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Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites
Life Sciences, Vol. 42, pp. 745-752 Printed in the U.S.A.
Pergamon Journals
PHARMACOLOGICAL SPECIFICITY OF SOME PSYCHOTOMIMETIC AND ANTIPSYCHOTIC AG~ITS FOR TSESIC, MAANDPCP BINDING SITES
YOSSEF ITZHAK Dept. of Pharmacology, Hadassah School of Medicine, The Hebrew University, Jerusalem, Israel (Received in final form December 8, 1987)
The pharmacological specificity of representative psychotomimetic agents such as phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with [3H]-I-[I(3-hydroxyphenyl) cyclohexyl] piperldine ([3H]PCP-3-OH), (+)[=H]-N-allylnormetazocine [(+)[3H]SKF 10047] and (+)[~H]-3-[3-hydroxy-phenyl]-N-(1-propyl)piperidine [(+)[3H]-3-PPP]. PCP analogs inhibit potently high affinity [3H]PCP-3-OH binding and (+)[3H]SKF 10047 binding, moderately the low affinity binding component of [3H]PCP-3-OH and very weakly (+)[3H]-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+)[3H]SKF 10047, high affinity [3H]PCP-3-OH and (+)[SH]-3-PPP binding, but extremely weak inhibitors of low affinity [sH]PCP-3-OH binding. The antipsychotic 3 agents display high affinity for (+)[ H]-3-PPP binding sites, moderate affinity for (+)[3H]SKF 10047 sites and have no effect on either the high or low affinity [3H]PCP-3-OH binding. The present data further support the existence of multiple sigma and PCP binding sites. Specific binding sites for the psychotomimetic agents phencyclidine (PCP) and opiate benzomorphans (N-allylnormetazocine; SKF 10047 and c~clzocine) were first labeled and identified, in rat CNS, by utilizing [~H]PCP (I-3). The rank order of potency of PCP analogs and opiate benzomorphans to inhibit [sH]PCP specific binding is consistent with their potency in behavioral experiments (4-5) related to psychotomimetic activity in man. Similarly, the drug specificity of (+)[3H]SKF 10047 binding sites is consistent with that of [sH]PCP binding sites (6,7). These radioreceptor binding studies along with drug discriminative experiments in rats and monkeys (8,9) have suggested the existence of a common dextrorotatoryopioid/PCP binding site for both PCP analogs and psychotomimetic opiates. Additional studies have revealed that (+)[3H]SKF 10047 specific binding is associated not only with a PCP-sensitive binding component (12,13), but also with an haloperidol-sensitive and PCP-insensitive binding component (10-13). The latter site was also labeled and visualized with the dopamine autorecepter agonist 3-(3-hydroxyphenyl)-N-(1-propyl) piperidine (3-PPP; Ref. 14) and displays drug specificit~ similar to that of the haloperidolsensitive binding component of (+)[~H]SKF 10047 (12,15). 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Journals Ltd.
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Furthermore, binding of one of the most potent analogs of PCP i.e. 1-[1(3-hydroxyphenyl)cyclohexyl]piperidine (PCP-3-OH) in rat brain membranes revealed the labeling of two distinct [~H]PCP-3-OH binding sites (16). High affinity [3H]PCP-3-OH binding is associated with one component of (+)SKF 10047 binding, whereas low affinity [3H]PCP-3-OH sites are insensitive to (+)SKF 10047. These two sites are apparently distinct from the haloperidolsensitive/ (+)[3H]SKF 10047 site (16). The present study describes the pharmacological specificity of several PCP analogs, psychotomimetic benzomorphans and antipsychotic drugs for: (i) High affinity [3H]PCP-3-OH/(+)[3H]SKF 10047 binding site, (ii) Low affinity [3H]PCP-3-OH binding site and (iii) (+)[3H]-3-PPP binding site(s). Differences and compatibilities in receptor specificity of the compounds tested further support the existence of multiple binding components of the sigma and PCP receptors. Materials and Methods (+)[~H]SKF 10047 (240i/mmole) and (+)[SH]-3-PPP (99 Ci/mmole) were purchased from New England Nuclear (Mass.). [sH]PCP-3-OH (9.8 Ci/mmole) was purchased from Israel Nuclear Research Center, Negev, Israel. The purity of [3H]PCP-3-OH, determined by U.V. and TLC was >99%. PCP, PCP-3-OH and N-ethylphenylcyclohexylamine (PCNHEt) were synthesized as previously described (17, 18). The two isomers of SKF 10047 were generously supplied by Dr. R. Hawaks, National Institute on Drug Abuse. All other drugs used are from commercial sources. Fresh whole brain (male Sabra rats 200-250g) were homogenized in 15 volumes of ice-cold Tris-HC1 buffer (50mM; pH 7.7 at 25°C) and centrifuged at 45,O00g for 15 min at 0-4~C. Pellets were suspended in 6 volumes of sucrose solution (0.32M) and membrane aliquots were stored at -80°C until needed. For binding assays membranes were resuspended in 10 volumes of appropriate buffer. This procedure resulted in a tissue homogenate containing 1±0.1mg protein/ml (6). The following buffers were used for binding assays: 5mM Tris-HCl buffer (pH 7.7) for [sH]PCP-3-OH binding, 10mM Tris-HC1 buffer (pH 7.8) for (+)[3H]-3-PPP binding and 5mM HEPES-KOH buffer (pH 7.7) containing ImM FOTA, for (+)[3H]SKF 10047 binding. One ml samples of brain homogenate suspension in triplicates, were incubated in the absence and presence of 1-5~M PCP-3-0H for 45 min [(+)[3H]SKF 10047 and [3H]PCP-3-OH] or 10uM of pentazocine for 90 min [(+)[3H]-3-PPP] at 25°C. The followin~ concentrations of tritiated ligands were used: 0.7-0.8nM and 20-24nM of [~H]PCP-3-OH; 4nM (+)[3H]SKF 10047 and I-1.5nM (+)[3H]-3-PPP. Samples were filtered through %Wnatman GF/B filters presoaked in 0.03% polyethyleneimine and washed twice with ice-cold Tris-HC1 buffer (SmM). Under the described conditions specifically bound [SH]PCP-3-OH at the low and high concentrations represents 95% and 78%, respectively, and that for (+)[3H]SKF 10047 and (+)[~H]-S-PPP 75±5% and 83±3% respectively~of total binding. Radioactivity adsorbed to GF/B filter blanks was less than 0.3% of total [3HI ligands added. Analysis of the binding data was carried out with the aid of the LIGAND computer program
(19). Results
[3H]PCp-3NOH: Site selective competition studies At concentration range between O.15-4ONM [sH]PCP-3-OH labels high (Kd= O.SnM) and low (Kd=16nM) affinity binding sites in rat brain membranes: low affinity binding site was identified in the presence of saturating concentrations of (+)SKF 10047 (16). Thus, site selective competition binding assays were carried out by utilizing low concentration (O.7nM) of
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Drug Specificity for Sigma and PCP Receptors
747
[SH]PCP-3-OH to label primarily high affinity sites and high concentration (24nM) of the tritiated ligand in the presence of (+)SKF 10047 (100nM) in order to label low affinity sites. Residual binding of [sH]PCP-3-OH under the latter conditions represents 75±5~ of total specific binding. PCP analogs, (+)SKF 10047 and cyclazocine potently inhibit [sH]PCP-3-OH (0.7nM) specific binding to high affinity sites, yielding ICon values between 2-65nM (Fig. I, Table I). Pseudo Hill coefficient values a~g all near unity (0.85-0.94), suggesting interaction with homogenous class of binding sites.
I
HIGHAFFI BinNdiITnYg
[3HJPCP-3-OH
g
site
ao 6°
u
40
~4K,~
)4--
"~
20 0
J -9
I
-8
J -7
I
-6
-5
[DRUG](LogM) FIG. 1 Inhibition of [SH]PCP-3-OH (0.7nM) specific binding to rat brain membranes by PCP (O), cyelazocine (~), (+)SKF 10047 (•) and haloperidol (D). Control specific binding was 950±50 cpm and represented 93±3~ of total binding. Results represent the mean of 3 to 4 determinations (SEM < 10%). In contrast to PCP analogs, the opiate benzomorphans tested are very poor inhibitors of [sH]PCP-3-OH binding to the low affinity site. Their ICon values are over 100 fold higher than those obtained for competition for t ~ high affinity site. However, the potency of PCP analogs to inhibit [SH]PCP-3-OH binding to the low affinity site is only 5-15 fold lower compared to the inhibition of binding to the high affinity site (Table I). Haloperidol and other antipsychotic agents (Table I) have no effect on [SH]PCP-3-OH specific binding to either high or low affinity sites, at concentration range between 1-10,OOOnM. _~[SHJSKF 10047 bindin~ sites --~CP -~a--~ogs,--sj---~ISK-F I0047, cyclazocine and pentazocine inhibit (÷)[3H]SKF 10047 (4nM) specific binding in a monophasic manner yielding pseudo Hill coefficient values between 0.81-0.95 and ICs0 values between 18-85nM (Table I, Fig. 2). Except for pentazocine the rank order of potency
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of the psychotomimetic drugs tested for the inhibition of (+)[3H]SKF 10047 binding is similar to that observed for the inhibition of [3H]PCP-3-OH high affinity binding (Table I). However, haloperidol, droperidol and chlerpromazine are able to inhibit partially (55-65%) (+)[~H]SKF 10047 specific binding. Complete inhibition of (+)[3H]SKF 10047 binding by these antips~chotic agents was not achieved even at high drug concentration (IxlO-~M).
~.~
0
[3H]SKF loo47
r- 100 uo ~0
80
~r -
60
._c
~0
u
~u
20
Q.
~
C
I I ~~
-9
-8
-7
-6
-5
[ D R U G ] (Log M ) FIG. 2 Inhibition of (+)[3H]SKF 10047 (inM) specific binding to rat brain membranes by (+)SKF 100~7 (A), PCP (O), cyclazocine (~) and haloperidol ([]). Control specific binding was 2200±1OO cpm and represented 75±5% of total binding. Results represent the mean of 3 to 4 determination (SEM < 10%). _~[~H]-3-PPP bindin~ sites Specific binding of (+)[3H]-3-PPP (I-1.5nM) to rat brain membranes represents 80-85% of total binding, as determined in the presence of pentazocine (lx10"SM). In contrast to [3H]PCP-3-OH high affinity binding and (+)[3H]SKF 10047 binding, (+)[3H]-3-PPP specific binding is rather poorly inhibited by PCP analogs; being 10 to 300 fold less potent at (+)[3H]-3-PPP binding sites. Moreover, PCP analogs display low pseudo IIill coefficient values (0.60-0.62). Similarly, the opiate benzomorphans tested compete for (+)[3H]-3-PPP binding in a manner indicative of heterogenous characteristics, exhibiting pseudo Hill coefficient values between 0.48-0.69 (Table I: Fig. 3).
Haloperidel is the most potent ligand, among the compounds tested, to inhibit (+)[3H]PPP specific binding (Table I). In contrast to (+)[3H]SKF 10047 binding, haloperidol (10OHM) completely inhibits (+)[3H]-3-PPP binding (Fig. 3 vs. Fig. 2). The tricyclic antidepressant, imipramine, displays also relative high affinity (ICs0=6OnM) for (+)[3H-3-PPP binding sites, but is completely inactive as an inhibitor of (+)[3H]SKF I00&7 and [3H]PCP-3-OH specific binding. The antipsychotic drugs and imipramine all compete ~n a
IC50 (nM) 2±0.3 21±1 48±3 61±5 28±2 90±8 !2,500±100 I> i0,000 I>i0,000 I>I0,000 >i0,000 >I0,000 --
I>I0,000
IC50 (nM)
--
31±2 25±2 32±3 450±30 15±2 600±50 500±40 >I0,000 >10,000
85±7
18±2 70±5
0.82 0.84 0.89 0.91 0.95 0.82 0.72
I ................................
IC50 (nM) InH
0.94 34±2 10.98 0.93 150±8 10.87 0.91 300±30 10.91 0.85 6,300±30010.62 0.95 4,800±50010.58 0.801>10,000 -0.81 5,000±200 --- >10,000 --- >10,000 --- >10,000 --- >10,000 --
nH
nH
(+)[3H]SKF 10047 (4nM)
650±40 750±50 6,000±100 70Z6 95±8 10±2 600±30 3±0.3 150±10 700±50 60±4 5,000±100
IC50 (nM)
0.60 0.61 0.62 0.48 0.69 0.65 0.67 1.07 0.82 0.85 0.94
nil
(+)[3H]-3-PPP (l-l.5nM)
Competition binding assays were carried out as described under Materials and Methods. The potency of the drugs for low affinity [3H]PCP-3-OH binding site was determined in the presence of (+)SKF 10047 (100nM) in order to eliminate high affinity [3H]pcP-3-OH binding. Under these conditions control specific binding was 4200±300 cpm. Control specific binding for the other radioligands used is given in the legends to figures. IC50 and pseudo Hill coefficient (nH) values were determined by using the computer LIGAND program and represent the mean ± SEM of four to five separate experiments.
PCP-3-OH PCP PCNHEt (+)SKF 10047 (±)Cyclazocine (i)Pentazocine (-)SKF 10047 Haloperidol Droperidol Chlorpromazine Imipramine Levomepromazine
[3H]PCP-3-OH (24nM) + (+)SKF 10047 (100nM) Low Affinity
................................................................
High Affinity
.......................................
Drug
[3H]PCP-3-OH (0.7nM)
(+)[3H]-3-PPP specific binding in rat brain membranes
Potency of various drugs in competition for [3H]PCP-3-OH, (+)[3H]SKF 10047 and
TABLE I
%0
O
O m
m O
£o
OQ
4=-
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Drug Specificity for Sigma and PCP Receptors
Vol. 42, No. 7, 1988
monophasic manner for (+)[~H]-3-PPP binding (Fig. 3), yielding pseudo Hill coefficient near unity (0.82-1.07).
0
Hatoperidol
,oo)
c.,sKF,=,,
0
•
u
if)
-9
-8 [DRUG]
-7
-6
-5
(Log M)
FIG. 3 Inhibition of (+)[3H]PPP (1.5nM) specific binding in rat brain membranes by haloperidol (O), (+)SKF 10047 (O) and PCP-3-OH (A). Control specific binding was 5200±500 cpm and represented 83±3Z of total binding. Results are the mean of to 5 determinations (SEM < IOZ). Discussion
PCP-3-OH, (+)SKF 10047 and (+)-3-PPP were shown to label multiple/heterogenous psychotomimetic binding sites in rat CNS. [3H]PCP-3-OH labels high (Kd=O.SnM) and low (Kd=16nM) affinity binding sites (16). (+)[3H]SKF 10047 labels a PCP-sensitive site and a PCP-insensitive-haloperidol-sensitive site (12,13). The dopamine autoreceptor agonist, (+)[3H]-3-PPP, was found to label the haloperidol-sensitive/(+)[3H]SKF 10047 site (12,15). The present study assessed the pharmacological specificity of a series of psychotomimetic and antipsychotic agents for the binding sites labeled with these three ligands. Table II summarizes the main findings of this study. The relative high affinity of PCP analogs and psychotomimetic benzomorphans for high affinity [sH]PCP-3-oH and (+)[3H]SKF 10047 binding sites further supports the existence of a common high affinity binding site for these agents (6,16). This site is apparently stereospecific for the (+) isomer of SKF 10047 as revealed by the diminished affinity of the (-) isomer for high affinity [3H]PCP-3-OH binding site and (+)[3H]SKF 10047 binding sites (Table I). Data from drug discriminative stimulus experiments showing cross-generalization between (+)SKF 10047 and PCP also support this hypothesis (8,9). However, the reduced relative potency of PCP analogs for (+)[3H]SKF I00~7 inhibition of binding and the ability of haloperidol to partially inhibit (+) [3H]SKF 10047 binding but not high- or Iow-[3H]PCP-3-OH binding components, support the existence of baloperidol-sensitive and PCP-insensitive component of (+)[3H]SKF 10047 binding (10-13).
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TABLE II Summary of the relative potencies of selected compounds for the inhibition of tritium labeled PCP-3-OH binding to high affinity (H.A.) and low affinity (L.A.) sites and (+)SKF 10047 and (+)-3-PPP specific binding Drug PCP-3-0H PCP Cyclazocine (+)SKF 10047 Pentazocine Haloperidol
Relative potency H.A. PCP-3-OH > (+)SKF 10047 > L.A. PCP-3-OH H.A. PCP-3-OH > (+)SKF 10047 > L.A. PCP-3-OH H.A. PCP-3-OH = (+)SKF 10047 > (+)-3-PPP >>> (+)SKF 10047 > (+)-3-PPP = H.A. PCP-3-OH >>> (+)-3-PPP > (+)SKF 10047 > H.A. PCP-3-OH >>> (+)-3-PPP > (+)SKF 10047; not active at H.A.
>>> (+)-3-PPP >>> (+)-3-PPP L.A. PCP-3-OH L.A. PCP-3-OH L.A. PCP-3-OH or L.A. PCP-3-OH
This presentation reflects the results from Table I. The discrimination between the high and low affinity [3H]PCP-3-OH binding sites by PCP analogs is 7-15 fold. Similar results were observed for the discrimination between high and low affinity [3H]thienyl-phencyclidine ([3H]TCP) binding sites by PCP analogs (20). However, the benzomorphans tested discriminate between high and low affinity [3H]PCP-3-OH sites over 100 fold, suggesting that the low affinity site may represent a PCP-selective site. These results now provide further characterization of the binding sites labeled by [sH]PCP-3-OH, a relatively new radioligand, compared to the more complete characterization of the binding sites labeled by [sH]PCP and [~H]TCP (I-5,7,12,13,20). The finding that the antipsychotic agents tested display the highest affinity for (+)[3H]-3-PPP binding sites, but having no effect on [3H]PCP-3-OH binding sites indicate the fundamental differences between the sites labeled by these two ligands. The existence of a common (+)[SH]-3-PPP/SKF 10047 binding site is supported by the ability of both benzomorphans and haloperidol, at rather low concentrations, to inhibit the binding of these two ligands. However, the finding that haloperidol inhibits only partially (+)[3 H]SKF 10047 binding, but it completely inhibits (+)[3H]-3-PPP binding, together with the observation of low pseudo-Hill coefficient values for the inhibition of (+)[SH]-3-PPP specific binding by the benzomorphans , suggest that (+)[SH]-3-PPP binding is associated with multiple state binding model distinct, at least partially, from (+)[3H]SKF 10047 - receptor complex. In summary, the differential selectivity of the psychotic and antipsychotic agents tested for the sigma and PCP binding sites and the mode of interaction with these sites, in binding experiments, further support the existence of multiple subtypes or states of the sigma and PCP receptors. Ackmowle4~emem%s This study was supported by the Hillston Foundation for Science, The Hebrew University of Jerusalem. The author wishes to thank Mr. M. Khouri and Ms. L. Benny for their excellent technical assistance.
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R.S. ZUKIN and S.R. ZUKIN, Proc. Natl. Acad. Sci. U.S.A. 76 5372-5376 (1979). J.P. VINCENT, B. KARTALOVSKI, P. GENES, J.M. KAME~{A and M. LAZDU~!SKI, Proc. Natl. Acad. Sci. U.S.A. 76 4678-4682 (1979). R. QUIRION, R. }MMMER, M. HERKENHAM and C.B. PERT, Proc. Natl. Acad. Sci.
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238, 739-748 (1986). 13. R. SIRCAR, R. NICHTENHAUSER, J.R. IENI and S.R. ZUKIN, J. Pharmacol. Exp. Ther. 237, 681-688 (1986). 14. S. HJORTH, A. CARLSSON, H. WIKSTROM, P. LINDBERG, D. SANCHEZ, H. HACKSELL, L.E. ARVIDSSON, U. SVENSSON, J.L.G. NILSSON, Life Sci. 28 1225-1238 (1981). 15. B.L. LARGENT. A.L. GUNDLACH and S.H. SNYDER, Proc. Natl. Acad. Sci.
U.S.A. 81 4983-4987 (1984). 16. Y. ITZHA--K, Eur. J. Pharmacol. 136 231-234 (1987). 17. Y. ITZHAK, A. KALIR, B.A. WEISSMAN and S. COHEN, J. Med. Chem. 2~ 496-499
(1981). 18. A. KALIR, S. MAAYANI, M. REHAVI, R. ELKAVETZ, I. PRI-BAR, O. BUC}~AH and M. SOKOLOVSKY, Eur. J. Med. Chem. 13 17-2& (1978). 19. P.J. MUNSON and D. RODBARD, Anal. Bioehem. 107 220-239 (1980). 20. J. VIGNON, A. PRIVAT, I. CHAUDIEU, A. THIERRY, J.M. KAMENKA and R. CHICHEPORTICHE, Brain Res. 378 133-141 (1986).
Pergamon Journals
PHARMACOLOGICAL SPECIFICITY OF SOME PSYCHOTOMIMETIC AND ANTIPSYCHOTIC AG~ITS FOR TSESIC, MAANDPCP BINDING SITES
YOSSEF ITZHAK Dept. of Pharmacology, Hadassah School of Medicine, The Hebrew University, Jerusalem, Israel (Received in final form December 8, 1987)
The pharmacological specificity of representative psychotomimetic agents such as phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with [3H]-I-[I(3-hydroxyphenyl) cyclohexyl] piperldine ([3H]PCP-3-OH), (+)[=H]-N-allylnormetazocine [(+)[3H]SKF 10047] and (+)[~H]-3-[3-hydroxy-phenyl]-N-(1-propyl)piperidine [(+)[3H]-3-PPP]. PCP analogs inhibit potently high affinity [3H]PCP-3-OH binding and (+)[3H]SKF 10047 binding, moderately the low affinity binding component of [3H]PCP-3-OH and very weakly (+)[3H]-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+)[3H]SKF 10047, high affinity [3H]PCP-3-OH and (+)[SH]-3-PPP binding, but extremely weak inhibitors of low affinity [sH]PCP-3-OH binding. The antipsychotic 3 agents display high affinity for (+)[ H]-3-PPP binding sites, moderate affinity for (+)[3H]SKF 10047 sites and have no effect on either the high or low affinity [3H]PCP-3-OH binding. The present data further support the existence of multiple sigma and PCP binding sites. Specific binding sites for the psychotomimetic agents phencyclidine (PCP) and opiate benzomorphans (N-allylnormetazocine; SKF 10047 and c~clzocine) were first labeled and identified, in rat CNS, by utilizing [~H]PCP (I-3). The rank order of potency of PCP analogs and opiate benzomorphans to inhibit [sH]PCP specific binding is consistent with their potency in behavioral experiments (4-5) related to psychotomimetic activity in man. Similarly, the drug specificity of (+)[3H]SKF 10047 binding sites is consistent with that of [sH]PCP binding sites (6,7). These radioreceptor binding studies along with drug discriminative experiments in rats and monkeys (8,9) have suggested the existence of a common dextrorotatoryopioid/PCP binding site for both PCP analogs and psychotomimetic opiates. Additional studies have revealed that (+)[3H]SKF 10047 specific binding is associated not only with a PCP-sensitive binding component (12,13), but also with an haloperidol-sensitive and PCP-insensitive binding component (10-13). The latter site was also labeled and visualized with the dopamine autorecepter agonist 3-(3-hydroxyphenyl)-N-(1-propyl) piperidine (3-PPP; Ref. 14) and displays drug specificit~ similar to that of the haloperidolsensitive binding component of (+)[~H]SKF 10047 (12,15). 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Journals Ltd.
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Vel. 42, No. 7, 1988
Furthermore, binding of one of the most potent analogs of PCP i.e. 1-[1(3-hydroxyphenyl)cyclohexyl]piperidine (PCP-3-OH) in rat brain membranes revealed the labeling of two distinct [~H]PCP-3-OH binding sites (16). High affinity [3H]PCP-3-OH binding is associated with one component of (+)SKF 10047 binding, whereas low affinity [3H]PCP-3-OH sites are insensitive to (+)SKF 10047. These two sites are apparently distinct from the haloperidolsensitive/ (+)[3H]SKF 10047 site (16). The present study describes the pharmacological specificity of several PCP analogs, psychotomimetic benzomorphans and antipsychotic drugs for: (i) High affinity [3H]PCP-3-OH/(+)[3H]SKF 10047 binding site, (ii) Low affinity [3H]PCP-3-OH binding site and (iii) (+)[3H]-3-PPP binding site(s). Differences and compatibilities in receptor specificity of the compounds tested further support the existence of multiple binding components of the sigma and PCP receptors. Materials and Methods (+)[~H]SKF 10047 (240i/mmole) and (+)[SH]-3-PPP (99 Ci/mmole) were purchased from New England Nuclear (Mass.). [sH]PCP-3-OH (9.8 Ci/mmole) was purchased from Israel Nuclear Research Center, Negev, Israel. The purity of [3H]PCP-3-OH, determined by U.V. and TLC was >99%. PCP, PCP-3-OH and N-ethylphenylcyclohexylamine (PCNHEt) were synthesized as previously described (17, 18). The two isomers of SKF 10047 were generously supplied by Dr. R. Hawaks, National Institute on Drug Abuse. All other drugs used are from commercial sources. Fresh whole brain (male Sabra rats 200-250g) were homogenized in 15 volumes of ice-cold Tris-HC1 buffer (50mM; pH 7.7 at 25°C) and centrifuged at 45,O00g for 15 min at 0-4~C. Pellets were suspended in 6 volumes of sucrose solution (0.32M) and membrane aliquots were stored at -80°C until needed. For binding assays membranes were resuspended in 10 volumes of appropriate buffer. This procedure resulted in a tissue homogenate containing 1±0.1mg protein/ml (6). The following buffers were used for binding assays: 5mM Tris-HCl buffer (pH 7.7) for [sH]PCP-3-OH binding, 10mM Tris-HC1 buffer (pH 7.8) for (+)[3H]-3-PPP binding and 5mM HEPES-KOH buffer (pH 7.7) containing ImM FOTA, for (+)[3H]SKF 10047 binding. One ml samples of brain homogenate suspension in triplicates, were incubated in the absence and presence of 1-5~M PCP-3-0H for 45 min [(+)[3H]SKF 10047 and [3H]PCP-3-OH] or 10uM of pentazocine for 90 min [(+)[3H]-3-PPP] at 25°C. The followin~ concentrations of tritiated ligands were used: 0.7-0.8nM and 20-24nM of [~H]PCP-3-OH; 4nM (+)[3H]SKF 10047 and I-1.5nM (+)[3H]-3-PPP. Samples were filtered through %Wnatman GF/B filters presoaked in 0.03% polyethyleneimine and washed twice with ice-cold Tris-HC1 buffer (SmM). Under the described conditions specifically bound [SH]PCP-3-OH at the low and high concentrations represents 95% and 78%, respectively, and that for (+)[3H]SKF 10047 and (+)[~H]-S-PPP 75±5% and 83±3% respectively~of total binding. Radioactivity adsorbed to GF/B filter blanks was less than 0.3% of total [3HI ligands added. Analysis of the binding data was carried out with the aid of the LIGAND computer program
(19). Results
[3H]PCp-3NOH: Site selective competition studies At concentration range between O.15-4ONM [sH]PCP-3-OH labels high (Kd= O.SnM) and low (Kd=16nM) affinity binding sites in rat brain membranes: low affinity binding site was identified in the presence of saturating concentrations of (+)SKF 10047 (16). Thus, site selective competition binding assays were carried out by utilizing low concentration (O.7nM) of
Vol. 42, No. 7, 1988
Drug Specificity for Sigma and PCP Receptors
747
[SH]PCP-3-OH to label primarily high affinity sites and high concentration (24nM) of the tritiated ligand in the presence of (+)SKF 10047 (100nM) in order to label low affinity sites. Residual binding of [sH]PCP-3-OH under the latter conditions represents 75±5~ of total specific binding. PCP analogs, (+)SKF 10047 and cyclazocine potently inhibit [sH]PCP-3-OH (0.7nM) specific binding to high affinity sites, yielding ICon values between 2-65nM (Fig. I, Table I). Pseudo Hill coefficient values a~g all near unity (0.85-0.94), suggesting interaction with homogenous class of binding sites.
I
HIGHAFFI BinNdiITnYg
[3HJPCP-3-OH
g
site
ao 6°
u
40
~4K,~
)4--
"~
20 0
J -9
I
-8
J -7
I
-6
-5
[DRUG](LogM) FIG. 1 Inhibition of [SH]PCP-3-OH (0.7nM) specific binding to rat brain membranes by PCP (O), cyelazocine (~), (+)SKF 10047 (•) and haloperidol (D). Control specific binding was 950±50 cpm and represented 93±3~ of total binding. Results represent the mean of 3 to 4 determinations (SEM < 10%). In contrast to PCP analogs, the opiate benzomorphans tested are very poor inhibitors of [sH]PCP-3-OH binding to the low affinity site. Their ICon values are over 100 fold higher than those obtained for competition for t ~ high affinity site. However, the potency of PCP analogs to inhibit [SH]PCP-3-OH binding to the low affinity site is only 5-15 fold lower compared to the inhibition of binding to the high affinity site (Table I). Haloperidol and other antipsychotic agents (Table I) have no effect on [SH]PCP-3-OH specific binding to either high or low affinity sites, at concentration range between 1-10,OOOnM. _~[SHJSKF 10047 bindin~ sites --~CP -~a--~ogs,--sj---~ISK-F I0047, cyclazocine and pentazocine inhibit (÷)[3H]SKF 10047 (4nM) specific binding in a monophasic manner yielding pseudo Hill coefficient values between 0.81-0.95 and ICs0 values between 18-85nM (Table I, Fig. 2). Except for pentazocine the rank order of potency
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Drug Specificity for Sigma and PCP Receptors
Vol. 42, No. 7, 1988
of the psychotomimetic drugs tested for the inhibition of (+)[3H]SKF 10047 binding is similar to that observed for the inhibition of [3H]PCP-3-OH high affinity binding (Table I). However, haloperidol, droperidol and chlerpromazine are able to inhibit partially (55-65%) (+)[~H]SKF 10047 specific binding. Complete inhibition of (+)[3H]SKF 10047 binding by these antips~chotic agents was not achieved even at high drug concentration (IxlO-~M).
~.~
0
[3H]SKF loo47
r- 100 uo ~0
80
~r -
60
._c
~0
u
~u
20
Q.
~
C
I I ~~
-9
-8
-7
-6
-5
[ D R U G ] (Log M ) FIG. 2 Inhibition of (+)[3H]SKF 10047 (inM) specific binding to rat brain membranes by (+)SKF 100~7 (A), PCP (O), cyclazocine (~) and haloperidol ([]). Control specific binding was 2200±1OO cpm and represented 75±5% of total binding. Results represent the mean of 3 to 4 determination (SEM < 10%). _~[~H]-3-PPP bindin~ sites Specific binding of (+)[3H]-3-PPP (I-1.5nM) to rat brain membranes represents 80-85% of total binding, as determined in the presence of pentazocine (lx10"SM). In contrast to [3H]PCP-3-OH high affinity binding and (+)[3H]SKF 10047 binding, (+)[3H]-3-PPP specific binding is rather poorly inhibited by PCP analogs; being 10 to 300 fold less potent at (+)[3H]-3-PPP binding sites. Moreover, PCP analogs display low pseudo IIill coefficient values (0.60-0.62). Similarly, the opiate benzomorphans tested compete for (+)[3H]-3-PPP binding in a manner indicative of heterogenous characteristics, exhibiting pseudo Hill coefficient values between 0.48-0.69 (Table I: Fig. 3).
Haloperidel is the most potent ligand, among the compounds tested, to inhibit (+)[3H]PPP specific binding (Table I). In contrast to (+)[3H]SKF 10047 binding, haloperidol (10OHM) completely inhibits (+)[3H]-3-PPP binding (Fig. 3 vs. Fig. 2). The tricyclic antidepressant, imipramine, displays also relative high affinity (ICs0=6OnM) for (+)[3H-3-PPP binding sites, but is completely inactive as an inhibitor of (+)[3H]SKF I00&7 and [3H]PCP-3-OH specific binding. The antipsychotic drugs and imipramine all compete ~n a
IC50 (nM) 2±0.3 21±1 48±3 61±5 28±2 90±8 !2,500±100 I> i0,000 I>i0,000 I>I0,000 >i0,000 >I0,000 --
I>I0,000
IC50 (nM)
--
31±2 25±2 32±3 450±30 15±2 600±50 500±40 >I0,000 >10,000
85±7
18±2 70±5
0.82 0.84 0.89 0.91 0.95 0.82 0.72
I ................................
IC50 (nM) InH
0.94 34±2 10.98 0.93 150±8 10.87 0.91 300±30 10.91 0.85 6,300±30010.62 0.95 4,800±50010.58 0.801>10,000 -0.81 5,000±200 --- >10,000 --- >10,000 --- >10,000 --- >10,000 --
nH
nH
(+)[3H]SKF 10047 (4nM)
650±40 750±50 6,000±100 70Z6 95±8 10±2 600±30 3±0.3 150±10 700±50 60±4 5,000±100
IC50 (nM)
0.60 0.61 0.62 0.48 0.69 0.65 0.67 1.07 0.82 0.85 0.94
nil
(+)[3H]-3-PPP (l-l.5nM)
Competition binding assays were carried out as described under Materials and Methods. The potency of the drugs for low affinity [3H]PCP-3-OH binding site was determined in the presence of (+)SKF 10047 (100nM) in order to eliminate high affinity [3H]pcP-3-OH binding. Under these conditions control specific binding was 4200±300 cpm. Control specific binding for the other radioligands used is given in the legends to figures. IC50 and pseudo Hill coefficient (nH) values were determined by using the computer LIGAND program and represent the mean ± SEM of four to five separate experiments.
PCP-3-OH PCP PCNHEt (+)SKF 10047 (±)Cyclazocine (i)Pentazocine (-)SKF 10047 Haloperidol Droperidol Chlorpromazine Imipramine Levomepromazine
[3H]PCP-3-OH (24nM) + (+)SKF 10047 (100nM) Low Affinity
................................................................
High Affinity
.......................................
Drug
[3H]PCP-3-OH (0.7nM)
(+)[3H]-3-PPP specific binding in rat brain membranes
Potency of various drugs in competition for [3H]PCP-3-OH, (+)[3H]SKF 10047 and
TABLE I
%0
O
O m
m O
£o
OQ
4=-
750
Drug Specificity for Sigma and PCP Receptors
Vol. 42, No. 7, 1988
monophasic manner for (+)[~H]-3-PPP binding (Fig. 3), yielding pseudo Hill coefficient near unity (0.82-1.07).
0
Hatoperidol
,oo)
c.,sKF,=,,
0
•
u
if)
-9
-8 [DRUG]
-7
-6
-5
(Log M)
FIG. 3 Inhibition of (+)[3H]PPP (1.5nM) specific binding in rat brain membranes by haloperidol (O), (+)SKF 10047 (O) and PCP-3-OH (A). Control specific binding was 5200±500 cpm and represented 83±3Z of total binding. Results are the mean of to 5 determinations (SEM < IOZ). Discussion
PCP-3-OH, (+)SKF 10047 and (+)-3-PPP were shown to label multiple/heterogenous psychotomimetic binding sites in rat CNS. [3H]PCP-3-OH labels high (Kd=O.SnM) and low (Kd=16nM) affinity binding sites (16). (+)[3H]SKF 10047 labels a PCP-sensitive site and a PCP-insensitive-haloperidol-sensitive site (12,13). The dopamine autoreceptor agonist, (+)[3H]-3-PPP, was found to label the haloperidol-sensitive/(+)[3H]SKF 10047 site (12,15). The present study assessed the pharmacological specificity of a series of psychotomimetic and antipsychotic agents for the binding sites labeled with these three ligands. Table II summarizes the main findings of this study. The relative high affinity of PCP analogs and psychotomimetic benzomorphans for high affinity [sH]PCP-3-oH and (+)[3H]SKF 10047 binding sites further supports the existence of a common high affinity binding site for these agents (6,16). This site is apparently stereospecific for the (+) isomer of SKF 10047 as revealed by the diminished affinity of the (-) isomer for high affinity [3H]PCP-3-OH binding site and (+)[3H]SKF 10047 binding sites (Table I). Data from drug discriminative stimulus experiments showing cross-generalization between (+)SKF 10047 and PCP also support this hypothesis (8,9). However, the reduced relative potency of PCP analogs for (+)[3H]SKF I00~7 inhibition of binding and the ability of haloperidol to partially inhibit (+) [3H]SKF 10047 binding but not high- or Iow-[3H]PCP-3-OH binding components, support the existence of baloperidol-sensitive and PCP-insensitive component of (+)[3H]SKF 10047 binding (10-13).
Vol. 42, No. 7, 1988
Drug Specificity for Sigma and PCP Receptors
751
TABLE II Summary of the relative potencies of selected compounds for the inhibition of tritium labeled PCP-3-OH binding to high affinity (H.A.) and low affinity (L.A.) sites and (+)SKF 10047 and (+)-3-PPP specific binding Drug PCP-3-0H PCP Cyclazocine (+)SKF 10047 Pentazocine Haloperidol
Relative potency H.A. PCP-3-OH > (+)SKF 10047 > L.A. PCP-3-OH H.A. PCP-3-OH > (+)SKF 10047 > L.A. PCP-3-OH H.A. PCP-3-OH = (+)SKF 10047 > (+)-3-PPP >>> (+)SKF 10047 > (+)-3-PPP = H.A. PCP-3-OH >>> (+)-3-PPP > (+)SKF 10047 > H.A. PCP-3-OH >>> (+)-3-PPP > (+)SKF 10047; not active at H.A.
>>> (+)-3-PPP >>> (+)-3-PPP L.A. PCP-3-OH L.A. PCP-3-OH L.A. PCP-3-OH or L.A. PCP-3-OH
This presentation reflects the results from Table I. The discrimination between the high and low affinity [3H]PCP-3-OH binding sites by PCP analogs is 7-15 fold. Similar results were observed for the discrimination between high and low affinity [3H]thienyl-phencyclidine ([3H]TCP) binding sites by PCP analogs (20). However, the benzomorphans tested discriminate between high and low affinity [3H]PCP-3-OH sites over 100 fold, suggesting that the low affinity site may represent a PCP-selective site. These results now provide further characterization of the binding sites labeled by [sH]PCP-3-OH, a relatively new radioligand, compared to the more complete characterization of the binding sites labeled by [sH]PCP and [~H]TCP (I-5,7,12,13,20). The finding that the antipsychotic agents tested display the highest affinity for (+)[3H]-3-PPP binding sites, but having no effect on [3H]PCP-3-OH binding sites indicate the fundamental differences between the sites labeled by these two ligands. The existence of a common (+)[SH]-3-PPP/SKF 10047 binding site is supported by the ability of both benzomorphans and haloperidol, at rather low concentrations, to inhibit the binding of these two ligands. However, the finding that haloperidol inhibits only partially (+)[3 H]SKF 10047 binding, but it completely inhibits (+)[3H]-3-PPP binding, together with the observation of low pseudo-Hill coefficient values for the inhibition of (+)[SH]-3-PPP specific binding by the benzomorphans , suggest that (+)[SH]-3-PPP binding is associated with multiple state binding model distinct, at least partially, from (+)[3H]SKF 10047 - receptor complex. In summary, the differential selectivity of the psychotic and antipsychotic agents tested for the sigma and PCP binding sites and the mode of interaction with these sites, in binding experiments, further support the existence of multiple subtypes or states of the sigma and PCP receptors. Ackmowle4~emem%s This study was supported by the Hillston Foundation for Science, The Hebrew University of Jerusalem. The author wishes to thank Mr. M. Khouri and Ms. L. Benny for their excellent technical assistance.
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Drug Specificity for Sigma and PCP Receptors
Vol. 42, No. 7, 1988
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