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Neuroleptics have identical potencies in human brain limbic and putamen regions
European Journal of Pharmacology, 94 (1983) 145-148
145
Elsevier
Short communication N E U R O L E P T I C S HAVE IDENTICAL P O T E N C I E S IN H U M A N BRAIN LIMBIC A N D P U T A M E N REGIONS PHILIP SEEMAN * and C A R L A U L P I A N
Department of Pharamcology, Medical Sciences Building, University of Toronto, Toronto, Canada M5S IA8 Received 27 July 1983, accepted 15 August 1983
P. SEEMAN and C. ULPIAN, Neuroleptics have identical potencies in human brain limbic and putamen regions, European J. Pharmacol. 94 (1983) 145-148. In order to examine whether some neuroleptic drugs were specifically more potent on human limbic dopamine receptors than on striatal dopamine receptors, we tested the potency of eight neuroleptics on their ability to inhibit the binding of [3H]spiperone to D 2 dopamine receptors in human putamen and nucleus accumbens. Each of the neuroleptics had an identical potency in both tissues, the IC50 values being 0.2 nM for spiperone, 2.5 nM for haloperidol, 2.6 nM for trifluperidol, 5 nM for fluphenazine, 20 nM for thioridazine, 25 nM for chlorpromazine, 100 nM for metoclopramide and 300 nM for clozapine. There is no evidence, therefore, for the concept of a limbic-specific dopamine receptor antagonist. [ 3H]Spiperone
D 2 dopamine receptors
1. Introduction
Although it has usually been assumed that D 2 dopamine receptors in different regions of the brain and pituitary all have the same sensitivities to neuroleptic drugs (Seeman, 1980, 1982; Creese and Left, 1982; Creese et al., 1983; Kebabian and Calne, 1979), it has been reported by Borison et al. (1981) that thioridazine and clozapine were 'sitespecific' for limbic dopamine receptors. Using human brain striatal tissue, Borison et al. found that haloperidol was potent in inhibiting the binding of [3H]spiperone while clozapine and thioridazine were 'several orders of magnitude less potent'. In human limbic tissue, however, Borison et al. found that the three neuroleptics were equally potent; they thus concluded that clozapine and thioridazinc were specifically potent toward limbic dopamine receptors. These findings of Borison et al. have been repeatedly cited as demonstrating that thioridazine * To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
has a 'minimal effect on striatal dopamine receptors' (Sandoz, 1983), implying that this compound has significantly less risk for causing tardive dyskinesia. Since the potencies of neuroleptics for dopamine receptors depend on the final concentration of brain tissue in the test-tube (Seeman et al., 1982), we re-examined this question of sitespecificity for neuroleptics using less than 1 mg tissue per ml. We found that the neuroleptic potencies in human putamen were identical to those in human limbic tissue, a result compatible with that found by Reynolds et al. (1982) for thioridazine.
2. Materials and methods
Post-mortem human brain tissues (putamen and nucleus accumbens) were obtained from the Canadian Brain Tissue Bank (Dr. Catherine Bergeron, Banting Institute, University of Toronto, 101 College St., Toronto). The interval between
146
[
I illllll]
"
I rllllli I
~
1PPlllii I
i Irill]11
~
i iiliiill_ j
HUMAN PUTAMEN
1 5 0 0 ~ ~
,ooo-oo,
~ lil~nt I
thioridazine
\
! "O
500
= O
"Q 1~ t'--
-
- with (+)-butaclamol~t ~
/
o.~
~
-
. . . .
.-
600:
800 --
._~
-i
thioridazine
--
4
50%~
400 200--
with (+)-butaclamol~
~tlHtl 10-11
10-10
i JtttntL t JiJlllil 10-9
L iiltillL
10-8
10-7
t lilillil 10-6
J ltlllilll 10-5
tool/Liter Fig. 1. The potency of haloperidol in inhibiting the binding of [3 H]spiperone to D, dopamine receptors in the human putamen was identical to that in the human nucleus accumbens (bottom). The haloperidol IC~ value was 2.5 nM in both tissues, while that for thioridazine was 20 nM in both tissues.
death (myocardial infarctions) and freezing of the brain ( - 7 0 ° C ) was 24 h or less. We have previously found that the density of D 2 dopamine receptors remains constant in the brains of rats removed up to 72 h post-mortem and where the dead rat had been kept at 15°C up to the time when the brain was removed and frozen (T. Lee and P. Seeman, unpublished). The dissected brain regions were weighed, and 9
ml of buffer medium were added for each g of original wet tissue. The buffer medium contained: 50 mM Tris-HCI (pH 7.4 at 20°C), 120 mM NaC1, 5 mM KCI, 1.5 mM CaC12, 4 mM MgCI z • 6HzO, 1 mM EDTA acid, 0.1% ascorbic acid and 12 #M nialamide. The suspension was homogenized in a glass homogenizer with a teflon piston rotating at 650 rpm (8 up-and-down strokes). One ml of homogenate was diluted with 35 ml buffer and
147
centrifuged (39000 × g ) for 30 min at 4°C. The pellet was resuspended in 35 ml buffer and recentrifuged. The resulting pellet was again resuspended in 35 ml buffer and centrifuged; the final pellet was resuspended in 35 ml buffer, yielding a concentration of 3 mg original tissue per ml. The suspension was then homogenized with a Polytron (Brinkmann instruments; PT-10; 20 s; setting at 7, full power being 10). The effects of eight neuroleptics on the binding of [3H]spiperone to D 2 dopamine receptors were done in triplicate, each of which received the following aliquots in the order listed: 0.5 ml buffer medium (with or without competing neuroleptic), 0.5 ml of [3H]spiperone (80 pM final concentration in the incubate; 31-34 C i / m m o l ; New England Nuclear Corp., Boston), and 0.5 ml of the Polytron-homogenized tissue suspension. Thus, the final tissue concentration was about 1 mg original tissue per ml (equivalent to about 70 #g tissue protein per ml of final incubate). The mixtures were incubated at room temperature (20°C) for 120 min and then filtered (12 tubes simultaneously) by means of a Titertak Cell Harvester (Skatron AS Instruments, Lier, Norway; Flow Laboratories, Ontario) using a glass fiber filter mat (Flow Lab. No. 78-105-05) and a vacuum of 400 to 500 mm Hg. The filter mat was then rinsed with a 15 s rinse (10 ml) of 50 mM Tris-HC1 (pH 7.4 at 20°C). The damp filters were removed from the mat and placed in liquid scintillation polyethylene vials to which 8 ml of Scint-A (Packard Instrument Co., Illinois) scintillation fluid was added. The samples were monitored (after chilling for 6 h at 4°C) for 3H at 43% efficiency (Packard 460C spectrometer). The specific binding of 3Hspiperone was defined as that inhibited by 1 #M (+)-butaclamol (Ayerst Research Laboratories, Montreal) which inhibits the binding of 3Hspiperone to both D 2 sites and Sz serotonergic sites (List and Seeman, 1981); in human striatum, however, only 4% of the [3H]spiperone sites are S2 in nature (unpublished). The IC50 value was defined, as that concentration which inhibited specific binding by 50%.
1000~1
, llllll, I
I ,llml I
, Jli~[,q~
I llil,,I I
E c,ozapine/ r100____-O
R = 0.999 /
-_ J -
pramide
/chlorpromazine
10 ..Q E
~ o metoclo-
== _-
thioridazine
fluphenazine o// . ~ trifluperidol ~ haloperidol
-
Y
- J° spiperone 0.1
1 0.1
I I IIIIIll I l l~nd I ~JlJml i i inml 1 10 100 1000 p u t a m e n IC50, n M
Fig. 2. The IC50 value (on [3H]spiperonebinding) for each of eight neuroleptics was identical in the human putamen and nucleus accumbens. Each IC50 value is the average (values within 10% of each other) of two independent experiments, each concentrationtested in triplicate.
3. Results
The potency of each of eight neuroleptics in the human putamen was identical to its potency in the human nucleus accumbens. Examples for haloperidol and thioridazine are shown in fig. 1, wherein the IC50 value for haloperidol was 2.5 nM in both tissues while that for thioridazine was 20 nM for both tissues. The results in fig. 2 illustrate the same findings for spiperone, trifluperidol, fluphenazine, chlorpromazine, metoclopramide and clozapine. Separate experiments, using 10 to 600 pM [3H]spiperone to saturate the D 2 receptors, indicated that the dissociation constant (KD) for [3H]spiperone was 80 + 12 pM (mean + S.E.M. for 10 human putamens). Thus, using the socalled Cheng-Prusoff equation (see Hartley and Seeman, 1978), the K i values were lower than the IC50 by a factor of 2.
4. Discussion
The results indicate that each of six neuroleptics had identical potencies in the human striatum and
148
nucleus accumbens. These data were obtained using very low concentrations of tissue such as to minimize the depletion of both [3H]spiperone and neuroleptic from the medium into the tissue membranes. We have earlier shown that this depletion can markedly alter the apparent K D or neuroleptic IC50 (Seeman et al., 1982; Hartley and Seeman, 1978). For example, our [3H]spiperone K D was 80 pM, while that found by Borison et al. (1981) was 800 pM. The very high K D seen by Borison et al. suggests that they used concentrations of tissue exceeding 1 mg per ml (final), as indicated by Seeman et al. (1982). Furthermore, it is possible that the final concentrations of striatal tissue and limbic tissue (used by Borison et al., 1981) were also different, thus accounting for the different neuroleptic potencies in the two regions. The present data do not in any way bear on the separate issue of the likelihood of the development of tardive dyskinesia that may be caused by the different neuroleptics. The data merely indicate that a given neuroleptic has the same affinity for D 2 dopamine receptors in two different brain regions. This observation is compatible with the hypothesis that the D 2 dopamine receptor may be the same protein in these different regions. The present data do not support the concept of a limbic-specific neuroleptic as widely claimed by others (Sandoz, 1983).
Acknowledgements We thank Dr. Catherine Bergeron and Maria Pataki (Canadian Brain Tissue Bank) for their excellent assistance,
and Dr. David Marshall (Ayerst Research Laboratories, Montreal) for ( * )-butaclamol. This work was supported by the C.K. Clarke Foundation, the Friends of Schizophrenics (Metro Toronto Chapter), the Ontario Mental Health Foundation and the Medical Research Council of Canada.
References Borison, R.L., J.Z. Fields and B.I. Diamond, 1981, Site-specific blockade of dopamine receptors by neuroleptic agents in human brain, Neuropharmacology 20, 1321. Creese, 1. and S.E. Left, 1982, Dopamine receptors: A classification, J. Clin. Psychopaharmacol. 2, 329. Creese, I., A.L. Morrow, S.E. Leff, D.R. Sibley and M.W. Hamblin, 1983, Dopamine receptors in the central nervous system, Int. Rev. Neurobiol. 23, 255. Hartley, E.J. and P. Seeman, 1978, The effect of varying 3H-spiperone concentration on its binding parameters, Life Sci. 23, 303. Kebabian, J.W. and D.B. Calne, 1979, Multiple receptors for dopamine, Nature (London) 277, 93. List, S.J. and P. Seeman, 1981, Resolution of dopamine and serotonin receptor components of 3H-spiperone binding to rat brain regions, Proc. Nat. Acad. SCi. U.S.A. 78, 2620. Reynolds, G.P., L. Cowey, M.N. Rutter and L.L. Iversen, 1982, Thioridazine is not specific for limbic dopamine receptors, Lancet ii, d99. Sandoz, Inc., 1983, Tardive dyskinesia, are some neuroleptics less likely to cause it? Arch. Gen. Psychiatr. 40, 394. Seeman, P., 1980, Brain dopamine receptors, Pharmacol. Rev. 32, 229. Seeman, P., 1982, Nomenclature of central and peripheral dopaminergic sites and receptors, Biochem. Pharmacol. 31, 2563. Seeman, P., C. Ulpian0 and J. Wells, 1982, Dopamine receptor parameters (detected by 3H-spiperone) depend on tissue concentration, Soc. Neurosci. Abstr. 8, 718.
145
Elsevier
Short communication N E U R O L E P T I C S HAVE IDENTICAL P O T E N C I E S IN H U M A N BRAIN LIMBIC A N D P U T A M E N REGIONS PHILIP SEEMAN * and C A R L A U L P I A N
Department of Pharamcology, Medical Sciences Building, University of Toronto, Toronto, Canada M5S IA8 Received 27 July 1983, accepted 15 August 1983
P. SEEMAN and C. ULPIAN, Neuroleptics have identical potencies in human brain limbic and putamen regions, European J. Pharmacol. 94 (1983) 145-148. In order to examine whether some neuroleptic drugs were specifically more potent on human limbic dopamine receptors than on striatal dopamine receptors, we tested the potency of eight neuroleptics on their ability to inhibit the binding of [3H]spiperone to D 2 dopamine receptors in human putamen and nucleus accumbens. Each of the neuroleptics had an identical potency in both tissues, the IC50 values being 0.2 nM for spiperone, 2.5 nM for haloperidol, 2.6 nM for trifluperidol, 5 nM for fluphenazine, 20 nM for thioridazine, 25 nM for chlorpromazine, 100 nM for metoclopramide and 300 nM for clozapine. There is no evidence, therefore, for the concept of a limbic-specific dopamine receptor antagonist. [ 3H]Spiperone
D 2 dopamine receptors
1. Introduction
Although it has usually been assumed that D 2 dopamine receptors in different regions of the brain and pituitary all have the same sensitivities to neuroleptic drugs (Seeman, 1980, 1982; Creese and Left, 1982; Creese et al., 1983; Kebabian and Calne, 1979), it has been reported by Borison et al. (1981) that thioridazine and clozapine were 'sitespecific' for limbic dopamine receptors. Using human brain striatal tissue, Borison et al. found that haloperidol was potent in inhibiting the binding of [3H]spiperone while clozapine and thioridazine were 'several orders of magnitude less potent'. In human limbic tissue, however, Borison et al. found that the three neuroleptics were equally potent; they thus concluded that clozapine and thioridazinc were specifically potent toward limbic dopamine receptors. These findings of Borison et al. have been repeatedly cited as demonstrating that thioridazine * To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
has a 'minimal effect on striatal dopamine receptors' (Sandoz, 1983), implying that this compound has significantly less risk for causing tardive dyskinesia. Since the potencies of neuroleptics for dopamine receptors depend on the final concentration of brain tissue in the test-tube (Seeman et al., 1982), we re-examined this question of sitespecificity for neuroleptics using less than 1 mg tissue per ml. We found that the neuroleptic potencies in human putamen were identical to those in human limbic tissue, a result compatible with that found by Reynolds et al. (1982) for thioridazine.
2. Materials and methods
Post-mortem human brain tissues (putamen and nucleus accumbens) were obtained from the Canadian Brain Tissue Bank (Dr. Catherine Bergeron, Banting Institute, University of Toronto, 101 College St., Toronto). The interval between
146
[
I illllll]
"
I rllllli I
~
1PPlllii I
i Irill]11
~
i iiliiill_ j
HUMAN PUTAMEN
1 5 0 0 ~ ~
,ooo-oo,
~ lil~nt I
thioridazine
\
! "O
500
= O
"Q 1~ t'--
-
- with (+)-butaclamol~t ~
/
o.~
~
-
. . . .
.-
600:
800 --
._~
-i
thioridazine
--
4
50%~
400 200--
with (+)-butaclamol~
~tlHtl 10-11
10-10
i JtttntL t JiJlllil 10-9
L iiltillL
10-8
10-7
t lilillil 10-6
J ltlllilll 10-5
tool/Liter Fig. 1. The potency of haloperidol in inhibiting the binding of [3 H]spiperone to D, dopamine receptors in the human putamen was identical to that in the human nucleus accumbens (bottom). The haloperidol IC~ value was 2.5 nM in both tissues, while that for thioridazine was 20 nM in both tissues.
death (myocardial infarctions) and freezing of the brain ( - 7 0 ° C ) was 24 h or less. We have previously found that the density of D 2 dopamine receptors remains constant in the brains of rats removed up to 72 h post-mortem and where the dead rat had been kept at 15°C up to the time when the brain was removed and frozen (T. Lee and P. Seeman, unpublished). The dissected brain regions were weighed, and 9
ml of buffer medium were added for each g of original wet tissue. The buffer medium contained: 50 mM Tris-HCI (pH 7.4 at 20°C), 120 mM NaC1, 5 mM KCI, 1.5 mM CaC12, 4 mM MgCI z • 6HzO, 1 mM EDTA acid, 0.1% ascorbic acid and 12 #M nialamide. The suspension was homogenized in a glass homogenizer with a teflon piston rotating at 650 rpm (8 up-and-down strokes). One ml of homogenate was diluted with 35 ml buffer and
147
centrifuged (39000 × g ) for 30 min at 4°C. The pellet was resuspended in 35 ml buffer and recentrifuged. The resulting pellet was again resuspended in 35 ml buffer and centrifuged; the final pellet was resuspended in 35 ml buffer, yielding a concentration of 3 mg original tissue per ml. The suspension was then homogenized with a Polytron (Brinkmann instruments; PT-10; 20 s; setting at 7, full power being 10). The effects of eight neuroleptics on the binding of [3H]spiperone to D 2 dopamine receptors were done in triplicate, each of which received the following aliquots in the order listed: 0.5 ml buffer medium (with or without competing neuroleptic), 0.5 ml of [3H]spiperone (80 pM final concentration in the incubate; 31-34 C i / m m o l ; New England Nuclear Corp., Boston), and 0.5 ml of the Polytron-homogenized tissue suspension. Thus, the final tissue concentration was about 1 mg original tissue per ml (equivalent to about 70 #g tissue protein per ml of final incubate). The mixtures were incubated at room temperature (20°C) for 120 min and then filtered (12 tubes simultaneously) by means of a Titertak Cell Harvester (Skatron AS Instruments, Lier, Norway; Flow Laboratories, Ontario) using a glass fiber filter mat (Flow Lab. No. 78-105-05) and a vacuum of 400 to 500 mm Hg. The filter mat was then rinsed with a 15 s rinse (10 ml) of 50 mM Tris-HC1 (pH 7.4 at 20°C). The damp filters were removed from the mat and placed in liquid scintillation polyethylene vials to which 8 ml of Scint-A (Packard Instrument Co., Illinois) scintillation fluid was added. The samples were monitored (after chilling for 6 h at 4°C) for 3H at 43% efficiency (Packard 460C spectrometer). The specific binding of 3Hspiperone was defined as that inhibited by 1 #M (+)-butaclamol (Ayerst Research Laboratories, Montreal) which inhibits the binding of 3Hspiperone to both D 2 sites and Sz serotonergic sites (List and Seeman, 1981); in human striatum, however, only 4% of the [3H]spiperone sites are S2 in nature (unpublished). The IC50 value was defined, as that concentration which inhibited specific binding by 50%.
1000~1
, llllll, I
I ,llml I
, Jli~[,q~
I llil,,I I
E c,ozapine/ r100____-O
R = 0.999 /
-_ J -
pramide
/chlorpromazine
10 ..Q E
~ o metoclo-
== _-
thioridazine
fluphenazine o// . ~ trifluperidol ~ haloperidol
-
Y
- J° spiperone 0.1
1 0.1
I I IIIIIll I l l~nd I ~JlJml i i inml 1 10 100 1000 p u t a m e n IC50, n M
Fig. 2. The IC50 value (on [3H]spiperonebinding) for each of eight neuroleptics was identical in the human putamen and nucleus accumbens. Each IC50 value is the average (values within 10% of each other) of two independent experiments, each concentrationtested in triplicate.
3. Results
The potency of each of eight neuroleptics in the human putamen was identical to its potency in the human nucleus accumbens. Examples for haloperidol and thioridazine are shown in fig. 1, wherein the IC50 value for haloperidol was 2.5 nM in both tissues while that for thioridazine was 20 nM for both tissues. The results in fig. 2 illustrate the same findings for spiperone, trifluperidol, fluphenazine, chlorpromazine, metoclopramide and clozapine. Separate experiments, using 10 to 600 pM [3H]spiperone to saturate the D 2 receptors, indicated that the dissociation constant (KD) for [3H]spiperone was 80 + 12 pM (mean + S.E.M. for 10 human putamens). Thus, using the socalled Cheng-Prusoff equation (see Hartley and Seeman, 1978), the K i values were lower than the IC50 by a factor of 2.
4. Discussion
The results indicate that each of six neuroleptics had identical potencies in the human striatum and
148
nucleus accumbens. These data were obtained using very low concentrations of tissue such as to minimize the depletion of both [3H]spiperone and neuroleptic from the medium into the tissue membranes. We have earlier shown that this depletion can markedly alter the apparent K D or neuroleptic IC50 (Seeman et al., 1982; Hartley and Seeman, 1978). For example, our [3H]spiperone K D was 80 pM, while that found by Borison et al. (1981) was 800 pM. The very high K D seen by Borison et al. suggests that they used concentrations of tissue exceeding 1 mg per ml (final), as indicated by Seeman et al. (1982). Furthermore, it is possible that the final concentrations of striatal tissue and limbic tissue (used by Borison et al., 1981) were also different, thus accounting for the different neuroleptic potencies in the two regions. The present data do not in any way bear on the separate issue of the likelihood of the development of tardive dyskinesia that may be caused by the different neuroleptics. The data merely indicate that a given neuroleptic has the same affinity for D 2 dopamine receptors in two different brain regions. This observation is compatible with the hypothesis that the D 2 dopamine receptor may be the same protein in these different regions. The present data do not support the concept of a limbic-specific neuroleptic as widely claimed by others (Sandoz, 1983).
Acknowledgements We thank Dr. Catherine Bergeron and Maria Pataki (Canadian Brain Tissue Bank) for their excellent assistance,
and Dr. David Marshall (Ayerst Research Laboratories, Montreal) for ( * )-butaclamol. This work was supported by the C.K. Clarke Foundation, the Friends of Schizophrenics (Metro Toronto Chapter), the Ontario Mental Health Foundation and the Medical Research Council of Canada.
References Borison, R.L., J.Z. Fields and B.I. Diamond, 1981, Site-specific blockade of dopamine receptors by neuroleptic agents in human brain, Neuropharmacology 20, 1321. Creese, 1. and S.E. Left, 1982, Dopamine receptors: A classification, J. Clin. Psychopaharmacol. 2, 329. Creese, I., A.L. Morrow, S.E. Leff, D.R. Sibley and M.W. Hamblin, 1983, Dopamine receptors in the central nervous system, Int. Rev. Neurobiol. 23, 255. Hartley, E.J. and P. Seeman, 1978, The effect of varying 3H-spiperone concentration on its binding parameters, Life Sci. 23, 303. Kebabian, J.W. and D.B. Calne, 1979, Multiple receptors for dopamine, Nature (London) 277, 93. List, S.J. and P. Seeman, 1981, Resolution of dopamine and serotonin receptor components of 3H-spiperone binding to rat brain regions, Proc. Nat. Acad. SCi. U.S.A. 78, 2620. Reynolds, G.P., L. Cowey, M.N. Rutter and L.L. Iversen, 1982, Thioridazine is not specific for limbic dopamine receptors, Lancet ii, d99. Sandoz, Inc., 1983, Tardive dyskinesia, are some neuroleptics less likely to cause it? Arch. Gen. Psychiatr. 40, 394. Seeman, P., 1980, Brain dopamine receptors, Pharmacol. Rev. 32, 229. Seeman, P., 1982, Nomenclature of central and peripheral dopaminergic sites and receptors, Biochem. Pharmacol. 31, 2563. Seeman, P., C. Ulpian0 and J. Wells, 1982, Dopamine receptor parameters (detected by 3H-spiperone) depend on tissue concentration, Soc. Neurosci. Abstr. 8, 718.