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The low affinity component of [3H]spiperone binding reflects association to a non-dopaminergic, neuroleptic site
Neuroscience Letters, 29 (1982) 147-151
147
Elsevier/North-HollandScientificPublishers Ltd.
T H E L O W AFFINITY C O M P O N E N T OF [3H]SPIPERONE BINDING REFLECTS ASSOCIATION T O A N O N - D O P A M I N E R G I C , N E U R O L E P T I C SITE
P.M. BEART, M. KRSTICH, D. McDONALDand A.L. GUNDLACH University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Vic. 3084 (Australia)
(Received December24th, 1981; Revised version receivedand accepted January 22nd, 1982) The pharmacologicalproperties of a low affinity site labelled by [3H]spiperonein membranesprepared from the rat nucleus accumbens were examined by investigating the ability of dopaminergic agonists, antagonists and various other drugs to displace the binding of 2 nM [3H]spiperone.Neurolepticdrugs were the most potent displacers of binding, and dopaminergic agonists, except for bromocryptine and lergotrile, had inhibition constants > 1/~M. The majority of drugs studied exhibiteda high selectivityfor the high affinity site labelled by [3H]spiperoneand low affinity sites probably represent a non-specific, non-dopaminergic site for neuroleptic drugs. Although [3H]spiperone has been suggested as the ligand of choice for studying neuroleptic receptors [9], one major problem in accepting [3H]spiperone binding as representing an association to dopamine receptors has always been the very low potency o f dopamine and dopamine agonists in inhibiting the binding [4, 15, 16]. Most o f the early equilibrium studies with [3H]spiperone gave binding data which yielded linear Scatchard plots, and which were interpreted as involving a single population of binding sites [4, 15, 16]. However, recent radioligand binding studies have reported multiple binding sites for [3H]spiperone [1, 2, 6, 9, 11-13, 17], and brain regions which receive a dense dopaminergic innervation appear to contain two binding sites, one of high (apparent K o < 0.1 nM) and a second o f lower affinity (KD > lnM) [2, 5, 6]. The high affinity site, which is more likely to have an apparent KD in the range 20-70 pM, has pharmacological properties consistent with a D-2 receptor in that dopamine agonists, ergots and antagonists (neuroleptic drugs) are all potent displacers of [3H]spiperone binding [2, 6, 7]. By contrast, the nature and importance o f the low affinity site has not been established, and the purpose of the present study was to delineate the pharmacological properties of this [3H]spiperone binding site. Rat nucleus accumbens septi (NAS) were dissected [2] and homogenized in 50 vol. o f ice-cold 50 mM Tris-HCl, p H 7.7. The homogenate was centrifuged at 45,000 × g for l 0 min and the resultant pellet was washed extensively before resuspension in icecold 50 mM Tris-HC1, pH 7.1, containing 120 mM NaCl, 5 mM KCI, 2 mM CaC12, 1 0304-3940/82/0000-0000/$ 02.75 © Elsevier/North-HollandScientificPublishers Ltd.
148 mM MgCI2, 10/zM pargyline and 0.1°70 EDTA. Homogenate (950/~1, equivalent to 0.8 mg wet wt. tissue), [3H]spiperone (50/zl, final concentration 2 riM; 25 Ci/mmol) and various displacing agents (10 td) were incubated for 20 min at 37°C. Nonspecific binding was determined in identical tubes containing l /zM unlabelled spiperone. The filtration, washing and counting of samples were as previously described [2]. Drugs were obtained as follows: N-n-propylnorapomorphine (Prof. J.L. Neumeyer, Boston); apomorphine (Macfarlan Smith); bromocryptine (Sandoz); (+)- and (-)-butaclamol (Ayerst); cinanserin, fluphenazine (Squibb); domperidone (Jannsen); dopamine, serotonin (Sigma); epinine (Aldrich); c/s- and trans-flupenthixol (Lundbeck); haloperidol (Searle); lergotrile, pergolide (Eli Lilly); spiperone (Ethnor); sulpiride (Delagrange). Specific [3H]spiperone binding to the crude membrane preparation of nucleus accumbens was rapid and saturable. Specific binding represented 45 +_ 3°7o (n -- 14) of total binding at 2 nM [3H]spiperone. Scatchard analysis of binding data from experiments employing spiperone concentrations in the nanomolor range has previously indicated a population of binding sites with an apparent KD of 4.1 nM and an apparent Bmax of 25 pmol/g wet wt. [2]. Thus at a concentration of [3H]spiperone of 2 nM, and taking into consideration the parameters for the low and the high affinity sites (KD 74 pM and Bmax 4 pmol/g wet wt. [2]), at least 67°70 of the binding will be to the low affinity site. All neuroleptic drugs, dopamine agonists and various miscellaneous drugs were capable of displacing [3H]spiperone binding. The concentration of drug displacing 50°7o of specific spiperone binding (ICs0) was determined by computer-assisted iterative curve fitting and the inhibition constant (Ki) w a s obtained using the Cheng-Prusoff equation [3]. The inhibition constants for the displacement of binding at 2 nM [aH]spiperone are given in Table I together with those determined at 25 pM [3H]spiperone, where > 85°7o of the binding is to the high affinity site [2]. Also shown is the ratio of the inhibition constants for each drug and a high ratio is indicative of greater selectivity for the high affinity binding site. The drug displacement data indicate that this site has pharmacological properties consistent with a D-2 receptor [2, 7], particularly since dopaminergic agonists, ergots and neuroleptic drugs are all potent displacers of the binding. Spiperone (K i 7 nM) was the most effective drug at displacing the binding of 2 nM [3H]spiperone, while other neuroleptic and antipsychotic drugs had K i values in the range 20-850 nM. [3H]Spiperone binding to the low affinity site was stereospecific as demonstrated by the results obtained with the racemic pairs of butaclamol and flupenthixol: for both neuroleptic drugs the active isomer was approximately 350 times more potent at displacing [3H]spiperone binding than the inactive isomer. All neuroleptic drugs were much weaker at displacing binding to the low affinity site than to the high affinity site. The butyrophenones (spiperone, haloperidol and domperidone) and pimozide were the drugs most selective for the high affinity site, but they were also among the drugs which were most potent at inhibiting
149 TABLE I DISPLACEMENT OF [3H]SPIPERONE BINDING FROM MEMBRANES PREPARED FROM RAT NUCLEUS ACCUMBENS BY DRUGS Inhibition constants (Ki) were determined from ICs0 values for the inhibition of binding using the Cheng-Prusoff [3] equation IC50 = Ki (1 + D/KD). Values are from 1-3 experiments employing 8 concentrations of drug in triplicate. The concentration of [3H]spiperone employed for drug displacement s~udies at the low affinity site was 2 nM, while that for high affinity studies was 25 pM. N-n-PA = N-npropylnorapomorphine; ADTN = 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene. site (nM)
Ki high affinity site a (nM)
Ki low affinity Ki high affinity
Spiperone Fluphenazine Pimozide Haloperidol ( + )-Butaclamol cis-Flupenthixol Domperidone ( ± )-Sulpiride Metoclopramide trans-Flupenthix ol ( - )-Butaclamol
7 21 49 74 76 120 300 390 850 4,000 27,000
0.05 1 0.4 1 4 17 2 100 230 600 12,000
140 21 123 74 19 7 148 4 4 7 2
Bromocryptine Lergotrile Apomorphine Pergolide Epinine N-n-PA ADTN Dopamine
260 540 l, 100 1,600 3,400 3,800 6,900 18,000
6 53 35 13 3,700 17 3,000 620
Serotonin Cinanserin
8,600 12,000
27,000 2,000
Drug
Ki low affinity
43 10 30 127 1 224 2 29 0.3 6
aFrom ref. 2.
[3H]spiperone binding to the low affinity site. Representatives from the phenothiazine, thioxanthene and dibenzcycloheptyl classes of neuroleptic drugs (fluphenazine, c/s-flupenthixol and (+)-butaclamol respectively) were also reasonably potent as displacers of the binding to the low affinity binding site for [3H]spiperone. Bromocryptine and lergotrile, two ergots which have high affinity for D-2 receptors [7, 8, 10], were the agonists most effective at displacing binding of 2 nM [3H]spiperone. The K i values for all other dopamine agonists were > 1 ~M. Epinine and ADTN showed virtually no selectivity for either binding site, while pergolide and N-n-propylnorapomorphine were selective and potent displacers at the high
150 affinity site. In general, d o p a m i n e agonists were weak displacers o f binding at the low affinity site, especially when c o m p a r e d with their affinity for the high affinity site. This low affinity o f the d o p a m i n e agonists for the binding site labelled by 2 nM [3H]spiperone makes it very unlikely that the binding represents association to a dopaminergic site. In further studies noradrenaline, phentolamine, prazosin, propranolol, histamine, mepyramine, and cimetidine were all very weak displacers o f the binding o f 2 nM [3H]spiperone, with K i values > 10/~M. Thus, the binding site labelled by n a n o m o l a r concentrations o f [3H]spiperone is not likely to involve c~-and t3-adrenoreceptors, histamine or serotonin receptors (Table I). Whereas the high affinity site labelled by low (picomolar) concentrations o f [3H]spiperone is likely to represent a D-2 receptor, the low affinity site by virtue o f the low potency o f d o p a m i n e agonists must be non-dopaminergic. Neuroleptic drugs also have relatively lower affinity for this site than for the D-2 receptor, although they were the m o s t effective drugs at displacing [3H]spiperone binding. Such low affinity sites exist in h o m o g e n a t e s prepared f r o m m a n y regions o f the central nervous system and have been suggested to be spirodecanone (butyrophenone) sites [5]. The present results indicate that other classes o f neuroleptic drugs also have appreciable affinity for these sites. In view o f the general ability o f neuroleptic drugs to concentrate in cell m e m b r a n e s [14], the low affinity site labelled by [3H]spiperone is m o r e likely to be a non-specific, nondopaminergic site for neuroleptic drugs in general. Supported by the National Health and Medical Research Council o f Australia. The authors acknowledge the assistance o f Dr. G. M c P h e r s o n . 1 Andorn, A.C. and Maguire, M.E., [3H]Spiroperidol binding in rat striatum: two high affinity sites of differing selectivities, J. Neurochem., 35 (1980) 1105-1113. 2 Beart, P.M. and McDonald, D., Neurochemical studies of the mesolimbic dopaminergic pathway. [3H]Spiperone labels two binding sites in homogenates of the nucleus accumbens of rat brain, J. Neurochem., in press. 3 Cheng, Y. and Prusoff, W.H., Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I5o) of an enzymatic reaction, Biochem. Pharmacol., 22 (1973) 3099-3108. 4 Creese, I., Sibley, D.R., Leff, S. and Hamblin, M., Dopamine receptors: subtypes, localization and regulation, Fed. Proc., 40 (1981) 1 4 7 1 5 2 . . ~i. 5 Howlett, D.R., Morris, H. and Nahorski, S.R., Anomalous properties of [3H]spipero~ bii~ling sites in various areas of the rat limbic system, Molec. Pharmacol., 15 (1979) 506-514. :.~. ~ i ~, 6 Howlett, D.R. and Nahorski, S.R., Quantitative assessment of heterogeneous [3H]spilberonebifiding to rat neostriatum and frontal cortex, Life Sci. 26 (1980) 511-517. ~ i; 7 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (Lond;), 277 (1979) 93-96. 8 Lew, J.Y., Nakamura, S., Battista, A.R. and Goldstein, M., Dopamine agonist potencies of ergolines, Commun. Psychopharmacol., 3 (1979) 179-183. 9 Leysen, J.E., Gommeren, W. and Laduron, P.M., Spiperone: a iigand of choice for neuroleptic receptors. I. Kinetics and characteristics of in vitro binding, Bioehem. Pharmacol., 27 (1978) 307-316. •
151
10 Marek, K.L. and Roth R.H., Ergot alkaloids: interaction with presynaptic dopamine receptors in the ncostriatum and olfactory tubercles, Europ. J. Pharmacol., 62 (1980) 137-146. 11 Near, J.A. and Mahler, H.R., Dopamine receptors in subcellular fractions from bovine caudate: enrichment of [3H]spipcrone binding in a postsynaptic fraction, J. Neurochem., 36 (1981) 1142-1151. 12 Pedigo, N.W., Reisine, T.D., Fields, J.Z. and Yamamura, H.I., [3H]Spiroperidol binding to two receptor sites in both the corpus striatum and frontal cortex of rat brain, Europ. J. Pharmacol., 50 (1978) 451-453. 13 Quik, M., Emson, P.C. and Joyce, E., Dissociation between the presynaptic dopamine sensitive adenylate cyclasc and [3H]spiperone binding in rat substantia nigra, Brain Res., 167 (1979) 355-365. 14 Seeman, P., Anti-schizophrenic drugs - membrane receptor site of action, Biochem. Pharmacol., 26 (1977) 1741-1748. 15 Seeman, P., Brain dopamine receptors, Pharmacol. Rev., 32 (1980) 229-313. 16 Seeman, P., Tedesco, J.L., Lee, T., Chau-Wong, M., Muller, P., Bowles, J., Whitaker, P.M., McManus, C., Titler, M., Wcinrcich, P., Friend, W.C. and Brown, G.M., Dopamine receptors in the central nervous system, Fed. Proc. 37 (1978) 130-136. 17 Sundcrmann, R.H. and Wootcn, G.F., Biochemical properties of spipcrone binding to rat brain membranes, Pharmacology, 21 (1980) 295-305.
147
Elsevier/North-HollandScientificPublishers Ltd.
T H E L O W AFFINITY C O M P O N E N T OF [3H]SPIPERONE BINDING REFLECTS ASSOCIATION T O A N O N - D O P A M I N E R G I C , N E U R O L E P T I C SITE
P.M. BEART, M. KRSTICH, D. McDONALDand A.L. GUNDLACH University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Vic. 3084 (Australia)
(Received December24th, 1981; Revised version receivedand accepted January 22nd, 1982) The pharmacologicalproperties of a low affinity site labelled by [3H]spiperonein membranesprepared from the rat nucleus accumbens were examined by investigating the ability of dopaminergic agonists, antagonists and various other drugs to displace the binding of 2 nM [3H]spiperone.Neurolepticdrugs were the most potent displacers of binding, and dopaminergic agonists, except for bromocryptine and lergotrile, had inhibition constants > 1/~M. The majority of drugs studied exhibiteda high selectivityfor the high affinity site labelled by [3H]spiperoneand low affinity sites probably represent a non-specific, non-dopaminergic site for neuroleptic drugs. Although [3H]spiperone has been suggested as the ligand of choice for studying neuroleptic receptors [9], one major problem in accepting [3H]spiperone binding as representing an association to dopamine receptors has always been the very low potency o f dopamine and dopamine agonists in inhibiting the binding [4, 15, 16]. Most o f the early equilibrium studies with [3H]spiperone gave binding data which yielded linear Scatchard plots, and which were interpreted as involving a single population of binding sites [4, 15, 16]. However, recent radioligand binding studies have reported multiple binding sites for [3H]spiperone [1, 2, 6, 9, 11-13, 17], and brain regions which receive a dense dopaminergic innervation appear to contain two binding sites, one of high (apparent K o < 0.1 nM) and a second o f lower affinity (KD > lnM) [2, 5, 6]. The high affinity site, which is more likely to have an apparent KD in the range 20-70 pM, has pharmacological properties consistent with a D-2 receptor in that dopamine agonists, ergots and antagonists (neuroleptic drugs) are all potent displacers of [3H]spiperone binding [2, 6, 7]. By contrast, the nature and importance o f the low affinity site has not been established, and the purpose of the present study was to delineate the pharmacological properties of this [3H]spiperone binding site. Rat nucleus accumbens septi (NAS) were dissected [2] and homogenized in 50 vol. o f ice-cold 50 mM Tris-HCl, p H 7.7. The homogenate was centrifuged at 45,000 × g for l 0 min and the resultant pellet was washed extensively before resuspension in icecold 50 mM Tris-HC1, pH 7.1, containing 120 mM NaCl, 5 mM KCI, 2 mM CaC12, 1 0304-3940/82/0000-0000/$ 02.75 © Elsevier/North-HollandScientificPublishers Ltd.
148 mM MgCI2, 10/zM pargyline and 0.1°70 EDTA. Homogenate (950/~1, equivalent to 0.8 mg wet wt. tissue), [3H]spiperone (50/zl, final concentration 2 riM; 25 Ci/mmol) and various displacing agents (10 td) were incubated for 20 min at 37°C. Nonspecific binding was determined in identical tubes containing l /zM unlabelled spiperone. The filtration, washing and counting of samples were as previously described [2]. Drugs were obtained as follows: N-n-propylnorapomorphine (Prof. J.L. Neumeyer, Boston); apomorphine (Macfarlan Smith); bromocryptine (Sandoz); (+)- and (-)-butaclamol (Ayerst); cinanserin, fluphenazine (Squibb); domperidone (Jannsen); dopamine, serotonin (Sigma); epinine (Aldrich); c/s- and trans-flupenthixol (Lundbeck); haloperidol (Searle); lergotrile, pergolide (Eli Lilly); spiperone (Ethnor); sulpiride (Delagrange). Specific [3H]spiperone binding to the crude membrane preparation of nucleus accumbens was rapid and saturable. Specific binding represented 45 +_ 3°7o (n -- 14) of total binding at 2 nM [3H]spiperone. Scatchard analysis of binding data from experiments employing spiperone concentrations in the nanomolor range has previously indicated a population of binding sites with an apparent KD of 4.1 nM and an apparent Bmax of 25 pmol/g wet wt. [2]. Thus at a concentration of [3H]spiperone of 2 nM, and taking into consideration the parameters for the low and the high affinity sites (KD 74 pM and Bmax 4 pmol/g wet wt. [2]), at least 67°70 of the binding will be to the low affinity site. All neuroleptic drugs, dopamine agonists and various miscellaneous drugs were capable of displacing [3H]spiperone binding. The concentration of drug displacing 50°7o of specific spiperone binding (ICs0) was determined by computer-assisted iterative curve fitting and the inhibition constant (Ki) w a s obtained using the Cheng-Prusoff equation [3]. The inhibition constants for the displacement of binding at 2 nM [aH]spiperone are given in Table I together with those determined at 25 pM [3H]spiperone, where > 85°7o of the binding is to the high affinity site [2]. Also shown is the ratio of the inhibition constants for each drug and a high ratio is indicative of greater selectivity for the high affinity binding site. The drug displacement data indicate that this site has pharmacological properties consistent with a D-2 receptor [2, 7], particularly since dopaminergic agonists, ergots and neuroleptic drugs are all potent displacers of the binding. Spiperone (K i 7 nM) was the most effective drug at displacing the binding of 2 nM [3H]spiperone, while other neuroleptic and antipsychotic drugs had K i values in the range 20-850 nM. [3H]Spiperone binding to the low affinity site was stereospecific as demonstrated by the results obtained with the racemic pairs of butaclamol and flupenthixol: for both neuroleptic drugs the active isomer was approximately 350 times more potent at displacing [3H]spiperone binding than the inactive isomer. All neuroleptic drugs were much weaker at displacing binding to the low affinity site than to the high affinity site. The butyrophenones (spiperone, haloperidol and domperidone) and pimozide were the drugs most selective for the high affinity site, but they were also among the drugs which were most potent at inhibiting
149 TABLE I DISPLACEMENT OF [3H]SPIPERONE BINDING FROM MEMBRANES PREPARED FROM RAT NUCLEUS ACCUMBENS BY DRUGS Inhibition constants (Ki) were determined from ICs0 values for the inhibition of binding using the Cheng-Prusoff [3] equation IC50 = Ki (1 + D/KD). Values are from 1-3 experiments employing 8 concentrations of drug in triplicate. The concentration of [3H]spiperone employed for drug displacement s~udies at the low affinity site was 2 nM, while that for high affinity studies was 25 pM. N-n-PA = N-npropylnorapomorphine; ADTN = 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene. site (nM)
Ki high affinity site a (nM)
Ki low affinity Ki high affinity
Spiperone Fluphenazine Pimozide Haloperidol ( + )-Butaclamol cis-Flupenthixol Domperidone ( ± )-Sulpiride Metoclopramide trans-Flupenthix ol ( - )-Butaclamol
7 21 49 74 76 120 300 390 850 4,000 27,000
0.05 1 0.4 1 4 17 2 100 230 600 12,000
140 21 123 74 19 7 148 4 4 7 2
Bromocryptine Lergotrile Apomorphine Pergolide Epinine N-n-PA ADTN Dopamine
260 540 l, 100 1,600 3,400 3,800 6,900 18,000
6 53 35 13 3,700 17 3,000 620
Serotonin Cinanserin
8,600 12,000
27,000 2,000
Drug
Ki low affinity
43 10 30 127 1 224 2 29 0.3 6
aFrom ref. 2.
[3H]spiperone binding to the low affinity site. Representatives from the phenothiazine, thioxanthene and dibenzcycloheptyl classes of neuroleptic drugs (fluphenazine, c/s-flupenthixol and (+)-butaclamol respectively) were also reasonably potent as displacers of the binding to the low affinity binding site for [3H]spiperone. Bromocryptine and lergotrile, two ergots which have high affinity for D-2 receptors [7, 8, 10], were the agonists most effective at displacing binding of 2 nM [3H]spiperone. The K i values for all other dopamine agonists were > 1 ~M. Epinine and ADTN showed virtually no selectivity for either binding site, while pergolide and N-n-propylnorapomorphine were selective and potent displacers at the high
150 affinity site. In general, d o p a m i n e agonists were weak displacers o f binding at the low affinity site, especially when c o m p a r e d with their affinity for the high affinity site. This low affinity o f the d o p a m i n e agonists for the binding site labelled by 2 nM [3H]spiperone makes it very unlikely that the binding represents association to a dopaminergic site. In further studies noradrenaline, phentolamine, prazosin, propranolol, histamine, mepyramine, and cimetidine were all very weak displacers o f the binding o f 2 nM [3H]spiperone, with K i values > 10/~M. Thus, the binding site labelled by n a n o m o l a r concentrations o f [3H]spiperone is not likely to involve c~-and t3-adrenoreceptors, histamine or serotonin receptors (Table I). Whereas the high affinity site labelled by low (picomolar) concentrations o f [3H]spiperone is likely to represent a D-2 receptor, the low affinity site by virtue o f the low potency o f d o p a m i n e agonists must be non-dopaminergic. Neuroleptic drugs also have relatively lower affinity for this site than for the D-2 receptor, although they were the m o s t effective drugs at displacing [3H]spiperone binding. Such low affinity sites exist in h o m o g e n a t e s prepared f r o m m a n y regions o f the central nervous system and have been suggested to be spirodecanone (butyrophenone) sites [5]. The present results indicate that other classes o f neuroleptic drugs also have appreciable affinity for these sites. In view o f the general ability o f neuroleptic drugs to concentrate in cell m e m b r a n e s [14], the low affinity site labelled by [3H]spiperone is m o r e likely to be a non-specific, nondopaminergic site for neuroleptic drugs in general. Supported by the National Health and Medical Research Council o f Australia. The authors acknowledge the assistance o f Dr. G. M c P h e r s o n . 1 Andorn, A.C. and Maguire, M.E., [3H]Spiroperidol binding in rat striatum: two high affinity sites of differing selectivities, J. Neurochem., 35 (1980) 1105-1113. 2 Beart, P.M. and McDonald, D., Neurochemical studies of the mesolimbic dopaminergic pathway. [3H]Spiperone labels two binding sites in homogenates of the nucleus accumbens of rat brain, J. Neurochem., in press. 3 Cheng, Y. and Prusoff, W.H., Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I5o) of an enzymatic reaction, Biochem. Pharmacol., 22 (1973) 3099-3108. 4 Creese, I., Sibley, D.R., Leff, S. and Hamblin, M., Dopamine receptors: subtypes, localization and regulation, Fed. Proc., 40 (1981) 1 4 7 1 5 2 . . ~i. 5 Howlett, D.R., Morris, H. and Nahorski, S.R., Anomalous properties of [3H]spipero~ bii~ling sites in various areas of the rat limbic system, Molec. Pharmacol., 15 (1979) 506-514. :.~. ~ i ~, 6 Howlett, D.R. and Nahorski, S.R., Quantitative assessment of heterogeneous [3H]spilberonebifiding to rat neostriatum and frontal cortex, Life Sci. 26 (1980) 511-517. ~ i; 7 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (Lond;), 277 (1979) 93-96. 8 Lew, J.Y., Nakamura, S., Battista, A.R. and Goldstein, M., Dopamine agonist potencies of ergolines, Commun. Psychopharmacol., 3 (1979) 179-183. 9 Leysen, J.E., Gommeren, W. and Laduron, P.M., Spiperone: a iigand of choice for neuroleptic receptors. I. Kinetics and characteristics of in vitro binding, Bioehem. Pharmacol., 27 (1978) 307-316. •
151
10 Marek, K.L. and Roth R.H., Ergot alkaloids: interaction with presynaptic dopamine receptors in the ncostriatum and olfactory tubercles, Europ. J. Pharmacol., 62 (1980) 137-146. 11 Near, J.A. and Mahler, H.R., Dopamine receptors in subcellular fractions from bovine caudate: enrichment of [3H]spipcrone binding in a postsynaptic fraction, J. Neurochem., 36 (1981) 1142-1151. 12 Pedigo, N.W., Reisine, T.D., Fields, J.Z. and Yamamura, H.I., [3H]Spiroperidol binding to two receptor sites in both the corpus striatum and frontal cortex of rat brain, Europ. J. Pharmacol., 50 (1978) 451-453. 13 Quik, M., Emson, P.C. and Joyce, E., Dissociation between the presynaptic dopamine sensitive adenylate cyclasc and [3H]spiperone binding in rat substantia nigra, Brain Res., 167 (1979) 355-365. 14 Seeman, P., Anti-schizophrenic drugs - membrane receptor site of action, Biochem. Pharmacol., 26 (1977) 1741-1748. 15 Seeman, P., Brain dopamine receptors, Pharmacol. Rev., 32 (1980) 229-313. 16 Seeman, P., Tedesco, J.L., Lee, T., Chau-Wong, M., Muller, P., Bowles, J., Whitaker, P.M., McManus, C., Titler, M., Wcinrcich, P., Friend, W.C. and Brown, G.M., Dopamine receptors in the central nervous system, Fed. Proc. 37 (1978) 130-136. 17 Sundcrmann, R.H. and Wootcn, G.F., Biochemical properties of spipcrone binding to rat brain membranes, Pharmacology, 21 (1980) 295-305.