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Graded footshock stress elevates pituitary cyclic AMP and plasma β-endorphin, β-LPH, corticosterone and prolactin
Life Sciences, Vol. 33, pp. 2657-2663 Printed in the U.S.A.
Pergamon Press
G R A D E D FOOTSHOCK STRESS ELEVATES P I T U I T A R Y C Y C L I C AMP AND P L A S M A 13-ENDORPHIN, 13-LPH, CORTICOSTERONE AND P R O L A C T I N G. Jean Kant, Edward H. Mougey, Lee L. Pennington and James L. M e y e r h o f f N e u r o e n d o c r i n o l o g y and N e u r o c h e m i s t r y Br., Dept of Med Neurosciences, D i v o f N e u r o p s y c h i a t r y , Walter Reed A r m y I n s t i t u t e f o r Research l, Washington DC 20307.
(Received in final form October
12, 1983)
Summary Male rats w e r e subjected to 15 min o f various i n t e n s i t i e s of f o o t s h o c k c u r r e n t (0.0, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2mA) on a v a r i a b l e i n t e r v a l schedule w i t h an average i n t e r s h o c k i n t e r v a l of 30 sec (30 shocks/15 min session). Each shock lasted 5 sec. Animals w e r e s a c r i f i c e d i m m e d i a t e l y a f t e r being r e m o v e d f r o m the shock box. Two s i m i l a r studies w e r e c o n d u c t e d . In the f i r s t e x p e r i m e n t , rats were s a c r i f i c e d by m i c r o w a v e i r r a d i a t i o n and p i t u i t a r y cyclic AMP levels were d e t e r m i n e d . In the second study, rats w e r e d e c a p i t a t e d and plasma hormones (prolactin, c o r t i c o s t e r o n e , 13endorphin, 8-LPH) were measured by r a d i o i m m u n o a s s a y . Although all b i o c h e m i c a l indices of stress measured increased as shock i n t e n s i t y increased, some d i f f e r e n c e s among t h e substances measured w e r e observed w i t h respect to t h r e s h o l d i n t e n s i t y , range of p r o p o r t i o n a l response and m a x i m a l response. We have previously r e p o r t e d t h a t a v a r i e t y of stressors including r e s t r a i n t , f o r c e d running, s.c. f o r m a l i n , c o n d i t i o n e d p s y c h o l o g i c a l stress, and f o o t s h o c k e l e v a t e d levels of p i t u i t a r y c y c l i c AMP as w e l l as plasma c o r t i c o s t e r o n e and plasma p r o l a c t i n (1-5). We have p o s t u l a t e d t h a t this p i t u i t a r y c y c l i c AMP increase may be i n v o l v e d in t h e r e g u l a t i o n of p i t u i t a r y h o r m o n a l o u t p u t in response t o stress. Although the e l e v a t i o n in p i t u i t a r y c y c l i c AMP in response to d i f f e r e n t stressors appeared to c o r r e l a t e w i t h the a p p a r e n t " s e v e r i t y ' of t h e stressor, t h e ranking of stressors was c l e a r l y s u b j e c t i v e . We t h e r e f o r e decided t o f u r t h e r c h a r a c t e r i z e the p i t u i t a r y c y c l i c AMP response t o stress using a stressor whose i n t e n s i t y could be m a n i p u l a t e d o b j e c t i v e l y and t o include 13endorphin and 13-LPH responses as w e l l as c o r t i c o s t e r o n e and p r o l a c t i n . An a d d i t i o n a l o b j e c t i v e was to c o m p a r e the response of these h o r m o n a l indices of stress f o l l o w i n g exposure t o the same graded stressor w i t h regard t o s e n s i t i v i t y , range of response and m a x i m a l response. Two p a r a l l e l studies w e r e p e r f o r m e d to p e r m i t s a c r i f i c e by e i t h e r m i c r o w a v e i r r a d i a t i o n (cyclic AMP e x p e r i m e n t ) or d e c a p i t a t i o n (plasma hormones). Methods Animals.
Male (250-350 g) S p r a g u e - D a w l e y rats w e r e purchased f r o m
Taconic
1The views of the author(s) do not p u r p o r t t o r e f l e c t t h e position of the D e p a r t m e n t of the A r m y or the D e p a r t m e n t of Defense, (para 4-3, AR 360-5).
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Farms 2 Rats w e r e i n d i v i d u a l l y housed under c o n d i t i o n s of c o n t r o l l e d l i g h t (lights on f r o m 700 to 1900) and t e m p e r a t u r e w i t h f o o d and w a t e r f r e e l y a v a i l a b l e . Rats w e r e handled f o r 4 days p r i o r to the e x p e r i m e n t t o m i n i m i z e n o n - s p e c i f i c stress e f f e c t s . Rats t o be s a c r i f i c e d by m i c r o w a v e i r r a d i a t i o n were p r e v i o u s l y h a b i t u a t e d t o t r a v e r s i n g an o p e n ended p l a s t i c c y l i n d e r s i m i l a r t o t h e p l a s t i c m i c r o w a v e a p p l i c a t o r tube. Footshock. Constant c u r r e n t s c r a m b l e d shock was d e l i v e r e d t o t h e f l o o r bars of an o p e r a n t box on a v a r i a b l e schedule. Shock d u r a t i o n was 5 sec and t h e i n t e r s h o c k i n t e r v a l averaged 30 sec. Shock i n t e n s i t y was v a r i e d b e t w e e n 0.0 and 3.2 mA of c u r r e n t as s p e c i f i e d for each e x p e r i m e n t . Each rat r e c e i v e d only one shock i n t e n s i t y during the e n t i r e session and shock i n t e n s i t i e s were d i s t r i b u t e d t h r o u g h o u t the e x p e r i m e n t t o randomize circadian effects. One group of animals was s a c r i f i c e d in each e x p e r i m e n t i m m e d i a t e l y upon r e m o v a l f r o m t h e i r home cage w i t h o u t being exposed t o t h e shock box envi r o n m e n t ( c o n t r o l s ) . B e h a v i o r a l stress observer who did not i n d i c a t e d no response score of 2 i n d i c a t e d vocalizing. Behavioral
response. Rats w e r e observed during f o o t s h o c k and scored by an know which shock i n t e n s i t y was being d e l i v e r e d . A score of 0 during shock " o n ' , a score of 1 i n d i c a t e d n o t i c e a b l e flinching, a a c t i v e jumping and a score of 3 i n d i c a t e d a c t i v e jumping and ratings w e r e only made f o r e x p e r i m e n t 1.
Sacrifice. In the f i r s t study, rats were s a c r i f i c e d by m i c r o w a v e i r r a d i a t i o n t o p r e v e n t p o s t - m o r t e m d e g r a d a t i o n of c y c l i c AMP (6,7). Since m i c r o w a v e i r r a d i a t i o n hemolyzes blood which can i n t e r f e r e w i t h the assay f o r 6-LPH and 13-endorphin, we p e r f o r m e d a second e x p e r i m e n t using d e c a p i t a t i o n t o measure 13-endorphin and 13-LPH. Plasma c o r t i c o s t e r o n e and plasma p r o l a c t i n were measured in both e x p e r i m e n t s . The m i c r o w a v e m e t h o d e m p l o y e d 5 sec of high p o w e r i r r a d i a t i o n using a m o d i f i e d Varian PPS-2.5 power g e n e r a t o r w i t h an o u t p u t of 2.5. KW at a f r e q u e n c y of 2450 MHz. F o l l o w i n g i r r a d i a t i o n , the rats w e r e d e c a p i t a t e d and t r u n k blood was c o l l e c t e d for m e a s u r e m e n t of c o r t i c o s t e r o n e and p r o l a c t i n . The heads w e r e c o o l e d on d r y ice and t h e p i t u i t a r i e s w e r e dissected, w e i g h e d and s o n i c a t e d in 1 rnl of 50 mM sodium a c e t a t e buffer, pH 6.2. The sonicates were c e n t r i f u g e d at 12,000 g for 15 rnin and t h e supernatants s t o r e d at -70 ° u n t i l assayed f o r c y c l i c AMP. In the second e x p e r i m e n t , rats w e r e s a c r i f i c e d by d e c a p i t a t i o n and t h e t r u n k blood was c o l l e c t e d in h e p a r i n i z e d beakers. F o l l o w i n g c e n t r i f u g a t i o n , plasma was t r a n s f e r r e d t o tubes c o n t a i n i n g A p r o t i n i n (0.45 T I U / t u b e ) and stored at -35 ° u n t i l assayed for plasma 13-endorphin, 6-L PH, p r o l a c t i n and c o r t i c o s t e r o n e . Assay Procedures. C y c l i c AMP was d e t e r m i n e d by r a d i o i m m u n o a s s a y (8,9). A h i g h l y specific antisera was used at a f i n a l d i l u t i o n of 1:400,000. The a n t i s e r u m e x h i b i t e d c r o s s - r e a c t i v i t i e s f o r ATP and c y c l i c GMP of less than 0.00007% and 0.14% r e s p e c t i v e l y . she samples w e r e assayed in t r i p l i c a t e and assay d a t a w e r e a n a l y z e d by c o m p u t e r (10). Within assay v a r i a t i o n was 7% and b e t w e e n assay v a r i a t i o n was 18%. M a t e r i a l s f o r the p r o l a c t i n assay w e r e p r o v i d e d by the N a t i o n a l I n s t i t u t e of H e a l t h through the Rat P i t u i t a r y H o r m o n e D i s t r i b u t i o n Program. P r o l a c t i n was r a d i o i o d i n a t e d as p r e v i o u s l y described (8). C o r t i c o s t e r o n e was measured using an a n t i b o d y produced in our l a b o r a t o r y in rabbits (11).
2In c o n d u c t i n g the research described in this r e p o r t , the i n v e s t i g a t o r ( s ) adhere t o the 'Guide f o r the Care and Use of L a b o r a t o r y Animals', as p r o m u l g a t e d by the C o m m i t t e e on Care and Use of L a b o r a t o r y Animals of t h e I n s t i t u t e of L a b o r a t o r y A n i m a l Resources, N a t i o n a l Research Council.
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For the assay of 13-endorphin and 6 - L P H , plasma samples w e r e t h a w e d and r e c e n t r i f u g e d at 12,000 g for 10 min. A 0.5 ml a l i q u o t was e x t r a c t e d w i t h 75 mg PrepPak 500/C18 (Waters Associates Inc.). The C18 absorbant was rinsed t h r e e t i m e s w i t h I ml o f d i s t i l l e d H 2 0 . A p o r t i o n of t h e 13-LPH f r a c t i o n was e l u t e d f r o m t h e absorbant w i t h t w o 1 ml rinses of a c e t o n e : w a t e r (1:1). These rinses w e r e c o m b i n e d , e v a p o r a t e d and assayed f o r i3-LPH c o n t e n t , using the 13-endorphin a n t i b o d y . R e c o v e r y studies of human 13-LPH and 13-endorphin added t o r a t plasma in v a r y i n g c o n c e n t r a t i o n s showed t h a t a p p r o x i m a t e l y 20% of t h e 13-LPH added was r e c o v e r e d in this f r a c t i o n w h i l e o n l y 5% of added 13-endorphin was e l u t e d at this step. The absorbant was then rinsed t w i c e w i t h 1 ml o f a c e t o n e : H 2 0 : T F A (80:19:1) which e l u t e d 60% of B-endorphin and 5% o f B-LPH. Similar r e c o v e r i e s of c a m e l 13-endorphin added t o rat plasma w e r e o b t a i n e d . E x t r a c t i o n of 0.4 t o 1.0 ml of r a t plasma produced l i n e a r results in the r a d i o i m m u n o a s s a y . The a n t i b o d y used f o r assay of 13-endorphin/13-L PH was produced in r a b b i t s a g a i n s t a c o n j u g a t e of human 13-endorphin and b o v i n e serum a l b u m i n . This a n t i b o d y , used at a f i n a l d i l u t i o n of 1:20,000, c r o s s - r e a c t e d 100~o (on a m o l a r basis) w i t h human 13-LPH and 95% (on a m o l a r basis) w i t h c a m e l B-endorphin but e x h i b i t e d no measurable cross r e a c t i o n w i t h ~e n d o r p h i n or t h e enkephalins. Although not s p e c i f i c a l l y tested, N - a c e t y l a t e d B-endorphin would be e x p e c t e d t o c r o s s - r e a c t w i t h this a n t i b o d y since t h e b i n d i n g d e t e r m i n a n t s a r e in the C - t e r m i n a l half of t h e m o l e c u l e . Human 13-endorphin was r a d i o i o d i n a t e d using c h l o r a m i n e - T and N a l ~ S l ( A m e r s h a m C o r p o r a t i o n ) . Sodium m e t a b i s u l f i t e was used t o s t o p t h e r e a c t i o n . T h e i o d i n a t e d p r o d u c t was p u r i f i e d on a 1.4 x 36 cm. c o l u m n of Sephadex G-2S, f i n e ( P h a r m a c i a Fine Chemicals) w i t h a solution of 0.25% bovine serum a l b u m i n in 0.5% a c e t i c acid as e l u e n t . Human IB-endorphin was used as standard. Free and bound antigen w e r e separated, f o l l o w i n g a four day, 4 ° C i n c u b a t i o n , w i t h d e x t r a n - c o a t e d c h a r c o a l and the bound f r a c t i o n was c o u n t e d in an LKB Rack G a m m a II. A l l values r e p o r t e d are c o r r e c t e d f o r r e c o v e r y using r e c o v e r y values o b t a i n e d on a separate plasma pool run in the same assay. The c o e f f i c e n t s of v a r i a t i o n f o r s e p a r a t e e x t r a c t i o n s f r o m the same rat plasma pool w e r e 20% for 13-LPH and 6% for 13-endorphin. The assay is s e n s i t i v e t o 3 pg of added 13-endorphin per tube (10 p g / m l plasma) and 9 pg of 13-L PH per t u b e (90 p g / m l plasma). Statistics: B i o c h e m i c a l d a t a w e r e f i r s t a n a l y z e d by one way analysis of v a r i a n c e . All measures d e m o n s t r a t e d s i g n i f i c a n t F scores. A d d i t i o n a l comparisons w e r e then made b e t w e e n the 0.0 c u r r e n t group and each o t h e r group. The b e h a v i o r a l ratings w e r e a n a l y z e d by a n o n - p a r a m e t r i c t e s t ( K r u s k a l - W a l l i s ) and a d d i t i o n a l comparisons w e r e t h e n made b e t w e e n t h e 0.0 c u r r e n t group and o t h e r groups by t h e M a n n - W h i t n e y t e s t . Results E x p e r i m e n t 1. Increasing c u r r e n t i n t e n s i t y increased t h e b e h a v i o r a l r a t i n g o f stress response, increased levels of p i t u i t a r y c y c l i c AMP, and increased levels of plasma p r o l a c t i n and c o r t i c o s t e r o n e as shown in Table I. A l t h o u g h all measures increased, t h e r e are clear d i f f e r e n c e s among these stress response m a r k e r s . Plasma c o r t i c o s t e r o n e increased t o near m a x i m a l levels a f t e r p l a c e m e n t of t h e r a t i n t o t h e u n f a m i l i a r shock box w i t h o u t any c u r r e n t being applied. The b e h a v i o r a l r a t i n g was s e n s i t i v e t o l o w levels o f shock but r e a c h e d its c e i l i n g at 1o6 m A . P r o l a c t i n was v e r y responsive o v e r most of t h e range t e s t e d (see also fig. 1). P i t u i t a r y cyclic AMP did n o t respond t o t h e l o w e r l e v e l s o f s t i m u l a t i o n , but was m a r k e d l y e l e v a t e d a f t e r t h e m o d e r a t e and high c u r r e n t i n t e n s i t i e s . As c o m p a r e d t o c o n t r o l s , t h e a p p a r e n t
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TABLE I .
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Effects of Graded Footshock on P i t u i t a r y C y c l i c AMP, Plasma Prolactin, Plasma C o r t i c o s t e r o n e and Behavior. Current (n) (mA) .
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Cyclic AMP (pmoles/mg wt) .
Controls(12) 0.0 (12) 0.2 (6) 0.4 (12) 0.8 (12) 1.6 (12) 2.4 (6) 3.2 (6)
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1.17 1.49 1.64 1.18 3.14 9.83 6.66 16.52
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-+ ± ± -+ ± -+ ± ±
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0.10 0.15 0.18 0.12 0.19 3.31 1.74 6.14
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Prolactin (ng/ml) .
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40 28 71 101 313 270 421 409
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Corticosterone (ug/100ml) .
-+ 9.4 ± 5.2 -+ 14 _+ 27 -+ 84 ± 62 ±160 ± 154
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6.2 17.3 19.5 20.2 22.6 22.8 21.6 27.8
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-+ ± -+ ± ± -+ ± ±
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0,8 2.4 4.0 1.8 2.0 2.2 2.4 6.1
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Behavior (rating) .
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0.0 0.2 1.0 1.3 1.6 2.9 2.8 3.0
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± ± -+ ± ± ± ± +
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0.0 0,2 0.5 0.3 0.3 0.8 0.2 0.0
range of response in this e x p e r i m e n t was 14-fold for p i t u i t a r y cyclic AMP, 10-fold for prolactin, and 4.5 fold for c o r t i c o s t e r o n e . E x p e r i m e n t 2. The responses of plasma 13-endorphin, 13-LPH, c o r t i c o s t e r o n e , and p r o l a c t i n are shown in Fig. 1. C o r t i c o s t e r o n e was the only measured hormone t o increase s i g n i f i c a n t l y a f t e r p l a c e m e n t into the box w i t h o u t shock as compared t o controls. Most of the c o r t i c o s t e r o n e response had occurred at or below 0.8 mA current. In contrast, B-endorphin d e m o n s t r a t e d very l i t t l e response at less than 0°8 mA, w i t h the greatest changes ocurring between 0.8 mA and 1.6 mA. 13-LPH changed m a r k e d l y between 0.4 and 1.6 mA. Prolactin was sensitive to small amounts of current and d e m o n s t r a t e d a broad range of response between 0.2 mA and 1.6 mA current. All measures reached m a x i m a l levels at 1.6 mA. Discussion A c u t e stress e l i c i t s numerous neuroendocrine responses in rats. Increased p i t u i t a r y release of p r o l a c t i n , a d r e n o c o r t i c o t r o p i c hormone ( A C T H ) , 13-endorphin, and !3-LPH has been f r e q u e n t l y reported and the release of cor t icosterone,, epinephrine and norepinephrine from the adrenals is also increased f o l l o w i n g acute stress (12-18). We have observed that acute stressors markedly e l e v a t e p i t u i t a r y c y c l i c AMP and have been i n v e s t i g a t i n g the role of stress-induced p i t u i t a r y c y c l i c AMP elevations (1-5). In the p i t u i t a r y gland, c y c l i c AMP appears to be involved in the release and/or synthesis of p i t u i t a r y hormones. Incubation of p i t u i t a r i e s in v i t r o w i t h cyclic AMP analogues increases the release of hormones i n t o the medium (19,20). Releasing factors such as CRF have been r e p o r t e d t o increase levels of p i t u i t a r y c y c l i c AMP in v i t r o (21). Thus there is reason to believe t h a t stress-induced p i t u i t a r y cyclic AM P elevations might have f u n c t i o n a l s i g n i f i c a n c e w i t h regard to c o n t r o l of stress-sensitive p i t u i t a r y hormones. In the present study, we have examined the p i t u i t a r y c y c l i c AMP response to a graded stressor and we have compared the response c h a r a c t e r i s t i c s of c y c l i c AMP w i t h four commonly employed hormonal indices of stress response and a behavioral index. We found that while 'stress' did increase all indices measured, the response c h a r a c t e r i s t i c s and the useful range of response f o r these markers were d i f f e r e n t . C o r t i c o s t e r o n e was a very sensitive responder not only to an obvious stressor (footshock) but also to the change in the e n v i r o n m e n t accompanying the procedure. Simply placing the animal in the shock box w i t h o u t d e l i v e r y of any current e l i c i t e d a strong c o r t i c o s t e r o n e response, as has been previously reported (22,23). While
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600 560 520 480 4,40 4OO 360 320 280 240 2OO 160
PROLACTIN
120 8O 4O 0
t
7200 6800 6400 6000 5600
m
E
5200 4800 4400
w
4000 3600 3200 2800 2400
- LPH
2O00 I 600 1200 8OO 400
I
1600 m
E
1200 800 400
ENDORPHIN I
~E*
24
"--
~CORTICOSTERONE
I
0L
I
I
I
I
I
I
0.0
0.8
1.6
2.4
3.2
CONTROL
SHOCK CURRENT INTENSITY ( m Amp ) Fig 1. E f f e c t s o f G r a d e d F o o t s h o c k on Plasma H o r m o n e s . Values r e p r e s e n t t h e mean % SEM o f 6 a n i m a l s . C o n t r o l s w e r e s a c r i f i c e d upon r e m o v a l f r o m t h e i r h o m e c a g e . O t h e r g r o u p s w e r e e x p o s e d t o 15 m i n o r s h o c k (see m e t h o d s ) at t h e i n d i c a t e d c u r r e n t i n t e n s i t i e s .
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c o r t i c o s t e r o n e is a useful index of m i l d stress or arousal, the l i m i t e d range of response b e t w e e n mild and m o d e r a t e c u r r e n t i n t e n s i t i e s suggests t h a t i t is a poor i n d i c a t o r f o r distinguishing b e t w e e n more severe stressors. In f a c t , we have found t h a t c o r t i c o s t e r o n e response t o cold exposure, f o r m a l i n s.c., r e s t r a i n t , and f o o t s h o c k are s i m i l a r w h i l e o t h e r i n d i c a t o r s of stress are able t o d i f f e r e n t i a t e b e t w e e n these stressors (1). On the o t h e r hand, p i t u i t a r y c y c l i c AMP was not a sensitive responder t o l o w c u r r e n t intensities, but did d e m o n s t r a t e a range of response o v e r the m o d e r a t e t o high i n t e n s i t i e s . Plasma p r o l a c t i n , 13-endorphin and 8-LPH did n o t respond t o the n o v e l t y of the shock box, but did respond p r o p o r t i o n a t e l y over a range of c u r r e n t i n t e n s i t y b e f o r e reaching a m a x i m a l plateau. P r o l a c t i n and 13-LPH showed l a r g e r responses t o c u r r e n t than did 6-endorphin. These indices w o u l d appear t o be useful in d i f f e r e n t i a t i n g m o d e r a t e t o s e v e r e st ressors. A l t h o u g h ACTH and 13-endorphin have been r e p o r t e d to be released by s i m i l a r s t i m u l i (24,25), the response p r o f i l e s of c o r t i c o s t e r o n e and 8-endorphin in the present study are q u i t e d i f f e r e n t . However, the c o r t i c o s t e r o n e levels r e f l e c t not only the ACTH released but also the adrenal s e n s i t i v i t y t o ACTH. Since, the adrenals are most s e n s i t i v e t o basal c o n c e n t r a t i o n s of ACTH as c o m p a r e d t o stress-induced levels (14), the d i f f e r e n c e s in c o r t i c o s t e r o n e and 13-endorphin measurable threshhold response are not surprising. The o b s e r v a t i o n t h a t t h e measured hormones responded t o l o w e r c u r r e n t levels than p i t u i t a r y c y c l i c AMP suggests t h a t m a r k e d p i t u i t a r y c y c l i c AMP e l e v a t i o n s m i g h t not be necessary f o r t h e i n i t i a l release of p i t u i t a r y hormones. Possibly, t h e stress-induced rise in p i t u i t a r y c y c l i c AMP r e f l e c t s t h e f e e d b a c k e f f e c t s of p i t u i t a r y or adrenal hormones upon t h e p i t u i t a r y for the purpose of r e g u l a t i n g f u r t h e r release or synthesis of p i t u i t a r y hormones. Acknowledgements The authors wish t o a c k n o w l e d g e the e x c e l l e n t t e c h n i c a l assistance p r o v i d e d by David R. Collins, Clyde C. Kenion, Golden C. D r i v e r , Willie G a m b l e , C l i n t o n B. Wormley, SP5 Leigh Landman Roberts, SP4 Terry Eggleston, Bruce Waskowicz and Steve Shiue. We also wish t o t h a n k Pat Conners f o r her assistance in p r e p a r i n g the m a n u s c r i p t .
References 1. 2.
3. 4.
5. 6. 7. 8.
G.J. KANT, J.Lo M E Y E R H O F F , B.N. B U N N E L L and R.H. LENOX, Pharmacol. B i o c h e m . Behav. 17 1067-1072 (1982). I . L . M E Y E R H O F F , G.J. K A N T , G.R. SESSIONS, E.H. M O U G E Y , L . L . PENNINGTON and R.H. L E N O X , Perspectives in B e h a v i o r a l Medicine~ vol 2, R. WILLIAMS (ed), A c a d e m i c Press, New York (in press). G.J. K A N T , G.R. SESSIONS, R.H. LENOX and J.L. M E Y E R H O F F , L i f e Sciences 29 2491-2499 (1981). J.L. M E Y E R H O F F , G.J. KANT and R.H. L E N O X , M i c r o w a v e I r r a d i a t i o n as as Tool to Study Labile M e t a b o l i t e s in Tissue, W.B. STAVINOHA, Y . M A R U Y A M A and C.L. B L A N K (eds), Pergamon Press, O x f o r d (in press). B.N. B U N N E L L , G . ] . KANT, R.H. L E N O X , L.L. PENNINGTON, D.R. COLLINS, J.L. M E Y E R H O F F and E.H. M O U G E Y , Neurosci. Abs. 7 869 (1981). D.J. JONES and W.B. S T A V I N O H A , N e u r o p h a r m a c o l o g y of C y c l i c Nucleotides, G.C. P A L M E R (ed.), 253-281, Urban & Schwarzenberg, B a l t i m o r e (1979). R.H. L E N O X , G.J. KANT and J.L. M E Y E R H O F F , Handbook of N e u r o c h e m i s t r y , vol 2, A. L A J T H A ( e d . ) i n press. R.H. L E N O X , G.J. K A N T , G.R. SESSIONS, L.L. P E N N I N G T O N , E.H. MOUGEY and J.L. M EYE RHOFF, N e u r o e n d o c r i n o l o g y 3.~0300-308 (1980).
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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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A.L. STEINER, A.W. PARKER and D.M. KIPNIS, J. Biol. Chem. 247 1106-1113 (1972). D. RODBARD, R.H. LENOX, W.L. WRAY and D. RAMSETH, Clin. Chem. 22 350-358 (1976)o E.H. MOUGEY, Analyt. Biochem. 91 566-582 (1978). L. KRULICH, E. HEFCO, P. ILLNER and C.B. READ, Neuroendocrinology 16 293-311 (1974). G.P. M U E L L E R , . L i f e Sciences 29 1669-1674 (1981). B. H. NATELSON, W.N. TAPP, J.E. ADAMUS, J.C. MITTLER and B.E. LEVIN, Pharmacol. Biochem. Behav.26 1049-1054 (1981). J. ROSSIER, E.D. FRENCH, C. RlVlER, N. LING, R. GUILLEMIN and F.E. BLOOM, Nature 270 618-620 (1977). J.L. M E Y E R H O F F , B.N. BUNNELL and E.H. MOUGEY, Neurosci. Abs. 7 166(1981). M.J. M I L L A N , R. PRZEWLOCKI, M. JERLICZ, C.H. GRAMSCH, V. HOLLT and A. HERZ, Brain Res. 208 325-338 (1981). R. M c C A R T Y , R. KVETNANSKY and I.J. KOPIN, Physiol. Behav. 26 27-31 (1981). G.T. PEAKE, Frontiers of Neuroendocrinology, vol 3, W.F. GANONG and L. MARTINI (ed) Oxford University Press, pp 173-208 (1973). G. PELLETIER, A. LEMAY, G. BERAUD and F. LABRIE, Endocrinology 91 1355-1370 (1972). F. LABRIE, R. VEILLEUX, G. LEFEVRE, D.H. COY, J. SUEIRAS-DIAZ and A.V. S C H A L L Y , Science 216 1007-1008 (1982). S.B. FRIEDMAN, R. ADER, L.J. GROTA and T. LARSON, Psychosom. Med. 29 323-328 (1967). M.B. HENNESSY and S. LEVINE, Physio. Behav. 21 295-297 (1978). J. ROSSIER, E. FRENCH, C. RIVIER, N. LING, R. GUlLLEMIN and F. BLOOM, N a t u r e 270 618-620 (1977). R. GUlLLEMIN, T. VARGO, J. ROSSIER, S. MINICK, N. LING, C. RIVIER, W. VALE and F. BLOOM, S c i e n c e 197 1 3 6 8 - 1 3 6 9 (1977).
Pergamon Press
G R A D E D FOOTSHOCK STRESS ELEVATES P I T U I T A R Y C Y C L I C AMP AND P L A S M A 13-ENDORPHIN, 13-LPH, CORTICOSTERONE AND P R O L A C T I N G. Jean Kant, Edward H. Mougey, Lee L. Pennington and James L. M e y e r h o f f N e u r o e n d o c r i n o l o g y and N e u r o c h e m i s t r y Br., Dept of Med Neurosciences, D i v o f N e u r o p s y c h i a t r y , Walter Reed A r m y I n s t i t u t e f o r Research l, Washington DC 20307.
(Received in final form October
12, 1983)
Summary Male rats w e r e subjected to 15 min o f various i n t e n s i t i e s of f o o t s h o c k c u r r e n t (0.0, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2mA) on a v a r i a b l e i n t e r v a l schedule w i t h an average i n t e r s h o c k i n t e r v a l of 30 sec (30 shocks/15 min session). Each shock lasted 5 sec. Animals w e r e s a c r i f i c e d i m m e d i a t e l y a f t e r being r e m o v e d f r o m the shock box. Two s i m i l a r studies w e r e c o n d u c t e d . In the f i r s t e x p e r i m e n t , rats were s a c r i f i c e d by m i c r o w a v e i r r a d i a t i o n and p i t u i t a r y cyclic AMP levels were d e t e r m i n e d . In the second study, rats w e r e d e c a p i t a t e d and plasma hormones (prolactin, c o r t i c o s t e r o n e , 13endorphin, 8-LPH) were measured by r a d i o i m m u n o a s s a y . Although all b i o c h e m i c a l indices of stress measured increased as shock i n t e n s i t y increased, some d i f f e r e n c e s among t h e substances measured w e r e observed w i t h respect to t h r e s h o l d i n t e n s i t y , range of p r o p o r t i o n a l response and m a x i m a l response. We have previously r e p o r t e d t h a t a v a r i e t y of stressors including r e s t r a i n t , f o r c e d running, s.c. f o r m a l i n , c o n d i t i o n e d p s y c h o l o g i c a l stress, and f o o t s h o c k e l e v a t e d levels of p i t u i t a r y c y c l i c AMP as w e l l as plasma c o r t i c o s t e r o n e and plasma p r o l a c t i n (1-5). We have p o s t u l a t e d t h a t this p i t u i t a r y c y c l i c AMP increase may be i n v o l v e d in t h e r e g u l a t i o n of p i t u i t a r y h o r m o n a l o u t p u t in response t o stress. Although the e l e v a t i o n in p i t u i t a r y c y c l i c AMP in response to d i f f e r e n t stressors appeared to c o r r e l a t e w i t h the a p p a r e n t " s e v e r i t y ' of t h e stressor, t h e ranking of stressors was c l e a r l y s u b j e c t i v e . We t h e r e f o r e decided t o f u r t h e r c h a r a c t e r i z e the p i t u i t a r y c y c l i c AMP response t o stress using a stressor whose i n t e n s i t y could be m a n i p u l a t e d o b j e c t i v e l y and t o include 13endorphin and 13-LPH responses as w e l l as c o r t i c o s t e r o n e and p r o l a c t i n . An a d d i t i o n a l o b j e c t i v e was to c o m p a r e the response of these h o r m o n a l indices of stress f o l l o w i n g exposure t o the same graded stressor w i t h regard t o s e n s i t i v i t y , range of response and m a x i m a l response. Two p a r a l l e l studies w e r e p e r f o r m e d to p e r m i t s a c r i f i c e by e i t h e r m i c r o w a v e i r r a d i a t i o n (cyclic AMP e x p e r i m e n t ) or d e c a p i t a t i o n (plasma hormones). Methods Animals.
Male (250-350 g) S p r a g u e - D a w l e y rats w e r e purchased f r o m
Taconic
1The views of the author(s) do not p u r p o r t t o r e f l e c t t h e position of the D e p a r t m e n t of the A r m y or the D e p a r t m e n t of Defense, (para 4-3, AR 360-5).
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Farms 2 Rats w e r e i n d i v i d u a l l y housed under c o n d i t i o n s of c o n t r o l l e d l i g h t (lights on f r o m 700 to 1900) and t e m p e r a t u r e w i t h f o o d and w a t e r f r e e l y a v a i l a b l e . Rats w e r e handled f o r 4 days p r i o r to the e x p e r i m e n t t o m i n i m i z e n o n - s p e c i f i c stress e f f e c t s . Rats t o be s a c r i f i c e d by m i c r o w a v e i r r a d i a t i o n were p r e v i o u s l y h a b i t u a t e d t o t r a v e r s i n g an o p e n ended p l a s t i c c y l i n d e r s i m i l a r t o t h e p l a s t i c m i c r o w a v e a p p l i c a t o r tube. Footshock. Constant c u r r e n t s c r a m b l e d shock was d e l i v e r e d t o t h e f l o o r bars of an o p e r a n t box on a v a r i a b l e schedule. Shock d u r a t i o n was 5 sec and t h e i n t e r s h o c k i n t e r v a l averaged 30 sec. Shock i n t e n s i t y was v a r i e d b e t w e e n 0.0 and 3.2 mA of c u r r e n t as s p e c i f i e d for each e x p e r i m e n t . Each rat r e c e i v e d only one shock i n t e n s i t y during the e n t i r e session and shock i n t e n s i t i e s were d i s t r i b u t e d t h r o u g h o u t the e x p e r i m e n t t o randomize circadian effects. One group of animals was s a c r i f i c e d in each e x p e r i m e n t i m m e d i a t e l y upon r e m o v a l f r o m t h e i r home cage w i t h o u t being exposed t o t h e shock box envi r o n m e n t ( c o n t r o l s ) . B e h a v i o r a l stress observer who did not i n d i c a t e d no response score of 2 i n d i c a t e d vocalizing. Behavioral
response. Rats w e r e observed during f o o t s h o c k and scored by an know which shock i n t e n s i t y was being d e l i v e r e d . A score of 0 during shock " o n ' , a score of 1 i n d i c a t e d n o t i c e a b l e flinching, a a c t i v e jumping and a score of 3 i n d i c a t e d a c t i v e jumping and ratings w e r e only made f o r e x p e r i m e n t 1.
Sacrifice. In the f i r s t study, rats were s a c r i f i c e d by m i c r o w a v e i r r a d i a t i o n t o p r e v e n t p o s t - m o r t e m d e g r a d a t i o n of c y c l i c AMP (6,7). Since m i c r o w a v e i r r a d i a t i o n hemolyzes blood which can i n t e r f e r e w i t h the assay f o r 6-LPH and 13-endorphin, we p e r f o r m e d a second e x p e r i m e n t using d e c a p i t a t i o n t o measure 13-endorphin and 13-LPH. Plasma c o r t i c o s t e r o n e and plasma p r o l a c t i n were measured in both e x p e r i m e n t s . The m i c r o w a v e m e t h o d e m p l o y e d 5 sec of high p o w e r i r r a d i a t i o n using a m o d i f i e d Varian PPS-2.5 power g e n e r a t o r w i t h an o u t p u t of 2.5. KW at a f r e q u e n c y of 2450 MHz. F o l l o w i n g i r r a d i a t i o n , the rats w e r e d e c a p i t a t e d and t r u n k blood was c o l l e c t e d for m e a s u r e m e n t of c o r t i c o s t e r o n e and p r o l a c t i n . The heads w e r e c o o l e d on d r y ice and t h e p i t u i t a r i e s w e r e dissected, w e i g h e d and s o n i c a t e d in 1 rnl of 50 mM sodium a c e t a t e buffer, pH 6.2. The sonicates were c e n t r i f u g e d at 12,000 g for 15 rnin and t h e supernatants s t o r e d at -70 ° u n t i l assayed f o r c y c l i c AMP. In the second e x p e r i m e n t , rats w e r e s a c r i f i c e d by d e c a p i t a t i o n and t h e t r u n k blood was c o l l e c t e d in h e p a r i n i z e d beakers. F o l l o w i n g c e n t r i f u g a t i o n , plasma was t r a n s f e r r e d t o tubes c o n t a i n i n g A p r o t i n i n (0.45 T I U / t u b e ) and stored at -35 ° u n t i l assayed for plasma 13-endorphin, 6-L PH, p r o l a c t i n and c o r t i c o s t e r o n e . Assay Procedures. C y c l i c AMP was d e t e r m i n e d by r a d i o i m m u n o a s s a y (8,9). A h i g h l y specific antisera was used at a f i n a l d i l u t i o n of 1:400,000. The a n t i s e r u m e x h i b i t e d c r o s s - r e a c t i v i t i e s f o r ATP and c y c l i c GMP of less than 0.00007% and 0.14% r e s p e c t i v e l y . she samples w e r e assayed in t r i p l i c a t e and assay d a t a w e r e a n a l y z e d by c o m p u t e r (10). Within assay v a r i a t i o n was 7% and b e t w e e n assay v a r i a t i o n was 18%. M a t e r i a l s f o r the p r o l a c t i n assay w e r e p r o v i d e d by the N a t i o n a l I n s t i t u t e of H e a l t h through the Rat P i t u i t a r y H o r m o n e D i s t r i b u t i o n Program. P r o l a c t i n was r a d i o i o d i n a t e d as p r e v i o u s l y described (8). C o r t i c o s t e r o n e was measured using an a n t i b o d y produced in our l a b o r a t o r y in rabbits (11).
2In c o n d u c t i n g the research described in this r e p o r t , the i n v e s t i g a t o r ( s ) adhere t o the 'Guide f o r the Care and Use of L a b o r a t o r y Animals', as p r o m u l g a t e d by the C o m m i t t e e on Care and Use of L a b o r a t o r y Animals of t h e I n s t i t u t e of L a b o r a t o r y A n i m a l Resources, N a t i o n a l Research Council.
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For the assay of 13-endorphin and 6 - L P H , plasma samples w e r e t h a w e d and r e c e n t r i f u g e d at 12,000 g for 10 min. A 0.5 ml a l i q u o t was e x t r a c t e d w i t h 75 mg PrepPak 500/C18 (Waters Associates Inc.). The C18 absorbant was rinsed t h r e e t i m e s w i t h I ml o f d i s t i l l e d H 2 0 . A p o r t i o n of t h e 13-LPH f r a c t i o n was e l u t e d f r o m t h e absorbant w i t h t w o 1 ml rinses of a c e t o n e : w a t e r (1:1). These rinses w e r e c o m b i n e d , e v a p o r a t e d and assayed f o r i3-LPH c o n t e n t , using the 13-endorphin a n t i b o d y . R e c o v e r y studies of human 13-LPH and 13-endorphin added t o r a t plasma in v a r y i n g c o n c e n t r a t i o n s showed t h a t a p p r o x i m a t e l y 20% of t h e 13-LPH added was r e c o v e r e d in this f r a c t i o n w h i l e o n l y 5% of added 13-endorphin was e l u t e d at this step. The absorbant was then rinsed t w i c e w i t h 1 ml o f a c e t o n e : H 2 0 : T F A (80:19:1) which e l u t e d 60% of B-endorphin and 5% o f B-LPH. Similar r e c o v e r i e s of c a m e l 13-endorphin added t o rat plasma w e r e o b t a i n e d . E x t r a c t i o n of 0.4 t o 1.0 ml of r a t plasma produced l i n e a r results in the r a d i o i m m u n o a s s a y . The a n t i b o d y used f o r assay of 13-endorphin/13-L PH was produced in r a b b i t s a g a i n s t a c o n j u g a t e of human 13-endorphin and b o v i n e serum a l b u m i n . This a n t i b o d y , used at a f i n a l d i l u t i o n of 1:20,000, c r o s s - r e a c t e d 100~o (on a m o l a r basis) w i t h human 13-LPH and 95% (on a m o l a r basis) w i t h c a m e l B-endorphin but e x h i b i t e d no measurable cross r e a c t i o n w i t h ~e n d o r p h i n or t h e enkephalins. Although not s p e c i f i c a l l y tested, N - a c e t y l a t e d B-endorphin would be e x p e c t e d t o c r o s s - r e a c t w i t h this a n t i b o d y since t h e b i n d i n g d e t e r m i n a n t s a r e in the C - t e r m i n a l half of t h e m o l e c u l e . Human 13-endorphin was r a d i o i o d i n a t e d using c h l o r a m i n e - T and N a l ~ S l ( A m e r s h a m C o r p o r a t i o n ) . Sodium m e t a b i s u l f i t e was used t o s t o p t h e r e a c t i o n . T h e i o d i n a t e d p r o d u c t was p u r i f i e d on a 1.4 x 36 cm. c o l u m n of Sephadex G-2S, f i n e ( P h a r m a c i a Fine Chemicals) w i t h a solution of 0.25% bovine serum a l b u m i n in 0.5% a c e t i c acid as e l u e n t . Human IB-endorphin was used as standard. Free and bound antigen w e r e separated, f o l l o w i n g a four day, 4 ° C i n c u b a t i o n , w i t h d e x t r a n - c o a t e d c h a r c o a l and the bound f r a c t i o n was c o u n t e d in an LKB Rack G a m m a II. A l l values r e p o r t e d are c o r r e c t e d f o r r e c o v e r y using r e c o v e r y values o b t a i n e d on a separate plasma pool run in the same assay. The c o e f f i c e n t s of v a r i a t i o n f o r s e p a r a t e e x t r a c t i o n s f r o m the same rat plasma pool w e r e 20% for 13-LPH and 6% for 13-endorphin. The assay is s e n s i t i v e t o 3 pg of added 13-endorphin per tube (10 p g / m l plasma) and 9 pg of 13-L PH per t u b e (90 p g / m l plasma). Statistics: B i o c h e m i c a l d a t a w e r e f i r s t a n a l y z e d by one way analysis of v a r i a n c e . All measures d e m o n s t r a t e d s i g n i f i c a n t F scores. A d d i t i o n a l comparisons w e r e then made b e t w e e n the 0.0 c u r r e n t group and each o t h e r group. The b e h a v i o r a l ratings w e r e a n a l y z e d by a n o n - p a r a m e t r i c t e s t ( K r u s k a l - W a l l i s ) and a d d i t i o n a l comparisons w e r e t h e n made b e t w e e n t h e 0.0 c u r r e n t group and o t h e r groups by t h e M a n n - W h i t n e y t e s t . Results E x p e r i m e n t 1. Increasing c u r r e n t i n t e n s i t y increased t h e b e h a v i o r a l r a t i n g o f stress response, increased levels of p i t u i t a r y c y c l i c AMP, and increased levels of plasma p r o l a c t i n and c o r t i c o s t e r o n e as shown in Table I. A l t h o u g h all measures increased, t h e r e are clear d i f f e r e n c e s among these stress response m a r k e r s . Plasma c o r t i c o s t e r o n e increased t o near m a x i m a l levels a f t e r p l a c e m e n t of t h e r a t i n t o t h e u n f a m i l i a r shock box w i t h o u t any c u r r e n t being applied. The b e h a v i o r a l r a t i n g was s e n s i t i v e t o l o w levels o f shock but r e a c h e d its c e i l i n g at 1o6 m A . P r o l a c t i n was v e r y responsive o v e r most of t h e range t e s t e d (see also fig. 1). P i t u i t a r y cyclic AMP did n o t respond t o t h e l o w e r l e v e l s o f s t i m u l a t i o n , but was m a r k e d l y e l e v a t e d a f t e r t h e m o d e r a t e and high c u r r e n t i n t e n s i t i e s . As c o m p a r e d t o c o n t r o l s , t h e a p p a r e n t
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TABLE I .
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Effects of Graded Footshock on P i t u i t a r y C y c l i c AMP, Plasma Prolactin, Plasma C o r t i c o s t e r o n e and Behavior. Current (n) (mA) .
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Cyclic AMP (pmoles/mg wt) .
Controls(12) 0.0 (12) 0.2 (6) 0.4 (12) 0.8 (12) 1.6 (12) 2.4 (6) 3.2 (6)
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1.17 1.49 1.64 1.18 3.14 9.83 6.66 16.52
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-+ ± ± -+ ± -+ ± ±
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0.10 0.15 0.18 0.12 0.19 3.31 1.74 6.14
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Prolactin (ng/ml) .
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40 28 71 101 313 270 421 409
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Corticosterone (ug/100ml) .
-+ 9.4 ± 5.2 -+ 14 _+ 27 -+ 84 ± 62 ±160 ± 154
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6.2 17.3 19.5 20.2 22.6 22.8 21.6 27.8
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-+ ± -+ ± ± -+ ± ±
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0,8 2.4 4.0 1.8 2.0 2.2 2.4 6.1
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Behavior (rating) .
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0.0 0.2 1.0 1.3 1.6 2.9 2.8 3.0
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± ± -+ ± ± ± ± +
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0.0 0,2 0.5 0.3 0.3 0.8 0.2 0.0
range of response in this e x p e r i m e n t was 14-fold for p i t u i t a r y cyclic AMP, 10-fold for prolactin, and 4.5 fold for c o r t i c o s t e r o n e . E x p e r i m e n t 2. The responses of plasma 13-endorphin, 13-LPH, c o r t i c o s t e r o n e , and p r o l a c t i n are shown in Fig. 1. C o r t i c o s t e r o n e was the only measured hormone t o increase s i g n i f i c a n t l y a f t e r p l a c e m e n t into the box w i t h o u t shock as compared t o controls. Most of the c o r t i c o s t e r o n e response had occurred at or below 0.8 mA current. In contrast, B-endorphin d e m o n s t r a t e d very l i t t l e response at less than 0°8 mA, w i t h the greatest changes ocurring between 0.8 mA and 1.6 mA. 13-LPH changed m a r k e d l y between 0.4 and 1.6 mA. Prolactin was sensitive to small amounts of current and d e m o n s t r a t e d a broad range of response between 0.2 mA and 1.6 mA current. All measures reached m a x i m a l levels at 1.6 mA. Discussion A c u t e stress e l i c i t s numerous neuroendocrine responses in rats. Increased p i t u i t a r y release of p r o l a c t i n , a d r e n o c o r t i c o t r o p i c hormone ( A C T H ) , 13-endorphin, and !3-LPH has been f r e q u e n t l y reported and the release of cor t icosterone,, epinephrine and norepinephrine from the adrenals is also increased f o l l o w i n g acute stress (12-18). We have observed that acute stressors markedly e l e v a t e p i t u i t a r y c y c l i c AMP and have been i n v e s t i g a t i n g the role of stress-induced p i t u i t a r y c y c l i c AMP elevations (1-5). In the p i t u i t a r y gland, c y c l i c AMP appears to be involved in the release and/or synthesis of p i t u i t a r y hormones. Incubation of p i t u i t a r i e s in v i t r o w i t h cyclic AMP analogues increases the release of hormones i n t o the medium (19,20). Releasing factors such as CRF have been r e p o r t e d t o increase levels of p i t u i t a r y c y c l i c AMP in v i t r o (21). Thus there is reason to believe t h a t stress-induced p i t u i t a r y cyclic AM P elevations might have f u n c t i o n a l s i g n i f i c a n c e w i t h regard to c o n t r o l of stress-sensitive p i t u i t a r y hormones. In the present study, we have examined the p i t u i t a r y c y c l i c AMP response to a graded stressor and we have compared the response c h a r a c t e r i s t i c s of c y c l i c AMP w i t h four commonly employed hormonal indices of stress response and a behavioral index. We found that while 'stress' did increase all indices measured, the response c h a r a c t e r i s t i c s and the useful range of response f o r these markers were d i f f e r e n t . C o r t i c o s t e r o n e was a very sensitive responder not only to an obvious stressor (footshock) but also to the change in the e n v i r o n m e n t accompanying the procedure. Simply placing the animal in the shock box w i t h o u t d e l i v e r y of any current e l i c i t e d a strong c o r t i c o s t e r o n e response, as has been previously reported (22,23). While
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Biochemical
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600 560 520 480 4,40 4OO 360 320 280 240 2OO 160
PROLACTIN
120 8O 4O 0
t
7200 6800 6400 6000 5600
m
E
5200 4800 4400
w
4000 3600 3200 2800 2400
- LPH
2O00 I 600 1200 8OO 400
I
1600 m
E
1200 800 400
ENDORPHIN I
~E*
24
"--
~CORTICOSTERONE
I
0L
I
I
I
I
I
I
0.0
0.8
1.6
2.4
3.2
CONTROL
SHOCK CURRENT INTENSITY ( m Amp ) Fig 1. E f f e c t s o f G r a d e d F o o t s h o c k on Plasma H o r m o n e s . Values r e p r e s e n t t h e mean % SEM o f 6 a n i m a l s . C o n t r o l s w e r e s a c r i f i c e d upon r e m o v a l f r o m t h e i r h o m e c a g e . O t h e r g r o u p s w e r e e x p o s e d t o 15 m i n o r s h o c k (see m e t h o d s ) at t h e i n d i c a t e d c u r r e n t i n t e n s i t i e s .
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c o r t i c o s t e r o n e is a useful index of m i l d stress or arousal, the l i m i t e d range of response b e t w e e n mild and m o d e r a t e c u r r e n t i n t e n s i t i e s suggests t h a t i t is a poor i n d i c a t o r f o r distinguishing b e t w e e n more severe stressors. In f a c t , we have found t h a t c o r t i c o s t e r o n e response t o cold exposure, f o r m a l i n s.c., r e s t r a i n t , and f o o t s h o c k are s i m i l a r w h i l e o t h e r i n d i c a t o r s of stress are able t o d i f f e r e n t i a t e b e t w e e n these stressors (1). On the o t h e r hand, p i t u i t a r y c y c l i c AMP was not a sensitive responder t o l o w c u r r e n t intensities, but did d e m o n s t r a t e a range of response o v e r the m o d e r a t e t o high i n t e n s i t i e s . Plasma p r o l a c t i n , 13-endorphin and 8-LPH did n o t respond t o the n o v e l t y of the shock box, but did respond p r o p o r t i o n a t e l y over a range of c u r r e n t i n t e n s i t y b e f o r e reaching a m a x i m a l plateau. P r o l a c t i n and 13-LPH showed l a r g e r responses t o c u r r e n t than did 6-endorphin. These indices w o u l d appear t o be useful in d i f f e r e n t i a t i n g m o d e r a t e t o s e v e r e st ressors. A l t h o u g h ACTH and 13-endorphin have been r e p o r t e d to be released by s i m i l a r s t i m u l i (24,25), the response p r o f i l e s of c o r t i c o s t e r o n e and 8-endorphin in the present study are q u i t e d i f f e r e n t . However, the c o r t i c o s t e r o n e levels r e f l e c t not only the ACTH released but also the adrenal s e n s i t i v i t y t o ACTH. Since, the adrenals are most s e n s i t i v e t o basal c o n c e n t r a t i o n s of ACTH as c o m p a r e d t o stress-induced levels (14), the d i f f e r e n c e s in c o r t i c o s t e r o n e and 13-endorphin measurable threshhold response are not surprising. The o b s e r v a t i o n t h a t t h e measured hormones responded t o l o w e r c u r r e n t levels than p i t u i t a r y c y c l i c AMP suggests t h a t m a r k e d p i t u i t a r y c y c l i c AMP e l e v a t i o n s m i g h t not be necessary f o r t h e i n i t i a l release of p i t u i t a r y hormones. Possibly, t h e stress-induced rise in p i t u i t a r y c y c l i c AMP r e f l e c t s t h e f e e d b a c k e f f e c t s of p i t u i t a r y or adrenal hormones upon t h e p i t u i t a r y for the purpose of r e g u l a t i n g f u r t h e r release or synthesis of p i t u i t a r y hormones. Acknowledgements The authors wish t o a c k n o w l e d g e the e x c e l l e n t t e c h n i c a l assistance p r o v i d e d by David R. Collins, Clyde C. Kenion, Golden C. D r i v e r , Willie G a m b l e , C l i n t o n B. Wormley, SP5 Leigh Landman Roberts, SP4 Terry Eggleston, Bruce Waskowicz and Steve Shiue. We also wish t o t h a n k Pat Conners f o r her assistance in p r e p a r i n g the m a n u s c r i p t .
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