Dopamine. (D2-type) receptors were solubilized from striatum of three species. (human, canine, calf) using digitonin. 2. The receptors were labeled with.
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SOLUIDLIZED DOPAMINE/NEUROLEPTIC RECEPTORS(D2-I’YPE)
BERTHA
K.
MADRAS,
ALAN
Psychopharmacology Section, Department of Pharmacolgy,
(Final
DAVIS,
BETTY
Clarke University
form,
CHAN
and
Institute of Toronto,
April
of
PHILIP
SEEMAN
Psychiatry Toronto,
and Canada
1981)
Abstract 1. 2. 3. 4. 5. 6.
Key
Dopamine (D2-type) receptors were solubilized from striatum of three species (human, canine, calf) using digitonin. The receptors were labeled with 3 H-spiperone and assayed by Sephadex G-50 columns or polyethylene glycol precipitation. The soluble receptors from canine and human tissue had similar Kd’s and rank order of drug affinities to the membrane-bound sites. Soluble calf recepLors showed reduced affinity for spiperone and chlorpromazine (12-fold). Non-specific binding also increased. Solubilized canine binding was insensitive to ascorbate, Mn++, and ethylenediamine tetracetic acid (EDTA) in contrast to the membrane binding sites. 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. words:
Abbreviation:
soluble
receptors,
ethylenediamine
dopamine, tetracetic
neuroleptics acid
(EDTA),
Tris-EDTA-ascorbate-nialamide
(TEAN)
Introduction Neuroleptic drugs bind their clinical doses for receptor and subsequent treatment, and clarification
to dopamine receptors schizophrenia (Seeman antibody formation may of the pathological
(D2) in an order of potencies et al. 1975a,1976b). Purification have clinical applications for mechanisms of the disease.
The success of receptor solubilization, a preliminary on the type of tissue used. Since considerable amounts purification and analysis, it is essential to determine canine) would provide the highest yield of soluble and
that
parallels of this diagnosis,
to purification, appears to depend of tissue are required for ultimate which species (human, bovine, 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 This is because a plentiful supply of calf tissue is generally with those of calf caudate. available and because much previous work has described the characteristics of the calf brain D2 site. provides an excellent source The solubilized canine caudate but not calf caudate, of D2 receptors because of its similarity to soluble human D2 receptors and its stability in solution.
Met hods Brain within house.
tissues. Dogs were anaesthesized with pentobarbital two or three hours of death. Fresh calf brains were The brains were harvested within two hours and striatal
and the striata obtained from membranes
were a local prepared
removed slaughter
544
B.K.
immediately human brains.
or
after
freezing
overnight.
et al.
Madras
Dr.
John
Deck
(Toronto
Methods of tissue preparation were essentially those described The brain regions were homogenized in sucrose (0.25 Ml, centrifuged resuspended in sucrose and centrifuged at 105,000 x g for 60 min. in TEAN buffer (tris-EDTA-ascorbate-nialamide) . The P?-P2 membrane fraction Caron e; ai. (1976; see also microscopy of the solubilized 1974) confirmed the complete Binding tions of
methods. standard
The specific using Sephadex 1980a,b; Davis was developed
Binding methods
was solubilized with 1978) Tam and Seeman, material (negatively absence of membrane-like of (Hartley
3 H-spiperone
binding of 3H-spiperone G-50 columns (Caron et al., 1980, 19811. in our laboratory as
and
to Seeman,
by
Hospital)
membrane 19781
fractions
provided
Madras et al. (1980a,b). at 1100 x g for 10 min, The pellet was resuspended
dieitonin (1%) usinn with minor modifications.’ stained by phosphotungstic fragments.
to the digitonin-solubilized Lefkowitz, 1976; Tam and In addition, a polyethylene a rapid screening assay (Ghan
and
Western
was
the
done
procedure Electron acid,
by
minor
of Seeman
modifica-
material was assayed Seeman, 1978; Madras et al., glycol precipitation method 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. and receptor recovery. The Kd values for both canine and human striatum were -Kd values 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 contained 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
Table Comparison
of
solubilized
A.
Ratio
D2 of
IC50
1
receptors
from
values
in
>lOO,OOO &lO,OOO increase arbitrarily 100,000
human,
soluble
(+)-Butaclamol (-)-Butaclamol Fluphenazine Spiperone Haloperidol Chlorpromazine Dopamine Apomorphine ADTN (-)Norepinephrine 5-Hydroxytryptamine Epinephrine a. b. c.
H-spiperone
Kd
calf
Calf
0.6 b 1.6 2.5 2.7 3.2 0.8 1.0 2.1 a a n.d.
0.2 b 0.9 2.0 2.0 3.9
1.2 0.32 3.4 12.0 0.8 15.0 2.2 1.0 1.9 0.25= 2.1 a
binding
to
1.0 2.0 1.6 a a n.d.
D2
striata
membrane
!i%
1B
membrane soluble
and
Human
Table
(nM)
canine
versus
nM in both membrane and soluble nM in both membrane and soluble in affinity may actually be greater because used because it was 100,000 nM and the nM
3
545
an IC50 of 100,000 highest concentration
nM is used
receptors
Human
&
Calf
0.37 0.95
0.4 0.8
0.25 3.00
73% 82%
72% 50%
% Specific Membrane soluble
82% 82%
Table Recovery
of
3
H-spiperone
binding
sites
1C
and
protein
Human
Assuming Assuming
digitonin
Eti
Calf
solubilization
Receptor density fmoleslmg
63%
48%
50yoa )
1 nb
Total
24%
22%
20%a,
J%b
3 7%
46%
40%
affinity low affinity
component. sites.
receptors
Protein a. b.
after
solubilization solubilization
of of
only both
the high
high and
was
546
B.K.
et
Madras
al.
Membrane
coluble
Mn+’ TEAN TAN Na+ 0
3
24
0
3
24
Hours
Hours
Effect of ions and Na2EDTA on stability of 3H-spiperone binding. Receptor preparaFig. 1. tions were incubated with ions (NaCl 100 mM=-m“ MgCl2 (1 mM x - x) MnC12 (1 mMA-A) (The CaC12 curve is omitted TAN buffer (0-O) TEAN (O-O) for the times (0, 3, 24 hr). but is almost identical to that of MgCl;.) Then 3H-spiperone (1 nM) or 3H-spiperone and Each (+)-butaclamol (1 PM) were added to measure total and non-specific binding (resp.). point is a mean of three experiments done in triplicate. Specific binding at 0 time (in At 3 hr presence of individual ions) is the denominator for the percentage calculations. and 24 hr the EDTA and Mn++ treated membrcres are significantly different than controls (p < 0.005).
Ion the
effects. soluble
Only preparation
Na+
enhanced (Table 2).
specific
3
H-spiperone
Table Effects
of
ions
on
binding
ion
Buffer NaCl (100 mM) CaC12 (1 mM) MgC12 (1 mM) MnC12 (1 mM) Na2EDTA (5 mM) Each value is the mean triplicate. The receptor O°C as described under by tissue and 3H-spiperone. a.
p < 0.1 p < 0.05
(two-tailed (single-tailed
H-Spiperone
Membrane 263 278 246 2332 232 294
f 44 f 10 + 23 7 +_ 15 f 25
S.E.M. of three experiments using 3 prepared in tris-ascorbate-nialamide Methods. The ions were added to the (+)-Butaclamol (1 PM) was used Students t-test) Students t-tests)
and
only
in
2 3
fmoleslmg Addit
significantly
Binding protein Soluble 94 128 88 95 95 91
? 2 f f f f
individual (TAN) incubation to define
13 9a 14 la 16 17 brains performed in buffer was incubated at buffer (TAN) followed non-specific binding.
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 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 preparations of the other species retains the same level of specific binding. The affinity of the calf soluble preparation whereas the affinity for some neuroleptics both serotonergic (Leysen et al., 1978) as 19801, these sites may have been solubilized The use of other the membrane preparation. istics of soluble calf receptors.
the
for norepinephrine increased about 4-fold decreased lo-fold. As H-spiperone binds to well as a-adrenergic sites (Andorn and Maguire, in a different ratio than their occurence in detergents may improve the binding character-
Modifications of the membrane matrix which surround neuroleptic receptors may affect Ascorbic acid, EDTA, Mn++ modulate neuroleptic binding to neuroleptic receptor binding. membrane receptors but not solubilized receptors. Ascorbic acid produces a profound decline binding (80% in 3 hrsl if t!e receptor is prepared in tris-ascorbate and in 3H-spiperone The ascot-bate effect is minimal if incubated in this buffer in the3absence of H-spiperone. introduced simultaneously with H-spiperone indicating that spiperone confers a protective Binding to the solubilized receptors shows no particular effect on the binding sites. 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 neuroleptic 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, It remains to be shown whether modulation of neuroleptic binding by on the membrane. is due to changes in receptor ascorbate, Mn+‘, or Na+ is of physiological relevance, is equally numbers or affinity and whether the binding of all antagonists and agonists effective. References ANDORN, A.C., MAGUIRE, M.E. (1980). 3H-Spiroperidol binding in rat striatum. Two highaffinity sites of differing selectivities. J. Neurochem. 35:1105-1113. CARON, M.G. and LEFKOWITZ, R.J. (1976). Solubilization and characterization of the J. Biol. Chem 251:2374-2384. 6-adrenergic receptor binding sites of frog erythrocytes. 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 Eur. J. Pharmacol. 70:321-329. receptors of human brain striatum. GORISSEN, H., AERTS, C., and LADURON, P. (1978). Characterization of solubilized dopamine Fed. Eur. Biochem. Sot. Lett. 100:281-285. receptors from dog striatum. The effect of varying 3Hziperone concentration on HARTLEY, E.J. and SEEMAN, P. (1978). Life Sci. 23:513-518. its binding parameters. DUNLOP C.E. III, COX B.M(1980). Ascorbate decreases ligand binding to LESLIE, F.M., 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 J. Neurochem. during Fe+ and ascorbate-stimulated lipid peroxidation -34: 1630. --in vitro.
’
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.
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