Dopamine Receptors - American Chemical Society

2 The D-2 Dopamine Receptor in the Intermediate Lobe of the Rat Pituitary Gland

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 3, 2015 | http://pubs.acs.org Publication Date: June 29, 1983 | doi: 10.1021/bk-1983-0224.ch002

Physiology, Pharmacology, and Biochemistry J. W . K E B A B I A N , M . B E A U L I E U , T . E . C O T E , R. L . E S K A Y , E . A . F R E Y , M . E. G O L D M A N , C . W. G R E W E , M . M U N E M U R A , J. C . S T O O F , and K . T S U R U T A National Institutes of Health, Experimental Therapeutics Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20205

Dopaminergic neurons synapse upon the parenchymal cells of the intermediate lobe ( I L ) o f the r a t p i t u i t a r y gland. Dopamine decreases the c a p a c i t y of the IL c e l l s to synthesize c y c l i c AMP and i n h i b i t s the r e l e a s e o f αMSH and other peptides from t h i s t i s s u e . The presence o f a D-2 receptor accounts f o r both o f these phenomena. This D-2 dopamine receptor can be studied i n a binding assay using [3H]-spiroperidol, a dopamine antagonist. These observations support the two dopamine receptor hypothesis. The hypothesis suggesting the existence o f two c a t e g o r i e s of dopamine receptor (1_, Figure 1 ) arose from the observation that l e r g o t r i l e blocks the dopamine-induced enhancement o f s t r i a t a l adenylate c y c l a s e a c t i v i t y (2, see a l s o Brown and Dawson-Hughes, t h i s volume) but mimicks the a c t i o n o f dopamine upon the mammotrophs o f the a n t e r i o r p i t u i t a r y gland (_3). When the hypothesis was i n i t i a l l y put forward, the dopamine receptor upon the mammotrophs was taken as the p r o t o t y p i c example o f a D-2 r e c e p t o r . According t o t h i s hypothesis, s t i m u l a t i o n o f the D-2 dopamine receptor does "not i n v o l v e e i t h e r the s t i m u l a t i o n o f adenylate c y c l a s e or the accumulation of intracellular c y c l i c AMP" (1_, 4-7)· Although the mammotroph provided an example o f a D-2 dopamine r e c e p t o r , the c e l l u l a r heterogeneity of the a n t e r i o r p i t u i t a r y gland l i m i t e d the p r e c i s i o n o f t h i s biochemical model o f the D-2 dopamine receptor (8; see a l s o Labrie et a l . , this volume). Indeed, many biochemical i n v e s t i g a t i o n s o f the a n t e r i o r p i t u i t a r y dopamine receptor make the untestable assumption that a biochemical s i g n a l not l i n k e d to p r o l a c t i n i s generated by the mammotrophs and none o f the other c e l l types i n the gland. The intermediate lobe (IL) o f the r a t p i t u i t a r y gland possesses a D-2 dopamine receptor amenable t o experimental i n v e s t i g a t i o n . The IL c o n s i s t s o f an homogeneous population o f c e l l s (£) possessing both a βadrenoceptor (U)) and a dopamine receptor (11_, 1_2). An This chapter not subject to U.S. copyright. Published 1983, American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


DOPAMINE

Name

D-l

D-2

Cyclase linkage Location of proto type receptor Dopamine

Yes Bovine parathyroid

No Mammotroph of anterior pituitary Agonist (nmolar potency) Agonist (nmolar potency) Agonist (nmolar potency)

Apomorphine Dopaminergic ergots

Selective antagonist Radiolabelled ligand

Figure 1.

Agonist ^molar potency) Partial agonist or antagonist Potent antagonist (nmolar potency) Weak agonist ^molar potency) None known as yet c/s-flupenthixol

RECEPTORS

Metoclopramide sulpiride Dihydroergocryptine

Criteria for the classification of dopamine receptors.

Radiolabelled cis-fiupenthixol can be used as a ligand specific for the dopamine receptor linked to adenylyl cyclase in the rat striatum (64). Its affinity for the dopamine receptor in the anterior pituitary has not been measured. (Previously (65) the two categories of dopamine receptors were designated as "α-dopaminergic" and "β-dopaminergic." This has led to confusion with the a and β adrenoreceptors. The new designations should prevent further confusion.)

In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

2.

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ET AL.

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35

u n d e r s t a n d i n g o f the b i o c h e m i c a l consequences o f s t i m u l a t i o n o f t h e D - 2 dopamine r e c e p t o r h a s come from i n v e s t i g a t i o n s o f the I L dopamine r e c e p t o r .


The C e l l B i o l o g y o f t h e I n t e r m e d i a t e

Lobe

The I L o f t h e r a t p i t u i t a r y g l a n d c o n s i s t s o f a n a r r o w band o f c e l l s a d h e r i n g t o t h e s l i g h t l y l a r g e r n e u r a l l o b e . The p a r e n c h y m a l c e l l s o f t h e I L s y n t h e s i z e p r o o p i o m e l a n o c o r t i n and then c l e a v e t h i s l a r g e molecule i n t o s m a l l e r fragments which a r e c o n v e r t e d i n t o t h e hormones o f t h e I L ( 1_4 ) . In rodents, t h e s e hormones a f f e c t c o a t c o l o r by s t i m u l a t i n g t h e d e r m a l m e l a n o c y t e s t o s y n t h e s i z e and s e c r e t e m e l a n i n (1_5, 1_6). The most w i d e l y s t u d i e d o f t h e m e l a n o t r o p i c I L hormones i s a l p h a m e l a n o c y t e - s t i m u l a t i n g hormone ( N - a c e t y l ACTH1-13 a m i d e , oMSH). However, r e c e n t r e p o r t s document t h a t N - , O - d i a c e t y l oMSH i s t h e p r e d o m i n a n t m o l e c u l a r s p e c i e s i n t h e r a t I L (1_7,J_8, Goldman et a l . , submitted). I n view o f the hormonal c o n t r o l o f coat color i n rodents, t h e r e l e a s e o f hormones from t h e r a t I L provides a convenient, physiologically relevant sign of a c t i v i t y i n the I L . The hormones r e l e a s e d from t h e I L c a n be q u a n t i f i e d by e i t h e r b i o a s s a y o r by radioimmunoassay. Several o M S H - l i k e m o l e c u l e s a r e r e l e a s e d from t h e I L ; h o w e v e r , i n t h e v a s t m a j o r i t y o f s t u d i e s , t h e amount o f m e l a n o t r o p h i c hormone o r i m m u n o r e a c t i v e , otMSH-like m a t e r i a l (IR-otMSH) i s q u a n t i f i e d i n a s s a y s u s i n g otMSH as a s t a n d a r d . C y c l i c AMP and t h e I n t e r m e d i a t e

Lobe

β-adrenoceptor

A β - a d r e n o c e p t o r o c c u r s upon t h e p a r e n c h y m a l c a l l s o f t h e r a t I L and c a n be s t u d i e d w i t h b i o c h e m i c a l p r o c e d u r e s . The β adrenoceptor itself can be identified with [1251]monoiodohydroxybenzylpindolol (IHYP) (19). Using IHYP to i d e n t i f y b i n d i n g s i t e s and p r o p r a n o l o l t o d e f i n e ' n o n - s p e c i f i c b i n d i n g * , s p e c i f i c b i n d i n g s i t e s f o r the r a d i o l a b e l e d l i g a n d c a n be d e t e c t e d . The a f f i n i t y o f t h e s p e c i f i c b i n d i n g s i t e f o r a n o n - r a d i o a c t i v e d r u g c a n be as d e t e r m i n e d by p e r m i t t i n g t h e d r u g t o compete w i t h IHYP f o r o c c u p a n c y o f t h e s p e c i f i c b i n d i n g sites. I n t h e I L ( a s i n many o t h e r t i s s u e s ) t h e β - a d r e n o c e p t o r regulates adenylate cyclase, the enzyme catalyzing the c o n v e r s i o n o f ATP i n t o cAMP ( 1_9 ) . The a d e n y l a t e cyclase activity of cell-free homogenates of the IL i s markedly stimulted (up t o 7 - f o l d ) by c a t e c h o l a m i n e s ; t h e s t i m u l a t o r y e f f e c t o f c a t e c h o l a m i n e s r e q u i r e s t h e p r e s e n c e o f GTP and c a n be b l o c k e d by s e v e r a l β - a d r e n e r g i c a n t a g o n i s t s ( F i g u r e 2 , l e f t ; V9, 2 0 ) ) . B e c a u s e t h e IHYP b i n d i n g a s s a y and t h e adenylate c y c l a s e assay u t i l i z e s i m i l a r assay c o n d i t i o n s , a comparison between the affinities obtained i n the two a s s a y s seems justified. The a p p r o x i m a t e agreement o f t h e a f f i n i t i e s from t h e two a s s a y s s u g g e s t s t h a t some o r a l l o f t h e b i n d i n g s i t e s are the receptors enhancing adenylate c y c l a s e a c t i v i t y i n the


In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983. I

1

1(H GTP (M)

I

10-5

I 0

L L y /

1

10-7

1

GTP (M)

10-6

1

10-5

L _

104

Guanosine 5'-triphosphate participation in the functioning of the IL β-adrenoceptor the D-2 dopamine receptor in the IL of the rat pituitary gland.

10-7

and

Key: left, effects of GTP on basal and isoproterenol (i)-stimulated adenylate cyclase activity in particulate material from a homogenate of fresh IL tissue; at the indicated concentrations, GTP was tested in the absence of drugs «>) or in the presence of 3 μΜ isoproterenol (φ) (data are from Reference 20); and right, effect of GTP upon adenylate cyclase activity in a homogenate of cholera toxin-treated (30 nM, 2 h) IL tissue; at the indicated concentrations, GTP was tested alone (0) or in combination with either 3 μΜ isoproterenol (+) or 10 μΜ apo morphine ( Δ ) (data are from Reference 22).

Figure 2.

0


2.

KEBABIAN

ET AL.

D-2 Dopamine

37

Receptor

c e l l - f r e e homogenates. The β - a d r e n o c e p t o r i n t h e I L i s a β a d r e n o c e p t o r (1_9, 21). C y c l i c AMP formed i n r e s p o n s e t o s t i m u l a t i o n o f t h e β a d r e n o c e p t o r may i n i t i a t e t h e i n t r a c e l l u l a r e v e n t s u l t i m a t e l y expressed as an enhanced release of OMSH. β-Adrenergic agonists ( F i g u r e 3) a s w e l l a s o t h e r a g e n t s i n c r e a s i n g cAMP content (e.g. theophylline, 3-isobuty-1 -methylxanthine or cholera toxin) increase t h e r e l e a s e o f otMSH (1_2, 2 2 , 23). Furthermore, c y c l i c n u c l e o t i d e analogues ( e . g . d i b u t y r y l cAMP o r 8 B r cAMP) a l s o i n c r e a s e t h e r e l e a s e o f oMSH (1_2, 2 4 ) . A "working hypothesis" accounting f o r the e f f e c t s o f β - a d r e n e r g i c a g o n i s t s and o t h e r d r u g s upon t h e I L i s a s f o l l o w s : occupancy o f t h e β - a d r e n o c e p t o r by an a g o n i s t e n h a n c e s a d e n y l a t e c y c l a s e a c t i v i t y and t h e r e b y i n c r e a s e s the i n t r a c e l l u l a r content of cAMP. I n t u r n , cAMP i n i t i a t e s t h e i n t r a c e l l u l a r e v e n t s l e a d i n g t o enhanced hormone s e c r e t i o n (Figure 4). The p r e s e n c e o f c a l c i u m i o n s i n t h e e x t r a c e l l u l a r medium i s o b l i g a t o r y f o r t h e stimulated release of otMSH (23, 25). At present the i n t r a c e l l u l a r s i t e ( s ) where c a l c i u m and cAMP i m p i n g e upon t h e r e l e a s e p r o c e s s i s unknown. The e v i d e n c e from t h e I L , t o g e t h e r w i t h t h e e v i d e n c e f r o m many o t h e r b i o l o g i c a l s y s t e m s , supports the h y p o t h e s i s that cAMP m e d i a t e s t h e e f f e c t s o f β - a d r e n e r g i c a g o n i s t s upon t h e I L . H o w e v e r , t h e r e a r e a number o f unanswered q u e s t i o n s a b o u t how this receptor, cAMP and c a l c i u m i o n s 'work* i n t h e I L . For example, isoproterenol enhances the release of otMSH from dispersed IL c e l l s at concentrations s u b s t a n t i a l l y lower than the concentrations required to either occupy the specific b i n d i n g s i t e i d e n t i f i e d w i t h IHYP, s t i m u l a t e adenylate c y c l a s e a c t i v i t y i n c e l l - f r e e homogenates o f t h e I L o r enhance cAMP f o r m a t i o n by i n t a c t I L c e l l s ( F i g u r e 5 , 19). At present no e x p e r i m e n t a l o b s e r v a t i o n upon I L t i s s u e e x p l a i n s t h i s a p p a r e n t discrepancy. However, t h e r e a r e a number o f i n d i c a t i o n s t h a t the I L g i v e s a maximal p h y s i o l o g i c a l response w h i l e u t i l i z i n g o n l y a f r a c t i o n o f i t s c a p a c i t y t o s y n t h e s i z e cAMP. Thus, m a x i m a l i s o p r o t e r e n o l - s t i m u l a t e d hormone r e l e a s e i s a c c o m p a n i e d by a n a c c u m u l a t i o n o f cAMP w h i c h i s o n l y 3% o f t h e cyclic n u c l e o t i d e a c c u m u l a t i o n e l i c i t e d by a c o m b i n a t i o n o f c h o l e r a t o x i n and a p h o s p h o d i e s t e r a s e i n h i b i t o r ( 2 3 ) . Furthermore, the I L c e l l s e x c r e t e cAMP i n t o t h e e x t r a c e l l u l a r medium so t h a t a f t e r a few m i n u t e s o f s t i m u l a t i o n o f t h e β - a d r e n o c e p t o r , more cAMP i s f o u n d o u t s i d e t h e c e l l s t h a n i s i n s i d e t h e m . This e v i d e n c e s u g g e s t s t h a t a m i n i m a l change i n i n t r a c e l l u l a r cAMP i s needed t o i n i t i a t e s e c r e t i o n and p o i n t s t o t h e need f o r a greater understanding of calcium-dependent secretion, in general, before significant progress can be made in understanding the β - a d r e n e r g i c s t i m u l a t i o n o f s e c r e t i o n from the I L , i n p a r t i c u l a r .


2



38

DOPAMINE

0

1

2

RECEPTORS

3

TIME (hours) Figure 3. Effect of certain drugs on lR-aMSH release from rat cells. Dispersed rat IL cells were exposed to isoproterenol, lisuride, or no drug (control) for the indicated periods of time. Isoproterenol enhances the release of IR-aMSH while lisuride inhibits the release of IR-aMSH. (Data are from Reference 12.)


KEBABIAN

E T AL.

D-2 Dopamine


ISOPROTERENOL

Receptor

DOPAMINE

Figure 4. A representation of the "working hypothesis" of the dual regulation of adenylate cyclase activity and hormone release by a β-adrenoceptor and a D-2 dopamine receptor in the IL of the rat pituitary gland. A β-adrenergic agonist (isoproterenol) occupies a β-adrenoceptor (β) to interact with a stimulatory guanyl nucleotide site (Ns); GTP interacts with Ν s to stimulate adenylate cyclase (cyclase) activity. A dopaminergic agonist (dopamine) occupies a D-2 dopamine receptor (D-2) to interact with the hypothetical inhibitory guanyl nucleotide component (Ni); GTP interacts with Ni to inhibit adenylate cyclase activity. GTP activates Ns only in the presence of α βadrenergic agonist and activates Ni only in the presence of a dopaminergic agonist. Cholera toxin selectively acts upon Ns. Increased formation of cAMP results in the enhanced release of IR-aMSH via an unknown mechanism(s). Newly formed cAMP can be hydrolyzed by intracellular phosphodiesterase(s) (PDE) to become 5'-AMP, or can be excreted into the extra cellular milieu.


DOPAMINE

RECEPTORS


τ

I-ISOPROTERENOL (log M)

Figure 5. Comparison of the potency of isoproterenol in eliciting physiological and biochemical responses from the rat IL. Substantially lower concentrations of iso proterenol stimulate the release of IR-aMSH (aMSH) than are required to enhance cAMP accumulation by intact IL cells (cAMP), stimulate adenylate cyclase activity in cell-free homogenates of IL tissue (cyclase), or occupy the specific binding sites defined with IHYP (binding) (19).


2.

KEBABIAN

ET

AL.

D-2 Dopamine


C y c l i c AMP and t h e I n t e r m e d i a t e

Receptor

4.

Lobe Dopamine R e c e p t o r

The r a t I L i s i n n e r v a t e d b y d o p a m i n e r g i c n e u r o n s . The somata o f t h e s e n e u r o n s a r e l o c a t e d i n t h e a r c u a t e n u c l e u s ; t h e i r a x o n s l e a v e t h e b r a i n v i a t h e p i t u i t a r y s t a l k and p r o j e c t t o t h e i n t e r m e d i a t e l o b e where t h e y t e r m i n a t e i n t h e v i c i n i t y o f the parenchymal I L c e l l s ( 2 6 ) . Baumgarten e t a l . d e s c r i b e 'synapse-like' connections between the dopaminergic nerve t e r m i n a l s and t h e p a r e n c h y m a l c e l l s o f t h e I L ( 2 7 ) . Several lines of evidence support the view that dopaminergic n e u r o t r a n s m i s s i o n o c c u r s w i t h i n t h e I L . F i r s t , dopamine i s t h e predominant catecholamine w i t h i n the I L (28, Saavedra, p e r s o n a l communication). S e c o n d , r a d i o l a b e l l e d dopamine i s t a k e n up and s t o r e d i n t h e I L ; s u b s e q u e n t l y , t h i s n e w l y s t o r e d dopamine c a n be r e l e a s e d from t h e n e r v e t e r m i n a l s ( 2 9 , 3 0 ) . The a v a i l a b l e evidence (which u n f o r t u n a t e l y does not include electrical r e c o r d i n g from t h e d o p a m i n e r g i c n e u r o n s t h e m s e l v e s ) suggests t h a t i n v i v o the dopaminergic neurons are spontaneously a c t i v e (30). Dopaminergic I n h i b i t i o n of Intermediate Lobe A d e n y l a t e Cyclase. The IL dopamine receptor can be studied with biochemical procedures. The r e c e p t o r i t s e l f c a n be i d e n t i f i e d in binding studies (31-33)* Using [3H]-spiroperidol, a dopamine a n t a g o n i s t from t h e b u t y r o p h e n o n e s e r i e s , t o i d e n t i f y binding sites and fluphenazine to define 'non-specific b i n d i n g ' , s p e c i f i c b i n d i n g s i t e s f o r the r a d i o l a b e l e d l i g a n d c a n be d e t e c t e d i n t h e i n t e r m e d i a t e l o b e ( F i g u r e 6 ) . The r a t I L c o n t a i n s 2 0 . 2 f m o l e o f h i g h a f f i n i t y ( K d = 0 . 3 nM) s p e c i f i c spiroperidol binding sites (33). The affinity of non radioactive dopaminergic agonists or antagonists can be i n f e r r e d from t h e i r a b i l i t y t o compete w i t h [ 3 H ] - s p i r o p e r i d o l f o r occupancy o f the s p e c i f i c b i n d i n g s i t e s ( e . g . see F i g u r e 7). L i s u r i d e i s t h e most p o t e n t o f t h e dopamine agonists t e s t e d and s p i r o p e r i d o l i s t h e most p o t e n t o f t h e dopamine antagonists tested. The dopamine receptor i n the IL regulates adenylate cyclase a c t i v i t y . S t i m u l a t i o n o f t h e I L dopamine receptor decreases the c a p a c i t y o f the parenchymal c e l l s to s y n t h e s i z e cAMP ( 1 2 , 1 9 - 2 2 , 3 7 ) . T h i s e f f e c t o f dopamine i s e s p e c i a l l y pronounced (and therefore amenable to experimental i n v e s t i g a t i o n ) when t h e I L a d e n y l a t e c y c l a s e a c t i v i t y h a s been increased with either isoproterenol or cholera t o x i n . The c h o l e r a t o x i n - t r e a t e d I L h a s p r o v e n t o be a n e s p e c i a l l y u s e f u l t i s s u e f o r i n v e s t i g a t i n g dopaminergic drugs s i n c e t h e i r e f f e c t upon t h e dopamine r e c e p t o r c a n be s e g r e g a t e d from any p o s s i b l e e f f e c t upon t h e β - a d r e n o c e p t o r ( F i g u r e 8 ) . Demonstration o f the dopaminergic i n h i b i t i o n o f I L adenylate c y c l a s e a c t i v i t y r e q u i r e s t h e p r e s e n c e o f GTP ( F i g u r e 2 , r i g h t ) .



DOPAMINE

RECEPTORS

3

[ H]-SPIROPERIDOL (nM)

Figure 6.

Binding of [3H]-spiroperidol to cell-free homogenates of the neurointermediate lobe of the rat pituitary gland.

Key: A, total (%) and nonspecific binding (i.e., binding in the presence of 2 μΜ fluphenazine) (O) was determined in the presence of the indicated concentrations of [3H]-spiroperidol; and B, specifically bound [3H]-spiroperidol (i.e., the difference between total and nonspecific bind ing) is shown as a function of the concentration of [3H]-spiroperidol. Left inset a plot of the data in Β according to Rosenthal's method', yields an apparent Kd of 0.2 nM and a maximal concentration of specific binding sites of 94.1 fmol/mg protein (this is equivalent to 20.2 fmol/NIL) (33).



2.

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Ο

D-2 Dopamine

6

10"

Receptor

5

10"

43

4

3

10"

10"

DOPAMINE (M)

Figure 7. Competition between [3H]-spiroperidol and dopamine for occupancy of binding sites in the NIL of the rat pituitary gland. The amount of [3H]-spiro peridol bound to NIL tissue was determined in the presence of 2 μΜ fluphenazine (0), 2 μΜ fluphenazine and 100 μΜ GTP (4) or dopamine (at the indicated concentrations) in the absence (O) or presence (Φ) of 100 Μ GTP (33). μ



DOPAMINE

0 10-6

10-5

10-4

(-)SULPIRIDE(M)

Figure 8.

10-6

10-5

RECEPTORS

10-4

APOMORPHINE (M)

Competitive interaction between apomorphine and (—)-sulpiride in regulating adenylate cyclase activity in the IL (33,).

IL tissue was treated with cholera toxin and adenylate cyclase activity was determined. Enzyme activity was determined in the presence of the indicated concentrations of (-)-sulpiride alone (O) or in combination with apomorphine (3 μΜ, φ; 10 μΜ, Δ ; or 30 μΜ, A) (left). In a separate experiment, enzyme activity was determined in the presence of the indicated concentrations of apomorphine alone (O) or in combination with (-)-sulpiride (3 μΜ, Δ»" 30 μΜ, A) (right). In both experiments, data represent the mean ± SE (n = 3).



2.

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ET

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D-2 Dopamine

Receptor

45

r3Hl-Spiroperidol Identified the Intermediate Lobe Dopamine R e c e p t o r . B e c a u s e t h e s p i r o p e r i d o l b i n d i n g a s s a y and the adenylate c y c l a s e assay are performed under equivalent a s s a y c o n d i t i o n s , a c o m p a r i s o n between t h e a f f i n i t i e s o f d r u g i n t h e two a s s a y s y s t e m s seems a p p r o p r i a t e . F i g u r e 9 shows t h e a p p r o x i m a t e agreement between t h e r e s u l t s o b t a i n e d from t h e two experimental protocols. The similarity between the values o b t a i n e d from t h e two a s s a y s y s t e m s s u p p o r t s t h e hypothesis t h a t some o r a l l o f t h e s p e c i f i c [ 3 H ] - s p i r o p e r i d o l b i n d i n g s i t e s a r e t h e dopamine r e c e p t o r s w h i c h when s t i m u l a t e d i n h i b i t adenylate cyclase a c t i v i t y . A "working hypothesis" o f the b i o c h e m i c a l o r g a n i z a t i o n o f t h e D - 2 dopamine r e c e p t o r i n t h e I L i s as f o l l o w s : t h e dopamine r e c e p t o r on t h e e x t e r i o r s u r f a c e o f a n I L c e l l i s c o u p l e d t o a d e n y l a t e c y c l a s e by an i n h i b i t o r y guanyl n u c l e o t i d e r e g u l a t o r y p r o t e i n ( N i ) , occupancy o f the r e c e p t o r by an a g o n i s t ( i . e . s t i m u l a t i o n ) a l t e r s t h e p r o p e r t i e s o f N i so t h a t i n t r a c e l l u l a r GTP c a n i n t e r a c t w i t h N i and t h e r e b y i n h i b i t t h e enzyme a c t i v i t y ( F i g u r e 4 ) . Dopamine a f f e c t s t h r e e p h y s i o l o g i c a l p r o c e s s e s i n t h e I L w h i c h , w i t h v a r y i n g d e g r e e s o f c e r t a i n t y , may be l i n k e d t o t h e a b i l i t y o f dopamine t o i n h i b i t t h e c a p a c i t y o f t h e I L t o s y n t h e s i z e cAMP. F i r s t the parenchymal c e l l s o f the r a t I L display spontaneous propagated e l e c t r i c a l spikes (38, 39). Dopamine d i m i n i s h e s t h e f r e q u e n c y , b u t n o t t h e a m p l i t u d e , o f t h e s e e l e c t r i c a l d i s c h a r g e s a p p a r e n t l y by s t i m u l a t i n g a D - 2 dopamine r e c e p t o r ( 4 0 ) . The p o s s i b l e i n v o l v e m e n t o f c y c l i c AMP i n t h i s i n h i b i t o r y p r o c e s s has not been i n v e s t i g a t e d . Second, dopamine i n h i b i t s t h e s p o n t a n e o u s r e l e a s e o f IR-aMSH ( F i g u r e 3) o r m e l a n o t r o p h i c hormones f r o m t h e I L ( 1 0 ) . The p r e s e n c e o f a r e c e p t o r f o r dopamine i n t h e I L o f many v e r t e b r a t e s p e c i e s h a s been i n f e r r e d from t h e a b i l i t y o f dopamine t o i n h i b i t the r e l e a s e o f m e l a n o t r o p h i c hormones o r IR-otMSH and t h e a b i l i t y o f dopaminergic antagonists to abolish t h i s effect o f dopamine (11 , 1 2 , 3 4 ) . The d o p a m i n e - i n d u c e d i n h i b i t i o n o f a d e n y l a t e cyclase activity and the dopamine-induced inhibition of electrical activity may participate in the dopaminergic i n h i b i t i o n o f the r e l e a s e o f IR-aMSH, T h i r d dopamine w i l l d i m i n i s h t h e a b i l i t y o f β - a d r e n e r g i c a g o n i s t s t o enhance e i t h e r a d e n y l a t e c y c l a s e a c t i v i t y o r t h e r e l e a s e o f IR-aMSH (1_2, _35, 37). The dopamine-induced decrease in the β-adrenergic enhancement o f cAMP s y n t h e s i s seems l i k e l y t o be i n v o l v e d i n t h i s phenomenon ( 3 8 ) . When the potency of dopaminergic agonists upon p r e p a r a t i o n s o f i n t a c t I L c e l l s i s compared t o t h e p o t e n c y o f t h e same compounds i n c e l l - f r e e a s s a y s y s t e m s ( i . e . a d e n y l a t e c y c l a s e o r s p i r o p e r i d o l b i n d i n g , a s t r i k i n g d i s c r e p a n c y emerges (Figure 10). F o r each o f the a g o n i s t s t e s t e d , the i n t a c t c e l l s respond to approximately 1/100th the c o n c e n t r a t i o n o f drug t h a t i s r e q u i r e d t o e l i c i t a comparable e f f e c t from the c e l l - f r e e homogenate. At the present time the hypothesis that the pituitary dopamine r e c e p t o r can e x i s t i n two " s t a t e s " of



DOPAMINE

-3

I ζ

-4

Ω Ζ

m

-5

RECEPTORS

1. Spiroperidol 2. Fluphenazine 3. Cis-Flupenthixol 4. Lisuride 5. Bromocriptine 6. Trans-Flupenthixol 7. ( - )-Sulpinde 8. Apomorphine 9. Dopamine 10. (+)-Sulpiride 11. LY 141865 12. Norepinephrine

—Ι

Ο g oc LU Ω

- 6 h

Ο

-7

Ο

ce

-10

- 9

- 8

- 7

- 6

- 5

AFFINITY FROM CYCLASE (log M)

Figure 9. Apparent affinity constants of agonists and antagonists for the dopamine receptor in the neurointermediate lobe of the rat pituitary gland. In studies of [3H]-spiroperidol binding, the apparent affinity constant of an agonist or antagonist was determined on the basis of the ability of the compound to compete with [3H]-spiroperidol for occupancy of the specific binding site (IL contributes 86% of the specific [3H]-spiroperidol binding sites in the NIL) and assuming a competitive interaction between the radiolabelled ligand and the nonradioactive compound. In studies of adenylate cyclase activity the apparent affinity constant of an agonist was the concentration of agonist producing half of its maximal inhibition of the enzyme activity in cholera toxin treated IL tissue. The apparent affinity constant of each antagonist was determined on the basis of the ability of the compound to reverse the apomorphine-induced inhibition of adenylate cyclase activity, by assuming a competitive interaction between apomorphine and each antagonist. Each value is the mean of determinations from three or more separate experiments (33).



2.

KEBABIAN

E T A L .

D-2 Dopamine

Receptor

47

APOMORPHINE (LOG M)

Figure 10.

Comparison of the potency of apomorphine as a dopaminergic agonist upon intact IL cells or cell-free homogenate of IL tissue.

For each response examined, inhibition of isoproterenol-stimulated cAMP accumulation by intact cells ( • ); inhibition of basal release of IR-aMSH by intact cells ( φ ); occupancy of specific [3H]-spiroperidol binding sites in a cell-free homogenate (O); and inhibition of isoproterenol-stimulated adenylate cyclase activity in a cell-free homogenate (M), the effect achieved with the indicated concentration of apomorphine is expressed as a percentage of the maximal effect of apomorphine (33J.

American Chemical Society Library 1155 13th St. N. w. Washington. 0. C. 20036 In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

48

DOPAMINE

RECEPTORS


differing affinity f o r agonists i s the most attractive explanation f o r t h i s discrepancy (4j_, 4 2 ; see a l s o Caron, t h i s volume). Concluding Remarks The IL o f the r a t p i t u i t a r y gland possesses a dopamine receptor amenable to experimental investigation. Two interesting points have emerged from the combined p h y s i o l o g i c a l , pharmacological and biochemical approach towards this tissue. F i r s t , the p h y s i o l o g i c a l a c t i o n s o f dopamine upon the IL "seem not to i n v o l v e e i t h e r the s t i m u l a t i o n o f an a d e n y l y l c y c l a s e or the accumulation of i n t r a c e l l u l a r c y c l i c AMP (1_)." Furthermore, the IL dopamine receptor i s stimulated by ergots (e.g. l i s u r i d e or bromocriptine) and blocked by the s u b s t i t u t e d benzamides (e.g. ( - ) - s u l p i r i d e ) . Therefore, the IL dopamine receptor ressembles the D-2 dopamine receptor i n the c l a s s i f i c a t i o n schema o f Kebabian and Calne ( 1_). Second, the IL has become a u s e f u l component o f a screening p r o t o c o l designed to i d e n t i f y s e l e c t i v e antagonists o f e i t h e r the D-1 or the D-2 r e c e p t o r . The a b i l i t y o f LY 141865 ( f o r s t r u c t u r e see F i g u r e 11) to s e l e c t i v e l y stimulate the D-2 receptor (36) and the a b i l i t y o f YM-09151-2 (Figure 11) t o s e l e c t i v e l y block t h i s receptor (43) were discovered i n experiments u t i l i z i n g the IL dopamine receptor as the model D-2 receptor and the g o l d f i s h r e t i n a as the model D-1 r e c e p t o r . These compounds together with the s e l e c t i v e D-2 a g o n i s t s , RU 24213 and RU 2 4 9 2 6 , and the s e l e c t i v e D-2 antagonist, domperidone, ( f o r s t r u c t u r e s see Figure 11 ) provide u s e f u l pharmacological t o o l s f o r i d e n t i f y i n g other D-2 receptors ( 4 4 - 4 8 ) . Two categories of dopamine receptor exist i n the cardiovascular system. These two receptors have been designated as the DA-1 and the DA-2 receptors ( 4 9 , see Goldberg and K o h l i , t h i s volume). The D-2 and the DA-2 receptors have very s i m i l a r pharmacological properties. However, the DA-1 receptor and the D-1 receptor are perceived by Goldberg and h i s colleagues as being d i s t i n c t e n t i t i e s . In p a r t i c u l a r , the DA-1 receptor i s blocked by ( + ) - s u l p i r i d e ; ( + ) - s u l p i r i d e i s the most potent and most s e l e c t i v e antagonist o f the DA-1 receptor ( 4 9 ) . In contrast, the D-1 receptor i s a t best only weakly antagonized by t h i s compound ( 4 6 , 4 7 ) . Although ( + ) - s u l p i r i d e i s the most potent and most s e l e c t i v e antagonist o f the DA-1 receptor i n i n v i t r o experiments, when i t i s t e s t e d i n v i t r o upon s t r i p s o f i s o l a t e d canine mesenteric a r t e r y , i t i s a weak dopamine antagonist ( 5 0 ) . S i m i l a r l y , ( + ) - s u l p i r i d e i s a weak antagonist o f the D-1 receptor i n the bovine p a r a t h y r o i d gland (see Brown and Dawson-Hughes, t h i s volulme). In the f u t u r e , i t w i l l be important to i d e n t i f y the b a s i s f o r the d i f f e r e n c e between the i n v i v o and i n v i t r o potencies o f s u l p i r i d e . However, i n the context o f the present d i s c u s s i o n about the number and p r o p e r t i e s o f the c a r d i o v a s c u l a r dopamine r e c e p t o r s ,



KEBABIAN

Figure IL

ET AL.

D-2 Dopamine

Receptor

Structures of agonists (top) and antagonists (bottom) of the D-2 receptor.



50

DOPAMINE

RECEPTORS

i t seems worthwhile to consider four s i m i l a r i t i e s between the DA-1 and the D-1 receptors. F i r s t , (+)-bulbocapnine i s an antagonist capable o f b l o c k i n g e i t h e r the D-1 or the DA-1 receptor; t h i s compound i s s u b s t a n t i a l l y l e s s potent as an antagonist o f the DA-2 receptor and i s devoid of antagonist a c t i v i t y upon the D-2 receptor (51, 52, Itoh, Goldman and Kebabian, unpublished o b s e r v a t i o n s ) . Second, SKF 38393 i s an agonist upon the D-1 and the DA-1 receptor, yet i t does not e l i c i t the p h y s i o l o g i c a l responses o c c u r r i n g as a consequence of s t i m u l a t i o n of e i t h e r the D-2 or the DA-2 receptor (53) * T h i r d , LY 141865, a s e l e c t i v e D-2 agonist (36), f a i l s to stimulate the DA-1 receptor i n the r e n a l v a s c u l a t u r e of the r a t (54). Fourth, domperidone i s an antagonist o f e i t h e r the D-2 or the DA-2 receptor which does not block e i t h e r the D-1 or the DA-1 receptors (45, 4B). These four s i m i l a r i t i e s between the D-1 and the DA-1 receptor suggest that they are extremely s i m i l a r i f not i d e n t i c a l i n terms of t h e i r pharmacological properties. C l e a r l y , i t w i l l a l s o be h e l p f u l to determine the b a s i s f o r the d i f f e r e n c e i n the i n v i v o and i n v i t r o potencies o f the s u b s t i t u t e d benzamides. However, at present i t may be premature to discount the s t r i k i n g s i m i l a r i t i e s between the D-1 and the DA-1 r e c e p t o r s . The IL also serves as a model for dopaminergic neurotransmission i n the b r a i n ; the concepts f i r s t e l u c i d a t e d i n the IL can be a p p l i e d to the b r a i n . For example, i n the neostriatum the i n t e r a c t i o n between dopamine and a D-2 receptor i n h i b i t s the stimulated e f f l u x of cAMP (and by i n f e r e n c e the enhancement of adenylate c y c l a s e a c t i v i t y ) o c c u r r i n g as a consequence of s t i m u l a t i o n of the D-1 receptor 55, 56). The types o f dopamine receptors i n the CNS and t h e i r p h y s i o l o g i c a l f u n c t i o n are discussed i n t h i s volume by Stoof.

Literature Cited 1. 2. 3.

4.

5. 6. 7.

Kebabian, J . W.; Calne, D. B. Nature 1979, 277, 93-96. Kebabian, J . W.; Calne, D. B.; Kebabian, P. R. Commun. i n Psychopharmacol. 1977, 1, 311-318. Kebabian, J. W. "Dopamine, Advances in Biochemical Psychopharmacology Volume 19"; Raven Press: New York, 1978; pp 131-154. Borgeat, P.; Chavancy, G.; Dupont, Α.; Labrie, F.; Arimura, Α.; S c h a l l y , Α. V. Proc. N a t l . Acad. S c i . USA 1972, 69, 2677-2681. D e C a m i l l i , P.; Macconi, D.; Spada, A. Nature 1979, 278, 252-254. G i a n n a t t a s i o , G.; De F e r r a r i , M. E.; Spada, A. L i f e S c i . 1981, 28, 1605-1612. Thorner, M. O.; Hackett, J . T.; Murad, F.; MacLeod, R. M. Neuroendocrinology 1980, 31, 309-402.


2. 8.

9. 10.


11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26.

27. 28. 29. 30. 31.

KEBABIAN

ET

AL.

D-2 Dopamine

Receptor

51

Baker, B. "Handbook o f Physiology: S e c t i o n 7, Volume 4, Part I , The Pituitary Gland and I t s Neuroendocrine Control"; American Physiological Society: Bethesda, Maryland, 1974; pp 45-80. Dube, D.; L i s s i t z k y , J . C.; L e c l e r c , R.; P e l l e t i e r , G. Endocrinology 1978, 102, 1283-1291. Bower, Α.; Hadley, M. E.; Hruby, V. J . Science 1974, 184, 70-72. Morgan, C. M.; Hadley, M. E. Neuroendocrinology 1976, 21, 10-19. Munemura, M.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1980, 106, 1795-1803. Baker, Β. I . J. E n d o c r i n o l . 1974, 63, 533-538. E i p e r , Β. Α.; Mains, R. E. Endocrine Rev. 1980, 1, 1-27. S i l v e r s , W. K. "The Coat Colors o f Mice"; S p r i n g e r - V e r l a g : New York, 1979; pp 1-5. Geschwind, I . I . ; Huseby, R. Α.; Nishioka, R. Rec. Prog. Horm. Res. 1972, 28, 91-130. Rudman, D.; Chawla, R. K.; H o l l i n s , B. M. J . Biol. Chem. 1979, 254, 10102-10108. Browne, C. Α.; Bennett, H. P. J . ; Solomon, S. Biochemistry 1981, 20, 4538-4546. Cote, T.; Munemura, M.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1980, 107, 108-116. Cote, T. E.; Grewe, C. W.; Kebabian, J . W. Endocrinology 1982, 110, 805-811. Muniere, H. and L a b r i e , F. Eur. J. Pharmacol. 1982, 81, 411-420. Cote, T. E.; Grewe, C. W.; Tsuruta, K.; Stoof, J . C.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1982, 110, 812-819. Tsuruta, K.; Grewe, C. W.; Cote, T. E.; Eskay, R. L.; Kebabian, J . W. Endocrinology 1982, 110, 1133-1139. Baker, B.I. J . E n d o c r i n o l . 1974, 63, 533-538. Tomiko, S. Α.; Taraskevich, P. S.; Douglas, W. W. Neuroscience 1981, 6, 2259-2267. L i n d v a l l , O.; Bjorklund, A. "Handbook o f Experimental Pharmacology, Volume 9"; Plenum Press: New York, 1978; pp 139-231. Baumgarten, H. G.; Bjorklund, Α.; H o l s t e i n , A. F.; Nobin, A. Z. Z e l l f o r s c h . 1972, 126, 483-517. Saavedra, J . M.; P a l k o v i t s , M.; K i z e r , J . S.; Brownstein, M.; Z i v i n , J . J. Neurochem. 1975, 25, 257-260. Tilders, F. J . H.; Mulder, A. H.; Smelik, P. G. Neuroendocrinology 1975, 18, 125-130. T i l d e r s , F. J . H.; Van Der Woude, Η. Α.; Swaab, D. F.; Mulder, A. H. B r a i n Res. 1979, 171, 425-435. S i b l e y , D. R.; Creese, I . Endocrinology 1980, 107, 14051419.


DOPAMINE

52 32. 33. 34. 35.


36. 37. 38. 39. 40. 41. 42. 43. 44.

45. 46. 47. 48. 49. 50.

51.

52. 53. 54. 55. 56.

RECEIVED

RECEPTORS

Stefanini, E.; Devoto, P.; Marchisio A . M.; V e r n a l e o n e , A . M.; Collu, R . Life Sci. 1 9 8 0 , 2 6 , 5 8 3 - 5 8 7 . F r e y , Ε . Α . ; C o t e , T . E.; Grewe, C . W . ; K e b a b i a n , J. W. Endocrinology 1 9 8 2 , 110, 1 8 9 7 - 1 9 0 4 . Munemura, M.; C o t e , T . E.; Tsuruta, K.; Eskay, R. L.; K e b a b i a n , J. W. Endocrinology 1980, 1 9 7 , 1676-1683. C o t e , T . E.; Grewe, C . W . ; K e b a b i a n , J. W. E n d o c r i n o l o g y 1981, 108, 420-426. Tsuruta, K.; F r e y , Ε . Α . ; Grewe, C . W . ; C o t e , T . E.; E s k a y , R . L.; K e b a b i a n , J. W. N a t u r e 1 9 8 1 , 2 9 2 , 4 6 3 - 4 6 5 . M u n i e r e , H.; Labrie, F. Life Sci. 1 9 8 2 , 3 0 , 9 6 3 - 9 6 8 . D o u g l a s , W. W . ; Taraskevich, P. S. J. Physiol. (London) 1978, 285, 171-184. D o u g l a s , W. W . ; Taraskevich, P. S. J. Physiol. (London) 1980, 209, 623-630. D o u g l a s , W. W . ; Taraskevich, P . S. J. Physiol. (London) 1 9 8 2 , 326, 2 0 1 - 2 1 1 . De L e a n , Α . ; Kilpatrick B . F.; C a r o n , M . G . E n d o c r i n o l o g y 1 9 8 2 , 110, 1 0 6 4 - 1 0 6 6 . S i b l e y , D . R . ; De L e a n , Α . ; C r e e s e , I. J. Biol. Chem. 1982, 257, 6351-6361. G r e w e , C . W . ; F r e y , Ε . Α . ; C o t e , T . E.; K e b a b i a n , J. W. Eur. J. P h a r m a c o l . 1 9 8 2 , 81, 1 4 9 - 1 5 2 . E u v a r d , C; Ferland, L.; Dipaolo, T.; Beaulieu, M.; Labrie, F.; Oberlander, C.; Raynaud, J.P.; Boissier, J.R. Neuropharmacology, 1980, 19, 379. Denef, C.; Follebouckt, J.-J. Life Sci. 1978, 23, 4 3 1 . W a t l i n g , K.J.; D o w l i n g , J.E.; Iversen, L.L. Nature, 1979, 281, 578. W a t l i n g , K.J.; D o w l i n g , J.E. J. Neurochem. 1 9 8 1 , 3 5 , 5 5 9 . Kohli, J.D.; Glock, D.; Goldberg, L.I. Eur. J. Pharmacol. 1983, in press. Goldberg, L.I.; Kohli, J.D. Commun. Psychopharmacol. 1 9 7 9 , 3, 4 4 7 - 4 5 6 . Kohli, J.D.; Takeda, H.; Ozaki, N.; Goldberg, L.I. "Advances in the Biosciences: Volume 20, Peripheral Dopaminergic Receptors"; Pergamon P r e s s : Oxford, U.K., 1 9 7 9 ; pp 1 4 3 - 1 4 9 . Goldberg, L.I.; Musgrave, G.E.; Kohli, J.D. "Sulpiride and O t h e r B e n z a m i d e s " ; Italian Brain Research Foundation Press: Milan, Italy 1 9 7 9 ; pp 7 3 - 8 1 . Shepperson, N.B.; D u v a l , N.; M a s s i n g h a m , R . ; L a n g e r , S . Ζ . J. P h a r m a c o l . E x p . T h e r . 1 9 8 0 , 2 2 1 , 7 5 3 - 7 6 1 . Setler, P.E.; Sarau, H.M.; Zirkle, C.L.; Saunders, H.L. Eur. J. P h a r m a c o l . 1 9 7 8 , 5 0 , 4 1 9 . H a h n , R.A.; M c D o n a l d , B.; Martin, M. J. Pharmacol. Exp. T h e r . 1983, 224, 206-214. S t o o f , J. C.; K e b a b i a n , J. W. N a t u r e 1 9 8 1 , 2 9 4 , 3 6 6 - 3 6 8 . S t o o f , J. C.; K e b a b i a n , J. W. Brain R e s . 1 9 8 2 , 2 5 0 , 2 6 3 270. November 4, 1982



Commentary: The D A _ Dopamine Receptor in the Anterior and Intermediate Lobes of the Pituitary Gland F . L A B R I E , H . M E U N I E R , M . G O D B O U T , R. V E I L L E U X , V . G I G U E R E , L . P R O U L X - F E R L A N D , T . DI P A O L O , and V . R A Y M O N D Le Centre Hospitalier de l'Université Laval, Department of Molecular Endocrinology, Quebec G 1 V 4G2, Canada

Dopamine released from nerve endings of tuberoinfund i b u l a r neurons is the main f a c t o r of hypothalamic o r i g i n which exerts a d i r e c t i n h i b i t o r y e f f e c t on p r o l a c t i n s e c r e t i o n in the a n t e r i o r p i t u i t a r y gland. The dopamine receptor on p i t u i t a r y mammotrophs is n e g a t i v e l y coupled to adenylate c y c l a s e . Estrogens i n h i b i t while androgens and progestins as w e l l as androgens facilitated the activity of dopamine on p r o l a c t i n s e c r e t i o n by an a c t i o n at a post-receptor site. The p e p t i d i c c o r t i c o t r o p i n - r e l e a s i n g f a c t o r (CRF) i s a potent s t i m u l a t o r of adenylate c y c l a s e activity and peptide s e c r e t i o n in pars intermedia cells. The intermediate lobe i s thus c o n t r o l l e d by two stimulatory r e c e p t o r s , namely CRF and β-adrenerg i c while it is i n h i b i t e d by a dopaminergic receptor negatively coupled to adenylate c y c l a s e . The nomen c l a t u r e DΑ+, DA_ and DA0 is proposed f o r dopamine receptors which are p o s i t i v e l y , n e g a t i v e l y or un coupled, r e s p e c t i v e l y , to adenylate c y c l a s e . The dopamine receptors i n both the a n t e r i o r and interme d i a t e lobes of the p i t u i t a r y gland are n e g a t i v e l y coupled to adenylate cyclase and are thus t y p i c a l of the DA_ type.

The predominant r o l e of dopamine i n normal b r a i n f u n c t i o n s as w e l l as i n diseases such as Parkinson's disease and schizophrenia i s a strong stimulus f o r research on the dopamine receptor and i t s mechanism of a c t i o n . Since dopamine appears to be the main i f not the e x c l u s i v e f a c t o r of hypothalamic o r i g i n a c t i n g as i n h i b i t o r of prolactin secretion 2) and changes of p r o l a c t i n s e c r e t i o n can be measured with p r e c i s i o n i n vivo and i n v i t r o , the a n t e r i o r p i t u i t a r y gland i s an a t t r a c t i v e model f o r a dopaminergic recep t o r . Another model of great i n t e r e s t i s the intermediate lobe of the p i t u i t a r y gland, a pure population of dopamine-sensitive c e l l s 0097-6156/83/0224-005 3 $06.00/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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s p e c i a l i z e d i n the s e c r e t i o n of proopiomelanocortin-related p e p t i des, s p e c i a l l y α-MSH (a-raelanotropin) and 3-endorphin ( 3 ) . Both mammotrophs of the r a t a n t e r i o r p i t u i t a r y gland and pars interme d i a c e l l s can e a s i l y be grown i n monolayer c u l t u r e where changes of dopamine receptors can be c o r r e l a t e d with biochemical parame ters such as changes i n c y c l i c AMP l e v e l s and peptide s e c r e t i o n i n t o the medium. Kebabian et a l . ( t h i s volume) present a c l e a r and p r e c i s e r e view of the l a r g e body of biochemical data obtained by t h e i r group on the c o n t r o l of pars intermedia c e l l a c t i v i t y by 3-adrenergic agents and dopamine during the l a s t years (4-13). Since the p e p t i d i c c o r t i c o t r o p i n - r e l e a s i n g f a c t o r (CRF) i s a potent s t i m u l a t o r of pars intermedia c e l l a c t i v i t y (14^ 15, 16), we w i l l complement t h i s review by summarizing recent data on the mechanism of a c t i o n of CRF i n the intermediate lobe as w e l l as i t s i n t e r a c t i o n with 3adrenergic and dopaminergic agents. Data w i l l a l s o be presented on the coupling of the a n t e r i o r p i t u i t a r y dopamine receptor con t r o l l i n g p r o l a c t i n s e c r e t i o n with adenylate c y c l a s e as w e l l as on the marked i n t e r a c t i o n s of t h i s receptor with gonadal s t e r o i d s . F i n a l l y , taking i n t o account a l l a v a i l a b l e evidence, we w i l l pro pose to use the nomenclature DA+, DA^ and DA f o r dopamine r e ceptors coupled p o s i t i v e l y , n e g a t i v e l y or uncoupled, r e s p e c t i v e l y , to adenylate c y c l a s e . The dopamine receptors i n the a n t e r i o r and intermediate lobes of the p i t u i t a r y gland are examples of dopamine receptors n e g a t i v e l y coupled to adenylate c y c l a s e (DA_ t y p e ) . Q

The dopamine receptor i n the a n t e r i o r p i t u i t a r y gland i s negative l y coupled to adenylate c y c l a s e The r o l e of c y c l i c AMP as modulator of p r o l a c t i n s e c r e t i o n was f i r s t suggested by the f i n d i n g of a s t i m u l a t o r y e f f e c t of c y c l i c AMP d e r i v a t i v e s (17-22) and i n h i b i t o r s of c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t y such as t h e o p h y l l i n e and IBMX (22-26) on the s e c r e t i o n of t h i s hormone. More convincing evidence supporting a r o l e of c y c l i c AMP i n the a c t i o n of dopamine on pro l a c t i n s e c r e t i o n had to be obtained, however, by measurement of adenohypophysial adenylate c y c l a s e a c t i v i t y or c y c l i c AMP accumu l a t i o n under the i n f l u e n c e of the catecholamine. As i l l u s t r a t e d i n F i g . 1, a d d i t i o n of 100 nM dopamine to male r a t h e m i p i t u i t a r i e s led to a rapid i n h i b i t i o n of c y c l i c AMP accumulation, a maximal e f f e c t (30% i n h i b i t i o n ) being already obtained 5 min a f t e r a d d i t i o n of the catecholamine. Thus, while dopamine i s w e l l known t o stimulate adenylate cyclase a c t i v i t y i n the striatum (27, 28), i t s e f f e c t at the adenohypophysial l e v e l i n i n t a c t c e l l s i s i n h i b i t o r y . Dopamine has also been found to exert p a r a l l e l i n h i b i t o r y e f f e c t s on c y c l i c AMP l e v e l s and p r o l a c t i n r e l e a s e i n ovine adeno hypophysial c e l l s i n c u l t u r e (29) and p u r i f i e d r a t mammotrophs (30). Using paired h e m i p i t u i t a r i e s obtained from female r a t s , Ray and W a l l i s (22) have found a r a p i d i n h i b i t o r y e f f e c t of dopamine on c y c l i c AMP accumulation to approximately 75% of c o n t r o l .



Figure 1. Time course of the effect of dopamine (100 nM) on cyclic AMP accumulation in male rat anterior pituitaries (54). Key: O — O, control; and ·—·, with dopamine.



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As much as higher concentrations of dopamine are required to stimulate s t r i a t a l adenylate cyclase a c t i v i t y as compared to the a f f i n i t y of the drug f o r the a g o n i s t i c s i t e s of the receptor, the s e n s i t i v i t y of the a n t e r i o r p i t u i t a r y adenylate cyclase to dopamine agonists i n both human prolactinoma (26) and r a t adenohypophys i a l homogenate (31) i s lower than the potency of the compounds to i n h i b i t p r o l a c t i n s e c r e t i o n i n i n t a c t c e l l s ( 2 ) . This v a r i a b l e loss of s e n s i t i v i t y upon c e l l homogeneization i s a phenomenon frequently observed i n other systems (32). The reports that DA d i d not show s i g n i f i c a n t changes of cyc l i c AMP l e v e l s i n the adenohypophysis (33, 34, 35) have l e d to the suggestion that the p i t u i t a r y DA receptor i s a prototype of D receptors (D ) not coupled to adenylate cyclase (36). These negat i v e r e s u l t s were however obtained using a very heterogeneous t i s s u e , the t o t a l a n t e r i o r p i t u i t a r y gland. The background of c y c l i c AMP contributed by the f i v e other c e l l types could e a s i l y mask the changes i n c y c l i c AMP o c c u r r i n g s p e c i f i c a l l y i n mammotrophs under the i n f l u e n c e of dopamine. Using p i t u i t a r y homogenate obtained from female r a t s containing a higher proportion of mammotrophs than the t i s s u e used i n previous studies (33, 34), Giannattasio et a l . (31) could demonstrate a c l e a r GTP-dependent i n h i b i t i o n of adenylate cyclase a c t i v i t y . Since c y c l i c AMP d e r i v a t i v e s and i n h i b i t o r s of c y c l i c nucleot i d e phosphodiesterase stimulate p r o l a c t i n r e l e a s e (17, 37, 38) and dopamine i s a potent i n h i b i t o r of p r o l a c t i n s e c r e t i o n (1_, 2> 39), i t i s not s u r p r i s i n g that the catecholamine does not s t i m u l a te the adenylate cyclase system. On the contrary, the data summar i z e d above show that the p i t u i t a r y DA receptor i s n e g a t i v e l y coupled to adenylate c y c l a s e . The p i t u i t a r y DA receptor i s thus a t y p i c a l DA_-receptor (40, 41). In view of the m u l t i p l i c i t y of f a c t o r s involved i n the c o n t r o l of p r o l a c t i n s e c r e t i o n , i n c l u d i n g sex s t e r o i d s , i t i s l i k e l y that mechanisms other than c y c l i c AMP are involved (39, 42)· It does however appear that i n h i b i t i o n of c y c l i c AMP formation by dopamine i s a key element i n a m u l t i f a c t o r i a l c o n t r o l system responsible f o r the f i n e tuning of p r o l a c t i n secretion.

2

2

Modulation of the a n t e r i o r p i t u i t a r y dopamine receptor by gonadal steroids Administration of estrogens i s w e l l known to cause an i n c r e a se i n plasma p r o l a c t i n (PRL) l e v e l s i n man (43, 44 ) as w e l l as i n the r a t (45-48). This stimulatory e f f e c t of estrogens i s a l s o observed i n v i t r o i n a n t e r i o r p i t u i t a r y gland expiants (49), tumor a l adenohypophysial c e l l s (50) and normal r a t a n t e r i o r p i t u i t a r y c e l l s i n primary c u l t u r e (39, 40, 42, 51). Seventeen-3-estradiol ( E ) does not only stimulate basal and TRH-induced PRL s e c r e t i o n i n r a t a n t e r i o r p i t u i t a r y c e l l s i n c u l t u r e but i t can also reverse almost completely the i n h i b i t o r y e f f e c t of dopamine (DA) agonists on PRL release (40). 2


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Our data have shown that the non-aromatisable androgen DHT and progestins can act d i r e c t l y at the p i t u i t a r y l e v e l at p h y s i o l o g i c a l concentrations to i n h i b i t spontaneous PRL s e c r e t i o n and reverse the well-known s t i m u l a t o r y e f f e c t of E (3^9, 40_, 4_2, 51). In a d d i t i o n , the e f f e c t of E , DHT and progestins i s observed, not only on spontaneous and TRH-induced PRL s e c r e t i o n , but a l s o i n the presence of IBMX. This l a s t observation suggests that the marked modulatory e f f e c t s of sex s t e r o i d s are exerted at a step f o l l o w i n g c y c l i c AMP formation. Ihese r e s u l t s prompted us to suggest a model f o r the s i t e of the modulatory r o l e of sex s t e r o i d s on PRL s e c r e t i o n i n the female r a t ( F i g . 2). TRH probably acts through a c t i v a t i o n of adenylate cyclase, the e f f e c t of TRH being mimicked by c y c l i c AMP d e r i v a t i ves and i n h i b i t o r s of c y c l i c n u c l e o t i d e phosphodiesterase (17, 52, 53) ( F i g . 2) while DA receptors are n e g a t i v e l y coupled to adenylate c y c l a s e (26, 54). Estrogens exert t h e i r stimulatory a c t i o n on TRH receptors as w e l l as at steps f o l l o w i n g cAMP formation while androgens and progestins apparently i n h i b i t PRL s e c r e t i o n at a post-receptor step. This schematic r e p r e s e n t a t i o n does not exclude other elements which are probably involved i n the c o n t r o l of the mammotroph c e l l but i t only i n d i c a t e s the main s i t e s of s t e r o i d a c t i o n using the p r e s e n t l y a v a i l a b l e evidence. C a and the phosp h a t i d y l i n o s i t o l response are also i n t i m a t e l y involved i n the s e c r e t i o n mechanisms i n mammotrophs. The present data i n d i c a t e that besides p r o v i d i n g a b e t t e r knowledge of the c o n t r o l of p r o l a c t i n s e c r e t i o n , the tuberoinfund i b u l a r system can a l s o be used as a model f o r other l e s s a c c e s s i b l e dopaminergic systems i n the c e n t r a l nervous system. The i n t e rest of such studies i n the r a t on the i n t e r a c t i o n of sex s t e r o i d s with b r a i n catecholaminergic systems i s strengthened by the observ a t i o n that estrogen a d m i n i s t r a t i o n to male or female p a t i e n t s r e c e i v i n g n e u r o l e p t i c s can f a c i l i t a t e the appearance of parkinsonian symptoms (55). Moreover, treatment with estrogens has been found to improve the symptoms of t a r d i v e dyskinesias induced by chronic treatment with L-Dopa or n e u r o l e p t i c s (56). The abovementioned e f f e c t s of estrogen treatment i n the human are compatible with an antidopaminergic a c t i o n and open the p o s s i b i l i t y of c l i n i c a l a p p l i c a t i o n s of sex hormones i n the treatment of neurolog i c a l as w e l l as p s y c h i a t r i c d i s e a s e s . 2


2

2 +

The dopamine receptor i n the intermediate lobe of the p i t u i t a r y gland i s n e g a t i v e l y coupled to adenylate c y c l a s e As i l l u s t r a t e d i n F i g . 3A, dopamine leads to a 30% (p < 0.01) i n h i b i t i o n of basal c y c l i c AMP l e v e l s i n pars intermedia c e l l s at an E D value of 5.0 nM. An almost i d e n t i c a l potency of dopamine i s observed on the elevated c y c l i c AMP concentration induced by simultaneous incubation with 30 nM ( - ) i s o p r o t e r e n o l ( F i g . 3B). S i m i l a r i n h i b i t o r y e f f e c t s of dopamine are observed i n the presence of a phosphodiesterase i n h i b i t o r , isobutylmethylxanthine, thus 5 Q



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Figure 2. Representation of the site of interaction of sex steroids, TRH, and dopamine on PRL secretion in the anterior pituitary gland according to the data obtained in the female rat.



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B

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-8

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DOPAMINE (LOG M)

Figure 3. Effect of increasing concentrations of dopamine on basal (A) and (—)isoproterenol-induced (B) cyclic AMP levels in rat pars intermedia cells in culture. Cells were incubated for 30 min in DM EM containing 5 mM HEPES, 100 μΜ ascorbic acid, and the indicated concentrations of dopamine alone (A). In B, 30 nM ( — isoproterenol was present during the last 4 min of incubation. Data are expressed as percent of control (in the absence of dopamine). Control cyclic AMP levels were 0.96 ± 0.10 and 5.29 ± 0.13 pmol/2 χ 10 cells in the absence (A) or presence (B) of 30 nM (—isoproterenol, respectively (4\). 5


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i n d i c a t i n g that the changes i n c y c l i c AMP l e v e l s are due to p a r a l l e l i n h i b i t i o n of adenylate cyclase a c t i v i t y rather than to stimul a t i o n of c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t y . Apomorphine, the prototype of dopaminergic agonists ( 5 7 ) , i s approximat e l y 3 times more potent than dopamine i n i n h i b i t i n g pars i n t e r media c y c l i c AMP l e v e l s at a K value of 1.5 nM ( 4 1 ) . Ergot a l k a l o i d s which act as potent dopaminergic agonists on p r o l a c t i n r e l e a s e i n the a n t e r i o r p i t u i t a r y gland ( 2 ) a l s o show potent agon i s t i c a c t i v i t y i n the intermediate lobe: l i s u r i d e , 2-bromo-cte r g o c r y p t i n e , p e r g o l i d e , dihydroergocryptine and l e r g o t r i l e i n h i b i t b a s a l c y c l i c AMP l e v e l s at KQ values of 0.05, 0.20, 0 . 2 7 , 0.50 and 2.7 nM, r e s p e c t i v e l y . (-)Norepinephrine and ( ^ e p i n e p h r i ne are l e s s potent, half-maximal i n h i b i t o r y e f f e c t s on c y c l i c AMP l e v e l s being measured at 3 0 and 5 0 nM, r e s p e c t i v e l y . P r o p r a n o l o l ( 1 0 0 nM) was present during i n c u b a t i o n with (-)epinephrine and (-)norepinephrine i n order to block t h e i r i n t e r a c t i o n with the 3adrenergic receptor which i s s t i m u l a t o r y on c y c l i c AMP accumulat i o n i n pars intermedia c e l l s ( 4 ) · The s p e c i f i c i t y of the dopamine receptor was f u r t h e r studied with a s e r i e s of dopaminergic antagonists of w e l l known pharmacological activity. The 3 0 - 4 0 % i n h i b i t o r y e f f e c t of 10 nM dopamine was completely reversed by the a d d i t i o n of i n c r e a s i n g concentrat i o n s of the potent n e u r o l e p t i c s (+)butaclamol ( K = 1.5 nM) and ( - ) s u l p i r i d e ( K = 0.5 nM) while t h e i r pharmacologically weak enantiomers (-)butaclamol and ( + ) s u l p i r i d e were 86 and 167 times l e s s potent, r e s p e c t i v e l y . The n e u r o l e p t i c s s p i r o p e r i d o l , t h i o properazine, domperidone, h a l o p e r i d o l , fluphenazine and pimozide completely reversed the i n h i b i t o r y e f f e c t of dopamine at low K values ranging from 0.02 to 0.8 nM ( 4 1 ) .


D

N

N

N

Coupling o f dopamine receptors with adenylate c y c l a s e : DA+, DA_ and PA receptors Q

Dopamine can thus be added to the l i s t of hormones and neurot r a n s m i t t e r s which can stimulate or i n h i b i t c y c l i c AMP formation, depending upon t h e i r t i s s u e of a c t i o n . Thus, while dopamine stimul a t e s c y c l i c AMP formation i n parathyroid c e l l s , s u p e r i o r c e r v i c a l g a n g l i a , r e t i n a and s t r i a t a l t i s s u e ( 2 7 , 5 8 - 6 1 ) , i t i n h i b i t s the accumulation of the c y c l i c n u c l e o t i d e i n c e l l s of the intermediate and a n t e r i o r lobes of the p i t u i t a r y gland. Opposite e f f e c t s on the c y c l i c AMP system are a l s o found with LHRH which stimulates and i n h i b i t s c y c l i c AMP l e v e l s i n the a n t e r i o r p i t u i t a r y gland ( 6 2 ) and ovary ( 6 3 ) , r e s p e c t i v e l y . S i m i l a r l y , alpha-adrenergic agents show opposite e f f e c t s on c y c l i c AMP formation i n b r a i n ( 6 4 ) and p l a t e l e t s ( 6 5 ) . PGE, stimulates c y c l i c AMP formation i n the anter i o r p i t u i t a r y gland ( 6 2 ) while i t i n h i b i t s the same parameter i n fat cells ( 6 6 ) . There i s much ambiguity i n the c u r r e n t l y used c l a s s i f i c a t i o n s of dopamine r e c e p t o r s . This complexity i s p a r t l y due to the combined use of a c l a s s i f i c a t i o n based on binding data ( 6 7 , 6 8 ) and



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Receptor

another one based on f u n c t i o n of the dopamine receptor (36, 41, 54). U n t i l more p r e c i s e data are a v a i l a b l e i n a l a r g e v a r i e t y of t i s s u e s (using homogeneous c e l l populations, i f p o s s i b l e ) , c l a s s i f i c a t i o n s of dopamine receptors based on the binding c h a r a c t e r i s t i c s of various ligands and c l a s s i f i c a t i o n s d e s c r i b i n g f u n c t i o n a l coupling to various c e l l u l a r a c t i v i t i e s such as a c t i v a t i o n of adenylate c y c l a s e , should be kept separate. Although c l i n i c a l i n t e r e s t has stimulated the synthesis of long s e r i e s of dopamine agonists and antagonists, there i s a r e l a t i v e l y small number of model systems where the binding c h a r a c t e r i s t i c s of dopaminergic agents can be c o r r e l a t e d with a t y p i c a l p r o f i l e of c e l l u l a r func t i o n . In f a c t , p r e c i s e parameters of i n t a c t c e l l u l a r a c t i v i t y under dopaminergic c o n t r o l can be measured with p r e c i s i o n only i n a few dopaminergic systems, namely p r o l a c t i n - s e c r e t i n g ( 2 ) , pars intermedia (4) and parathyroid (60) c e l l s . Unfortunately, turning and s t e r e o t y p i c behavior i n r a t s , p s y c h i a t r i c and n e u r o l o g i c a l symptoms i n man and other dopaminergic functions of the c e n t r a l nervous system are r e l a t i v e l y imprecise. While the c l a s s i f i c a t i o n of α-adrenergic receptors i n t o o^and ou (69, 70) and 3-adrenergic receptors i n t o 3 and 3 -subtypes (71, 72) i s based on the rank order of potency of a s e r i e s of catecholamines and analogs to e l i c i t a l a r g e s e r i e s of b i o l o g i c a l responses, there i s no such l a r g e s c a l e basis f o r c l a s s i f i c a t i o n of dopamine r e c e p t o r s . One of the e a r l i e s t biochemical a c t i o n s of dopamine to have been described i s the s t i m u l a t i o n of s t r i a t a l adenylate cyclase a c t i v i t y . Although i t has the disadvantage of a h i g h l y heterogenous c e l l population and i t s assay i s performed i n a broken c e l l preparation, s t r i a t a l dopamine-induced a d e n y l y l c y c l a s e has been much u s e f u l f o r c h a r a c t e r i z a t i o n of the dopamine receptor (27, 58, 73). However, not a l l dopaminergic responses appear to be mediated by c y c l i c AMP (74, 75, 76). This has l e d to the proposal by Kebabian and Calne (36) of two categories of dopa mine receptors, namely D, ( l i n k e d to adenylate cyclase) and D (not l i n k e d to cyclase) ( Table 1, 36). The parathyroid and the p i t u i t a r y mammotroph were taken as examples of Dl and D2 recep t o r s , r e s p e c t i v e l y . While few data were then a v a i l a b l e on the coupling of the a n t e r i o r p i t u i t a r y dopamine receptor to adenylate cyclase (26^ 29), i t i s now quite c l e a r that the D1-D2 terminology can no longer adequately describe the f u n c t i o n a l coupling between the dopamine receptors and adenylate cyclase i n various t i s s u e s . It has i n f a c t been convincingly demonstrated that the ante r i o r p i t u i t a r y dopamine receptor i s n e g a t i v e l y coupled to adenyla te c y c l a s e i n both tumoral (26), and normal (54 ) adenohypophysial t i s s u e . Thus, the adenohypophysis cannot be taken, as o r i g i n a l l y proposed (36), as example of a receptor not coupled to adenylate c y c l a s e . Moreover, i t i s w e l l known that some dopaminergic respon ses appear independent of c y c l i c AMP (74, 75, 76)· In order to take i n t o account a l l the a v a i l a b l e data, i t appears p r e f e r a b l e to use the terminology DA+, DA- and DA^ (54, F i g . 4 ) . Other advan tages of t h i s terminology are i t s c l e a r separation from the nomen2

2



0

+

Figure 4. Representation of the classification of the dopamine receptor based on its coupling with adenylate cyclase activity. DA receptors (left) are coupled to adenylate cyclase through the Νs GTP-binding protein (9\) with secondary activation of adenylate cyclase. DA. receptors (middle) are coupled through the Ni GTP-binding protein, thus resulting in inhibition of cyclic AMP for mation. DA receptors (right) are those uncoupled to cyclic AMP formation, the example being possibly some autoreceptors on nigrostriatal dopaminergic neurons.


LABRIE

ET AL.

DA- Dopamine

63

Receptor

c l a t u r e D to based on binding data (68), the use of the w e l l accepted acronym DA f o r dopamine and the l a c k of p o s s i b l e confu s i o n with the vitamin Ό receptor. 1


C o r t i c o t r o p i n - r e l e a s i n g f a c t o r stimulates adenylate c y c l a s e i n the intermediate lobe of the p i t u i t a r y gland Although the f i r s t evidence suggesting the presence of hypo thalamic substances c o n t r o l l i n g p i t u i t a r y gland a c t i v i t y was that of a c o r t i c o t r o p i n - r e l e a s i n g f a c t o r (CRF) (77_, 78), i t i s only r e c e n t l y that the s t r u c t u r e of ovine CRF could be e l u c i d a t e d (79, 80). The 41-amino a c i d peptide i s a potent stimulator of adrenoc o r t i c o t r o p i n (ACTH) s e c r e t i o n i n vivo i n the r a t as w e l l as i n adenohypophysial c e l l s i n c u l t u r e (79, 81, 82, 83)· Somewhat unex pectedly, we have r e c e n t l y found that the a d m i n i s t r a t i o n of CRF leads to a p a r a l l e l increase i n plasma l e v e l s , not only of ACTH, a peptide secreted mainly by the a n t e r i o r p i t u i t a r y gland, but also of α-melanocyte-stimulating hormone (a-MSH) (14), a s e c r e t o r y product o r i g i n a t i n g almost e x c l u s i v e l y from the intermediate lobe of the p i t u i t a r y gland O , 84)· In order to obtain d i r e c t evidence that CRF i s a c t i n g d i r e c t l y on c e l l s of the pars intermedia rather than i n d i r e c t l y at the hypothalamic l e v e l , we have studied the e f f e c t of CRF on α-MSH s e c r e t i o n i n pars intermedia c e l l s i n c u l ture. A f t e r a 6-h incubation with pars intermedia c e l l s i n c u l t u r e , a maximal concentration of ovine CRF (300 nM) causes a 2 - f o l d s t i m u l a t i o n of α-MSH release at an E D value of 1 nM ( F i g . 5B). It can also be seen that preincubation with the potent glucocor t i c o i d dexamethasone under conditions which lead to an almost complete i n h i b i t i o n of ACTH s e c r e t i o n i n c o r t i c o t r o p h s of the a n t e r i o r p i t u i t a r y gland (85), has no i n h i b i t o r y e f f e c t on e i t h e r spontaneous or CRF-induced α-MSH s e c r e t i o n . In r a t pars intermedia c e l l s , the rate of s e c r e t i o n of the proopiomelanocortin-derived peptides (α-MSH being the major secre t o r y product) (3_, 85) was so f a r known to r e s u l t from a balance between the stimulatory e f f e c t of 3-adrenergic agents and the i n h i b i t o r y i n f l u e n c e of dopaminergic substances (14, 41, 86-89). The present data c l e a r l y demonstrate that i n a d d i t i o n to 3-adre n e r g i c agents, a second substance, namely CRF, could w e l l be involved as p h y s i o l o g i c a l stimulator of the a c t i v i t y of pars intermedia c e l l s . Since the adenylate cyclase system has been w e l l demonstrated to play a mediatory r o l e i n c o n t r o l l i n g the a c t i o n of 3-adrenergic and dopaminergic agents i n pars intermedia c e l l s (5-13, 41), we have studied the p o s s i b i l i t y of a s i m i l a r r o l e of c y c l i c AMP i n CRF a c t i o n . A f t e r a 10-min incubation with i n c r e a s i n g CRF concen t r a t i o n s , an approximately 6-fold increase i n c y c l i c AMP content i s measured at an E D value of 6 nM ( F i g . 5A). A maximal stimu l a t o r y e f f e c t of CRF on c y c l i c AMP accumulation i s observed 2 min a f t e r a d d i t i o n of CRF. As observed on α-MSH s e c r e t i o n , preincuba5 Q

5 Q



0

0.15

0.30

0.45

J

-11

-10

-7 CRF (LOG

I CO

GC

Q LU CO

4^

OS


LABRIE

ET

AL.

DA-

Dopamine

65

Receptor

t i o n with dexamethasone has no i n h i b i t o r y e f f e c t on b a s a l or CRFinduced c y c l i c AMP accumulation. The above-described data show that CRF added to c e l l s of the rat intermediate lobe i n c u l t u r e causes a r a p i d s t i m u l a t i o n of ot MSH r e l e a s e and c y c l i c AMP accumulation, thus demonstrating a d i r e c t a c t i o n of the peptide on pars intermedia c e l l s (15). I t i s however d i f f i c u l t , using i n t a c t c e l l s , to d i s s o c i a t e between increases i n c y c l i c AMP l e v e l s due to s t i m u l a t i o n of adenylate c y c l a s e a c t i v i t y or to i n h i b i t i o n of c y c l i c n u c l e o t i d e phospho d i e s t e r a s e or to a combination of both e f f e c t s . D e f i n i t i v e proof of the r o l e of adenylate c y c l a s e i n the a c t i o n of CRF i n the intermediate lobe of the p i t u i t a r y gland i s provided by the f o l lowing f i n d i n g s of a CRF-induced s t i m u l a t i o n of adenylate c y c l a s e a c t i v i t y i n homogenate of r a t and bovine pars intermedia c e l l s . As i l l u s t r a t e d i n F i g . 6, maximal concentrations of s y n t h e t i c ovine CRF cause a 100% s t i m u l a t i o n of adenylate cyclase a c t i v i t y i n bovine pars intermedia p a r t i c u l a t e f r a c t i o n at an E D value of 150 nM. Since guanyl n u c l e o t i d e s have been demonstrated to play a c e n t r a l r o l e i n mediating the a c t i v a t i o n of adenylate c y c l a s e by many hormones (90, 91), we next i n v e s t i g a t e d the p o s s i b i l i t y of such a r o l e of GTP i n CRF a c t i o n . We thus studied the i n t e r a c t i o n of CRF with GTP and the two p r e v i o u s l y known r e g u l a t o r s of pars intermedia c e l l a c t i v i t y using the 3-adrenergic agonist (-)isopro t e r e n o l and dopamine. As i l l u s t r a t e d i n F i g . 7, 3 μΜ CRF and 1 uM ( - ) i s o p r o t e r e n o l cause a 190 and 110% s t i m u l a t i o n of adenylate cyclase a c t i v i t y i n r a t pars intermedia p a r t i c u l a t e f r a c t i o n , r e s p e c t i v e l y . An a d d i t i v e e f f e c t i s observed when both s t i m u l a t o r y agents are present. Dopamine (30 μΜ), on the other hand, has no s i g n i f i c a n t e f f e c t alone. However, i n the presence of GTP, the catecholamine causes a 40 to 60% i n h i b i t i o n of adenylate c y c l a s e a c t i v i t y stimulated by CRF, ISO or CRF + ISO. It can also be seen that while 0.3 mM GTP alone causes a 100% increase i n basal adenylate c y c l a s e a c t i v i t y , i t leads to a marked p o t e n t i a t i o n of the e f f e c t of ISO and CRF on [ P ] c y c l i c AMP accumulation. I t should be n o t i c e d that i n the absence of the guanyl n u c l e o t i d e , dopamine has no i n h i b i t o r y e f f e c t on adenylate c y c l a s e a c t i v i t y i n any of the groups s t u d i e d . The present data c l e a r l y demonstrate that the 41-amino a c i d ovine CRF i s a potent s t i m u l a t o r of adenylate cyclase a c t i v i t y i n r a t and bovine pars intermedia t i s s u e . Our previous data have shown that CRF causes a r a p i d and marked s t i m u l a t i o n of c y c l i c AMP accumulation i n r a t pars intermedia c e l l s i n c u l t u r e (15). The f i n a l proof of the r o l e of adenylate cyclase i n the observed chan ges of c y c l i c AMP l e v e l s had to be obtained by d i r e c t measurement of adenylate cyclase a c t i v i t y . As mentioned e a r l i e r , guanyl n u c l e o t i d e s have been found to play an important r o l e i n the a c t i v a t i o n of adenylate c y c l a s e a c t i v i t y by many hormones (90, 91). The present observations show that i n pars intermedia t i s s u e , GTP causes an almost doubling of the s t i m u l a t o r y e f f e c t of CRF while that of the 3-adrenergic ago5 Q

32


66

DOPAMINE

RECEPTORS


7 0 0 -ι

[CRF] L O G M

Figure 6.

Effect of increasing concentrations of CRF on adenylate cyclase activity in bovine pars intermedia pituitary homogenate. ζ LU

3000 •

\-

O DC Ο-

2500 H

M

CONTROL G T P (0.3 m M )

Ο 2000 H

Û LU Έ

ce ο

J

1500

1000

LL CL

Dopamine Receptors - American Chemical Society

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