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Dopamine-inhibited adenylate cyclase in female rat adenohypophysis
Life Sciences, Vol. 28, pp. 1605-1612 Printed in the U.S.A.
Pergamon Press
DOPAMINE-INHIBITED ADENYLATE CYCLASE IN FEMALE RAT ADENOHY POPHYS I S Giuliana Giannattasio, Maria Elisabetta De F e r r a r i and Anna Spada* CNR Center of Cytopharmacology and *Endocrine Unit, II Medical C l i n i c , University of Milano, Via V a n v i t e l l i 3 2 , 20129 Milano, Italy. (Received in final form January 28, 1981)
Summary A dopamine-inhibited adenylate cyclase has been demonstrated in a n t e r i o r pituitary gland of adult female r a t s , lactating and not lactating. This i n h i bitory effect was completely GTP dependent. In c o n t r a s t , in the adenohypophysis of male r a t s , dopamine had no detectable effect on adenylate cyclase activity. In female rats the inhibition of the enzyme appears mediated by specific dopaminergic r e c e p t o r s : the effect of dopamine was mimicked by the dopaminergic agonists apomorphine and the ergot derivative CH 29-717, while norepinephrine was much l e s s potent. On the other hand, the dopaminergic antagonists t r i f l u o p e r a z i n e and sulpiride competitively antagonized the dopamine inhibition of the adenylate c y c l a s e . The p o s s i bility that the dopamine-inhibited enzyme is located in mammotrophs appears supported 1) by its observation in the female rat p i t u i t a r y , which contains this type of cells in much l a r g e r proportion than the male gland (33-38% vs. < 5%); 2) by the pharmacological similarity between the dopaminergic r e ceptors mediating the adenylate cyclase inhibition (this work) and those regulating prolactin r e l e a s e (which have been c h a r a c t e r i z e d in previous studies). The well known inhibition of prolactin r e l e a s e brought about by dopamine could therefore be mediated, at least in p a r t , by a d e c r e a s e in the i n t r a c e l l u l a r level of cAMP. Dopamine (DA) r e l e a s e d by the tuberoinfundibular hypothalamic n e u r o n s into the hypophyseal portal blood is known to play a key role in the regulation of the pituitary function, p a r t i c u l a r l y as an inhibitor of prolactin (PRL) s e c r e t i o n (1). It is conceivable therefore that mammotrophic cells possess specific DA r e c e p t o r s . Binding studies c a r r i e d out on pituitary homogenates and membrane p r e p a r a t i o n s , using labeled agonists and a n t a g o n i s t s , have indeed revealed the existence in the gland of specific dopaminergic sites (2-7). Although this kind of study does not provide informations about the cell type containing these s i t e s , the similarity between the pharmacological p r o p e r ties of the sites identified by the use of dopaminergic r a d i o l i g a n d s , such as [3H] d i h y d r o e r g o c r y p t i n e , and the c h a r a c t e r i s t i c s of the dopaminergic inhibit{on of PRL s e c r e tion, strongly suggests a localization of DA r e c e p t o r s in mammotrophic cells (5). This conclusion is also supported by immunocytochemical data (8). 0024-3205/81/141605-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Pituitary DA-Inhibited Adenylate Cyclase
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An i n t e r e s t i n g problem r a i s e d by the existence of DA r e c e p t o r s in mammotrophs concerns the i n t r a c e l l u l a r mechanisms of transduction or r e c e p t o r activation. Previous studies failed to reveal the existence of a DA-linked adenylate cyclase (AC) in the a n t e r i o r pituitary gland (9-12). As a c o n s e q u e n c e , the DA r e c e p t o r s of mammotrophs labeled with[3H]dihydroergocryptine have been r e c e n t l y proposed by Kebabian and Calne as prototypes or a c l a s s of DA r e c e p t o r s (designed a s D-2 r e c e p t o r s ) whose activation is not coupled to an i n c r e a s e of cyclic AMP (cAMP) synthesis (11). However, it should be considered that these negative r e s u l t s were obtained using the total pituitary gland, i . e . a very heterogeneous tissue; if DA did affect AC activity of a single cell type, this effect could have gone undetected because basal AC activity was contributed p r i m a r i l y by the other cell types. It is known that the relative number of mammotrophs in the pituitary gland v a r i e s according to age, sex and physiological conditions of the animal (13). Thus the relative contribution of these cells to the AC activity of the gland may be expected to vary in parallel with their number. On this b a s i s , the possible existence of a D A - s e n s i t i v e AC in mammotrophs has been here reinvestigated by studying the effect of DA on the AC a c tivity of a n t e r i o r pituitary homogenates obtained from animals with a physiologically high number of P R L - s e c r e t i n g ceils: mature female r a t s , lactating and not lactating (mammotrophs = 38% and 33% of total c e l l s , respectively) ( t ~ , 15). These data on female rats have been compared with other data obtained in p a r a l l e l on male rat adenohypophysis, in which mammotrophs are quite s p a r s e (13, 1L) and which had been used in the previous studies of the l i t e r a t u r e (9-12). Materials and Methods Materials. N , N - d i m e t h y l - N ' - ( 6 - m e t h y l - e r g o l i n e - 8 0 ( - y l ) - s u l f a m i d e hydrochloride (CH 29-717), t r i f l u o p e r a z i n e and ( - ) s u l p i r i d e were kind gifts of Sandoz, B a s e l , S w i t z e r land, Ravizza, Milan, Italy and Maggioni, Milan, I t a l y , respectively; apomorphine was purchased from S1FAC, Milan, Italy; DA, ( - ) n o r e p i n e p h r i n e (NE), adenosine - 3 ' , 5 ' cyclic monophosphate (cAMP), adenosine 5'-triDhosphate (ATP), c r e a t i n e phosphate, c r e a t i n e phosphokinase, g u a n o s i n e - 5 ' - t r i p h o s p h a t e (GTP), ethylene glycol-his (2-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA) and theophylline from Sigma, St Louis, MO. USA; [ 8 - 1 ~ C ] A T P and cyclic [8-3H]AMP from the Radiochemical C e n t e r , Amersham, U.K. Animals. Adult female (220-250 g), lactating or not l a c t a t i n g , and adult male (200220 g) Sprague-Dawley r a t s were purchased from Charles R i v e r , Calco, Italy. They were housed in a light, temperature and humidity controlled room, with food and water ad libitum . Adenylate cyclase a s s a y . Rats were killed by decapitation, the hypophyses quickly removed and the n e u r o i n t e r m e d i a t e lobes d i s c a r d e d . A n t e r i o r lobes were homogenized by a g l a s s - T e f l o n Potter homogenizer in 20 vol or 1 mM T r i s - m a l e a t e buffer (pH 7.&) containing 1 mM EGTA. Most enzyme a s s a y s were c a r r i e d out on the total homogenate, filtered through a nylon gauze. The a s s a y mixture (0.1 ml) contained: 80 mM T r i s - m a l e a t e burrer, pH 7.~,10mM theophylline, 1 mM cAMP, 0.2 mM EGTA, 0.15mM [ 8 - 1 & C ] A T P (&0 dpm/pmol), 1.5 mM MgSO&, 7 mM phosphocreatine, c r e a t i n e phosphokinase (20 U/ml) and 0.01 mM G T P . P r o t e i n concentration was about 1 mg/ml. The r e a c t i o n , started by the addition of the homogenate, was c a r r i e d out for 8 rain at 30°C and stopped by adding
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a solution c o n t a i n i n g 2% sodium d o d e c y l s u l f a t e , 1.~ mM cAMP and 40 mM A T P . In the e x p e r i m e n t s p e r f o r m e d to t e s t the e f f e c t of G T P on the AC a c t i v i t y , a c r u d e membrane p r e p a r a t i o n , sediment ed from the homogenate by c e n t r i f u g a t i o n at 20,000 x g for 10 min, was u s e d . The p e l l e t was r e s u s p e n d e d in T r i s - m a l e a t e b u f f e r - E G T A at a c o n c e n t r a t i o n of about 2 . 5 mg p r o t e i n / m l . The r e a c t i o n m i x t u r e c o n t a i n e d G T P at the c o n c e n t r a t i o n s s p e c i f i e d in the " R e s u l t s " . The final p r o t e i n c o n c e n t r a t i o n was 0 . 5 m g / m l . C y c l i c [ 8 - 1 ~ C ] AMP was i s o l a t e d and m e a s u r e d a c c o r d i n g to Salomon et a l . (16). P r o t e i n was e v a l u a t e d a c c o r d i n g to Lowry et a l . (17). Results The AC a c t i v i t y was i n v e s t i g a t e d in homogenates of a n t e r i o r p i t u i t a r y gland of adult male and a d u l t , l a c t a t i n g and n o n l a c t a t i n g female r a t s . The b a s a l AC a c t i v i t y was s i g n i ficantly h i g h e r in female than in male r a t s . In non l a c t a t i n g female r a t s and, even more, in l a c t a t i n g ones 10-5M DA e l i c i t e d a highly s i g n i f i c a n t d e c r e a s e in the AC a c t i v i t y , while in male r a t s no inhibition was o b s e r v e d (Table I). As shown in figs. i - £ , the i n h i b i t o r y effect of DA on AC a c t i v i t y of female r a t adeno_ h y p o p h y s i s was d o s e d e p e n d e n t . The a p p a r e n t K D was 6. 2 5 + I .3 O x 10 - 7M • The hl"ghest enzyme inhibition o c c u r r e d between 10 -5 and 5 x 10-SM, whi'~e at h i g h e r c o n c e n t r a t i o n s DA e l i c i t e d a slight s t i m u l a t o r y e f f e c t . The effect of DA was mimicked by NE ( F i g . i ) , apomorphine and the w a t e r - s o l u b l e d o p a m i n e r g i c e r g o t CH 29-717 (18) ( F i g . 2), but NE was much l e s s potent ( a p p a r e n t K D = 2.5"< 10-SM) and a p o m o r p h i n e and CH 29-717 more potent than D A ( a p p a r e n t K D ~ 1 . 5 x I0-7M and 1 . 5 x 10-tSM, r e s p e c t i v e l y ) .
TABLE I Effect of Dopamine on Adenyi.ate C y c l a s e A c t i v i t y of Rat A n t e r i o r P i t u i t a r y Gland
Animals
Adenylate C y c l a s e A c t i v i t y pmol c A M P / m g prot x min % inhibition basal IO-SM DA
P
Adult male r a t s (3)
11.7 + 0 . £
10.9 + 0.L
6.8
NS
Adult female r a t s (10)
2 8 . 6 + 1.3 a
17.6 + 0 . 8
38.5
< 0.0001
Lactating r a t s (2)
3 0 . 5 + 1.0 a
15.9 + 0.1
L7.8
< 0.001
A s s a y s w e r e c a r r i e d out on total h o m o g e n a t e . Number of e x p e r i m e n t s , each c a r r i e d out in t r i p l i c a t e , in p a r e n t h e s e s . Values a r e m e a n s + SEM. a p < o . o 0 1 v s . male r a t s .
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22. E
t-
-- 20 E
Fig. 1
x
Effect of dopamine and (-) n o r e p i n e p h r i n e on a d e n y l ate c y c l a s e activity in the total homogenate of non l a c t a t i n g female rat adenoh y p o p h y s i s . The values given a r e the means of 3 determinations + S Eb,I.
18
X u 16
E 14
12
o
1o'-'
10-~
lo'-'
M
32-
~
~
,
.E
•
DOPAMINE
o
APOMORPHINE
~~
CH
E
x 28
29-717
Effect of dopamine, a p o morphine and CH 29-717 on adenylate c y c l a s e a c tivity in the total homogenate of non lactating female rat adenohypop h y s i s . The values given a r e the means of 3 d e t e r minations + SEb4.
~24 ~ 2o 16
r--~/ 0
10 -s
10 -7
Fig. 2
10 -5
1(~-3
M
The dopaminergic antagonist t r i f l u o p e r a z l n e , at the dose of 10 -7 I'd, had no effect on b a s a l AC activity but competitively antagonized the DA action ( F i g . 3). The same r e s u l t was6obtained with another dopaminergic a n t a g o n i s t , (-) s u l p i r i d e , at the c o n c e n t r a t i o n of 10- M ( F i g . g).
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Pituitary DA-Inhibited Adenylate Cyclase
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38l~/~--~...,,p.,~
+
TRIFLUOPERAZINE
C
Fig. 3
E x 34
Effect of d o p a m i n e in the a b s e n c e and p r e s e n c e of 10 - 7 M t r i f l u o p e r a z i n e on adenylate cyclase activity in t h e t o t a l h o m o g e n a t e of non l a c t a t i n g female r a t adenohypophysis. The v a l u e s g i v e n a r e the m e a n s of 3 d e t e r m i n a t i o n s -+ SEN
gm. o.
3o < u
-g 2s O
E
22 r"V/
DOPAMINE
(M)
34~
Fig.
32
\
x 30
28
u 26-
(-) SULPIRIDE
10 -6 M
\
24
E f f e c t of d c p a m i n e in the a b s e n c e and p r e s e n c e of 10 - 6 M ( - ) s u l p i r i d e on adenylate cyclase activity in the t o t a l h o m o g e n a t e of non l a c t a t i n g f e m a l e r a t a d e n o h y p o p h y s i s . The v a l u e s g i v e n a r e the m e a n s of 3 d e t e r m i n a t i o n s + SEM
22 ,.../!
0
14 .7
10 -s DOPAMINE
10 -3 (M)
DA i n h i b i t i o n of AC r e q u i r e d the p r e s e n c e of G T P ( T a b l e I I ) . In fact the c y c l a s e a c t i v i t y of c r u d e m e m b r a n e p r e p a r a t i o n s o b t a i n e d from non l a c t a t i n g f e m a l e r a t p i t u i t a r y h o m o g e n a t e s was not s i g n i f i c a n t l y i n h i b i t e d by DA when G T P was a b s e n t from the i n c u b a t i o n m i x t u r e . When G T P was a d d e d , t h e b a s a l AC a c t i v i t y was g r e a t l y e n h a n c e d and a s i g n i f i c a n t DA i n h i b i t i o n a p p e a r e d ( - g 3 % a t 3 x 10-5M G T P ) . In male r a t s t h e G T P i n c r e a s e of b a s a l a c t i v i t y w a s much s m a l l e r and no s i g n i f i c a n t i n h i b i tion was b r o u g h t aEout by DA at a n y d o s e of G T P .
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TABLE 11 E f f e c t of G T P on A d e n y l a t e C y c l a s e A c t i v i t y of Male and F e m a l e Rat A n t e r i o r P i t u i t a r y G l a n d
Adenylate Cyclase Activity pmol c A M P / m g p r o t x min GTP
(M)
Male r a t s basal
DA
0
/4.2 -+ 0 . 1
4 . 0 -+ 0 . 1
3 x 10 . 7
6 . 2 -+ 0 . 2
6 . 7 -+ 0 . 7
I0 -6
8.7 + -0.6
3 x i0 -6
Female r a t s % inhibition
/4.8 a
basal
DA
% inhibition
9 . 6 -+ 0 . 7
8 . 9 -+ 0 . 7
7.3 a
-
1 6 . 3 -+ 0 . 3
1 4 . 3 + 0./4
12.3 b
+ 7.7 -0.1
ii .5a
2 2 . 9 -+0 . 6
19.7 -+ 0 . 6
1~.0 b
I0.i -+ O.&
9.3 + 0.6
7.9 a
30.2 -+ 0.2
19.0 -+ O.&
37.1
10 - 5
10.5-+ 1.0
9.3 +0.5
11.4 a
32.5 +0.6
1 9 . 3 -+0.1
~0.6 c
3 x 10 - 5
1 0 . 2 -+ 0 . 6
9 . 6 -+ 1 . 3
5.9 a
3 3 . 6 -+ 0 . 6
19.1 2 0 . 3
43.1
10 . 4
10.0 + 0.3
9 . 6 +- 0 . 7
4.0 a
3 2 . 7 -+ 0 . 7
1 9 . 2 -+ 0 . 7
L1.3 c
c
c
A s s a y s w e r e c a r r i e d out on c r u d e m e m b r a n e p r e p a r a t i o i q s . DA was 2 . 5 x 10 - 5 M. •
V a l u e s a r e the m e a n s o f 3 d e t e r m i n a n o n s
+
- SEM.
a
NS;
D
P<'0.02;
C
P<0.001.
Discussion O u r d a t a d e m o n s t r a t e c l e a r d i f f e r e n c e s in t h e p i t u i t a r y AC of male and f e m a l e r a t s . In m a l e s t h e b a s a l a c t i v i t y i s low a n d , in a g r e e m e n t with the r e s u l t s p r e v i o u s l y r e p o r t e d by o t h e r s ( 9 - 1 2 ) , is not s i g n i f i c a n t l y m o d i f i e d by DA. In f e m a l e s the a c t i v i t y is h i g h e r t h a n in m a l e s and i s c o n s i d e r a b l y i n h i b i t e d by D A . To o u r k n o w l e d g e , t h i s is the f i r s t time t h a t a D A - i n h i b i t e d AC is d e m o n s t r a t e d in the r a t a n t e r i o r p i t u i t a r y g l a n d . A l a r g e n u m b e r of h o r m o n e s and n e u r o t r a n s m i t t e r s a r e now known to i n h i b i t the AC a c t i v i t y in d i f f e r e n t s y s t e m s ( s e e f o r r e v i e w s r e f s . 19 a n d 20). A common p r o p e r t y of h o r m o n e - i n d u c e d i n h i b i t i o n s of AC is the G T P d e p e n d e n c e , v e r y s i m i l a r to the known G T P r e q u i r e m e n t f o r h o r m o n a l AC s t i m u l a t i o n ; h o w e v e r h i g h e r c o n c e n t r a t i o n s a r e u s u a l ly r e q u i r e d f o r AC i n h i b i t i o n t h a n f o r s t i m u l a t i o n (19). O u r r e s u l t s show t h a t t h i s property is a l s o s h a r e d by t h e D A - i n h i b i t i o n of AC in f e m a l e a d e n o h y p o p h y s i s . The d o s e s of G T P r e q u i r e d to d e t e c t t h e i n h i b i t i o n by DA a r e c l o s e to t h o s e r e p o r t e d f o r the i n h i b i t i o n of AC a c t i v i t y by a d e n o s i n e , m u s c a r i n i c a n d C ¥ - a d r e n e r g i c a g o n i s t s in o t h e r systems (21-23). A d m i t t e d l y , t h e r e is no d i r e c t p r o o f that the D A - i n h i b i t e d AC is l o c a l i z e d in m a m m o t r o p h i c c e i l s . H o w e v e r the fact that t h i s e n z y m e was d e t e c t e d in p i t u i t a r y g l a n d s of n o r m a l o r l a c t a t i n g f e m a l e r a t s , w h e r e t h e n u m b e r of m a m m o t r o p h s is much g r e a t e r than
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Pituitary DA-Inhibited Adenylate Cyclase
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in male r a t s , might be c o n s i s t e n t with t h i s l o c a l i z a t i o n . A c c o r d i n g t o t h i s hypothesis, the f a i l u r e in d e t e c t i n g the D A - i n h i b i t e d c y c l a s e in male r a t s c o u l d be due to the low n u m b e r of m a m m o t r o p h s in the p i t u i t a r y gland of t h e s e a n i m a l s . The p o s s i b i l i t y of a d i f f e r e n t c e l l u l a r l o c a l i z a t i o n of t h e e n z y m e must be t a k e n i n t o a c c o u n t but s e e m s q u i t e r e m o t e , a s the e x i s t e n c e of DA r e c e p t o r s in p i t u i t a r y c e l l s o [ h e r t h a n m a m m o t r o p h s is s t i l l c o n t r o v e r s i a l ( 2 4 - 2 6 ) and the p r o p o r t i o n of t h e s e c e l l s d o e s not v a r y d r a m a t i c a l l y d e p e n d i n g on the sex of t h e a n i m a l s (27). On the c o n t r a r y , the view t h a t o u r r e s u l t s a r e due to the m a m m o t r o p h i c c e l l s is s u p p o r t e d by o u r f i n d i n g on h u m a n P R L - s e c r e t i n g p i t u i t a r y a d e n o m a s , i . e . h o m o g e n e o u s p o p u l a t i o n s ot m a m m o t r o p h s , in w h i c h the p r e s e n c e of a n AC s i g n i f i c a n t l y i n h i b i t e d by DA was c l e a r l y d e m o n s t r a t e d (28). Along the same l i n e B a r n e s et a l . h a v e r e c e n t l y s h o w n t h a t in p o p u l a t i o n s of a n t e r i o r p i t u f t a r y c e l l s e n r i c h e d i n m a m m o t r o p h s D A i n h i b i t e d the i n c r e a s e in i n t r a c e l l u l a r cAMP l e v e l s i n d u c e d by t r e a t m e n t with TRH (29). The r e s u l t s o b t a i n e d with d o p a m i n e r g i c a g o n i s t s a n d a n t a g o n i s t s d e m o n s t r a t e t h a t the DA i n h i b i t i o n of AC i s m e d i a t e d by s p e c i f i c DA r e c e p t o r s . T h u s , the e f f e c t of DA on c y c l a s e is m i m i c k e d by a p o m o r p h i n e and CH 2 9 - 7 1 7 and c o m p e t i t i v e l y a n t a g o n i z e d by t r i f l u o p e r a z i n e a n d a l s o by s u l p i r i d e , w h i c h is a s e l e c t i v e a n t a g o n i s t of p i t u i t a r y DA r e c e p t o r s (11); NE was much l e s s p o t e n t . T h e o r d e r of p o t e n c y of a g o n i s t s , a s well a s the much l o w e r e f f e c t of N E , a r e in full a g r e e m e n t with the r e s u l t s of t h e p r e v i o u s in v i t r o binding studies using "3H]dihydroergocryptine as ligand(5). Since the receptors id e n t i f i e d with t h i s l i g a n d a r e a s s u m e d to m e d i a t e the DA i n h i b i t i o n o f PRL s e c r e t i o n ( 5 ) , o u r r e s u l t s r a i s e the q u e s t i o n w h e t h e r t h e s e r e c e p t o r s , so f a r c o n s i d e r e d u n c o u p l e d to an A C - c A M P s y s t e m , a r e l i n k e d to AC by an i n h i b i t o r y c o u p l i n g and w h e t h e r a d e c r e a s e of the i n t r a c e l l u l a r c o n c e n t r a t i o n of cAMP p l a y s a r o l e in e l i c i t i n g the i n h i b i t o r y e f f e c t of DA on PRL s e c r e t i o n . T h i s h y p o t h e s i s i s s u p p o r t e d by the o b s e r v a t i o n s t h a t cAMP is s t i m u l a t o r y to PRL s e c r e t i o n ( 3 0 , 31) and t h a t a g e n t s w h i c h s t i m u l a t e PRL r e l e a s e , s u c h a s V a s o a c t i v e I n t e s t i n a l P o l y p e p t i d e , a c t i v a t e a p i t u i t a r y AC ( 3 2 ) . T h e fact t h a t h i g h e r c o n c e n t r a t i o n of DA a r e r e q u i r e d f o r h a l f - m a x i m a l AC i n h i b i t i o n ( a p p a r e n t K D = 6 . 2 5 x 10-7M) t h a n f o r h a l f - m a ~ ' i m a l e f f e c t on PRL r e l e a s e ( a p p a r e n t K D = 3 . 5 x 1 0 - 8 M , r e f . 5) s e e m s to be in c o n t r a d i c t i o n with the a b o v e h y p o t h e s i s . H o w e v e r t h i s i s not n e c e s s a r i l y the c a s e , s i n c e the s a m e d i s c r e p a n c y is o f t e n o b s e r v e d when a h o r m o n e - m e d i a t e d AC i n h i b i t i o n i s d e t e c t e d in c e l l f r e e s y s t e m (19). Of c o u r s e , t h e p o s s i b i l i t y t h a t i n t r a c e l l u l a r m e c h a n i s m s o t h e r t h a n cAMP a r e i n v o l v e d in the D A i n h i b i t i o n of PRL s e c r e t i o n r e m a i n s e n t i r e l y o p e n . In t h i s c a s e the D A i n d u c e d m o d i f i c a t i o n s in AC a c t i v i t y would c o n s t i t u t e only one s t e p of a m o r e c o m p l e x , p o s s i b l y m u l t i f a c t o r i a l p r o c e s s r e g u l a t i n g the s e c r e t i o n of the h o r m o n e .
Acknowledgements We thank Drs. J.Meldolesi, G.Faglia, S.Nicosia and D.Borgese for discussion and critical reading of the manuscriptand Mrs.M. Bassetti for technical assistance. References
1. 2. 3.
R.M.MACLEOD, Frontiers in Neuroendocrinology,L.Martiniand W.F. Ganong(Eds.) Vol.4, pp. 169-194, Raven Press, N.Y. (1976). G.M.BROWN, P.SEEMAN and T.LEE, Endocrinology99 1407-1410 (1976). I.CREESE, R.SCHNEIDER and S.H.SNYDER, Eur. J.Pharmacol. 46 377-381 (1977).
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M.A.CALABRO and R.M.MACLEOD, Neuroendocrinology 2._5532-46 (1978). M.G.CARON, M.BEAULIEU, V.RAYMOND, B . G A G N E ' , J . D R O U I N , R . J . L E F K O WITZ and F . L A B R I E , J . B i o l . C h e m . 253 22/*~-2253 (1978). M . J . C R O N I N , J . M . R O B E R T S and R . I . W E I N E R , Endocrinology 103 302-309 (1978). M . J . C R O N I N and R . W E I N E R , Endocrinology 101+ 307-312 (1979). P . G . G O L D S M I ' I H , M.J .CRONIN, R.J .RUBIN and R . I . W E I N E R , Fifth Annual Meet. Soc . N e u r o s c i e n c e , A b s t r a c t 1098 (1978). M.G.SCHMIDT and L . E . H I L L , Life S c i . 20 789-797 (1977). P . F . S P A N O , S.GOVONI and M.TRABUCCHI, Advances in Biochemical F s y c h o p h a r m a c o l o g y , P . J . R o b e r t s , G .N.Woodruff and L . L . I v e r s e n (Eds .) Vol. 19, pp. 155-165, Raven P r e s s , N . Y . (1978). J . W . K E B A B I A N and D . B . C A L N E , Nature 277 93-96 (1979). E . S T E F A N I N I , A . M . M A R C H I S I O , F.VERNALEONE, P . D E V O T O a n d R.COLLU, Life S c i . 26 589-59/* (19 80). M . G . F A R Q U H A R , E . H . S K U T E L S K Y and C . R . H O P K I N S , T h e A n t e r i o r P i t u i t a r y , A . T i x i e r - V i d a l and M . G . F a r q u h a r ( E d s . ) pp. 83-135, Academic P r e s s , N.Y.(1975). W.C.HYMER, W . H . E V A N S , J . K R A I C E R , A.MASTRO, J . D A V I S and E.GRISWOLD, Endocrinology 92 275-287 (1973). W.C.HYMER, J . S N Y D E R , W.WILFINGER, N.SWANSON and J . A . D A V I S , E n d o c r i n o l o g y 9_.5 107-122 (1971+). Y.SALOMON, C.LONDOS and M.RODBELL, A n a l . B i o c h e m . 58 5l+1-5l+8 (197/+). O.H.LOWRY, N . J . R O S E E R O U G H , A . L . F A R R and R.J.RANDALL, J . B i o l . C h e m . 193 265-275 (1951) E . F L U C K I G E R , U . B R I N E R , W . D O E P F N E R , E . K O V A C S , P.MARBACH and H.'R. WAGNER, E x p e r i e n t i a 3__~g 1330-1331 (1978). K . H . j A K O B S , M o l . C e l l . E n d o c r i n o l . 16 1/.7-156 (1979). M.RODBELL, Nature 28/* 17-22 (1980). C.LONDOS, D . I ' 4 . F . C O O P E R , W.SCHLEGEL and M.RODBELL, P r o c . N a t l . A c a d . S c i . USA 75 5362-5366 (1978). K . H . J A K O B S , K . A K T O R I E S and G . S C H U L T Z , 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 . 310 113-119 (1979). K . H . J A K O B S and G . S C H U L T Z , 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 . 310 121-127 (1979). P . G . C H I O D I N I and A . L I U Z Z I , The Regulation of Growth Hormone S e c r e t i o n , D. F . H o r r o b i n ( E d . ) V o l 1, pp. 19-25 Eden P r e s s , Montreal (1979). T . R A N T A , P . M ) ~ N N I S T O a n d J . T U O M I S T O , J . E n d o c r i n o l . 72 329-335 (1977). H . P . G . S C H N E I D E R and S.M.MAC CANN, Endocrinology 85 121-132 (1969). C . D E N E F , E . H A U T E K E E T E , A.DE WOLF and B.VANDERSCHUEREN, E n d o c r i n ology 103 72/.-735 (1978). P . D E CAMILLI, D.MACCONI and A . S P A D A , Nature 278 252-251+ (1979). G . D . B A R N E S , B.L.BROWN, T . G . G A R D , D . A T K I N S O N and R . P . E K I N S , Mol. Cell E n d o c r i n o l . 12 273-28/* (1978). M.K.HILL, R.M.MAC LEOD and P . O R C U T T , Endocrinology 99 1612-1617 (1976). F . L A B R I E , P . B O R G E A T , A.LEMAY, S . L E M A I R E , N.BARDEN, J . D R O U I N , I . LEMAIRE, P . J O L I C O E UR and A.BELANGER, Advances in Cyclic Nucleotide Re s e a r c h , G . I .Drummond, P . G r e e n g a r d and G . A . R o b i n s o n ( E d s . ) V o l . 5, pp. 787801, Raven P r e s s , N . Y . (1975). S . N I C O S I A , A . S P A D A , C . B O R G H I , L.CORTELAZZI and G . G I A N N A T T A S I O , F E B S Lett. 112 159-162 (1980).
Pergamon Press
DOPAMINE-INHIBITED ADENYLATE CYCLASE IN FEMALE RAT ADENOHY POPHYS I S Giuliana Giannattasio, Maria Elisabetta De F e r r a r i and Anna Spada* CNR Center of Cytopharmacology and *Endocrine Unit, II Medical C l i n i c , University of Milano, Via V a n v i t e l l i 3 2 , 20129 Milano, Italy. (Received in final form January 28, 1981)
Summary A dopamine-inhibited adenylate cyclase has been demonstrated in a n t e r i o r pituitary gland of adult female r a t s , lactating and not lactating. This i n h i bitory effect was completely GTP dependent. In c o n t r a s t , in the adenohypophysis of male r a t s , dopamine had no detectable effect on adenylate cyclase activity. In female rats the inhibition of the enzyme appears mediated by specific dopaminergic r e c e p t o r s : the effect of dopamine was mimicked by the dopaminergic agonists apomorphine and the ergot derivative CH 29-717, while norepinephrine was much l e s s potent. On the other hand, the dopaminergic antagonists t r i f l u o p e r a z i n e and sulpiride competitively antagonized the dopamine inhibition of the adenylate c y c l a s e . The p o s s i bility that the dopamine-inhibited enzyme is located in mammotrophs appears supported 1) by its observation in the female rat p i t u i t a r y , which contains this type of cells in much l a r g e r proportion than the male gland (33-38% vs. < 5%); 2) by the pharmacological similarity between the dopaminergic r e ceptors mediating the adenylate cyclase inhibition (this work) and those regulating prolactin r e l e a s e (which have been c h a r a c t e r i z e d in previous studies). The well known inhibition of prolactin r e l e a s e brought about by dopamine could therefore be mediated, at least in p a r t , by a d e c r e a s e in the i n t r a c e l l u l a r level of cAMP. Dopamine (DA) r e l e a s e d by the tuberoinfundibular hypothalamic n e u r o n s into the hypophyseal portal blood is known to play a key role in the regulation of the pituitary function, p a r t i c u l a r l y as an inhibitor of prolactin (PRL) s e c r e t i o n (1). It is conceivable therefore that mammotrophic cells possess specific DA r e c e p t o r s . Binding studies c a r r i e d out on pituitary homogenates and membrane p r e p a r a t i o n s , using labeled agonists and a n t a g o n i s t s , have indeed revealed the existence in the gland of specific dopaminergic sites (2-7). Although this kind of study does not provide informations about the cell type containing these s i t e s , the similarity between the pharmacological p r o p e r ties of the sites identified by the use of dopaminergic r a d i o l i g a n d s , such as [3H] d i h y d r o e r g o c r y p t i n e , and the c h a r a c t e r i s t i c s of the dopaminergic inhibit{on of PRL s e c r e tion, strongly suggests a localization of DA r e c e p t o r s in mammotrophic cells (5). This conclusion is also supported by immunocytochemical data (8). 0024-3205/81/141605-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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An i n t e r e s t i n g problem r a i s e d by the existence of DA r e c e p t o r s in mammotrophs concerns the i n t r a c e l l u l a r mechanisms of transduction or r e c e p t o r activation. Previous studies failed to reveal the existence of a DA-linked adenylate cyclase (AC) in the a n t e r i o r pituitary gland (9-12). As a c o n s e q u e n c e , the DA r e c e p t o r s of mammotrophs labeled with[3H]dihydroergocryptine have been r e c e n t l y proposed by Kebabian and Calne as prototypes or a c l a s s of DA r e c e p t o r s (designed a s D-2 r e c e p t o r s ) whose activation is not coupled to an i n c r e a s e of cyclic AMP (cAMP) synthesis (11). However, it should be considered that these negative r e s u l t s were obtained using the total pituitary gland, i . e . a very heterogeneous tissue; if DA did affect AC activity of a single cell type, this effect could have gone undetected because basal AC activity was contributed p r i m a r i l y by the other cell types. It is known that the relative number of mammotrophs in the pituitary gland v a r i e s according to age, sex and physiological conditions of the animal (13). Thus the relative contribution of these cells to the AC activity of the gland may be expected to vary in parallel with their number. On this b a s i s , the possible existence of a D A - s e n s i t i v e AC in mammotrophs has been here reinvestigated by studying the effect of DA on the AC a c tivity of a n t e r i o r pituitary homogenates obtained from animals with a physiologically high number of P R L - s e c r e t i n g ceils: mature female r a t s , lactating and not lactating (mammotrophs = 38% and 33% of total c e l l s , respectively) ( t ~ , 15). These data on female rats have been compared with other data obtained in p a r a l l e l on male rat adenohypophysis, in which mammotrophs are quite s p a r s e (13, 1L) and which had been used in the previous studies of the l i t e r a t u r e (9-12). Materials and Methods Materials. N , N - d i m e t h y l - N ' - ( 6 - m e t h y l - e r g o l i n e - 8 0 ( - y l ) - s u l f a m i d e hydrochloride (CH 29-717), t r i f l u o p e r a z i n e and ( - ) s u l p i r i d e were kind gifts of Sandoz, B a s e l , S w i t z e r land, Ravizza, Milan, Italy and Maggioni, Milan, I t a l y , respectively; apomorphine was purchased from S1FAC, Milan, Italy; DA, ( - ) n o r e p i n e p h r i n e (NE), adenosine - 3 ' , 5 ' cyclic monophosphate (cAMP), adenosine 5'-triDhosphate (ATP), c r e a t i n e phosphate, c r e a t i n e phosphokinase, g u a n o s i n e - 5 ' - t r i p h o s p h a t e (GTP), ethylene glycol-his (2-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA) and theophylline from Sigma, St Louis, MO. USA; [ 8 - 1 ~ C ] A T P and cyclic [8-3H]AMP from the Radiochemical C e n t e r , Amersham, U.K. Animals. Adult female (220-250 g), lactating or not l a c t a t i n g , and adult male (200220 g) Sprague-Dawley r a t s were purchased from Charles R i v e r , Calco, Italy. They were housed in a light, temperature and humidity controlled room, with food and water ad libitum . Adenylate cyclase a s s a y . Rats were killed by decapitation, the hypophyses quickly removed and the n e u r o i n t e r m e d i a t e lobes d i s c a r d e d . A n t e r i o r lobes were homogenized by a g l a s s - T e f l o n Potter homogenizer in 20 vol or 1 mM T r i s - m a l e a t e buffer (pH 7.&) containing 1 mM EGTA. Most enzyme a s s a y s were c a r r i e d out on the total homogenate, filtered through a nylon gauze. The a s s a y mixture (0.1 ml) contained: 80 mM T r i s - m a l e a t e burrer, pH 7.~,10mM theophylline, 1 mM cAMP, 0.2 mM EGTA, 0.15mM [ 8 - 1 & C ] A T P (&0 dpm/pmol), 1.5 mM MgSO&, 7 mM phosphocreatine, c r e a t i n e phosphokinase (20 U/ml) and 0.01 mM G T P . P r o t e i n concentration was about 1 mg/ml. The r e a c t i o n , started by the addition of the homogenate, was c a r r i e d out for 8 rain at 30°C and stopped by adding
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Pituitary DA-Inhlblted Adenylate Cyclase
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a solution c o n t a i n i n g 2% sodium d o d e c y l s u l f a t e , 1.~ mM cAMP and 40 mM A T P . In the e x p e r i m e n t s p e r f o r m e d to t e s t the e f f e c t of G T P on the AC a c t i v i t y , a c r u d e membrane p r e p a r a t i o n , sediment ed from the homogenate by c e n t r i f u g a t i o n at 20,000 x g for 10 min, was u s e d . The p e l l e t was r e s u s p e n d e d in T r i s - m a l e a t e b u f f e r - E G T A at a c o n c e n t r a t i o n of about 2 . 5 mg p r o t e i n / m l . The r e a c t i o n m i x t u r e c o n t a i n e d G T P at the c o n c e n t r a t i o n s s p e c i f i e d in the " R e s u l t s " . The final p r o t e i n c o n c e n t r a t i o n was 0 . 5 m g / m l . C y c l i c [ 8 - 1 ~ C ] AMP was i s o l a t e d and m e a s u r e d a c c o r d i n g to Salomon et a l . (16). P r o t e i n was e v a l u a t e d a c c o r d i n g to Lowry et a l . (17). Results The AC a c t i v i t y was i n v e s t i g a t e d in homogenates of a n t e r i o r p i t u i t a r y gland of adult male and a d u l t , l a c t a t i n g and n o n l a c t a t i n g female r a t s . The b a s a l AC a c t i v i t y was s i g n i ficantly h i g h e r in female than in male r a t s . In non l a c t a t i n g female r a t s and, even more, in l a c t a t i n g ones 10-5M DA e l i c i t e d a highly s i g n i f i c a n t d e c r e a s e in the AC a c t i v i t y , while in male r a t s no inhibition was o b s e r v e d (Table I). As shown in figs. i - £ , the i n h i b i t o r y effect of DA on AC a c t i v i t y of female r a t adeno_ h y p o p h y s i s was d o s e d e p e n d e n t . The a p p a r e n t K D was 6. 2 5 + I .3 O x 10 - 7M • The hl"ghest enzyme inhibition o c c u r r e d between 10 -5 and 5 x 10-SM, whi'~e at h i g h e r c o n c e n t r a t i o n s DA e l i c i t e d a slight s t i m u l a t o r y e f f e c t . The effect of DA was mimicked by NE ( F i g . i ) , apomorphine and the w a t e r - s o l u b l e d o p a m i n e r g i c e r g o t CH 29-717 (18) ( F i g . 2), but NE was much l e s s potent ( a p p a r e n t K D = 2.5"< 10-SM) and a p o m o r p h i n e and CH 29-717 more potent than D A ( a p p a r e n t K D ~ 1 . 5 x I0-7M and 1 . 5 x 10-tSM, r e s p e c t i v e l y ) .
TABLE I Effect of Dopamine on Adenyi.ate C y c l a s e A c t i v i t y of Rat A n t e r i o r P i t u i t a r y Gland
Animals
Adenylate C y c l a s e A c t i v i t y pmol c A M P / m g prot x min % inhibition basal IO-SM DA
P
Adult male r a t s (3)
11.7 + 0 . £
10.9 + 0.L
6.8
NS
Adult female r a t s (10)
2 8 . 6 + 1.3 a
17.6 + 0 . 8
38.5
< 0.0001
Lactating r a t s (2)
3 0 . 5 + 1.0 a
15.9 + 0.1
L7.8
< 0.001
A s s a y s w e r e c a r r i e d out on total h o m o g e n a t e . Number of e x p e r i m e n t s , each c a r r i e d out in t r i p l i c a t e , in p a r e n t h e s e s . Values a r e m e a n s + SEM. a p < o . o 0 1 v s . male r a t s .
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Vol. 28, No. 14, 1981
22. E
t-
-- 20 E
Fig. 1
x
Effect of dopamine and (-) n o r e p i n e p h r i n e on a d e n y l ate c y c l a s e activity in the total homogenate of non l a c t a t i n g female rat adenoh y p o p h y s i s . The values given a r e the means of 3 determinations + S Eb,I.
18
X u 16
E 14
12
o
1o'-'
10-~
lo'-'
M
32-
~
~
,
.E
•
DOPAMINE
o
APOMORPHINE
~~
CH
E
x 28
29-717
Effect of dopamine, a p o morphine and CH 29-717 on adenylate c y c l a s e a c tivity in the total homogenate of non lactating female rat adenohypop h y s i s . The values given a r e the means of 3 d e t e r minations + SEb4.
~24 ~ 2o 16
r--~/ 0
10 -s
10 -7
Fig. 2
10 -5
1(~-3
M
The dopaminergic antagonist t r i f l u o p e r a z l n e , at the dose of 10 -7 I'd, had no effect on b a s a l AC activity but competitively antagonized the DA action ( F i g . 3). The same r e s u l t was6obtained with another dopaminergic a n t a g o n i s t , (-) s u l p i r i d e , at the c o n c e n t r a t i o n of 10- M ( F i g . g).
Vol. 28, No. 14, 1981
Pituitary DA-Inhibited Adenylate Cyclase
1609
38l~/~--~...,,p.,~
+
TRIFLUOPERAZINE
C
Fig. 3
E x 34
Effect of d o p a m i n e in the a b s e n c e and p r e s e n c e of 10 - 7 M t r i f l u o p e r a z i n e on adenylate cyclase activity in t h e t o t a l h o m o g e n a t e of non l a c t a t i n g female r a t adenohypophysis. The v a l u e s g i v e n a r e the m e a n s of 3 d e t e r m i n a t i o n s -+ SEN
gm. o.
3o < u
-g 2s O
E
22 r"V/
DOPAMINE
(M)
34~
Fig.
32
\
x 30
28
u 26-
(-) SULPIRIDE
10 -6 M
\
24
E f f e c t of d c p a m i n e in the a b s e n c e and p r e s e n c e of 10 - 6 M ( - ) s u l p i r i d e on adenylate cyclase activity in the t o t a l h o m o g e n a t e of non l a c t a t i n g f e m a l e r a t a d e n o h y p o p h y s i s . The v a l u e s g i v e n a r e the m e a n s of 3 d e t e r m i n a t i o n s + SEM
22 ,.../!
0
14 .7
10 -s DOPAMINE
10 -3 (M)
DA i n h i b i t i o n of AC r e q u i r e d the p r e s e n c e of G T P ( T a b l e I I ) . In fact the c y c l a s e a c t i v i t y of c r u d e m e m b r a n e p r e p a r a t i o n s o b t a i n e d from non l a c t a t i n g f e m a l e r a t p i t u i t a r y h o m o g e n a t e s was not s i g n i f i c a n t l y i n h i b i t e d by DA when G T P was a b s e n t from the i n c u b a t i o n m i x t u r e . When G T P was a d d e d , t h e b a s a l AC a c t i v i t y was g r e a t l y e n h a n c e d and a s i g n i f i c a n t DA i n h i b i t i o n a p p e a r e d ( - g 3 % a t 3 x 10-5M G T P ) . In male r a t s t h e G T P i n c r e a s e of b a s a l a c t i v i t y w a s much s m a l l e r and no s i g n i f i c a n t i n h i b i tion was b r o u g h t aEout by DA at a n y d o s e of G T P .
1610
Pituitary DA-Inhibited Adenylate
Cyclase
Vol. 28, No. 14, 1981
TABLE 11 E f f e c t of G T P on A d e n y l a t e C y c l a s e A c t i v i t y of Male and F e m a l e Rat A n t e r i o r P i t u i t a r y G l a n d
Adenylate Cyclase Activity pmol c A M P / m g p r o t x min GTP
(M)
Male r a t s basal
DA
0
/4.2 -+ 0 . 1
4 . 0 -+ 0 . 1
3 x 10 . 7
6 . 2 -+ 0 . 2
6 . 7 -+ 0 . 7
I0 -6
8.7 + -0.6
3 x i0 -6
Female r a t s % inhibition
/4.8 a
basal
DA
% inhibition
9 . 6 -+ 0 . 7
8 . 9 -+ 0 . 7
7.3 a
-
1 6 . 3 -+ 0 . 3
1 4 . 3 + 0./4
12.3 b
+ 7.7 -0.1
ii .5a
2 2 . 9 -+0 . 6
19.7 -+ 0 . 6
1~.0 b
I0.i -+ O.&
9.3 + 0.6
7.9 a
30.2 -+ 0.2
19.0 -+ O.&
37.1
10 - 5
10.5-+ 1.0
9.3 +0.5
11.4 a
32.5 +0.6
1 9 . 3 -+0.1
~0.6 c
3 x 10 - 5
1 0 . 2 -+ 0 . 6
9 . 6 -+ 1 . 3
5.9 a
3 3 . 6 -+ 0 . 6
19.1 2 0 . 3
43.1
10 . 4
10.0 + 0.3
9 . 6 +- 0 . 7
4.0 a
3 2 . 7 -+ 0 . 7
1 9 . 2 -+ 0 . 7
L1.3 c
c
c
A s s a y s w e r e c a r r i e d out on c r u d e m e m b r a n e p r e p a r a t i o i q s . DA was 2 . 5 x 10 - 5 M. •
V a l u e s a r e the m e a n s o f 3 d e t e r m i n a n o n s
+
- SEM.
a
NS;
D
P<'0.02;
C
P<0.001.
Discussion O u r d a t a d e m o n s t r a t e c l e a r d i f f e r e n c e s in t h e p i t u i t a r y AC of male and f e m a l e r a t s . In m a l e s t h e b a s a l a c t i v i t y i s low a n d , in a g r e e m e n t with the r e s u l t s p r e v i o u s l y r e p o r t e d by o t h e r s ( 9 - 1 2 ) , is not s i g n i f i c a n t l y m o d i f i e d by DA. In f e m a l e s the a c t i v i t y is h i g h e r t h a n in m a l e s and i s c o n s i d e r a b l y i n h i b i t e d by D A . To o u r k n o w l e d g e , t h i s is the f i r s t time t h a t a D A - i n h i b i t e d AC is d e m o n s t r a t e d in the r a t a n t e r i o r p i t u i t a r y g l a n d . A l a r g e n u m b e r of h o r m o n e s and n e u r o t r a n s m i t t e r s a r e now known to i n h i b i t the AC a c t i v i t y in d i f f e r e n t s y s t e m s ( s e e f o r r e v i e w s r e f s . 19 a n d 20). A common p r o p e r t y of h o r m o n e - i n d u c e d i n h i b i t i o n s of AC is the G T P d e p e n d e n c e , v e r y s i m i l a r to the known G T P r e q u i r e m e n t f o r h o r m o n a l AC s t i m u l a t i o n ; h o w e v e r h i g h e r c o n c e n t r a t i o n s a r e u s u a l ly r e q u i r e d f o r AC i n h i b i t i o n t h a n f o r s t i m u l a t i o n (19). O u r r e s u l t s show t h a t t h i s property is a l s o s h a r e d by t h e D A - i n h i b i t i o n of AC in f e m a l e a d e n o h y p o p h y s i s . The d o s e s of G T P r e q u i r e d to d e t e c t t h e i n h i b i t i o n by DA a r e c l o s e to t h o s e r e p o r t e d f o r the i n h i b i t i o n of AC a c t i v i t y by a d e n o s i n e , m u s c a r i n i c a n d C ¥ - a d r e n e r g i c a g o n i s t s in o t h e r systems (21-23). A d m i t t e d l y , t h e r e is no d i r e c t p r o o f that the D A - i n h i b i t e d AC is l o c a l i z e d in m a m m o t r o p h i c c e i l s . H o w e v e r the fact that t h i s e n z y m e was d e t e c t e d in p i t u i t a r y g l a n d s of n o r m a l o r l a c t a t i n g f e m a l e r a t s , w h e r e t h e n u m b e r of m a m m o t r o p h s is much g r e a t e r than
Vol. 28, No. 14, 1981
Pituitary DA-Inhibited Adenylate Cyclase
1611
in male r a t s , might be c o n s i s t e n t with t h i s l o c a l i z a t i o n . A c c o r d i n g t o t h i s hypothesis, the f a i l u r e in d e t e c t i n g the D A - i n h i b i t e d c y c l a s e in male r a t s c o u l d be due to the low n u m b e r of m a m m o t r o p h s in the p i t u i t a r y gland of t h e s e a n i m a l s . The p o s s i b i l i t y of a d i f f e r e n t c e l l u l a r l o c a l i z a t i o n of t h e e n z y m e must be t a k e n i n t o a c c o u n t but s e e m s q u i t e r e m o t e , a s the e x i s t e n c e of DA r e c e p t o r s in p i t u i t a r y c e l l s o [ h e r t h a n m a m m o t r o p h s is s t i l l c o n t r o v e r s i a l ( 2 4 - 2 6 ) and the p r o p o r t i o n of t h e s e c e l l s d o e s not v a r y d r a m a t i c a l l y d e p e n d i n g on the sex of t h e a n i m a l s (27). On the c o n t r a r y , the view t h a t o u r r e s u l t s a r e due to the m a m m o t r o p h i c c e l l s is s u p p o r t e d by o u r f i n d i n g on h u m a n P R L - s e c r e t i n g p i t u i t a r y a d e n o m a s , i . e . h o m o g e n e o u s p o p u l a t i o n s ot m a m m o t r o p h s , in w h i c h the p r e s e n c e of a n AC s i g n i f i c a n t l y i n h i b i t e d by DA was c l e a r l y d e m o n s t r a t e d (28). Along the same l i n e B a r n e s et a l . h a v e r e c e n t l y s h o w n t h a t in p o p u l a t i o n s of a n t e r i o r p i t u f t a r y c e l l s e n r i c h e d i n m a m m o t r o p h s D A i n h i b i t e d the i n c r e a s e in i n t r a c e l l u l a r cAMP l e v e l s i n d u c e d by t r e a t m e n t with TRH (29). The r e s u l t s o b t a i n e d with d o p a m i n e r g i c a g o n i s t s a n d a n t a g o n i s t s d e m o n s t r a t e t h a t the DA i n h i b i t i o n of AC i s m e d i a t e d by s p e c i f i c DA r e c e p t o r s . T h u s , the e f f e c t of DA on c y c l a s e is m i m i c k e d by a p o m o r p h i n e and CH 2 9 - 7 1 7 and c o m p e t i t i v e l y a n t a g o n i z e d by t r i f l u o p e r a z i n e a n d a l s o by s u l p i r i d e , w h i c h is a s e l e c t i v e a n t a g o n i s t of p i t u i t a r y DA r e c e p t o r s (11); NE was much l e s s p o t e n t . T h e o r d e r of p o t e n c y of a g o n i s t s , a s well a s the much l o w e r e f f e c t of N E , a r e in full a g r e e m e n t with the r e s u l t s of t h e p r e v i o u s in v i t r o binding studies using "3H]dihydroergocryptine as ligand(5). Since the receptors id e n t i f i e d with t h i s l i g a n d a r e a s s u m e d to m e d i a t e the DA i n h i b i t i o n o f PRL s e c r e t i o n ( 5 ) , o u r r e s u l t s r a i s e the q u e s t i o n w h e t h e r t h e s e r e c e p t o r s , so f a r c o n s i d e r e d u n c o u p l e d to an A C - c A M P s y s t e m , a r e l i n k e d to AC by an i n h i b i t o r y c o u p l i n g and w h e t h e r a d e c r e a s e of the i n t r a c e l l u l a r c o n c e n t r a t i o n of cAMP p l a y s a r o l e in e l i c i t i n g the i n h i b i t o r y e f f e c t of DA on PRL s e c r e t i o n . T h i s h y p o t h e s i s i s s u p p o r t e d by the o b s e r v a t i o n s t h a t cAMP is s t i m u l a t o r y to PRL s e c r e t i o n ( 3 0 , 31) and t h a t a g e n t s w h i c h s t i m u l a t e PRL r e l e a s e , s u c h a s V a s o a c t i v e I n t e s t i n a l P o l y p e p t i d e , a c t i v a t e a p i t u i t a r y AC ( 3 2 ) . T h e fact t h a t h i g h e r c o n c e n t r a t i o n of DA a r e r e q u i r e d f o r h a l f - m a x i m a l AC i n h i b i t i o n ( a p p a r e n t K D = 6 . 2 5 x 10-7M) t h a n f o r h a l f - m a ~ ' i m a l e f f e c t on PRL r e l e a s e ( a p p a r e n t K D = 3 . 5 x 1 0 - 8 M , r e f . 5) s e e m s to be in c o n t r a d i c t i o n with the a b o v e h y p o t h e s i s . H o w e v e r t h i s i s not n e c e s s a r i l y the c a s e , s i n c e the s a m e d i s c r e p a n c y is o f t e n o b s e r v e d when a h o r m o n e - m e d i a t e d AC i n h i b i t i o n i s d e t e c t e d in c e l l f r e e s y s t e m (19). Of c o u r s e , t h e p o s s i b i l i t y t h a t i n t r a c e l l u l a r m e c h a n i s m s o t h e r t h a n cAMP a r e i n v o l v e d in the D A i n h i b i t i o n of PRL s e c r e t i o n r e m a i n s e n t i r e l y o p e n . In t h i s c a s e the D A i n d u c e d m o d i f i c a t i o n s in AC a c t i v i t y would c o n s t i t u t e only one s t e p of a m o r e c o m p l e x , p o s s i b l y m u l t i f a c t o r i a l p r o c e s s r e g u l a t i n g the s e c r e t i o n of the h o r m o n e .
Acknowledgements We thank Drs. J.Meldolesi, G.Faglia, S.Nicosia and D.Borgese for discussion and critical reading of the manuscriptand Mrs.M. Bassetti for technical assistance. References
1. 2. 3.
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1612
1+. 5. 6. 7. 8. 9. 10.
11. 12. 13. 1/*. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2/*. 25. 26. 27. 28. 29. 30. 31.
32.
Pituitary DA-Inhibited Adenylate Cyclase
Vol. 28, No. 14, 1981
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