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Dopamine receptor binding of 3H-ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) regulated by guanyl nucleotides
European Journal of Pharmacology, 50 (1978) 459--461
459
© Elsevier/North-HollandBiomedicalPress Rapid c o m m u n i c a t i o n D O P A M I N E RECEPTOR BINDING OF 3H-ADTN
(2-AMINO-6,7-DIHYDROXY-1,2,3,4-TETRAHYDRONAPHTHALENE) REGULATED BY GUANYL NUCLEOTIDES
IAN CREESE and SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
Received 14 July 1978, accepted 14 July 1978
ADTN (2-amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene), a rigid analogue of dopamine, stimulates the dopamine-sensitive adenylate cyclase and has potent dopaminelike behavioral effects when injected centrally (Iversen, 1975). Low affinity binding to brain membranes by 3H-ADTN of low specific activity lacks many properties expected of dopamine receptors (Roberts et al., 1977). With 3H-ADTN of higher specific radioactivity (3 Ci/mmole, Amersham) we now describe specific labeling of dopamine receptors. Homogenates of rat striatum (100 vols w/v), prepared in 50 mM Tris-HCl buffer (pH 7.7 at 25°C) by sonication, were centrifuged twice (48,000 × g) at 4°C with resuspension in fresh buffer. Final resuspension was in Tris buffer containing 0.1% ascorbic acid and 1 mM MnC12 (pH 7.1 at 37°C). After incubation with 3H-ADTN in the presence or absence of 1 pM (+)-butaclamol and unlabeled drugs at 37°C for 10 min, triplicate samples were filtered rapidly under vacuum over Whatman GF/B filters, rinsed with 3 × 5 ml ice-cold Tris-HC1 buffer, and radioactivity trapped on the filters counted by liquid scintillation spectroscopy. In typical experiments with 8 nM 3HADTN total binding is about 600 cpm and nonspecific binding is about 300 cpm. Specific 3H-ADTN binding reaches equilibrium by 10 min at 37°C. Specific binding is saturable, Scatchard analysis showing two components
of binding with apparent dissociation constants (KD) of 6 nM and 45 nM and binding site number (Bronx) of 15 and 35 pmoles/g wet weight. Scatchard analysis reveals preponderant labeling of high affinity sites at the concentration of 3H-ADTN routinely employed (8 riM). Drug specificity of 3H-ADTN binding fulfills characteristics expected of dopamine receptors (table 1) and resembles the binding of the dopamine agonist 3H-apomorphine (Thal et al., 1978; Seeman et al., 1976). ADTN and apomorphine are the most potent dopamine agonists, about 10 times more potent than dopamine itself which in turn is 3 times more potent than norepinephrine. Serotonin is virtually inactive. Fluphenazine is the most potent phenothiazine neuroleptic with an affinity 3 times greater than that of chlorpromazine and 50 times greater than promazine. Butaclamol effects are stereospecific favoring the (+)-isomer by more than 1000 fold. The geometric isomers of flupenthixol also exhibit marked stereospecificity with a-flupenthixol being 60 fold more potent than the ~-isomer. The butyrophenone neuroleptics, spiroperidol and haloperidol, are relatively weak contrasting to their great pharmacological potency, resembling results obtained with other dopamine 3H-agonists (Burt et al., 1976). These properties differ markedly from low affinity binding of 3HADTN (Roberts et al., 1977), in which
460 TABLE 1 Properties of 3H-ADTN binding to rat striatal membranes. ICs0 's were determined by log-probit analysis of drug displacements of 8 nM 3H-ADTN. Non-specific binding was determined in the presence of 1 uM (+)butaclamol. Nucleotide concentrations were 50 tiM. Data presented as the mean of 2--6 independent replications. (A) Drug specificity IC~o (nM) Dopamine Apomorphine ADTN Norepinephrine Epinephrine Serotonin (+)-Butaclamol (--)-Butaclamol a-Flupenthixol t3-Flupenthixol Fluphenazine Chlorpromazine Promazine Spiroperidol Haloperidol Phentolamine Propranolol
300 30 25 900 1,200 > 10,000 6 > 10,000 24 1,600 13 40 500 90 40 2,000 > 10,000
(B) Guanyl nucleotide effects 3H-ADTN binding % control GTP GppNHp GDP GMP ATP ADP AMP
50 63 58 95 92 96 98
b o t h agonists and a n t a g o n i s t s were less t h a n o n e - t h o u s a n d t h as p o t e n t as o b s e r v e d in the p r e s e n t s t u d y . T h e u b i q u i t o u s regional dist r i b u t i o n r e p o r t e d for low a f f i n i t y 3H-ADTN binding also m i t i g a t e s against a selective labeling o f d o p a m i n e r e c e p t o r s . In the a b s e n c e of a d d e d ions o n l y a b o u t
10% o f t o t a l 3H-ADTN binding is displaceable b y 1 pM ( + ) - b u t a c l a m o l . Manganese signifi c a n t l y increases specific b i n d i n g at 1 pM and progressively increases specific binding to m a x i m a l e f f e c t s at 1 m M , t h e c o n c e n t r a t i o n used in r o u t i n e assays. Calcium and m a g n e s i u m are m u c h less effective, p r o d u c i n g no influence on specific binding at 1 pM and increasing specific binding at 1 m M b y o n l y h a l f as m u c h as m a n g a n e s e . S o d i u m ( 1 0 - 100 mM} decreases n o n s p e c i f i c b i n d i n g 1 0 - - 3 0 % with little change in specific binding. Guanyl nucleotides enhance effects of s o m e h o r m o n e s and n e u r o t r a n s m i t t e r s o n a d e n y l a t e cyclase b y facilitating r e c e p t o r cyclase c o u p l i n g a n d l o w e r i n g agonist affinity o f the r e c o g n i t i o n site. G T P , G p p ( N H ) p and G D P diminish d o p a m i n e r e c e p t o r binding of 3H-ADTN and 3 H - a p o m o r p h i n e ( d a t a n o t shown}. T h e specificity o f this e f f e c t is e v i d e n t in t h e failure o f GMP, ATP, ADP a n d AMP to alter agonist binding. G T P has no influence on t o t a l r e c e p t o r b i n d i n g o f the d o p a m i n e a n t a g o n i s t 3H-spiroperidol. H o w ever, the ability of agonists to inhibit 3Hs p i r o p e r i d o l binding is r e d u c e d in the prese n c e of 10 pM GTP. A c o m p o n e n t of 3HA D T N binding m a y thus be associated with the d o p a m i n e - s e n s i t i v e a d e n y l a t e cyclase.
Acknowledgements Tim Prosser, Ted Usdin and Kim Stewart are thanked for their assistance. Research was supported by grants to S.H.S. from USPHS MH-18501, The John A. Hartford Foundation, The McKnight Foundation, Sandoz Pharmaceuticals and to I.C. from Merck, Sharpe and Dohme, and an Institutional grant. Pam Morgan and Susan M. Garonski are thanked for manuscript preparation.
References Burt, D.R., I. Creese and S.H. Snyder, 1976, Properties of [3H]-haloperidol and [3H]-dopamine binding associated with dopamine receptors in calf brain membranes, Mol. Pharmacol. 12, 800.
461
Iversen, L.L., 1975, Dopamine receptors in the brain, Science 188, 1084. Roberts, P.J., G.N. Woodruff and J.A. Poat, 1977, Binding of a conformationaUy restricted dopamine analogue, 2-amino-6,7-dihydroxy-l,2,3,4tetrahydronaphthalene to receptors on rat brain synaptic membranes, Mol. Pharmacol. 13, 541. Seeman, P., T. Lee, M. Chau-Wong, J. Tedesco and
K. Wong, 1976, Dopamine receptors in human and calf brains using [3H]-apomorphine and an antipsychotic drug, Proc. Nat. Acad. Sci. U.S.A., 73, 4354. Thai, L., I. Creese and S.H. Snyder, 1978, 3H-Apomorphine interactions with dopamine receptors in calf brain, European J. Pharmacol. 49, 295.
459
© Elsevier/North-HollandBiomedicalPress Rapid c o m m u n i c a t i o n D O P A M I N E RECEPTOR BINDING OF 3H-ADTN
(2-AMINO-6,7-DIHYDROXY-1,2,3,4-TETRAHYDRONAPHTHALENE) REGULATED BY GUANYL NUCLEOTIDES
IAN CREESE and SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
Received 14 July 1978, accepted 14 July 1978
ADTN (2-amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene), a rigid analogue of dopamine, stimulates the dopamine-sensitive adenylate cyclase and has potent dopaminelike behavioral effects when injected centrally (Iversen, 1975). Low affinity binding to brain membranes by 3H-ADTN of low specific activity lacks many properties expected of dopamine receptors (Roberts et al., 1977). With 3H-ADTN of higher specific radioactivity (3 Ci/mmole, Amersham) we now describe specific labeling of dopamine receptors. Homogenates of rat striatum (100 vols w/v), prepared in 50 mM Tris-HCl buffer (pH 7.7 at 25°C) by sonication, were centrifuged twice (48,000 × g) at 4°C with resuspension in fresh buffer. Final resuspension was in Tris buffer containing 0.1% ascorbic acid and 1 mM MnC12 (pH 7.1 at 37°C). After incubation with 3H-ADTN in the presence or absence of 1 pM (+)-butaclamol and unlabeled drugs at 37°C for 10 min, triplicate samples were filtered rapidly under vacuum over Whatman GF/B filters, rinsed with 3 × 5 ml ice-cold Tris-HC1 buffer, and radioactivity trapped on the filters counted by liquid scintillation spectroscopy. In typical experiments with 8 nM 3HADTN total binding is about 600 cpm and nonspecific binding is about 300 cpm. Specific 3H-ADTN binding reaches equilibrium by 10 min at 37°C. Specific binding is saturable, Scatchard analysis showing two components
of binding with apparent dissociation constants (KD) of 6 nM and 45 nM and binding site number (Bronx) of 15 and 35 pmoles/g wet weight. Scatchard analysis reveals preponderant labeling of high affinity sites at the concentration of 3H-ADTN routinely employed (8 riM). Drug specificity of 3H-ADTN binding fulfills characteristics expected of dopamine receptors (table 1) and resembles the binding of the dopamine agonist 3H-apomorphine (Thal et al., 1978; Seeman et al., 1976). ADTN and apomorphine are the most potent dopamine agonists, about 10 times more potent than dopamine itself which in turn is 3 times more potent than norepinephrine. Serotonin is virtually inactive. Fluphenazine is the most potent phenothiazine neuroleptic with an affinity 3 times greater than that of chlorpromazine and 50 times greater than promazine. Butaclamol effects are stereospecific favoring the (+)-isomer by more than 1000 fold. The geometric isomers of flupenthixol also exhibit marked stereospecificity with a-flupenthixol being 60 fold more potent than the ~-isomer. The butyrophenone neuroleptics, spiroperidol and haloperidol, are relatively weak contrasting to their great pharmacological potency, resembling results obtained with other dopamine 3H-agonists (Burt et al., 1976). These properties differ markedly from low affinity binding of 3HADTN (Roberts et al., 1977), in which
460 TABLE 1 Properties of 3H-ADTN binding to rat striatal membranes. ICs0 's were determined by log-probit analysis of drug displacements of 8 nM 3H-ADTN. Non-specific binding was determined in the presence of 1 uM (+)butaclamol. Nucleotide concentrations were 50 tiM. Data presented as the mean of 2--6 independent replications. (A) Drug specificity IC~o (nM) Dopamine Apomorphine ADTN Norepinephrine Epinephrine Serotonin (+)-Butaclamol (--)-Butaclamol a-Flupenthixol t3-Flupenthixol Fluphenazine Chlorpromazine Promazine Spiroperidol Haloperidol Phentolamine Propranolol
300 30 25 900 1,200 > 10,000 6 > 10,000 24 1,600 13 40 500 90 40 2,000 > 10,000
(B) Guanyl nucleotide effects 3H-ADTN binding % control GTP GppNHp GDP GMP ATP ADP AMP
50 63 58 95 92 96 98
b o t h agonists and a n t a g o n i s t s were less t h a n o n e - t h o u s a n d t h as p o t e n t as o b s e r v e d in the p r e s e n t s t u d y . T h e u b i q u i t o u s regional dist r i b u t i o n r e p o r t e d for low a f f i n i t y 3H-ADTN binding also m i t i g a t e s against a selective labeling o f d o p a m i n e r e c e p t o r s . In the a b s e n c e of a d d e d ions o n l y a b o u t
10% o f t o t a l 3H-ADTN binding is displaceable b y 1 pM ( + ) - b u t a c l a m o l . Manganese signifi c a n t l y increases specific b i n d i n g at 1 pM and progressively increases specific binding to m a x i m a l e f f e c t s at 1 m M , t h e c o n c e n t r a t i o n used in r o u t i n e assays. Calcium and m a g n e s i u m are m u c h less effective, p r o d u c i n g no influence on specific binding at 1 pM and increasing specific binding at 1 m M b y o n l y h a l f as m u c h as m a n g a n e s e . S o d i u m ( 1 0 - 100 mM} decreases n o n s p e c i f i c b i n d i n g 1 0 - - 3 0 % with little change in specific binding. Guanyl nucleotides enhance effects of s o m e h o r m o n e s and n e u r o t r a n s m i t t e r s o n a d e n y l a t e cyclase b y facilitating r e c e p t o r cyclase c o u p l i n g a n d l o w e r i n g agonist affinity o f the r e c o g n i t i o n site. G T P , G p p ( N H ) p and G D P diminish d o p a m i n e r e c e p t o r binding of 3H-ADTN and 3 H - a p o m o r p h i n e ( d a t a n o t shown}. T h e specificity o f this e f f e c t is e v i d e n t in t h e failure o f GMP, ATP, ADP a n d AMP to alter agonist binding. G T P has no influence on t o t a l r e c e p t o r b i n d i n g o f the d o p a m i n e a n t a g o n i s t 3H-spiroperidol. H o w ever, the ability of agonists to inhibit 3Hs p i r o p e r i d o l binding is r e d u c e d in the prese n c e of 10 pM GTP. A c o m p o n e n t of 3HA D T N binding m a y thus be associated with the d o p a m i n e - s e n s i t i v e a d e n y l a t e cyclase.
Acknowledgements Tim Prosser, Ted Usdin and Kim Stewart are thanked for their assistance. Research was supported by grants to S.H.S. from USPHS MH-18501, The John A. Hartford Foundation, The McKnight Foundation, Sandoz Pharmaceuticals and to I.C. from Merck, Sharpe and Dohme, and an Institutional grant. Pam Morgan and Susan M. Garonski are thanked for manuscript preparation.
References Burt, D.R., I. Creese and S.H. Snyder, 1976, Properties of [3H]-haloperidol and [3H]-dopamine binding associated with dopamine receptors in calf brain membranes, Mol. Pharmacol. 12, 800.
461
Iversen, L.L., 1975, Dopamine receptors in the brain, Science 188, 1084. Roberts, P.J., G.N. Woodruff and J.A. Poat, 1977, Binding of a conformationaUy restricted dopamine analogue, 2-amino-6,7-dihydroxy-l,2,3,4tetrahydronaphthalene to receptors on rat brain synaptic membranes, Mol. Pharmacol. 13, 541. Seeman, P., T. Lee, M. Chau-Wong, J. Tedesco and
K. Wong, 1976, Dopamine receptors in human and calf brains using [3H]-apomorphine and an antipsychotic drug, Proc. Nat. Acad. Sci. U.S.A., 73, 4354. Thai, L., I. Creese and S.H. Snyder, 1978, 3H-Apomorphine interactions with dopamine receptors in calf brain, European J. Pharmacol. 49, 295.