 E

A -6

.~ g

200

qtto

100

C

HS

HS+NS

HS+WD

Fig. 3. Effect of 14-days i.p. hypertonic saline injection on C R H and VP m R N A levels in the P V N in rats drinking water (HS), drinking 0.9% NaCI ( H S + N S ) or subjected to water restriction for 8 h ( H S + W D ) after the i.p. injection. Data are the m e a n and SE of values obtained by in situ hybridization in 4 sections of 5 rats per group. * P < 0.01 vs. controls (C); • P < 0.05 vs. HS.

CONTROL

i.p. HYPERTONIC SALINE

Fig. 4. Effect of 14 days hypertonic saline injection on the content of immunoreactive VP in the PVN, SON and median eminence. Panels A to D are representative of results in serial coronal sections of the hypothalamic region of 6 rats treated with colchieine. E and F are representative of observations in 8 median eminence sections corresponding to 8 rats non-treated with colchicine.

saline injected rats, VP immunostaining in parvicellular terminals of the external zone, analyzed in 8 sections throughout the median eminence in 8 rats, was markedly increased compared with controls (Fig. 4). Injection of colchicine, 75 /zg i.c.v.) caused marked depletion of irVP from the external zone of the median eminence. However, it was not possible to detect increases in immunostaining in parviceIlular cell bodies of the PVN in the presence or in the absence of colchicine (not shown). CRH mRNA in the PVN was increased by 35 + 4.7 after 14 days hypertonic saline injection (P<0.05) (Figs. 2 and 3). To determine whether enhancement of the osmotic component of the stress can reveal the recognized inhibitory effect of osmotic stimulation on hypothalamic CRH expression, CRH mRNA levels were measured in rats receiving hypertonic saline injection for 14 days while subjected to partial water deprivation or saline intake. As shown in Fig. 3, lower panel, prolonging the osmotic stimulation did not prevent the increases in CRH mRNA observed after 14 days hypertonic saline injection (35 + 4.7, 34 5:4.6 and 73 5: 3.5% in controls drinking water, rats drinking normal saline and 8 h water restriction, respectively). The increase in CRH mRNA levels induced by hypertonic saline injection in rats subjected to 8 h daily water restriction was significantly higher than those in the groups with access to water or isotonic saline (P < 0.05).

266

-

~

..'~,,..

i

A

B

Fig. 5. Effect of repeated i.p. hypertonic saline injection on irCRH in the PVN (A and B) and median eminence (C and D). irCRH in PVN cell bodies was measured 8 h after i.c.v, injection of 2 0 / z g colchicine. Figures are representative of the observations in 16 sections from 4 rats per group for the PVN and of 8 sections throughout the median eminence in 4 rats.

In the absence of colchicine, irCRH in cell bodies of the PVN was almost undetectable in both controls and repeatedly injected rats (not shown). Similarly, CRH immunostaining in the external zone of the median eminence was comparable in both groups of rats (Fig. 5C,D). However, consistent with the marked increase in CRH mRNA, 8 h after i.c.v, injection of 20 /xg of colchicine used as a stressor, the apparent content of irCRH measured by immunohistochemistry was higher in the group receiving repeated hypertonic saline injection (Fig. 5A,B), indicating that the capacity of parvicellular neurons to synthesize CRH is increased.

HPA responses to novel stimuli To study the responsiveness of the HPA axis to a novel stimulus, plasma ACTH and corticosterone responses to acute immobilization were measured in controls and chronically stressed rats, 24 h after the last hypertonic saline injection. As shown in Fig. 7 and

200 [] []

%

Effect of hypertonic saline injection on POMC mRNA and CRH receptors As shown in Fig. 6, hypertonic saline injection for 14 days caused a marked, 95% increase in POMC mRNA in the anterior pituitary lobe (5,000 + 450 and 9,750 + 1,500 c p m / m g for controls and stressed groups, respectively). In contrast, POMC mRNA levels were unchanged in the intermediate pituitary lobe. The effects of repeated hypertonic saline injection for 14 days on pituitary CRH receptors are shown in Table II. In three experiments, Scatchard analysis of the binding data revealed a 33% decrease in CRH receptor concentration after 14 days hypertonic saline injection (P < 0.01, n = 3). Receptor affinity (K d) was similar in both groups.

Controt Hypertonic Saline

1O0

x

E

m

E O

o_

T A B L E II

Effect of i.p. hypertonic saline injection for 14 days on anterior pituitary CRH receptors Anterior Pituitary

Intermediate Pituitary

Fig. 6. Effect of 14 days i.p. hypertonic saline injection on P O M C m R N A levels in the anterior and intermediate pituitary lobes measured by in situ hybridization. Bars are the m e a n and S.E. of values obtained in 18 to 22 pituitary sections of 5 rats per group. * P < 0.001 vs. control

Experimental condition Controls Hypertonic saline injection (14 days) • P < 0.01 compared with controls.

[ ~251]Tyr o_CRH binding B,,,~x (fmol / rag)

K a (nM)

567 _+9.5 380 _+4.3 *

1.6 + 0.2 1.3 + 0.1

267 3000 ~ ] -r
ontrol

++#

Acu t e stress

2000

1000

0

*# tu Z o ul Io _t 2 Io

60 40 20-

I.p. NORMAL SALINE

l.p. HYPERTONIC SALINE

Fig. 7. Plasma A C T H (upper panel) and corticosterone (lower panel) levels in response to 15 rain immobilization stress in control rats and rats injected with hypertonic saline i.p. for 14 days (n = 8 in each group); * P < 0.001 vs. basal; # P < 0.01 vs. control; + P < 0.05 vs. basal saline control.

Table III, in hypertonic saline injected rats, 24 h after the last injection, basal 'plasma ACTH levels were similar to those in control rats. However, the responses to 15 min immobilization were significantly elevated in the chronically stressed group (Fig. 7, upper panel, Table 3). Despite the normal basal plasma ACTH levels, 24 h after the last injection, basal plasma corticosterone levels were significantly elevated compared with controls (P < 0.05). Similar to ACTH, the increases in plasma corticosterone following 15 min immobilization were significantly higher in the chronically stressed group. As shown in Table III, the increased pituitary ACTH responses to acute immobilization were not prevented by enhancing the osmotic component of the chronic stress. Basal plasma ACTH levels and the responses to acute immobilization stress were similar in rats receiving water or normal saline (Table III).

Twenty-four hours after the last hypertonic saline injection, basal plasma corticosterone was increased in both groups with respect to normal controls, an effect which was more marked in the group drinking saline. Similar to ACTH, the increases in plasma corticosterone following 15 min immobilization were significantly increased in the chronically stressed groups (Fig. 7, Table III). In an additional experiment, increasing the duration of the osmotic stimulation by daily water restriction for 8 h after the injection also failed to prevent the enhancement of the response to acute immobilization stress. In control rats, plasma ACTH levels increased from a basal value of 70.1 + 4.1 (n -- 6) to 786.3 + 71.6 p g / m l (n = 7) after 15 min immobilization, whereas in the hypertonic saline injected group increased from 89.9 + 12.3 (n --- 5) to 1201.3 + 166.4 p g / m l (n = 7), a response significantly higher than in controls (P < 0.05). Basal plasma corticosterone levels were increased from 0.75 + 0.01 in controls to 4.4 + 2.7 in the hypertonic saline injected group. The absolute increases in plasma corticosterone in response to immobilization were also higher in the hypertonic saline injected group (49.5 -/4.6 /zg/dl compared with 38.6 + 4.6 in controls, P < 0.05). However, the increase was not apparent when data were expressed as increase over basal. DISCUSSION

This study provides further information on the diversity of the responses of the HPA axis during adaptation to chronic stress. Previous studies have shown that while osmotic stress inhibits hypothalamic and pituitary responses 4't°'25, several types of physical and psychological stress cause transient hypothalamic and pituitary activation and increased ACTH responsiveness to a novel stress despite sustained elevations in plasma glucocorticoid levels 3'2°-22'38. In the present stress model, with physical-psychological and osmotic components, there is no desensitization of the ACTH response to repeated exposure to the stress, and as in other models of physical or psychological stress, the responses to a

T A B L E III

Plasma ACTH and corticosterone responses to 15 min immobilization in rats injected with hypertonic saline i.p. for 14 days (HS) while receiving water or 0.9% NaCI as drinking fluid. Data represents the mean and S.E.of the values obtained in the number of rats indicated in parenthesis Experimental condition

Plasma ACTH (pg / ml) Basal

Stress

Plasma corticosterone (Iz g / dl) Basal Stress

Controls HS drinking water HS drinking 9% NaC!

78.7+ 4.5(16) 102.4+ 8.8(17) 113.8+ 14.1 (9)

1045+146 * (13) 1984+184 *'# (11) 2638+291 *'# (10)

1.9+0.9 (16) 6.3+1.3 * (24) 17.1+3.5 . , o (9)

• P < 0.01 vs. basal; # P < 0.01 vs. controls; o p < 0.05 vs. water drinking.

41.4+3.0 * (13) 59.7+4.7 *'# (13) 52.8+4.5 *'# (10)

268 novel stress are enhanced. Although the increases in plasma osmolarity and VP after the injection are transient, the marked increases in VP mRNA and irVP following repeated injection indicate a strong osmotic component in this stress paradigm. Since osmotic stress is known to inhibit CRH mRNA and ACTH responses to a novel stress 4'1°'25'43, it was unexpected to find that increasing the duration of the osmotic stimulus by water restriction or saline intake, enhanced rather than prevented the stimulatory effects of hypertonic saline injection on CRH mRNA and ACTH secretion. Moreover, previous studies 29 and the present experiments show that repeated hypertonic saline injection, not only did not decrease, but caused a sustained increase in CRH mRNA levels in the PVN. Since predominantly psychological stress paradigms, such as repeated immobilization, cause desensitization of the ACTH responses and transient increases in CRH mRNA21'23, it is likely that the sustained responses in the present model are due to the physical component of the stress. Further studies will be needed to determine the mechanism by which the physical component of the stress overcomes inhibitory effect of the osmotic stimulation. The mechanism by which chronic stress increases the pituitary sensitivity to a novel stress is still unclear and probably involves diverse neural pathways, hypothalamic and pituitary changes. As in other chronic stress paradigms, the increased pituitary POMC mRNA levels and secretory responses in hypertonic saline injected rats occur despite sustained elevations in circulating corticosterone, suggesting an alteration in the feedback mechanism. It has been shown that in cultured pituitary cells from chronically stressed rats, glucocorticoids are stimulatory rather than inhibitory for ACTH release 18'4z, suggesting functional changes in the interaction of the glucocorticoid receptor with the regulatory elements of the POMC gene. In this regard there is evidence that a number of transcription factors can modulate the genomic effects of the glucocorticoid receptor ~5. The expression of some of these factors, such as the proto-oncogene c-jun is stimulated by protein kinase C, which mediates the effects of VP. There is also evidence indicating that VP prevents the inhibitory effect of glucocorticoids on CRH-stimulated ACTH release in cultured pituitary cells 1. In the present experiments, the presence of elevated levels of irVP in the external zone of the median eminence in rats with repeated i.p. saline injection suggests activation of parvicellular VP expression. The fact that irVP pools in the external zone of the median eminence are rapidly depleted following acute stress or colchicine administration, indicates that it is unlikely that the increase in irVP is due to decreased peptide

release. The inability to detect any increase in irVP content in parvicellular cell bodies, was probably due to the low sensitivity of the immunohistochemical procedure employed, since other investigators using immunofluorescent techniques have shown increases in parvicellular VP after repeated immobilization after 24 h colchicine treatment H. Since activation of the parvicellular vasopressinergic system is a consistent finding in chronic stress paradigms with increased pituitary ACTH responsiveness to novel stimuli 3'12'~3, it is probable that increased exposure of the pituitary corticotroph to VP contributes to the sensitization of the ACTH responses to a novel stress. On the other hand, it is unlikely that the increase in parvicellular vasopressin is responsible for the lack of desensitization to the repeated injection, because similar activation of the system is observed in stress paradigms, such as repeated immobilization in which there is rapid desensitization to the primary stress 3'2°'21. On the other hand, it is possible that the acute increases in magnocellular VP secretion following each injection may contribute to the preservation of the ACTH responses. In contrast to chronic osmotic stimulation which inhibits pituitary ACTH responses, acute increases in plasma VP following saline infusion have been shown to enhance the increases in plasma ACTH in response to CRH infusion3°. As shown by the present experiments and previous reports 29, repeated hypertonic saline injection causes marked and sustained increases in CRH mRNA levels in parvicellular neurons, a response not usually observed in stress paradigms showing the characteristic desensitization of the ACTH responses to the primary repeated stress. Also, the higher increase in irCRH after colchicine administration following 14 days repeated hypertonic saline injections indicate that the capacity of the PVN cells to synthesize CRH is increased and suggest that the release of the peptide to the portal circulation is increased. Therefore, it is possible that an increase in hypothalamic CRH may account for the lack of desensitization to the primary stress. However, it has been shown that increasing pituitary exposure to CRH or CRH plus VP by continuous or intermittent administration of the peptides results in desensitization rather than enhancement of the pituitary ACTH responses in rats 24'35'37'41. Although the circulating levels of CRH and VP in the latter experiments are similar to those reported in pituitary portal blood 32, it is possible that a different pattern of secretion of the endogenous peptides during stress accounts for increasing corticotroph responsiveness. The lack of desensitization to repeated injections could also involve the participation of other factors,

269 such as enkephalins, which expression in the hypothalamus is markedly increased in this, but not other chronic stress models 23'28. A lack of desensitization of pituitary AC-qT-I responses to repeated stimulation has also been reported after repeated insulin hypoglycemia in the rat ~2. In this model, parvicellular VP is increased and there are biphasic changes in irCRH with a decrease after 7 days and latter restoration to basal levels 12. The extent to which insulin hypoglycemia and hypertonic saline injection cause any common responses in plasma VP, enkephalin mRNA, CRH mRNA or irCRH expression or others, which may explain the conserved ACTH responses remain to be elucidated. As observed in adrenalectomy and other stress paradigms, in the present experiments there was no relationship between pituitary ACTH responsiveness and changes in pituitary CRH receptors 3A4'4°. This indicates that post receptor events and interaction of CRH with other regulators are more likely to determine the corticotroph responsiveness. Several factors may contribute to the pituitary CRH receptor downregulation in the present experimental model. These include CRH, VP and glucocorticoids, which secretion is increased in hypertonic saline injected rats and are known to induce pituitary CRH receptor downregulation. In conclusion, the responses of the HPA axis to repeated exposure to a combined physical-psychological and osmotic stress are characterized by marked and sustained activation of the hypothalamic parvicellular system and hypersensitivity of the pituitary ACTH responses to a novel stress, without desensitization to the repeated primary stress. Although the mechanism by which physical stress overcomes the inhibitory effect of osmotic stimulation on the HPA axis, the data provides further evidence for a strong relationship between activation of the parvicellular vasopressinergic system and increased corticotroph responsiveness during chronic stress. REFERENCES 1 Abou-Samra, A.-B., Catt, K.J. and Aguilera, G., Biphasic inhibition of adrenocorticotropin release by corticosterone in cultured anterior pituitary cells, Endocrinology, 119 (1986) 972-977. 2 Abou-Samra, A.-B., Harwood, J.P., Catt, K.J. and Aguilera, G., Mechanisms of action of CRF and other regulators of ACTH release in pituitary corticotrophs, Ann. NYAcad. Sci., 512 (1987) 67-84. 3 Aguilera, G., Kiss, A., Hauger, R.L. and Tizabi, Y. Regulation of the hypothalamic-pituitary-adrenal axis during stress: role of neuropeptides and neurotransmitters. In R. Kvetnansky, R. McCarty and J. Axelrod (Eds.) Stress: Neuroendocrine and Molecular Approaches, Gordon and Breach, New York, 1993, pp. 365-381. 4 Aguilera, G., Lightman, S.L. and Kiss, A., Regulation of the

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