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3H-spiroperidol binding and dopamine-stimulated adenylate cyclase: Evidence for multiple classes of receptors in primate brain regions
Life Sciences, Vol . 23, pp . 629-634 Printed in the U .S .A .
Pergamon Press
3H-SPIROPERIDOL BINDING AND DOPAMINE-STIMULATED ADENYLATE CYCLASE : EVIDENCE FOR MULTIPLE CLASSES OF RECEPTORS IN PRIMATE BRAIN REGIONS L .J . Thal, M .H . Makman, H .S . Ahn, R .K . Mishra,~ S .G . Horowitz, B . Dvorkin and R . Katzman Departments of Neurology and Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N .Y . 10461 * Department of Psychiatry, McMaster University, Hamilton, Ontario (Current Address) SUMMARY Studies of displacement by agonist and antagonist drugs of 3 Hspiroperidol binding in brain regions of Cebus and rhesus monkeys revealed one type of receptor Fn caudate nûc~us and a second type Compared of receptor to both frontal and anterior limbic cortex . with caudate, the cortical regions were more sensitive to clozapine and loxaptne, equally sensitive to fluphenazine and relatively less sensitive to haloperidol . Also, the cortical regions were insensitive to molindone . Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex . In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of 3H-spiroperidol receptors are coupled to adenylate cyclase . Evidence exists Indicating that neuroleptic drugs exert their main This evipharmacological effects by blockade of dopamine receptors (1,2) . dence includes the observation that behavior provoked by dopamtnergtc com pounds can be readily blocked by neuroleptic drug administration (3,4) " Neuroleptic drugs have also been shown to inhibit dopamine-sensitive adenylate cyclase activity present in various regions of the CNS (5,6,7,) . Recently, direct binding studies with radiolabeled dopamine agonists and antagonists have supported the view that neuroleptics act primarily by blockade of dopamine receptors (8,9,10) . However, most of the binding studies have been carried out with striatal tissue, a brain region believed to be more closely involved with extrapyramidal side effects of neuroleptics th n with antipsychotic activity . Also, with the ligands used in these studies, ~H-haloperidol, 3H-dopamine and 3H-apomorphine, receptors could be detected only in regions with a high density of receptors such as caudate, olfactory tubercle, and nucleus accumbens, while cortical receptors could not be readily identified (10,11) . Using 3 H-spiroperidol, a butyrophenone with high affinity for the striatal dopamine receptor and low non-specific binding (12,13), receptors in rat frontal cortex (FC) could be demonstrated (14) . We have utilized 3H-spiroperidol to study receptors in primate caudate nucleus (CN), (FC), and anterior limbic cortex (ALC) to determine the binding characteristics of receptors in these regions and to determine whether neuroleptics exert differential effects on these three regions as indicated by our previous studies using the adenylate cyclase system (5,6,7) . The adenylate cyclase studies, to be reported in detail in a separate communication, revealed three types of dopamine receptors coupled to adenylate cyclase in monkey CNS : one type in ALL, a second In FC, and a third in several regions including CN, retina and hypothalamus . 0300-9653/78/0814-0629$02 .00/0 Copyright (c) 1978 Pergamon Press
63 0
Neuroleptic Binding and Adenylate Cyclase
Vol . 23, No . 6, 1978
MATERIALS AND METHODS For the receptor binding studies, fresh or frozen rhesus or Cebus monkey brain tissue was homogenized at a concentration of 10 mg/ml In icecord 50 mM Trts HC1 buffer, pH 7 .7 at 25° C, with a Polytron, (speed 6, 10 seconds) . The homogenate was centrifuged twice at 50,000 x g for 10 minutes (Sorvall RC 2-B, 20,000 RPM) with resuspenslon in fresh buffer . The final pellet was suspended at a concentration of 4 mg of wet wt ./ml in fresh 50 mM Tris HCl buffer containing : 1 .0 uM pargyline, 0 .1$ ascorbic acid, 120 mM NaCI, 5 mM KCI, 2 mM CaC12, and 1 mM MgC12 at a final pH of 7 .1 at 37° C . This suspension was warmed to 37° C for ten minutes then returned to ice . 3H-sptroperidol, 20-26 Ci/mnole was obtained from New England Nuclear and Amersham . Assays utilized a final volume of 1 ml in triplicate tubes . To 100 ul of 3H-sptroperidol solution at varying concentrations and 100 ul of either 0 .1$ ascorbic acid or unlabeled drug to ascorbic acid were added 0 .8 ml of tissue suspension . Tubes were incubated at 37° C for 20 minutes then rapidly filtered through Whatman GF/B filters followed by three 5 ml rinses with ice cold 50 mM Tris buffer, pH 7 .7 at 25° C . The radioactivity on the filters was counted by liquid scintillation spectrometry in 8 ml of New England Nuclear Formula 950 at efficiencies of 35-37$ after overnight extraction . Saturable or specific 3H-sptroperidol binding was defined as excess over non-specific binding determined by blank incubations with 1 uM unlabeled spiropertdol . This blank resulted in the same value as was obtained with 10 uM(+) butaclamol . At 10 uM, the pharmacologically inactive Isomer (-)-butaclamol did not displace 3H-sptroperidol . Adenylate cyclase assays of homogenates of monkey brain regions were tattled out as previously described (15, 16) . Incubation of FC was for 5 minutes, ALC either 5 minutes or 2 .5 minutes and caudate 2 .5 minutes . Dopamine was studied over a concentration range of 10 - 9 M to 10' 4 M for determination of For determination of relative potencies of antagonists, dopamine EC50 values . was generally present at a fixed concentration of 5 uM and the concentration of antagonist drug varied over an appropriate range . RESUL7S Specific 3H-sptroperidol binding was saturable in all three regions studied . The number of binding sites in monkey CN was 17 .8 pmoles/gm wet weight with a Kp of 1 .1 nM . Both FC and ALC contained about one-half as many binding sites as caudate with virtually identical KD~ s of 1 nM . In typical displacement experiments using 0 .5 nM 3H-spiropertdol, specific binding represented 50-85$ of total binding in the CN and 40-50$ of total binding in ALC and FC . In the CN, clinically potent phenothlazlne and butyrophenone neurolepttcs displaced 3H-sptroperidol at nanomolar concentrations(Table l, Fig . la) . Clozapine, a dibenzodiazepine antipsychotic drug with low extrapyramidal side effects, was only 1/40 to 1/80 as potent In displacing 3H-spiropertdol . However, loxapine, a substituted dibenzodiazepine, which has been found to produce extrapyramidal side effects, had an affinity similar to clozaplne for spiropertdol binding sites . Each drug tested had a similar affinity for spiropertdol binding sites in each cortical area tested, but the affinity for binding in the cortical areas
Vol . 23, No . 6, 1978
Neuroleptic Binding and Adenylate Cyclase
631
was quite different from that found in the CN (Table 1, Ftg, lb, lc) . The most striking differences were the relatively low affinity of haloperidol in the cortex (only 1/25 that in caudate) and the markedly increased affinities of clozapine (20 fold) and loxapine (4-15 fold) in cortical areas as compared with caudate . Apomorphine, a dopamine agonist, was considerably more potent in caudate than in cortical regions while methysergide, a putative serotonin antagonist was 2-5 times more potent in cortical regions than in caudate .
Fig .
l.
Competition for 3H-spiroperidoi binding in : (a) CN, (b) FC and (c) ALC . Increasing concentration of unlabeled drug was added to tubes containing 0 .5 nM 3 H-spiroperidoi and membranes . Each point represents the average percent of specific spiroperidoi binding displaced by drug at each concentration as determined in 3-5 separate experiments each performed in triplicate . Legend : ~ - spiroperidoi, haloperidol, Q - fluphenazine, []- clozapine . In contrast, results obtained with use of the dopamine-stimulated adenylate cyclase system in these three brain regions (Table 2) indicated that the ALC had the greatest sensitivity to each of the neuroleptic drugs studied In addition, each of the regions studied exas well as to dopamine itself . hibited a different hierarchy of sensitivity to neuroleptics . The CN dopaminestimulated adenylate cyclase was most sensitive to fluphenazine, significantly less sensitive to haloperidol and clozapine, and quite insensitive to pimozide . Both cortical areas were similar to each other but different from CN in that to cortex haloperidol was approximately equal to fluphenazine in potency and clozapine was closer to fluphenazine than in caudate . However, the two cortical regions differed markedly from one another to their sensitivity to pimozide . We have previously reported that the dopamine-stimulated adenylate cyclase in the two cortical regions differed in response to agonists (15) . In contrast, we have not thus far observed pharmacological differences for 3H-spiroperi .dol binding to these two cortical regions . DISCUSSION The relative potencies of drugs for displacement of 3H-spiroperidoi from binding sites in monkey CN were similar to those previously reported for this ligand in calf (17) and rat CN (12) as well as for 3H-haloperidol in calf (10) . However, the potencies of drugs found in the present study for the primate cortical regions differed markedly from those found for caudate . On first in-
63 2
Neuroleptic
BindinE
and Adenylate Cyclase
yol . 23, No . 6, 1978
TABLE 1 REGIONAL DIFFERENCES IN DRUG CONCENTRATION REQUIRED FOR INHIBITION OF 3H -SPIROPERIDOL BINDING Ki Drug
Caudate
+ Butaclamol Spiroperidol Haloperidol Fluphenazine Clozapine Loxapine Apomorphine Methysergide Molindone
21 .2 + 4 (4) 5 .2 + 2 .4 (5) 4 .8 + .52 (4) 12 .7 + 1,8 (3) 414 + 146 (3) 204 _+ 108 (2) 207 (1) 552 (1) -
(nM)
Frontal Cortex 19 .1 5 .7 122 + 14 .E 20 .3
+ 11 .3 (3) + 3 .8 (3) 11,2 (3) + 3 .9 (3) + 3 .4 (3) 60 (1) 733 (1) 233 (1) -
Anterior Limbic Cortex 12 .7 + 5 .8 (3) 7 .0 + 3 .2 (3) 125 +51 .6 (4) 28 .9+ 6 .3 (3) 27 .8 + 16 .5 (3) 13+0 (2) 1333 (1) 100 (1) >10,000 (1)
Valûes are _+SEM number of separâte experiments in parenthesis Kl = IC50/I + C/KD where C is the concentration of the ligand, KD is the dissociation constant determined by saturation studies and IC50 is the concentration of drug required to inhibit specific binding by 50~ . Table 2 REGIONAL DIFFERENCES IN DRUG CONCENTRATION REQUIRED FOR INHIBITION OF DOPAMINE-STIMULATED ADENYLATE CYCLASE ACTIVITY Region
Caudate Nucleus 0 .2 Haloperidol Fluphenazine Clozapine Pimozide Molindone
50
_+
15
Anterior Limbic Frontal Cortex Cortex for dopamine E 50 uM Ki
(6)
1 (6) 5 + 4 + 10 (6) 1,000 +300 (3) -
0 .4 (nM) for antagonist
30 20 60 5,000
+ 5 (3) + 7 (3) + 20(3) +500(3) -
0 .7 0 .9 3 .6 3 .0 >2,000
0 .l + + + +
0 .3 0 .4 1 .0 0 .7
Tl)
(6) (6) (6) (3}
K~~ÎC 5 0/(I + S/EC ) wheré IC50 represents the concentration required for 50$ inhibition ~~ the response to dopamine, EC50 the concentration of dopamine for half-maximal stimulation and 5 the concentration of dopamine present in the assay for the Ki studies as described in the Methods section . Values are + SEM (number of separate experiments in parenthesis) . spection, it appears that the markedly greater affinity of clozapine to cortical compared with CN receptors might explain its antipsychotic potency coupled with a low incidence of extrapyramidal side effects . However, the finding that loxaplne, a drug that does induce extrapyramidal side effects, has a similar high affinity for cortical as compared to CN spiroperidol binding sites, appears to invalidate the theory that the existence of these side effects can be correlated with affinity for spiroperidol binding in CN . Within each cortical region, neuroleptics bound with high affinity except for haloperidol which appears to be considerably less potent than fluphenazine or clozapine . The rank order of neuroleptic potency in cortex determined by spiroperidol binding did not match the clinical potency of neuroleptics as reported in CN with haloperidol binding (8,9) . Affinities for 3H-
Yol . 23, No . 6, 1978
Neuroleptic Binding and Adenylate Cyclase
63 3
spiroperidol binding in rat FC similar to those we have obtained in primate have recently beef reported (14), although in our studies both apomorphine and methysergide are clearly less potent than neuroleptics in d(splacing 3H-sptroperidol . Molindone, an antipsychotic containing an indole group, did not displace spiroperidol at a concentration of 10 mM to ALC nor was it active In blocking the dopamine-stimulated adenylate Cyclase in this cortical region (as reported separately in this volume by Ahn and Makman) . The differences in affinity of neuroleptics in cortex and CN, the low affinity for apomorphine in cortex, and the relatively higher affinity of methysergide in cortex suggests that the receptor measured by 3H-spiroperidol in cortex differs substantially from the receptor measured by 3H-spiroperidol in CN . Drugs binding to cortical receptors with the highest affinities are the neuroleptics . Perhaps the most appropriate name at present for this cortical receptor is a neuroleptic receptor since Its properties do not appear to clearly fit that of any known transmitter system . A more complete definition of this receptor and an evaluation of possible differences between FC and ALC are currently being pursued . In contrast to finding one pattern of binding in CN, and a second pattern in both cortical regions, the adenylate Cyclase studies demonstrated a different type of receptor in each region with the ALC having the greatest sensitivity both to stimulation by dopamine and to Inhibition by neuroleptics . In additional studies we have found both monkey hypothalamus and retina to contain receptors coupled to adenylate Cyclase similar to those in the CN (5,18) . In each region, binding and adenylate Cyclase studies demonstrated a different rank order for the drugs studied suggesting that only a relatively small portion of the 3H-spiroperidol receptors are likely to be coupled to the dopamine-stimulated adenylate Cyclase . The best match between in vivo antipsychotic drug potency and in vitro activity occurred in the ALC fôr the Cyclase studies and to CN for btnd~fng studies . However, it does not seem valid at present to attempt to explain the in vivo antipsychotic activity of these drugs on the basis of their interactionwith only one of the in vitro assay systems . The studies presented here indicate that primate cortices reg~ns contain both spiroperidol binding sites and dopamine sensitive adenylate Cyclase with sensitivity to neuroleptic drugs which differ from those In the CN . Also, in the cortex Itself, as well as In the CN, spiroperidol binding sites and dopamine receptors coupled to adenylate Cyclase differ from one another . In view of the high affinity of the antipsychotic drugs to the spiroperidol binding sites and dopamine-adenylate Cyclase in cortex, these receptors might play an Important role in the action of these drugs, a role which remains to be elucidated . (NIH NS 09649) 1. 2. 3" 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
REFERENCES A . Carlsson and M .Lindqutst : Acta Pharmacol . Toxicol . _20 : 140-143 1963 " L . L . Iversen : Science 188 : 10 -10 9, 1975 . G .Sedvall : Anti s choticDru s, Pergamon Press, London, 1976 . F .C .Colpaert, .M . Van Beaver, and J .E . Leysen : Int . Rev . Neurobiol . 1~: 225-267, 1976 . R .K .Mishra, M .H .Makman, H .S .Ahn et .al : Neurosci .Abstr . 2 : 1134, 1976 . R .Katzman, M .H .Makman, H .S .Ahn et .al . : TANA 102 : 1- , 1977 . M .H .Makman : In Biochem .Actions of Hormones , IV, G .Litwack(Ed .)p408-496,1977 . P .Seeman, T .Lee, M .Chau-Wong, and K .Wong : Nature 261 : 717-718, 1976 . I .Creese, D .R .Burt, and S .H .Snyder : Science 192 : SST-483, 1976 . D .R .Burt, I .Creese, and S .H .Snyder : M~arm . 12 : 800-812, 1976 . P .Seeman, T .Lee, M .Chau-along et .al . : PNAS U A 73 : 4354-4358, 1976 . J .Z .Fields, T .D .ReTsine, and H .I .Yamamura : Brain Research , In press, 1978 . J .E .Leysen, W .Gammeren, and P .M .Laduron : Biochem .Pharm . 27 : 307-316, 1978 . J .E .Leysen, C .J .E .Neimegeers, J .P .Tollengere et .a : Nature 272 : 168-171,1978 H .S .Ahn, R . K .Mishra, C .DemirJlan et .al : Brain Res . 1Tb~~7-Tî54, 1976 . R .K .Mishra, C .DemIrJian, R .Katzman et .al : Braes . _96 : 395 - 399, 1975 . I .Creese, R .Schnelder, and S .H .Snyder :Eur . .PhTarmacol 46 : 377-381, 1977 . H .S .Ahn, M .H .Makman : Brain Res . 138 : 125-13 , 1977 .
Pergamon Press
3H-SPIROPERIDOL BINDING AND DOPAMINE-STIMULATED ADENYLATE CYCLASE : EVIDENCE FOR MULTIPLE CLASSES OF RECEPTORS IN PRIMATE BRAIN REGIONS L .J . Thal, M .H . Makman, H .S . Ahn, R .K . Mishra,~ S .G . Horowitz, B . Dvorkin and R . Katzman Departments of Neurology and Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N .Y . 10461 * Department of Psychiatry, McMaster University, Hamilton, Ontario (Current Address) SUMMARY Studies of displacement by agonist and antagonist drugs of 3 Hspiroperidol binding in brain regions of Cebus and rhesus monkeys revealed one type of receptor Fn caudate nûc~us and a second type Compared of receptor to both frontal and anterior limbic cortex . with caudate, the cortical regions were more sensitive to clozapine and loxaptne, equally sensitive to fluphenazine and relatively less sensitive to haloperidol . Also, the cortical regions were insensitive to molindone . Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex . In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of 3H-spiroperidol receptors are coupled to adenylate cyclase . Evidence exists Indicating that neuroleptic drugs exert their main This evipharmacological effects by blockade of dopamine receptors (1,2) . dence includes the observation that behavior provoked by dopamtnergtc com pounds can be readily blocked by neuroleptic drug administration (3,4) " Neuroleptic drugs have also been shown to inhibit dopamine-sensitive adenylate cyclase activity present in various regions of the CNS (5,6,7,) . Recently, direct binding studies with radiolabeled dopamine agonists and antagonists have supported the view that neuroleptics act primarily by blockade of dopamine receptors (8,9,10) . However, most of the binding studies have been carried out with striatal tissue, a brain region believed to be more closely involved with extrapyramidal side effects of neuroleptics th n with antipsychotic activity . Also, with the ligands used in these studies, ~H-haloperidol, 3H-dopamine and 3H-apomorphine, receptors could be detected only in regions with a high density of receptors such as caudate, olfactory tubercle, and nucleus accumbens, while cortical receptors could not be readily identified (10,11) . Using 3 H-spiroperidol, a butyrophenone with high affinity for the striatal dopamine receptor and low non-specific binding (12,13), receptors in rat frontal cortex (FC) could be demonstrated (14) . We have utilized 3H-spiroperidol to study receptors in primate caudate nucleus (CN), (FC), and anterior limbic cortex (ALC) to determine the binding characteristics of receptors in these regions and to determine whether neuroleptics exert differential effects on these three regions as indicated by our previous studies using the adenylate cyclase system (5,6,7) . The adenylate cyclase studies, to be reported in detail in a separate communication, revealed three types of dopamine receptors coupled to adenylate cyclase in monkey CNS : one type in ALL, a second In FC, and a third in several regions including CN, retina and hypothalamus . 0300-9653/78/0814-0629$02 .00/0 Copyright (c) 1978 Pergamon Press
63 0
Neuroleptic Binding and Adenylate Cyclase
Vol . 23, No . 6, 1978
MATERIALS AND METHODS For the receptor binding studies, fresh or frozen rhesus or Cebus monkey brain tissue was homogenized at a concentration of 10 mg/ml In icecord 50 mM Trts HC1 buffer, pH 7 .7 at 25° C, with a Polytron, (speed 6, 10 seconds) . The homogenate was centrifuged twice at 50,000 x g for 10 minutes (Sorvall RC 2-B, 20,000 RPM) with resuspenslon in fresh buffer . The final pellet was suspended at a concentration of 4 mg of wet wt ./ml in fresh 50 mM Tris HCl buffer containing : 1 .0 uM pargyline, 0 .1$ ascorbic acid, 120 mM NaCI, 5 mM KCI, 2 mM CaC12, and 1 mM MgC12 at a final pH of 7 .1 at 37° C . This suspension was warmed to 37° C for ten minutes then returned to ice . 3H-sptroperidol, 20-26 Ci/mnole was obtained from New England Nuclear and Amersham . Assays utilized a final volume of 1 ml in triplicate tubes . To 100 ul of 3H-sptroperidol solution at varying concentrations and 100 ul of either 0 .1$ ascorbic acid or unlabeled drug to ascorbic acid were added 0 .8 ml of tissue suspension . Tubes were incubated at 37° C for 20 minutes then rapidly filtered through Whatman GF/B filters followed by three 5 ml rinses with ice cold 50 mM Tris buffer, pH 7 .7 at 25° C . The radioactivity on the filters was counted by liquid scintillation spectrometry in 8 ml of New England Nuclear Formula 950 at efficiencies of 35-37$ after overnight extraction . Saturable or specific 3H-sptroperidol binding was defined as excess over non-specific binding determined by blank incubations with 1 uM unlabeled spiropertdol . This blank resulted in the same value as was obtained with 10 uM(+) butaclamol . At 10 uM, the pharmacologically inactive Isomer (-)-butaclamol did not displace 3H-sptroperidol . Adenylate cyclase assays of homogenates of monkey brain regions were tattled out as previously described (15, 16) . Incubation of FC was for 5 minutes, ALC either 5 minutes or 2 .5 minutes and caudate 2 .5 minutes . Dopamine was studied over a concentration range of 10 - 9 M to 10' 4 M for determination of For determination of relative potencies of antagonists, dopamine EC50 values . was generally present at a fixed concentration of 5 uM and the concentration of antagonist drug varied over an appropriate range . RESUL7S Specific 3H-sptroperidol binding was saturable in all three regions studied . The number of binding sites in monkey CN was 17 .8 pmoles/gm wet weight with a Kp of 1 .1 nM . Both FC and ALC contained about one-half as many binding sites as caudate with virtually identical KD~ s of 1 nM . In typical displacement experiments using 0 .5 nM 3H-spiropertdol, specific binding represented 50-85$ of total binding in the CN and 40-50$ of total binding in ALC and FC . In the CN, clinically potent phenothlazlne and butyrophenone neurolepttcs displaced 3H-sptroperidol at nanomolar concentrations(Table l, Fig . la) . Clozapine, a dibenzodiazepine antipsychotic drug with low extrapyramidal side effects, was only 1/40 to 1/80 as potent In displacing 3H-spiropertdol . However, loxapine, a substituted dibenzodiazepine, which has been found to produce extrapyramidal side effects, had an affinity similar to clozaplne for spiropertdol binding sites . Each drug tested had a similar affinity for spiropertdol binding sites in each cortical area tested, but the affinity for binding in the cortical areas
Vol . 23, No . 6, 1978
Neuroleptic Binding and Adenylate Cyclase
631
was quite different from that found in the CN (Table 1, Ftg, lb, lc) . The most striking differences were the relatively low affinity of haloperidol in the cortex (only 1/25 that in caudate) and the markedly increased affinities of clozapine (20 fold) and loxapine (4-15 fold) in cortical areas as compared with caudate . Apomorphine, a dopamine agonist, was considerably more potent in caudate than in cortical regions while methysergide, a putative serotonin antagonist was 2-5 times more potent in cortical regions than in caudate .
Fig .
l.
Competition for 3H-spiroperidoi binding in : (a) CN, (b) FC and (c) ALC . Increasing concentration of unlabeled drug was added to tubes containing 0 .5 nM 3 H-spiroperidoi and membranes . Each point represents the average percent of specific spiroperidoi binding displaced by drug at each concentration as determined in 3-5 separate experiments each performed in triplicate . Legend : ~ - spiroperidoi, haloperidol, Q - fluphenazine, []- clozapine . In contrast, results obtained with use of the dopamine-stimulated adenylate cyclase system in these three brain regions (Table 2) indicated that the ALC had the greatest sensitivity to each of the neuroleptic drugs studied In addition, each of the regions studied exas well as to dopamine itself . hibited a different hierarchy of sensitivity to neuroleptics . The CN dopaminestimulated adenylate cyclase was most sensitive to fluphenazine, significantly less sensitive to haloperidol and clozapine, and quite insensitive to pimozide . Both cortical areas were similar to each other but different from CN in that to cortex haloperidol was approximately equal to fluphenazine in potency and clozapine was closer to fluphenazine than in caudate . However, the two cortical regions differed markedly from one another to their sensitivity to pimozide . We have previously reported that the dopamine-stimulated adenylate cyclase in the two cortical regions differed in response to agonists (15) . In contrast, we have not thus far observed pharmacological differences for 3H-spiroperi .dol binding to these two cortical regions . DISCUSSION The relative potencies of drugs for displacement of 3H-spiroperidoi from binding sites in monkey CN were similar to those previously reported for this ligand in calf (17) and rat CN (12) as well as for 3H-haloperidol in calf (10) . However, the potencies of drugs found in the present study for the primate cortical regions differed markedly from those found for caudate . On first in-
63 2
Neuroleptic
BindinE
and Adenylate Cyclase
yol . 23, No . 6, 1978
TABLE 1 REGIONAL DIFFERENCES IN DRUG CONCENTRATION REQUIRED FOR INHIBITION OF 3H -SPIROPERIDOL BINDING Ki Drug
Caudate
+ Butaclamol Spiroperidol Haloperidol Fluphenazine Clozapine Loxapine Apomorphine Methysergide Molindone
21 .2 + 4 (4) 5 .2 + 2 .4 (5) 4 .8 + .52 (4) 12 .7 + 1,8 (3) 414 + 146 (3) 204 _+ 108 (2) 207 (1) 552 (1) -
(nM)
Frontal Cortex 19 .1 5 .7 122 + 14 .E 20 .3
+ 11 .3 (3) + 3 .8 (3) 11,2 (3) + 3 .9 (3) + 3 .4 (3) 60 (1) 733 (1) 233 (1) -
Anterior Limbic Cortex 12 .7 + 5 .8 (3) 7 .0 + 3 .2 (3) 125 +51 .6 (4) 28 .9+ 6 .3 (3) 27 .8 + 16 .5 (3) 13+0 (2) 1333 (1) 100 (1) >10,000 (1)
Valûes are _+SEM number of separâte experiments in parenthesis Kl = IC50/I + C/KD where C is the concentration of the ligand, KD is the dissociation constant determined by saturation studies and IC50 is the concentration of drug required to inhibit specific binding by 50~ . Table 2 REGIONAL DIFFERENCES IN DRUG CONCENTRATION REQUIRED FOR INHIBITION OF DOPAMINE-STIMULATED ADENYLATE CYCLASE ACTIVITY Region
Caudate Nucleus 0 .2 Haloperidol Fluphenazine Clozapine Pimozide Molindone
50
_+
15
Anterior Limbic Frontal Cortex Cortex for dopamine E 50 uM Ki
(6)
1 (6) 5 + 4 + 10 (6) 1,000 +300 (3) -
0 .4 (nM) for antagonist
30 20 60 5,000
+ 5 (3) + 7 (3) + 20(3) +500(3) -
0 .7 0 .9 3 .6 3 .0 >2,000
0 .l + + + +
0 .3 0 .4 1 .0 0 .7
Tl)
(6) (6) (6) (3}
K~~ÎC 5 0/(I + S/EC ) wheré IC50 represents the concentration required for 50$ inhibition ~~ the response to dopamine, EC50 the concentration of dopamine for half-maximal stimulation and 5 the concentration of dopamine present in the assay for the Ki studies as described in the Methods section . Values are + SEM (number of separate experiments in parenthesis) . spection, it appears that the markedly greater affinity of clozapine to cortical compared with CN receptors might explain its antipsychotic potency coupled with a low incidence of extrapyramidal side effects . However, the finding that loxaplne, a drug that does induce extrapyramidal side effects, has a similar high affinity for cortical as compared to CN spiroperidol binding sites, appears to invalidate the theory that the existence of these side effects can be correlated with affinity for spiroperidol binding in CN . Within each cortical region, neuroleptics bound with high affinity except for haloperidol which appears to be considerably less potent than fluphenazine or clozapine . The rank order of neuroleptic potency in cortex determined by spiroperidol binding did not match the clinical potency of neuroleptics as reported in CN with haloperidol binding (8,9) . Affinities for 3H-
Yol . 23, No . 6, 1978
Neuroleptic Binding and Adenylate Cyclase
63 3
spiroperidol binding in rat FC similar to those we have obtained in primate have recently beef reported (14), although in our studies both apomorphine and methysergide are clearly less potent than neuroleptics in d(splacing 3H-sptroperidol . Molindone, an antipsychotic containing an indole group, did not displace spiroperidol at a concentration of 10 mM to ALC nor was it active In blocking the dopamine-stimulated adenylate Cyclase in this cortical region (as reported separately in this volume by Ahn and Makman) . The differences in affinity of neuroleptics in cortex and CN, the low affinity for apomorphine in cortex, and the relatively higher affinity of methysergide in cortex suggests that the receptor measured by 3H-spiroperidol in cortex differs substantially from the receptor measured by 3H-spiroperidol in CN . Drugs binding to cortical receptors with the highest affinities are the neuroleptics . Perhaps the most appropriate name at present for this cortical receptor is a neuroleptic receptor since Its properties do not appear to clearly fit that of any known transmitter system . A more complete definition of this receptor and an evaluation of possible differences between FC and ALC are currently being pursued . In contrast to finding one pattern of binding in CN, and a second pattern in both cortical regions, the adenylate Cyclase studies demonstrated a different type of receptor in each region with the ALC having the greatest sensitivity both to stimulation by dopamine and to Inhibition by neuroleptics . In additional studies we have found both monkey hypothalamus and retina to contain receptors coupled to adenylate Cyclase similar to those in the CN (5,18) . In each region, binding and adenylate Cyclase studies demonstrated a different rank order for the drugs studied suggesting that only a relatively small portion of the 3H-spiroperidol receptors are likely to be coupled to the dopamine-stimulated adenylate Cyclase . The best match between in vivo antipsychotic drug potency and in vitro activity occurred in the ALC fôr the Cyclase studies and to CN for btnd~fng studies . However, it does not seem valid at present to attempt to explain the in vivo antipsychotic activity of these drugs on the basis of their interactionwith only one of the in vitro assay systems . The studies presented here indicate that primate cortices reg~ns contain both spiroperidol binding sites and dopamine sensitive adenylate Cyclase with sensitivity to neuroleptic drugs which differ from those In the CN . Also, in the cortex Itself, as well as In the CN, spiroperidol binding sites and dopamine receptors coupled to adenylate Cyclase differ from one another . In view of the high affinity of the antipsychotic drugs to the spiroperidol binding sites and dopamine-adenylate Cyclase in cortex, these receptors might play an Important role in the action of these drugs, a role which remains to be elucidated . (NIH NS 09649) 1. 2. 3" 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
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