0022-3565/81/2161-0142502.00/0 THE JOURNALOr PHARMACOLOGYAND EXPERIMENTAl.THERAPEUTIC8 Vo], 216, No, I Copyr_ht © 1981 by The American Society for Pharrnscology and Expenrnenta] Therapeutics Printed in U.S.A
[3H]Mianserin" Differential Labeling of Serotonin2 and Histamine, Receptors in Rat Brain' f":, , i _.-F_ i "r I
STEPHEN J. PEROUTKA 2 and SOLOMON H. SNYDER Departments of Neuroscience, Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland Accepted for publication October 14, 1980
ABSTRACT
IPeroutka, / Stephen J. and Solomon H. Snyder: [3H]Mianserin: branes. By using [3H]mianserin, both 5-HT2 and histamine H1 _)itterentlal labeling of serotonin2 and histamine1 receptors in receptors can be specifically labeled in rat cerebral cortex _ rat brain. J. Pharmacol. Exp. Ther. 216: 142-148, 1981. membranes. Simultaneous incubation of brain membranes with 300 nM triprolidine or 30 nM spiroperidol enables the selective Mianserin, a tetracyclic antidepressant, is a potent serotonin labeling of 5-HT2 or histamine H1 receptors, respectively. In the (5-HT) and histamine H_ antagonist in peripheral smooth mus- guinea-pig cerebellum, [3H]mianserin exclusively labels hista- cle systems. Mianserin was found to possess high affinity for mine H_ receptors, since 5-HT2 sites are virtually absent in this 5-HT2 and histamine H_ receptor binding sites in brain mem- area.
Mianserin is a tetracyclic piperazine derivative that was only a small portion of [3H]spiroperidol binding labels 5-HT2 developed as an antiallergic compound (van der Burg et al., receptors as most of the binding is associated with dopamine 1970). In peripheral smooth muscle systems, it displays potent receptors (Leysen et al., 1978; Creese and Snyder, 1978; Per- antiserotoninergic and antihistaminic activity as well as some outka and Snyder, 1980). Moreover, in portions of the limbic a-adrenolytic effects (Vargaftig et al., 1971). Mianserin is also system [3H]spiroperidol binding is largely associated with an a clinically effective antidepressant (Itil et al., 1972; Fell et al., anomalous site with selectivity for the spirodecanone structure 1973) despite relatively weak inhibition of norepinephrine and (Howlett et al., 1979). Because of these problems, we explored serotonin reuptake (Kofoe and Leonard, 1973; Zis and Goodwin, [3H]mianserin as an alternative ligand for 5-HT_ receptors. 1979}, failure to_everse reserpine-induced hypothermia (van We now report that [3H]mianserin does label 5-HT2 receptors Riezen, 1972} and inability to block monoamine oxidase (Fell et in rat brain. Additionally, [3H]mianserin binds to histamine H_ al., 1973). Although central physiological effects of mianserin receptors with a selectivity similar to that of [3H]mepyramine have been reported (Maj et al., 1978), mianserin's interactions (Tran et al., 1978; Hill et al., 1978). The binding of [3H]mian- with neurotransmitter receptor sites have not been thoroughly serin to 5-HT2 and histamine H_ receptors can be differentiated evaluated, by pharmacologic or anatomic manipulations. Two distinct serotonin (5-HT) receptor sites can be labeled in brain membranes (Peroutka and Snyder, 1979). Receptors Materials and Methods referred to as 5-HT_ are labeled by ['3H]-5-HT, regulated by guanine nucleotides and appear to be linked to the 5-HT Animals. Adult male Sprague-Dawley rats were obtained from sensitive adenylate cyclase in brain membranes (Peroutka et Sprague-Dawley Laboratories (Madison, WI) and were used at weights al., 1979, 1980). 5-HT2 receptors, labeled by [3H]spiroperidol, of 150 to 200 g. Rats were maintained under a 12-hr light-dark cycle and had free access to food and water. After decapitation, brains were mediate the behavioral syndrome associated with central sere- removed and specific regions dissected over ice immediately before toninergic stimulation (Jacobs, 1976) since the potencies of assay. Frozen guinea-pig brains were purchased from Pel-Freez Biolog- numerous drugs in preventing the behavioral effects correlate icals, Inc. (Rogers, AK) and were defrosted at room temperature in closely with their affinities for 5-HT2, but not 5-HTI receptors saline before use. (Peroutka et al., 1980). Binding assays. Rat frontal cerebral cortices or guinea-pig cerebella There are some serious limitations in the use of ['_H]spiro- were homogenized in Tris-HCl buffer (pH 7.7 at 25°C) using a Brink- peridol for labeling 5-HT2 receptors. In the corpus striatum, mann Polytron for 10 sec. The homogenate was centrifuged at 50,000 X g for l0 rain, resuspended in Tris-HCl buffer and centrifuged again at 50,000 x g for 10 vain. The pellet was suspended in the standard Received forpublicationAprill, 1980. assay buffer (Tris-HC! buffer and 0.1% ascorbic acid) and stored on ice This work was supportedby U.S. Public Health Service GrantsDA-00266, until used. MH-18501and a grantof the McKnightFoundation. Recipient of MedicalScientist TrainingGrant 5T32GM0309from the Na- Incubation tubes received 0.1 ml of _H-ligand, 0.1 ml of various drugs tional Institutesof Health. and 0.8 ml of tissue suspension. All assays were performed in triplicate. 142 1981 Mianserin Receptor Interactions 143
The final tissue concentration was l0 mg of wet weight ti._sue per ml substantial affinity for alpha-2 receptors labeled byp-[:_H]ami - for all brain regions except caudate (3.5 mg of wet weight tissue per noclonidine (19 nM) and alpha-1 sites labeled by [:_H]-WB-4101 ml). After incubation at 37°C for 15 rain, the samples were rapidly (63 nM),although these effects are about an order of magnitude filtered under vacuum through Whatman GF/B filters with three 5-ml less than its affinity for 5-HT2 and histamine H_ sites. Mianserin rinses of ice-cold Tris-HCl buffer (pH 7.7 at 25°C). The filters were counted by liquid scintillation spectrometry in 10 ml of Formula 947 is quite weak at muscarinic cholinergic, beta adrenergic, opiate (New England Nuclear; Boston, MA) after 18 hr extraction at 4°C. and benzodiazepine receptor binding sites. Drugs. [3H]Mianserin (16 Ci/mmol) was purchased from Amersham Properties of [3H]mianserin binding to rat cerebral {Arlington Heights, IL). All other 3H-ligands were obtained from New cortex membranes. The binding of [3H]mianserin to brain England Nuclear. aH-ligands were stored at -20°C and diluted in membranes is saturable (fig. 1). Specific binding accounts for standard assay buffer immediately before assay. Unlabeled drugs were approximately 50% of total binding at a concentration of 0.7 obtained from the following sources: mianserin (Organon, Amsterdam, nM, the amount of [3H]mianserin used in routine drug compe- Holland), cyproheptadine (Merck Sharp & Dohme; Rahway, NJ), d- tition studies. At ligand concentrations above 1.0 riM, specific and l-chlorpheniramine (Smith Kline and French Laboratories, Phila- binding represents a decreasing percentage of total binding and delphia, PAL triprolidine (Burroughs-Wellcome, Research Triangle is only 25% of total binding at 10 nM [aH]mianserin. Specific aPacidrk,diethylamideNC), iprindole (LSD)(Wyeth(NLaboratoriational Institues, Philadelphia,te on Drug PAAbuse), d-)ly, se5-rgicHT [3H]mianserin binding reaches plateau levels at about 6 nM (Sigma Chemical Co., St. Louis, MO), spiroperidol (Janssen Pharma- and attains half maximal binding at approximately 3 nM. By ceutica, Beerse, Belgium) and cinanserin (Squibb, Princeton, NJ). contrast, nonspecific binding increases linearly with increasing [aH]mianserin concentrations. This nonspecific binding is not Results altered by 30 nM spiroperidol or 300 nM triprolidine, which are employed (below) to differentiate [3H]mianserin interactions - Influence of mianserin on various neurotransmitter with histamine H_ and 5-HT2 receptors. Scatchard analysis receptor binding sites. Mianserin has been reported to pos- indicates the presence of a single population of binding sites sess blocking activity at 5-HT, histamine and alpha adrenergic with an apparent dissociation constant (KD) of 3.7 nM. receptors in peripheral smooth muscle systems (Vargaftig et Specific [aH]mianserin binding is linear between 2 and 20 al., 1971). To evaluate the influence ofmianserin on neurotrans- mg/ml of brain membrane, with all experiments performed mitter receptors in the brain, we determined its potency in using 10 mg wet weight tissue per ml. The specific binding of competing for the binding of various 3H-ligands (table 1). Mian- [3H]mianserin displays a relatively broad pH optimum with serin is most potent at 5-HT2 receptors labeled by [aH]spiro- about the same levels of binding between pH 7 and 8. The peridol and histamine H] receptors labeled by [aH]mepyramine amount of [3H]mianserin binding obtained in standard Tris- with Ki values of 2.4 and 3.3 nM, respectively. In contrast to its HCI assay buffer is not significantly altered by the addition of high affinity for 5-HT2 receptors, mianserin is about 400 times 4 mM calcium chloride, 100 mM sodium ions or 10 mM potas- less potent at 5-HT_ receptors labeled by [aH]-5-HT (860 riM). sinm ions. Unlike spiroperidol, which has equally high affinity for both Kinetics of[aH]mianserin binding. Specific [3H]mianserin dopamine and 5-HT2 receptors, mianserin is 1000 times less binding is time-dependent (fig. 2A). At 37°C, binding plateaus potent at dopamine receptors labeled by [aH]spiroperidol in the between 7 and 30 min and attains half-maximal binding at caudate (2200 nM) than at 5-HT_ receptors labeled by [3H]- approximately 1 min. The rate constant for association (k0 is spiroperidol in the cerebral cortex. Mianserin also displays 0.10 nM-_min -_ at 37°C determined from the equation
TABLE I ...... k,,_ - k_, Apparent affinity (K,) of mianserin for neurotransmitter receptor k_ = [L] binding sites Standard assay conditions were used in all experiments. The aH4igand concen- where k_] equals the f'Lrst order dissociation rate constant, [L] trations were 0.25 nM [aH]spiroperidol, 2.0 nM laH}-5-HT. 2.0 nM [aH]mepyra- mine, 0.6 nM/>_aH]aminoclonidine, 0.9 nM [3H]-WB-410), 0.2 nM laH]cluinucti - equals the steady-state free ligand concentration and kobs is the dinyl beflzilate (QNB). 0.8 nM [aH]dihydroalprenolol, 1.g nM [aH]naloxone, 1.4 observed rate of association. nM d-[aH}-ala-leu-enkephalin and 0.7 nM laH]flunitrazepam. Specific binding was To determine the dissociation rate of specifically bound
recdefinedeptorsas, the1 /.teMxcetrilssarolidineover blafornkshitstakeaminen in threeceptorspresen,c0e.1 ofmM1 #Moxotrd-LSeDmorinefor 5-HTfor [3H]-mianserin, we conducted incubations to equilibrium, at muscarinic cholinergic receptors, 1 #M (±)-propranolol for beta adrenergic which time 1/_M cyproheptadine was added and residual bind-
azepinereceptors,receptors1 #M levaliando10rphap.Mn (-)for-norepineopiate prhrineeceptors,for1alpp.haM -diazepa1 andmalpforha-2berecepnzodi-- ing was examined at various intervals. The dissociation appears tors_ IC_o values were determined from Iog-probit analysis of drug competition to be a first-order process, as represented by a straight line data using 4 to 5 concentrations of mianserin. K, values were calculated from the when plotted on a semi-logarithnfic scale (fig. 2B). The half- equation K, = ICso/(1 + [aH ligand]/Krj). The KD values.were obtained from time for dissociation is about 2.5 rain and the rate constant for previous reports (Bennett, 1978; Peroutka and Snyder, 197g). Values given are the means ± S.E. of three or more experiments, each performed in triplicate, dissociation (k-l) is 0.31 min -]. The apparent dissociation con-
Receptor 3H-Ligand gp0arentK, stant (KD) determined from the ratio k__/k_ is 3.1 nM, which nM agrees well with the KD value determined in saturation experi- 5-HT_ laHlSpiroperidol (cortex) 2.4 ± 0.51 ments. 5-HT, [aH}-5-HT 860 ± t l0 Displacement of [aH]mianserin binding by 5-HT and HistamineHt [_H]Mepyramine 3.3 ± 0.63 histamine-related agents. Displacement of specifically AIDha-2 adrenergic p-{aHlAmino¢lonidine Ig ± 5.0 A/pha-1adrenergic [aH}-WB410t 63 ± 7.4 bound [3H]mianserin by the antihistamines d-ch]orphenira- Muscarinic cholinergic [aH]-QNB 2,100 ± 380 mine and triprolidine and by the 5-HT-related agents d-LSD
BetaDopamadrenergine ic [a{aH]DlSpiihyrodroalpperidorlenolo(claudate) 4,42,20000 ± 96t100 and spiroperidol yield extremely shallow displacement curves Opiate [aHlNaloxone lO,000 + t,700 (fig. 3A). At least five orders of magnitude are required to Opiate d-[3H}-Ala4eu-enkephalin 12,000± 2,0oo displace 90% of the bound [3H]mianserin. Clearcut biphasic Benzodiazepine [aH]Flunitrazepam >100,000 displacement is apparent for triprolidine, whose displacement 144 Peroutka and Snyder Vol. 216
I0,000
8,000
-_ TOTAL
123 Z
_Do 6,000
Z
Z
4,000 NON-SPECIFIC T
SPECIFIC ______.._m 2,000
o _ _ _ _ ,o
[3H] MIANSERIN (nM) Fig. 1. [3H]Mianserinbindingas a functionof increasingconcentrationsof [3H]mianserin.Rat frontalcerebral cortexmembraneswere incubated under standard bindingassay conditionswith increasingconcentrationsof [3H]mianserin.Nonspecific binding was defined as the amount ot [3H]mianserinbound inthepresenceof 1 p.Mcyproheptadine.Specific bindingrepresentsthe difference between total and nonspecificbinding. The experimentwas replicatedthree times.
curve is essentially flat between 0.1 and 1.0/aM concentrations. Hill coefficients of about 1.0. At the same time, the potencies of Hill coefficients for d-chlorpheniramine, triprolidine, d-LSd and d-LSD and spiroperidol are decreased while their Hill coeffi- spiroperidol are 0.33, 0.23, 0.52 and 0.32, respectively, cients are raised to 1.0. The shallow displacement curves and similar potencies of 5- The 50% decrease in [3H]mianserin binding in the presence HT and histamine antagonists could indicate that [aH]mian- of 300 nM triprolidine or 30 nM spiroperidol derives from 50% serin binds to a unique receptor site displaying considerable site occupancy and therefore should alter the apparent Bma= cooperativity. Alternatively, it is possible that [aH]mianserin with no change in apparent KD. To test this prediction, we labels both 5-HT2 and histamine H_ receptors. The low Hill conducted saturation studies of [aH]mianserin in the presence coefficients may reflect drug competition for the binding of of 300 nM triprolidine or 30 nM spiroperidol (fig. 4). Addition [3H]-mianserin to the two receptor sites with markedly different of triprolidine or spiroperidol elicits no change in KD of specific .... affinities. If this were the case, it might be possible to obtain [3H]mianserin binding. However, both drugs reduce the maxi- [3H]mianserin binding selectively to 5-HT2 receptors ff the real number of binding sites (Bin,x) by approximately 50%. Thus, binding to histamine H_ sites were pharmacologically blocked, total specific [3H]mianserin binding in the rat cerebral cortex Accordingly, we examined the displacement of [3H]mianserin appears to be a composite of the labeling of two distinct sites, binding in the presence of 300 nM triprolidine, a concentration half of which involve 5-HTe receptors and the other half his- of unlabeled antagonist which saturates all HI receptors (Tran tamine H_ receptors. The Bma, values for apparent 5-HT_ and et al., 1978). Under these conditions, d-LSD and spiroperidol histamine H_ sites (13.7 and 13.3 pmol/g of wet weight tissue) become substantially more potent with about 50% inhibition of determined in this way agree well with parallel determinations binding at 10 and 3 nM, respectively (fig. 3B). Moreover, d- using [3H]mepyramine for histamine H_ sites (12.8 pmol/g) and chlorpheniramine and triprolidine became much weaker in [3H]spiroperidol for 5-HT2 receptors (14 pmol/g). inhibiting [3H]mianserin binding with half-maximal displace- Differential effects of drugs on [sI-I]mianserin binding ment at approximately l0 #M. For all four drugs, the Hill in the presence or absence of 300 nM triprolidine or 30 coefficients are significantly increased in the presence of 300 nM spiroperidol. To further determine whether the binding nM triprolidine to about 1.0. sites labeled by [3H]mianserin represent both 5-HT2 and his- Conversely, possible labeling of histamine H_ receptors by tamine H1 receptors, we evaluated a series of 5-HT- and hista- [3H]mianserin should be apparent in the presence of 30 nM mine-related agents (table 2). We compared drug potencies in spiroperidol, a concentration sufficient to fully occupy 5-HT2 inhibiting [3H]mianserin binding in the absence and presence receptors (Peroutka and Snyder, 1979). Under these conditions, of 300 nM triprolidine or 30 nM spiroperidol. These values were d-chlopheniramine and triprolidine become substantially more also compared to the drug potencies at [_H]spiroperidol labeled potent with 50% displacement at 5 to 8 nM for both drugs (fig. 5-HT2 receptors and [aH]mepyramine labeled H_ receptors in 3C). Moreover, displacement curves for the antihistamines are the rat cerebral cortex. significantly steeper in the presence of 30 nM spiroperidol with Mianserin and cyproheptadine are potent blockers of both 5- 1981 Mianserin Receptor Interactions 145
r_ varies more than 2000-fold between its affinity for [3H]mian- z t_ . serin sites in the presence of 300 nM triprolidine (11,000 nM) m I00 _ _- : = and in the presence of 30 nM spiroperidol (5.0 nM). The same
.J of pattern of potency differences also occurs with d-chlorpheni- Uo. A A reciprocal pattern occurs with drugs active at 5-HT recep- tors. d-LSD is only one-fortieth as potent at [3H]mepyramine _- K 60 (390 nM) as at [aH]spiroperidol (10 nM) sites. Its potency at 03 z m [aH]mianserin sites in the presence of 300 nM triprolidine (12 - fo", 40 nM) resembles its effects at 5-HT2 receptors while it competes (tuD -- for ['_H]mianserin binding in the presence of 30 nM spiroperidol z < 20 (180 riM) with a potency similar to its affinity for histamine HI _; receptors. This pattern of differential drug potencies also occurs cinanserin.Ofthe "1- ,_ 0 5 It) 15 2() 2'5 3'0 with 5-HT itself and the 5-HT antagonist ft Z TIME (MINUTES) _ A ...... |00 _ :. _- 0-CHLORPHENIR_,MLNE 2t"t B _ _.,.___ X--X TRIPROL,D,NE 80 O u . _ >. 70 • _ 4 _ am 0 so 50 _o x _'- F.,"_ 40 - 3o rrm_z_ 20 - * • 1" , , , i j L x_ W_ _ 0 I0 9 8 7 6 5 4 Z =--_E I0 - LOG {DRUG] "r 0 2 5 5 7.5 I0 12.5 mO IOO B " x _ x TIME (MINUTES) B, dissociation of [aH]mianserin from its receptor at 37°C. The sample "-E 0 Io 9 8 7 s .5 4 volumes were incubated for 15 min at 37°C under standard binding - assay conditions at which time 1 #M cyproheptadine was added (time - LOG [DRUG] zero) to initiate dissociation. The samples were then filtered at various ,._ time intervals. The experiment was replicated three times. 0 C m HT2 and histamine HI receptors as shown by their high affmi- _ __ ties for [aH]spiroperidol and [3H]mepyramine binding, respec- _ _ 8o tively. These two drugs also have nanomolar affinity for [aH]- =o_60 mianserin binding whether assayed alone or in the presence of == triprolidine or spiroperidol. By contrast, d-chlorpheniramine z =_ 40 has 1000 times greater affinity for histamine receptors labeled 0: by [3H]mepyramine (8.0 nM) than for 5-HT_ sites labeled by _ o drugs tested, spiroperidol is best able to differentiate 5-HT2 and little, if any, binding in the guinea-pig cerebellum (S. J. Per- histamine H_ receptors. Its affinities for [:_H]spiroperidol (1.0 outka and S. H. Snyder, manuscript in preparation). Accord- nM) and ['aH]mepyramine (670 nM) are approximated by spi- ingly, we evaluated the saturation of ['_H]mianserin binding to roperidors affinity for ['_H]mianserin sites in the presence of membranes prepared from guinea-pig cerebellum (fig. 5). Spe- triprolidine (3.7 nM) or spiroperidol (570 nM). cific [3H]mianserin binding to guinea-pig cerebellum is triple The differential effects of the 5-HT2 and histamine H_ selec- nonspecific binding and is saturable with a Ko value of 2.4 nM tire drugs indicate that the binding sites labeled by [3H]mian- for a single population of binding sites. The Bin,, for [3H]- serin represent both 5-HT2 and histamine H_ receptors in rat mianserin (23.1 pmol/g of wet weight tissue) is the same as the frontal cerebral cortex. B_x in the same tissue determined in parallel for [aH]mepyra- Specific [SH]mianserin binding in the guinea-pig cere- mine labeling of histamine H_ sites (21 pmol/g). In marked bellum. It would be desirable to evaluate [_H]mianserin bind- contrast to the results obtained in rat cerebral cortex, [3H]- hag to 5-HT or histamine receptors in isolation. Histamine H_ mianserin binding in guinea-pig cerebellum is totally abolished receptors are relatively ubiquitous in the brains of various by 300 nM triprolidine, while 30 nM spiroperidol has no effect species, including the guinea pig (Chang et al., 1979). By con- on either KD or B_,. values. These results suggest that [3H]- trast, preliminary studies have shown that [3H]spiroperidol- mianserin exclusively labels histamine H_ receptors in this brain labeled 5-HT2 receptors display marked regional variations with region. A A 3.000 A _o 3,000 Q CONTROL Z0 CONTROL • Z "_ D x .J J ,_ 2,000 . < _ 2Doe z_ z Iv nr 1,000 '" z tO00 d*300 nM TRIPROLIDINE O 2 4 6 8 I0 0 _ e_2 , 4. __ . 6 _ ' _o ,o Fig. 4. Saturation of [3H]mianserin binding in rat frontal cerebral cortex Fig. 5. Saturation of [3H]mianserin binding in guinea-pig cerebellar membranes. At each [aH]mianserin concentration, total binding was membranes. At each [3H]mianserin concentration, total binding was measured in the absence of inhibitor (O), in the presence of 300 nM measured in the absence of inhibitor (O), in the presence of 300 nM triprolidine (O) or in the presence of 30 nM spiroperidol (x). Specific triprolidine (O) and in the presence o( 30 nM spiroperidol (×). Specific binding was defined as the excess over blanks taken in the presence binding was defined as the excess over blanks taken in the presence of 1 _.M cyproheptadine. Points shown are the means of triplicate of 1 #M cyproheptadine. Points shown are the means of triplicate _ assays performed in a single experiment. The experiment was per- assays performed in a single experiment. The experimenl was repli- formed three times with values which varied less than 15%. cared three times with values which varied less than 20%. TABLE 2 Drug affinities for 3H-ligandbinding sites in rat frontal cerebral cortex Binding was conducted as described in "Materials and Methods." Values are means ----.S.E.M. for three to six independent ICso determinations. Each ICso value was obtained from Iog-probit analysis of the effects of four to six drug concentrations. IC_o Drug [3H]Spiroperidol [3H]MianserlR + 300 nM [3H]Mianseri n [:_'-I]Mianserin + 30 nM [3H]Mepyramine Trilxolidme Spiroperidol nM nM aM nM nM 5-HT2 and H_active Mianserin 2.5 ± 0.51 7.9 + 1.1 2.4 + 0.53 5.8 ± 0.14 3.3 ± 0.63 Cyproheptadine 3.0 ± 0.27 4.2 ± 1.6 3.7 ± 0.25 5.8 ± 1.5 4.1 + O.81 H, selective d-Chlorpheniramine 8,200 ± 2,400 4,200 ± 1,300 260 ± 70 7.8 ± 1.5 8.0 ± 2.7 I-Chlorpheniramine 14,000 ± 2,000 12,000 ± 1,400 3,500 ± 700 790 ± 250 700 ± 400 Triprolidine 11 ,O00 ± 1,500 11,0OO± 820 190 ± 85 5.O ± 1.6 4.7 ± 1.2 Iprindole 2,900 ± 640 2,000 ± 500 580 ± 49 370 ± 200 200 ± 51 5-HT2 selective d-LSD 10 ± 3.6 12 ± 3.0 70 ± 7.9 180 ± 16 390 ± 23 5-HT 3,200 ± 610 1,000 ± 360 22,000 ± 5,000 35,000 ± 6,600 90,000 ± 25,000 Cinanserin 36 ± 9.2 75 ± 2.6 300 ± 89 1,100 ± 190 1,000 ± 210 Spiroperidol 1.0 ± 0.12 3.7 ± 0.22 44 ± 19 570 ± 76 670 ± 120 1981 Mianserin Receptor Interactions 147 To further examine this possibility, we analyzed displacement Discussion curves of d-chlorpheniramine, triprolidine, d-LSD and spiro- The present study demonstrates that [_H]mianserin has high peridol in the guinea-pig cerebellum (fig. 6). d-Chlorphenira- affinity for both 5-HTe and histamine H_ receptors in brain mine and triprolidine potently displace [:_H]mianserin binding membranes. Central anti-5-HT effects of mianserin have been with ICr_ values between 1 and 5 nM. By contrast, d-LSD and reported on the basis of its blockade of 5-hydroxytryptophan- spiroperidol are much weaker displacing agents, requiring about induced head twitches (van Riezen, 1972; Maj et al., 1978) and 1 pM concentrations to displace 50% of [3H]mianserin binding, inhibition of 5-HT-induced hyperthermia (Maj et al., 1978). Additionally, the displacement curves for each of these drugs Mianserin also causes a uniquely complex set of electroen- are monophasic with Hill coefficients of approximately 1.0. cephalographic changes that may relate to central neurotrans- Relative affinities of various drugs for guinea-pig cerebellum mitter interactions (Itil et al., 1972). Clinically, the sedative binding sites of [3H]mianserin also confmn the notion that side-effects of mianserin (Fell et al., 1973) may be consistent [aH]mianserin selectively labels histamine H_ receptors in this with its potency at central a-noradrenergic receptors brain region (table 3). For all classes of drugs tested, potencies (U'Prichard et al., 1978). The present study provides the first in reducing [aH]mianserin binding in guinea-pig cerebellum direct demonstration of the interactions of mianserin with closely resemble potencies at sites in rat cerebral cortex labeled central nervous system receptors. by [3H]mepyramine or by [aH]mianserin in the presence of 30 As a radioligand, [3H]mianserin may prove useful in 5-HT_ nM spiroperidol. Interestingly, mianserin, cyproheptadine, d- and histamine H] receptor binding studies. All the binding of and /-chlorpheniramine and triprolidine are somewhat more [3H]mianserin observed here can be accounted for by histamine potent in guinea-pig cerebellum than in rat cortex, a character- H_ and 5-HT2 receptors. No selective reduction of binding by istic noted in earlier studies (Chang et al., 1979). alpha-1 or alpha-2 drugs is demonstratable (S. J. Peroutka, ( unpublished observations). Until the present time, [3H]spiro- peridol was the only ligand available for the study of 5-HT2, but not 5-HT] receptors. However, in brain regions such as the ___ A _- : d ° CHLORPHIFNIRAMINI corpus striatum, nucleus accumbens and olfactory tubercle, the [OO -_,,. _ Xo--ox TRIPROLId-LSO FOIN extremely high density of doparnine receptors make it techni- x_ "_0%_ -- -- SPIROPERIDOL cally difficult to study selectively [aH]spiroperidol-labeled 5- _) eo \\ _ HT2 receptors. In addition, [aH]spiroperidol may bind to a 60 affinity for the spirodecanone class of drugs (Howlett et al., _ 40 triprolidine, [3H]mianserin appears to label 5-HT2 binding sites z exclusively. In the present study, only about 50% of total ._ 20 [aH]mianserin binding is specific. This limitation is related to _-__oz °xm o ' ,__ _ thi1th9e79rd,)lo. wasBspyyeecificctontrast,undefinactiviedwhty, enr(ec16assayeptoCi/rmmoed sitl)ine tthhatofe thpresenceheas3H-a ligaselectindof ve300usehdighnMin IO 9 0 7 fi 5 4 this study. With new preparations of [3H]mianserin with higher specific activity, up to 75% of total binding is associated with 5- -LOG [DRUG] HT and histamine receptors (S. J. Peroutka, unpublished ob- Fig. 6. Displacemen__tof [aHlmianserinbindingto guinea-pigcerebellar servations). membranesby d-chlorphenirarnine(D), triprolidine(x), d-LSD (C))and [3H]Mianserin has proved valuable in assessing relative spiroperidol(I). Theexperimentwasperformedthree timeswithresults amounts of 5-HT2 receptor binding in different brain regions, varying less than 20%. especially those in which [3H]spiroperidol binding largely in- volves sites other than 5-HTe receptors (Peroutka and Snyder, 1980). Numbers of 5-HT2 receptors quantified with [aH]mian- TABLE 3 serin are similar to levels obtained using [aH]LSD with appro- Drug affinities for aH-ligandbinding in guinea-pig cerebellum priate displacers. Binding was assayed as described in "Materials and Methods." Values are 3 means :t: SE.M. for three to six independent IC5o determinations. Each ICso value [ H]Mianserin represents the first labeled antidepressant that wasobtainedfromIog-probitanalysisusingfourtosevendrugconcentrations, has been demonstrated to bind to previously characterized IC_ neurotransmitter receptor sites. Raisman et al. (1979) obtained Drug specific high affinity binding of [aH]imipramine that displays a laH]Mepyramine [aH]Mianserin unique pharmacological prof'de which does not fit with any nM known receptor site nor with the equipotent clinical effects of 5HTMi2aansernd H,in active 2.1 ± 0.72 2.8 ± 0.66 antidepressants. [aH]Desipramine binding displays biphasic Cyproheptadine 1.2 ± 0.24 1.8 2:0.24 saturation and a unique pharmacological profile, suggesting H, selective that multiple neurotransmitter sites may be labeled (Biegon d-Chlorpheniramine 2.4 ± 0.8 2.7 ± 0.26 and Samuel, 1979). Similarly, the multiphasic saturation of I-Chlorpheniramine 230 :t:50 230 ± 48 [3H]amitriptyline also suggests the labeling of multiple receptor ITriprinprdooliledine 531.20 ± 07.36 432.50 ± 20.786 sites (Rehavi and Sokolovsky, 1978). It remains unclear whether 5-HT2 selective any of the binding of the above antidepressants involves 5-HT2 d-LSD 480 ± 110 570 ± 130 or histamine H1 receptors. 5-HT 6000 ± 950 5500 ± 200 Cinanserin 4200 ± 640 35100 ± 110 Acknowledgments Spiroperidol 3200 ± 120 1700 ± 27 The authors wishto thank RichardLebovitzfortechnicalassistanceandDawn Hanksand NancyBruceforexcellentmanuscriptpreparation. 148 Peroutka and Snyder Vo/. 216 References serotonin receptors with different physiological functions. Science (Wash. DC}, BENNE'rr, J. P., JR.: Methods in binding studies. In Neurotransmitter Receptor in pre._% 1980. Binding, ed. by I-i. 1. Yamamura, S. ,1. Enna, and M. J. Kuhar, pp. 57-.'90, Raven PEROUTKA, S. J- ANn SNYDER, S. H.: Multiple serotonin receptors: Differential Press, New York, 1978. binding of [:_H]serotonin, [ 'H]lysergic acid diethylamide and [:'H ]spiroperidol. BIEGON, A. AND SAMUEL, D.: Binding of a labeled antidepre_nt to rat brain Mol. Pharrnacol. 16: 687-699, 1979. tissue. Biochem. Pharmacol. 28: 3361-3366, 1979. PEROUTRA, S. J. AND SNVDER, S. H.: Two distinct serotonin receptors: Regional CRANG, R. S. L., TRAN, V. T. AND SNYDER, S. H.: Heterogeneity of histamine H,- variations in receptor binding in mammalian brain. Brain Res., in pre._% 1980. receptors: Species variations in [_H]mepyramine binding to brain membranes. RAISMAN, R., RRILEY, M. AND LANGER, S. Z.: Specific trieyclic antidepressant J. Neuroehem. 32: 1653-1663, 1979. binding sites in rat brain. Nature ILond.) 281: 148-150, 1979. CREESE, I. AND SNYDER, S. H.: :'H-Spiroperidol labels serotonin receptors in rat RIEHAVl, M. AND SOKOLOVSK¥, M.: Multiple binding sites of tricyclic antidepres- cerebral cortex and hippocampus. Eur. J. Pharmacol. 29: 201-202, 1978. sants to mammalian brain receptors. Brain Res. 149: 525-529, 1978. FELL, P. J., QUANTOCK, D. C. AND VAN DER BURG, W. J.: The human pharrna- TSAN, V. T., CHANG, R. S. L. AND SNYDER, S. H.: Histamine H_ receptors cology of GB 94--A new psychotropic agent. Eur. J. Clin. Pharmacol. 5: 161- identified in mammalian brain membranes with [:'H]mepyramine. Proc. Natl. 173, 1973. Acad. Sci. U.S.A., 75: 6290-6294, 1978. HILL, S. J., EMSON, P. C. AND YOUNG, J. M.: The binding of _H-mepyramine to U'PPACHARD, D. C., GREENBIr_RG,D. A., SHEEHAN, P. P. AND SNYDER, S. H.: histamine Hvreceptors in guinea pig brain. J. Neurochem. 31: 997-1004, 1978. Tricyclic antidepressants: Therapeutic properties and affinity for a-noradre- HOWLETT, D. R, MORRIS, H. AND NAHOI_SKI, S. R.: Anom.alous properties of nergie receptor binding sites in the brain. Science (Wash. DC) 199: 197-198, [:*H]spiperone binding sites in rat limbic system. Mo]. Pharmacol.15: 506-514, 1978. 1979. VAN DER BURG, W. J., BONTA, I. L., DELOBELLE, J., RAMON, C. AND VARGAFTIG, B.: A novel type of substituted piperazine with high antiserotonin potency. J. 1TIL, T. M., POLVAN, N. AND HSU, W.: Clinical and EEG effects of GB-94, a Med. Pharrn. Chem. 13: 3,5-39, 1970. "Tetracyclic'" antidepressant (EEG model in discovery of a new psychotropic drug). CurT. Ther. Res. 14: 395-413, 1972. VAN RIEZEN, H.: Different central effects of 5-HT antagonists mianserine and JACOBS, B. L.: An animal behavior model for studying central serotonergic cyproheptadine. Arch. Int. Pharmacodyn. Ther. 198: 256-269, 1972. synapses. Life Sci. 19: 777-786, 1976. VARCArrIo, B. B., COICNET, J. L., DE VOS, C. J., GRIJSEN, H. AND BONTA, I. L.: KOFOE, W. F. AND LEONARD, B. E.: The effect of a new trieyclic antidepressant Mianserin hydroch]oride: Peripheral and central effects in relation to antago- compound, ORG GB 94, on the turnover of doparnine noradrenalin and sero- nism against 5-hydroxytryptamine and tryptamine. Eur. J. Pharmacol. 16: 336- tonin in rat brain. Arch. Int. Pharmacodyn. Ther. 206: 389-391, 1973. 346, 1971. LEYSEN, J. E.,NIEMEGEERS, C. J. E.,TOLLENAERE, J. P. AND LADURON, P.M.: ZIs,A. P. AND GOODWIN, F. K.: Novel antidepressantsand the biogenicamine Serotonergiccomponent of neurolepticreceptors.Nature (Lond.)272: 16,3-166, hypothesis of depression.The case for iprindoleand mianserin.Arch. Gen. ( - 1978. Psychiatry 36: 1097-1107, 1979. MAJ, J.,SOWINSKA, H., BARAN, L, GANCARCZYK, L. AND ROWKOW, A.: The centralantiserotonergJactic onof mianserin.Psychopharmacology 59: 79-84, 1978. Send reprint requests to: Solomon H. Snyder, M.D., Departments of Neuro- PEROUTKA, S. J.,LEBOVITZ, R. M. AND SNYDER, S. H.: Serotonin receptors science,Pharmacology and Experimental Therapeutics and Psychiatryand Be- affecteddifferentiallyby guanine nucleotides.Mol. Pharmacol. 16: 700-708, havioralSciences,Johns Hopkins UniversitySchool of Medicine,725 N. Wolfe 1979. St.,Baltimore,MD 21205. PEROUTKA, S. J.,LEBOVITZ, R. M. AND SNYDER, S. I-l.Tw: o distinctcentral