Differential binding of 3H-imipramine and 3H-mianserin in rat cerebral cortex

Life Sciences, Vol. 29, pp. 2049-2058 Printed in the U.S.A.

Pergamon Press

DIFFERENTIAL BINDING OF 3H-IMIPRAMINE AND 3H-MIANSERIN IN RAT CEREBRAL CORTEX Ante Dumbr~lle-Ross, S1u W. Tang and Donald V. Cosc~na Departments of Psychopharmacology and B~opsychology, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario, Canada M5T IR8 (Received in final form September 14, 1981) Summary Drug competltlon proflles, effect of raph~ leslon, and sodlum dependency of the blndlng of two antldepressant drugs 3H-Imlpramlne and 3H-mlanserln to rat cerebral cortex homogenate were compared to examlne whether the drugs bound to a common "antldepressant receptor." Of the neurotransmltters tested, only serotonln dlsplaced blndlng of both 3H-imlpramlne and 3H-mlanserln. 3H-M1anserln blndlng was potently dlsplaced by serotonln S 2 antagonlsts and exhlblted a profile slmllar to that of 3H-splperone blndlng. In the presence of the serotonln S 2 antagonlst splperone, antlhlstamlnes (H I ) potently dlsplaced 3H-mlanserln blndlng. 3H-Imlpramlne blndlng was dlsplaced potently by serotonln uptake inhlbltors. The order of potency of serotonerglc drugs in dlsplaclng 3H-imlpramlne blndlng was not slmllar to thelr order in d~splaclng 3H-sp~perone or 3H-serotonln blnd~ng. Prior m~dbra~n raph~ leslons greatly decreased the b~nd~ng of 3H-im~pram~ne but d~d not alter blnd~ng of 3H-m~anserln. B~nd~ng of 3H-~m~pram~ne but not 3H-mlanser~n was sodlum dependent. These results show that 3H-~m~pram1~e and 3H-m~anser~n b~nd to d~fferent receptors. 3H-Imlpram~ne b~nds to a presynapt~c seroton~n receptor which ~s probably related to a seroton~n uptake recogn~tlon s~te, the b~nd~ng of whlch ~s sodium dependent. 3H-M~anser~n b~nds to postsynaptlc receptors, possibly both seroton~n S 2 and h~stam~ne H 1 receptors, the b~nd~ng of whlch ~s sodium ~ndependent. Antldepressant drugs have been shown to have multlple actlons on CNS neurotransmltter systems. To mentlon a few, they Inhlblt noreplnephrlne and serotonln neuronal uptake (i, 2), reduce turnover of noreplnephrlne (3) and decrease the frequency of dlscharge of noradrenerglc and serotonerglc neurons (4, 5). They interact wlth muscarlnlc receptors (6), hlstamlnerglc receptors, (7, 8, 9), adrenerglc and serotonln receptors (I0, ii, 12). The posslblllty that all antldepressant drugs exert thelr therapeutlc actlon through a common "antldepressant receptor" is a polnt of controversy. Recently, the dlscovery of the blndlng sltes for the trlcycllc antldepressant 3H-imlpramlne (13, 14) and the atyplcal antldepressant 3H-mlanserln (15, 16, 17) ralsed the posslb111ty that these blndlng sltes may be related to the therapeutlc slte of actlon of antldepressant drugs.

0024-3205/81/202049-10502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

2050

~H-m~ansermn and ~H-~m~pram~ne

B~nd~ng

Vol.

29, No. 20, 1981

In thls study, the blndlng of 3H-Imlpr~mlne and 3H-mlanserln to rat brain tlssues was studled and compared. The sodium dependency and the effect of prior serotonln-depletlng mldbraln raph@ lesions on the receptor binding of these two antidepressant drugs were examined. Methods Blndln$ Assay. Male Wlstar rats (200-250 g) were decapitated and the brains removed. The cortical grey matter was gently scraped from the dorsal surface of the cerebrum. The braln tissue was homogenized in 50 volumes of ice cold buffer by using a glass homogenizer wlth a Teflon piston (0.13-0.18 mm clearance) rotated at 500 rpm wlth 15 up-and-down strokes. After centrlfugatlon at 30,000 g for iO mln a[ 4 ° , the supernatant was discarded and the pellet resuspended in 50 volumes of cold buffer and agaln centrifuged at 30,000 g. The pellet was then resuspended in 50 volumes cold buffer, homogenized wlth a Brlnkman Polytron at setting 6 for 5 sec, recentrlfuged at 30,000 g, reconstituted in 50 volumes of buffer and further homogenized slmllarly for 20 sec. The tlssues were kept on ice until used. TEAN buffer (15 mM Trls HCI, pH 7.4, 5 mM Na2EDTA , I.i mM ascorblc acld and 12.5 DM nlalamlde) was used for 3H-mlanserln, 3H-splperone and 3H-serotonln blndln~ assays and tissue preparations for these assay. The buffer used in JH-imlpramlne assays and respective tlssue preparatlon was 50 mM Trls HCI (pH 7.4), lO0 mM NaCI, and 5 mM KCI as per Ralsman et al. (!3). Binding was determlned by incubatlng 0.2 ml of 3H-llgand (3H-mlanserln, 14-16 C1/mmole, Amersham, or 57.7 C1/mmole, New England Nuclear [NEN], 3H-splperone, 34-45 Ci/mmole NEN, 3 H-se r otonln~32 Ci/mmole, NEN), 0.2 m i of varlous unlabelled drugs and 0.2 ml of membrane homogenate (flnal concentratlon: 6.5 mg wet welght/ml). After incubatlon at room temperature (23 ° ) for 30 minutes, 0.5 ml of thls mlxture was rapldly filtered under vacuum through Whatman GF/B f11ters. The f.lters were washed once wlth i0 ml of buffer. 3H-Imlpramlne blndlng was assayed in a method slmIlar to that of Ralsman et al. (13). Incubatlon tubes contained 0.035 ml 3H-imlpramlne (29.8 C1/mmole, NEN), 0.035 ml cold drug, and O.18 ml of membrane (final concentration: 6.5 mg wet welght/ml). After incubatlon at 0 ° for 1 hour, 5 ml of ice cold buffer was added to the incubatlon tube and the total volume rapldly fl]tered under vacuum through Whatman GF/B filters and rlnsed twice wlth 7.5 ml cold buffer. The radloactlvlty remalnlng on the filters was counted by iiquld sclntillatlon spectrometry in Aquasol. Inhibition of 3H-mlanserln blndlng by various drugs was tested uslng 0.5 nM 3H-mlanserln (flnal concentration). Speclf~c b~nd~ng was deflned as that displaced by 500 nM mlanserln. For 3H-~m~pram~ne b~nd~ng, 2.0 nM 3H-imlpram~ne was used and spec~flc b~ndlng was defined by i00 ~M des~pramlne. Specific b~ndlng of 3H-splperone (0.2 nM) was defined by 1 ~M (+)-butaclamol, and of 3H-seroton~n (0.5 nM) by 100 nM serotonln. Raph@ Lesions. As described prevlously (18), slmultaneous dorsal and medlal raph@ leslons were made in the brains of pentobarbltal anesthetlzed male Wlstar rats (50 mg/kg Nembutal, body weight 250 g) by generatlng 55 ° for i mlnute through a Radlonlcs thermal probe at the followlng stereotaxlc coordinates: anterlor posterior -0.35 mm, medial lateral 0 mm, and dorsalventral -5 and -7 mm. Histological verlflcatlon of correCt lesion placement was made. Levels of endogenous serotonln, noreplnephrlne, and dopamlne in

Vol. 29, No. 20, 1981

3H-mianserzn and 3H-imzpramine Bznding

2051

the hypothalamus of control and lesloned rats were measured by HPLC (J.J. Warsh, zn preparation) to conf~rm the expected depletion of seroton~n. Rats wezghed 400-500 g at the tzme of sacrifice, about 5-6 weeks after operation. 3H-Seroton~n Uptake Study. For determznat~on of 3H-seroton~n uptake, the cortices of 2 raph~ leszoned rats were pooled. The pool was dzv~ded znto two al~quots: one for measuring 3H-~m~pram~ne and 3H-m~anser~n b~nd~ng, and the second for seroton~n uptake studzes. Cortzces from wezght-matched control rats were s~mllarly alzquoted, and uptake and b~nd~ng measured zn parallel wzth les~oned t~ssue. Synaptosomes were prepared ~n a method s~mllar to that descrzbed by Kolde and Uyemura (19). The cerebral cortex was suspended ~n 20 volumes of ~ce-cold sucrose (0.32 M) and gently homogenized by 8 up-and-down strokes of a Teflon glass homogenizer drzven at about 500 rpm. The homogenate was centrifuged at I000 g for IO m~n. The resulting supernatant was recentrzfuged at 15,000 g for 30 mzn. The f~nal pellet was suspended zn 0.32 M sucrose to a f~nal concentration of iO0 mg orlgznal wet weight per ml and was used ~mmed~ately ~n measurzng 3H-serotonzn uptake. Incubatzon of tubes contalnzng 50 ~i of the synaptosomal suspenslon and 500 ~,1 of zncubatlon medlum (130 mM NaCI, 3 mM KC1, 2 mM CaC12, 2 mM MgCI 2 and 20 mM Trls HCI [pH 7.4]) were prezncubated for 5 mln at 37°C. To inztlate uptake, 50 ~I of 3H-serotonln zn concentratlons ranglng from 4-40 nM were added and the incubatzon contznued for 3 mzn. Incubatlon was termlnated by f11terlng 500 ~I of thls mzxture under vacuum through Whatman GF/B f11ters. The fzlters were washed once wzth iO ml of zce-cold washlng solutzon (260 mM sucrose, 2 mM CaCI2, 2 mM MgCI 2 and 20 mM Trls-HCl, pH 7.4). Identlcally prepared samples incubated at O ° were used as blanks. The radloactlvlty left on the filters was counted by llquzd sczn[lllatzon countzng ~n 9 ml of Aquasol. Kznetzc parameters (Km and Vma x) were determlned accordlng to the method of L1neweaver and Burk (20). Results Characterlstzcs of 3H-Mlanserzn and 3H-ImIpramzne Bzndzn$ to Rat Cerebral Cortex Membranes. The speclflc blndlng of 3H-mzanserzn to rat cortlcal membranes is saturable. Scatchard analysls gave a strazght Izne wlth an apparent dzssoclatlon constant (K d) of 1.4 nM and a maxzmal blndzng (Bma x) of 250 fmoles/mg proteln. The rate constant for assoclatlon (K I) Is 0.07 nM -I mzn -1 at 23 ° . D1ssoczatzon appeared to be bzphaszc when represented on a semllogarlthmzc scale, wlth the steeper part of the curve showzng a dlssoclatzon constant (K_ 1) of 0.13 mln -I. The apparent dlssoclatzon constant determlned from ~ ~ ~ e I ~ ~.~ ~ . w h ~ zs very close to the K d value determined in u n x r m n K d calculated from the shallow phase of the dlssoclatzon curve (Kd=0.1 nM) was not zndlcated on the Scatchard plot. Of the neurotransmltters tested, only serotonzn inhlblted blndzng of 3H-mzanserzn. The S 2 antagonlsts R43 448 and LSD competed wlth blnd~ng of 3H-mlanserzn at low concentratlons wzth HIll slopes 0.9 to 1.0; the speclflc S 2 antagonlst R41 468 competed wlth a Hzll slope of 0.4. Spzperone dzsplaced 3H-mlanserln bzndlng at h~gher concentratzons (IC50 = IOO nM), but the dzsplacement curve was clearly blphaslc, suggestlng that 3H-mzanserln mzght be blndlng to more than one slte (Fig. i). The serotonln uptake inhzbztors, fluoxetlne and cltalopram, were less potent wlth IC50 values about 2 ~M. H~stamzne H I antagonlsts mepyramzne, chlorphenlramlne and

2052

3H-mmanserzn and 3H-zmzpramine Binding

Vol. 29, No. 20, 1981

d~phenhydram~ne d~splaced the bzndzng wzth IC50's of 100-400 nM and shallow H~11 slopes. In order to examine whether the second component in the spzperone d~splacement curve represented b~ndzng of 3H-mzanserzn to a hzstam~ne H 1 receptor, dzsplacement of the 11gand by hzstamzne antagonzsts ~n the presence of i00 nM spzperone (blocking 3H-mzanserzn b~nd~ng to serotonln s~tes) was examzned. Under these cond~tzons, the ant~h~stamznes potently d~splaced 3H-m~anserzn blnd~ng wzth IC50's of 10-35 nM and H~II slopes

100

~

~

"~

ImO

"'-.

°

\

,°4

o.o

[ PIOI.AR] FIG. 1 Dzsplacement of speclflcally bound 3H-mlansenn from rat cerebral cortex hcmogenate by splperone (•), LSD (•), R43 468 (o), R41 448 (x), andmepyr~zne (D). Speclflc blndlngwas deflned by 500 r~ mlanserln. All polnts represent means + standard errors of 3 separate exper~nents. about 0.5. These IC50 values are slmllar to those recently reported by Peroutka and Snyder (17), but under slmllar condltlons, those authors reported that antzhlstamlnes dlsplaced 3H-mlanserln wlth HIll slopes about 1.0. We can not explaln thzs dlfference in results from the two laboratorles. 3H-Imlpramzne blndlng to rat cerebral cortex has prevlously been studled by Ralsman et al. (13) and Paul et al. (14). S1mllar to these groups, we found speczflc blndlng of 3H-imzpramzne to be saturable and of hlgh affznlty. Scatchard analysls zndlcated a slngle populatlon of sltes wlth an apparent K d of 7.4 nM and Bma x of 340 fmoles/mg proteln. S1mllar to 3H-mlanserln blndlng, that of 3H-zmlpramlne was dlsplaced by serotonln (IC50 = 4 BM). In contrast to 3H-mlanserln bzndlng, the serotonln uptake antagonzsts cztalopram and fluoxetzne potently dzsplaced 3H-Imlpramlne bzndlng wzth IC50 values of about 20 nM and HIll slopes of about 0.5. The serotonln antagonlsts splperone, R43 448, and LSD dld not dlsplace the blndlng at low concentratlons (IC50 values 3, 6 and >i0 ~M respectlvely). Sodlum Dependency. The uptake of serotonln by synaptosomes is sodzum dependent (21). In the absence of sodlum zons (Trls HCI, pH 7.4), the blndlng of 3H-mlanserln was not altered. Scatchard analysls showed the K d and Bma x unchanged, and dlsplacement of 3H-mzanserln by R43 448, splperone, LSD and mepyramlne was not affec[ed by incubatlon In sodlum free buffer.

Vol. 29, No. 20, 1981

3H-mianserin and 3H-imipramine Binding

2053

TABLE 1 IC50 (nM) 3H-M1anserln

3H-Imlpramlne

3H-Splperone

3H-Serotonln

Drug Antidepressant Mlanserln Amltrlptyline Imlpramlne Deslpramlne Iprlndole

3 14 70 180 500

20,000 25* lO 18" 5,500*

4 28 140 460 730

67 250 650 440 7,1OO

3,800 ii 20 i 2,000 i,i00

4 170 4 21 >20,000 >20,000

Serotonln Active Serotonln R43 448 LSD Splperone Citalopram Fluoxetlne

17,000 4 32 I00 1,200 2,500

1,800 6,500 >20,000 4,050 38 23

Bindlng assays were performed m the presence of 0.5 nM 3H-mlanserln, 2.0 nM 3H-Imlpranlne, 0.2 r~ 3H-splperone or 0.5 nM 3H-serotonln. Speclflc blndlng was defined by 500 ~M mianserln, I00 ~M deslprannne, I ~M (+)-butaclamol and i00 r~ serotomn, respectively. All values are the means of 3 independent determmatlons Standard errors are In all cases less than 14% of the mean. * frumPalsman et al., 1980(~). However, blndlng of 3H-Imlpramlne was sodlum dependent. In the absence of sodlum ions (Trls HCI, pH 7.4 or Trls HCI, pH 7.4 and KC1 5 nM), no saturable speclflc blndlng could be shown. When sodium was absent, the IC50 of lmlpramlne in'dlsplaclng 3H-imlpramlne Increased from 15 nM to 5000 raM; fluoxetlne, cltalopram, R43 448 and splperone dld not displace the llgand at 10,OOO nM. TABLE II Effect of Sodium on Speclflc Binding of 3H-Imlpramlne and 3H-M1anserln [Na +] mM 3H-Imlpramlne

iOO 0

3H-M1anserln

i0 0

Kd (nM)

Bma x (fmoles /mg protein)

7.50 ± 0.52 -- ND --

328 ± 14

1.42 + 0.06 1.32 ± 0.Ii

250 + 232 +

7 8

Number of Experlments 6 5 12 5

All values are means + standard errors. Assay procedures are described In the text. Membranes were incubated In standard buffer solutions (contamlng sodium) or m Trls ~uffer (sodium free). Specific blndlng of 3H-~nlpramlne was defined by I00 ~M deslpramlne and of H-mlanserln was defined by 500 nM nuansenn. ND = hlgh afflnlty bzndlng was not detectable. Raph~ Lesioned Rats. The leslon of presynaptlc serotonerglc flbres was verlfled by HPLC examination of neurotransmltter levels and by examlnlng

2054

~H-mianser~n

and 3H-rm~pramine

Binding

Vol.

29, No. 20, 1981

cortical seroton~n uptake in vrtro. Raph~ les~ons decreased seroton~n levels by 55-80%, but drd not alter levels of noradrenallne or dopam~ne ~n the hypothalamus as measured by HPLC. Uptake of seroton~n was reduced by 50-82% ~n the cerebral cortex of les~oned rats (Table Ill). TABLE III 3 Effect of Raph~ Les~ons on Specific B~nd~ng of H-M~anserln and 3H-Im~pram~ne and on Uptake of 3H-Seroton~n

(a) Specific B~nd~ng 3H-Im~pramlne

3H-M~anserln

control lesroned les~oned

~ d (nM)

Bma x (fmoles/mg protexn)

7.75 ± 0.55 419 ± 15 -- ND -8.70 ± 1.3 241 ± 13

Number of Experiments iO 8 2

control lesloned

1.27 ± 0.IO 1.23 ± 0.18

191 ± 204 ±

8 7

6 6

control* les~oned*

1.17 ± 0.07 1.46 ± 0.20

132 ± 129 ±

7 9

4 4

(b) 3H-Seroton~n

Uptake

control lesloned

Km (nM)

Vma x (pmoles/mg prote~n/3 m~n)

16.8 ± 2.3 14.6 ± 0.4

3.18 ± O.41 0.78 ± O.11

6 6

All values aremeans i standard errors. Assay condltlons are descrlbed ~n the test. Specific brndrng of 3H-n-nlpramrne was deflned by I ~ cles~pra~nne and for 3H-mlanser~n by5OOnMm~anser~n. tone to ~wo annnal cortices were used In each expern~ent. ND - hrgh affinity b~ndlngwas not detectable. "Spec~flc b~nd~ngwas defined by 80 nM sp~perone

After raph~ leslons, blndlng of 3H-imlpramlne dlmlnlshed substantlally whlle 3H-mlanserln blndlng dld not change (Table III). In elght of the ten rats examlned in whlch serotonln uptake was decreased to 18-22% of normal, no saturable speclflc blndlng could be shown for 3H-imlpramlne in the cerebral cortex. Scatchard analysls of raph~ lesloned tlssue was not dlfferent from that of heat denatured tlssue (i00 °, 60 mln). In two rats, when uptake decreased to 50 and 37% of normal respectlvely, saturable blnding wlth Bma x values of 250 and 228 fmoles/mg proteln respectlvely were obtalned (Fig. 2~ In vlew of the posslblllty that 3H-mlanserln may blnd to both hlstamlne H I and seroton~n S 2 s~tes, we verlf~ed that nelther s~te was decreased after the leslon by measurlng the IC50 of the H l an[agonlst, mepyramlne and S 2 antagonlst, R43 448, in control and lesloned tlssue. Nelther value was altered by leslonlng. Also, uslng 80 nM splperone as basellne to select for serotonln sltes, the blndlng of 3H-mlanserln to lesloned and control anlmals was agaln not dlfferent. Dlscusslon One approach in the search for an "antldepressant receptor" is a dlrect llgand blndlng. Blndlng studles wlth 3H-amltrlptyllne and OH-deslpramlne have not been very rewardlng. 3H-Amltrlptyllne was found to blnd to muscarlnlc, hlstamlne H 2 and posslbly other sltes (23), whlle 3H-deslpramlne, blndlng only to unlysed synaptosomal preparatlons, did not have the characterlstlcs of hlgh-afflnlty blndlng to cell surface receptors (24). Recently,

Vol. 29, No. 20, 1981

3H-mlanserln and 3H-imlpramlne Blndlng

2055

8O

A

2O

ii

, 100

!

200

300

3H-IMI BOUND

5OO

(fmoL/mg protein)

8°I ~

400

B

60

4o

100

200

300

400

500

3 H - I M I BOUND ( f m o l / m g protein) FIG.

2

Effect of raph6 lesion on binding of 3H-zrnlpramlne (3H-IMI) to rat cerebral cortex homogenate. F~ch point represents the rnean ± standard e r r o r frcrnScatchard analyses (3tblmlpraralne blndzng, ~ange O.1 - 1 nM) of normal ( • ) and raph6 lesioned ( × ) t i s s u e (n : 8 1hA and n = 2 in B). Speczflc binding is defined by 1OO ~M deszpramlne.

2056

3H-mlanserln

and

3H-imlpramlne

B1ndlng

Vol.

29, No.

20,

1981

2OO

i

A

160

120

80

40

50

100

150

200

3H.,-,bfIAN (frocking protein)

2OO

B A

160

120

8O

40

o 50

lOO

~50

200

3H...MU~ BOUND U/~l/uqE ~ e ~

FIG.

3

Effect of raph~ leslons on blndlng of 3H-mlanserm (3H-MIAN) to rat cerebral cortex ha-nogenate. Each polnt is the mean + standard error from Scatchard analyses of norrrml ( I ) and raph~-lesloned (x) tlssue. Speclflc blndlng Is deflned m A by 500 r~m~anserln (n = 6), and in B by 80 r~ splperone (n = 4) in order to select for blndlng to serotonm S 2 sltes.

Vol.

29, No. 20, 1981

3H-mzanserin and 3H-imipramine Binding

2057

3H-zmlpramlne was found to bind wlth hlgh-afflnlty to both brain tissue and blood platelets. Thls blndln$ sltes has some characteristics of the serotonzn uptake slte (13, 14). JH-M1anserln was discovered to blnd wlth hlgh-afflnlty to serotonzn S 2 receptors (15, 16, 17) and also to hlstamlne H I receptors (17). The results of the present study clearly show that the bzndlng sltes for 3H-imlpramlne and 3H-mlanserln are dlstlnct entltles. 3H-Imlpramlne bzndlng is located presynaptzcally as it is greatly reduced by raph~ lesion. Its blndzng is sodium dependent, as ~s the serotonln re-uptake process. The seroton~n uptake inhlbltors potently d~splaced Its binding. These results strongly suggest that 3H-~mzpramzne is binding to a slte that zs very much related to the serotonln re-uptake process. 3H-Mianserln, on the other hand, appears to b~nd to a s~te to which 3H-splperone also binds. Shallow compet~tlon curves of serotonln antagonists and antlh~stamznes (even in the presence of splperone) indlcate that 3H-m~anserln may bind to other s~tes ~n addlt~on to the serotonln S 2 and histamine H I sites. Although 3H-m~anser~n b~nd~ng was not decreased by raph~ lesion, neither was an increase in binding observed. Thls failure of the serotonln S 2 receptors to become supernumerous followlng a decrease in serotonerg~c ~nput ensuing the raph~ leslon is similar to that previously reported for 3H-spzperone b~nd~ng ~n the hzppocampus (25). It seems that either the serotonln S 2 receptor as labelled by 3H-mzanserln or 3H-sp~perone does not respond to a decrease in serotonerg~c input, or the development of supersenslt~v~ty on the serotonln S 2 sites ~s not generally detectable by l~gand blndlng experiments. Previous b~ndlng experiments (26-29) have failed to detect supersensitivity as indicated by neurophyslologlcal (30) and behav~oural (31) studies. Although mlanserzn and imlpramlne appear to blnd to two dlfferent serotonzn sites, thls does not preclude the posslb11ity that the net effect might be the same on neurotransmlsslon. Imlpramlne blocks presynaptlc serotonln re-uptake and might consequently ~nduce a reductlon In the number of postsynaptlc serotonln S 2 sites. M~anserzn, blocklng the postsynaptzc serotonln S 2 site directly, mlght also result in a reduction In neurotransmzsslon through the serotonzn S 2 receptor. Thus, the net effect mlght be slmzlar although the mechanl~m of actlon could be quite different. It zs apparent that different antidepressant drugs vary tremendously in their ability to dlsplace elther 3H-mlanserzn or 3H-imlpramlne from thelr blndlng sites. As trlcyclzc antidepressant drugs and some of the novel antidepressant drugs are clznlcally effective in a slmzlar dose range (150-250 mgm daily), it is unlikely that either of these two sltes represents the hypothetlcal "antidepressant receptor." In fact, there Is both clznlcal and blochemlcal evidence that depression is not a homogeneous dlsease. For example, previous work in thls area (32-34) has revealed at least two depressive subgroups, one wlth low pretreatment urlne levels of the major norepznephrzne metabollte 3-methoxy-4-hydroxyphenylglycol (MHPG) for whom imlpramlne is the drug of choice, and another group wlth hlgh or normal MHPG levels, for whom amltrlptyllne is the more effectlve drug. In anlma] studles, chronlc treatment with deslpramlne and amltrlptylzne induced a differential reduction in B-adrenerglc and serotonln S 2 receptor number (29), whlch substantlates the notlon that successful treatment in depresslon may be subserved by more than one slngle mechanism. Acknowledgement We thank Dr. S. L. Lee, Dr. J. Chambers and Mrs. J. Chang for their excellent technical help. HPLC assays were supervised by Mr. A. Chlu, Bzochemlcal Psychiatry, Clarke Instltute of Psvchlatry. We thank Clba-Gelgy,

2058

3H-mianserin and 3H-imlpramlne Binding

Vol. 29, No. 20, 1981

Janssen Pharmaceutlcal, H. Lunbeck and Co., Merck, Sharp and Dohme, Organon, Parke Davls and Wyeth Ltd. for thelr supply of drugs. References I. 2 3 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15.

16.

J. GLOWINSKI and J. AXELROD, Nature 204 1318-1319 (1964). A. CARLSSON, H. CORRODI, K. FUXE and T. HOKFELT, Eur. J. Pharmacol. 5 367-373 (1969). J.J. SCHILDKRAUT, M. ROFFMAN, R.P. ORSULAK, A.F. SCHATZBERG and M.A. KING, Pharmakopsych. 9 193-202 (1976). H.V. NYBACK, J.R. WALTERS, G.K. AGHAJANIAN and R.H.ROTH, Eur. J. Pharmacol. 32 302-312 (1975). M.H. SHEARD, A. ZOLOVICK and G.K. AGHAJANIAN, Braln Res. 43 690-694 (1970) S.H. SNYDER and H.I. YAMAMURA, Arch. Gen. Psychlat. 34 236-239 (1977). E. RICHELSON, Nature 274 176-177 (1978). E. RICHELSON, Mayo Clln. Proc. 54 669-674 (1979). J.P. GREEN and S. MAAYANI, Nature 269 163-165 (1977). D.C. U'PRICHARD, D.A. GREENBERG, P.P. SHEENAN and S.H. SNYDER, Sclence 199 197-198 (1978). J.L. BENNETT and G.K. AGHAJANIAN, Life Scl. 15 1935-1944 (1975). S.W. TANG and P. SEEMAN, Naunyn-Schmledeberg's Arch. Pharmacol. 311 255-261 (1980). R. RAISMAN, M.S. BRILEY and S.Z. LANGER, Eur. J. Pharmacol. 61 373-380 (1980). S.M. PAUL, P. REHAVI, P. SKOLNICK and F.K. GOODWIN, Life Scl. 26 953-959 (1980). P.M. WHITAKER and A . J . CROSS, B l o c h e m . P h a r m a c o l . 29 2 7 0 9 - 2 7 1 2 ( 1 9 8 0 ) . A. DUMBRILLE-ROSS, S.W. TANG a n d P. SEEMAN, E u r . J . P h a r m a c o l . 68 3 9 5 - 3 9 6

(198o). 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

S.J. PEROUTK~ and S.H. SNYDER, J. Pharmacol. Exp. Thor. 216 142-148 (1981). D.V. COSCINA and H.C. STANCER, Sclence 195 416-419 (1977). T. KOIDE and K. UYEMURA, Neuropharmacology 19 349-354 (1980). H. LINEWEAVER and D. BURK, J. Am. Chem. Soc. 56 658-666 (1934). D.F. BOGDANSKI, A. TISSARI and B.B. BRODIE, Life Scl. 7 419-428 (1968). J.E. LEYSEN, W. GOMMEREN and P.M. LADURON, B1ochem. Pharmacol. 27 307-316 (1978). M. REHAVI and M. SOKOLOVSKY, Braln Res. 149 525-529 (1978). A. BIEGON and D. SAMUEL, B1ochem. Pharmacol. 28 3361-3366 (1979). P. SEEMAN, K. WESTMAN, D. COSCINA and J.J. WARSH, Eur. J. Pharmacol. 66 179-191 (1980). D.A. BERGSTROM and K.J. KELLAR, J. Pharmacol. Exp. Ther. 209 256-261 (1979) T. SEGAWA, T. MIZUTA and Y. NOMURA, Eur. J. Pharmacol. 58 75-83 (1979). S.J.PEROUTKA and S.H. SNYDER, Sclence 210 88-90 (1980). S.W. TANG, P. SEEMAN, and S. KWAN, Psychlat. Res. 4 129-138 (1981). C. DE MONTIGY and G.K. AGHAJANIAN, Sclence 202 1307-1306 (1978). E. FRIEDMAN and A. DALLOB, Comm. Psychopharmacol. 3 89-92 (1979). J.J. SCHILDKRAUT, Am. J. Psychlat. 130 696-698 (1973). J.W. MAAS, J.A. FAWCETT and H. DEKIRMENJIAN, Arch. Gen. Psychlat. 26 252-262 (1972). H. BEC~MANN and F.K. GOODWIN, Arch. Gen. Psychlat. 32 17-21 (1975).