3H-spiroperidol labels dopamine receptors in pituitary and brain

European Journal of Pharmacology, 46 (1977) 377--381

377

© Elsevier/North-Holland Biomedical Press

Short communication aH-SPIROPERIDOL LABELS DOPAMINE RECEPTORS IN PITUITARY AND BRAIN I.AN CREESE, ROBERT SCHNEIDER 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 5 October 1977, accepted 11 October 1977

I. CREESE, R. SCHNEIDER and S.H. SNYDER, 3H-Spiroperidol labels dopamine receptors in pituitary and brain, European J. Pharmacol. 46 (1977) 377--381. 3H-Spiroperidol of high specific radioactivity labels dopamine receptors in membranes of bovine caudate nucleus and anterior pituitary. The saturation and kinetic properties of 3H-spiroperidol binding are similar in the two tissues. In both caudate and pituitary 3H-spiroperidol displays very high affinity with a dissociation constant of 0.2--0.3 nM. The relative potencies of numerous dopamine agonists and antagonists in competing for 3Hspiroperidol binding are closely similar in anterior pituitary and caudate. 3H-Spiroperidol Dopamine receptor

Anterior pituitary

1. Introduction

Dopamine receptor sites in the brain have been labeled with agonists such as 3H-dopamine (Burt et al., 1976b; Seeman et al., 1975), and 3H-apomorphine (Seeman et al., 1976), the antagonist 3H-haloperidol (Burt et al., 1976b; Seeman et al., 1975) and the mixed agonist-antagonist 3H-LSD (Burt et ai., 1976a). A difficulty with the use of all of these 3H-ligands is that nonspecific binding is relatively high so that specific binding is difficult to detect in areas with low densities of dopamine receptors. Dopamine agonists and antagonists are thought to regulate the secretion of prolactin by actions on specific dopamine receptors in the anterior pituitary gland (Sachar, 1976; Meltzer et al., 1977). Preliminary reports have described attempts to label pituitary dopamine receptors with 3Hdihydroergokryptine (Caron et ai., 1977) and 3H-haloperidol (Brown et al., 1976). However the saturable binding of 3H-haloperidol in the pituitary is only 10% of total binding which makes detailed evaluation of its charac-

Neuroleptic

Caudate nucleus

teristics difficult. Experiments in our own laboratory indicate that only about 50% of SH-dihydroergokryptine binding to anterior pituitary membranes involves dopamine receptors, while the remainder involves a-noradrenergic receptor sites, being displaced with high affinity by a-agonist drugs such as oxymetazoline. Because of the therapeutic importance of drugs which influence pituitary function through actions at the dopamine receptor we sought to compare properties of pituitary and brain dopamine receptors in some detail as pituitary or brain specific dopaminergic drugs might have unique therapeutic value. 3H-Spiroperidol has been found to label dopamine receptors in vivo (Hollt et al., 1977; Laduron and Leysen, 1977). In the present study we describe properties of dopamine receptors in the brain and pituitary as labeled in vitro with 3H-spiroperidol. Spiroperidol is a butyrophenone neuroleptic whose affinity for dopamine receptors in brain tissue is greater than that of any other known drug (Burt et ai., 1976b) and has

378 recently become available labeled to a high specific activity.

I. C R E E S E

ET AL.

E.R. Squibb and Sons; promazine -- Wyeth; clonidine -- Boehringer Ingelheim; isoproterenol -- Sterling Winthrop.

2. Materials and methods 3. Results Calf caudate or steer anterior pituitary tissue frozen at --70°C for 1--25 days was homogenized in 100 vols (w/v) of ice cold 50 mM Tris-HC1 buffer, pH 7.7 at 25 °, with a Tekmar SDT homogenizer. The anterior pituitary homogenate was filtered through a Nitex 40 micron (nylon) mesh. The homogenates were centrifuged twice at 50,000 × g for 10 min (Sorvall RC-2-B) with rehomogenization of the intermediate pellet in fresh Tris buffer. The final pellet was homogenized in 150 volumes of cold 50 mM Tris buffer containing 0.1% ascorbic acid, 10 pM pargyline, and ions as follows: 120 mM NaC1, 5 m M KCI, l m M CaC12 and l m M MgC12, to give a final pH of 7.1 at 37°C. The homogenates were incubated at 37°C for 10 min and returned to ice. 3H-Spiroperidol, 26 Ci/mMol (Amersham and New England Nuclear) was diluted on the day of use with 0.1% ascorbic acid solution. Incubation tubes in triplicate received 100 gl of diluted 3H-spiroperidol, 100 pl of various concentrations of drugs diluted in 0.1% ascorbic acid, and 1.8 ml of tissue homogenate. Caudate samples were incubated for 20 min and anterior pituitary samples for 10 min at 37°C and rapidly filtered under vacuum through Whatman GF/B filters with three 5 ml rinses of ice cold 50 mM Tris buffer, pH 7.7, at 25°C. The filters were counted by liquid scintillation spectrometry in 9 ml of Formula 947 fluor (New England Nuclear) at efficiencies of 37--44%. The sources of drugs were as follows: dopamine, norepinephrine, epinephrine -- Sigma Chemical Co.; haloperidol -- McNeil Laboratories; apomorphine -- Merck and Co.; butaclamol and propranolol -- Ayerst Labs.; spiroperidol -- Janssen Pharmaceutica; serotonin -- Regis Chemical Co.; chlorpromazine - - Smith, Kline and French; fluphenazine --

Binding of 3H-spiroperidol to calf caudate membranes is saturable (fig. la). Binding plateaus at a b o u t 1 nM and reaches halfmaximal levels at about 0.3 nM. At a concentration of 0.15 nM 3H-spiroperidol which is used in routine experiments, non-specific binding measured in the presence of 1 pM (+)-butaclamol, is a b o u t 1/10th of total binding. At these concentrations a b o u t 30% of added 3H-spiroperidol is specifically bound to dopamine receptors in the membranes. Nonspecific binding increases linearly up to at least 2.5 nM. Scatchard analysis of the saturation data indicate the presence of only 1 c o m p o n e n t of specific 3H-spiroperidol binding (fig. lb). The dissociation constant (KD) is a b o u t 0.3 nM while the maximal number of binding sites (Bmax) is about 25 pmole/g weight of calf caudate tissue. This value is similar to the Br~ax value for the 3H-haloperidol binding to calf caudate dopamine receptors (Butt et al., 1976b). The characteristics of 3H-spiroperidol binding to steer anterior pituitary membranes are similar to interactions in calf caudate (fig. lc). Nonspecific binding, measured in the presence of 1 pM (+)-butaclamol, is linear up to a concentration of a b o u t 2 nM 3H-spiroperidol. Specific binding is saturable, plateaus at a b o u t 1 nM and attains half-maximal values at a b o u t 0.2 nM. At a concentration of 0.15 nM 3H-spiroperidol total binding is a b o u t 5 times the nonspecific level of binding. At this concentration of 3H-spiroperidol a b o u t 10% of added radioactivity is specifically b o u n d to apparent dopamine receptors. Scatchard analysis of these data (fig. l d ) indicate only a single population of binding sites with a dissociation constant of 0.2 nM and Bm~x of a b o u t 7 pmol/g wet weight.

BRAIN AND PITUITARY DOPAMINE RECEPTORS

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3H-SPtROPERIDOL BOUND( pmole/g wet weight) Fig. 1. Saturation of 3H-spiroperidol binding in caudate and anterior pituitary (a) increasing concentrations of 3H-spiroperidol were incubated with frozen calf caudate membranes in the presence (nonspecific binding),and absence (total binding) of 1 pM (+)-butaclamol. Specific binding was determined as total minus nonspecific binding. Data shown are the results of a single experiment in triplicate which was replicated twice. (b) Scatchard analysis of specific 3H-spiroperidol binding to calf caudate membranes. (c) 3H-Spiroperidol binding to frozen steer anterior pituitary membranes, method as in (a). (d) Scatchard analysis of specific 3H-spiroperidol binding to steer anterior pituitary membranes. CONC.3H-SPIROPERIDOL (nM)

3H-Spiroperidol binding in calf caudate membranes associates rapidly reaching equilibrium within 20 min at 37°C. The association rate constant (ki) is 0.4/nM/min, calculated from the initial slope of the association curve, the known concentration of 3Hspiroperidol and receptor density determined from Scatchard plots. Dissociation rate of 3H-spiroperidol from calf caudate membranes was measured by incubating membranes with 0.15 nM 3H-spiroperidol for 20 min after which 1 pM (+)-butaclamol was added and samples filtered at various time intervals. The dissociation of bound 3H-spiroperidol takes place according to simple first order kinetics with a half-life at 37°C of about 9 min corresponding to a rate constant for dissociation (k2) of 0.075/min. The dissociation constant

(KD) determined by the ratio of k2 to k, is 0.2 nM which agrees well with the K D value determined in saturation experiments. A s s o c i a t i o n , o f 3H-spiroperidol to steer anterior pituitary membranes reaches equilibrium by 10 min with a k, value of 0.8/nM/ min. ~The dissociation o f 3H-spiroperidol determined as in the: experiments with cau, date :membranes, has a half-life o f about 7 min corresponding to a k2 value of 0.1/min. The K D values of 0.1 nM, determined by the ratios of k2 to kt, also agrees well with the value obtained in equilibrium studies. We evaluated the influence of numerous agonists and antagonists of dopamine receptors on the binding of 3H-spiroperidol in caudate and pituitary membranes (table 1). Of the various neuroleptics examined spiro-

380

I. CREESE ET AL.

TABLE 1 Inhibition of 3H-spiroperidol binding to bovine caudate and anterior pituitary membranes. Frozen caudate or anterior pituitary was assayed with three or more concentrations of drug in triplicate. The concentrations of drug required to inhibit specific binding by 50% (ICs0) were determined from log probit plots and converted to Ki values according to the equation K i -- ICs0/(1 + C/KD) where c is the concentration of 3H-spiroperidol (0.15 nM) and K D its dissociation constant (0.2 nM in the pituitary and 0.3 nM in the caudate). Because butyrophenones and other drugs may adsorb to test tube surfaces lowering the free drug concentration, absolute values for K i may not be quantitatively accurate. Results are the mean + S.E.M. for the number of experiments indicated in the parentheses. Drugs which had significantly different Ki's between the two regions are indicated 1 p < 0.05, 2 p < 0.025, 3 p < 0.01 (Student's t-test). Drugs Spiroperidol Haloperidol Fluphenazine Chlorpromazine Promazine (+)-Butaclamol (--)-Butaclamol Apomorphine Dopamine 3 2-Bromo-~-ergokryptine (--)-Epinephrine 2 (--)-Norepinephrine 1 Clonidine (--)-Propranolol (--)-Isoproterenol Serotonin

Caudate, Ki (nM) 0.52 6.7 2.4 15 230

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peridol was the most potent, displaying about 10 times the affinity of haloperidol for the binding sites, while the phenothiazine, fluphenazine appears to be about twice as potent as haloperidol. Chlorpromazine is 2--3% as potent as spiroperidol in competing for binding sites while promazine, a relatively weak phenothiazine neuroleptic, is less than 0.2% as potent as spiroperidol. Receptor sites in both caudate and pituitary display stereospecificity towards the isomers of butKclamol with about 100,000 times greater potency of the pharmacologically active (+)-isomer. Apomorphine, a potent classical agonist, is more than 20 times as potent as dopamine itself. Other catecholamines such as epinephrine, norepinephrine and isoproterenol are much less potent than dopamine. Bromokryptine, which acts as a pharmacological agonist at dopamine receptors in both brain and

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pituitary, displays a Ki value of about 3 nM in both pituitary and caudate. The inactivity of isoproterenol and propranolol indicates that spiroperidol binding does not involve ~-receptors, and the failure to detect any influence of the potent ~-noradrenergic agonist clonidine indicates that 3H-spiroperidol does not label s-receptors. The extremely low potency of serotonin indicates that its receptors are not labeled by 3H-spiroperidol. The absolute potencies of drugs are similar in pituitary and caudate except for dopamine, epinephrine and norepinephrine, which are 2.4--6 times more potent in caudate than pituitary. In experiments in which various parts of the pituitary have been compared, specific 3H-spiroperidol binding is demonstrable only in the anterior pituitary with negligible specific binding in the posterior or intermediate lobes.

B R A I N A N D PITUITARY D O P A M I N E R E C E P T O R S

381

4. Discussion The d r u g affinities for 3H-spiroperidol binding sites to caudate and pituitary indicate that 3H-spiroperidol is labeling dopamine receptors in both regions. In caudate membranes more than 90% of 3H-spiroperidol binding is specific compared to only 40--50% of 3H-haloperidol binding (Burr et al., 1976b). Moreover its dissociation rate is one tenth that of 3H-haloperidol, which decreases variability in filtering. Thus 3H-spiroperidol has proved to be the most suitable labeled ligand yet to be investigated for the study of dopamine receptors in vitro. Its utility has been reported for labeling dopamine receptors in vivo (Hollt et al., 1977; Laduron and Leysen, 1977). The 3H-spiroperidol binding sites in caudate and anterior pituitary have a similar affinity for 3H-spiroperidol although there are about one third as many binding sites in the pituitary. Neuroleptics display similar potencies in eliciting effects mediated by the pituitary, such as prolactin release, and behavioral effects, in animals and man (Meltzer et al., 1977; Sachar, 1976). Binding studies indicate that neuroleptic affinities for dopamine receptors in both regions are indeed identical and similar to those found previously with 3Hhaloperidol binding in calf caudate (Butt et al., 1976b). However the catecholamine agonists are about one third weaker in the pituitary. The significance of this result is unclear. Bromokryptine, which is a pharmacological agonist, displays very high affinity for 3H-spiroperidol binding sit~s in both regions.

Acknowledgements Susan M. Garonski is thanked for manuscript preparation. This research was supported by USPHS grant MH-18501 to S.H.S.

References Brown, G.M., P. Seeman and T. Lee, 1976, Dopamine/neuroleptic receptors in basal hypothalamus and pituitary, Endocrinology 99, 1407. Burt, D.R., I. Creese and S.H. Snyder, 1976a, Binding interactions of lysergic acid diethylamide and related agents with dopamine receptors in the brain, Mol. Pharmacol. 12,631. Burt, D.R., I. Creese and S.H. Snyder, 1976b, Properties of [3H]-haloperidol and [ZH]dopamine binding associated with dopamine receptors in calf brain membranes, Mol. Pharmacol. 12, 800. Caron, M.G., V. Raymond, R.J. Lefkowitz and F. Labrie, 1977, Identification of dopaminergic receptors in anterior pituitary: correlation with the dopaminergic control of prolactin release, Federation Proc. 36, 278. Hollt, V., A. Calankowski and A. Herz, 1977, The demonstration in vivo of specific binding sites for neuroleptic drugs in mouse brain, Brain Res. 130, 176. Laduron, P. and J. Leysen, 1977, Specific in vivo binding of neuroleptic drugs in rat brain, Biochem. Pharmacol. 26, 1003. Meltzer, H.Y., S.M. Paul and K.S. Gang, 1977, Effect of flupenthixol and butaclamol isomers on prolactin secretion in rats, Psychopharmacology 51, 181. Sachar, E., 1976, The use of neuroendocrine techniques in psychopharmacological research, in: Hormones, Behavior and Psychopathology, ed. E. Sachar (Raven Press, New York) p. 161. Seeman, P., M. Chau-Wong, J. Tedesco and K. Wong, 1975, Brain receptors for antispychotic drugs and dopamine: direct binding assays, Proc. Nat. Acad. Sci. U.S.A. 72, 4376. 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.