Prog. Nemo-PsychopharmacoZ. 1981, Vo1.5, pp.543-54s Printed in Great Britain. All rights reserved.

0364-7722/81/060543-06.$Q3.oo/o Copyright @1981Pergamon Press Ltd.

SOLUIDLIZED DOPAMINE/NEUROLEPTIC RECEPTORS (D2-I’YPE)

BERTHA K. MADRAS, ALAN DAVIS, BETTY CHAN and PHILIP SEEMAN

Psychopharmacology Section, Clarke Institute of Psychiatry and Department of Pharmacolgy, University of Toronto, Toronto, Canada

(Final form, April 1981)

Abstract

1. Dopamine (D2-type) receptors were solubilized from striatum of three species (human, canine, calf) using digitonin.

2. The receptors were labeled with 3 H-spiperone and assayed by Sephadex G-50 columns or polyethylene glycol precipitation.

3. The soluble receptors from canine and human tissue had similar Kd’s and rank order of drug affinities to the membrane-bound sites.

4. Soluble calf recepLors showed reduced affinity for spiperone and chlorpromazine (12-fold). Non-specific binding also increased.

5. Solubilized canine binding was insensitive to ascorbate, Mn++, and ethylenediamine tetracetic acid (EDTA) in contrast to the membrane binding sites.

6. Solubilized canine striatum serves as an excellent source of D2 receptors because these receptors are stable in solution and are a prototype of human D2 receptors.

Key words: soluble receptors, dopamine, neuroleptics

Abbreviation: ethylenediamine tetracetic acid (EDTA), Tris-EDTA-ascorbate-nialamide (TEAN)

Introduction

Neuroleptic drugs bind to dopamine receptors (D2) in an order of potencies that parallels their clinical doses for schizophrenia (Seeman et al. 1975a,1976b). Purification of this receptor and subsequent antibody formation may have clinical applications for diagnosis, treatment, and clarification of the pathological mechanisms of the disease.

The success of receptor solubilization, a preliminary to purification, appears to depend on the type of tissue used. Since considerable amounts of tissue are required for ultimate purification and analysis, it is essential to determine which species (human, bovine, canine) would provide the highest yield of soluble and specific dopamine receptors of the D2

type*

Although our earlier work indicated that D2-type receptors could be successfully solubilized from human and dog striata (Gorissen et al. 1979; Madras et al. 1980a,b; Davis et al. 1980, 1981), it remained necessary to compare the yields and properties of the sites with those of calf caudate. This is because a plentiful supply of calf tissue is generally available and because much previous work has described the characteristics of the calf brain D2 site. The solubilized canine caudate but not calf caudate, provides an excellent source of D2 receptors because of its similarity to soluble human D2 receptors and its stability in solution.

Met hods

Brain tissues. Dogs were anaesthesized with pentobarbital and the striata were removed within two or three hours of death. Fresh calf brains were obtained from a local slaughter house. The brains were harvested within two hours and striatal membranes prepared

544 B.K. Madras et al.

immediately or after freezing overnight. Dr. John Deck (Toronto Western Hospital) provided human brains.

Methods of tissue preparation were essentially those described by Madras et al. (1980a,b). The brain regions were homogenized in sucrose (0.25 Ml, centrifuged at 1100 x g for 10 min, resuspended in sucrose and centrifuged at 105,000 x g for 60 min. The pellet was resuspended in TEAN buffer (tris-EDTA-ascorbate-nialamide) .

The P?-P2 membrane fraction was solubilized with dieitonin (1%) usinn the procedure of Caron e; ai. (1976; see also Tam and Seeman, 1978) with minor modifications.’ microscopy of the solubilized material (negatively stained by phosphotungstic 1974) confirmed the complete absence of membrane-like fragments.

Binding methods. Binding of 3 H-spiperone to membrane fractions was done by tions of standard methods (Hartley and Seeman, 19781

Electron acid, Seeman

minor modifica-

The specific binding of 3H-spiperone to the digitonin-solubilized material was assayed using Sephadex G-50 columns (Caron and Lefkowitz, 1976; Tam and Seeman, 1978; Madras et al., 1980a,b; Davis et al., 1980, 19811. In addition, a polyethylene glycol precipitation method

was developed in our laboratory as a rapid screening assay (Ghan et al., in press).

Results

E50 values. A comparison of the solubilized D2 receptors from human, canine, and calf striata is given in Table 1. The IC50 values for various drugs on the solubilized D2 receptors correlate closely with values derived from the membrane preparations of human and canine striatum. The IC50 values for calf, however, are distinctly different for spiperone, chlorpromazine, and norepinephrine. Norepinephrine and dopamine are equipotent on the calf soluble receptor.

Kd values and receptor recovery. The Kd values for both canine and human striatum were - changed minimally whereas the Kd for calf increased at least lo-fold (Table 1B). In addition, the percent of total binding that is specific (as defined by (+)-butaclamol) fell from 72% to 50% in the calf soluble preparation but remained high (82%) for the other preparations. The recovery of the receptor binding sites was about 20% for all three species. However, as Scatchard analysis indicated that the calf membrane preparation con- tained at least two sites, the recovery of receptor numbers from both high and low affinity sites was only 7%.

Ascorbate and EDTA effects. Neuroleptic binding to both receptor preparations was reduced after preincubation at 22OC in the absence of 3H-spiperone. Preincubation of the membrane prepared in tris-ascorbate-nialamide buffer for three hours resulted in a rapid loss (80%) of neuroleptic binding (Fig. 1). Na2EDTA (5 mM) and Mn++ prevented the loss of binding whereas MgC12, NaCl and CaC12 ions did not. The extent and rate of decline of the solubilized receptor differed significatly from the membrane-bound receptor. Preincubation of the soluble preparation resulted in decreased 3H-spiperone binding in the presence of all the ions. EDTA and Mn++ did not prevent this loss of binding ability. The rateof decline was more gradual than that of the membrane-bound preparation.

Soluble dopamine receptors 545

Table 1

Comparison of solubilized D2 receptors from human, canine and calf striata

A. Ratio of IC50 values in soluble versus membrane

Human !i% Calf

(+)-Butaclamol (-)-Butaclamol Fluphenazine Spiperone Haloperidol Chlorpromazine Dopamine Apomorphine ADTN (-)Norepinephrine 5-Hydroxytryptamine Epinephrine

0.6 0.2 b b

1.6 0.9 2.5 2.0 2.7 2.0 3.2 3.9 0.8 1.0 1.0 2.0 2.1 1.6

a a a a

n.d. n.d.

1.2 0.32 3.4

12.0 0.8

15.0 2.2 1.0 1.9

0.25= 2.1

a

a. >lOO,OOO nM in both membrane and soluble b. &lO,OOO nM in both membrane and soluble c. increase in affinity may actually be greater because an IC50 of 100,000 nM is

arbitrarily used because it was 100,000 nM and the highest concentration used was 100,000 nM

Table 1B

3 H-spiperone binding to D2 receptors

(nM) Kd Human & Calf

membrane 0.37 0.4 0.25 soluble 0.95 0.8 3.00

% Specific

Membrane soluble

82% 82%

73% 82%

72% 50%

Table 1C

Recovery of 3

H-spiperone binding sites and protein after digitonin solubilization

Human Eti Calf

Receptor density fmoleslmg

63% 48% 50yoa ) 1 nb

Total receptors

Protein

24% 22% 20%a, J%b

3 7% 46% 40%

a. Assuming solubilization of only the high affinity component. b. Assuming solubilization of both high and low affinity sites.

546 B.K. Madras et al.

Membrane coluble

Mn+’ TEAN TAN

Na+

0 3 24 0 3 24

Hours Hours

Fig. 1. Effect of ions and Na2EDTA on stability of 3H-spiperone binding. Receptor prepara- tions were incubated with ions (NaCl 100 mM=-m“ MgCl2 (1 mM x - x) MnC12 (1 mMA-A) TAN buffer (0-O) TEAN (O-O) for the times (0, 3, 24 hr). (The CaC12 curve is omitted but is almost identical to that of MgCl;.) Then 3H-spiperone (1 nM) or 3H-spiperone and (+)-butaclamol (1 PM) were added to measure total and non-specific binding (resp.). Each point is a mean of three experiments done in triplicate. Specific binding at 0 time (in presence of individual ions) is the denominator for the percentage calculations. At 3 hr and 24 hr the EDTA and Mn++ treated membrcres are significantly different than controls (p < 0.005).

Ion effects. Only Na+ enhanced specific 3 H-spiperone binding significantly and only in the soluble preparation (Table 2).

Table 2

Effects of ions on 3

H-Spiperone Binding

fmoleslmg protein

Addit ion Membrane Soluble

Buffer 263 f 44 94 ? 13 NaCl (100 mM) 278 f 10 128 2 9a CaC12 (1 mM) 246 + 23 88 f 14 MgC12 (1 mM) 2332 7 95 f la MnC12 (1 mM) 232 +_ 15 95 f 16 Na2EDTA (5 mM) 294 f 25 91 f 17

Each value is the mean S.E.M. of three experiments using 3 individual brains performed in triplicate. The receptor prepared in tris-ascorbate-nialamide (TAN) buffer was incubated at O°C as described under Methods. The ions were added to the incubation buffer (TAN) followed by tissue and 3H-spiperone. (+)-Butaclamol (1 PM) was used to define non-specific binding.

a. p < 0.1 (two-tailed Students t-test) p < 0.05 (single-tailed Students t-tests)

Soluble dopamine receptors 547

Discussion

Canine striatum is an excellent source of soluble D2 receptors because of its similarity to soluble human D2 receptors and its stability in solution. The binding properties of the solubilized calf preparation, however, do not parallel those of the membrane preparation. Non-specific binding c3H-spiperone binding not displaceable by (+)-butaclamol) of soluble calf receptors is considerably higher than the membrane preparation. The soluble prepara- tions of the other species retains the same level of specific binding.

The affinity of the calf soluble preparation for norepinephrine increased about 4-fold whereas the affinity for some neuroleptics decreased lo-fold. As H-spiperone binds to both serotonergic (Leysen et al., 1978) as well as a-adrenergic sites (Andorn and Maguire, 19801, these sites may have been solubilized in a different ratio than their occurence in the membrane preparation. The use of other detergents may improve the binding character- istics of soluble calf receptors.

Modifications of the membrane matrix which surround neuroleptic receptors may affect neuroleptic receptor binding. Ascorbic acid, EDTA, Mn++ modulate neuroleptic binding to membrane receptors but not solubilized receptors. Ascorbic acid produces a profound decline in 3H-spiperone binding (80% in 3 hrsl if t!e receptor is prepared in tris-ascorbate and incubated in this buffer in the3absence of H-spiperone. The ascot-bate effect is minimal if introduced simultaneously with H-spiperone indicating that spiperone confers a protective ’ effect on the binding sites. Binding to the solubilized receptors shows no particular susceptibility to ascorbate indicating that the ascorbate effect is on the membrane itself.

Both EDTA and Mn++ prevent the decline in neuroleptic binding to membrane receptors, but are without effect on solubilized receptors, indicating that EDTA and Mn++ act indirectly possibly by inhibiting ascorbate and Fe++ catalyzed lipid peroxidation of the membranes (Shaefer et al., 1975; Leslie et al., 1980; Rehncrona et al., 1980). The neuro- leptic receptor D2-type should be prepared either in tris or tris-ascorbate EDTA buffer, but not tris-ascorbate buffer. The solubilized receptors provides an excellent preparation for differentiating the effects of compounds directly on receptor binding or indirectly,

on the membrane. It remains to be shown whether modulation of neuroleptic binding by ascorbate, Mn+‘, or Na+ is of physiological relevance, is due to changes in receptor

numbers or affinity and whether the binding of all antagonists and agonists is equally effective.

References

ANDORN, A.C., MAGUIRE, M.E. (1980). 3H-Spiroperidol binding in rat striatum. Two high- affinity sites of differing selectivities. J. Neurochem. 35:1105-1113.

CARON, M.G. and LEFKOWITZ, R.J. (1976). Solubilization and characterization of the 6-adrenergic receptor binding sites of frog erythrocytes. J. Biol. Chem 251:2374-2384.

DAVIS, A., MADRAS, B.K. and SEEMAN, P. (19801. Solubilization of the neuro=tic binding site of human brain. sot. Neurosci. Abstr. 6:500.

DAVIS, A., MADRAS, B.K. and SEEMAN, P. (1981).- Solubilization of neurolepticldopamine receptors of human brain striatum. Eur. J. Pharmacol. 70:321-329.

GORISSEN, H., AERTS, C., and LADURON, P. (1978). Characterization of solubilized dopamine receptors from dog striatum. Fed. Eur. Biochem. Sot. Lett. 100:281-285.

HARTLEY, E.J. and SEEMAN, P. (1978). The effect of varying 3Hziperone concentration on its binding parameters. Life Sci. 23:513-518.

LESLIE, F.M., DUNLOP C.E. III, COX B.M- (1980). Ascorbate decreases ligand binding to neurotransmitter receptors. J. Neurochem %:219.

LEYSEN, J.E., NIEMEGEERS, C.J.E., TOLLENAERE, J.P. and LADURON, P.M. (1978). Serotonergic component of neuroleptic receptors. Nature (London) 272:168-171.

MADRAS, B.K., DAVIS, A. and SEEMAN, P. (1980al. SolubiKation of the neuroleptic/ dopamine (D-2) from dog striatum. Sot. Neurosci. Abstr. 6:240.

MADRAS, B.K. DAVIS, A., KUNASHKO, P. and SEEMAN, P. (1980b). Solubilization of dopamine receptors from dog and human brains. In: Psychopharmacology and Biochemistry of Neurotransmitter Receptors, ed. by H. Yamamura, R.W. Olsen, and E. Usdin, pp. 411-419, Elsevier North Holland, New York.

REHNCRONA, S., SMITH, D.S., AKESSON, B., WETERBERG, E. and SIESJO, B.K. (1980). Peroxidative changes in brain cortical fatty acids and phospholipids. as characterized during Fe+ and ascorbate-stimulated lipid peroxidation in vitro. J. Neurochem. 34: 1630. -- -

548 .

B.K. Madras et al.

SCHAEFER, A., KOMLOS, M. and SEREGI, A. (1975). Lipid pet-oxidation as a cause of the ascorbic acid induced decrease of adenosine triphosphatase activities of rat brain microsomes and its inhibition by biogenic amines and psychotropic drugs. Biochem. Pharmacol. 24:1781.

SEEMAN, P. (1974). Ultrastructure of membrane lesions in immune lysis, osmotic lysis and drug-induced lysis. Fed. Proc. 33:2116-2124.

SEEMAN, P., LEE, T., CHAU-WONG, M.,and WONG, K. (1976a). Antipsychotic drug doses and neurolepticldopamine receptors. Nature (London) 261:717-719.

SEEMAN, P., LEE, T., CHAU-WONG, M., and WONG, K. (1976b). Correlat ion of ant ipsychot ic drug potency and neuroleptic receptor block. sot . Neurosci. Abst r. 2:87a.

TAM, S., and SEEMAN, P. (1978). Neuroleptic receptors in calf caudatey Solubilization by digitonin. Eur. J. Pharmacol. z:151-152.

Inquiries and reprint requests should be addressed to:

Dr. B.K. Madras Psychopharmacology Section, Clarke Institute of Psychiatry, 250 College, Street, Toronto, Ontario, M5T 1RB