D-1 Dopamine Receptor-Mediated Activation of Adenylate Cyclase

1 D-1 Dopamine Receptor-Mediated Activation of Adenylate Cyclase, cAMP Accumulation, and PTH Release in Dispersed Bovine Parathyroid Cells

Downloaded by 179.106.190.62 on November 3, 2015 | http://pubs.acs.org Publication Date: June 29, 1983 | doi: 10.1021/bk-1983-0224.ch001

EDWARD M. BROWN and BESS DAWSON-HUGHES Brigham and Women's Hospital, Endocrine-Hypertension Unit, Boston, MA 02115 The evidence supporting the existence of a s p e c i f i c category o f dopamine receptor on the parenchymal c e l l s of the bovine parathyroid gland and the p o s s i b l e biochemical mechanisms by which dopamine stimulates the release of parathyroid hormone are reviewed. The dopamine receptor on the bovine parathyroid cell i s compared to other dopamine receptors. The parathyroid glands play a major r o l e i n normal calcium homeostasis (±). Calcium i s g e n e r a l l y recognized as the p r i n c i p a l p h y s i o l o g i c a l r e g u l a t o r of the r e l e a s e of parathyroid hormone (PTH) (2.). When the plasma concentration o f i o n i z e d calcium decreases, PTH s e c r e t i o n increases. In turn, t h i s PTH acts to r a i s e plasma calcium by three mechanism: f i r s t , PTH increases r e n a l tubular reabsorption of calcium; second, PTH enhances the release of s k e l e t a l calcium; and t h i r d , PTH increases g a s t r o i n t e s t i n a l absorption o f calcium by s t i m u l a t i n g r e n a l formation of 1,25 dihydroxyvitamin D. These three mechanisms elevate the plasma concentration of ionized calcium and consequently reduce the augmented s e c r e t i o n of PTH, thereby c l o s i n g a negative feedback loop. The i n h i b i t o r y e f f e c t of calcium upon PTH s e c r e t i o n contrasts with the stimulatory e f f e c t of calcium upon most other secretory systems (3.)· In a d d i t i o n to calcium, other f a c t o r s a l s o modify PTH s e c r e t i o n . Many o f these f a c t o r s change c e l l u l a r c y c l i c adenosine 3 5 , monophosphate (cAKP) l e v e l s at the same time that they modify PTH s e c r e t i o n This volume provides a forum i n which i t i s appropriate to d i s c u s s the bovine parathyroid gland and the e f f e c t s of dopamine upon t h i s t i s s u e . When administered intravenously to cows, dopamine r a i s e s the plasma content of immunoreactive PTH (X). This stimulatory e f f e c t of dopamine i s p a r t i a l l y blocked by pimozide, a dopamine antagonist, but i s unaffected by p r o p r a n o l o l , a beta-adrenergic antagonist. An understanding of !

0097-6156/83/0224-0001$06.25/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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2

DOPAMINE

RECEPTORS

the c e l l u l a r mechanisms involved i n mediating t h i s dopamine-induced s t i m u l a t i o n of PTH s e c r e t i o n i s l i m i t e d by the c e l l u l a r heterogeneity of the bovine parathyroid gland (2.) as w e l l as by the temporal and s p a t i a l imprecision of intravenous infusions. Nevertheless, the I n v i v o r e s u l t s provide a standard against which i n v i t r o r e s u l t s can be compared. The c e l l u l a r and molecular events involved i n the dopamine-stimulated release of PTH can be c l a r i f i e d i n experiments u t i l i z i n g bovine parathyroid c e l l s dispersed with collagenase and DNase (&). This d i s p e r s i o n procedure y i e l d s parenchymal c e l l s with only a s l i g h t contamination by red blood cells. The parenchymal c e l l s exclude trypan blue and appear normal by l i g h t and e l e c t r o n microscopy (&). These c e l l s r e l e a s e PTH i n a l i n e a r f a s h i o n f o r s e v e r a l hours; the release i s i n h i b i t e d by calcium and stimulated by dopamine and beta-adrenergic agonists at concentrations comparable to those used to e l i c i t p h y s i o l o g i c a l responses j j i v i v o (A,&) · Dopamine Enhances PTH Cells

S e c r e t i o n i n Dispersed

Bovine Parathyroid

Dopamine (1 μΜ) causes a t r a n s i e n t 2 to 4 - f o l d increase i n the rate of release of immunoreactive PTH (IR-PTH) from dispersed bovine parathyroid c e l l s (ϋ). The stimulatory e f f e c t of dopamine i s maximal a f t e r 5 minutes exposure and p e r s i s t s f o r approximately 30 minutes (Figure 1). Several compounds mimicking the e f f e c t s of dopamine i n other systems mimic the stimulatory e f f e c t of dopamine on IR-PTH r e l e a s e . Both 2-amino, 6,7-dihydroxy t e t r a l i n (6,7-ADTN) and SKF 38393 increase the r e l e a s e of IR-PTH to the same degree as does dopamine. The release of IR-PTH i s half-maximally stimulated by dopamine, 6,7-ADTN and SKF 38393 at 0.2 μΜ, 0.15 μΗ and 0.3 μΜ, r e s p e c t i v e l y (Figure 2) (1Q_). In c o n t r a s t , other dopaminergic agonists are s u b s t a n t i a l l y l e s s potent than dopamine; both apomorphine and l e r g o t r i l e e l i c i t no more than 25% of the maximal response to dopamine, and l i s u r i d e i s devoid of agonist a c t i v i t y . The dopamine-stimulated r e l e a s e of IR-PTH i s i n h i b i t e d i n a s t e r e o s p e c i f i c manner by the isomers of f l u p e n t h i x o l (£) ; c i s - f l u p e n t h i x o l i s approximately 100-fold more potent than i t s trans-isomer ( c a l c u l a t e d K s of 33 nM and 3,300 nM, r e s p e c t i v e l y ) (Figure 3)· l

i

Dopamine Enhances cAHP Accumulation i n Dispersed Parathyroid C e l l ?

Bovine

Dopamine causes a 20 to 30-fold increase i n the content of cAMP i n dispersed bovine parathyroid c e l l s (Figure 4) (ϋ.). Like the dopamine-stiraulated enhancement of PTH r e l e a s e , the dopamine-stimulated increase i n cAMP content i s maximal a f t e r 5 to 10 minutes of exposure to 10 μΜ dopamine (S.) and

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


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TIME, MINUTES Figure 1. Stimulation of PTH release from dispersed bovine parathyroid cells by dopamine. Cells were incubated with (M) or without (%) 1 μΜ dopamine, and PTH release was determined by radioimmunoassay.

[AGONIST], M

Figure 2. Stimulation of PTH release from dispersed bovine parathyroid cells by varying concentrations of dopamine (O), 6,7-ADTN SKF 38393 (A), apomorphine or dihydroergocryptine (M)> (Reproduced with permission from Ref. 10. Copyright 1980, American Society for Pharmacology and Experimental Therapeutics.)



4

DOPAMINE

RECEPTORS

10-6 [ANTAGONIST], M

Figure 3.

Inhibition of PTH-release stimulated by 1 μΜ dopamine by a-flupenthixol (Φ), β-flupenthixol (0),or(—) propranolol ( Α λ

0) ο

i ο 5 α οΓ

< ο

ιο ί AgonistI, Μ

Figure 4. Stimulation of cAMP accumulation in dispersed bovine parathyroid cells by varying concentrations of dopamine (O), 6,7-ADTN ( Π λ SKF 38393 (A), or apomorphine (Reproduced with permission from Ref. 10. Copyright 1980, American Society for Pharmacology and Experimental Therapeutics.)


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subsequently decreases (Figure 5). S i g n i f i c a n t q u a n t i t i e s of cAMP are excreted from the dispersed c e l l s during the f i r s t 15 minutes of exposure to dopamine. As i s the case f o r the dopanine-stimulated enhancement of IR-PTH r e l e a s e , s e v e r a l dopaminergic agonists mimic the a b i l i t y of dopamine to increase cAMP accumulation i n the dispersed bovine parathyroid c e l l s (Table I ) . For each compound tested, the concentration of agonist half-maximally enhancing cAMP accumulation i s approximately the same as the concentration of agonist required to half-maximally stimulate the release of IR-PTH. Dopamine antagonists block the dopamine-stimulated increase i n cAMP (Figure 6) (Table I ) . I n t e r e s t i n g l y , apomorphine, l i s u r i d e , l e r g o t r i l e and bromocriptine each block the dopamine receptor i n t h i s system (Figure 6 ) . Dopamine Stimulates Adenylate Cyclase A c t i v i t y o f Dispersed gpvine Parathyroid C e l l s When tested on o s m o t i c a l l y - l y s e d bovine parathyroid c e l l s , dopamine enhances the a c t i v i t y o f adenylate c y c l a s e , the enzyme converting ATP to cAKP (JUL). In comparison with i t s e f f e c t on cAMP accumulation, the e f f e c t of dopamine on adenylate cyclase a c t i v i t y i s r e l a t i v e l y modest, only a 2 - f o l d increase i n enzyme a c t i v i t y (Figure 7 ) . Guanosine 5'-triphosphate (GTP) increases the stimulatory e f f e c t of dopamine; i n the presence of GTP, there i s 3 to 4-fold s t i m u l a t i o n of enzyme a c t i v i t y (JL1). In other c e l l types, guanine nucleotides i n t e r a c t with a guanine nucleotide subunit (G- or N -subunit) to t r a n s l a t e receptor s t i m u l a t i o n i n t o increased adenylate c y c l a s e a c t i v i t y (12.). Cholera t o x i n i n h i b i t s a s p e c i f i c GTPase on t h i s guanine n u c l e o t i d e subunit and thereby increases adenylate cyclase a c t i v i t y (13.)· In dispersed c e l l s from the bovine parathyroid gland, c h o l e r a t o x i n markedly increases cAWP formation and causes a 3 to 10-fold increase i n the apparent a f f i n i t y c f dopamine f o r i t s receptor (as determined by cAMP accumulation or IR-PTH s e c r e t i o n (14). The e f f e c t s of guanine nucleotides and c h o l e r a t o x i n on cAMP accumulation i n parathyroid c e l l s r e s u l t from i n t e r a c t i o n s with the guanine nucleotide subunit i n this c e l l . In e i t h e r the presence or absence of GTP, half-maximal s t i m u l a t i o n of enzyme a c t i v i t y i s achieved with 3 yM dopamine. Both 6,7-ADTN and epinine (N-methyl dopamine) stimulate adenylate cyclase a c t i v i t y to the same degree as does dopamine (Figure 8 ) . In c o n t r a s t , apomorphine i s a p a r t i a l agonist e l i c i t i n g only 30? o f the maximal e f f e c t of dopamine. The dopamine-stimulated adenylate cyclase a c t i v i t y i s s e l e c t i v e l y blocked by c i s - f l u p e n t h i x o l rather than the trans-isomer of t h i s antagonist (11). Among the antagonists tested, the order of potency i s : c i s - f l u p e n t h i x o l = fluphenazine > chlorpromazine > h a l o p e r i d o l > t r a n s - f l u p e n t h i x o l (Table I ) . g


5


6

DOPAMINE

15

30

RECEPTORS

60 120

TIME, MINUTES Figure 5. Stimulation of intracellular (%) and extracellular (O) cAMP by 10 μΜ dopamine in dispersed bovine parathyroid cells. cAMP was determined by radio immunoassay.


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7

TABLE I


A f f i n i t y of drugs f o r the dopamine receptor i n bovine parat h y r o i d c e l l s determined i n experiments measuring cAMP accumulation, adenylate c y c l a s e , or PTH r e l e a s e . K cAMP Accumulation

a

or K i (yM) Adenylate Cyclase

PTH Release

Agonists Dopamine ADTN Epinine

0.6 0.5 0.6

3 4 10

0.2 0.15

P a r t i a l Agonists SKF 38393 Apomorphine

1 1

3 3

0.3 1

Antagonists d-Butaclamol a-Flupenthixol Fluphenazine Lisuride Lergotrile Bromoergocryptine YM-09151-2 (+) S u l p i r i d e (-) S u l p i r i d e 20% i n h i b i t i o n at 3 χ 10"

.005 .03 .04 .015 .7 21 15 13 Ï 5

.011 .07 .16 .37 2.1

^


.033 .09 .9

DOPAMINE

8

^fo*

io^e

iô^T jô^ë

RECEPTORS

10-5 10-4


ANTAGONIST^ Figure 6. Inhibition of cAMP accumulation stimulated by 1 μΜ dopamine by lisuride (O), a-flupenthixol ([2), β-flupenthixol (M)> lergotrile (A), a-bromoergocryptine (Φ), or fluphenazine (A)-

1200 ι

1000 h c

ε ο 'à

ε < υ "δ

ε α

(Dopamine), Μ

Figure 7. Dopamine-stimulated adenylate cyclase activity in lysates of bovine parathyroid cells in the absence (O) or presence (O) of 100 μΜ guanosine triphos phate (GTP). (Reproduced with permission from Ref. 11. Copyright 1980, The Endocrine Society.)


A N D DAWSON-HUGHES

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BROWN

Figure 8. Stimulation of adenylate cyclase in lysates of bovine parathyroid cells by 6,7-ADTN (A), epinine (O), or apomorphine in the presence of 100 μΜ GTP. (Reproduced with permission from Ref. 11. Copyright 1980, The Endocrine Society.)


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Does Dopamine I n t e r a c t with Beta- or Alpha-adrenergic receptors? The bovine parathyroid gland possesses a beta-adrenergic receptor. Like dopamine, beta-adrenergic agonists enhance adenylate cyclase a c t i v i t y , cAMP accumulation, and the release of IR-PTH ( 1 5 . ) . The beta-adrenergic receptor, however, can be d i f f e r e n t i a t e d from the receptor f o r dopamine with s e l e c t i v e antagonists (Figure 9 ) · For example, p r o p r a n o l o l , a potent antagonist of the beta-adrenergic receptor, causes a nearly complete i n h i b i t i o n of cAMP accumulation stimulated by i s o p r o t e r e n o l , epinephrine, or norepinephrine. Propranolol, on the other hand, does not diminish dopamine-stimulated cAMP accumulation. Conversely, c i s - f l u p e n t h i x o l blocks the dopamine- and epinine-stiraulated accumulation o f cAMP but does not reduce the i s o p r o t e r e n o l - s t i m u l a t e d accumulation o f cAMP. The bovine parathyroid gland a l s o possesses an alpha-adrenergic receptor ( 1 6 . ) . However, i t seems improbable that an i n t e r a c t i o n between dopamine and the alpha-adrenergic receptor accounts f o r the p h y s i o l o g i c a l and biochemical e f f e c t s of dopamine upon t h i s t i s s u e . Phentolamine, an alpha-adrenergic antagonist, has no e f f e c t on dopamine-stimulated cAMP accumulation at concentrations as high as 10 μΜ, a concentration t o t a l l y blocking alpha-adrenergic e f f e c t s i n t h i s system (JLQ.). Furthermore, u n l i k e the dopaminergic receptor, s t i m u l a t i o n o f the parathyroid alpha-adrenergic receptor i n h i b i t s the agonist-stimulated a c t i v a t i o n o f accumulation o f cAMP and r e l e a s e of IR-PTH (l£L). Several dopaminergic drugs i n t e r a c t not only with the dopamine receptor but a l s o with alpha- and beta-adrenergic receptors i n the bovine parathyroid gland. For example, l i s u r i d e , a potent dopamine agonist upon the a n t e r i o r p i t u i t a r y gland, blocks the alpha-adrenergic receptor, the beta-adrenergic receptor, and the receptor f o r dopamine i n parathyroid c e l l s ( H ) . Does cAM? T r i g g e r the Release of IR-PTH? The dopamine-stimulated formation o f cAMP may i n i t i a t e the dopamine-induced r e l e a s e of IR-PTH. A l i n e a r r e l a t i o n s h i p e x i s t s between the dopamine-induced release o f IR-PTH and the logarithm of the dopamine-induced accumulation of cAMP ( H ) . S i m i l a r l y , other agents i n c r e a s i n g cAMP accumulation and IR-PTH release (e.g. beta-adrenergic agonists, s e c r e t i n and phosphodiesterase i n h i b i t o r s , a l s o d i s p l a y such a l o g - l i n e a r r e l a t i o n s h i p . A d d i t i o n a l support f o r the p o s s i b i l i t y that i n t r a c e l l u l a r cAMP might i n i t i a t e PTH s e c r e t i o n comes from the observations that c h o l e r a t o x i n (14), phosphodiesterase i n h i b i t o r s ( H ) and d i b u t y r y l cAMP (IS.), agents known to increase i n t r a c e l l u l a r cAMP or mimic the biochemical e f f e c t s of cAMP, increase the release of IR-PTH.



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(-)ISO

(-)EPI

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(-)NOREPI DOPAMINE EPININE

Figure 9. Specificity of β-adrénergie and dopaminergic stimulation of cAMP accumulation. Dispersed parathyroid cells were incubated with the indicated β-adrenergic or dopaminergic agonists either alone (open bars), with 1 μΜ ( — ) propranolol (solid bars), or with 10 μΜ. a-flupenthixol (stippled bars).



12

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A cAMP-dependent phosphorylation o f s p e c i f i c c e l l u l a r substrates i s hypothesized to i n i t i a t e t i s s u e - s p e c i f i c p h y s i o l o g i c a l responses. C e r t a i n elements o f t h i s hypothesis can be a p p l i e d to the bovine parathyroid gland. Dispersed bovine parathyroid c e l l s contain predominantly the type 2 isozyme(s) o f cAMP-dependent p r o t e i n kinase (19.). The a c t i v a t i o n s t a t e of t h i s form of the kinase remains r e l a t i v e l y constant when t i s s u e i s extracted i n t o b u f f e r s c o n t a i n i n g high concentrations o f s a l t . This s i t u a t i o n permits an estimation of the drug-induced a c t i v a t i o n of the cAMP-dependent p r o t e i n kinase a c t i v i t y i n i n t a c t c e l l s . In a recent s e r i e s of experiments u t i l i z i n g dispersed bovine parathyroid c e l l s , we compared the dopamine-induced r e l e a s e of IR-PTH with dopamine-induced a l t e r a t i o n s i n the a c t i v i t y r a t i o of the cAMP-dependent p r o t e i n kinase (an estimate of the f r a c t i o n a l a c t i v a t i o n of t h i s enzyme) (20.) · A c l o s e c o r r e l a t i o n e x i s t s between dopamine-induced changes i n the a c t i v i t y r a t i o and dopamine-induced IR-PTH s e c r e t i o n (Figure 10). This observation i s c o n s i s t e n t with a mediatory r o l e f o r cAMP-dependent p r o t e i n phosphorylation i n dopamine-stimulated hormonal s e c r e t i o n . A d d i t i o n a l evidence supporting t h i s p o s s i b i l i t y i s the observation that cAMP promotes the phosphorylation o f s e v e r a l endogenous p r o t e i n s i n sonicates of dispersed bovine parathyroid c e l l s (21) and that dopamine stimulates the phosphorylation o f two p r o t e i n s o f molecular weight 15,000 and 19,000 i n i n t a c t bovine parathyroid c e l l s (22.). The biochemical mechanisms by which cAMP-dependent phosphorylation leads to enhanced IR-PTH r e l e a s e remain to be determined. I t i s of i n t e r e s t , however, that i s o p r o t e r e n o l a c t i v a t e s phosphorylation of p r o t e i n s o f s i m i l a r molecular weight i n the r a t p a r o t i d gland (21), while glucagon stimulates phosphorylation o f a p r o t e i n of molecular weight 19,000 i n c a l c i t o n i n - s e c r e t i n g c u l t u r e d c e l l s from a medullary carcinoma of the r a t t h y r o i d (2H) · I t i s conceivable that i n a l l three t i s s u e s , a c t i v a t i o n of exocytosis r e s u l t s from a cAMP-dependent phosphorylation of a c r i t i c a l c e l l u l a r s u b s t r a t e . Receptors I n h i b i t i n g Dopamine-stimulated cAMP Accumulation and

Both alpha-adrenergic agonists (16) and prostaglandin F2a (PGF2a) (25.) d i m i n i s h dopamine-stimulated cAMP accumulation and hormonal s e c r e t i o n . The i n h i b i t o r y e f f e c t s o f these agents on hormonal s e c r e t i o n can be q u a n t i t a t i v e l y accounted f o r by t h e i r i n h i b i t o r y e f f e c t on cAMP accumulation (11). The mechanism(s) by which these compounds lower c e l l u l a r cAMP has not been i n v e s t i g a t e d ; i n other systems alpha-adrenergic agonists i n h i b i t adenylate cyclase through a GTP-dependent mechanism


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Activation

0.75 h

200 0.375 Η ιοο Û.

< ιο"'

ιο"°

10"'


[DOPAMINE] , Μ

100

at 10"^ M.


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17

c i s - f l u p e n t h i x o l , and l i s u r i d e are most potent. Phenothiazines, such as fluphenazine and chlorpromazine, have K s i n the range of 4 χ 10" -4 χ 10~ M. Most ergots (e.g. l e r g o t r i l e , dihydroergocryptine, and bromocryptine) are of moderate to low potency 1-10 uM). (+) and (-) Butaclamol and c i s - and t r a n s - f l u p e n t h i x o l show marked s t e r e o s p e c i f i c i t y , with the a c t i v e isomer being of high potency. Agents p u t a t i v e l y s p e c i f i c f o r dopamine receptors i n the a n t e r i o r p i t u i t a r y or intermediate lobe of the r a t p i t u i t a r y are e i t h e r weak antagonists [(+)- and ( - ) - s u l p i r i d e , YM-09151-2 ( 3 1 ) ] or have no e f f e c t [LY-141865 ( 3 2 ) ] i n bovine parathyroid c e l l s . Several features of the dopamine receptor i n the bovine parathyroid gland a l s o occur i n other mammalian t i s s u e s as w e l l as i n lower vertebrates and even i n v e r t e b r a t e s (Table I I I ) . These dopamine-sensitive adenylate c y c l a s e systems are c h a r a c t e r i z e d by dopamine, epinine and 6,7-ADTN being f u l l agonists of micromolar potency, apomorphine being a p a r t i a l agonist, and ergots such as l i s u r i d e , l e r g o t r i l e , or bromocriptine d i s p l a y i n g minimal agonist a c t i v i t y but being antagonists of moderate potency. In the preparations o f i n t a c t c e l l s which have been t e s t e d , dopamine agonists increase cAMP accumulation. This dopamine-stimulated increase i n cAMP can be l i n k e d to a c t i v a t i o n of a cAMP-dependent p r o t e i n kinase ( 3 1 ) , or to phosphorylation of s p e c i f i c c e l l u l a r substrates ( 4 1 ) . In some cases, exogenous cAMP mimics the p h y s i o l o g i c a l e f f e c t s of dopamine ( 1 2 ) . Thus, these dopamine receptors (designated as D-1 receptors, ( 1 3 . ) appear to act, at l e a s t i n part, through e l e v a t i o n of c e l l u l a r cAMP, a c t i v a t i o n o f cAMP-dependent p r o t e i n kinases, and phosphorylation of s p e c i f i c c e l l u l a r substrates. f

7


i

Dopamine Receptors J n f r i f r i t i n g Adenylate

Cyclase A c t i v i t y

C e r t a i n dopamine receptors do not resemble the dopamine receptor i n the bovine parathyroid gland. For example, the dopamine receptor on the mammotroph of the p i t u i t a r y gland i s c h a r a c t e r i z e d by dopamine, apomorphine, and ergot a l k a l o i d s being agonists with nanomolar potency. Butyrophenones are potent antagonists o f t h i s receptor. While t h i s c l a s s of receptor was o r i g i n a l l y c l a s s i f i e d on the b a s i s of the lack of any s t i m u l a t o r y e f f e c t s on cAMP accumulation or adenylate cyclase a c t i v i t y ( 1 3 . ) , i t has become c l e a r that s t i m u l a t i o n of t h i s receptor i n h i b i t s cAMP metabolism ( 1 4 ) · An a d d i t i o n a l example of t h i s second category of receptor (designated as the D-2 receptor) occurs i n the intermediate lobe of the r a t p i t u i t a r y gland. In t h i s l a t t e r system, dopamine i n h i b i t s basal and p a r t i c u l a r l y agonist-stimulated cAMP accumulation and adenylate c y c l a s e a c t i v i t y ( 1 5 L , 1 & ) ; these i n h i b i t o r y e f f e c t s are GTP-dependent ( U ) . The changes i n c e l l u l a r cAHP c o r r e l a t e with the i n h i b i t i o n of hormone release from the IL (45,46 f

Kebabian

a l . , t h i s volume).



a

40 4-8 67 5 3.9 3 ^50 20

4.8 5

1

Fluphenazine

11 1 0.48

a-Flupenthixol

K i , nM

600

3,000 95 5.32 2002

Spiroperidol

9,10 33 34 35 36 37 38 39 40

Reference

Kj[ f o r h a l o p e r i d o l . S p i r o p e r i d o l and h a l o p e r i d o l are of comparable potency i n s e v e r a l dopamines e n s i t i v e adenylate c y c l a s e systems.

Kj_ f o r chlorpromazine. Fluphenazine i s about 7-10-fold more potent than chlorpromazine i n s e v e r a l dopamine-sensitive adenylate c y c l a s e systems.

3 10 2-10 10 6-10 10 1-2 2 1.7

Dopamine

a

K , yM

f o r dopamine and K i f o r s e v e r a l dopamine antagonists on dopamine-sensitive adenylate c y c l a s e

Bovine Parathyroid C e l l Rat Candate Nucleus Substantia Nigra Rat O l f a c t o r y Tubercle Superior C e r v i c a l Ganglion Guinea P i g Retina Carp Retina S n a i l Nervous System Cockroach B r a i n

Tissue

K

TABLE I I I



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Activation

19

There are d i s t i n c t p a r a l l e l s , t h e r e f o r e , between the D-1 and D-2 receptors. Both i n t e r a c t with adenylate c y c l a s e , one through a stimulatory guanine nucleotide subunit and the other, presumably, through an i n h i b i t o r y subunit (UZ). Both may exert t h e i r b i o l o g i c a l e f f e c t s through changes i n c e l l u l a r cAMP (as was i n i t i a l l y hypothesized by Sutherland and h i s c o l l e a g u e s ) . The r e l a t i o n s h i p between the D-1 and the D-2 receptors i s very analogous to that between the beta- and the o^-adrenergic receptors. These receptors a l s o a c t upon adenylate c y c l a s e through stimulatory and i n h i b i t o r y guanine nucleotide subunits, r e s p e c t i v e l y (J2.,4S.) · The ^ - a d r e n e r g i c receptor, on the other hand, i s thought to a c t through changes i n c e l l u l a r calcium dynamics, without appreciable e f f e c t s on adenylate cyclase (4Q). By analogy, i t may be speculated that an a d d i t i o n a l c l a s s o f dopamine receptor a c t i n g through changes i n c y t o s o l i c calcium (and, t h e r e f o r e , equivalent to the α-j- adrenergic receptor) might e x i s t . Conceivably, t h i s hypothesized category of dopamine receptor would correspond to the D receptor postulated to e x i s t by Meunier and Labrie (50). At present, no example of e i t h e r of these t h e o r e t i c a l c o n s t r u c t s has been identified. Q

guaaary Dispersed bovine parathyroid c e l l s contain a dopamine receptor which increases c e l l u l a r cAMP through a guanine nucleotide-stimulated a c t i v a t i o n of adenylate c y c l a s e . The dopamine-stimulated increase i n c e l l u l a r cAMP c o r r e l a t e s c l o s e l y with a c t i v a t i o n o f cAMP-dependent p r o t e i n kinase, phosphorylation o f endogenous c e l l u l a r p r o t e i n s , and s e c r e t i o n of IR-PTH. The potency o f various dopaminergic agonists and antagonists i n modifying these b i o l o g i c a l e f f e c t s as w e l l as the r o l e of cAMP i n modifying p h y s i o l o g i c a l f u n c t i o n suggest that the bovine parathyroid dopamine receptor i s a D-1 dopamine receptor. This system may be of use i n studying the i n t e r a c t i o n of r a d i o l a b e l e d l i g a n d s with the D-1 receptor.

The authors g r a t e f u l l y acknowledge the e x c e l l e n t t e c h n i c a l help o f Joseph Thatcher and Edward Watson and s e c r e t a r i a l work of Mrs. Nancy O r g i l l . This work was supported by USPHS Grants AM25910 and AM30028.

Literature Cited 1. 2.

Parsons, J.A. "Endocrinology"; Grune and S t r a t t o n : New York, 1979; V o l 2, p 621. Sherwood, L.M.; Potts, J r . , J.T.; Care, A.D.; Mayer, G.P.; Aurbach, G.D. Nature 1966, 209, 52.


DOPAMINE

20

3. 4. 5. 6. 7. 8. 9.


10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

RECEPTORS

Douglas, W.W. Ciba Foundation Symposium 1978, 54, 61. Brown, E . M . Mineral Electrolyte Metabolism 1982, 130, 3. Peck, W.A.; Klahr, S. "Advances i n Cyclic Nucleotides Research"; Raven Press: New York, 1979; Vol 11, p 89. Heath, I I I , H. Endocrine Reviews 1980, 1, 319. Blum, J.W.; Kunz, P.; Fischer, J . Α . ; Binswanger, U . ; Lichtensteiger, W.; DaPrada, M. Am. J. Physiol. 1980, 239, E255. Brown, E . M . ; Hurwitz, S.; Aurbach, G.D. Endocrinology 1976, 99, 1582. Brown, E . M . ; C a r r o l l , R.; Aurbach, G.D. Proc. Nat'l. Acad. S c i . USA 1977, 74, 4210. Brown, E . M . ; A t t i e , M . F . ; Reen, S.; Gardner, D.G.; Kebabian, J.; Aurbach, G.D. Molec. Pharmacol 1980, 18, 335. A t t i e , M.F.; Brown, E . M . ; Gardner, D.G.; Spiegel, A . M . ; Aurbach, G.D. Endocrinology 1980, 107, 1776. Rodbell, M. Nature 1980, 284, 17. Cassel, D.; Selinger, Z. Proc. Nat'l. Acad. S c i . USA 1977, 74, 3307. Brown, E . M . ; Gardner, D.G.; Windeck, R.A.; Aurbach, G.D. Endocrinology 1979, 104, 218. Brown, E . M . ; Hurwitz, S.; Aurbach, G.D. Endocrinology 1977, 100, 1696. Brown, E . M . ; Hurwitz, S.H.; Aurbach, G.D. Endocrinology 1978, 103, 893. Brown, E . M . ; Gardner, D.G.; Windeck, R.A.; Aurbach, G.D. Endocrinology 1978, 101, 2323. Morrissey, J.J.; Cohn, D.V. J. C e l l B i o l . 1979, 83, 521. Thatcher, J . G . ; Gardner, D.G.; Brown, E.M. Endocrinology 1982, 110, 1367. Brown, E . M . ; Thatcher, J.G. Endocrinology 1982, 110, 1374. Brown, E . M . ; Thatcher, J.G. Program and Abstracts. Fourth Annual S c i e n t i f i c Meeting of the American Society for Bone and Mineral Research, San Francisco, CA, 1982, p S-37. Lasker, R.D.; Spiegel, A.M. Clin. Res. 1982, 30, 398A. Baum, B.J.; Freiberg, J . M . ; Ito, H . ; Roth, G.S.; Filburn, C.R. J . B i o l . Chem. 1981, 256, 9731. Gagel, R . F . ; Andrews, K . L . Abstracts of the 4th Annual S c i e n t i f i c Meeting of the American Society for Bone and Mineral Research 1982, p S-18. Gardner, D.G.; Brown, E . M . ; Windeck, R.; Aurbach, G.D. Endocrinology 1979, 104, 1. Jakobs, K . H . ; Saur, W.; Schulz, G. FEBS Letters 1978, 85, 167. Brown, E . M . ; Gardner, D.G.; Aurbach, G.D. Endocrinology 1980, 106, 133. Jacobowitz, D.; Brown, E.M. Experentia 1980, 36, 115. Brown, E . M . ; Aurbach, G.D. Vitamins and Hormones 1980, 38, 205.


1.

BROWN

30. 31. 32. 33. 34. 35.


36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

A N D DAWSON-HUGHES

D-l

Mediated

Activation

Brown, E.M. Endocrinology 1980, 107, 1998. Grewe, G.W.; Frey, E.A.; Cote, T.E.; Kebabian, J.W. Eur. J. Pharmacol. 1982, 81, 149. Tsuruta, K.; Frey, E.A.; Grewe, C.W.; Cote, T.E.; Eskay, R.L.; Kebabian, J.W. Nature, 1981, 292, 463. Kebabian, J.W. Petzgold, G.L.; Greengard, P. Proc. Nat'l. Acad. Sci. 1972, 69, 2145. Phillipson, O.T.; Horn, A.S. Nature 1976, 261, 418. Horn, A.S.; Cuello, A.C.; M i l l e r , R.J. J. Neurochem. 1974, 22, 265. Kebabian, J.W. Greengard, P. Science 1971, 174, 1346. Brown, J . H . ; Makman, M.Η. Proc. Nat'l. Acad. Sci. USA 1972, 69, 539. Watling, K.J.; Dowling, J . E . J. Neurochem, 1981, 36, 559. Osborne, N.N. Experentia 1977, 33, 917. Harmar, A.J.; Horn, A.S. Mol. Pharmacol. 1977, 13, 512. Nestler, E.J.; Greengard, P. Proc. Nat'l. Acad. Sci. USA 1980, 77, 7479. Greengard, P.; McAfee, D.A.; Kebabian, J.W. Adv. Cyclic. Nucl. Res. 1972, 1, 373. Kebabian, J.W.; Calne, D.B. Nature 1979, 277, 93. Camilli, P.D.; Macconi, D.; Spada, A. Nature (London) 1979, 278, 252. Munemura, M.; Eskay, R.L.; Kebabian, J.W. Endocrinology 1980, 106, 1795. Cote, T.E.; Grewe, C.W.; Kebabian, J.W. Endocrinology 1981, 108, 420. Cote, T . E . ; Grewe, C.W., Tsuruta, K.; Stoof, J . C . ; Eskay, R.L.; Kebabian, J.W. Endocrinology 1982, 110, 812. Brown, E.M.; Aurbach, G.D. "Contemporary Metabolism"; Plenum: New York, 1982; Vol 2, p 247. Exton, J.H. Am. J. Physiol. 1980, Vol., E3. Meunier, H.; Labrie, F. Life Science 1982, 30, 963.

RECEIVED

March 25, 1983


Commentary: Dopamine-Sensitive Adenylate Cyclase as a Receptor Site


PIERRE M . LADURON Janssen Pharmaceutica, Department of Biochemical Pharmacology, B-2340 Beerse, Belgium There i s no doubt that dopamine-sensitive adenylate c y c l a s e (D1 s i t e ) is not involved i n the a n t i p s y c h o t i c e f f e c t of n e u r o l e p t i c drugs. All the pharmacological and behavioural e f f e c t s elicited by dopamine agonists and antagonists i n the b r a i n can only be explained if such an i n t e r a c t i o n occurs a t the l e v e l of the dopamine receptor (D2 receptor site); the D1 s i t e still remains i n search of a f u n c t i o n . Bovine parathyroid cells were reported to possess dopamine D1 s i t e s which should be involved i n the c o n t r o l of parathormone s e c r e t i o n . However, the very poor pharmacological c h a r a c t e r i z a t i o n and the lack of i n v i v o evidence do not allow to assess the dopaminergic nature of t h i s hormone s e c r e t i o n . Dopamine-sensitive adenylate c y c l a s e is thus not a receptor d i r e c t l y i m p l i c a t e d i n the dopaminergic neurotransmission; it is an enzyme which could have an important r o l e i n the c o n t r o l of long term metabolic e f f e c t s such as the synthesis of neuronal c o n s t i t u e n t s .

In the l a s t decade, the term receptor has been used by so many people i n so many d i f f e r e n t ways that we are, now, f a r from the o r i g i n a l d e f i n i t i o n proposed by Langley (1) i n the e a r l y twentieth century. In f a c t the receptor concept arose from p h y s i o l o g i c a l and pharmacological experiments; t h e r e f o r e , a p h y s i o l o g i c a l response i s one of the most e s s e n t i a l elements d e f i n i n g a receptor. According to Langley, a receptor i s a s i t e of competition f o r agonist and antagonist; the agonist produces a stimulus which leads to a p h y s i o l o g i c a l response and t h i s i s blocked by the antagonist. One can e a s i l y apply such a concept to the dopaminergic system; f i r s t i t i s necessary to c l e a r l y d e f i n e the p h y s i o l o g i c a l e f f e c t s of dopamine. Nausea, emesis, f

0097-6156/83/0224-0022$06.00/0 © 1983 American Chemical Society In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


LADURON

Adenylate

Cyclase as Receptor Site

23

stereotypy, h y p e r m o t i l i t y , decrease o f p r o l a c t i n s e c r e t i o n , stomach r e l a x a t i o n , neurogenic v a s o d i l a t a t i o n , a n t i p a r k i n s o n e f f e c t s , psychosis e t c . . . are the main p h y s i o l o g i c a l or pharmacological responses t o dopamine and i t s a g o n i s t s . Às a r u l e , these e f f e c t s are measured under i n v i v o c o n d i t i o n s : t h i s i s a p r e r e q u i s i t e f o r c a l l i n g those p h y s i o l o g i c a l responses. Sometimes one can detect a p h y s i o l o g i c a l e f f e c t i n v i t r o as on i s o l a t e d organs f o r instance; however to be r e l e v a n t such an e f f e c t must correspond to a process o c c u r r i n g i n the whole body. One of the major sources of confusion around dopamine receptor o r i g i n a t e d with the idea that the increase o f the c y c l i c AMP production by dopamine i s a p h y s i o l o g i c a l e f f e c t o f dopamine {2); s t a r t i n g from t h i s viewpoint, i t i s not necessary to t r y to c o r r e l a t e the data obtained i n v i t r o with p h y s i o l o g i c a l e f f e c t s i n v i v o . In f a c t , the s t i m u l a t i o n of c y c l i c AMP by dopamine i s a biochemical e f f e c t f o r which one needs t o f i n d a p h y s i o l o g i c a l response i n v i v o . J u s t because an enzyme i s stimulated or i n h i b i t e d by a given neurotransmitter does not i p s o - f a c t o prove that such a process i s involved i n neurotransmission. As we w i l l see f u r t h e r , numerous c r i t e r i a must be f u l f i l l e d before an enzyme or a binding s i t e may be c a l l e d a receptor s i t e . The e f f e c t s of dopamine quoted above, are antagonized by n e u r o l e p t i c or antiemetic drugs (3yj4) and a l l are mediated through the dopamine D2 receptor s i t e (5,6,7). This D2 subtype (we p r e f e r to c a l l i t the dopamine receptor) i s the binding s i t e l a b e l l e d by dopamine antagonists l i k e h a l o p e r i d o l and spiperone a t nanomolar concentrations and by dopamine agonists a t micromolar concentrations (7) and i s not coupled t o adenylate c y c l a s e . More than 17 pharmacological, behavioural and biochemical parameters r e l a t e d to the e f f e c t s o f dopamine agonists and antagonists n i c e l y c o r r e l a t e with ICsQ-values obtained i n the i n v i t r o binding assay (5,8). How the problem a r i s e s whether or not the dopamines e n s i t i v e adenylate c y c l a s e (D^ s i t e ) ( 2 ) a l s o answers these c r i t e r i a or other c r i t e r i a which j u s t i f y i t being c a l l e d a dopamine receptor l i k e the D2 receptor s i t e (j>-8.). The purpose o f the present paper i s to d i s c u s s t h i s problem e s p e c i a l l y with regard to parathormone s e c r e t i o n . S p e c i a l a t t e n t i o n w i l l be paid to the pharmacological c h a r a c t e r i z a t i o n of t h i s hormone s e c r e t i o n . Is Dopamine-Sensitive Adenylate

Cyclase a Dopamine Receptor ?

As r e c e n t l y quoted by Briggs and McAfee (9): "Rigorous q u a n t i t a t i v e pharmacology i s required i n equating the receptor u t i l i z e d i n s y n a p t i c transmission. Unfortunately the a p p l i c a t i o n of t h i s pharmacology when a v a i l a b l e i s o f t e n superficial . As a r u l e , a too small number of drugs, sometimes even one or two and o f t e n given a t a s i n g l e high c o n c e n t r a t i o n M



24

DOPAMINE

RECEPTORS

have been used to c h a r a c t e r i z e , pharmacologically, the production of c y c l i c AMP stimulated by neurotransmitters. The dopamine-sensitive adenylate c y c l a s e d i d not escape t h i s r u l e ; when Greengard's group reported the occurrence of dopamine-sensitive adenylate c y c l a s e i n r a t caudate nucleus as a p o s s i b l e t a r g e t f o r a n t i p s y c h o t i c drugs (10,11), i t r a p i d l y became evident that the potent n e u r o l e p t i c drugs l i k e h a l o p e r i d o l and pimozide d i s p l a y e d a too low a f f i n i t y f o r the c y c l a s e with regard to t h e i r high potency i n pharmacological t e s t s and i n the c l i n i c . The discrepancy was most apparent f o r pimozide which was found to be 10 times l e s s a c t i v e than chlorpromazine on the c y c l a s e whereas i t i s known t o be 30 to 50 times more potent than chlorpromazine i n v i v o (3,4). Thereafter c e r t a i n drugs l i k e s u l p i r i d e or domperidone were reported t o be p r a c t i c a l l y i n a c t i v e on the c y c l a s e (5,12). Table I shows c l e a r l y that numerous potent dopamine antagonists are poorly a c t i v e or i n a c t i v e on the c y c l a s e although they compete i n the binding assay, sometimes even at nanomolar c o n c e n t r a t i o n . Only the phenothiazines and the thioxanthenes are approximatively e q u i a c t i v e i n both t e s t s . I t became obvious that the a n t i p s y c h o t i c e f f e c t s of n e u r o l e p t i c drugs were not mediated through the c y c l a s e (D^ s i t e ) (j>-8) . There was a complete lack of c o r r e l a t i o n between the i n h i b i t i o n of the dopamine-sensitive adenylate c y c l a s e and 17 behavioural biochemical, pharmacological and c l i n i c a l parameters {6,&) . Two other pieces o f evidence i n d i c a t e that the s i t e i s not involved i n dopaminergic neurotransmission i n the b r a i n . First the i n v i v o accumulation of c y c l i c adenosine monophosphate induced by apomorphine i n the striatum was not blocked by s u l p i r i d e and h a l o p e r i d o l whereas the behavioural e f f e c t s were blocked by both drugs (12). Secondly, when l a b e l l e d n e u r o l e p t i c s were i n j e c t e d i n t o r a t s , the r a d i o a c t i v i t y was found i n a s s o c i a t i o n with the D2 receptor, but never on the D 1 s i t e s (13); indeed the dopamine s e n s i t i v e adenylate c y c l a s e and the binding s i t e (D2) possess a completely d i f f e r e n t s u b c e l l u l a r l o c a l i z a t i o n (14) , a f a c t which g i v e s r i s e to the idea that they are two d i f f e r e n t e n t i t i e s not r e l a t e d to each other. From these c o n s i d e r a t i o n s , one may conclude that the D^ s i t e i s not d i r e c t l y implicated i n dopaminergic neurotransmission; t h i s does not exclude a p o s s i b l e f u n c t i o n a l r o l e for the c y c l a s e but h i t h e r t o i t remains unknown. A p o s s i b l e hypothesis i s that the c y c l a s e may c o n t r o l long term metabolic e f f e c t s such as the synthesis of neuronal c o n s t i t u e n t s . One may argue that the pharmacology of the D^ s i t e does not n e c e s s a r i l y have to be the same as that of the D 2 receptor s i t e ; t h i s i s true but the problem i s to get an i n v i v o pharmacology f o r t h i s D^ s i t e which e n t i r e l y f i t s the data obtained i n v i t r o on the c y c l a s e . Up t o now there i s no answer to t h i s problem. I t i s g e n e r a l l y b e l i e v e d that parathormone


LADURON

Adenylate


25

Table I IC5Q-values f o r various drugs on dopamine s e n s i t i v e adenylate c y c l a s e (D^) and % - h a l o p e r i d o l binding ( D 2 ) IC


Drug

α-Flupenthixol Chlorpromaz ine (+)-Butaclamol Haloperidol Pimozide Spiperone Sulpiride Halopemide Domperidone

D± s i t e A 10'-8 10'-6 X 10'-7 X 10" -7 X 10" -5 X 10" -6 > 10" -3 > 10" -3 2. 4 X 10" -4

2. 3 1. 1 2 7. 5 1. 5 2. 2

X X

5 0

D

2

(M) receptor s i t e Β 2 1.3 1 3.6 3.2 4.4 8 1 1.4

X 10" -8 X 10" -7 X 10" -8 X 10" -9 X 10" -9 X 10- -10 X 10" -8 X 10'-8 X 10" -9

Ratio A/B

1.2 8.5 20 208 4.,687 5,,500 > 10.,000 >100 ,000 142, ,857 l

s e c r e t i o n i s mediated v i a the D^ s i t e (2) ; we w i l l now examine the c r i t e r i a v a l i d a t i n g or i n v a l i d a t i n g t h i s hypothesis. Is Parathormone S e c r e t i o n Mediated through a Dopamine D] S i t e ? In 1977, Brown e t a l . (15) reported that dopamine (10~ M) s t i m u l a t e s by 2-4 f o l d the s e c r e t i o n of parathormone from dispersed bovine parathyroid c e l l s . ADTN and other dopamine agonists mimicked t h i s e f f e c t which was antagonized by a - and β-flupenthixol, the α-isomer being 100 times more potent. In a s i m i l a r way, dopamine caused a r a p i d 20-30-fold increase i n c e l l u l a r cAMP i n dispersed bovine parathyroid c e l l s . The potency of a s e r i e s of dopaminergic agonists and antagonists on adenylate c y c l a s e a c t i v i t y p a r a l l e l e d the e f f e c t s of these l i g a n d s on cAMP accumulation and parathormone s e c r e t i o n (16). I t was concluded that bovine parathyroid c e l l s possess dopamine D^ s i t e s which are involved i n the c o n t r o l of parathormone s e c r e t i o n . This needs some comments; f i r s t , s e v e r a l other agents that are not dopaminergic agonists, such as β-adrenergic catecholamines, s e c r e t i n , phosphodiesterase i n h i b i t o r s , histamine, protaglandin ( P G E 2 ) and cholera t o x i n were a l s o found to enhance cAMP accumulation and parathormone s e c r e t i o n i n human and bovine parathyroid c e l l s (17,18,19) ( c f r . Table I I ) . Moreover, calcium has been known f o r a long time, to play an important r o l e i n the parathormone s e c r e t i o n ; therefore the e f f e c t s of dopamine agonists are not s e l e c t i v e or s p e c i f i c f o r a given neurotransmitter. Somewhat s u r p r i s i n g i s the f a c t that dopamine i s i n e f f e c t i v e i n human dispersed parathyroid c e l l s ; t h i s c e r t a i n l y 6


26

DOPAMINE

RECEPTORS

Table I I Compounds which can regulate (+) or not (-) cAMP production and parathormone s e c r e t i o n i n parathyroid c e l l s


Epinephrine Dopamine Histamine Secretin Prostaglandin Phosphodiesterase Choieratoxin

inhibitors

Bovine parathyroid

Human parathyroid

+ +

+

-+

-+ -+

-? +

+

l i m i t s the importance of t h i s s o - c a l l e d D^ dopamine s i t e , more e s p e c i a l l y as the present r e s u l t s do not allow the p o s s i b i l i t y of a non s p e c i f i c e f f e c t of dopamine on parathormone s e c r e t i o n to be excluded. One could assume, f o r instance that dopamine l i k e the other agonists can increase the membrane p e r m e a b i l i t y so that more hormone can be released a f t e r a d d i t i o n of these compounds. Compatible with t h i s hypothesis i s the f a c t that the increase of parathormone s e c r e t i o n e l i c i t e d by dopamine i s a r e l a t i v e l y slow process and that i t can l a s t as long as 60 minutes. As a r u l e the presence of agonists on a receptor s i t e for a long p e r i o d of time leads to a d e s e n s i t i z a t i o n phenomenon; t h i s i s not the case here. More i n t r i g u i n g i s the f a c t that the parathormone s e c r e t i o n occurs i n the absence of dopamine; what dopamine i s doing, i s t o enhance a phenomenon already present; t h i s i s a l s o a t variance with the normal p h y s i o l o g i c a l response to a neurotransmitter which i s g e n e r a l l y an a l l or none process. In f a c t the most important p o i n t concerns the very poor pharmacological c h a r a c t e r i z a t i o n of the cAMP formation enhanced by dopamine and of the parathormone s e c r e t i o n . F i r s t l y , apomorphine i s much l e s s potent than dopamine, a f a c t which i s not compatible with what we know from pharmacological, behavioural and even biochemical s t u d i e s (3,4^,2) · ^ b e l i e v e d that apomorphine i s a p a r t i a l antagonist, but t h i s has never been found i n i n v i v o c o n d i t i o n s . The higher potency o f apomorphine i s a l s o r e f l e c t e d by i t s high a f f i n i t y i n H - h a l o p e r i d o l and H-spiperone b i n d i n g . Secondly ergot d e r i v a t i v e s which r e v e a l a c l e a r c u t a g o n i s t i c a c t i v i t y on p r o l a c t i n s e c r e t i o n and as antiparkinson agents (20) were i n a c t i v e on the c y c l a s e . S u r p r i s i n g l y , l i s u r i d e and l e r g o t r i l e were found to be weak antagonists of dopamine stimulated cAMP accumulation, but they could a l s o antagonize the cAMP production stimulated by i s o p r o t e r e n o l ; as I f c

3

s

3



LADURON

Adenylate


27

both compounds are completely i n a c t i v e on the β-receptors, t h i s f i n d i n g c o n s t i t u t e s a strong argument a g a i n s t the s p e c i f i c i t y o f the s o - c a l l e d dopaminergic and β-adrenergic s i t e s i n v o l v e d i n the cAMP accumulation i n the bovine p a r a t h y r o i d c e l l s . Another point concerns the K i ' s o f 1- and d-butaclamol on dopamine s e n s i t i v e adenylate c y c l a s e which are 1 /uM and 2.5 juM respectively. I t i s well-known that the d-form i s the a c t i v e enantiomer and 1- the i n a c t i v e one; t h e r e f o r e i f d-butaclamol i s l e s s a c t i v e than 1-butaclamol, and a l s o that i t i s l e s s a c t i v e than β-flupenthixol, again an i n a c t i v e enantiomer, the e f f e c t s of these drugs on the c y c l a s e are thus i r r e l e v a n t p h y s i o l o g i c a l l y . In f a c t t o assess the dopaminergic nature f o r the c o n t r o l o f parathormone s e c r e t i o n , the f o l l o w i n g c r i t e r i a should be f u l f i l l e d : 1) a l a r g e number o f dopamine antagonists should be t e s t e d i n c l u d i n g compounds belonging t o d i f f e r e n t chemical c l a s s e s , such as phenothiazines, butyrophenones, thioxanthenes, diphenylbutylamines, domperidone, d- and 1-butaclamol; 2) drugs belonging t o other pharmacological c l a s s e s should be tested (a and β-adrenergic, a n t i s e r o t o n e r g i c , a n t i h i s t a m i n e , a n t i c h o l i n e r g i c , and lysosomotropic drugs l i k e c l o r o q u i n e f o r instance; 3) the a f f i n i t y o f dopamine agonists should be compared t o that of non dopaminergic a g o n i s t s ; 4) a good c o r r e l a t i o n should be found between the i n h i b i t i o n o f the parathormone s e c r e t i o n measured under i n v i t r o as w e l l as i n v i v o c o n d i t i o n s and the decrease i n cAMP and the i n h i b i t i o n o f dopamine-sensitive adenylate c y c l a s e ; i n t h i s regard, a l a r g e number o f drugs with a broad range o f a c t i v i t y should be t e s t e d ; 5) the increase o f cAMP production e l i c i t e d by dopamine antagonists and i t s i n h i b i t i o n by dopamine antagonists should be examined i n the p a r a t h y r o i d under i n v i v o c o n d i t i o n s . Only such an a n a l y t i c a l approach can decide whether the c o n t r o l o f the parathormone s e c r e t i o n i n v o l v e s a dopaminergic receptor. In my o p i n i o n , one may assume a p r i o r i that these c r i t e r i a w i l l not be f u l f i l l e d ; f i r s t , i f the dopamine-sensitive adenylate c y c l a s e was r e a l l y involved i n the parathormone s e c r e t i o n , the p a t i e n t s treated with n e u r o l e p t i c s and e s p e c i a l l y with the most potent drugs on the s i t e s (phenothiazine and thioxanthene d e r i v a t i v e s ) would have normally revealed marked changes i n t h e i r parathormone s e c r e t i o n , j u s t l i k e as i s the case f o r the p r o l a c t i n s e c r e t i o n ; i n f a c t such changes have never been observed; secondly, a recent report c l e a r l y i n d i c a t e s that the i n j e c t i o n o f dopamine i n man does not modify parathormone s e c r e t i o n although a marked decrease i n p r o l a c t i n was observed (21) . There i s no receptor without p h y s i o l o g i c a l response; the study o f receptor r e q u i r e s a m u l t i d i s c i p l i n a r y approach. H i t h e r t o , i t has not been proved t h a t the ϋχ s i t e i s


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r e a l l y involved i n parathormone s e c r e t i o n ; the D^ s i t e i s thus an enzyme but not a receptor s i t e s i n c e a p h y s i o l o g i c a l r o l e has not been demonstrated. Acknowledgments Part o f t h i s work was supported by I.W.O.N.L. I thank David Ashton f o r h i s h e l p i n preparing the manuscript.


Literature

Cited

1. Langley, J.N. P r o c . Roy. S o c . S e r . Β 1906, 78, 107-194. 2. Kebabian, J . W . ; Calne, D . B . Nature 1979, 277, 93-96. 3. Niemegeers, C.J.E.; Janssen, P.A.J. L i f e Sci. 1979, 24, 2201-2216. 4. Costall, B.; N a y l o r , R.J. L i f e Sci. 1981, 28, 215-229. 5. Laduron, P . Trends Pharmacol. Sci. 1980, 1, 471-474. 6. Laduron, P . "Advances in Dopamine Research" Kohsaka, Ed.; Pergamon, 1982, Vol. 37; p. 71. 7. Seeman, P . Pharmacol. Rev. 1980, 32, 229-313. 8. Laduron, P . "Apomorphine and Other Dopaminomimetics" Gessa and Corsini, E d s . ; Raven P r e s s , 1981, Vol. 1; p . 85. 9. B r i g g s , C.A.; McAfee, D . A . Trends Pharmacol. Sci. 1982, 3, 241-244. 10. Kebabian, J . W . ; P e t z o l d , G.L.; Greengard, P . P r o c . Natl. Acad. Sci. USA 1972, 69, 2145-2149. 11. Clement-Cormier, Y.C.; Kebabian, J . W . ; P e t z o l d , G.L.; Greengard, P . Proc. Natl. Acad. Sci. USA , 71, 1113-1117. 12. T r a b u c c h i , M.; Longoni, R . ; Fresia, P.; Spano, P . F . L i f e Sci. 1975, , 1551-1556. 13. Laduron, P . M . ; Janssen, P.F.M.; Leysen, J.E. Biochem. Pharmacol. 1978, 27, 323-328. 14. Leysen, J.E.; Laduron, P . L i f e Sci. 1977, 20, 281-288. 15. Brown, E.M.; Carroll, R . ; Aurbach, G.D. P r o c . Natl. Acad. Sci. USA 1977, 74, 4210-4213. 16. Brown, E.M.; Attie, M.F.; Reen, S . ; Gardner, D . G . ; Kebabian, J.; Aurbach, G . D . M o l . Pharmacol. 1980, 18, 335-340. 17. Brown, E.M.; H u r w i t z , S . , Aurbach, G.D. Endocrinology 1977, 100, 1696-1702. 18. Brown, E.M.; Gardner, D . G . ; Windeck, R.A.; Aurbach, G . D . Endocrinology 1979, 104, 218-224. 19. Brown, E.M.; Gardner, D . G . ; Windeck, R . A . ; H u r w i t z , S . ; Brennan, M.F.; Aurbach, G.D. J. Clin. E n d o c r i n o l . Metab. 1979, 48, 618-626. 20. Schachter, M.; B é d a r d , P.; Debono, A.G.; Jenner, P.; Marsden, C.D.; P r i c e , P.; Parkes, J.D.; Keenan, J.; Smith, B.; Rosenthaler, J.; Horowski, R . ; Dorow, R. Nature 1980, 286, 157-159. 21. B a n s a l , S . ; Woolf, P . D . ; F i s c h e r , J.A.; Caro, J.F. J. Clin. E n d o c r i n o l . Metab. 1982, 54, 651-652. R E C E I V E D February 18, 1983



D r . Brown's Replies to D r . Laduron's Comments D r . L a d u r o n raises a number of issues w h i c h r e q u i r e c o m m e n t . H e r e f e r s t o the w o r k of L a n g l e y (1) t o p r o v i d e support for a p r e d o m i n a n t l y i n v i v o d e f i n i t i o n of r e c e p t o r s . B y n e c e s s i t y , o f c o u r s e , these e a r l y e x p e r i m e n t s w e r e based s o l e l y on p h y s i o l o g i c a l responses, such as m u s c l e c o n t r a c t i o n . It is of i n t e r e s t , h o w e v e r , t h a t L a n g l e y was p r e s c i e n t i n p o s t u l a t i n g t h a t i n t e r a c t i o n of agonists w i t h a " r e c e p t i v e substance", w h i c h r e c e i v e s and t r a n s m i t s i n f o r m a t i o n , leads t o a change i n i n t r a c e l l u l a r substances (?second messengers) and u l t i m a t e l y to a change in c e l l u l a r f u n c t i o n . M o d e l systems such as b o v i n e p a r a t h y r o i d c e l l s and c e l l s of the i n t e r m e d i a t e lobe of the r a t p i t u i t a r y g l a n d have a l l o w e d for a d i r e c t d e m o n s t r a t i o n of such a s e q u e n c e . D r . L a d u r o n is c o r r e c t t h a t a p h y s i o l o g i c a l r o l e for D - l d o p a m i n e r g i c r e c e p t o r s has not y e t been d e m o n s t r a t e d i n the b r a i n , but, o f c o u r s e , t h i s does not m e a n t h a t such a f u n c t i o n does not e x i s t . A s e c r e t o r y response to d o p a m i n e has been d e m o n s t r a t e d i n v i v o i n the c o w (2) w h i c h was not b l o c k e d by p r o p r a n o l o l . It should also be p o i n t e d out t h a t m u c h of t h e a c c u m u l a t e d k n o w l e d g e about w e l l - d e f i n e d r e c e p t o r systems such as the betaa d r e n e r g i c r e c e p t o r have c o m e f r o m models l i k e the t u r k e y or the f r o g e r y t h r o c y t e , i n w h i c h the p h y s i o l o g i c a l r o l e of the r e c e p t o r is u n k n o w n . B y D r . L a d u r o n ' s d e f i n i t i o n , these s y s t e m s do not have a b e t a r e c e p t o r . T h i s " s h o r t c o m i n g " has c e r t a i n l y not lessened t h e u t i l i t y of these s y s t e m s for c a r r y i n g out d e t a i l e d b i o c h e m i c a l c h a r a c t e r i z a t i o n of the b e t a r e c e p t o r . F i n a l l y , a major weakness o f a p u r e l y p h a r m a c o l o g i c a p p r o a c h or e v e n one w h i c h c o r r e l a t e s binding s i t e s w i t h p h y s i o l o g i c a l responses is t h a t i t c a n be o n l y c o r r e l a t i v e . W i t h such an a p p r o a c h , i t is a l w a y s possible t h a t i f a d d i t i o n a l drugs w e r e t e s t e d , e x c e p t i o n s w o u l d a r i s e w h i c h w o u l d not support the p o s t u l a t e d r e c e p t o r - m e d i a t e d l i n k a g e t o a p h y s i o l o g i c a l response. A c a u s a l r e l a t i o n s h i p b e t w e e n the i n t e r a c t i o n of an agonist w i t h a r e c e p t o r and a p h y s i o l o g i c a l response c a n o n l y be e s t a b l i s h e d by w o r k i n g out the d e t a i l e d m o l e c u l a r m e c h a n i s m s by w h i c h the r e c e p t o r - m e d i a t e d changes i n c e l l u l a r f u n c t i o n t a k e p l a c e . D r . L a d u r o n also feels t h a t the d o p a m i n e - s t i m u l a t e d i n c r e a s e i n c y c l i c A M P a c c u m u l a t i o n and P T H s e c r e t i o n m a y be " n o n - s p e c i f i c " and has not been shown to be m e d i a t e d by a d o p a m i n e ( D - l ) r e c e p t o r . We w o u l d s i m p l y l i k e t o r e i t e r a t e the e v i d e n c e w e p r e s e n t e d p r e v i o u s l y and t o point out a d d i t i o n a l e v i d e n c e for a s p e c i f i c r e c e p t o r - m e d i a t e d p r o c e s s . The e f f e c t s o f d o p a m i n e a r e r a p i d (less t h a n a m i n u t e i n v i v o and n e a r l y as fast i n v i t r o ) and are b l o c k e d s p e c i f i c a l l y by d o p a m i n e r g i c antagonists of the p h e n o t h i a z i n e , t h i o x a n t h i n e , and butyrophenone classes as w e l l as by d - b u t a c l a m o l (see b e l o w ) . T h e y are t o t a l l y u n a f f e c t e d , o n the o t h e r h a n d , by the b e t a - a d r e n e r g i c a n t a g o n i s t p r o p r a n o l o l and the a l p h a a d r e n e r g i c b l o c k e r p h e n t o l a m i n e at c o n c e n t r a t i o n s w h i c h t o t a l y i n h i b i t responses due to beta-adrenergic or alpha-adrenergic agonists, r e s p e c t i v e l y . In a d d i t i o n , i n p i l o t studies we h a v e found t h a t h i s t a m i n e , s e r o t o n i n , o c t o p a m i n e and c a r b a m y l c h o l i n e h a v e not c o n s i s t e n t e f f e c t on c y c l i c A M P a n d / o r P T H r e l e a s e . F i n a l l y , t h e e f f e c t s o f P G E are not b l o c k e d by p r o p r a n o l o l , a l p h a - f l u p e n t h i x o l , or p h e n t o l a m i n e (3), the i n h i b i t o r y e f f e c t s of p r o s t a g l a n d i n F a r e not i n h i b i t e d by p h e n t o l a m i n e 29 In Dopamine Receptors; Kaiser, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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(4), and the s t i m u l a t o r y e f f e c t s of s e c r e t i n are not i n h i b i t e d by propranolol, alpha-flupenthixol, or phentolamine (5). Since m e t h y l i s o b u t y l x a n t h i n e and other phosphodiesterase i n h i b i t o r s as w e l l as c h o l e r a t o x i n a c t i n t r a c e l l u l a r ^ through a n o n - r e c e p t o r m e d i a t e d p r o c e s s , w e do not f e e l i t l i k e l y t h a t these agents a c t t h r o u g h any of the r e c e p t o r s n o t e d a b o v e . We f e e l , t h e r e f o r e , t h a t the s p e c i f i c i t y of the d o p a m i n e r g i c response of b o v i n e p a r a t h y r o i d c e l l s speaks for i t s e l f . We agree w i t h D r . L a d u r o n , h o w e v e r , t h a t the use of further drugs t o t e s t s p e c i f i c i t y w o u l d s t r e n g t h e n these d a t a e v e n f u r t h e r . It should be p o i n t e d out t h a t the response o f a c e l l t o o n l y a single c l a s s o f agonists w o u l d be the e x c e p t i o n r a t h e r t h a n the r u l e . In the c e n t r a l nervous s y s t e m , i t is b e c o m i n g i n c r e a s i n g l y c l e a r t h a t the i n t e r p l a y of a number o f p h a r m a c o l o g i c i n f l u e n c e s m a y d e t e r m i n e the i n t e g r a t e d response o f g i v e n c e l l t y p e . T o a v o i d c o n f u s i o n o n the p a r t of the r e a d e r , s e v e r a l i n a c c u r a c i e s o n the p a r t of D r . L a d u r o n should be p o i n t e d o u t . (1) It is i n c o r r e c t t o s t a t e t h a t d e s e n s i t i z a t i o n does not o c c u r i n the d o p a m i n e r g i c response of b o v i n e p a r a t h y r o i d cells. We d i d not, i n f a c t , e x a m i n e this p o i n t d i r e c t l y . D e s e n s i t i z a t i o n , h o w e v e r , probably a c t u a l l y does o c c u r a t m o r e t h a n one l o c u s i n this s y s t e m . F i r s t , the secretory response o f the p a r a t h y r o i d c e l l r a p i d l y b e c o m e s r e f r a c t o r y t o agents such as d o p a m i n e and i s o p r o t e r e n o l w h i c h produce l a r g e e l e v a t i o n s i n c y c l i c A M P (see r e f . 2). S e c o n d l y , t h e r e is a p r o g r e s s i v e d e c r e a s e i n c e l l u l a r c y c l i c A M P despite the c o n t i n u e d presence of dopamine (see F i g u r e 5 i n our m a n u s c r i p t ) possibly due t o d e s e n s i t i z a t i o n of the r e c e p t o r adenylate cyclase compex. (2) D r . L a d u r o n uses the r e l a t i v e p o t e n c y of d - and 1b u t a c l a m o l as a major a r g u m e n t against the s p e c i f i c i t y of t h e p a r a t h y r o i d dopamine r e c e p t o r . We a p o l o g i z e for the confusion w h i c h we a p p a r e n t l y caused h i m i n t h i s r e g a r d . In our o r i g i n a l m a n u s c r i p t , the s y m b o l w h i c h D r . L a d u r o n took t o indicate a potency of 1 m i c r o m o l a r for 1-butaclamol a c t u a l l y r e f e r r e d to F o o t n o t e 1 w h i c h s t a t e d t h a t 1 - b u t a c l a m o l had no e f f e c t a t 100 m i c r o m o l a r . In a d d i t i o n , b e c a u s e of the r e l a t i v e l y l o w p o t e n c y o f d - b u t a c l a m o l i n our o r i g i n a l studies (k. = 2.5 m i c r o m o l a r ) , w e c a r r i e d out a d d i t i o n a l studies w i t h fresh samples o f d - a n d 1 - b u t a c l a m o l . The p o t e n c y of t h e s e n e w e r samples are r e f l e c t e d i n T a b l e I o f the present m a n u s c r i p t (k for d - b u t a c l a m o l 5 = n a n o m o l a r ; 1 - b u t a c l a m o l a g a i n had no e f f e c t at 10 m o l a r ) . S i n c e we do not have d a t a on the e f f e c t s o f the newer samples o n a d e n y l a t e c y c l a s e , w e h a v e not i n c l u d e d d a t a for the e f f e c t s of b u t a c l a m o l o n c y c l a s e i n the newer m a n u s c r i p t . (3) D r . L a d u r o n s t a t e s t h a t b o t h l i s u r i d e and l e r g o t r i l e are a n t a g o n i s t s a t the b e t a - r e c e p t o r i n b o v i n e p a r a t h y r o i d cells. H e a g a i n uses this p i e c e o f e v i d e n c e as a s t r o n g a r g u m e n t against the s p e c i f i c i t y o f the d o p a m i n e r e c e p t o r i n t h i s s y s t e m . We k n o w of no e v i d e n c e , h o w e v e r , e i t h e r i n our w o r k or t h a t of o t h e r s t h a t l e r g o t r i l e has such a n e f f e c t o n


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Cyclase as Receptor

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31

p a r a t h y r o i d c e l l s . M o r e o v e r , the k. f o r l i s u r i d e at the b e t a r e c e p t o r d i f f e r s b y 3 t o 4 - f o l d f r o m hhat for i t s e f f e c t s on the dopamine r e c e p t o r , suggesting t h a t i t i s , i n f a c t , a c t i n g at t w o different receptors. (4) In T a b l e II o f his c o m m e n t s , D r . L a d u r o n points out d i f f e r e n c e s b e t w e e n b o v i n e and h u m a n p a r a t h y r o i d c e l l s . S e v e r a l of these are i n e r r o r . Prostaglandins affect both h u m a n and b o v i n e p a r a t h y r o i d c e l l s . Phosphodiesterase i n h i b i t o r s have not been t e s t e d d i r e c t l y i n h u m a n p a r a t h y r o i d c e l l s , although d i b u t y r y l c y c l i c A M P , which may act i n part by i n h i b i t i n g phosphodiesterase, stimulated P T H release in f r a g m e n t s o f h u m a n p a r a t h y r o i d glands (6). (5) It is v e r y l i k e l y an i n a c c u r a c y to c a l l the D - l dopamine receptor an e n z y m e . B e c a u s e of the e f f e c t s o f guanine nucleotides on dopamine-stimulated adenylate c y c l a s e , i t is l i k e l y by a n a l o g y w i t h o t h e r r e c e p t o r s (i.e. the 3 r e c e p t o r ) t h a t the D - l r e c e p t o r is a d i s t i n c t m o l e c u l a r e n t i t y w h i c h is 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 a guanine n u c l e o t i d e b i n d i n g subunit. P r o o f o f this p o i n t , o f c o u r s e , w i l l r e q u i r e p h y s i c a l s e p a r a t i o n of these e n t i t i e s . (6) D r . L a d u r o n c a t e g o r i c a l l y s t a t e s t h a t the D - 2 r e c e p t o r is not 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 . R e c e n t w o r k , h o w e v e r , u t i l i z i n g b o t h the e f f e c t s o f d o p a m i n e on the m a m m o t r o p h as w e l l as o n dispersed c e l l s o f the i n t e r m e d i a t e l o b e of the r a t p i t u i t a r y gland (the f o r m e r o f w h i c h D r . L a d u r o n appears t o f e e l i t is an e x a m p l e of t h e D - 2 r e c e p t o r ) suggests t h a t the D - 2 r e c e p t o r is, i n f a c t , 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 i n a n e g a t i v e w a y . It m a y w e l l t u r n o u t , t h e r e f o r e , t h a t , l i k e the alpha-2 a d r e n e r g i c r e c e p t o r , the D - 2 r e c e p t o r is 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 through a n i n h i b i t o r y guanine n u c l e o t i d e subunit. W h e t h e r or not o t h e r D - 2 r e c e p t o r s i n the c e n t r a l nervous s y s t e m are l i n k e d t o the c y c l a s e i n t h i s fashion r e m a i n s t o be d e t e r m i n e d .

Literature Cited 1. 2. 3. 4. 5. 6.

L a n g l e y , J.N. Proc. R o y . S o c . Ser. B . 1906, 78, 170-194. B l u m , J . W . ; K u n z , P . ; F i s c h e r , J.A.; B i n s w a n g e r , U.; L i c h t e n s t e i g e r , W . ; D a P r a d a , M. Am. J. P h y s i o l . 1980, 239, E 2 5 5 . Gardner, D . B . ; Brown, E . M . ; Windeck, R . ; Aurbach, G . D . E n d o c r i n o l o g y 1978, 103, 577. Gardner, D . B . ; Brown, E.M.; Windeck, R . ; Aurbach, G . D . E n d o c r i n o l o g y 1979, 104, 1. Windeck, R . ; Brown, E.M.; Gardner, D . B . ; Aurbach, G . D . E n d o c r i n o l o g y 1978, 103, 2020. D i e t e l , M.; D o r n , G.; M o n t z , R . ; A l t e n a k r , E. Acta E n d o c r i n o l o g i c a (Kbh) 1977, 8 5 , 5 4 1 .


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