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Binding of [3H]FH-510 to σ ligand recognition sites in guinea-pig brain membranes
European Journal of Pharmacology, 238 (1993) 93-100
93
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53153
Binding of [3H]FH-510 to or ligand recognition sites in guinea-pig brain membranes M a k o t o T a n a k a , Shinsuke Kaku, M a k o t o M u r a m a t s u and S u s u m u O t o m o Department of Pharmacology, Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan Received 24 March 1993; accepted 13 April 1993
We examined the characteristics of the binding of radiolabeled 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride ([3H]FH-510), a highly potent and selective tr ligand, to guinea-pig brain membranes. [3H]FH-510 showed saturable and reversible binding to cr binding sites. The association rate constant (k+l) and dissociation rate constant (k_ 1) of [3H]FH-510 were 0.023 min -1- nM -1 and 0.081 min -l, respectively. Scatchard plot analysis showed a dissociation constant (K d) and maximal number of binding sites (B,~ax) of 6.0 + 0.63 nM and 1763.3 -t- 177.4 fmol/mg protein (n = 7), respectively. The rank order of potency (K i) of several structurally dissimilar tr ligands obtained for the displacement of [3H]FH-510 binding was highly correlated with that determined for [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3HI(+)-3-PPP) binding. The binding of [3H]FH-510 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic or cholinergic agents at 10 -7 M. Higher [3H]FH-510 binding to brain regions was observed in the cerebellum and pons-plus-medulla. Except for the nuclear fraction, the highest level of [3H]FH-510 and [3H](+)-3-PPP binding to subceilular fractions was observed in the microsomal fraction. From these results, it is suggested that FH-510 selectively binds with high affinity to tr binding sites in guinea-pig membranes. tr Binding sites; FH-510 (5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochioride); Radiolabeled ligand binding; Brain (guinea-pig)
1. Introduction
The existence of or receptors was first postulated by Martin et al. (1976), to account for the psychotomimetic effects of N-allylnormetazocine (SKF10,047) and related racemic benzomorphans. Although tr binding sites were originally thought to be a type of traditional opiate receptor, several subsequent studies have revealed that they are not: naloxone is ineffective against the effects of or ligands, and enantiomers of benzomorphan opiates exhibit opposite effects against or and traditional opiate receptors (Su, 1982; Tam, 1983; Vaupel, 1983; Katz et al., 1985, Weber et al., 1986). Furthermore, in earlier reports, the binding sites of tr and of the psychotomimetic agent phencyclidine (PCP) were thought to be identical, based on the ability of ( + ) SKF10,047 to displace bound [3H]PCP (Zukin et al., 1984; Mendelsohn et al., 1985). However, substantial
Correspondence to: M. Tanaka, Department of Pharmacology, Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan. Tel. 048 (663) 1111, fax 048 (652) 7254.
evidence now indicates that or and PCP binding sites are distinct physiological entities (Gundlach et al., 1985; Quirion et al., 1987). In addition, the neuroleptic agent haloperidol, which possesses low affinity for PCP binding sites, displays high affinity for [3H](+)-SKF10,047 binding sites (Tam and Cook, 1984; Itzhak, 1988). The binding sites of tr ligands may play a role in schizophrenia, since many antipsychotic drugs bind to the sites with high affinity (Tam and Cook, 1984). Although haloperidol is the most potent inhibitor of [3H](+)-SKF10,047 binding (Tam and Cook, 1984; Itzhak, 1988), because it has approximately the same affinity for the dopaminergic receptor (Tam and Cook, 1984; Itzhak, 1988) it is not a good drug to verify the involvement of tr ligand binding sites in schizophrenia. In recent years, more selective or ligands, such as ( + )-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)3-PPP) and 1,3-di-o-tolylguanidine (DTG), have been identified, and radiolabeled compounds of these ligands are available and are used to label or binding sites in brain tissues (Largent et al., 1984; Weber et al., 1986; Itzhak, 1988). These agents have moderate affinity for or binding sites, but novel potent and selective or
94
ligands arc needed to explore the relationship between ~r binding sites and schizophrenia, l)cCosta ct al. (1989) have reported that [3H](+)-pcntazocine has potent and selective affinity for o- binding sites, with a potency comparable to that of haloperidol. In our previous study, wc also reported the identification of a novel potent and selective ~r ligand, 5,8-dimcthyl-4-(2-di-npropylaminoethyl)carbazol monohydrochloridc (FH510), obtained from carbazol derivatives (Tanaka et al., 1993). In the study, FH-510 showed a very potent inhibitory effect on (+)-SKF10,047-induccd behavior in mice (Tanaka ct al., 1993). In the present study, wc characterized the binding of radiolabelcd FH-510 to guinea-pig brain membranes.
2. Materials and methods
2.1. Materials The following drugs were purchased from Research Biochemicals Inc. (Natick, MA): haloperidol, ( - ) - and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine hydrochloride (3-PPP), ( - ) - and (+)-N-allylnormetazocine hydrochloridc (SKF10,047), 1,3-di-o-tolylguanidine (DTG), rimcazoic dihydrochloridc, (+)-7-chloro-8-hydroxy-3-methyl- 1-phcnyl-2,3,4,5-tet rahydro- 1H-3-benzazcpine hydrochloride (SCH23390), (+)-8-hydroxy-2(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), ritanserin and 5,5-diphenylhydantoin sodium (phenytoin). Dcxtromethorphan hydrobromide, pyrilamine maleate (mepyramine), tiapridc hydrochloride, phentolamine, dl-propranolol hydrochloride, and atropine were purchased from Sigma Chemical Co. (St. louis, MO). (+)-Pcntazocine and famotidine were prepared from commercially available medicines, Pcntagin and Gastcr, which were obtained from Sankyo Co. (Tokyo, Japan) and Yamanouchi Pharmaceutical Co. (Tokyo, Japan), respectively. 5,8-Dimcthyl-4-(2-din-propylaminoethyl)carbazol monohydrochloride (FH510, fig. 1), ( - ) - and (+)-a-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-l-piperazinc butanol (BMY14802), and phencyclidine (PCP) were synthesized in the Department of Organic Chemistry at our Research Center. [3H]FH-510 (specific activity: 29.1 Ci/mmol) was
.HCI
Fig. 1. Chemical structure of 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride (FH-510).
synthesized at Chemsyn Science Laboratories (Lcncxa. KA). [~H]( + )-3-PPP (117.0 Ci/mmol) was purchased from Du Pont/New England Nuclear (Wilmington, DE). All other chemicals wcrc of the highest purity available.
2,2. Preparation of guinea-pig brain membranes for receptor binding Male Hartley guinea-pigs (Japan SLC, Hamamatsu, Japan; 350-400 g) were decapitated and thc whole brains or seven dissected sites (cerebral cortex, hippocampus, striatum, thalamus-plus-midbrain, hypothalamus, pons-plus-medulla, and cerebellum) were homogenized in 10 volumes of ice-cold 10 mM Tris-HCI buffer (pH 7.4), using a Physcotron homogenizer (NitiOn Medical and Physiological Instruments Mfg., Funabashi, Japan), and centrifuged in a Beckman L8-70 centrifuge at 48,000 × g for 10 rain at 4°C. The pellets obtained were rehomogenized in the same buffer and rccentrifuged. The final membrane suspension for use in the receptor binding assay was diluted in 40 volumes of 10 mM Tris-HCl buffer (pH 7.4) to a protein concentration of 0.8-1.5 mg/ml. Radioligand binding was found to be linear with respect to protein concentration within this range. Protein concentration was determined by the method of lowry et al. (1951).
2.3. Preparation of guinea-pig brain subcellular fractions Subcellular fractions of guinea-pig brain were obtained by differential centrifugation and discontinuous sucrose gradient, as described elsewhere (Bylund and Snyder, 1976). Whole brain was homogenized in 10 volumes of ice-cold 0.32 M sucrose, using a motordriven Teflon pestle glass homogenizer. After centrifugation at 900 x g for 10 min, the crude nuclear pellet was rehomogenized in 0.32 M sucrose and centrifuged at the same speed. The combined supernatant was centrifuged at 17,500 x g for 20 min, and the pellet obtained was the crude mitochondrial fraction. The supernatant was centrifuged at 100,000 x g for 60 min to obtain the crude microsomal pellet. To separate myelin, synaptosomes, and mitochondria, the crude mitochondrial fraction was resuspended in 0.32 M sucrose and layered o n t o a discontinuous gradient of 0.8 and 1.2 M sucrose (1:1). After centrifugation at 100,000 x g for 90 min at 4°C, the following fractions were collected: 0.32-0.8 M sucrose (myelin), 0.8-1.2 M sucrose (synaptosomes), and the pellet at the bottom of 1.2 M sucrose (mitochondria). All fractions except microsomes were diluted 5-fold with 10 mM Tris-HCl buffer (pH 7.4) and centrifuged at 48,000 x g for 15 min at 4°C. The resulting pellets were resuspended in 10 mM Tris-HCl buffer (pH 7.4) and were used in
95 receptor binding assays to study subcellular distribution.
2. 4. Receptor binding assay For a typical binding experiment, the reaction was initiated by adding 1 ml of membrane suspension to polypropylene test tubes containing 2.0-2.5 nM of [3H]FH-510. The reaction mixture was incubated for 30 min at 25°C, a time period that allowed equilibrium (see kinetic experiments in the Results section). Nonspecific binding was determined in the presence of unlabeled FH-510 (10 /~M). Specific binding was defined as the difference between the total binding and nonspecific binding, and was 55-65% of the total binding. The reaction was terminated by rapid filtration through Whatman G F / B glass fiber filters that were presoaked in 0.5% polyethyleneimine for 4 h, after which the filters were washed 3 times with 5 ml of ice-cold 10 mM Tris-HCl buffer (pH 7.4). These steps were performed with a multiple cell harvester M-24R (Brandel Biomedical Research and Development Laboratories, Gaithersburg, MD). Aquazol-2-scintillator (Du Pont/New England Nuclear) (8 ml) was added and filter-bound radioactivity was quantified in a liquid scintillation spectrometer (Aloka, LSC-3500, Tokyo, Japan). The [3H]( +)-3-PPP binding assay was carried out as described above, except that nonspecific binding was determined in the presence of 10/xM (+)-3-PPP. For determination of the equilibrium dissociation constant (Ko), 0.5-25 nM of [3H]FH-510 was used. Saturation binding data were analyzed by Scatchard plot analysis, and the K d and the maximal number of binding sites (Bmax) were calculated using a computer program, SP123, developed by Dr. H. Ono of the University of Tokyo for the PC-9801 (NEC, Tokyo, Japan) personal computer. Association kinetics were determined using two concentrations of [3H]FH-510 (1.77 and 5.03 nM), and pseudo first-order association rates were calculated as described previously (Bylund and Yamamura, 1990). After equilibrium had been reached (30 min, 25°C), dissociation experiments were initiated by adding excess unlabeled FH-510 (10 ~M). Dissociation rate constants were calculated from the slope obtained after linear transformation of the data (Bylund and Yamamura, 1990). In the competition binding assay, the concentration of test compound causing 50% inhibition of specific [3H]FH-510 or [3H](+)-3-PPP binding (IC50) was determined from concentration-response curves, in which 10 concentrations (from 3 × 10 -t° to 1 × 10 -5 M) of test compound were examined. After determination of IC50 values by the Marquardt-Levenberg nonlinear curve-fitting procedure of the RS/1 program (BBN Research System, Cambridge, MA) running on a
VAX/VMS system, K i values for each test compound were calculated according to the equation of Cheng and Prusoff (1973), using the K d value obtained by Scatchard analysis ([3H]FH-510; 6.0 nM) and from published data ([3HI( +)-3-PPP; 43 nM) (Karbon et al., 1991).
3. Results
3.1. Time course of [3H]FH-510 binding to guinea-pig brain membranes At 25°C, the binding of [3H]FH-510 (1.77 nM) to guinea-pig brain membranes was increased in a timedependent manner and reached equilibrium at 20 min (fig. 2). After [3H]FH-510 binding reached a plateau, an excess of unlabeled FH-510 (10/xM) was added. As shown in fig. 2, displacement of [3H]FH-510 from its binding sites occurred immediately. Almost complete displacement was achieved 30 min after the addition of unlabeled FH-510.
3.2. Association and dissociation kinetics of [ 3H]FH-510 binding to guinea-pig brain membranes Association experiments were performed using two concentrations of [3H]FH-510 (1.77 and 5.03 nM). The association rate constant (k+t) and the dissociation rate constant (k_l) were determined by using the following equation: kobS= k+l • [L] + k t (ko~, observed apparent rate constant; [L], concentration of ligand). The k÷l and k ~ values calculated by using 2 kob, derived from the slope of pseudo first-order rate plots (fig. 3A) were 0.023 + 0.005 min -1 •nM -1 and 0.114 -t0.025 min-i, respectively (table 1). The K d value given by K a = k_ J k + i was 4.96 nM. The k_ l value was also
10 pM FH-510
l 20(3
0
'100
z ___o ,?,. o 03
~) 1
i
i
i
20
30
40
i
SO
i
60
Time (rain)
Fig. 2. Time course of [3H]FH-510 binding to guinea-pig brain membranes. Guinea-pig brain membranes were incubated with 1.77 nM [3H]FH-510 at 25°C. After equilibrium has been reached (30 min), 10/~M of unlabeled ligand was added to initiate displacement of [3H]FH-510from its specificbinding sites. The data are means of duplicate determinations.
96
3
O
(A)
T~me (rain) 10 20
"
'
(A) 30
'
3
o
o o-
--2 I•o
c2
},
~,
o
o
~E I- 6 u- E
0
10
20
13HIFH-510 (nM) ,
,
,
5
10
15
I(B) (B)
04i°
Time (rmn)
Fig. 3. Kinetic analysis of [31-I]FH-510 binding to guinea-pig brain membranes. (A) Guinea-pig brain membranes were incubated with 1.77 nM (©) and 5.73 nM (e) [3H]FH-510 at 25°C for the indicated times. B~ is the specific binding at equilibrium, and B is the amount of [3H]FH-510 specifically hound at any indicated time. Each point is the mean of two separate experiments, each done in duplicate. (B) After equilibrium had been reached (30 rain), 10 /~M of unlabeled ligand was added to initiate displacement of [3HlFH-510 from its specific binding sites. B 0 is the amount of []H]FH-510 at time zero. Each point is the mean of four separate experiments, each done in duplicate. Vertical bars show S.E. of mean.
determined from the slope obtained after linear transformation of the data for the dissociation time course in fig. 2 (fig. 3B and table 1). The k_ ~ value obtained from the dissociation rate experiment was 0.081 + 0.002 min--1. The K d value was calculated to be 3.52 nM.
3.3. Saturation binding of [3H/FH-510 to guinea-pig brain membranes The characteristics of the binding of [3H]FH-510 to guinea-pig brain membranes were studied further. The saturation studies of the binding of [3H]FH-510 revealed that nonspecific binding increased linearly with increasing concentrations of [3H]FH-510, whereas specific binding, determined by subtracting nonspecific binding from total binding, was saturable (fig. 4A).
TABLE 1 Association and dissociation rate experiments of [3H]FH-510 binding to guinea-pig brain membranes. Experiments were performed as described in the legend of fig. 3. Values are m e a n s ± S.E. of 3 - 4 separate experiments, each done in duplicate. Association rate e r p e H m e n t (n = 3)
k + ~ ( m i n 1. n M k l(min-1) k d (k_ ~ / k + l)
i)
0.023 ± 0.005 0.114+0.025 4.96
Dissociation rate experiment (n = 4)
k 1( m i n - 1 ) K d (k_i/k.l)
0.081±0.(X)2 3.52
E
"5 ,'n ~ 0 2
i
0
1 B (pmol/mg
2
protein)
Fig. 4. Saturation isotherm and Scatchard plot of [~H]FH-5]0 binding to guinea-pig brain membranes. Guinea-pig brain membranes were incubated with increasing concentrations of [3H]FH-5]0 at 25°C for 30 min. (A) Representative saturation curves for the equilibrium binding of [3H]FH-5]0 arc shown. Essentially the same results were obtained from seven independent experiments, done in duplicate. Total binding, nonspecific binding, and specific binding are indicated as o, O, and o, respectively. (B) Scatchard plot of the same data as in (A). Mean values o f K d and Bm~, are 6.00+0.63 nM and 1763.3 _+ ]77.4 fmol/mg protein, respectively.
Scatchard analysis of these data indicated the existence of a single class of binding sites in guinea-pig brain membranes (fig. 4B). The K a and Bmax values were calculated to be 6.00+0.63 nM and 1763.3_+ 177.4 f m o l / m g protein (n = 7), respectively.
3.4. Displacement of [SH]FH-510 binding by various cr ligands and neurotransmitter-related compounds Several structurally dissimilar ~ ligands were examined for their abilities to compete with the specific binding of [3H]FH-510 (fig. 5) and [3H](+)-3-PPP to guinea-pig brain membranes (table 2). [3H]FH-510 binding was concentration dependently inhibited by unlabeled FH-510. The Hill coefficient (nrt) and the K i value of FH-510 were 0.86 and 15.1 + 2.7 nM, respectively. The K i value and n H for FH-510 were close to that of haloperidol (13.1 + 3.8 nM and 0.82). The n u values of other o- ligands for [3H]FH-510 binding were as follows: (+)-3-PPP, 0.66; DTG, 0.72; ( + ) - S K F 10,047, 0.63. Markedly different K i values between ( + ) and ( - ) enantiomers of benzomorphan were observed. Competition of various ~ ligands against [3H]FH-510
97 100
¢.
TABLE 3
~5 t-
Effect of various neurotransmitter-related compounds on [3H]FH-510 binding to guinea-pig brain membranes.
A
9
Binding experiments were carried out as described in Materials and methods. The [3H]FH-510 concentration was 2.0-2.5 nM. Values are means + S.E. of four separate experiments, each done in duplicate.
"1" o5(] "5 0
% Inhibition of [3H]FH-510 binding
O
10-9 M
10-7 M
10-s M
-2.1+ 6.4 8.25:3.7 2.05:3.3 -1.9+ 6.7 - 1.9 + 6.8 0.6:t:15.1 -0.6+ 2.7 - 2.6 5:4.3 -10.15:3.1
4.0+ 2.2 8.7+ 4.1 9.05:6.0 -4.4+10.9 5.5 + 8.9 -0.6-1-10.1 1.4+- 3.2 - 3.7 5:4.4 -8,9+ 4.2
67.85:6.3 3.25:3.8 25.6+ 2.8 42.2+- 2.0 10.5 + 10.7 66.55:4.6 63.25:2.2 35.9 5:7.5 16.1+- 3.3
t'~
O9
9
B
7
~)
5
-log Concentration (M)
Fig. 5. Effects of several representative o- ligands on [3H]FH-510 binding to guinea-pig brain membranes. Guinea-pig brain membranes were incubated with increasing concentrations of sigma ligands in the presence of 2 nM [3H]FH-510. The results indicated are the mean value of three separate experiments, each done in duplicate. Key: FH-510 (o), haloperidol (e), DTG (a), (+)-3-PPP (v), and ( + )-SKFI0,047 ( [] ).
b i n d i n g revealed the following r a n k o r d e r of p o t e n cy: FH-510 = haloperidol > ( + ) - p e n t a z o c i n e > D T G > ( + )-3-PPP = ( + )-BMY14802 > ( + )-SKF10,047 >> dextromethorphan = progesterone > (-)-BMY14802 > rimcazole > PCP > ( - ) - 3 - P P P >> ( - ) - S K F 1 0 , 0 4 7 . A similar tr iigand selectivity of the c o m p o u n d s was observed for [ 3 H ] ( + ) - 3 - P P P b i n d i n g to g u i n e a - p i g b r a i n m e m b r a n e s (table 2). M a n y kinds of n e u r o t r a n s m i t t e r - r e l a t e d c o m p o u n d s were e x a m i n e d to identify the b i n d i n g sites of [3H]FH510, using c o n c e n t r a t i o n s of 10 -9, 10 -7 a n d 10 -5 M
(table 3). T h e r e were no c o m p o u n d s that i n h i b i t e d or e n h a n c e d the b i n d i n g of [3H]FH-510 to g u i n e a - p i g brain m e m b r a n e s a m o n g the histaminergic, d o p a m i n e r gic, adrenergic, serotonergic a n d cholinergic agents tested at 10 -9 a n d 10 -7 M. However, m e p y r a m i n e (H~ r e c e p t o r antagonist), p r o p r a n o l o l (/3-receptor antagonist) a n d 8 - O H - D P A T (5-HT1A r e c e p t o r agonist) inhibited [3H]FH-510 b i n d i n g by approximately 60% at 10-5 M. P h e n y t o i n (10 - 6 ~ 10 -4 M) did not e n h a n c e or suppress [3H]FH-510 b i n d i n g to g u i n e a - p i g b r a i n m e m b r a n e s (data not shown).
3.5. Regional and subcellular distribution of [ 3H ] FH- 510 binding to guinea-pig brain membranes
TABLE 2 Effect of various ~r ligands on [3H]FH-510 and [3H](+)-3-PPP binding to guinea-pig brain membranes. Competition binding was carried out as described in Materials and methods. The K i values were determined according to the equation of Cheng and Prusoff (1973), using the ICs0 values derived from the inhibition data and Ka values. The K d values for FH-510 (6.0 nM) and (+)-3-PPP (43 nM) were obtained by Scatchard analysis indicated in fig. 4 and from Karbon et al. (1991), respectively. Values are means5: S.E. of 2-9 separate experiments indicated in parentheses, each done in duplicate. Compounds
Mepyramine Famotidine SCH23390 Tiapride Phentolamine Propranolol 8-OH-DPAT Ritanserin Atropine
K i (nM) [3H]FH-510
I n o r d e r to investigate the characteristics of [3H]FH-510 b i n d i n g in g u i n e a - p i g b r a i n m e m b r a n e s further, the regional a n d subcellular d i s t r i b u t i o n of [3H]FH-510 b i n d i n g was examined. In these experim e n t s the cortex, h i p p o c a m p u s , s t r i a m m , t h a l a m u s p l u s - m i d b r a i n , hypothalamus, p o n s - p l u s - m e d u l l a a n d the c e r e b e l l u m were used. [3H]FH-510 b i n d i n g was TABLE 4
[3HI( + )-3-PPP
FH-510 15.1:t: 2.7 (9) 2 . 7 8 5 : 0.05 Haloperidol 13.1:1: 3.8 (3) 1.55 + 0.34 (+)-3-PPP 194.2+ 49.3 (4) 12.3 (-)-3-PPP 4865.8+ 742.3 (4) (+)-SKF10,047 337.6+ 85.7 (5) 67.4:1:14.5 ( - )-SKF10,047 11324.3+-2820 (4) (+)-BMYI4802 207.75: 5.8 (3) 23.4 5 : 4 . 8 (-)-BMY14802 2359.8+ 634.0 (4) DTG 145.0+ 64.6 (3) 23.1 5: 3.1 (+)-Pentazocine 100.8+ 22.0 (3) 9.90+ 0.55 Dextromethorphan 1238.8+- 363.4 (5) 120.1 5: 8.8 Rimcazole 3042.5+ 797.6 (4) 1269.7 +-176.6(3) PCP 3822.05:584.2 (3) Progesterone 1325.7+ 244.0 (3)
(3) (3) (2)
Regional distribution of [3H]FH-150 binding in guinea-pig brain membranes. Binding experiments were carried out as described in Materials and methods. The [3H]FH-510 concentration was 2.0-2.5 nM. Values are means+ S.E. of three separate experiments, each done in triplicate.
(3)
Specific binding (fmol/mg protein)
(3) (3) (3) (3)
Cortex Hippocampus Striatum Thalamus + midbrain Hypothalamus Ports+medulla Cerebellum
358.2+- 15.8 369.2 +- 6.0 320.0 +- 13.0 485.8 5:20.5 527.9 5:40.4 571.1 +- 8.7 603.5 +-43.1
9~ TAHt.E 5 Subccllular distribution of [ 3HJFI I-510 and [ 3 t-t]( + )-3-PPP binding to guinea-pig brain membranes. Binding experiments were carried out as described in Materials and methods. The [ 3H]FH-5111 and [ 3 H}( + )-3-PPP concentrations were 2.0-2.5 nM. Nonspecific binding of [3H}( 3-)-3-PPP was determined in the presence of 1 ;zM haloperidol. Values are m e a n s + S.E. of 5 - 6 determinations. Binding ( f m o l / m g protein)
Nuclei Myelin Synaptosomes Mitochondria Microsomes
[ 3H]Ft I-510
[3ti}( + )-3-PPP
815.9 _+212.4 277.9+ 38.8 287.5 + 44.5 321.1 5- 21.1 603.6 + 127.1
21.88 ~- 1.67 9.22+ 1.18 13.59 +_2.38 13.42+ 1.21 35.63 + 5.[YO
rich in the cerebellum as well as in the pons-plusmedulla (table 4). However, the lowest and highest binding densities differed by about 50%. As indicated in table 5, the highest level of [3H]FH510 binding observed was in the nuclear fraction, followed by the microsomal fraction. Despite the diversity of order in these fractions, higher levels of [3H]( + )-PPP binding were also observed in these fractions compared with other fractions. The binding of [3H]FH-510 and [3H]( + )-3-PPP to the other fractions (myelin, synaptosomes and mitochondria) was only 40-50% of that detected in the microsomal fraction.
4. Discussion
In the present study, the characteristics of [3H]FH510 binding to guinea-pig brain membranes were examined. The binding of [3H]FH-510 was time dependent and reached a plateau at 20 min, and the immediate displacement of bound [3H]FH-510 on the addition of unlabeled FH-510 demonstrated the reversible nature of [3H]FH-510 binding. Moreover, [3H]FH-510 bound to guinea-pig brain membranes in a saturable manner, and Scatchard plot analysis showed a single class of binding sites for [3H]FH-510. The K d values (4.96 and 3.52 nM) calculated using the k+ t and k_ 1 (Ka = k_ l / k+~), each obtained from association and dissociation experiments, were quite similar to those obtained from Scatchard plot analysis (6.0 nM). These values are about 10 times lower than those reported for [3H]( + )3-PPP and [3H]DTG (Largent ct al., 1984; Weber et al., 1986; Karbon et al., 1991). In order to verify that the [3H]FH-510 binding site in guinea-pig brain membranes is a t r binding site, the inhibitory effects of a number of structurally dissimilar cr ligands on [3H]FH-510 binding were studied and compared with their effects on [3H]( + )-3-PPP binding. Of these ligands, haioperidol was the most potent
compound (K, = 13.1 nM), and FH-510 showed almost the same effect ( K i = 15.1 nM) against [3H]FH-510 binding to guinea-pig brain membranes. Furthermore, differences in the binding affinities of stereoisomers of benzomorphan (SKF10,047), 3-PPP and BMY14802 for the binding site of [3H]FH-510 were studied, since a clear difference between the affinities of these stereoisomers ( ( + ) > > ( - ) ) is a typical criterion for confirming that the site is a o" binding site (Walker et al., 1990). There was a large difference between the potencies of the ( + ) and ( - ) stereoisomers, q'he rank order of potency to inhibit [3H]FH-510 binding was: FH-510 = haloperidol > (+)-pentazocine > DTG > ( + )-3-PPP = ( + )-BMYI4802 > ( + )-SKF10,047 >> dextromethorphan = progesterone > ( - ) - B M Y 1 4 8 0 2 > rimcazole > PCP > ( - )-3-PPP >> ( - )-SKFI0,047. The potency of these compounds was quite similar to that for [3H](+)-3-PPP binding obtained in the present study, and was consistent with that reported previously (Largent et al., 1984; Tam and Cook, 1984; Weber et at., 1986; Itzhak, 1988; Karbon et al., 1991). These results clearly suggest that FH-510 binds to o- binding sites with higher affinity than o- ligands, including ( + ) - 3 - P P P and DTG. In addition, the marked difference in affinity between ( + ) and (-)-SKF10,047 suggests that [3H]FH-510 is relatively selective for o-1 type (Quirion et al., 1992) binding sites. Neurotransmitter-related compounds, including histaminergic, dopaminergic, adrenergic, serotonergic and cholinergic agents, had little influence on [3H]FH-510 binding to guinea-pig brain membranes at 10 - 9 ~ 10 ...7 M. Although at 10 -5 M, mepyramine, propranolol and 8-OH-DPAT inhibited [3H]FH-510 binding by approximately 60%, these effects do not detract from the high specificity of [3H]FH-510 for cr binding sites. These compounds have low to moderate affinity for o- binding sites, as reported previously: (1) Tam (1983) and Craviso and Musacchio (1983b) reported inhibition of [3H]ethylketocyclazocine and [3H]dextromethorphan binding by both mepyramine and propranolol, (2) H~ antihistamines probably interact with o- binding sites due to a strong structural resemblance to the tricyclic antidepressants and neuroleptic agents (Gray et al., 1990), and (3) due to the similar structural resemblance of 8-OH-DPAT to benzomorphan. Considering these data together, the present results strongly suggest that the binding site of [3H]FH-510 is a ~r binding site. Autoradiographic studies with [ 3 H ] ( + ) - 3 - P P P (Gundlach et al., 1986) and [3H]DTG (McLean and Weber, 1988) revealed that o- binding sites in guineapig brains are more concentrated in motor areas and neurons, including the cerebellum and cranial nerve nuclei, than in limbic areas. [3H]Dextromethorphan binding sites are also distributed in the pons-medulla and cerebellum (Craviso and Musacchio, 1983a). In the present study, the regional distribution of [3H]FH-510
99
binding in seven dissected brain regions was investigated. Although no marked difference in the density of the binding sites between the highest and the lowest regions was observed, the cerebellum had the highest number of [3H]FH-510 binding sites, followed by the pons-plus-meduila. These findings also suggest the possibility that FH-510 binds to o" binding sites. The subcellular localization of tr binding sites has been reported by Craviso and Musacchio (1983a) and McCann et al. (1989), using [3H]dextromethorphan and [3H](+)-SKF10,047, respectively. Specific [3H]dextromethorphan binding to the subcellular fractions from guinea-pig brain was found in the nuclear, mitochondrial, synaptosomal and microsomal fractions, but high-affinity sites were found almost exclusively in the microsomal fractions (Craviso and Musacchio, 1983a). Furthermore, the level of [3H](+)-SKF10,047 binding in rat brain microsomes was 4-fold higher than that in synaptosomes (McCann et al., 1989). The present resuits show a higher density of both [3H]FH-510 and [3H](+)-3-PPP binding sites in the nuclear and microsomal fractions. The affinity of [3H]FH-510 for each subcellular fraction was not determined, and because the relative purity of these fractions was not assessed by assay of marker enzymes (5'-nucleotidase, choline acetyltransferase, and cytochrome c reductase) or DNA content, a clear explanation for the higher levels in the nuclear fractions cannot be given. The possibility that the higher [3H]FH-510 binding in the nuclear fraction was caused by nonspecific binding to DNA or chromosomes cannot be ruled out. However, considering the present data together, it is conceivable that FH-510 and the tr ligand share the same high-affinity binding sites. In conclusion, FH-510 was found to be a specific, selective and potent tr ligand. Radiolabeled FH-510 could be used as a ligand for labeling tr binding sites in the central nervous system. Thus FH-510 might be a good tool to clarify the function of o- binding sites, and moreover, to investigate the relationship between obinding sites and schizophrenia. References Bylund, D.B. and S.H. Snyder, 1976, Beta-adrenergic receptor binding in membrane preparations from mammalian brain. Mol. Pharmacol. 12, 568. Bylund, D.B. and H.I. Yamamura, 1990, Methods for receptor binding, in: Methods in Neurotransmitter Receptor Analysis, Eds. H.I. Yamamura, S.J. Enna and M.J. Kuhar (Raven Press, New York) p. 1. Cheng, Y.C. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 percent inhibition (Is0) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Craviso, G.L. and J.M. Musacchio, 1983a, High-affinity dextromethorphan binding sites in guinea pig brain. 1. Initial characterization, Mol. Pharmacol. 23, 619.
Craviso, G.L. and J.M. Musacchio, 1983b, High-affinity dextromethorphan binding sites in guinea pig brain. If. Competition experiments, Mol. Pharmacol. 23, 629. De Costa, B.R., W.D. Bowen, S.B. Hellewell, K.M. Walker, A. Thurkauf, A.E. Jacobson and K.C. Rice, 1989, Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for tr receptors, FEBS Lett. 251, 53. Gray, N.M., P.C. Contreras, S.E. Allen and D.P. Taylor, 1990, HI antihistamines interact with central sigma receptors, Life Sci. 47, 175. Gundlach, A.L., B.L. Largent and S.H. Snyder, 1985, Phencyclidine and a opiate receptors in brain: biochemical and autoradiographic differentiation, Eur. J. Pharmacol. 113, 465. Gundlach, A.L., B.L. Largent and S.H. Snyder, 1986, Autoradiographic localization of o--receptor binding sites in guinea pig and rat central nervous system with (+)-3H-3-(3-hydroxyphenyl)-N(1-propyl)piperidine, J. Neurosci. 6, 1575. Itzhak, Y., 1988, Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites, Life Sci. 42, 745. Karhon, E.W., K. Naper and M.J. Pontecorvo, 1991, [3H]DTG and [3H](+ )-3-PPP label pharmacologically distinct o- binding sites in guinea pig brain membranes, Eur. J. Pharmacol. 193, 21. Katz, J.L., R.D. Spealman and R.D. Clark, 1985, Stereoselective behavioral effects on N-allylnormetazocine in pigeons and squirrel monkeys, J. Pharmacol. Exp. Ther. 232, 452. Largent, B.L., A.U Gundlach and S.H. Snyder, 1984, Psychotomimetic opiate receptors labeled and visualized with ( + ) [3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, Proc. Natl. Acad. Sci. USA 81, 4983. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Martin, W.R., C.G. Eades, J.A. Thompson, R.E. Huppler and P.E. Gilbert, 1986, The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog, J. Pharmacol. Exp. Ther. 197, 517. McCann, D.J., R.A. Rabin, S. Rens-Domiano and J.C. Winter, 1989, Phencyclidine/SKF-10,047 binding sites: evaluation of function, Pharmacol. Biochem. Behav. 32, 87. McLean, S. and E. Weber, 1988, Autoradiographic visualization of haloperidol-sensitive sigma receptors in guinea-pig brain, Neuroscience 25, 259. Mendelsohn, L.G., V. Kalra, B.G. Johnson and G.A. Kerchner, 1985, Sigma opioid receptor: characterization and co-identity with the phencyclidine receptor, J. Pharmacol. Exp. Ther. 233, 597. Quirion, R., R. Chicheportich, P.C. Contreras, K.M. J o h n ~ n , D. Lodge, S.W. Tam, J.H. Woods and S.R. Zukin, 1987, Classification and nomenclature of phencyclidine and sigma receptor sites, Trends. Neurosci. 10, 444. Quirion, R., W.D. Bowen, Y. Itzhak, J.L. Junien, J.M. Musacchio, R.B. Rothman, T.P. Su, S.W. Tam and D.P. Taylor, 1992, A proposal for the classification of sigma binding sites, Trends. Pharmacol. Sci. 13, 85. Su, T.P., 1982, Evidence for sigma opioid receptor: binding of [3H]SKF-10047 to etorphine-inaccessible sites in guinea-pig brain, J. Pharmacol. Exp. Ther. 223, 284. Tam, S.W., 1983, Naloxone-inaccessible sigma receptor in rat central nervous system, Prec. Natl. Acad. Sci. USA 80, 6703. Tam, S.W. and L. Cook, 1984, o--Opiates and certain antipsychotic drugs mutually inhibit (+)-[3H]SKF10,047 and [3H]haloperidol binding in guinea pig brain membranes, Proc. Natl. Acad. Sci. USA 81, 5618. Tanaka, M., S. Chaki, Y. Imagawa, S. Okuyama, N. Muramatsu and S. Otomo, 1993, FH-510, a potent and selective ligand for rat brain a recognition sites. Eur. J. Pharmacol. 238, 89.
Vaupel, D.B., 1983, Naltrexone fails to antagonize the o effects of PCP and SKF 10,047 in the dog, Eur. J. Pharmacol. 92. 269. Walker, J.M., W.D. Boven, F.O. Walker. R.R. Matsumoto, B. De Costa and K.C. Rice, 1990, Sigma receptors: biology and function, Pharmacol. Rev. 42, 355. Weber. E., M. Sonders, M. Quarum, S. McLean, S. Pou and J.F.
Keana, 1986, 1,3-Di(2-[5-31-t]tolyl)guanidine: a selective ligand that labels sigma-type receptors for psychotomimetic opiates and antipsychotic drugs, Proc. Natl. Acad. Sci. USA 83, 8784. Zukin, S.R., K.T. Brady, B.L. Slifer and R.L. Balster, 1984, Behavioral and biochemical stereoselectivity of sigma opiate/PCP receptors, Brain Res. 294, 174.
93
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53153
Binding of [3H]FH-510 to or ligand recognition sites in guinea-pig brain membranes M a k o t o T a n a k a , Shinsuke Kaku, M a k o t o M u r a m a t s u and S u s u m u O t o m o Department of Pharmacology, Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan Received 24 March 1993; accepted 13 April 1993
We examined the characteristics of the binding of radiolabeled 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride ([3H]FH-510), a highly potent and selective tr ligand, to guinea-pig brain membranes. [3H]FH-510 showed saturable and reversible binding to cr binding sites. The association rate constant (k+l) and dissociation rate constant (k_ 1) of [3H]FH-510 were 0.023 min -1- nM -1 and 0.081 min -l, respectively. Scatchard plot analysis showed a dissociation constant (K d) and maximal number of binding sites (B,~ax) of 6.0 + 0.63 nM and 1763.3 -t- 177.4 fmol/mg protein (n = 7), respectively. The rank order of potency (K i) of several structurally dissimilar tr ligands obtained for the displacement of [3H]FH-510 binding was highly correlated with that determined for [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3HI(+)-3-PPP) binding. The binding of [3H]FH-510 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic or cholinergic agents at 10 -7 M. Higher [3H]FH-510 binding to brain regions was observed in the cerebellum and pons-plus-medulla. Except for the nuclear fraction, the highest level of [3H]FH-510 and [3H](+)-3-PPP binding to subceilular fractions was observed in the microsomal fraction. From these results, it is suggested that FH-510 selectively binds with high affinity to tr binding sites in guinea-pig membranes. tr Binding sites; FH-510 (5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochioride); Radiolabeled ligand binding; Brain (guinea-pig)
1. Introduction
The existence of or receptors was first postulated by Martin et al. (1976), to account for the psychotomimetic effects of N-allylnormetazocine (SKF10,047) and related racemic benzomorphans. Although tr binding sites were originally thought to be a type of traditional opiate receptor, several subsequent studies have revealed that they are not: naloxone is ineffective against the effects of or ligands, and enantiomers of benzomorphan opiates exhibit opposite effects against or and traditional opiate receptors (Su, 1982; Tam, 1983; Vaupel, 1983; Katz et al., 1985, Weber et al., 1986). Furthermore, in earlier reports, the binding sites of tr and of the psychotomimetic agent phencyclidine (PCP) were thought to be identical, based on the ability of ( + ) SKF10,047 to displace bound [3H]PCP (Zukin et al., 1984; Mendelsohn et al., 1985). However, substantial
Correspondence to: M. Tanaka, Department of Pharmacology, Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan. Tel. 048 (663) 1111, fax 048 (652) 7254.
evidence now indicates that or and PCP binding sites are distinct physiological entities (Gundlach et al., 1985; Quirion et al., 1987). In addition, the neuroleptic agent haloperidol, which possesses low affinity for PCP binding sites, displays high affinity for [3H](+)-SKF10,047 binding sites (Tam and Cook, 1984; Itzhak, 1988). The binding sites of tr ligands may play a role in schizophrenia, since many antipsychotic drugs bind to the sites with high affinity (Tam and Cook, 1984). Although haloperidol is the most potent inhibitor of [3H](+)-SKF10,047 binding (Tam and Cook, 1984; Itzhak, 1988), because it has approximately the same affinity for the dopaminergic receptor (Tam and Cook, 1984; Itzhak, 1988) it is not a good drug to verify the involvement of tr ligand binding sites in schizophrenia. In recent years, more selective or ligands, such as ( + )-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)3-PPP) and 1,3-di-o-tolylguanidine (DTG), have been identified, and radiolabeled compounds of these ligands are available and are used to label or binding sites in brain tissues (Largent et al., 1984; Weber et al., 1986; Itzhak, 1988). These agents have moderate affinity for or binding sites, but novel potent and selective or
94
ligands arc needed to explore the relationship between ~r binding sites and schizophrenia, l)cCosta ct al. (1989) have reported that [3H](+)-pcntazocine has potent and selective affinity for o- binding sites, with a potency comparable to that of haloperidol. In our previous study, wc also reported the identification of a novel potent and selective ~r ligand, 5,8-dimcthyl-4-(2-di-npropylaminoethyl)carbazol monohydrochloridc (FH510), obtained from carbazol derivatives (Tanaka et al., 1993). In the study, FH-510 showed a very potent inhibitory effect on (+)-SKF10,047-induccd behavior in mice (Tanaka ct al., 1993). In the present study, wc characterized the binding of radiolabelcd FH-510 to guinea-pig brain membranes.
2. Materials and methods
2.1. Materials The following drugs were purchased from Research Biochemicals Inc. (Natick, MA): haloperidol, ( - ) - and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine hydrochloride (3-PPP), ( - ) - and (+)-N-allylnormetazocine hydrochloridc (SKF10,047), 1,3-di-o-tolylguanidine (DTG), rimcazoic dihydrochloridc, (+)-7-chloro-8-hydroxy-3-methyl- 1-phcnyl-2,3,4,5-tet rahydro- 1H-3-benzazcpine hydrochloride (SCH23390), (+)-8-hydroxy-2(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), ritanserin and 5,5-diphenylhydantoin sodium (phenytoin). Dcxtromethorphan hydrobromide, pyrilamine maleate (mepyramine), tiapridc hydrochloride, phentolamine, dl-propranolol hydrochloride, and atropine were purchased from Sigma Chemical Co. (St. louis, MO). (+)-Pcntazocine and famotidine were prepared from commercially available medicines, Pcntagin and Gastcr, which were obtained from Sankyo Co. (Tokyo, Japan) and Yamanouchi Pharmaceutical Co. (Tokyo, Japan), respectively. 5,8-Dimcthyl-4-(2-din-propylaminoethyl)carbazol monohydrochloride (FH510, fig. 1), ( - ) - and (+)-a-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-l-piperazinc butanol (BMY14802), and phencyclidine (PCP) were synthesized in the Department of Organic Chemistry at our Research Center. [3H]FH-510 (specific activity: 29.1 Ci/mmol) was
.HCI
Fig. 1. Chemical structure of 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride (FH-510).
synthesized at Chemsyn Science Laboratories (Lcncxa. KA). [~H]( + )-3-PPP (117.0 Ci/mmol) was purchased from Du Pont/New England Nuclear (Wilmington, DE). All other chemicals wcrc of the highest purity available.
2,2. Preparation of guinea-pig brain membranes for receptor binding Male Hartley guinea-pigs (Japan SLC, Hamamatsu, Japan; 350-400 g) were decapitated and thc whole brains or seven dissected sites (cerebral cortex, hippocampus, striatum, thalamus-plus-midbrain, hypothalamus, pons-plus-medulla, and cerebellum) were homogenized in 10 volumes of ice-cold 10 mM Tris-HCI buffer (pH 7.4), using a Physcotron homogenizer (NitiOn Medical and Physiological Instruments Mfg., Funabashi, Japan), and centrifuged in a Beckman L8-70 centrifuge at 48,000 × g for 10 rain at 4°C. The pellets obtained were rehomogenized in the same buffer and rccentrifuged. The final membrane suspension for use in the receptor binding assay was diluted in 40 volumes of 10 mM Tris-HCl buffer (pH 7.4) to a protein concentration of 0.8-1.5 mg/ml. Radioligand binding was found to be linear with respect to protein concentration within this range. Protein concentration was determined by the method of lowry et al. (1951).
2.3. Preparation of guinea-pig brain subcellular fractions Subcellular fractions of guinea-pig brain were obtained by differential centrifugation and discontinuous sucrose gradient, as described elsewhere (Bylund and Snyder, 1976). Whole brain was homogenized in 10 volumes of ice-cold 0.32 M sucrose, using a motordriven Teflon pestle glass homogenizer. After centrifugation at 900 x g for 10 min, the crude nuclear pellet was rehomogenized in 0.32 M sucrose and centrifuged at the same speed. The combined supernatant was centrifuged at 17,500 x g for 20 min, and the pellet obtained was the crude mitochondrial fraction. The supernatant was centrifuged at 100,000 x g for 60 min to obtain the crude microsomal pellet. To separate myelin, synaptosomes, and mitochondria, the crude mitochondrial fraction was resuspended in 0.32 M sucrose and layered o n t o a discontinuous gradient of 0.8 and 1.2 M sucrose (1:1). After centrifugation at 100,000 x g for 90 min at 4°C, the following fractions were collected: 0.32-0.8 M sucrose (myelin), 0.8-1.2 M sucrose (synaptosomes), and the pellet at the bottom of 1.2 M sucrose (mitochondria). All fractions except microsomes were diluted 5-fold with 10 mM Tris-HCl buffer (pH 7.4) and centrifuged at 48,000 x g for 15 min at 4°C. The resulting pellets were resuspended in 10 mM Tris-HCl buffer (pH 7.4) and were used in
95 receptor binding assays to study subcellular distribution.
2. 4. Receptor binding assay For a typical binding experiment, the reaction was initiated by adding 1 ml of membrane suspension to polypropylene test tubes containing 2.0-2.5 nM of [3H]FH-510. The reaction mixture was incubated for 30 min at 25°C, a time period that allowed equilibrium (see kinetic experiments in the Results section). Nonspecific binding was determined in the presence of unlabeled FH-510 (10 /~M). Specific binding was defined as the difference between the total binding and nonspecific binding, and was 55-65% of the total binding. The reaction was terminated by rapid filtration through Whatman G F / B glass fiber filters that were presoaked in 0.5% polyethyleneimine for 4 h, after which the filters were washed 3 times with 5 ml of ice-cold 10 mM Tris-HCl buffer (pH 7.4). These steps were performed with a multiple cell harvester M-24R (Brandel Biomedical Research and Development Laboratories, Gaithersburg, MD). Aquazol-2-scintillator (Du Pont/New England Nuclear) (8 ml) was added and filter-bound radioactivity was quantified in a liquid scintillation spectrometer (Aloka, LSC-3500, Tokyo, Japan). The [3H]( +)-3-PPP binding assay was carried out as described above, except that nonspecific binding was determined in the presence of 10/xM (+)-3-PPP. For determination of the equilibrium dissociation constant (Ko), 0.5-25 nM of [3H]FH-510 was used. Saturation binding data were analyzed by Scatchard plot analysis, and the K d and the maximal number of binding sites (Bmax) were calculated using a computer program, SP123, developed by Dr. H. Ono of the University of Tokyo for the PC-9801 (NEC, Tokyo, Japan) personal computer. Association kinetics were determined using two concentrations of [3H]FH-510 (1.77 and 5.03 nM), and pseudo first-order association rates were calculated as described previously (Bylund and Yamamura, 1990). After equilibrium had been reached (30 min, 25°C), dissociation experiments were initiated by adding excess unlabeled FH-510 (10 ~M). Dissociation rate constants were calculated from the slope obtained after linear transformation of the data (Bylund and Yamamura, 1990). In the competition binding assay, the concentration of test compound causing 50% inhibition of specific [3H]FH-510 or [3H](+)-3-PPP binding (IC50) was determined from concentration-response curves, in which 10 concentrations (from 3 × 10 -t° to 1 × 10 -5 M) of test compound were examined. After determination of IC50 values by the Marquardt-Levenberg nonlinear curve-fitting procedure of the RS/1 program (BBN Research System, Cambridge, MA) running on a
VAX/VMS system, K i values for each test compound were calculated according to the equation of Cheng and Prusoff (1973), using the K d value obtained by Scatchard analysis ([3H]FH-510; 6.0 nM) and from published data ([3HI( +)-3-PPP; 43 nM) (Karbon et al., 1991).
3. Results
3.1. Time course of [3H]FH-510 binding to guinea-pig brain membranes At 25°C, the binding of [3H]FH-510 (1.77 nM) to guinea-pig brain membranes was increased in a timedependent manner and reached equilibrium at 20 min (fig. 2). After [3H]FH-510 binding reached a plateau, an excess of unlabeled FH-510 (10/xM) was added. As shown in fig. 2, displacement of [3H]FH-510 from its binding sites occurred immediately. Almost complete displacement was achieved 30 min after the addition of unlabeled FH-510.
3.2. Association and dissociation kinetics of [ 3H]FH-510 binding to guinea-pig brain membranes Association experiments were performed using two concentrations of [3H]FH-510 (1.77 and 5.03 nM). The association rate constant (k+t) and the dissociation rate constant (k_l) were determined by using the following equation: kobS= k+l • [L] + k t (ko~, observed apparent rate constant; [L], concentration of ligand). The k÷l and k ~ values calculated by using 2 kob, derived from the slope of pseudo first-order rate plots (fig. 3A) were 0.023 + 0.005 min -1 •nM -1 and 0.114 -t0.025 min-i, respectively (table 1). The K d value given by K a = k_ J k + i was 4.96 nM. The k_ l value was also
10 pM FH-510
l 20(3
0
'100
z ___o ,?,. o 03
~) 1
i
i
i
20
30
40
i
SO
i
60
Time (rain)
Fig. 2. Time course of [3H]FH-510 binding to guinea-pig brain membranes. Guinea-pig brain membranes were incubated with 1.77 nM [3H]FH-510 at 25°C. After equilibrium has been reached (30 min), 10/~M of unlabeled ligand was added to initiate displacement of [3H]FH-510from its specificbinding sites. The data are means of duplicate determinations.
96
3
O
(A)
T~me (rain) 10 20
"
'
(A) 30
'
3
o
o o-
--2 I•o
c2
},
~,
o
o
~E I- 6 u- E
0
10
20
13HIFH-510 (nM) ,
,
,
5
10
15
I(B) (B)
04i°
Time (rmn)
Fig. 3. Kinetic analysis of [31-I]FH-510 binding to guinea-pig brain membranes. (A) Guinea-pig brain membranes were incubated with 1.77 nM (©) and 5.73 nM (e) [3H]FH-510 at 25°C for the indicated times. B~ is the specific binding at equilibrium, and B is the amount of [3H]FH-510 specifically hound at any indicated time. Each point is the mean of two separate experiments, each done in duplicate. (B) After equilibrium had been reached (30 rain), 10 /~M of unlabeled ligand was added to initiate displacement of [3HlFH-510 from its specific binding sites. B 0 is the amount of []H]FH-510 at time zero. Each point is the mean of four separate experiments, each done in duplicate. Vertical bars show S.E. of mean.
determined from the slope obtained after linear transformation of the data for the dissociation time course in fig. 2 (fig. 3B and table 1). The k_ ~ value obtained from the dissociation rate experiment was 0.081 + 0.002 min--1. The K d value was calculated to be 3.52 nM.
3.3. Saturation binding of [3H/FH-510 to guinea-pig brain membranes The characteristics of the binding of [3H]FH-510 to guinea-pig brain membranes were studied further. The saturation studies of the binding of [3H]FH-510 revealed that nonspecific binding increased linearly with increasing concentrations of [3H]FH-510, whereas specific binding, determined by subtracting nonspecific binding from total binding, was saturable (fig. 4A).
TABLE 1 Association and dissociation rate experiments of [3H]FH-510 binding to guinea-pig brain membranes. Experiments were performed as described in the legend of fig. 3. Values are m e a n s ± S.E. of 3 - 4 separate experiments, each done in duplicate. Association rate e r p e H m e n t (n = 3)
k + ~ ( m i n 1. n M k l(min-1) k d (k_ ~ / k + l)
i)
0.023 ± 0.005 0.114+0.025 4.96
Dissociation rate experiment (n = 4)
k 1( m i n - 1 ) K d (k_i/k.l)
0.081±0.(X)2 3.52
E
"5 ,'n ~ 0 2
i
0
1 B (pmol/mg
2
protein)
Fig. 4. Saturation isotherm and Scatchard plot of [~H]FH-5]0 binding to guinea-pig brain membranes. Guinea-pig brain membranes were incubated with increasing concentrations of [3H]FH-5]0 at 25°C for 30 min. (A) Representative saturation curves for the equilibrium binding of [3H]FH-5]0 arc shown. Essentially the same results were obtained from seven independent experiments, done in duplicate. Total binding, nonspecific binding, and specific binding are indicated as o, O, and o, respectively. (B) Scatchard plot of the same data as in (A). Mean values o f K d and Bm~, are 6.00+0.63 nM and 1763.3 _+ ]77.4 fmol/mg protein, respectively.
Scatchard analysis of these data indicated the existence of a single class of binding sites in guinea-pig brain membranes (fig. 4B). The K a and Bmax values were calculated to be 6.00+0.63 nM and 1763.3_+ 177.4 f m o l / m g protein (n = 7), respectively.
3.4. Displacement of [SH]FH-510 binding by various cr ligands and neurotransmitter-related compounds Several structurally dissimilar ~ ligands were examined for their abilities to compete with the specific binding of [3H]FH-510 (fig. 5) and [3H](+)-3-PPP to guinea-pig brain membranes (table 2). [3H]FH-510 binding was concentration dependently inhibited by unlabeled FH-510. The Hill coefficient (nrt) and the K i value of FH-510 were 0.86 and 15.1 + 2.7 nM, respectively. The K i value and n H for FH-510 were close to that of haloperidol (13.1 + 3.8 nM and 0.82). The n u values of other o- ligands for [3H]FH-510 binding were as follows: (+)-3-PPP, 0.66; DTG, 0.72; ( + ) - S K F 10,047, 0.63. Markedly different K i values between ( + ) and ( - ) enantiomers of benzomorphan were observed. Competition of various ~ ligands against [3H]FH-510
97 100
¢.
TABLE 3
~5 t-
Effect of various neurotransmitter-related compounds on [3H]FH-510 binding to guinea-pig brain membranes.
A
9
Binding experiments were carried out as described in Materials and methods. The [3H]FH-510 concentration was 2.0-2.5 nM. Values are means + S.E. of four separate experiments, each done in duplicate.
"1" o5(] "5 0
% Inhibition of [3H]FH-510 binding
O
10-9 M
10-7 M
10-s M
-2.1+ 6.4 8.25:3.7 2.05:3.3 -1.9+ 6.7 - 1.9 + 6.8 0.6:t:15.1 -0.6+ 2.7 - 2.6 5:4.3 -10.15:3.1
4.0+ 2.2 8.7+ 4.1 9.05:6.0 -4.4+10.9 5.5 + 8.9 -0.6-1-10.1 1.4+- 3.2 - 3.7 5:4.4 -8,9+ 4.2
67.85:6.3 3.25:3.8 25.6+ 2.8 42.2+- 2.0 10.5 + 10.7 66.55:4.6 63.25:2.2 35.9 5:7.5 16.1+- 3.3
t'~
O9
9
B
7
~)
5
-log Concentration (M)
Fig. 5. Effects of several representative o- ligands on [3H]FH-510 binding to guinea-pig brain membranes. Guinea-pig brain membranes were incubated with increasing concentrations of sigma ligands in the presence of 2 nM [3H]FH-510. The results indicated are the mean value of three separate experiments, each done in duplicate. Key: FH-510 (o), haloperidol (e), DTG (a), (+)-3-PPP (v), and ( + )-SKFI0,047 ( [] ).
b i n d i n g revealed the following r a n k o r d e r of p o t e n cy: FH-510 = haloperidol > ( + ) - p e n t a z o c i n e > D T G > ( + )-3-PPP = ( + )-BMY14802 > ( + )-SKF10,047 >> dextromethorphan = progesterone > (-)-BMY14802 > rimcazole > PCP > ( - ) - 3 - P P P >> ( - ) - S K F 1 0 , 0 4 7 . A similar tr iigand selectivity of the c o m p o u n d s was observed for [ 3 H ] ( + ) - 3 - P P P b i n d i n g to g u i n e a - p i g b r a i n m e m b r a n e s (table 2). M a n y kinds of n e u r o t r a n s m i t t e r - r e l a t e d c o m p o u n d s were e x a m i n e d to identify the b i n d i n g sites of [3H]FH510, using c o n c e n t r a t i o n s of 10 -9, 10 -7 a n d 10 -5 M
(table 3). T h e r e were no c o m p o u n d s that i n h i b i t e d or e n h a n c e d the b i n d i n g of [3H]FH-510 to g u i n e a - p i g brain m e m b r a n e s a m o n g the histaminergic, d o p a m i n e r gic, adrenergic, serotonergic a n d cholinergic agents tested at 10 -9 a n d 10 -7 M. However, m e p y r a m i n e (H~ r e c e p t o r antagonist), p r o p r a n o l o l (/3-receptor antagonist) a n d 8 - O H - D P A T (5-HT1A r e c e p t o r agonist) inhibited [3H]FH-510 b i n d i n g by approximately 60% at 10-5 M. P h e n y t o i n (10 - 6 ~ 10 -4 M) did not e n h a n c e or suppress [3H]FH-510 b i n d i n g to g u i n e a - p i g b r a i n m e m b r a n e s (data not shown).
3.5. Regional and subcellular distribution of [ 3H ] FH- 510 binding to guinea-pig brain membranes
TABLE 2 Effect of various ~r ligands on [3H]FH-510 and [3H](+)-3-PPP binding to guinea-pig brain membranes. Competition binding was carried out as described in Materials and methods. The K i values were determined according to the equation of Cheng and Prusoff (1973), using the ICs0 values derived from the inhibition data and Ka values. The K d values for FH-510 (6.0 nM) and (+)-3-PPP (43 nM) were obtained by Scatchard analysis indicated in fig. 4 and from Karbon et al. (1991), respectively. Values are means5: S.E. of 2-9 separate experiments indicated in parentheses, each done in duplicate. Compounds
Mepyramine Famotidine SCH23390 Tiapride Phentolamine Propranolol 8-OH-DPAT Ritanserin Atropine
K i (nM) [3H]FH-510
I n o r d e r to investigate the characteristics of [3H]FH-510 b i n d i n g in g u i n e a - p i g b r a i n m e m b r a n e s further, the regional a n d subcellular d i s t r i b u t i o n of [3H]FH-510 b i n d i n g was examined. In these experim e n t s the cortex, h i p p o c a m p u s , s t r i a m m , t h a l a m u s p l u s - m i d b r a i n , hypothalamus, p o n s - p l u s - m e d u l l a a n d the c e r e b e l l u m were used. [3H]FH-510 b i n d i n g was TABLE 4
[3HI( + )-3-PPP
FH-510 15.1:t: 2.7 (9) 2 . 7 8 5 : 0.05 Haloperidol 13.1:1: 3.8 (3) 1.55 + 0.34 (+)-3-PPP 194.2+ 49.3 (4) 12.3 (-)-3-PPP 4865.8+ 742.3 (4) (+)-SKF10,047 337.6+ 85.7 (5) 67.4:1:14.5 ( - )-SKF10,047 11324.3+-2820 (4) (+)-BMYI4802 207.75: 5.8 (3) 23.4 5 : 4 . 8 (-)-BMY14802 2359.8+ 634.0 (4) DTG 145.0+ 64.6 (3) 23.1 5: 3.1 (+)-Pentazocine 100.8+ 22.0 (3) 9.90+ 0.55 Dextromethorphan 1238.8+- 363.4 (5) 120.1 5: 8.8 Rimcazole 3042.5+ 797.6 (4) 1269.7 +-176.6(3) PCP 3822.05:584.2 (3) Progesterone 1325.7+ 244.0 (3)
(3) (3) (2)
Regional distribution of [3H]FH-150 binding in guinea-pig brain membranes. Binding experiments were carried out as described in Materials and methods. The [3H]FH-510 concentration was 2.0-2.5 nM. Values are means+ S.E. of three separate experiments, each done in triplicate.
(3)
Specific binding (fmol/mg protein)
(3) (3) (3) (3)
Cortex Hippocampus Striatum Thalamus + midbrain Hypothalamus Ports+medulla Cerebellum
358.2+- 15.8 369.2 +- 6.0 320.0 +- 13.0 485.8 5:20.5 527.9 5:40.4 571.1 +- 8.7 603.5 +-43.1
9~ TAHt.E 5 Subccllular distribution of [ 3HJFI I-510 and [ 3 t-t]( + )-3-PPP binding to guinea-pig brain membranes. Binding experiments were carried out as described in Materials and methods. The [ 3H]FH-5111 and [ 3 H}( + )-3-PPP concentrations were 2.0-2.5 nM. Nonspecific binding of [3H}( 3-)-3-PPP was determined in the presence of 1 ;zM haloperidol. Values are m e a n s + S.E. of 5 - 6 determinations. Binding ( f m o l / m g protein)
Nuclei Myelin Synaptosomes Mitochondria Microsomes
[ 3H]Ft I-510
[3ti}( + )-3-PPP
815.9 _+212.4 277.9+ 38.8 287.5 + 44.5 321.1 5- 21.1 603.6 + 127.1
21.88 ~- 1.67 9.22+ 1.18 13.59 +_2.38 13.42+ 1.21 35.63 + 5.[YO
rich in the cerebellum as well as in the pons-plusmedulla (table 4). However, the lowest and highest binding densities differed by about 50%. As indicated in table 5, the highest level of [3H]FH510 binding observed was in the nuclear fraction, followed by the microsomal fraction. Despite the diversity of order in these fractions, higher levels of [3H]( + )-PPP binding were also observed in these fractions compared with other fractions. The binding of [3H]FH-510 and [3H]( + )-3-PPP to the other fractions (myelin, synaptosomes and mitochondria) was only 40-50% of that detected in the microsomal fraction.
4. Discussion
In the present study, the characteristics of [3H]FH510 binding to guinea-pig brain membranes were examined. The binding of [3H]FH-510 was time dependent and reached a plateau at 20 min, and the immediate displacement of bound [3H]FH-510 on the addition of unlabeled FH-510 demonstrated the reversible nature of [3H]FH-510 binding. Moreover, [3H]FH-510 bound to guinea-pig brain membranes in a saturable manner, and Scatchard plot analysis showed a single class of binding sites for [3H]FH-510. The K d values (4.96 and 3.52 nM) calculated using the k+ t and k_ 1 (Ka = k_ l / k+~), each obtained from association and dissociation experiments, were quite similar to those obtained from Scatchard plot analysis (6.0 nM). These values are about 10 times lower than those reported for [3H]( + )3-PPP and [3H]DTG (Largent ct al., 1984; Weber et al., 1986; Karbon et al., 1991). In order to verify that the [3H]FH-510 binding site in guinea-pig brain membranes is a t r binding site, the inhibitory effects of a number of structurally dissimilar cr ligands on [3H]FH-510 binding were studied and compared with their effects on [3H]( + )-3-PPP binding. Of these ligands, haioperidol was the most potent
compound (K, = 13.1 nM), and FH-510 showed almost the same effect ( K i = 15.1 nM) against [3H]FH-510 binding to guinea-pig brain membranes. Furthermore, differences in the binding affinities of stereoisomers of benzomorphan (SKF10,047), 3-PPP and BMY14802 for the binding site of [3H]FH-510 were studied, since a clear difference between the affinities of these stereoisomers ( ( + ) > > ( - ) ) is a typical criterion for confirming that the site is a o" binding site (Walker et al., 1990). There was a large difference between the potencies of the ( + ) and ( - ) stereoisomers, q'he rank order of potency to inhibit [3H]FH-510 binding was: FH-510 = haloperidol > (+)-pentazocine > DTG > ( + )-3-PPP = ( + )-BMYI4802 > ( + )-SKF10,047 >> dextromethorphan = progesterone > ( - ) - B M Y 1 4 8 0 2 > rimcazole > PCP > ( - )-3-PPP >> ( - )-SKFI0,047. The potency of these compounds was quite similar to that for [3H](+)-3-PPP binding obtained in the present study, and was consistent with that reported previously (Largent et al., 1984; Tam and Cook, 1984; Weber et at., 1986; Itzhak, 1988; Karbon et al., 1991). These results clearly suggest that FH-510 binds to o- binding sites with higher affinity than o- ligands, including ( + ) - 3 - P P P and DTG. In addition, the marked difference in affinity between ( + ) and (-)-SKF10,047 suggests that [3H]FH-510 is relatively selective for o-1 type (Quirion et al., 1992) binding sites. Neurotransmitter-related compounds, including histaminergic, dopaminergic, adrenergic, serotonergic and cholinergic agents, had little influence on [3H]FH-510 binding to guinea-pig brain membranes at 10 - 9 ~ 10 ...7 M. Although at 10 -5 M, mepyramine, propranolol and 8-OH-DPAT inhibited [3H]FH-510 binding by approximately 60%, these effects do not detract from the high specificity of [3H]FH-510 for cr binding sites. These compounds have low to moderate affinity for o- binding sites, as reported previously: (1) Tam (1983) and Craviso and Musacchio (1983b) reported inhibition of [3H]ethylketocyclazocine and [3H]dextromethorphan binding by both mepyramine and propranolol, (2) H~ antihistamines probably interact with o- binding sites due to a strong structural resemblance to the tricyclic antidepressants and neuroleptic agents (Gray et al., 1990), and (3) due to the similar structural resemblance of 8-OH-DPAT to benzomorphan. Considering these data together, the present results strongly suggest that the binding site of [3H]FH-510 is a ~r binding site. Autoradiographic studies with [ 3 H ] ( + ) - 3 - P P P (Gundlach et al., 1986) and [3H]DTG (McLean and Weber, 1988) revealed that o- binding sites in guineapig brains are more concentrated in motor areas and neurons, including the cerebellum and cranial nerve nuclei, than in limbic areas. [3H]Dextromethorphan binding sites are also distributed in the pons-medulla and cerebellum (Craviso and Musacchio, 1983a). In the present study, the regional distribution of [3H]FH-510
99
binding in seven dissected brain regions was investigated. Although no marked difference in the density of the binding sites between the highest and the lowest regions was observed, the cerebellum had the highest number of [3H]FH-510 binding sites, followed by the pons-plus-meduila. These findings also suggest the possibility that FH-510 binds to o" binding sites. The subcellular localization of tr binding sites has been reported by Craviso and Musacchio (1983a) and McCann et al. (1989), using [3H]dextromethorphan and [3H](+)-SKF10,047, respectively. Specific [3H]dextromethorphan binding to the subcellular fractions from guinea-pig brain was found in the nuclear, mitochondrial, synaptosomal and microsomal fractions, but high-affinity sites were found almost exclusively in the microsomal fractions (Craviso and Musacchio, 1983a). Furthermore, the level of [3H](+)-SKF10,047 binding in rat brain microsomes was 4-fold higher than that in synaptosomes (McCann et al., 1989). The present resuits show a higher density of both [3H]FH-510 and [3H](+)-3-PPP binding sites in the nuclear and microsomal fractions. The affinity of [3H]FH-510 for each subcellular fraction was not determined, and because the relative purity of these fractions was not assessed by assay of marker enzymes (5'-nucleotidase, choline acetyltransferase, and cytochrome c reductase) or DNA content, a clear explanation for the higher levels in the nuclear fractions cannot be given. The possibility that the higher [3H]FH-510 binding in the nuclear fraction was caused by nonspecific binding to DNA or chromosomes cannot be ruled out. However, considering the present data together, it is conceivable that FH-510 and the tr ligand share the same high-affinity binding sites. In conclusion, FH-510 was found to be a specific, selective and potent tr ligand. Radiolabeled FH-510 could be used as a ligand for labeling tr binding sites in the central nervous system. Thus FH-510 might be a good tool to clarify the function of o- binding sites, and moreover, to investigate the relationship between obinding sites and schizophrenia. References Bylund, D.B. and S.H. Snyder, 1976, Beta-adrenergic receptor binding in membrane preparations from mammalian brain. Mol. Pharmacol. 12, 568. Bylund, D.B. and H.I. Yamamura, 1990, Methods for receptor binding, in: Methods in Neurotransmitter Receptor Analysis, Eds. H.I. Yamamura, S.J. Enna and M.J. Kuhar (Raven Press, New York) p. 1. Cheng, Y.C. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 percent inhibition (Is0) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Craviso, G.L. and J.M. Musacchio, 1983a, High-affinity dextromethorphan binding sites in guinea pig brain. 1. Initial characterization, Mol. Pharmacol. 23, 619.
Craviso, G.L. and J.M. Musacchio, 1983b, High-affinity dextromethorphan binding sites in guinea pig brain. If. Competition experiments, Mol. Pharmacol. 23, 629. De Costa, B.R., W.D. Bowen, S.B. Hellewell, K.M. Walker, A. Thurkauf, A.E. Jacobson and K.C. Rice, 1989, Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for tr receptors, FEBS Lett. 251, 53. Gray, N.M., P.C. Contreras, S.E. Allen and D.P. Taylor, 1990, HI antihistamines interact with central sigma receptors, Life Sci. 47, 175. Gundlach, A.L., B.L. Largent and S.H. Snyder, 1985, Phencyclidine and a opiate receptors in brain: biochemical and autoradiographic differentiation, Eur. J. Pharmacol. 113, 465. Gundlach, A.L., B.L. Largent and S.H. Snyder, 1986, Autoradiographic localization of o--receptor binding sites in guinea pig and rat central nervous system with (+)-3H-3-(3-hydroxyphenyl)-N(1-propyl)piperidine, J. Neurosci. 6, 1575. Itzhak, Y., 1988, Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites, Life Sci. 42, 745. Karhon, E.W., K. Naper and M.J. Pontecorvo, 1991, [3H]DTG and [3H](+ )-3-PPP label pharmacologically distinct o- binding sites in guinea pig brain membranes, Eur. J. Pharmacol. 193, 21. Katz, J.L., R.D. Spealman and R.D. Clark, 1985, Stereoselective behavioral effects on N-allylnormetazocine in pigeons and squirrel monkeys, J. Pharmacol. Exp. Ther. 232, 452. Largent, B.L., A.U Gundlach and S.H. Snyder, 1984, Psychotomimetic opiate receptors labeled and visualized with ( + ) [3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, Proc. Natl. Acad. Sci. USA 81, 4983. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Martin, W.R., C.G. Eades, J.A. Thompson, R.E. Huppler and P.E. Gilbert, 1986, The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog, J. Pharmacol. Exp. Ther. 197, 517. McCann, D.J., R.A. Rabin, S. Rens-Domiano and J.C. Winter, 1989, Phencyclidine/SKF-10,047 binding sites: evaluation of function, Pharmacol. Biochem. Behav. 32, 87. McLean, S. and E. Weber, 1988, Autoradiographic visualization of haloperidol-sensitive sigma receptors in guinea-pig brain, Neuroscience 25, 259. Mendelsohn, L.G., V. Kalra, B.G. Johnson and G.A. Kerchner, 1985, Sigma opioid receptor: characterization and co-identity with the phencyclidine receptor, J. Pharmacol. Exp. Ther. 233, 597. Quirion, R., R. Chicheportich, P.C. Contreras, K.M. J o h n ~ n , D. Lodge, S.W. Tam, J.H. Woods and S.R. Zukin, 1987, Classification and nomenclature of phencyclidine and sigma receptor sites, Trends. Neurosci. 10, 444. Quirion, R., W.D. Bowen, Y. Itzhak, J.L. Junien, J.M. Musacchio, R.B. Rothman, T.P. Su, S.W. Tam and D.P. Taylor, 1992, A proposal for the classification of sigma binding sites, Trends. Pharmacol. Sci. 13, 85. Su, T.P., 1982, Evidence for sigma opioid receptor: binding of [3H]SKF-10047 to etorphine-inaccessible sites in guinea-pig brain, J. Pharmacol. Exp. Ther. 223, 284. Tam, S.W., 1983, Naloxone-inaccessible sigma receptor in rat central nervous system, Prec. Natl. Acad. Sci. USA 80, 6703. Tam, S.W. and L. Cook, 1984, o--Opiates and certain antipsychotic drugs mutually inhibit (+)-[3H]SKF10,047 and [3H]haloperidol binding in guinea pig brain membranes, Proc. Natl. Acad. Sci. USA 81, 5618. Tanaka, M., S. Chaki, Y. Imagawa, S. Okuyama, N. Muramatsu and S. Otomo, 1993, FH-510, a potent and selective ligand for rat brain a recognition sites. Eur. J. Pharmacol. 238, 89.
Vaupel, D.B., 1983, Naltrexone fails to antagonize the o effects of PCP and SKF 10,047 in the dog, Eur. J. Pharmacol. 92. 269. Walker, J.M., W.D. Boven, F.O. Walker. R.R. Matsumoto, B. De Costa and K.C. Rice, 1990, Sigma receptors: biology and function, Pharmacol. Rev. 42, 355. Weber. E., M. Sonders, M. Quarum, S. McLean, S. Pou and J.F.
Keana, 1986, 1,3-Di(2-[5-31-t]tolyl)guanidine: a selective ligand that labels sigma-type receptors for psychotomimetic opiates and antipsychotic drugs, Proc. Natl. Acad. Sci. USA 83, 8784. Zukin, S.R., K.T. Brady, B.L. Slifer and R.L. Balster, 1984, Behavioral and biochemical stereoselectivity of sigma opiate/PCP receptors, Brain Res. 294, 174.