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Adrenal-mediated depression of n-acetyltransferase activity and melatonin levels in the rat pineal gland
Life Sciences, Vol. 38, pp. 1573-1580 Printed in the U.S.A.
Pergamon Press
ADRENAL-MEDIATED DEPRESSION OF N-ACETYLTRANSFERASE ACTIVITY AND MELATONIN L E V E L S IN T H E R A T P I N E A L G L A N D Bhaskar N. Joshi, M a u r a e n E. Trolani, Josif Milin, Frank N~Irnburger, and Russel J. Relier Department of Cellular and Structural Biology, T h e University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas (Received in final form February ii, 1986) Summary
N-acetyltransferase (NAT) is believed to be the rate-llmiting enzyme in the synthesis of melatonin from serotonln in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h - lights on at 07.00 h). To test this more thoroughly, a time course study Was conducted during the dark phase (at 02.00 h 5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Soma factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland. Melatonin, which seems to be the major endocrine product of the pineal (1), is produced in the gland from the amino acid tryptophan (2). Tryptophan is taken u p from the blood a n d converted into serotonin (5-hydroxytryptemine). Serotonin is then converted to N-acetyiserotonin (3). This step utilizes the e n z y m e N-acetyltransferase ( N A T ) , which seems to be the rate-limiting e n z y m e in melatonin synthesis (3). T h e last step, utilizing the e n z y m e hydroxyindole-O-methyltransferase (HIOMT), consists of the O-methylation of N-acetylserotonin to form melatonin (4). Since it has been s h o w n that norepinephrine (NE) released from sympathetic nerve endings within the rat pineal gland act on D-receptors to 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
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s t i m u l a t e NAT a c t i v i t y a n d , t h e r e f o r e , m e l a t o n i n s y n t h e s i s ( 5 , 6 ) , i t h a s b e e n of g r e a t i n t e r e s t to s e e if c a t e c h o l a m i n e s f r o m t h e a d r e n a l m e d u l l a c a n e f f e c t pineal melatonin synthesis. I t w o u l d b e e x p e c t e d t h a t t h e i n c r e a s e in p l a s m a c a t e c h o l a m i n e s o r i g i n a t i n g from t h e a d r e n a l d u r i n g s t r e s s ( 7 , 8 ) w o u l d r e a c h t h e p i n e a l g l a n d a n d a c t on t h e B - r e c e p t o r s to i n c r e a s e p i n e a l m e l a t o n i n synthesis. D u r i n g t h e l i g h t p h a s e of t h e l i g h t / d a r k c y c l e in t h e r a t , t h e e f f e c t of s t r e s s on t h e p i n e a l g l a n d h a d n o t b e e n a s c l e a r c u t a s was e x p e c t e d . P h y s i c a l immobilization f o r 2 h o u r s h a s b e e n s h o w n b o t h to i n c r e a s e NAT a c t i v i t y a n d m e l a t o n i n c o n t e n t ( 9 , 1 0 ) a n d to d e c r e a s e NAT a c t i v i t y ( 1 1 ) . I n s u l i n i n d u c e d h y p o g l y c e m i a h a s b e e n s h o w n to i n c r e a s e NAT a c t i v i t y a n d m e l a t o n i n c o n t e n t b y 2 h o u r s a f t e r i n s u l i n i n j e c t i o n ( 9 , 8 ) , while t h e same t r e a t m e n t was s h o w n to d e c r e a s e NAT a c t i v i t y in a n o t h e r s t u d y ( 1 1 ) . This treatment has been interpreted as a stressor because plasma catecholamines are e l e v a t e d ( 8 ) . A h i g h c a r b o h y d r a t e d i e t , w h i c h c a u s e s t h e animal to h a v e h i g h i n s u l i n l e v e l s w i t h o u t h y p o g l y c e m i a , h a s no e f f e c t on p i n e a l NAT a c t i v i t y ( 9 ) . F o o d d e p r i v a t i o n , w h i c h c a u s e s h y p o g l y c e m i a w i t h low i n s u l i n l e v e l s h a s b e e n s h o w n to e l i c i t a d e c r e a s e in NAT a c t i v i t y ( 1 1 ) . A t 1-4 h o u r s a f t e r l i g h t s o n , a n i m a l s s u b j e c t e d to 3 rain of cold w a t e r , n o i s e , n o v e l e n v i r o n m e n t , o r e t h e r v a p o r s h o w e d a n i n c r e a s e in p i n e a l m e l a t o n i n c o n t e n t ( 1 2 ) , a n d a n i n c r e a s e in NAT a c t i v i t y a f t e r 20 rain of e t h e r s t r e s s ( 1 3 ) . At 1 hour or 5 hours after l i g h t s o u t , h o w e v e r , a s l i g h t b u t n o n - s i g n i f i c a n t d e c r e a s e in NAT a c t i v i t y was o b s e r v e d a f t e r 20 rain of e t h e r s t r e s s ( 1 3 ) . R e c e n t l y , Milin e t al s h o w e d t h a t a 1 . 5 c c i n j e c t i o n of p h y s i o l o g i c a l s a l i n e i n t o t h e h i n d l e g c a u s e d m o r p h o l o g i c a l c h a n g e s in t h e p i n e a l g l a n d w h i c h w e r e i n t e r p r e t e d a s e v i d e n c e t h a t t h e s e c r e t o r y a c t i v i t y of t h e p i n e a l g l a n d was increased (14). T h i s t r e a t m e n t was p r e s u m a b l y a s t r e s s o r b e c a u s e t h e s k i n of t h e r a t ' s b a c k l e g is t i g h t a n d a l a r g e volume of f l u i d i n j e c t e d s u b c u t a n e o u s l y in t h i s a r e a w o u l d p r o b a b l y c a u s e d i s c o m f o r t . A p r e l i m i n a r y s t u d y d o n e in t h i s l a b o r a t o r y s h o w e d , u n e x p e c t e d l y , t h a t t h i s t r e a t m e n t l a t e in t h e l i g h t p h a s e d i d in f a c t e l i c i t a d e c r e a s e in m e l a t o n i n c o n t e n t of t h e p i n e a l b y 10 rain after the saline injection (unpublished observations). S i n c e i t is n o t k n o w n , in g e n e r a l , w h a t t h e e f f e c t s of s t r e s s a r e on t h e p i n e a l g l a n d , a n d s p e c i f i c a l l y , w h a t e f f e c t s t r e s s h a s on t h e p i n e a l g l a n d d u r i n g t h e d a r k p h a s e of t h e l i g h t / d a r k c y c l e , i t was d e e m e d i m p o r t a n t to e x a m i n e t h e e f f e c t of t h i s model of s t r e s s on t h e p i n e a l g l a n d of t h e r a t w h e n m e l a t o n i n s y n t h e s i s is n e a r i t s p e a k , d u r i n g t h e d a r k p h a s e of t h e l i g h t / d a r k c y c l e . Materials and Methods A d u l t male S p r a g u e - D a w l e y r a t s , w e i g h i n g a p p r o x i m a t e l y 150 g r a t t h e time of t h e e x p e r i m e n t , w e r e o b t a i n e d from H a r l a n S p r a g u e - D a w l e y , H o u s t o n , Texas. Animals w e r e h o u s e d 4 p e r c a g e a n d r e c e i v e d food a n d w a t e r a d l i b i t u m . T h e y w e r e e x p o s e d to a 14/10 l i g h t / d a r k c y c l e ( l i g h t s on a t 07.00 h ) w i t h c o n s t a n t t e m p e r a t u r e (22 + 2 ° C ) . For each experiment, the experimental a n i m a l s r e c e i v e d a 1 . 5 c c s u b c u t a n e o u s i n j e c t i o n of n o r m a l (0.85%) s a l i n e on t h e d o r s a l a s p e c t of t h e h i n d l e g . C o n t r o l animals r e m a i n e d u n t r e a t e d . I n e x p e r i m e n t 1, 51 r a t s w e r e i n j e c t e d w i t h s a l i n e a t 02.20 h . Eight u n t r e a t e d animals w e r e k i l l e d b y d e c a p i t a t i o n a s c o n t r o l s a t 02.00 a n d 9 w e r e k i l l e d a s c o n t r o l s a t 03.00. T h e i n j e c t e d a n i m a l s w e r e d e c a p i t a t e d in g r o u p s ( 8 - 9 p e r g r o u p ) a t 10, 20, 30, 40, 60, a n d 90 rain a f t e r s a l i n e i n j e c t i o n . P i n e a l g l a n d s w e r e r e m o v e d b y r a p i d d i s s e c t i o n a n d f r o z e n on s o l i d CO 2 . T h e y w e r e p l a c e d in p r e c o o l e d 1500 ul m i c r o f u g e t u b e s a n d s t o r e d a t -70°C u n t i l a s s a y e d f o r NAT a c t i v i t y a n d m e l a t o n i n c o n t e n t .
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In the second experiment, 24 rats were bilaterally adrenalectomized and maintained on a drinking water solution of 1% saline until the time of the experiment. Eight untreated intact and 8 untreated adrenalectomized animals were killed by decapitation at 03.00 h as controls. The remJ~ining adrenalectomized rats as well as 16 intact animals were injected at 02.30 h and killed (8 per group) 10 and 20 minutes later. Pineals were dissected, stored, and assayed for N A T activity and meiatonin content. In experiment 3, 20 animals were injected with saline at 01.15 h. Ten untreated controls were killed at 01.30. Injected animals were killed (in groups of 10) 15 and 30 min after injection. Pineals were dissected, stored and assayed for N A T activity and melatonin content. T r u n k blood was coliected at the time of decapitation in heparinized 12 X 75 nun glass tubes and centrifuged at 3,000 r p m for 15 min to separate the plasma. Plasma was transferred to fresh glass 12 X 75 m m tubes and frozen at -20°C until assayed for corticosterone concentration. Pineal N A T activity and melatonin content were determined in the same pineal gland b y the method of C h a m p n e y and colleagues (15). Pineal glands were homogenized b y sonication in 100 ul of 0.05 M phosphate buffered saline. Twenty ul of this homogenate were added to a 1500 ul microfuge tube and assayed for N A T activity (16,17). Fifty ul of the homogenate were used for duplicate measurements of melatonin content b y radioimmunoassay (18,19). Piasma corticosterone was assayed using a radioimmunoassay kit from Radioassay Systems Laboratories, Carson, CA. Five hundred ~I of diluted plasma (1:250) or standard were added to glass 10 X 75 m m tubes. The 7 standards ranged from 0 to 2.0 n g corticosteroneJml. Four additional tubes were labeled total counts and nonspecific binding (NSB) in duplicate. The N S B tubes contained 600 ul of the 0 ng corticosterone/ml standard. The total tubes had 500 ul of the 0 n g corticosterone/mi standard. The samples and standards were run in duplicate. All of the tubes were incubated at 98°C for 10 min to denature the corticosterone binding 3Proteins. The tubes were allowed to cool to room temperature and 100 ~I of H-corticosterene were added to all of the tubes. All tubes except the N S B tubes then had 100 ul of corticosterone antiserum added. All tubes were then incubated overnight at 4°C and the free corticosterone was separated out with charcoal-dextran. The supernatant (bound corticosterone-antibody complex) was decanted and dpms were counted in a scintillation counter. Statistical analyses of the data were performed using a one w a y analysis of variance with significant differences between groups determined b y the Student-Newman-Keuls test. Values are expressed as means + standard errors. Results Experiment 1." Pineal N A T activity was depressed b y 20 min after injection (p<.025) and remained so through 60 min after the saline injection (p<.001). B y 90 min after the saline injection, pineal N A T activity had returned to control values (fig. 1). Pineal melatonin was depressed similarly with its lowest value at 20 min (p<.01) and a return to control values b y 60 rain (fig. 2). E x p e r i m e n t 2__L T h e s a l i n e - i n j e c t e d i n t a c t a n i m a l s e x h i b i t e d a s i g n i f i c a n t d e p r e s s i o n i n NAT a c t i v i t y ( p ( . 0 5 ) b y 20 rain a s c o m p a r e d to n o n - i n j e c t e d intact controls. A d r e n a l e c t o m i z e d a n i m a l s s h o w e d no r e s p o n s e in t e r m s of NAT a c t i v i t y to t h e s a l i n e i n j e c t i o n ( f i g . 3). P i n e a l m e l a t o n i n c o n t e n t of
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s a i i n e - i n j e c t e d i n t a c t animals showed a s l i g h t b u t n o n - s i g n i f i c a n t d e c r e a s e . This was not o n l y p r e v e n t e d b y adrenaiectomy~ b u t t h e r e was a c t u a l l y a s i g n i f i c a n t i n c r e a s e in melatonin c o n t e n t b y 20 rain in a d r e n a l e c t o m i z e d animals in r e s p o n s e to the saline injection ( p ( . 0 0 5 ) ( f i g . 4 ) . E x p e r i m e n t 3_.3: Control animals showed an NAT a c t i v i t y level of 5574 + 388 pM p r o d u c t / p i n e a l / h. I n j e c t e d animals were d e p r e s s e d to 3107 + 715 pM p r o d u c t / p i n e a l / h b y 15 rain a f t e r i n j e c t i o n . This is s i g n i f i c a n t at p<.005 vs c o n t r o l s By 30 rain NAT a c t i v i t y was b a c k up to c o n t r o l v a l u e s . Melatonin showed a s l i g h t b y n o n - s i g n i f i c a n t d e p r e s s i o n b y 15 rain which was r e v e r s e d b y 30 rain. Plasma c o r t i c o s t e r o n e r o s e in the e x p e r i m e n t from the c o n t r o l c o n c e n t r a t i o n of 273 + 146 p g / m l to 456 + 190 p g / m i b y 15 rain a n d to 550 + 191 p g / m l b y 30 r a i n . - T h e s e a r e signfficarTt from c o n t r o l s at p<.025 a n d p < . 0 1 . respectively. Discussion This model of a c u t e s t r e s s p r o d u c e s a d e p r e s s i o n in melatonin p r o d u c t i o n b y t h e p i n e a l g l a n d as m e a s u r e d b y a d e p r e s s i o n in NAT a c t i v i t y ( f i g . 1) a n d melatonin c o n t e n t ( f i g . 2 ) in the p i n e a l . This is i n t e r p r e t e d as a s t r e s s r e s p o n s e b e c a u s e it is a b o l i s h e d in a d r e n a l e c t o m i z e d animals ( f i g s . 3 & 4) a n d it c a u s e s an e l e v a t i o n in plasma c o r t i c o s t e r o n e c o n c e n t r a t i o n . Melatonin, in r e s p o n s e to this s t r e s s o r , does not seem to be as d r a m a t i c a l l y d e p r e s s e d as NAT a c t i v i t y . Since NAT is the p r o p o s e d r a t e - l i m l t i n g enzyme in melatonin s y n t h e s i s , t h i s a b o v e - m e n t i o n e d f i n d i n g seems to be an i n c o n g r u i t y . It may b e , h o w e v e r , t h a t the a c t i v i t y of HIOMT is e f f e c t e d b y this t r e a t m e n t a n d p a r t i a l l y c o u n t e r a c t s the d e p r e s s i o n in NAT activity so that melatonin synthesis is depressed only slightly. A d r e n a i e c t o m i z e d animals seem to have an i n c r e a s e in p i n e a l melatonin a f t e r saline i n j e c t i o n . This also s u p p o r t s the c o n j e c t u r e t h a t HIOMT a c t i v i t y is e f f e c t e d b y t h e t r e a t m e n t to i n c r e a s e melatonin s y n t h e s i s . In a d r e n a l e c t o m i z e d animals t h i s e f f e c t would be u n o p p o s e d b y the f a c t o r from the a d r e n a l c a u s i n g NAT a c t i v i t y d e p r e s s i o n , so melatonin c o n t e n t would i n c r e a s e . Other e x p l a n a t i o n s also e x i s t . It is p o s s i b l e t h a t r e l e a s e of melatonin is d e p r e s s e d b y s t r e s s so t h a t p i n e a l c o n t e n t of melatonin would remain only s l i g h t l y c h a n g e d even t h o u g h s y n t h e s i s is d r a m a t i c a l l y r e d u c e d . These possible e x p l a n a t i o n s have y e t to be i n v e s t i g a t e d . D u r i n g immobilization s t r e s s , plasma e p i n e p h r i n e (E) i n c r e a s e s 40-fold b y 15-20 rain a f t e r o n s e t of the s t r e s s o r ( 7 ) . NE is i n c r e a s e d 6-fold while dopamine (DA) is i n c r e a s e d 3-fold b y the same t r e a t m e n t (7). It has b e e n shown b y a d r e n a i e c t o m y a n d chemical s y m p a t h e c t o m y e x p e r i m e n t s t h a t n e a r l y all c i r c u l a t i n g E comes from t h e a d r e n a l g l a n d s while only 30% of c i r c u l a t i n g NE d e r i v e s from the a d r e n a l s , the r e m a i n d e r b e i n g s e c r e t e d b y s y m p a t h e t i c n e r v e s (7). Plasma c o r t i c o s t e r o n e i n c r e a s e s b y 15 min a f t e r the o n s e t of e i t h e r c o n t i n u o u s e t h e r s t r e s s , 2.5 rain of e t h e r s t r e s s a n d 30 sec of e t h e r s t r e s s (20) and after 3 rain of novel e n v i r o n m e n t , noise or cold (21). A d r e n o c o r t i c o t r o p h i n (ACTH) (20), p r o l a c t i n a n d g r o w t h hormone (21) from t h e p i t u i t a r y gland a r e i n c r e a s e d b y t h e same t r e a t m e n t s . It seems t h a t one of t h e s e s u b s t a n c e s from t h e a d r e n a l s is a c t i n g e i t h e r d i r e c t l y on the p i n e a l g l a n d , c e n t r a l l y to a l t e r the n e u r a l s i g n a l s t r a v e l i n g to the p i n e a l g l a n d , or a c t i n g on the p i t u i t a r y g l a n d which, in t u r n , could a l t e r its s e c r e t i o n of an e n d o c r i n e p r o d u c t which may a c t c e n t r a l l y or d i r e c t l y on the p i n e a l . Likewise, t h e r e a r e o t h e r p o s s i b l e e x p l a n a t i o n s f o r the o b s e r v e d r e s u l t s .
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Male Rats 6000
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M i n u t e s A f t e r Saline injection
FIG. i Effect of saline injection on pineal NAT activity. Con i was a non-injected control killed at 02.00. Con 2 was a non-injected control killed at 03.00. All other groups were injected at 02.20 and killed the indicated min after injection, a=p<.025 versus Con i. b=p<.001 versus Con 2. Eight to 9 animals per group. Values expressed as means + standard error.
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M i n u t e s A f t e r Saline iniecfion FIG. 2 Effect of saline injection on pineal melatonin content. Con I is the non-injected control group killed at 02.00 h. All experimental groups were injected at 02.20 h. a=p<.05 from Con 2 (non-injected animals killed at 03.00 h). b=p<.005 from Con 2. Eight to 9 animals per group. Values are expressed as means ~ standard errors.
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10000
Male Rats o
I--I Intact
~
AdrenalX
80006000-
"0 ~0_
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(D_
2000 Z
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Minutes After Saline Injection
FIG. 3 Effect of saline injection on pineal NAT activity in intact and adrenalectomized rats. a=p<.05 versus non-injected intact animals. Eight animals per group. Values are expressed as means ~ standard error.
8000-
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r~1 Intact
5v7,1AdrenalX
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.% . ~
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.G
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2000 -
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Minutes After Saline Injection FIG. 4 Effect of saline injection on pineal melatonin content in intact and adrenalectomized rats. a=p<.005 versus adrenalectomized control animals. Eight animals per group. Values are expressed as means standard errors.
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The p i n e a l g l a n d is g e n e r a l l y c o n s i d e r e d to be o u t s i d e of t h e b l o o d - b , ~ a i n - b a r r i e r . T h e r e seems to b e , h o w e v e r , a specific u p t a k e s y s t e m in t h e s y m p a t h e t i c n e r v e e n d i n g s t h a t p r o t e c t t h e p i n e a i o c y t e s from c i r c u l a t i n g catecholamines (22). A f t e r 20 rain of immobilization s t r e s s , it was shown t h a t t h e r e is no c h a n g e in NE, E, a n d DA c o n t e n t of t h e p i n e a l (23). Stimulation of t h e B - r e c e p t o r s in t h e p i n e a l t y p i c a l l y i n c r e a s e s p i n e a l NAT a c t i v i t y , so t h e i n c r e a s e in c i r c u l a t i n g catecholamines p r o d u c e d b y s t r e s s would s e e m i n g l y not a c t on t h e s e r e c e p t o r s to d e c r e a s e the a c t i v i t y of the a c e t y l a t i n g enzyme. a - A d r e n e r g i c r e c e p t o r s h a v e b e e n i d e n t i f i e d in t h e p i n e a l , b u t t h e s e seem to a c t to p o t e n t i a t e B - r e c e p t o r s t i m u l a t e d NAT a c t i v i t y (24). It may b e , h o w e v e r , t h a t catecholamines a r e a c t i n g t h r o u g h an u n e x p e c t e d mechanism to d e p r e s s melatonin c o n t e n t a n d NAT a c t i v i t y in the r a t p i n e a l . S t e r o i d s , s u c h as c o r t i c o s t e r o n e , a r e lipid soluble a n d c r o s s t h e blood-brain-barrier easily. If c o r t i c o s t e r o n e ( o r a n o t h e r s t e r o i d from t h e a d r e n a l ) is e l i c i t i n g t h e d e p r e s s i o n in NAT a c t i v i t y s e e n a f t e r saline i n j e c t i o n , t h e n it may a c t e i t h e r c e n t r a l l y or d i r e c t l y on t h e p i n e a l . It is well e s t a b l i s h e d t h a t plasma ACTH i n c r e a s e s a f t e r e t h e r s t r e s s (20,25). A f t e r i n t r a p e r i t o n e a l injection of 1 ml s a l i n e , h o w e v e r , p i n e a l ACTH l e v e l s a r e d r a m a t i c a l l y r e d u c e d (26) b y 10 rain. a f t e r i n j e c t i o n . The s o u r c e of p i n e a l ACTH is not known a n d what r o l e , if a n y it has in d e t e r m i n i n g s e r o t o n i n metabolism in t h e p i n e a l gland remains e s s e n t i a l l y u n i n v e s t i g a t e d . Acknowledgements This w o r k was s u p p o r t e d b y NSF g r a n t #PCM 8410592. B. Joshi was s u p p o r t e d b y a g r a n t from t h e g o v e r n m e n t of I n d i a . F. N ~ i r n b u r g e r was s u p p o r t e d b y a g r a n t from D e u t s c h e s F o r s c h u n g e m e i n s c h a f t . The a u t h o r s wish to t h a n k Mrs. V a l e n t i n a Golovko f o r h e r help with t h e a s s a y s . P o r t i o n s of t h e s e r e s u l t s were p r e s e n t e d a t t h e S o c i e t y f o r N e u r o s c i e n c e meeting (1985) in Dallas, T e x a s . S e n d r e p r i n t r e q u e s t s to Dr. R. J. R e i t e r , D e p a r t m e n t of Cellular a n d S t r u c t u r a l Biology, The U n i v e r s i t y of T e x a s Health Science C e n t e r a t San Antonio, San Antonio, T e x a s 78284.
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R. J. R e i t e r , The H a m s t e r , p p . 99-118 Plenum, H. I. Siegal, e d . , New York (1985). J. A x e l r o d , H. M. S h e i n , a n d R. J. Wurtman, Proc. Natl. A c a d . Sci. U . S . A . 62 655-659 (1969). D. C. Klein, a n d J. C. Weller, Science 169 1093-1094 (1970). J. A x e l r o d , a n d H. Weissbach, Science 131 1312 (1960). T. D e g u c h i , a n d J. A x e l r o d , Proc. Natl. A c a d . Sci. U . S . A . 69 2547-2550 (1972). A. P a r f i t t , J. L. Weller, a n d D. C. Klein, N e u r o p h a r m . 1_55353 (1976). R. K v e t n a n s k y , Catecholamlnes a n d S t r e s s : R e c e n t A d v a n c e s , E. U s d i n , R. K v e t n a n s k y a n d I. J. Kopin, e d s . , p p 7-20 E l s e v i e r / N o r t h Holland, New York (1980). T. H. C h a m p n e y , R. W. S t e g e r , D. S. C h r i s t i e , a n d R. J. R e i t e r , B r a i n Res. 338 25-32 (1985). H. J. L y n c h , J. P. Eng, a n d R. J. Wurtman, Proc. Natl. A c a d . Sci. U . S . A . 70 1704-1707 (1973). H. J. L y n c h , M. Ho, a n d R. J. Wurtman, J. N e u r a l T r a n s m . 40 87-97 (1977). H. A. Welker, a n d L. V o l l r a t h , J. N e u r a l T r a n s m . 59 69-80 (1994).
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39-49 (1985). G. M. Vaughan, J. P. Alien, W. Tullis, J. W. Sackman and M. K. Vaughan, Neurosci. Lett. 9 83-97 (1978). J. Milin, J. Martinovic, and M. Demajo, A r c h . d ' A n a t . Micro. 73 159-165
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T. H. Champney, A. P. Holtorf, R. W. Steger, and R. J. Reiter, J__uNeurosci. Res. 1159-66 (1984). T. Deguchi, and J. Axelrod, Anal. Biochem. 50174-178 (1972). P. U. Witte, and H. Matthaei, Mikrochemische Methoden f~ir Neurobiologische Untersuchungen, 90pp Springer-Verlag, Berlin (1980). E. S. Panke, M. D. Rollag, and R. J. Reiter, EndocrinoloLrv 104194-197 (1979). M. D. Rollag, and G. D. Niswender, Endocrinology9__88482-499 (1976). D. M. Cook, J. W. Kendall, M. A. Greer, and R. M. Kramer, Endocrinology 9/3 1019-1024 (1973). J. Seggie, and G. M. Brown, Brain Peptides an.d Hormones, R. Col/u, e d . , pp 277-285 Raven, New York (1992). A. G. Parfitt, and D. C. Klein, EndocrinologT 99 840-851 (1976). J. M. Saavedra, N. Barden, C. Chevillard, and J. F e r n a n d e z - P a r d a l , Cell. Mol. Neurobiol. 2 1-10 (1982). D. Sudgen, J. L. Welier, D. C. Klein, K. L. Kirk, and C. R. Creveling, Biochem. Pharmacol. 33 3947-3950 (1984). M. M. Wilson, S. E. Greer, and M. A. Greer, Am. J. Physiol. 244 E186-E189 (1983). O. Vuolteenaho, and J. Leppaluoto, Acta Physiol. Scand. 112 491-492 (1981).
Pergamon Press
ADRENAL-MEDIATED DEPRESSION OF N-ACETYLTRANSFERASE ACTIVITY AND MELATONIN L E V E L S IN T H E R A T P I N E A L G L A N D Bhaskar N. Joshi, M a u r a e n E. Trolani, Josif Milin, Frank N~Irnburger, and Russel J. Relier Department of Cellular and Structural Biology, T h e University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas (Received in final form February ii, 1986) Summary
N-acetyltransferase (NAT) is believed to be the rate-llmiting enzyme in the synthesis of melatonin from serotonln in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h - lights on at 07.00 h). To test this more thoroughly, a time course study Was conducted during the dark phase (at 02.00 h 5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Soma factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland. Melatonin, which seems to be the major endocrine product of the pineal (1), is produced in the gland from the amino acid tryptophan (2). Tryptophan is taken u p from the blood a n d converted into serotonin (5-hydroxytryptemine). Serotonin is then converted to N-acetyiserotonin (3). This step utilizes the e n z y m e N-acetyltransferase ( N A T ) , which seems to be the rate-limiting e n z y m e in melatonin synthesis (3). T h e last step, utilizing the e n z y m e hydroxyindole-O-methyltransferase (HIOMT), consists of the O-methylation of N-acetylserotonin to form melatonin (4). Since it has been s h o w n that norepinephrine (NE) released from sympathetic nerve endings within the rat pineal gland act on D-receptors to 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
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s t i m u l a t e NAT a c t i v i t y a n d , t h e r e f o r e , m e l a t o n i n s y n t h e s i s ( 5 , 6 ) , i t h a s b e e n of g r e a t i n t e r e s t to s e e if c a t e c h o l a m i n e s f r o m t h e a d r e n a l m e d u l l a c a n e f f e c t pineal melatonin synthesis. I t w o u l d b e e x p e c t e d t h a t t h e i n c r e a s e in p l a s m a c a t e c h o l a m i n e s o r i g i n a t i n g from t h e a d r e n a l d u r i n g s t r e s s ( 7 , 8 ) w o u l d r e a c h t h e p i n e a l g l a n d a n d a c t on t h e B - r e c e p t o r s to i n c r e a s e p i n e a l m e l a t o n i n synthesis. D u r i n g t h e l i g h t p h a s e of t h e l i g h t / d a r k c y c l e in t h e r a t , t h e e f f e c t of s t r e s s on t h e p i n e a l g l a n d h a d n o t b e e n a s c l e a r c u t a s was e x p e c t e d . P h y s i c a l immobilization f o r 2 h o u r s h a s b e e n s h o w n b o t h to i n c r e a s e NAT a c t i v i t y a n d m e l a t o n i n c o n t e n t ( 9 , 1 0 ) a n d to d e c r e a s e NAT a c t i v i t y ( 1 1 ) . I n s u l i n i n d u c e d h y p o g l y c e m i a h a s b e e n s h o w n to i n c r e a s e NAT a c t i v i t y a n d m e l a t o n i n c o n t e n t b y 2 h o u r s a f t e r i n s u l i n i n j e c t i o n ( 9 , 8 ) , while t h e same t r e a t m e n t was s h o w n to d e c r e a s e NAT a c t i v i t y in a n o t h e r s t u d y ( 1 1 ) . This treatment has been interpreted as a stressor because plasma catecholamines are e l e v a t e d ( 8 ) . A h i g h c a r b o h y d r a t e d i e t , w h i c h c a u s e s t h e animal to h a v e h i g h i n s u l i n l e v e l s w i t h o u t h y p o g l y c e m i a , h a s no e f f e c t on p i n e a l NAT a c t i v i t y ( 9 ) . F o o d d e p r i v a t i o n , w h i c h c a u s e s h y p o g l y c e m i a w i t h low i n s u l i n l e v e l s h a s b e e n s h o w n to e l i c i t a d e c r e a s e in NAT a c t i v i t y ( 1 1 ) . A t 1-4 h o u r s a f t e r l i g h t s o n , a n i m a l s s u b j e c t e d to 3 rain of cold w a t e r , n o i s e , n o v e l e n v i r o n m e n t , o r e t h e r v a p o r s h o w e d a n i n c r e a s e in p i n e a l m e l a t o n i n c o n t e n t ( 1 2 ) , a n d a n i n c r e a s e in NAT a c t i v i t y a f t e r 20 rain of e t h e r s t r e s s ( 1 3 ) . At 1 hour or 5 hours after l i g h t s o u t , h o w e v e r , a s l i g h t b u t n o n - s i g n i f i c a n t d e c r e a s e in NAT a c t i v i t y was o b s e r v e d a f t e r 20 rain of e t h e r s t r e s s ( 1 3 ) . R e c e n t l y , Milin e t al s h o w e d t h a t a 1 . 5 c c i n j e c t i o n of p h y s i o l o g i c a l s a l i n e i n t o t h e h i n d l e g c a u s e d m o r p h o l o g i c a l c h a n g e s in t h e p i n e a l g l a n d w h i c h w e r e i n t e r p r e t e d a s e v i d e n c e t h a t t h e s e c r e t o r y a c t i v i t y of t h e p i n e a l g l a n d was increased (14). T h i s t r e a t m e n t was p r e s u m a b l y a s t r e s s o r b e c a u s e t h e s k i n of t h e r a t ' s b a c k l e g is t i g h t a n d a l a r g e volume of f l u i d i n j e c t e d s u b c u t a n e o u s l y in t h i s a r e a w o u l d p r o b a b l y c a u s e d i s c o m f o r t . A p r e l i m i n a r y s t u d y d o n e in t h i s l a b o r a t o r y s h o w e d , u n e x p e c t e d l y , t h a t t h i s t r e a t m e n t l a t e in t h e l i g h t p h a s e d i d in f a c t e l i c i t a d e c r e a s e in m e l a t o n i n c o n t e n t of t h e p i n e a l b y 10 rain after the saline injection (unpublished observations). S i n c e i t is n o t k n o w n , in g e n e r a l , w h a t t h e e f f e c t s of s t r e s s a r e on t h e p i n e a l g l a n d , a n d s p e c i f i c a l l y , w h a t e f f e c t s t r e s s h a s on t h e p i n e a l g l a n d d u r i n g t h e d a r k p h a s e of t h e l i g h t / d a r k c y c l e , i t was d e e m e d i m p o r t a n t to e x a m i n e t h e e f f e c t of t h i s model of s t r e s s on t h e p i n e a l g l a n d of t h e r a t w h e n m e l a t o n i n s y n t h e s i s is n e a r i t s p e a k , d u r i n g t h e d a r k p h a s e of t h e l i g h t / d a r k c y c l e . Materials and Methods A d u l t male S p r a g u e - D a w l e y r a t s , w e i g h i n g a p p r o x i m a t e l y 150 g r a t t h e time of t h e e x p e r i m e n t , w e r e o b t a i n e d from H a r l a n S p r a g u e - D a w l e y , H o u s t o n , Texas. Animals w e r e h o u s e d 4 p e r c a g e a n d r e c e i v e d food a n d w a t e r a d l i b i t u m . T h e y w e r e e x p o s e d to a 14/10 l i g h t / d a r k c y c l e ( l i g h t s on a t 07.00 h ) w i t h c o n s t a n t t e m p e r a t u r e (22 + 2 ° C ) . For each experiment, the experimental a n i m a l s r e c e i v e d a 1 . 5 c c s u b c u t a n e o u s i n j e c t i o n of n o r m a l (0.85%) s a l i n e on t h e d o r s a l a s p e c t of t h e h i n d l e g . C o n t r o l animals r e m a i n e d u n t r e a t e d . I n e x p e r i m e n t 1, 51 r a t s w e r e i n j e c t e d w i t h s a l i n e a t 02.20 h . Eight u n t r e a t e d animals w e r e k i l l e d b y d e c a p i t a t i o n a s c o n t r o l s a t 02.00 a n d 9 w e r e k i l l e d a s c o n t r o l s a t 03.00. T h e i n j e c t e d a n i m a l s w e r e d e c a p i t a t e d in g r o u p s ( 8 - 9 p e r g r o u p ) a t 10, 20, 30, 40, 60, a n d 90 rain a f t e r s a l i n e i n j e c t i o n . P i n e a l g l a n d s w e r e r e m o v e d b y r a p i d d i s s e c t i o n a n d f r o z e n on s o l i d CO 2 . T h e y w e r e p l a c e d in p r e c o o l e d 1500 ul m i c r o f u g e t u b e s a n d s t o r e d a t -70°C u n t i l a s s a y e d f o r NAT a c t i v i t y a n d m e l a t o n i n c o n t e n t .
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In the second experiment, 24 rats were bilaterally adrenalectomized and maintained on a drinking water solution of 1% saline until the time of the experiment. Eight untreated intact and 8 untreated adrenalectomized animals were killed by decapitation at 03.00 h as controls. The remJ~ining adrenalectomized rats as well as 16 intact animals were injected at 02.30 h and killed (8 per group) 10 and 20 minutes later. Pineals were dissected, stored, and assayed for N A T activity and meiatonin content. In experiment 3, 20 animals were injected with saline at 01.15 h. Ten untreated controls were killed at 01.30. Injected animals were killed (in groups of 10) 15 and 30 min after injection. Pineals were dissected, stored and assayed for N A T activity and melatonin content. T r u n k blood was coliected at the time of decapitation in heparinized 12 X 75 nun glass tubes and centrifuged at 3,000 r p m for 15 min to separate the plasma. Plasma was transferred to fresh glass 12 X 75 m m tubes and frozen at -20°C until assayed for corticosterone concentration. Pineal N A T activity and melatonin content were determined in the same pineal gland b y the method of C h a m p n e y and colleagues (15). Pineal glands were homogenized b y sonication in 100 ul of 0.05 M phosphate buffered saline. Twenty ul of this homogenate were added to a 1500 ul microfuge tube and assayed for N A T activity (16,17). Fifty ul of the homogenate were used for duplicate measurements of melatonin content b y radioimmunoassay (18,19). Piasma corticosterone was assayed using a radioimmunoassay kit from Radioassay Systems Laboratories, Carson, CA. Five hundred ~I of diluted plasma (1:250) or standard were added to glass 10 X 75 m m tubes. The 7 standards ranged from 0 to 2.0 n g corticosteroneJml. Four additional tubes were labeled total counts and nonspecific binding (NSB) in duplicate. The N S B tubes contained 600 ul of the 0 ng corticosterone/ml standard. The total tubes had 500 ul of the 0 n g corticosterone/mi standard. The samples and standards were run in duplicate. All of the tubes were incubated at 98°C for 10 min to denature the corticosterone binding 3Proteins. The tubes were allowed to cool to room temperature and 100 ~I of H-corticosterene were added to all of the tubes. All tubes except the N S B tubes then had 100 ul of corticosterone antiserum added. All tubes were then incubated overnight at 4°C and the free corticosterone was separated out with charcoal-dextran. The supernatant (bound corticosterone-antibody complex) was decanted and dpms were counted in a scintillation counter. Statistical analyses of the data were performed using a one w a y analysis of variance with significant differences between groups determined b y the Student-Newman-Keuls test. Values are expressed as means + standard errors. Results Experiment 1." Pineal N A T activity was depressed b y 20 min after injection (p<.025) and remained so through 60 min after the saline injection (p<.001). B y 90 min after the saline injection, pineal N A T activity had returned to control values (fig. 1). Pineal melatonin was depressed similarly with its lowest value at 20 min (p<.01) and a return to control values b y 60 rain (fig. 2). E x p e r i m e n t 2__L T h e s a l i n e - i n j e c t e d i n t a c t a n i m a l s e x h i b i t e d a s i g n i f i c a n t d e p r e s s i o n i n NAT a c t i v i t y ( p ( . 0 5 ) b y 20 rain a s c o m p a r e d to n o n - i n j e c t e d intact controls. A d r e n a l e c t o m i z e d a n i m a l s s h o w e d no r e s p o n s e in t e r m s of NAT a c t i v i t y to t h e s a l i n e i n j e c t i o n ( f i g . 3). P i n e a l m e l a t o n i n c o n t e n t of
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s a i i n e - i n j e c t e d i n t a c t animals showed a s l i g h t b u t n o n - s i g n i f i c a n t d e c r e a s e . This was not o n l y p r e v e n t e d b y adrenaiectomy~ b u t t h e r e was a c t u a l l y a s i g n i f i c a n t i n c r e a s e in melatonin c o n t e n t b y 20 rain in a d r e n a l e c t o m i z e d animals in r e s p o n s e to the saline injection ( p ( . 0 0 5 ) ( f i g . 4 ) . E x p e r i m e n t 3_.3: Control animals showed an NAT a c t i v i t y level of 5574 + 388 pM p r o d u c t / p i n e a l / h. I n j e c t e d animals were d e p r e s s e d to 3107 + 715 pM p r o d u c t / p i n e a l / h b y 15 rain a f t e r i n j e c t i o n . This is s i g n i f i c a n t at p<.005 vs c o n t r o l s By 30 rain NAT a c t i v i t y was b a c k up to c o n t r o l v a l u e s . Melatonin showed a s l i g h t b y n o n - s i g n i f i c a n t d e p r e s s i o n b y 15 rain which was r e v e r s e d b y 30 rain. Plasma c o r t i c o s t e r o n e r o s e in the e x p e r i m e n t from the c o n t r o l c o n c e n t r a t i o n of 273 + 146 p g / m l to 456 + 190 p g / m i b y 15 rain a n d to 550 + 191 p g / m l b y 30 r a i n . - T h e s e a r e signfficarTt from c o n t r o l s at p<.025 a n d p < . 0 1 . respectively. Discussion This model of a c u t e s t r e s s p r o d u c e s a d e p r e s s i o n in melatonin p r o d u c t i o n b y t h e p i n e a l g l a n d as m e a s u r e d b y a d e p r e s s i o n in NAT a c t i v i t y ( f i g . 1) a n d melatonin c o n t e n t ( f i g . 2 ) in the p i n e a l . This is i n t e r p r e t e d as a s t r e s s r e s p o n s e b e c a u s e it is a b o l i s h e d in a d r e n a l e c t o m i z e d animals ( f i g s . 3 & 4) a n d it c a u s e s an e l e v a t i o n in plasma c o r t i c o s t e r o n e c o n c e n t r a t i o n . Melatonin, in r e s p o n s e to this s t r e s s o r , does not seem to be as d r a m a t i c a l l y d e p r e s s e d as NAT a c t i v i t y . Since NAT is the p r o p o s e d r a t e - l i m l t i n g enzyme in melatonin s y n t h e s i s , t h i s a b o v e - m e n t i o n e d f i n d i n g seems to be an i n c o n g r u i t y . It may b e , h o w e v e r , t h a t the a c t i v i t y of HIOMT is e f f e c t e d b y this t r e a t m e n t a n d p a r t i a l l y c o u n t e r a c t s the d e p r e s s i o n in NAT activity so that melatonin synthesis is depressed only slightly. A d r e n a i e c t o m i z e d animals seem to have an i n c r e a s e in p i n e a l melatonin a f t e r saline i n j e c t i o n . This also s u p p o r t s the c o n j e c t u r e t h a t HIOMT a c t i v i t y is e f f e c t e d b y t h e t r e a t m e n t to i n c r e a s e melatonin s y n t h e s i s . In a d r e n a l e c t o m i z e d animals t h i s e f f e c t would be u n o p p o s e d b y the f a c t o r from the a d r e n a l c a u s i n g NAT a c t i v i t y d e p r e s s i o n , so melatonin c o n t e n t would i n c r e a s e . Other e x p l a n a t i o n s also e x i s t . It is p o s s i b l e t h a t r e l e a s e of melatonin is d e p r e s s e d b y s t r e s s so t h a t p i n e a l c o n t e n t of melatonin would remain only s l i g h t l y c h a n g e d even t h o u g h s y n t h e s i s is d r a m a t i c a l l y r e d u c e d . These possible e x p l a n a t i o n s have y e t to be i n v e s t i g a t e d . D u r i n g immobilization s t r e s s , plasma e p i n e p h r i n e (E) i n c r e a s e s 40-fold b y 15-20 rain a f t e r o n s e t of the s t r e s s o r ( 7 ) . NE is i n c r e a s e d 6-fold while dopamine (DA) is i n c r e a s e d 3-fold b y the same t r e a t m e n t (7). It has b e e n shown b y a d r e n a i e c t o m y a n d chemical s y m p a t h e c t o m y e x p e r i m e n t s t h a t n e a r l y all c i r c u l a t i n g E comes from t h e a d r e n a l g l a n d s while only 30% of c i r c u l a t i n g NE d e r i v e s from the a d r e n a l s , the r e m a i n d e r b e i n g s e c r e t e d b y s y m p a t h e t i c n e r v e s (7). Plasma c o r t i c o s t e r o n e i n c r e a s e s b y 15 min a f t e r the o n s e t of e i t h e r c o n t i n u o u s e t h e r s t r e s s , 2.5 rain of e t h e r s t r e s s a n d 30 sec of e t h e r s t r e s s (20) and after 3 rain of novel e n v i r o n m e n t , noise or cold (21). A d r e n o c o r t i c o t r o p h i n (ACTH) (20), p r o l a c t i n a n d g r o w t h hormone (21) from t h e p i t u i t a r y gland a r e i n c r e a s e d b y t h e same t r e a t m e n t s . It seems t h a t one of t h e s e s u b s t a n c e s from t h e a d r e n a l s is a c t i n g e i t h e r d i r e c t l y on the p i n e a l g l a n d , c e n t r a l l y to a l t e r the n e u r a l s i g n a l s t r a v e l i n g to the p i n e a l g l a n d , or a c t i n g on the p i t u i t a r y g l a n d which, in t u r n , could a l t e r its s e c r e t i o n of an e n d o c r i n e p r o d u c t which may a c t c e n t r a l l y or d i r e c t l y on the p i n e a l . Likewise, t h e r e a r e o t h e r p o s s i b l e e x p l a n a t i o n s f o r the o b s e r v e d r e s u l t s .
Vol. 38, No. 17, 1986
Adrenal-mediated
Depression of NAT Activity
8000
Male Rats 6000
'o
4000
8
13._ o_
2000
a,b
a,b
20
30
b
7
Con 1
10
Con 2
40
60
90
M i n u t e s A f t e r Saline injection
FIG. i Effect of saline injection on pineal NAT activity. Con i was a non-injected control killed at 02.00. Con 2 was a non-injected control killed at 03.00. All other groups were injected at 02.20 and killed the indicated min after injection, a=p<.025 versus Con i. b=p<.001 versus Con 2. Eight to 9 animals per group. Values expressed as means + standard error.
4000
o
Male Rats
| T
3000
2000 E
~o -~ :E
1000
i
Con 1
10
20
30
Con 2
40
60
90
M i n u t e s A f t e r Saline iniecfion FIG. 2 Effect of saline injection on pineal melatonin content. Con I is the non-injected control group killed at 02.00 h. All experimental groups were injected at 02.20 h. a=p<.05 from Con 2 (non-injected animals killed at 03.00 h). b=p<.005 from Con 2. Eight to 9 animals per group. Values are expressed as means ~ standard errors.
1577
1578
Adrenal-mediated Depression of NAT Activity
Vol. 38, No. 17, 1986
10000
Male Rats o
I--I Intact
~
AdrenalX
80006000-
"0 ~0_
4000-
(D_
2000 Z
10
Con
20
Minutes After Saline Injection
FIG. 3 Effect of saline injection on pineal NAT activity in intact and adrenalectomized rats. a=p<.05 versus non-injected intact animals. Eight animals per group. Values are expressed as means ~ standard error.
8000-
Male Rats
r~1 Intact
5v7,1AdrenalX
6000-
.% . ~
4000-
.G
_,go -~
2000 -
Con
10
20
Minutes After Saline Injection FIG. 4 Effect of saline injection on pineal melatonin content in intact and adrenalectomized rats. a=p<.005 versus adrenalectomized control animals. Eight animals per group. Values are expressed as means standard errors.
Vol. 38, No. 17, 1986
Adrenal-mediated Depression of NAT Activity
1579
The p i n e a l g l a n d is g e n e r a l l y c o n s i d e r e d to be o u t s i d e of t h e b l o o d - b , ~ a i n - b a r r i e r . T h e r e seems to b e , h o w e v e r , a specific u p t a k e s y s t e m in t h e s y m p a t h e t i c n e r v e e n d i n g s t h a t p r o t e c t t h e p i n e a i o c y t e s from c i r c u l a t i n g catecholamines (22). A f t e r 20 rain of immobilization s t r e s s , it was shown t h a t t h e r e is no c h a n g e in NE, E, a n d DA c o n t e n t of t h e p i n e a l (23). Stimulation of t h e B - r e c e p t o r s in t h e p i n e a l t y p i c a l l y i n c r e a s e s p i n e a l NAT a c t i v i t y , so t h e i n c r e a s e in c i r c u l a t i n g catecholamines p r o d u c e d b y s t r e s s would s e e m i n g l y not a c t on t h e s e r e c e p t o r s to d e c r e a s e the a c t i v i t y of the a c e t y l a t i n g enzyme. a - A d r e n e r g i c r e c e p t o r s h a v e b e e n i d e n t i f i e d in t h e p i n e a l , b u t t h e s e seem to a c t to p o t e n t i a t e B - r e c e p t o r s t i m u l a t e d NAT a c t i v i t y (24). It may b e , h o w e v e r , t h a t catecholamines a r e a c t i n g t h r o u g h an u n e x p e c t e d mechanism to d e p r e s s melatonin c o n t e n t a n d NAT a c t i v i t y in the r a t p i n e a l . S t e r o i d s , s u c h as c o r t i c o s t e r o n e , a r e lipid soluble a n d c r o s s t h e blood-brain-barrier easily. If c o r t i c o s t e r o n e ( o r a n o t h e r s t e r o i d from t h e a d r e n a l ) is e l i c i t i n g t h e d e p r e s s i o n in NAT a c t i v i t y s e e n a f t e r saline i n j e c t i o n , t h e n it may a c t e i t h e r c e n t r a l l y or d i r e c t l y on t h e p i n e a l . It is well e s t a b l i s h e d t h a t plasma ACTH i n c r e a s e s a f t e r e t h e r s t r e s s (20,25). A f t e r i n t r a p e r i t o n e a l injection of 1 ml s a l i n e , h o w e v e r , p i n e a l ACTH l e v e l s a r e d r a m a t i c a l l y r e d u c e d (26) b y 10 rain. a f t e r i n j e c t i o n . The s o u r c e of p i n e a l ACTH is not known a n d what r o l e , if a n y it has in d e t e r m i n i n g s e r o t o n i n metabolism in t h e p i n e a l gland remains e s s e n t i a l l y u n i n v e s t i g a t e d . Acknowledgements This w o r k was s u p p o r t e d b y NSF g r a n t #PCM 8410592. B. Joshi was s u p p o r t e d b y a g r a n t from t h e g o v e r n m e n t of I n d i a . F. N ~ i r n b u r g e r was s u p p o r t e d b y a g r a n t from D e u t s c h e s F o r s c h u n g e m e i n s c h a f t . The a u t h o r s wish to t h a n k Mrs. V a l e n t i n a Golovko f o r h e r help with t h e a s s a y s . P o r t i o n s of t h e s e r e s u l t s were p r e s e n t e d a t t h e S o c i e t y f o r N e u r o s c i e n c e meeting (1985) in Dallas, T e x a s . S e n d r e p r i n t r e q u e s t s to Dr. R. J. R e i t e r , D e p a r t m e n t of Cellular a n d S t r u c t u r a l Biology, The U n i v e r s i t y of T e x a s Health Science C e n t e r a t San Antonio, San Antonio, T e x a s 78284.
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