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Growth hormone secretion of the neonatal rat pituitaries is stimulated by gamma-aminobutyric acid in vitro
Life Sciences, Vol. 34, pp. 1505-1511 Printed in the U.S.A.
Pergamon Press
GROWTH HORMONE SECRETION OF THE NEONATAL RAT PITUITARIES IS STIMULATED BY GAMMA-AMINOBUTYRIC ACID IN VITRO
Z s u z s a n n a /~cs, G ~ b o r B. M a k a r a a n d E r v i n S t a r k I n s t i t u t e of E x p e r i m e n t a l Medicine of t h e H u n g a r i a n A c a d e m y o f S c i e n c e s H- ] 4 50 B u d a p e s t P . O . B ; 67. H u n g a r y (Received in final form November 8, 1984)
SUMMARY Growth hormone secretion from pituitaries of neonatal rats was stimulated by gamma-aminobutyric acid (GABA) and the GABA agonist muscimol in vitro. This response to GABA was absent after the 9th postnatal day. The stimulation of growth hormone secretion by GABA was antagonized by bicuculline-methiodide and by picrotoxin. Diazepam stimulated while baclophen had no effect on growth hormone secretion. This stimulatory GABA effect might be related to a certain developmental stage of the pituitary GABA receptors or to the lack of hypothalamic regulatory influence(s) in the newborn. T h e p a t t e r n of g r o w t h h o r m o n e (GH) s e c r e t i o n in n e o n a t a l r a t s is s i g n i f i c a n t l y d i f f e r e n t from t h a t o b s e r v e d in a d u l t s . In n e o n a t e s s e r u m GH l e v e l s a r e r e l a t i v e l y h i g h ( 1 - 6 ) a n d f a i r l y c o n s t a n t (4 , 7 , 8 ) . T h e h i g h plasma GH l e v e l of n e w b o r n r a t s p r o b a b l y r e f l e c t s a g e n e r a l i z e d p h e n o m e n o n s i n c e si m i l ar o b s e r v a t i o n s h a v e b e e n made in t h e mouse ( 9) , d o g ( 1 0 ) , s h e e p ( 1 1 ) , p i g (12) a n d in man ( 1 3 ) . F a c t o r s r e g u l a t i n g t h e p a t t e r n of GH s e c r e t i o n in e a r l y i n f a n c y a r e still l a r g e l y u n k n o w n . S u b s t a n c e s k n o w n to a f f e c t GH s e c r e t i o n in t h e r a t become e f f e c t i v e o n l y a f t e r c e r t a i n s t a g e s of d e v e l o p m e n t , o r t h e y elicit c h a n g e s in t h e 6:H s e c r e t i o n of n e o n a t e s q u a l i t a t i v e l y d i f f e r e n t from t h o s e s e e n in t h e a d u l t s . F o r e x a m p l e , in c o n t r a s t to t h e a d u l t r a t , t h y r o t r o p i n r e l e a s i n g h o r m o n e s t i m u l a t e s (8, 14-16) while s o m a t o s t a t i n is w i t h o u t e f f e c t (15,16) on GH s e c r e t i o n o f n e o n a t a l r a t s . C l o n i d i n e a n d p e n t o b a r b i t a l a r e i n h i b i t o r y in n e o n a t e s a n d s t i m u l a t o r y in a d u l t s ( 7 ) . Gamma-aminobutyric acid (GABA), the putative inhibitory neurotransmitter, influences GH secretion in the rat (for review see 17, 18) . In adult rats, GABA has been shown both to stimulate (19-22) and to inhibit (22-24) GH secretion. The hypothalamusis believed tobe the main site of action of GABA on Gll secretion since GABA affected neither basal nor stimulated GH secretion of anterior pituitary lobes in vitro (10, 24, 25) . A f t e r t h e u n e x p e c t e d o b s e r v a t i o n (/~cs, u n p u b l i s h e d ) t h a t t w e n t y m i n u t e s a f t e r s u b c u t a n e o u s i n j e c t i o n of 0 . 5 mg GABA to n e w b o r n r a t s , plasma GH l e v e l s w e r e s i g n i f i c a n t l y h i g h e r t h a n in c o n t r o l s , it s e e m e d of i n t e r e s t to s t u d y t h e e f f e c t o f GABA on t h e p i t u i t a r y GH s e c r e t i o n in t h e p o s t n a t a l p e r i o d .
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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MATERIALS AND METHODS Two days old female CFY r a t s of our i n b r e d colony were used in these studies. In the experiment shown in Fig.2 r a t s of various ages were used. The morning on which the pups were found in the cage of the mother was considered f i r s t postnatal day. The neonates were left u n d i s t u r b e d with t h e i r mothers until the experiment.
Whole p i t u i t a r y ffl~nds from t h e d e c a p i t a t e d n e w b o r n r a t s w e r e i n c u b a t e d at 37°C in o x y g e n / c a r b o n d i o x i d e ( 9 5 : 5 v / v ) a t m o s p h e r e with g e n t l e s h a k i n g in 0.5 ml of K r e b s - R i n g e r b i c a r b o n a t e b u f f e r c o n t a i n i n g D - g l u c o s e (2 g / L ) a n d b o v i n e s e r u m albumin (0.5%: C a l b i o c h e m ) , pH 7 . 4 . A f t e r two e q u i l i b r a t i o n p e r i ods of 60 rain e a c h , t h e p i t u i t a r y g l a n d s w e r e i n c u b a t e d f o r one h o u r with t h e a p p r o p r i a t e c o n c e n t r a t i o n of t h e t e s t s u b s t a n c e s in t h e same b u f f e r . T h e t e s t s u b s t a n c e s w e r e a d d e d to t h e p i t u i t a r i e s a c c o r d i n g to a r a n d o m i z e d b l o c k d e s i g n . In some of t h e e x p e r i m e n t s with b i c u c u l l i n e m e t h i o d i d e ( p r e p a r e d a c c o r d i n g to 26) a n d p i c r o t o x i n ( B H D ) , a f t e r two e q u i l i b r a t i o n p e r i o d s t h e p i t u i t a r i e s w e r e p r e i n c u b a t e d f i r s t with t h e a n t a g o n i s t f o r one h o u r , a n d t h e n i n c u b a t e d f o r one h o u r with t h e a g o n i s t a n d th e a n t a g o n i s t p r e s e n t s i m u l t a n e o u s l y in c o n c e n t r a t i o n s i n d i c a t e d in T a b l e s II a n d I l l . At t h e e n d of t h e i n c u b a t i o n p e r i o d t h e medium was t r a n s f e r e d to c h i l l e d t u b e s , c e n t r i f u g e d at 4 ° C at 2500 r p m f o r 5 m i n u t e s . Th e s u p e r n a t a n t was a s p i r a t e d a n d s t o r e d at -20°C u n t i l a s s a y e d for GH. T h e amount of GH s e c r e t e d was d e t e r m i n e d b y r a d i o i m m u n o a s s a y u s i n g t h e r e a g e n t s k i n d l y p r o v i d e d b y NIAMDD a n d G H - R P - 1 as r e f e r e n c e p r e p a r a t i o n . B o u n d a n d f r e e h o r m o n e w e r e s e p a r a t e d b y a 20% w / v s u s p e n s i o n of h e a t - a n d f o r m a l d e h y d e i n a c t i v a t e d S t a p h y l o c o c c u s a u r e u s ( B a c t - A - S o r b , Human I n s t i t u t e for S e r o b a c t e r i o l o g i c a l R e s e a r c h an d P r o d u c t i o n , B u d a p e s t , H u n g a r y ) . Hormone a s s a y data s h o w e d an a p p r o x i m a t e l y l o g normal d i s t r i b u t i o n a n d w e r e f u r t h e r a n a l y s e d a f t e r log t r a n s f o r m a t i o n ( 2 7 ) . T w o - or t h r e e - w a y a n a l y s i s of v a r i a n c e followed b y D u n n e t t ' s t e s t was u s e d to evaluate statistical significance. FIG. 1 A
~ >-
100-
50-
g 20-
i//+ I
l~
¢
]
10-6
I
10-$
r
10-/.
I
10-3 rnot/k
B NM
2oo
+I
_
100
~
so
';
r
I
I
10-7 I0-6 I0-5 II0-4 I~)-3 rnol/L
Growth hormone secretion by t h e p i t u i t a r i e s of two d a y s old female r a t s in t h e p r e s e n c e of various concentrations of GABA (A) or muscimol ( B ) . Each point r e p r e s e n t s 6-10 determinations. Values are geometric means of n g GH secreted/ pituitary/ hour with RSEM (see Methods). * :p<0.05 ** : p < 0.01
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GABA Stimulates Neonatal GH Secretion
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RESULTS B o t h G A B A a n d t h e p o t e n t G A B A a g o n i s t m u s c i m o l e n h a n c e d GH s e c r e t i o n of the neonatal pituitary glands in vitro (Fig; 1). The effect of both drugs was dose dependent. Not o n l y p i t u i t a r i e s f r o m f e m a l e ( F i g . 1) b u t a l s o t h o s e f r o m m a l e n e o n a t e s ( d a t a n o t s h o w n ) r e s p o n d e d t o G A B A w i t h e n h a n c e d GH s e c r e t i o n . GABA antagonists, in equal concentrations to GABA were tested for their e f f e c t o n t h e GH r e s p o n s e t o G A B A . P i c r o t o x i n s h o w e d s i g n i f i c a n t ( p < 0 . 0 1 ) i n h i b i t i o n , b u t s t r y c h n i n e a n d b i c u c u l l i n e m e t h i o d i d e h a d n o e f f e c t ( T a b l e 1) . B i cuculline methiodide rather than bicuculline was used since the latter is hydrolyzed to the relatively i n a c t i v e b i c u c i n e a t 37°C a n d p h y s i o l o g i c a l p H , w i t h a half-life of only a few minutes (28) . TABLE I Effect of GABA on GH Secretion of Neonatal Pituitaries in the Presence of GABA Antagonists
n Bicuculline
16
Buffer without with antagonist
GABA without with antagonist
63.5
82.9
165.2
140.5
(1.19)
(1.18)
(1.09)
(1.15)
Picrotoxin
10
38.1 (1.21)
56.6 (1.19)
185.2 (1.12)
112.5 (1.14)
Strychnine
14
65.8 ( 1. ]4)
71.5 ( ] . 20)
153.3 (1.14)
193.6 (1.05)
All d r u g s w e r e t e s t e d in a c o n c e n t r a t i o n o f 10 . 4 m o l / L . V a l u e s a r e g e o m e t r i c m e a n s o f n g GH s e c r e t e d / p i t u i t a r y / h o u r w i t h t h e RSEM ( s e e M e t h o d s ) in p a r e n t h e s e s . Statistics: two way ANOVA revealing GABA effect (p<0.01) in all three experiments and its interaction with picrotoxin (p<0.01) . The effect of bicuculline methiodide and strychnine is not significant. T h e e f f e c t o f G A B A o n GH s e c r e t i o n w a s p r e v e n t e d b y b o t h b i c u c u l l i n e m e t h i o d i d e a n d p i c r o t o x i n if t h e c o n c e n t r a t i o n o f t h e a n t a g o n i s t s e x c e e d e d t h a t of the agonist tenfold and if the antagonists w e r e p r e s e n t f o r o n e h o u r p r i o r to G A B A a d m i n i s t r a t i o n a s well a s d u r i n g t h e i n c u b a t i o n p e r i o d ( T a b l e II a n d I I I ) . T A B L E II Effect of GABA on Growth Hormone Secretion of Neonatal Pituitaries after Preincubation and Coincubation with Bicuculline Methiodide GABA concentration n
@
(mol/L)
10 - 5
10 - 4
Control
12
88.3 ( 1. ] 9)
143.7 (1.13)
132.9 (1.07)
Bicuculline (10-4mol/L)
12
97.0 (1.21)
81.7 (1.13)
150.7 (1.08)
V a l u e s a r e g e o m e t r i c m e a n s of n g GH s e c r e t e d / p i t u i t a r y / h o u r with RSEM ( s e e M e t h o d s ) in p a r e n t h e s e s . Statistics: ANOVA revealing a significant interaction between GABA and bicuculline methiodide (p
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GABA Stimulates Neonatal GH Secretion
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TABLE III Effect of GABA on Growth Hormone Secretion of Neonatal Pituitaries a f t e r Preincubation and Coincubation with Picrotoxin Picrotoxin concentration (mol/L) ~ i0- ~ 3.33 i0- ~
n Cont rol
I0
GABA (10-~mol/L)
I0
10-3
129.4
148.3
96.7
126,4
(1.17)
(1.21)
(1.15)
(1.12)
295.2 ( i. 12)
182.8 (1. i0)
142.6 (1.09)
123.6 (1.12)
Values are geometric m e a n s of n g G H secreted/pituitary/hour with R S E M (see Methods) in parentheses. Statistics: A N O V A revealing a significant interaction between G A B A a n d picrotoxin (p<0.05). T h e Ctl s e c r e t i o n o f t h e to a similar extent to that of e f f e c t i v e o n t h e p i t u i t a r y GH s a l GH s e c r e t i o n increased shown ) .
seven and nine days old rats was enhanced by GABA the two days old animals. GABA, however, was ins e c r e t i o n i I a n d 13 d a y s p o s t p a r t u m ( F i g . 2 ) . B a w i t h a g e ( F i g . 2) a n d p i t u i t a r y w e i g h t ( d a t a n o t
From the GABAergic d r u g s t e s t e d diazepam increased GH secretion of t h e neonatal p i t u i t a r i e s ; Baclophen was without effect (Table I V ) . The putative n e u r o t r a n s m i t t e r amino acids, glycine, L-alanine, t a u r i n e and glutamic acid in concentrations of 10-4 mol/L were ineffective (data not s h o w n ) .
T
L,O0 0 "1-
ill
3O0
-
i
YX
//. Y/
200-
V/
-ii i11
VZ
Y/ i11 i11 .-/i i/i i11 i11
i//A
,//.~
..
50Y/, 2
..f
,,,
.f
9
7 DAY S
. f l / ,~r
11
1"
"f,,/,f
13
POSTPARTUM
FIG. 2 Growth hormone secretion of p i t u i t a r i e s of r a t s of various ages in the presence of 10-4 tool GABA/L. Values are geometric means of n g GH s e c r e t e d / p i t u i t a r y / h o u r with RSEM (see Methods). Open b a r s r e p r e s e n t controls, h a t c h e d b a r s GABA effect. Number in t h e columns denote number of determinations. * : p < 0.05; ** : p < 0.01
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TABLE IV E f f e c t of G A B A e r g i c D r u g s on G r o w t h Hormone S e c r e t i o n o f P i t u i t a r i e s of Two D a y s Old Female R a t s Expt. No.
n 6
i0
Control
GABA
306.9 (1.25)
651.5"* (1.23)
686.6** (1.12)
264.5**
N .D .
95.0
(1.06)
Diazepam
Baclophen
(1.08)
N.D.
89.5 (i .08)
V a l u e s a r e g e o m e t r i c m e a n s of n g GH s e c r e t e d / p i t u i t a r y / h o u r with RSEM ( s e e M e t h o d s ) in p a r e n t h e s e s . All d r u g s w e r e t e s t e d in a c o n c e n t r a t i o n of ]0 -4 m o l / L . * * : p < 0 . 0 1 v . s . r e s p e c t i v e c o n t r o l . N . D . : not determined. DISCUSSION T h e r e s u l t s show t h a t GABA h a s a d i r e c t s t i m u l a t o r y e f f e c t on p i t u i t a r y GH s e c r e t i o n in t h e f i r s t d a y s of p o s t n a t a l d e v e l o p m e n t , an e f f e c t a b s e n t in t h e a d u l t p i t u i t a r y (10, 2 4 , 2 5 ) . T h e m e c h a n i s m of t h i s u n u s u a l GABA e f f e c t d e m o n s t r a t e d in t h e n e o n a t a l p i t u i t a r i e s , r e m a i n s to be c l a r i f i e d , a n d a n y e x p l a n a t i o n is h i g h l y s p e c u l a t i v e . It is u n l i k e l y t h a t t h e e f f e c t of GABA was m e d i a t e d b y some n e u r o h y p o p h y s e a l m e c h a n i s m , s i n ce GABA e f f e c t i v e l y s t i m u l a t e d GH s e c r e t i o n of t h e i s o l a t e d a n t e r i o r p i t u i t a r y l o b e s of two d a y s old r a t s ( d a t a n o t s h o w n ) . T h e r e a r e at l e a s t t h r e e e n d o g e n o u s i n h i b i t o r s of GABA b i n d i n g which a r e n a t u r a l l y i n c o r p o r a t e d in cell m e m b r a n e s a n d m o d u l a t e t h e a f f i n i t y a n d t h e t o t al n u m b e r of a v a i l a b l e r e c e p t o r s i t e s f o r G A B A. T h e p r o t e i n i n h i b i t o r GABAmodulin h a s b e e n s h o w n to be a b s e n t from t h e c e n t r a l n e r v o u s s y s t e m GABA r e c e p t o r s in e a r l y s t a g e s of d e v e l o p m e n t ( 2 9 ) . T h e r e f o r e GABAmodulin m i g h t not b e p r e s e n t a f t e r b i r t h on t h e GABA r e c e p t o r s of t h e p i t u i t a r y GH s e c r e t i n g c e l l s , p r o v i d i n ~ GABA with a f r e e a c c e s s to t h e b i n d i n g s i t e s of t h e GH c e l l s . In t h e a d u l t , GABA b i n d i n g s i t e s of t h e GH c e l l s m i g h t be o c c u p i e d b y G A BA m o d u l i n p r e v e n t i n g GABA b i n d i n g a n d c o n s e q u e n t l y t h e d i r e c t GABA e f f e c t on GH s e c r e t i o n . On t h e o t h e r h a n d , GABA m ig h t i n d u c e i n t r a c e l l u l a r e h a n ~ e s in t h e n e o n a t a l GH s e c r e t i n g cells wh i c h d i f f e r e n t from t h o s e in a d u l t s . E f f e c t s of GABA a r e in g e n e r a l i n h i b i t o r y ; h o w e v e r GABA r e l e a s e s c a t e c h o l a m i n e s from t h e i s o l a t e d , p e r f u s e d b o v i n e a d r e n a l m e d u l l a (30) a n d from t h e p o s t e r i o r a r e a of t h e c a t ' s h y p o t h a l a m u s ( 3 1 ) . T h e s e GABA a c t i o n s w h i c h a r e r e v e r s i b l y a n t a g o n i z e d b y p i c r o t o x i n a n d b i c u c u l l i n e b u t n o t b y s t r y c h n i n e (30, 3 1 ) , r e s e m b l e t h e p r o p e r t i e s of t h e GH s t i m u l a t i n g e f f e c t of GABA in n e o n a t a l pituitaries. Some f e a t u r e s of t h e n e o n a t a l GH s e c r e t o r y p a t t e r n r e s e m b l e t h o s e s e e n a f t e r p l a c i n g an a n t e r o l a t e r a l c u t a r o u n d t h e medial b a s a l h y p o t h a l a m u s . A f t e r t h i s s u r g i c a l i n t e r v e n t i o n , similar to t h a t in t h e n e o n a t a l r a t s , h y p o t h a l a m i c so m a t o s t a t i n c o n t e n t is low ( 3 2 , 3 3 ) , plasma GH l e v e l is e l e v a t e d ( 1 - 6 , 34) with no s e c r e t o r y e p i s o d e s (4 , 7, 8,34 ) . P e n t o b a r b i t a l k n o w n to i n c r e a s e GH s e c r e t i o n in t h e i n t a c t a d u l t r a t (35) i n h i b i t s GH s e c r e t i o n b o t h in n e o n a t a l r a t s (7) a n d a f t e r a n t e r o l a t e r a l d e a f f e r e n t a t i o n of t h e medial b a s a l h y p o t h a l a m u s of a d u l t r a t s ( K a k u c s k a et al. in p r e p a r a t i o n ) . One e x p l a n a t i o n f o r o u r r e s u l t s may b e t h a t
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GABA Stimulates Neonatal GH Secretion
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34, No. 16, 1984
in t h e n e o n a t a l r a t i n h i b i t o r y h y p o t h a l a m i c f a c t o r s may n o t y e t be r e g u l a t i n g t h e p i t u i t a r y GH s e c r e t i o n (] 5, 16) , allowing t h e s t i m u l a t o r y m ech an i sm o f GABA to be u n m a s k e d . T h i s e f f e c t will be a b o l i s h e d d u r i n g d e v e l o p m e n t , b y t h e m a n i f e s t a tion of h y p o t h a l a m i c r e g u l a t o r y m e c h a n i s m s alone or in c o n j u n c t i o n with t h e i n c o r p o r a t i o n of GABA modulin i n t o t h e m e m b r a n e GABA r e c e p t o r s . It is n o t k n o w n at t h i s s t a ~ e w h e t h e r t h e r e m o v a l of h y p o t h a l a m i c f a c t o r s al o n e will a l t e r t h e a b i l i t y of t h e a n t e r i o r p i t u i t a r y to r e s p o n d to GABA with i n c r e a s e d GH s e c r e t i o n . The physiological significance of this GABA effect in the postnatal development of GH regulation remains to be elucidated. ACKNOWLEDGEME NTS The authors are grateful to Ms M. Pongrhcz, Ms K. Edes and Ms I. R6th~ti for their skillful technical assistance and to Mr G. Folly for mathematical analysis. Bicuculline methiodide was synthetized by Ms ~. Sz~n~ssy. We thank Dr A.F. Parlow and the NIAMDD for GH radioimmunoassay reagents and Dr I.L. Martin Medical Research Council, Cambridge, England for benzodiazepine. REFERENCES 1. 2. 3, 4. 5. 60 7. 8. 9. I0. 11. ]2. 13. 14. 15. 16. ]7. 18. 19. 20. 21. 22.
E. BLAZQUEZ, F.A. SIMON, M. BLAZQUEZand P.P. F O A , Proc. Soc. exptl. Biol. Med. 147 780-783 (1974). M. RIEUTORT, J. Endocr. 60 261-268 (1974). M.T. STROSSER and P. MIALHE, Hormone Metab. Res. 7 275-278 (1975). D. COCCHI, I. GIL-AD, A.E. PANERAI, V. LOCATELLTand E.E. MULLER, Life Sci. 19 825-836 (1976). S.L. KAPLAN, M.M. GRUMBACH and M.L. AUBERT, Rec. Prog. Hormone Res. 32 ]61-243 (1976). S. EDEN, Endocrinology 105 555-560 (1979). C . M . KUHN and S.M. SCILIANBERG, J. Pharmacol. exptl. Ther. 217 152156 (1981). V. STRBAK, J. JURCOVICOVAand M. VIGAS, Endocrinol. Experimentalis 15 243-259 (1981). Y~.N.SINHA, F.W. SELBY, U.J. LEWIS and W.P. VANDERLOON, Endocrinology 91 784-792 (1972). T. TSUSHIMA, M. IRIE and M. SAKUMA, Endocrinology 82 685-699 (1971). J.M. BASSETT and G. ALEXANDER, Biologia Neonat. 17 112-125 (1971). K.R. SWIATEK, D.M. KIPNIS, G. MASON, KUEN LAN CHAO and M. CORNBLATH, Am. J. Physiol. 214 400-405 (1968). S . M . GLICK, J. ROTH, R.S. YALOWand S.A. BERSON, Nature 199 784787 (1963). I. GIL-AD, D. COCCHI, A.E. PANERAI, V. LOCATELLI, P. MANTEGAZZA and E.E. MULLER, Neuroendocrinology 21 366-378 (1976). M. RIEUTORT, J. Endocr. 89 355-363 (1981). O. KHORRAM, L.R. DEPALATIS and S.M. MCCANN, Fed. Proc. 41 1488 Abst. No. 7120 (1982). G.B. MAKARAand E. STARK, Interactions between Putative Neurotransmitters in the Brain, EDS. S. GARATTINI, J . F . PUJOL and R. SAMANIN, 263-283, Raven Press, N.Y. (1978). G. RACAGNI, J.A. APUD, D. COCCHI, V. LOCATELLIand E.E. MULLER, Life Sci. 31 823-838 (1982). H. ABE, Y. KATO, K. CHIHARA, S. OGHO, Y. IWASAKIand H. IMURA, Endocrinol. Japon. 24 229-231 (1977). E. VIJAYAN and S~M. MCCANN, Endocrinology 103 1888-1893 (1978). J. TAKAHARA, S. YUNOKI, H. HOSOGI, W. YAKUSHIJI, J. KAGEYAMA and T. OFUJI, Endocrinology 106 343-347 (1980). D. COCCHI, F. CASENUEVA, V. LOCATELLI, J. APUD, A. MARTINEZCAMPOS, C. CIVATI, G. RACAGNI and E.E. MULLER, GABA and Benzo-
Vol. 34, No. 16, 1984
23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33. 34. 35.
GABA Stimulates Neonatal GH Secretion
1511
d i a z e p i n e R e c e p t o r s , EDS. E. C O S T A , G. D I C H I A R A a n d G . L . G E S S A , 247-259, R a v e n P r e s s , N . Y . ( 1 9 8 1 ) . J . F . B R U N I , R . J . MIODUSZEWSKI, L . J . GRANDISON, J . W . SIMPKINS a n d J . MEITES, F e ~ . P r o c . 36 323 A b s t . No. 285 ( 1 9 7 7 ) . J . FIOK, ZS; ACS a n d E; S T A R K , J . E n d o c r . 91 391-397 ( 1 9 8 1 ) . A. N E G R O - V I L A R , E. VIJAYAN a n d S.M. MCCANN, B r a i n R e s . Bu l l . 5, 239-244 ( 1 9 8 0 ) . S . F . PONG a n d L . T . GRAHAM, B r a i n R e s . 58 266-267 ( 1 9 7 3 ) . L. K R U L I C H , E. HEFCO a n d P . ILLNER, N e u r o e n d o c r i n o l o g y 16 293-311 ( 1974 ) . R.W. OLSEN, M. BAU, T . MILLER a n d G . A . R . J O H N T O N , B r a i n R e s . 98 383-387 ( 1 9 7 5 ) . J . M . P A L A C I O S , D . L . NICHOFF a n d M . J . KUHAR, B r a i n R e s . 179 390-387 (1979). S. SANGIAH, J . L . BOROWITZ a n d G . K . W . YIM, E u r . J . P h a r m a c o l . 27 130-135 ( 1 9 7 4 ) . A. P HI LI P P U , H. PRZUNTEK a n d W. R O S E N B E R G , N a u n y n - S c h m i e d e b e r g ' s A r c h . P h a r m a c o l . e x p t l . P a t h o l . 276 103-118 ( 1 9 7 3 ) . M. BROWNSTEIN, A. ARIMURA, R. F E R N A N D E Z - D U R A N G O , A.V. S C H A L LY, M. PALKOVITS a n d J . S . KIZER, E n d o c r i n o l o g y 100 24 6 - 2 4 9 ( 1 9 7 7 ) . P. WALKER, J.H. DUSSAULT, G. ALVARADO-URBIN-A--and A. DUPONT, Endocrinology 10] 782-787 (1977). M. KARTESZI, J. FIOK and G.B. MAKARA, J. Endocr. 94 77-81 (]982). J.B. MARTIN, Neuroendocrinology13 339-350 (1973-74).
Pergamon Press
GROWTH HORMONE SECRETION OF THE NEONATAL RAT PITUITARIES IS STIMULATED BY GAMMA-AMINOBUTYRIC ACID IN VITRO
Z s u z s a n n a /~cs, G ~ b o r B. M a k a r a a n d E r v i n S t a r k I n s t i t u t e of E x p e r i m e n t a l Medicine of t h e H u n g a r i a n A c a d e m y o f S c i e n c e s H- ] 4 50 B u d a p e s t P . O . B ; 67. H u n g a r y (Received in final form November 8, 1984)
SUMMARY Growth hormone secretion from pituitaries of neonatal rats was stimulated by gamma-aminobutyric acid (GABA) and the GABA agonist muscimol in vitro. This response to GABA was absent after the 9th postnatal day. The stimulation of growth hormone secretion by GABA was antagonized by bicuculline-methiodide and by picrotoxin. Diazepam stimulated while baclophen had no effect on growth hormone secretion. This stimulatory GABA effect might be related to a certain developmental stage of the pituitary GABA receptors or to the lack of hypothalamic regulatory influence(s) in the newborn. T h e p a t t e r n of g r o w t h h o r m o n e (GH) s e c r e t i o n in n e o n a t a l r a t s is s i g n i f i c a n t l y d i f f e r e n t from t h a t o b s e r v e d in a d u l t s . In n e o n a t e s s e r u m GH l e v e l s a r e r e l a t i v e l y h i g h ( 1 - 6 ) a n d f a i r l y c o n s t a n t (4 , 7 , 8 ) . T h e h i g h plasma GH l e v e l of n e w b o r n r a t s p r o b a b l y r e f l e c t s a g e n e r a l i z e d p h e n o m e n o n s i n c e si m i l ar o b s e r v a t i o n s h a v e b e e n made in t h e mouse ( 9) , d o g ( 1 0 ) , s h e e p ( 1 1 ) , p i g (12) a n d in man ( 1 3 ) . F a c t o r s r e g u l a t i n g t h e p a t t e r n of GH s e c r e t i o n in e a r l y i n f a n c y a r e still l a r g e l y u n k n o w n . S u b s t a n c e s k n o w n to a f f e c t GH s e c r e t i o n in t h e r a t become e f f e c t i v e o n l y a f t e r c e r t a i n s t a g e s of d e v e l o p m e n t , o r t h e y elicit c h a n g e s in t h e 6:H s e c r e t i o n of n e o n a t e s q u a l i t a t i v e l y d i f f e r e n t from t h o s e s e e n in t h e a d u l t s . F o r e x a m p l e , in c o n t r a s t to t h e a d u l t r a t , t h y r o t r o p i n r e l e a s i n g h o r m o n e s t i m u l a t e s (8, 14-16) while s o m a t o s t a t i n is w i t h o u t e f f e c t (15,16) on GH s e c r e t i o n o f n e o n a t a l r a t s . C l o n i d i n e a n d p e n t o b a r b i t a l a r e i n h i b i t o r y in n e o n a t e s a n d s t i m u l a t o r y in a d u l t s ( 7 ) . Gamma-aminobutyric acid (GABA), the putative inhibitory neurotransmitter, influences GH secretion in the rat (for review see 17, 18) . In adult rats, GABA has been shown both to stimulate (19-22) and to inhibit (22-24) GH secretion. The hypothalamusis believed tobe the main site of action of GABA on Gll secretion since GABA affected neither basal nor stimulated GH secretion of anterior pituitary lobes in vitro (10, 24, 25) . A f t e r t h e u n e x p e c t e d o b s e r v a t i o n (/~cs, u n p u b l i s h e d ) t h a t t w e n t y m i n u t e s a f t e r s u b c u t a n e o u s i n j e c t i o n of 0 . 5 mg GABA to n e w b o r n r a t s , plasma GH l e v e l s w e r e s i g n i f i c a n t l y h i g h e r t h a n in c o n t r o l s , it s e e m e d of i n t e r e s t to s t u d y t h e e f f e c t o f GABA on t h e p i t u i t a r y GH s e c r e t i o n in t h e p o s t n a t a l p e r i o d .
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Stimulates
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GH S e c r e t i o n
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MATERIALS AND METHODS Two days old female CFY r a t s of our i n b r e d colony were used in these studies. In the experiment shown in Fig.2 r a t s of various ages were used. The morning on which the pups were found in the cage of the mother was considered f i r s t postnatal day. The neonates were left u n d i s t u r b e d with t h e i r mothers until the experiment.
Whole p i t u i t a r y ffl~nds from t h e d e c a p i t a t e d n e w b o r n r a t s w e r e i n c u b a t e d at 37°C in o x y g e n / c a r b o n d i o x i d e ( 9 5 : 5 v / v ) a t m o s p h e r e with g e n t l e s h a k i n g in 0.5 ml of K r e b s - R i n g e r b i c a r b o n a t e b u f f e r c o n t a i n i n g D - g l u c o s e (2 g / L ) a n d b o v i n e s e r u m albumin (0.5%: C a l b i o c h e m ) , pH 7 . 4 . A f t e r two e q u i l i b r a t i o n p e r i ods of 60 rain e a c h , t h e p i t u i t a r y g l a n d s w e r e i n c u b a t e d f o r one h o u r with t h e a p p r o p r i a t e c o n c e n t r a t i o n of t h e t e s t s u b s t a n c e s in t h e same b u f f e r . T h e t e s t s u b s t a n c e s w e r e a d d e d to t h e p i t u i t a r i e s a c c o r d i n g to a r a n d o m i z e d b l o c k d e s i g n . In some of t h e e x p e r i m e n t s with b i c u c u l l i n e m e t h i o d i d e ( p r e p a r e d a c c o r d i n g to 26) a n d p i c r o t o x i n ( B H D ) , a f t e r two e q u i l i b r a t i o n p e r i o d s t h e p i t u i t a r i e s w e r e p r e i n c u b a t e d f i r s t with t h e a n t a g o n i s t f o r one h o u r , a n d t h e n i n c u b a t e d f o r one h o u r with t h e a g o n i s t a n d th e a n t a g o n i s t p r e s e n t s i m u l t a n e o u s l y in c o n c e n t r a t i o n s i n d i c a t e d in T a b l e s II a n d I l l . At t h e e n d of t h e i n c u b a t i o n p e r i o d t h e medium was t r a n s f e r e d to c h i l l e d t u b e s , c e n t r i f u g e d at 4 ° C at 2500 r p m f o r 5 m i n u t e s . Th e s u p e r n a t a n t was a s p i r a t e d a n d s t o r e d at -20°C u n t i l a s s a y e d for GH. T h e amount of GH s e c r e t e d was d e t e r m i n e d b y r a d i o i m m u n o a s s a y u s i n g t h e r e a g e n t s k i n d l y p r o v i d e d b y NIAMDD a n d G H - R P - 1 as r e f e r e n c e p r e p a r a t i o n . B o u n d a n d f r e e h o r m o n e w e r e s e p a r a t e d b y a 20% w / v s u s p e n s i o n of h e a t - a n d f o r m a l d e h y d e i n a c t i v a t e d S t a p h y l o c o c c u s a u r e u s ( B a c t - A - S o r b , Human I n s t i t u t e for S e r o b a c t e r i o l o g i c a l R e s e a r c h an d P r o d u c t i o n , B u d a p e s t , H u n g a r y ) . Hormone a s s a y data s h o w e d an a p p r o x i m a t e l y l o g normal d i s t r i b u t i o n a n d w e r e f u r t h e r a n a l y s e d a f t e r log t r a n s f o r m a t i o n ( 2 7 ) . T w o - or t h r e e - w a y a n a l y s i s of v a r i a n c e followed b y D u n n e t t ' s t e s t was u s e d to evaluate statistical significance. FIG. 1 A
~ >-
100-
50-
g 20-
i//+ I
l~
¢
]
10-6
I
10-$
r
10-/.
I
10-3 rnot/k
B NM
2oo
+I
_
100
~
so
';
r
I
I
10-7 I0-6 I0-5 II0-4 I~)-3 rnol/L
Growth hormone secretion by t h e p i t u i t a r i e s of two d a y s old female r a t s in t h e p r e s e n c e of various concentrations of GABA (A) or muscimol ( B ) . Each point r e p r e s e n t s 6-10 determinations. Values are geometric means of n g GH secreted/ pituitary/ hour with RSEM (see Methods). * :p<0.05 ** : p < 0.01
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GABA Stimulates Neonatal GH Secretion
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RESULTS B o t h G A B A a n d t h e p o t e n t G A B A a g o n i s t m u s c i m o l e n h a n c e d GH s e c r e t i o n of the neonatal pituitary glands in vitro (Fig; 1). The effect of both drugs was dose dependent. Not o n l y p i t u i t a r i e s f r o m f e m a l e ( F i g . 1) b u t a l s o t h o s e f r o m m a l e n e o n a t e s ( d a t a n o t s h o w n ) r e s p o n d e d t o G A B A w i t h e n h a n c e d GH s e c r e t i o n . GABA antagonists, in equal concentrations to GABA were tested for their e f f e c t o n t h e GH r e s p o n s e t o G A B A . P i c r o t o x i n s h o w e d s i g n i f i c a n t ( p < 0 . 0 1 ) i n h i b i t i o n , b u t s t r y c h n i n e a n d b i c u c u l l i n e m e t h i o d i d e h a d n o e f f e c t ( T a b l e 1) . B i cuculline methiodide rather than bicuculline was used since the latter is hydrolyzed to the relatively i n a c t i v e b i c u c i n e a t 37°C a n d p h y s i o l o g i c a l p H , w i t h a half-life of only a few minutes (28) . TABLE I Effect of GABA on GH Secretion of Neonatal Pituitaries in the Presence of GABA Antagonists
n Bicuculline
16
Buffer without with antagonist
GABA without with antagonist
63.5
82.9
165.2
140.5
(1.19)
(1.18)
(1.09)
(1.15)
Picrotoxin
10
38.1 (1.21)
56.6 (1.19)
185.2 (1.12)
112.5 (1.14)
Strychnine
14
65.8 ( 1. ]4)
71.5 ( ] . 20)
153.3 (1.14)
193.6 (1.05)
All d r u g s w e r e t e s t e d in a c o n c e n t r a t i o n o f 10 . 4 m o l / L . V a l u e s a r e g e o m e t r i c m e a n s o f n g GH s e c r e t e d / p i t u i t a r y / h o u r w i t h t h e RSEM ( s e e M e t h o d s ) in p a r e n t h e s e s . Statistics: two way ANOVA revealing GABA effect (p<0.01) in all three experiments and its interaction with picrotoxin (p<0.01) . The effect of bicuculline methiodide and strychnine is not significant. T h e e f f e c t o f G A B A o n GH s e c r e t i o n w a s p r e v e n t e d b y b o t h b i c u c u l l i n e m e t h i o d i d e a n d p i c r o t o x i n if t h e c o n c e n t r a t i o n o f t h e a n t a g o n i s t s e x c e e d e d t h a t of the agonist tenfold and if the antagonists w e r e p r e s e n t f o r o n e h o u r p r i o r to G A B A a d m i n i s t r a t i o n a s well a s d u r i n g t h e i n c u b a t i o n p e r i o d ( T a b l e II a n d I I I ) . T A B L E II Effect of GABA on Growth Hormone Secretion of Neonatal Pituitaries after Preincubation and Coincubation with Bicuculline Methiodide GABA concentration n
@
(mol/L)
10 - 5
10 - 4
Control
12
88.3 ( 1. ] 9)
143.7 (1.13)
132.9 (1.07)
Bicuculline (10-4mol/L)
12
97.0 (1.21)
81.7 (1.13)
150.7 (1.08)
V a l u e s a r e g e o m e t r i c m e a n s of n g GH s e c r e t e d / p i t u i t a r y / h o u r with RSEM ( s e e M e t h o d s ) in p a r e n t h e s e s . Statistics: ANOVA revealing a significant interaction between GABA and bicuculline methiodide (p
1508
GABA Stimulates Neonatal GH Secretion
Vol. 34, No. 16, 1984
TABLE III Effect of GABA on Growth Hormone Secretion of Neonatal Pituitaries a f t e r Preincubation and Coincubation with Picrotoxin Picrotoxin concentration (mol/L) ~ i0- ~ 3.33 i0- ~
n Cont rol
I0
GABA (10-~mol/L)
I0
10-3
129.4
148.3
96.7
126,4
(1.17)
(1.21)
(1.15)
(1.12)
295.2 ( i. 12)
182.8 (1. i0)
142.6 (1.09)
123.6 (1.12)
Values are geometric m e a n s of n g G H secreted/pituitary/hour with R S E M (see Methods) in parentheses. Statistics: A N O V A revealing a significant interaction between G A B A a n d picrotoxin (p<0.05). T h e Ctl s e c r e t i o n o f t h e to a similar extent to that of e f f e c t i v e o n t h e p i t u i t a r y GH s a l GH s e c r e t i o n increased shown ) .
seven and nine days old rats was enhanced by GABA the two days old animals. GABA, however, was ins e c r e t i o n i I a n d 13 d a y s p o s t p a r t u m ( F i g . 2 ) . B a w i t h a g e ( F i g . 2) a n d p i t u i t a r y w e i g h t ( d a t a n o t
From the GABAergic d r u g s t e s t e d diazepam increased GH secretion of t h e neonatal p i t u i t a r i e s ; Baclophen was without effect (Table I V ) . The putative n e u r o t r a n s m i t t e r amino acids, glycine, L-alanine, t a u r i n e and glutamic acid in concentrations of 10-4 mol/L were ineffective (data not s h o w n ) .
T
L,O0 0 "1-
ill
3O0
-
i
YX
//. Y/
200-
V/
-ii i11
VZ
Y/ i11 i11 .-/i i/i i11 i11
i//A
,//.~
..
50Y/, 2
..f
,,,
.f
9
7 DAY S
. f l / ,~r
11
1"
"f,,/,f
13
POSTPARTUM
FIG. 2 Growth hormone secretion of p i t u i t a r i e s of r a t s of various ages in the presence of 10-4 tool GABA/L. Values are geometric means of n g GH s e c r e t e d / p i t u i t a r y / h o u r with RSEM (see Methods). Open b a r s r e p r e s e n t controls, h a t c h e d b a r s GABA effect. Number in t h e columns denote number of determinations. * : p < 0.05; ** : p < 0.01
Vol.
34, No.
16, 1984
GABA Stimulates
Neonatal
GH Secretion
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TABLE IV E f f e c t of G A B A e r g i c D r u g s on G r o w t h Hormone S e c r e t i o n o f P i t u i t a r i e s of Two D a y s Old Female R a t s Expt. No.
n 6
i0
Control
GABA
306.9 (1.25)
651.5"* (1.23)
686.6** (1.12)
264.5**
N .D .
95.0
(1.06)
Diazepam
Baclophen
(1.08)
N.D.
89.5 (i .08)
V a l u e s a r e g e o m e t r i c m e a n s of n g GH s e c r e t e d / p i t u i t a r y / h o u r with RSEM ( s e e M e t h o d s ) in p a r e n t h e s e s . All d r u g s w e r e t e s t e d in a c o n c e n t r a t i o n of ]0 -4 m o l / L . * * : p < 0 . 0 1 v . s . r e s p e c t i v e c o n t r o l . N . D . : not determined. DISCUSSION T h e r e s u l t s show t h a t GABA h a s a d i r e c t s t i m u l a t o r y e f f e c t on p i t u i t a r y GH s e c r e t i o n in t h e f i r s t d a y s of p o s t n a t a l d e v e l o p m e n t , an e f f e c t a b s e n t in t h e a d u l t p i t u i t a r y (10, 2 4 , 2 5 ) . T h e m e c h a n i s m of t h i s u n u s u a l GABA e f f e c t d e m o n s t r a t e d in t h e n e o n a t a l p i t u i t a r i e s , r e m a i n s to be c l a r i f i e d , a n d a n y e x p l a n a t i o n is h i g h l y s p e c u l a t i v e . It is u n l i k e l y t h a t t h e e f f e c t of GABA was m e d i a t e d b y some n e u r o h y p o p h y s e a l m e c h a n i s m , s i n ce GABA e f f e c t i v e l y s t i m u l a t e d GH s e c r e t i o n of t h e i s o l a t e d a n t e r i o r p i t u i t a r y l o b e s of two d a y s old r a t s ( d a t a n o t s h o w n ) . T h e r e a r e at l e a s t t h r e e e n d o g e n o u s i n h i b i t o r s of GABA b i n d i n g which a r e n a t u r a l l y i n c o r p o r a t e d in cell m e m b r a n e s a n d m o d u l a t e t h e a f f i n i t y a n d t h e t o t al n u m b e r of a v a i l a b l e r e c e p t o r s i t e s f o r G A B A. T h e p r o t e i n i n h i b i t o r GABAmodulin h a s b e e n s h o w n to be a b s e n t from t h e c e n t r a l n e r v o u s s y s t e m GABA r e c e p t o r s in e a r l y s t a g e s of d e v e l o p m e n t ( 2 9 ) . T h e r e f o r e GABAmodulin m i g h t not b e p r e s e n t a f t e r b i r t h on t h e GABA r e c e p t o r s of t h e p i t u i t a r y GH s e c r e t i n g c e l l s , p r o v i d i n ~ GABA with a f r e e a c c e s s to t h e b i n d i n g s i t e s of t h e GH c e l l s . In t h e a d u l t , GABA b i n d i n g s i t e s of t h e GH c e l l s m i g h t be o c c u p i e d b y G A BA m o d u l i n p r e v e n t i n g GABA b i n d i n g a n d c o n s e q u e n t l y t h e d i r e c t GABA e f f e c t on GH s e c r e t i o n . On t h e o t h e r h a n d , GABA m ig h t i n d u c e i n t r a c e l l u l a r e h a n ~ e s in t h e n e o n a t a l GH s e c r e t i n g cells wh i c h d i f f e r e n t from t h o s e in a d u l t s . E f f e c t s of GABA a r e in g e n e r a l i n h i b i t o r y ; h o w e v e r GABA r e l e a s e s c a t e c h o l a m i n e s from t h e i s o l a t e d , p e r f u s e d b o v i n e a d r e n a l m e d u l l a (30) a n d from t h e p o s t e r i o r a r e a of t h e c a t ' s h y p o t h a l a m u s ( 3 1 ) . T h e s e GABA a c t i o n s w h i c h a r e r e v e r s i b l y a n t a g o n i z e d b y p i c r o t o x i n a n d b i c u c u l l i n e b u t n o t b y s t r y c h n i n e (30, 3 1 ) , r e s e m b l e t h e p r o p e r t i e s of t h e GH s t i m u l a t i n g e f f e c t of GABA in n e o n a t a l pituitaries. Some f e a t u r e s of t h e n e o n a t a l GH s e c r e t o r y p a t t e r n r e s e m b l e t h o s e s e e n a f t e r p l a c i n g an a n t e r o l a t e r a l c u t a r o u n d t h e medial b a s a l h y p o t h a l a m u s . A f t e r t h i s s u r g i c a l i n t e r v e n t i o n , similar to t h a t in t h e n e o n a t a l r a t s , h y p o t h a l a m i c so m a t o s t a t i n c o n t e n t is low ( 3 2 , 3 3 ) , plasma GH l e v e l is e l e v a t e d ( 1 - 6 , 34) with no s e c r e t o r y e p i s o d e s (4 , 7, 8,34 ) . P e n t o b a r b i t a l k n o w n to i n c r e a s e GH s e c r e t i o n in t h e i n t a c t a d u l t r a t (35) i n h i b i t s GH s e c r e t i o n b o t h in n e o n a t a l r a t s (7) a n d a f t e r a n t e r o l a t e r a l d e a f f e r e n t a t i o n of t h e medial b a s a l h y p o t h a l a m u s of a d u l t r a t s ( K a k u c s k a et al. in p r e p a r a t i o n ) . One e x p l a n a t i o n f o r o u r r e s u l t s may b e t h a t
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in t h e n e o n a t a l r a t i n h i b i t o r y h y p o t h a l a m i c f a c t o r s may n o t y e t be r e g u l a t i n g t h e p i t u i t a r y GH s e c r e t i o n (] 5, 16) , allowing t h e s t i m u l a t o r y m ech an i sm o f GABA to be u n m a s k e d . T h i s e f f e c t will be a b o l i s h e d d u r i n g d e v e l o p m e n t , b y t h e m a n i f e s t a tion of h y p o t h a l a m i c r e g u l a t o r y m e c h a n i s m s alone or in c o n j u n c t i o n with t h e i n c o r p o r a t i o n of GABA modulin i n t o t h e m e m b r a n e GABA r e c e p t o r s . It is n o t k n o w n at t h i s s t a ~ e w h e t h e r t h e r e m o v a l of h y p o t h a l a m i c f a c t o r s al o n e will a l t e r t h e a b i l i t y of t h e a n t e r i o r p i t u i t a r y to r e s p o n d to GABA with i n c r e a s e d GH s e c r e t i o n . The physiological significance of this GABA effect in the postnatal development of GH regulation remains to be elucidated. ACKNOWLEDGEME NTS The authors are grateful to Ms M. Pongrhcz, Ms K. Edes and Ms I. R6th~ti for their skillful technical assistance and to Mr G. Folly for mathematical analysis. Bicuculline methiodide was synthetized by Ms ~. Sz~n~ssy. We thank Dr A.F. Parlow and the NIAMDD for GH radioimmunoassay reagents and Dr I.L. Martin Medical Research Council, Cambridge, England for benzodiazepine. REFERENCES 1. 2. 3, 4. 5. 60 7. 8. 9. I0. 11. ]2. 13. 14. 15. 16. ]7. 18. 19. 20. 21. 22.
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