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Antagonism by neuroleptics of serotonin 5-HT1A and 5-HT2 receptors of normal human brain in vitro
European Journal of Pharmacology, 143 (1987) 279-282
279
Elsevier EJP 20022 Short communication
Antagonism by neuroleptics of serotonin 5-HTIAand 5-HT 2 receptors of normal human brain in vitro T h e o d o r e J. W a n d e r , A l b e r t N e l s o n , H a r u o O k a z a k i a a n d Elliott R i c h e l s o n * Departments of Psychiatry and Psychology, Pharmacology, and 1 Pathology, Mayo Foundation, Rochester, MN 55905, U.S.A.
Received 17 September 1987, accepted 22 September 1987
Using radioligand binding techniques and human frontal cortex, we determined the equilibrium dissociation constants (KDS) of 17 neuroleptics at the serotonin 5-HTIA and serotonin 5-HT2 receptors with [3H]WB4101 and [3H]ketanserin, respectively. At the serotonin 5-HTaA receptor, the most and least potent neuroleptics were chlorprothixene (K D = 230 nM) and fluphenazine (KD = 40 #M), respectively. At the serotonin 5-HT2 receptor, the most and least potent neuroleptics were spiperone (K D = 0.38 nM) and molindone, (K D = 5/aM), respectively. Neuroleptics; Brain; Serotonin receptors; (Human)
1. Introduction
Psychotherapeutic drugs antagonize m a n y different receptors in the central nervous system. This antagonism m a y explain a drug's therapeutic and adverse effects as well as its interactions with other drugs. In addition, there appears to be a relationship between a drug's receptor affinity (or its reciprocal, the equilibrium dissociation constant) and its potency or likelihood of causing an effect. Specifically for neuroleptics, there is a strong correlation between dopamine D-2 receptor affinity and daily dose for treating schizophrenia (Richelson and Nelson, 1984b). The radioligand binding assay is presently the method of choice in assessing a drug's affinity for certain receptors. Most assays of this type have utilized animal brain tissue, and some investigators have found good correlations between these results and those obtained with the use of h u m a n brain tissue (Richelson and Nelson, 1984a, b; Fowler, 1986). Because there are examples of dif* To whom all correspondence should be addressed: Guggenheim 7, Mayo Foundation, Rochester, MN 55905, U.S.A.
ferences in results between species (Pazos et al., 1984; Schnellmann et al., 1984), we have routinely used human brain tissue in our radioligand binding studies, and here we report the dissociation constants (KDS) of 17 neuroleptics at the serotonin 5-HTIA and 5-HT 2 receptors of h u m a n brain.
2. Materials and methods 2.1. Tissue preparation
H u m a n brain tissue was obtained at the time of autopsy (range 1.3-12.5 h after death) from donors with no known history of neuropsychiatric illness. Cardiovascular events were responsible for death in all five donors: myocardial infarction in two and cardiac dysrhythmia, ruptured thoraco-abdominal aortic aneurysm, and pulmonary embolism in the others. Specimens appeared grossly normal. Tissue was stored in a liquid nitrogen refrigerator until its use. G r a y matter was homogenized in 10 volumes of ice cold buffer using a Brinkmann Polytron PT-45 (45 s, setting 8). The buffer used for the serotonin 5-HTaA assays was
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
280 50 mM Tris HC1 (pH 7.7 at 25 o C) containing 5 mM MgSO4 and 0.5 mM EDTA, and that used for the serotonin 5-HT2 assays was 50 mM Tris HC1 (pH 7.7 at 25°C). The homogenate was centrifuged at 38 000 × g for 10 rain. The pellets were resuspended in buffer to give 30 and 10 mg initial wet weight/ml for the serotonin 5-HTIA and 5-HT2 assays, respectively. Aliquots of homogenate were stored at -33 ° C.
2.2. Receptor assays Into glass culture tubes (12 X 75 mm) were placed the tritiated ligand (0.05 ml), drug solution, and buffer. For the serotonin 5-HTaA assays, 0.10 ml of 9 gM prazosin was also added to each tube. The solution was stirred using a vortex mixer. Homogenate (0.25 ml) was added to each tube, and the suspension (1.0 rnl) was again stirred using a vortex mixer. The final concentration of each radioligand was 0.51 and 0.26 nM for the serotonin 5-HT1A ([3H]WB4101) and 5-HT2 ([3H]ketanserin) assays, respectively. Duplicate samples were incubated at 37 °C for 1 h and the suspensions were then filtered under vacuum using Whatman G F / B filter strips on a Brandel M-24 R cell harvester. The tubes and filters were rinsed with 3 x 5 ml of ice-cold buffer. The filter circles were placed in 6 ml plastic mini-vials (Research Products International) and 4 ml scintillation fluid (Safety Solve, Research Products International) was added to each. After the tubes stood 5 h, the radioactivity was measured using either a Searle Isocap 300 or a Beckman LS 7800 liquid scintillation counter at an efficiency of 30 and 42%, respe~..tively~iSpecific binding was defined as total biodirtg mi.~us non-specific binding, which for the .s¢rot0nin-5;~TrA assays was determined with 1 gM-,~ys~rgid.~i~LSD) and for the serotonin 5-HT2 ass¢y~ with,~]~i t~M methysergide. Using a ~wlett~Eackard: 9845B~ computer connected by ng~em t~a~Cy~er ~ m p m e r (Control Data Corp., Min!~eal~li~_)~e:ar!a|yz~ the data using the L,[GAND!,p~o.gr~ (Munson and Rodbard, 1980) to proyide valu~ fore,he equilibrium constant K D. ~,W¢have::jnodif~ed~this program in order to obtain theHitl ,~co.efficient (nil) as well ~he mean K D and n H values (__+S,E,M.) in tables 1 and 2 are
from at least three independent experiments, each determined in duplicate.
2.3. Drugs [3H]WB4101 (27.0 Ci/mmol) and [3H]ketanserin (76.7 Ci/mmol) were from New England Nuclear (Boston, MA); WB4101 from Amersham (Arlington Heights, IL); lysergide (LSD) from National Institute of Drug Abuse (Rockville, MD); ketanserin and spiperone from Janssen Pharmaceutica Inc. (New Brunswick, NJ); mesoridazine from Boehringer Ingelheim Ltd. (Ridgefield, CT); chlorpromazine from Sigma Chemical Co. (St. Louis, MO); thioridazine, clozapine and methysergide from Sandoz Pharmaceuticals (East Hanover, NJ); haloperidol from McNeil Pharmaceutical (Spring House, PA); fluphenazine and triflupromazine from E.R. Squibb & Sons, Inc. (Princeton, N J); perphenazine from Schering Corp. (Kenilworth, N J); prochlorperazine and trifluoperazine from Smith Kline & French Laboratories (Philadelphia, PA); molindone from Dupont Pharmaceuticals (Wilmington, DE); chlorprothixene from Hoffman-LaRoche Inc. (Nutley, N J); prazosin and cis-thiothixene from Pfizer Inc. (New York, NY); d-butaclamol from Ayerst Laboratories (New York, NY); loxapine from Lederle Laboratories (Wayne, NJ); and promazine from Wyeth Laboratories (Philadelphia, PA).
3. Results
3.1. Serotonin 5-HTIA receptor of human frontal cortex Although [3H]WB4101 has been considered to label al-adrenoceptors, it has been shown that this radioligand may be used to label the serotonin 5-I-IT1 (probably, 5-HT1A) site under certain experimental conditions (Norman et al., 1985). Because [3H]WB4101 has high affinity for al-adrenergic sites, prazosin (900 nM), a potent and selective al-adrenoceptor antagonist, was used in each assay to block its binding to these receptors. Binding to serotonin 5-HTIA receptors was rapid, reached steady state within several min and
281 TABLE 1 Neuroleptics: equilibrium dissociation constants (KDS) for the serotonin 5-HT1A receptor of human brain frontal cortex K D + S.E.M. (nM)
Hill coefficient + S.E.M.
230 5: 20 320 5: 90 350 5: 70 500 5: 100 800 + 300 12005: 300 1400 + 400 18005: 300 2 800 5: 600 2 900 5: 400 3 200 + 800 3 600 5: 400 5 900___ 700 120005:2000 2 3 0 0 0 + 1000 230005:5000 40000 + 10000
1.12 5:0.06 1.2 + 0.2 0.62 + 0.09 1.0 _+0.1 1.00 5:0.09 1.0 5:0.1 0.8 5:0.1 1.1 5:0.2 0.8 + 0.2 0.99 5:0.07 1.0 5:0.1 1.7 + 0.2 1.0 5:0.1 0.8 +0.1 0.7 5:0.1 0.7 +0.1 0.58 __.0.03
Neuroleplics Chlorprothixene Spiperone Thioridazine Mesoridazine d-Butaclamol Molindone cis-Thiothixene Clozapine Haloperidol Loxapine Chlorpromazine Perphenazine Prochlorperazine Promazine Trifluoperazine Triflupromazine Fluphenazine
Serotoninergic compounds WB4101 Lysergide (LSD)
3.1 5:0.3 0.64 5:0.08
0.94 + 0.04 0.90 + 0.08
maintained this level of binding for at least 1 h. Competition experiments revealed that WB4101 possessed a K D + S.E.M. equal to 3.1 + 0.3 nM (n = 29), which is similar to that (3.8 nM) reported using rat cortical homogenates (Norman et al., 1985). The Hill coefficient did not differ significantly from unity, indicating that the compound bound to a single class of receptor sites under these conditions. Of the 17 neuroleptics studied at the 5-HT~A receptor, chlorprothixene was the most potent with a K D = 230 ___20 nM and a Hill coefficient essentially equal to unity (table 1). The weakest binding was exhibited by fluphenazine, with a K D = 40 + 10 btM. N o correlation existed between the log of the KDS and the log of the average daily doses for treating schizophrenia (r = 0.15, p = 0.58). 3.2. Serotonin 5 - H T 2 receptor of human frontal cortex
[3H]Ketanserin and methysergide can be used to study the serotonin 5-HT2 receptor site (Leysen
TABLE 2 Neuroleptics: equilibrium dissociation constants (KDS) for the serotonin 5-HT2 receptor of human brain frontal cortex. K D _ S.E.M. (nM)
Hill coefficient 5: S.E.M.
Ratio K D 5 - H T I A / K D 5-HT2
0.38 + 0.05 0.4 + 0.1 0.43 + 0.08 1.4 + 0.4 1.6 + 0.4 1.7 + 0.4 4 + 1 4.8 + 0.3 5.6 + 0.6 14 + 1 15 + 2 16 + 4 19 + 5 22 + 8 36 + 5 130 + 30 5 000 + 1000
0.96 + 0.08 1.10 + 0.06 1.06 + 0.07 0.76 + 0.07 1.1 +0.2 1.1 +0.1 1.2 + 0.1 1.2 +0.2 1.22 + 0.06 1.1 +0.1 1.2 +0.1 1.3 +0.2 1.04 + 0.08 0.78 + 0.02 1.2 +0.1 1.04 + 0.09 0.84 + 0.07
840 2 000 540 2 300 1100 1700 5 800 100 640 1600 390 750 2100 16 78 11 0.24
0.56 + 0.04 7 +2
1.03 + 0.02 0.81 + 0.07
Neuroleptics Spiperone d-Butaclamol Chlorprothixene Chlorpromazine Clozapine Loxapine Triflupromazine Mesoridazine Perphenazine Trifluoperazine Prochlorperazine Promazine Fluphenazine Thioridazine Haloperidol cis-Thiothixene Molindone
Serotoninergic compounds Ketanserin Methysergide
282 et al., 1982). Ketanserin had a K D = 0.56 + 0.04 n M (n = 29), which is comparable to previous studies by Leysen et al. (1982) using rat prefrontal cortex ( K D = 0.42 nM). Hill coefficients for ketanserin and the neuroleptics were essentially equal to one (table 2), indicating binding to a single population of receptor sites. KDS for the 17 neuroleptics were relatively low (table 2), and spiperone (K D = 0.38 + 0.05 nM), d-butaclamol (K D = 0.4 + 0.1 nM) and chlorprothixene (K D = 0.43 _ 0.08 nM) all b o u n d m o r e strongly than ketanserin. Molindone had a K D = 5 + 1 #M, which was greatly different from the values for the other compounds. Again, no correlation existed between the log of the KDS and the log of the average daily doses (r = 0.097, P = 0.71).
4. Discussion Here we have presented the data for 17 neuroleptics binding at the serotonin 5 - H T 1 (probably, 5-HTIA ) and 5 - H T 2 receptors present in n o r m a l h u m a n brain frontal cortex. F o r these compounds, there was no significant correlation between the log of their KDS at the 5-HT1A receptor and the log of their KDS at the 5 - H T 2 receptor. However, all but one c o m p o u n d , molindone, b o u n d m o r e potently to the 5 - H T 2 receptor (table 2). We c o m p a r e d our data for six c o m p o u n d s in c o m m o n with the study of Leysen et al. (1982) who also used [3H]ketanserin as a radioligand but used rat prefrontal cortex. A significant correlation (r = 0.98, P = 0.0005) existed between the two sets of data. We have cited in this paper other comparisons between our results and those of others using animal brain. These comparisons suggest that there are no large species differences between h u m a n and animal brain serotonin receptors, although Pazos et al. (1984) reported species differences for mesulergine, an apparently selective serotonin 5 - H T 2 ligand. The clinical relevance of serotonin receptor
blockade b y neuroleptics is unknown. Certainly, the lack of correlation between receptor affinity and neurleptic dose indicates that serotonin receptor blockade alone does not explain the m o d e of action of these drugs. However, vascular side-effects of these c o m p o u n d s m a y be mediated by blockade of serotonin receptors (Peroutka, 1984).
Acknowledgements This work was supported by Mayo Foundation and U.S.P.H.S. Grant MH27692 from N.I.M.H.
References Fowler, C.J., 1986, The pros and cons of using human brain autopsy samples for radioligand binding experiments, Trends Pharmacol. Sci. 7, 9. Leysen, J.E., C.J.E. Niemegeers, J.M. Van Nueten and P.M. Laduron, 1982, [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin 2 receptor binding sites, Mol. Pharmacol. 21, 301. Munson, P.J. and D. Rodbard, 1980, LIGAND: A versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107, 220. Norman, A.B., G. Battaglia, A.L. Morrow and I. Creese, 1985, [3H]WB410t labels Si serotonin receptors in rat cerebral cortex, European J. Pbarmacol. 106, 461. Pazos, A., D. Hoyer and J.M. Palacios, 1984, Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: Evidence for species differences in brain serotonin-2 receptors, European J. Pharmacol. 106, 531. Peroutka, S.J., 1984, Vascular serotonin receptors-correlation with 5-HT1 and 5-HT2 binding sites, Biochem. Pharmacol. 33, 2349. Richelson, E. and A. Nelson, 1984a, Antagonism by neuroleptics of neurotransmitter receptors of normal human brain in vitro, European J. Pharmacol. 103, 197. Richelson, E. and A. Nelson, 1984b, Antagonism by antidepressants of neurotransmitter receptors of normal human brain in vitro, J. Pharmacol. Exp. Ther. 230, 94. Schnellmann, R.G., S.J. Waters and D.L. Nelson, 1984, [3H]5Hydroxytryptamine binding sites: Species and tissue variation, J. Neurochem. 42, 65.
279
Elsevier EJP 20022 Short communication
Antagonism by neuroleptics of serotonin 5-HTIAand 5-HT 2 receptors of normal human brain in vitro T h e o d o r e J. W a n d e r , A l b e r t N e l s o n , H a r u o O k a z a k i a a n d Elliott R i c h e l s o n * Departments of Psychiatry and Psychology, Pharmacology, and 1 Pathology, Mayo Foundation, Rochester, MN 55905, U.S.A.
Received 17 September 1987, accepted 22 September 1987
Using radioligand binding techniques and human frontal cortex, we determined the equilibrium dissociation constants (KDS) of 17 neuroleptics at the serotonin 5-HTIA and serotonin 5-HT2 receptors with [3H]WB4101 and [3H]ketanserin, respectively. At the serotonin 5-HTaA receptor, the most and least potent neuroleptics were chlorprothixene (K D = 230 nM) and fluphenazine (KD = 40 #M), respectively. At the serotonin 5-HT2 receptor, the most and least potent neuroleptics were spiperone (K D = 0.38 nM) and molindone, (K D = 5/aM), respectively. Neuroleptics; Brain; Serotonin receptors; (Human)
1. Introduction
Psychotherapeutic drugs antagonize m a n y different receptors in the central nervous system. This antagonism m a y explain a drug's therapeutic and adverse effects as well as its interactions with other drugs. In addition, there appears to be a relationship between a drug's receptor affinity (or its reciprocal, the equilibrium dissociation constant) and its potency or likelihood of causing an effect. Specifically for neuroleptics, there is a strong correlation between dopamine D-2 receptor affinity and daily dose for treating schizophrenia (Richelson and Nelson, 1984b). The radioligand binding assay is presently the method of choice in assessing a drug's affinity for certain receptors. Most assays of this type have utilized animal brain tissue, and some investigators have found good correlations between these results and those obtained with the use of h u m a n brain tissue (Richelson and Nelson, 1984a, b; Fowler, 1986). Because there are examples of dif* To whom all correspondence should be addressed: Guggenheim 7, Mayo Foundation, Rochester, MN 55905, U.S.A.
ferences in results between species (Pazos et al., 1984; Schnellmann et al., 1984), we have routinely used human brain tissue in our radioligand binding studies, and here we report the dissociation constants (KDS) of 17 neuroleptics at the serotonin 5-HTIA and 5-HT 2 receptors of h u m a n brain.
2. Materials and methods 2.1. Tissue preparation
H u m a n brain tissue was obtained at the time of autopsy (range 1.3-12.5 h after death) from donors with no known history of neuropsychiatric illness. Cardiovascular events were responsible for death in all five donors: myocardial infarction in two and cardiac dysrhythmia, ruptured thoraco-abdominal aortic aneurysm, and pulmonary embolism in the others. Specimens appeared grossly normal. Tissue was stored in a liquid nitrogen refrigerator until its use. G r a y matter was homogenized in 10 volumes of ice cold buffer using a Brinkmann Polytron PT-45 (45 s, setting 8). The buffer used for the serotonin 5-HTaA assays was
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
280 50 mM Tris HC1 (pH 7.7 at 25 o C) containing 5 mM MgSO4 and 0.5 mM EDTA, and that used for the serotonin 5-HT2 assays was 50 mM Tris HC1 (pH 7.7 at 25°C). The homogenate was centrifuged at 38 000 × g for 10 rain. The pellets were resuspended in buffer to give 30 and 10 mg initial wet weight/ml for the serotonin 5-HTIA and 5-HT2 assays, respectively. Aliquots of homogenate were stored at -33 ° C.
2.2. Receptor assays Into glass culture tubes (12 X 75 mm) were placed the tritiated ligand (0.05 ml), drug solution, and buffer. For the serotonin 5-HTaA assays, 0.10 ml of 9 gM prazosin was also added to each tube. The solution was stirred using a vortex mixer. Homogenate (0.25 ml) was added to each tube, and the suspension (1.0 rnl) was again stirred using a vortex mixer. The final concentration of each radioligand was 0.51 and 0.26 nM for the serotonin 5-HT1A ([3H]WB4101) and 5-HT2 ([3H]ketanserin) assays, respectively. Duplicate samples were incubated at 37 °C for 1 h and the suspensions were then filtered under vacuum using Whatman G F / B filter strips on a Brandel M-24 R cell harvester. The tubes and filters were rinsed with 3 x 5 ml of ice-cold buffer. The filter circles were placed in 6 ml plastic mini-vials (Research Products International) and 4 ml scintillation fluid (Safety Solve, Research Products International) was added to each. After the tubes stood 5 h, the radioactivity was measured using either a Searle Isocap 300 or a Beckman LS 7800 liquid scintillation counter at an efficiency of 30 and 42%, respe~..tively~iSpecific binding was defined as total biodirtg mi.~us non-specific binding, which for the .s¢rot0nin-5;~TrA assays was determined with 1 gM-,~ys~rgid.~i~LSD) and for the serotonin 5-HT2 ass¢y~ with,~]~i t~M methysergide. Using a ~wlett~Eackard: 9845B~ computer connected by ng~em t~a~Cy~er ~ m p m e r (Control Data Corp., Min!~eal~li~_)~e:ar!a|yz~ the data using the L,[GAND!,p~o.gr~ (Munson and Rodbard, 1980) to proyide valu~ fore,he equilibrium constant K D. ~,W¢have::jnodif~ed~this program in order to obtain theHitl ,~co.efficient (nil) as well ~he mean K D and n H values (__+S,E,M.) in tables 1 and 2 are
from at least three independent experiments, each determined in duplicate.
2.3. Drugs [3H]WB4101 (27.0 Ci/mmol) and [3H]ketanserin (76.7 Ci/mmol) were from New England Nuclear (Boston, MA); WB4101 from Amersham (Arlington Heights, IL); lysergide (LSD) from National Institute of Drug Abuse (Rockville, MD); ketanserin and spiperone from Janssen Pharmaceutica Inc. (New Brunswick, NJ); mesoridazine from Boehringer Ingelheim Ltd. (Ridgefield, CT); chlorpromazine from Sigma Chemical Co. (St. Louis, MO); thioridazine, clozapine and methysergide from Sandoz Pharmaceuticals (East Hanover, NJ); haloperidol from McNeil Pharmaceutical (Spring House, PA); fluphenazine and triflupromazine from E.R. Squibb & Sons, Inc. (Princeton, N J); perphenazine from Schering Corp. (Kenilworth, N J); prochlorperazine and trifluoperazine from Smith Kline & French Laboratories (Philadelphia, PA); molindone from Dupont Pharmaceuticals (Wilmington, DE); chlorprothixene from Hoffman-LaRoche Inc. (Nutley, N J); prazosin and cis-thiothixene from Pfizer Inc. (New York, NY); d-butaclamol from Ayerst Laboratories (New York, NY); loxapine from Lederle Laboratories (Wayne, NJ); and promazine from Wyeth Laboratories (Philadelphia, PA).
3. Results
3.1. Serotonin 5-HTIA receptor of human frontal cortex Although [3H]WB4101 has been considered to label al-adrenoceptors, it has been shown that this radioligand may be used to label the serotonin 5-I-IT1 (probably, 5-HT1A) site under certain experimental conditions (Norman et al., 1985). Because [3H]WB4101 has high affinity for al-adrenergic sites, prazosin (900 nM), a potent and selective al-adrenoceptor antagonist, was used in each assay to block its binding to these receptors. Binding to serotonin 5-HTIA receptors was rapid, reached steady state within several min and
281 TABLE 1 Neuroleptics: equilibrium dissociation constants (KDS) for the serotonin 5-HT1A receptor of human brain frontal cortex K D + S.E.M. (nM)
Hill coefficient + S.E.M.
230 5: 20 320 5: 90 350 5: 70 500 5: 100 800 + 300 12005: 300 1400 + 400 18005: 300 2 800 5: 600 2 900 5: 400 3 200 + 800 3 600 5: 400 5 900___ 700 120005:2000 2 3 0 0 0 + 1000 230005:5000 40000 + 10000
1.12 5:0.06 1.2 + 0.2 0.62 + 0.09 1.0 _+0.1 1.00 5:0.09 1.0 5:0.1 0.8 5:0.1 1.1 5:0.2 0.8 + 0.2 0.99 5:0.07 1.0 5:0.1 1.7 + 0.2 1.0 5:0.1 0.8 +0.1 0.7 5:0.1 0.7 +0.1 0.58 __.0.03
Neuroleplics Chlorprothixene Spiperone Thioridazine Mesoridazine d-Butaclamol Molindone cis-Thiothixene Clozapine Haloperidol Loxapine Chlorpromazine Perphenazine Prochlorperazine Promazine Trifluoperazine Triflupromazine Fluphenazine
Serotoninergic compounds WB4101 Lysergide (LSD)
3.1 5:0.3 0.64 5:0.08
0.94 + 0.04 0.90 + 0.08
maintained this level of binding for at least 1 h. Competition experiments revealed that WB4101 possessed a K D + S.E.M. equal to 3.1 + 0.3 nM (n = 29), which is similar to that (3.8 nM) reported using rat cortical homogenates (Norman et al., 1985). The Hill coefficient did not differ significantly from unity, indicating that the compound bound to a single class of receptor sites under these conditions. Of the 17 neuroleptics studied at the 5-HT~A receptor, chlorprothixene was the most potent with a K D = 230 ___20 nM and a Hill coefficient essentially equal to unity (table 1). The weakest binding was exhibited by fluphenazine, with a K D = 40 + 10 btM. N o correlation existed between the log of the KDS and the log of the average daily doses for treating schizophrenia (r = 0.15, p = 0.58). 3.2. Serotonin 5 - H T 2 receptor of human frontal cortex
[3H]Ketanserin and methysergide can be used to study the serotonin 5-HT2 receptor site (Leysen
TABLE 2 Neuroleptics: equilibrium dissociation constants (KDS) for the serotonin 5-HT2 receptor of human brain frontal cortex. K D _ S.E.M. (nM)
Hill coefficient 5: S.E.M.
Ratio K D 5 - H T I A / K D 5-HT2
0.38 + 0.05 0.4 + 0.1 0.43 + 0.08 1.4 + 0.4 1.6 + 0.4 1.7 + 0.4 4 + 1 4.8 + 0.3 5.6 + 0.6 14 + 1 15 + 2 16 + 4 19 + 5 22 + 8 36 + 5 130 + 30 5 000 + 1000
0.96 + 0.08 1.10 + 0.06 1.06 + 0.07 0.76 + 0.07 1.1 +0.2 1.1 +0.1 1.2 + 0.1 1.2 +0.2 1.22 + 0.06 1.1 +0.1 1.2 +0.1 1.3 +0.2 1.04 + 0.08 0.78 + 0.02 1.2 +0.1 1.04 + 0.09 0.84 + 0.07
840 2 000 540 2 300 1100 1700 5 800 100 640 1600 390 750 2100 16 78 11 0.24
0.56 + 0.04 7 +2
1.03 + 0.02 0.81 + 0.07
Neuroleptics Spiperone d-Butaclamol Chlorprothixene Chlorpromazine Clozapine Loxapine Triflupromazine Mesoridazine Perphenazine Trifluoperazine Prochlorperazine Promazine Fluphenazine Thioridazine Haloperidol cis-Thiothixene Molindone
Serotoninergic compounds Ketanserin Methysergide
282 et al., 1982). Ketanserin had a K D = 0.56 + 0.04 n M (n = 29), which is comparable to previous studies by Leysen et al. (1982) using rat prefrontal cortex ( K D = 0.42 nM). Hill coefficients for ketanserin and the neuroleptics were essentially equal to one (table 2), indicating binding to a single population of receptor sites. KDS for the 17 neuroleptics were relatively low (table 2), and spiperone (K D = 0.38 + 0.05 nM), d-butaclamol (K D = 0.4 + 0.1 nM) and chlorprothixene (K D = 0.43 _ 0.08 nM) all b o u n d m o r e strongly than ketanserin. Molindone had a K D = 5 + 1 #M, which was greatly different from the values for the other compounds. Again, no correlation existed between the log of the KDS and the log of the average daily doses (r = 0.097, P = 0.71).
4. Discussion Here we have presented the data for 17 neuroleptics binding at the serotonin 5 - H T 1 (probably, 5-HTIA ) and 5 - H T 2 receptors present in n o r m a l h u m a n brain frontal cortex. F o r these compounds, there was no significant correlation between the log of their KDS at the 5-HT1A receptor and the log of their KDS at the 5 - H T 2 receptor. However, all but one c o m p o u n d , molindone, b o u n d m o r e potently to the 5 - H T 2 receptor (table 2). We c o m p a r e d our data for six c o m p o u n d s in c o m m o n with the study of Leysen et al. (1982) who also used [3H]ketanserin as a radioligand but used rat prefrontal cortex. A significant correlation (r = 0.98, P = 0.0005) existed between the two sets of data. We have cited in this paper other comparisons between our results and those of others using animal brain. These comparisons suggest that there are no large species differences between h u m a n and animal brain serotonin receptors, although Pazos et al. (1984) reported species differences for mesulergine, an apparently selective serotonin 5 - H T 2 ligand. The clinical relevance of serotonin receptor
blockade b y neuroleptics is unknown. Certainly, the lack of correlation between receptor affinity and neurleptic dose indicates that serotonin receptor blockade alone does not explain the m o d e of action of these drugs. However, vascular side-effects of these c o m p o u n d s m a y be mediated by blockade of serotonin receptors (Peroutka, 1984).
Acknowledgements This work was supported by Mayo Foundation and U.S.P.H.S. Grant MH27692 from N.I.M.H.
References Fowler, C.J., 1986, The pros and cons of using human brain autopsy samples for radioligand binding experiments, Trends Pharmacol. Sci. 7, 9. Leysen, J.E., C.J.E. Niemegeers, J.M. Van Nueten and P.M. Laduron, 1982, [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin 2 receptor binding sites, Mol. Pharmacol. 21, 301. Munson, P.J. and D. Rodbard, 1980, LIGAND: A versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107, 220. Norman, A.B., G. Battaglia, A.L. Morrow and I. Creese, 1985, [3H]WB410t labels Si serotonin receptors in rat cerebral cortex, European J. Pbarmacol. 106, 461. Pazos, A., D. Hoyer and J.M. Palacios, 1984, Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: Evidence for species differences in brain serotonin-2 receptors, European J. Pharmacol. 106, 531. Peroutka, S.J., 1984, Vascular serotonin receptors-correlation with 5-HT1 and 5-HT2 binding sites, Biochem. Pharmacol. 33, 2349. Richelson, E. and A. Nelson, 1984a, Antagonism by neuroleptics of neurotransmitter receptors of normal human brain in vitro, European J. Pharmacol. 103, 197. Richelson, E. and A. Nelson, 1984b, Antagonism by antidepressants of neurotransmitter receptors of normal human brain in vitro, J. Pharmacol. Exp. Ther. 230, 94. Schnellmann, R.G., S.J. Waters and D.L. Nelson, 1984, [3H]5Hydroxytryptamine binding sites: Species and tissue variation, J. Neurochem. 42, 65.