D-1 Dopamine Receptor-Mediated Activation of Adenylate Cyclase

by dopamine, 6,7-ADTN and SKF 38393 at 0.2 μΜ, 0.15 μΗ and. 0.3 μΜ ... Figure 4. Stimulation of cAMP accumulation in dispersed bovine parathyroi...
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1 D-1 Dopamine Receptor-Mediated Activation of Adenylate Cyclase, cAMP Accumulation, and PTH Release in Dispersed Bovine Parathyroid Cells

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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.)

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

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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.)

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

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D-l Mediated

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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

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

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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.

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

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7

TABLE I

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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

^

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

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DOPAMINE

8

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RECEPTORS

10-5 10-4

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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.)

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

A N D DAWSON-HUGHES

D-l

Mediated

Activation

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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.)

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

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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.

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

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BROWN

A N D DAWSON-HUGHES

(-)ISO

(-)EPI

D-l

Mediated

Activation

(-)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).

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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

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

1.

BROWN

A N D DAWSON-HUGHES

D-l Mediated

13

Activation

0.75 h

200 0.375 Η ιοο Û.

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ιο"°

10"'

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[DOPAMINE] , Μ


100

at 10"^ M.

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

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A N D DAWSON-HUGHES

D-l Mediated

Activation

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

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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).

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

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

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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1.

BROWN

A N D DAWSON-HUGHES

D-l Mediated

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.

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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.

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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

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

Commentary: Dopamine-Sensitive Adenylate Cyclase as a Receptor Site

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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.

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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

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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

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

LADURON

Adenylate

Cyclase as Receptor Site

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

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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

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

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

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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

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

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LADURON

Adenylate

Cyclase as Receptor Site

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

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

DOPAMINE

28

RECEPTORS

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.

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Literature

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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

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

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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

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

Adenylate

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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 .

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