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Br. J. Pharmacol. (1988), 95, 95-102 Tricyclic antidepressants block N-methyl-D-aspartate receptors: similarities to the action of zinc 'Ian J. Reynolds & Richard J. Miller Department of Pharmacological and Physiological Sciences, The University of Chicago, 947 E. 58th Street, Chicago, IL 60615, U.S.A. 1 Using the radioligand [3H]-MK801, we have examined drug interactions with the phencyclidine recognition site of the N-methyl-D-aspartate receptor. 2 The trycycic antidepressants desmethylimipramine and imipramine inhibited [3H]-MK801 binding with ICjo values of 7.4 and 22.5 um, respectively. Other related tricyclic antidepressants and neuroleptics were also effective but less potent. 3 Desmethylimipramine, imipramine and chlorimipramine slowed the dissociation rate of [3H]- MK801 in a similar manner to Zn2+. Phencyclidine and related compounds had no effect on the dissociation rate of [3H]-MK801. 4 Desmethylimipramine, imipramine and ketamine also prevented the Ca2+ influx into cultured cortical neurones of the rat produced by N-methyl-D-aspartate. 5 As the actions of tricyclic antidepressants in this system are not competitive with respect to N-methyl-D-aspartate, glycine or MK-801, and as they slow the dissociation of [3H]-MK801, we conclude that tricyclic antidepressants may be acting at the Zn2 + recognition site on the N-methyl- D-aspartate receptor. Introduction The N-methyl-D-aspartate (NMDA-selective subtype of the glutamate receptor consists of several components. There is a recognition site for agonists such as glutamate and NMDA, which also recog- nizes antagonists, such as aminophosphonovalerate (AP5), that are competitive with respect to glutamate (Foster & Fagg, 1984). This recognition site is coupled to a cation selective ion channel (Mayer & Westbrook, 1987). Phenycyclidine-like drugs, includ- ing ketamine (Anis et al., 1983; Snell & Johnson, 1985; MacDonald et al., 1987) and the novel agent MK801 (Wong et al., 1986) are non-competitive NMDA antagonists, as is the divalent cation Mg2+ (Mayer et al., 1984; Nowak et al., 1984). As these inhibitors exhibit use- and voltage-dependent effects, their binding sites appear to be within the receptor ionophore (Mayer et al., 1984; Nowak et al., 1984; MacDonald et al., 1987). However, Mg2 + and phencyclidine probably act at different sites within the ion channel (Reynolds and Miller, 1988). In addition to drug recognition sites located within the ion channel there are at least two super- Author for correspondence. ficial sites which appear to modulate receptor func- tion. Thus, it has been shown that a novel glycine-selective site can increase the effects of NMDA-like agonists in a strychnine insensitive fashion (Johnson & Ascher, 1987; Reynolds et al., 1987). Finally, Zn2+ and related divalent cations can prevent NMDA-induced channel activation in a non-competitive fashion at a site independent of the locus of glycine action (Peters et al., 1987; West- brook & Mayer, 1987). The physiological signifi- cance of the recognition sites for glycine and Zn2+ is unknown. Elucidating the role of these sites in glutamate-mediated neurotransmission would be greatly facilitated by the availability of specific probes. It should be noted that all NMDA inhibi- tors, regardless of the site of action, appear to protect against glutamate-induced, calcium- dependent neurotoxicity as measured in cultured brain neurones (Peters et al., 1987; Choi, 1987; Rothman et al., 1987). Recent studies have shown that phencyclidine like drugs, including thienylphencyclidine (TCP) and MK801 bind to the activated state of the NMDA receptor (Loo et al., 1986; Foster & Wong, 1987; Fagg, 1987; Reynolds et al., 1987; Heuttner & Bean, I The Macmillan Press Ltd 1988
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Br. J. Pharmacol. (1988), 95, 95-102

Tricyclic antidepressants block N-methyl-D-aspartatereceptors: similarities to the action of zinc'Ian J. Reynolds & Richard J. Miller

Department of Pharmacological and Physiological Sciences, The University of Chicago, 947 E. 58th Street,Chicago, IL 60615, U.S.A.

1 Using the radioligand [3H]-MK801, we have examined drug interactions with the phencyclidinerecognition site of the N-methyl-D-aspartate receptor.2 The trycycic antidepressants desmethylimipramine and imipramine inhibited [3H]-MK801binding with ICjo values of 7.4 and 22.5 um, respectively. Other related tricyclic antidepressants andneuroleptics were also effective but less potent.3 Desmethylimipramine, imipramine and chlorimipramine slowed the dissociation rate of [3H]-MK801 in a similar manner to Zn2+. Phencyclidine and related compounds had no effect on thedissociation rate of [3H]-MK801.4 Desmethylimipramine, imipramine and ketamine also prevented the Ca2+ influx into culturedcortical neurones of the rat produced by N-methyl-D-aspartate.5 As the actions of tricyclic antidepressants in this system are not competitive with respect toN-methyl-D-aspartate, glycine or MK-801, and as they slow the dissociation of [3H]-MK801, we

conclude that tricyclic antidepressants may be acting at the Zn2 + recognition site on the N-methyl-D-aspartate receptor.

Introduction

The N-methyl-D-aspartate (NMDA-selectivesubtype of the glutamate receptor consists of severalcomponents. There is a recognition site for agonistssuch as glutamate and NMDA, which also recog-nizes antagonists, such as aminophosphonovalerate(AP5), that are competitive with respect to glutamate(Foster & Fagg, 1984). This recognition site iscoupled to a cation selective ion channel (Mayer &Westbrook, 1987). Phenycyclidine-like drugs, includ-ing ketamine (Anis et al., 1983; Snell & Johnson,1985; MacDonald et al., 1987) and the novel agentMK801 (Wong et al., 1986) are non-competitiveNMDA antagonists, as is the divalent cation Mg2+(Mayer et al., 1984; Nowak et al., 1984). As theseinhibitors exhibit use- and voltage-dependent effects,their binding sites appear to be within the receptorionophore (Mayer et al., 1984; Nowak et al., 1984;MacDonald et al., 1987). However, Mg2 + andphencyclidine probably act at different sites withinthe ion channel (Reynolds and Miller, 1988).

In addition to drug recognition sites locatedwithin the ion channel there are at least two super-

Author for correspondence.

ficial sites which appear to modulate receptor func-tion. Thus, it has been shown that a novelglycine-selective site can increase the effects ofNMDA-like agonists in a strychnine insensitivefashion (Johnson & Ascher, 1987; Reynolds et al.,1987). Finally, Zn2+ and related divalent cations canprevent NMDA-induced channel activation in anon-competitive fashion at a site independent of thelocus of glycine action (Peters et al., 1987; West-brook & Mayer, 1987). The physiological signifi-cance of the recognition sites for glycine and Zn2+ isunknown. Elucidating the role of these sites inglutamate-mediated neurotransmission would begreatly facilitated by the availability of specificprobes. It should be noted that all NMDA inhibi-tors, regardless of the site of action, appear toprotect against glutamate-induced, calcium-dependent neurotoxicity as measured in culturedbrain neurones (Peters et al., 1987; Choi, 1987;Rothman et al., 1987).

Recent studies have shown that phencyclidine likedrugs, including thienylphencyclidine (TCP) andMK801 bind to the activated state of the NMDAreceptor (Loo et al., 1986; Foster & Wong, 1987;Fagg, 1987; Reynolds et al., 1987; Heuttner & Bean,

I The Macmillan Press Ltd 1988

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96 I.J. REYNOLDS & R.J. MILLER

1988). Thus, binding of [3H]-TCP and [3H]-MK801is increased by the addition of glutamate or NMDA,can be further enhanced by the addition of glycine,and can be eliminated by NMDA antagonist drugs(including AP5) and by Mg2+ and Zn2+ (Reynoldset al., 1987; Reynolds & Miller, 1988). Using equi-librium and kinetic approaches to study [3H]-MK801 binding, we have found that it is possible todistinguish clearly between drug action at the fivedifferent binding sites described above. For example,if a drug decreases binding, one can determine thereversibility of the inhibition by adding an excess ofglutamate or glycine. This detects drug action at the'agonist' sites normally occupied by glutamate orglycine. Alternatively, the inability of drugs to alter,increase or decrease the dissociation rate of [3H]-MK801 can be used to indicate drug action at thephencyclidine, Mg2+ and Zn2+ binding sites, respec-tively (Reynolds & Miller, 1988). Thus, the appropri-ate use of these biochemical tests can preciselylocalize the site of interaction of drugs with theNMDA receptor.

In this study we demonstrate that various tricyclicantidepressant drugs, including desmethyl-imipramine and imipramine, inhibit [3H]-MK801binding at modest concentrations. Using the prin-ciples described above we have found that thesedrugs interact with the MK801 recognition site in aZn2 +-like manner. Furthermore the inhibition ofbinding is correlated with the ability of these drugsto inhibit NMDA-mediated Ca2+ fluxes into cul-tured neurones. The possible therapeutic relevance ofthese actions is discussed.

Methods

Radioreceptor binding assays

Binding assays were performed using well washedbrain membranes from female Wistar rats (35 daysold, Charles River, Wilmington MA.) using pre-viously described methods (Reynolds et al., 1987).Briefly, brains were homogenized with a polytron in20mM HEPES/NaOH buffer containing 1 mmEDTA. Homogenates were then centrifuged at50,000g and resuspended six times and frozen at-20'C for 24 h. Following thawing the centrifu-gation and resuspending steps were repeated threemore times, this time without EDTA. The mem-branes were then frozen in aliquots. On the day ofuse the membranes were centrifuged andresuspended in binding buffer before use. Typicalassays contained 0.5mg protein, 1 nm [3H]-MK801,100pM glutamate and 30pM glycine (unless other-wise noted) to maximize binding in a volume of

0.5 ml of 20mm HEPES/NaOH buffer at pH 7.4.Drugs were added as appropriate. Assays wereallowed at least 90min to attain equilibrium, afterwhich they were terminated by filtration using aBrandel cell harvester (Brandel Inc., Gaithersburg,MD) and Schleicher and Schuell number 32 glassfibre filters. Radioactivity was extracted into 4mlBudgetsolve and determined by liquid scintillationspectrometry using a Beckman 1801 scintillationcounter with an efficiency of approximately 50%.Specific binding was defined as the differencebetween total [3H]-MK801 binding and binding inthe presence of 10pM unlabelled MK-801. Values forthe IC5o of drugs against [3H]-MK801 binding weredetermined from linear regression analysis of Hillplots.

In order to determine dissociation rate constants,brain membranes at a concentration of 10mgml'protein were incubated with 12nm [3H]-MK801 forat least 90min. The association reaction was termin-ated by the addition of 25y1 aliquots to tubes con-taining 3 ml buffer with 100piM glutamate, 30pMglycine (unless otherwise noted) and drugs as appro-priate, after which the tubes were vortexed. Drugswere added in concentrations sufficient to inhibit atleast 90% of equilibrium [3H]-MK801 binding.After the appropriate period of time, between 1 and90 min, the reaction was terminated and the radioac-tivity determined as described above. Data wereplotted as in (Bt/Bo) against time, where Bt and Borepresent binding at time t and time zero respec-tively. Dissociation rate constants were derived fromlinear regression analysis of this plot.

NMDA-induced Ca2 + influx

Ca2+ influx into individual neurones of rat cortex inprimary culture was measured essentially asdescribed previously (Thayer et al., 1986; Murphy etal., 1987). Briefly, brains were removed from rat pupsat embryonic day 17-18. The meninges, brainstem,midbrain and hippocampus were removed, and thetissue dispersed following treatment with 0.1%trypsin for 15-20min. Cells were then plated ontoglass coverslips that had been pretreated with10pgml-' polylysine in deionized water for 12h, ina solution that contained Dulbecco's ModifiedEagles Medium, 10% foetal bovine serum,100pgmlP1 penicillin and 5pgml-P streptomycin,and 3 pg ml- 1 laminin. This medium was replaced bya similar medium containing 10% donor horseserum in place of foetal bovine serum, and withoutlaminin after 24 h. Cells were then fed every 2-3 daysuntil use between days 13 and 17 in vitro. Cytosinearabinoside (Cytosar-U, 1OpM) was added from days6-9 to reduce non-neuronal cell growth.

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Table 1 Effects of phencyclidine-like drugs, adepressants, neuroleptics and other agents[3H]-MK801 binding to rat brain membranes

Drug

Phencyclidine-like drugsCyclazocineDextromethorphanKetamine(±+-SKF 10,047

IC50(gM)

0.54 + 0.011.88 + 0.181.02 + 0.33

0.392 + 0.03

Antidepressants and neuroleptic drugsAmitriptyline 57.25 + 5.42Chlorpromazine 45.87 ± 11.5Chlorimipramine 44.87 + 12.53Desmethylimipramine 7.41 + 1.32Imipramine 22.52 + 2.44Nortriptyline 20.98 ± 0.67Protriptyline 24.90 + 2.59Thioridazine 92.45 ± 0.78

Inhibition parameters without exogenouslyadded glutamate and glycine in the assayCyclazocine 0.091 + 0.01Ketamine 0.83 ± 0.31Desmethylimipramine 13.25 + 1.14Imipramine 27.17 + 2.62

0.911.0,1.011.02

1.5E1.61.341.0E1.121.2:1.252.3'

1.0(0.921.2(1.11

Results represent mean values + s.e.mean, for 3-6determinations performed in duplicate. Assayscontained 100pM glutamate and 30 uM glycineexcept where noted. IC50 and nH values were

determined as described in Methods.

Intracellular calcium concentrations ([Ca2 +]i)were determined using the fluorescent dye fura-2 as

previously described (Thayer et al., 1986; Murphy etal., 1987). Cells were loaded with 5 pM of the cell per-meant form of the dye, fura-2 AM for 1 h in HEPES-Hanks (HH) buffer (composition, in mM: NaCl 137,KCI 5, CaCl2 1.26, MgCl2 0.41, MgSO4 0.49,NaHCO3 3.0, NaHPO4 0.6, K2PO4 0.4, glucose 5.6and HEPES 20, pH adjusted to 7.4 with NaOH),containing 5mgml-1 bovine serum albumin. Fol-lowing washing with HH, [Ca2+]i was determinedas described (Thayer et al., 1986; Murphy et al.,1987). In order to measure the effects of NMDA,cells were perfused with 12 chamber volume changesof Mg2+-free HH, after which 100pM NMDA was

added. Glycine (30pM) was added simultaneously asprevious studies have shown that it enhances theaction of NMDA and is necessary to achieve themaximum effect of NMDA (Johnson & Ascher,1987; Reynolds et al., 1987). When the response hadstabilized inhibitors were added in successivelyincreasing concentrations, following the re-

establishment of a new Ca2" level. Using this tech-

Lfti- nique it was usually possible to reverse the Ca2+on change to within 2 fold of the basal value, following

increases often exceeding 20 times basal. Inhibitionwas expressed as a percentage of the Ca2+ concen-

nH tration in the presence of NMDA and glycine but inthe absence of inhibitor. In some experiments cellswere briefly exposed to NMDA and glycine, after

I + 0.03 which the cells were washed with Mg2"-free HH7+ 0.01 containing inhibitor until the Ca2 + concentration[+ 0.07 had returned to basal levels. NMDA and glycine+ 0.02 were then added again. After this, cells were again

washed with Mg2 +-free HH, and NMDA andB + 0.18 glycine added without inhibtor.3 + 0.33 The response of cells treated with NMDA and4 + 0.42 glycine but no inhibitors was constant for 10min orI ± 0.07 more. It is interesting to note that at longer times2+ 0.09 over half the cells challenged with NMDA and3 + 0.06 glycine (4 of 6) showed a second phase of Ca2+ rise) + 0.07 that was very large (in excess of 3 yM), and was not5 + 0.17 reversible by the addition of inhibitors. This was

accompanied by cell swelling, and presumably rep-resents Ca2+-dependent neurotoxicity as previously

D + 0.07 described (Peters et al., 1987; Choi, 1987; Rothman7 + 0.05 et al., 1987). This phenomenon was observed in 3 outD + 0.06 of 20 cells treated with inhibitors.1 + 0.11

Drugs and chemicals

Labelled and unlabelled MK801 ((+)-5-methyl,10,11 -dihydro - 5H -dibenzo [a,d] cyclohept - 5, 10-iminemaleate) were obtained from Dr Geoffrey Woodruff,Merck, Sharp and Dohme, Terlings Park, U.K.Imipramine and chlorimipramine were obtainedfrom Ciba Geigy, Summit, NJ, U.S.A. Other anti-depressants were the gift of Dr L. Seiden, Universityof Chicago. Ketamine, cyclazocine, SKF 10,047 N-allylnormetazocine, phencyclidine were gifts of DrW. Woolverton, University of Chicago. All otherdrugs and chemicals were obtained from commercialsources.

Results

Binding studies

Drugs that are believed to bind to the phencyclidinerecognition site inhibited the binding of [3H]-MK801 in an apparently competitive fashion. Thuscyclazocine, (±)-SKF 10,047, ketamine and dextro-methorphan all completely inhibited binding withHill slopes approximating unity (Table 1). The site ofaction of these compounds is independent of the a-receptor as haloperidol was not active at 100pM(Table 2) (Largent et al., 1986). A range of tricyclicantidepressants and neuroleptics also inhibitedbinding (Figure 1). Desmethylimipramine (DMI) wasthe most potent, with an IC50 of approximately 7pM.

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98 I.J. REYNOLDS & R.J. MILLER

Table 2 Inhibitory effects of miscellaneous drugsat 100pM

Drug

Carbamazepineu-AmphetamineDopamineFluoxetineHaloperidolIprindolMaprotilineMianserinNaloxoneNitrazepamNoradrenaline(+)-Oxaprolidine(-)-OxaprolidinePargylinePentylenetetrazolPhenytoin5-HydroxytryptamineTrazodoneZimelidine

Binding(% control)

105 ± 1.250 + 2.082 + 6.995 + 1.1100 + 0.092 + 2.686 + 5.177 + 2.498 + 1.4105 ± 1.499 + 9.1101 + 1.5101 + 1.197 + 5.198 + 2.8102 ± 3.180 + 4.6100+0.096 + 7.3

Results represent mean values ± s.e.mean, for 3-6determinations performed in duplicate. Assayscontained 100pM glutamate and 30pM glycine.

However, in contrast to drugs that were competitivewith [3H]-MK801, the Hill slopes for these com-pounds were in general somewhat greater than unity(Table-1). These effects were specific for tricyclic

, 120 -

0o 100

80 -

V60-

40-00li 20-

.0.-8 -7 -6 -5 -4

log [Drug] (M)

Figure 2 Effect of phencyclidine-like compounds, andantidepressants on specific equilibrium [3H]-MK801binding to rat brain membranes in the absence of addedglutamate and glycine. The results shown represent themean of 3-6 experiments performed in duplicate. Stan-dard errors were omitted for clarity, and representedless than 15% of the values shown. (0) Cyclazocine,(*) desmethylimipramine, (0) imipramine and (C>)ketamine.

compounds, as a range of non-tricyclic anti-depressants was ineffective at 100pM (Table 2). Like-wise, this is not a general site of action for

C-0.5

40 60Time (min)

100

log [Drug] (M)

Figure 1 Effect of antidepressant and neurolepticdrugs on specific equilibrium [3H]-MK801 binding torat brain membranes. The results shown represent themean of 3-6 experiments performed in duplicate. Stan-dard errors were omitted for clarity, and representedless than 15% of the values shown. (x) Amitryptiline,(*) chlorpromazine, (+) chlorimipramine, (O) des-methylimipramine, (El) imipramine, (A) nortryptiline,(A) protryptiline and (O) thioridazine.

Figure 3 Effects of drugs on the dissociation of [3H]-MK801. Following equilibration with [3H]-MK80125 p1 aliquots of tissue were added to 3 ml of buffer con-taining glutamate, glycine and drugs as noted inTable 3, except for (U) control with no glutamate orglycine, where the tissue was diluted into buffer alone.Drugs were present in the diluting buffer at 10(cyclazocine) or 30/uM (chlorimipramine anddesmethylimipramine). The results represent curvesfrom a typical experiment performed in duplicate, andshow that antidepressants, but not cyclazocine, slow thedissociation of [3H]-MK801. (El) Control, (*) chlori-mipramine, (A) cyclazocine and (O>) desmethylimipra-mine.

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Table 3 Effects of phencyclidine-like drugs andtricyclic antidepressants on the dissociation of[3H]-MK801

Drug

ControlNo glutamate or glycineCyclazocineDextromethorphanKetaminePhencyclidine(±)-SKF 10,047ChlorimipramineDesmethylimipramineImipramine

Dissociation rate(min-' x 103)

6.25 + 0.611.85 + 0.22**7.38 + 0.735.70 + 0.697.68 + 0.767.68 + 0.767.68 + 0.601.70 + 0.15**4.10 + 0.28**3.60 + 0.33**

Data represent the mean values ± s.e.mean forseparate determinations performed in duplicate.Dissociation rates were determined as described inthe Methods section. All assays contained 100yMglutamate and 30pM glycine except where noted.Drugs were present in the diluting buffer at 10(cyclazocine and phencyclidine) or 30pM (allothers). ** Significantly different from control,P < 0.01, unpaired t test.

anticonvulsants, as the benzodiazepines, phenytoinand carbamazepine were also ineffective at 100yM(Table 2).

If DMI and related compounds do not competedirectly with [3H]-MK801 what might their site ofaction be? In order to determine whether DMI was

acting at the glutamate or glycine binding site, weexamined the ability of tricyclic antidepressant drugsand phencyclidine-like agents to inhibit binding inthe absence of added glutamate and glycine.However, this procedure did not significantlyincrease the apparent potency of DMI, imipramine,(±)-cyclazocine or ketamine (Figure 2, Table 1),indicating that these drugs do not act competitivelyat the glutamate or glycine binding sites. Indeed, thepotencies of DMI and imipramine were actuallyincreased somewhat in the presence of excess

agonist. We have previously shown that it is possibleto distinguish between NMDA antagonists acting atthe phencyclidine-, Mg2+- and Zn2+-sites based ontheir effects on the dissociation rate of [3H]-MK801(Reynolds & Miller, 1988). Phencyclidine, cyclazo-cine, (±)-SKF 10,047 and ketamine had virtually noeffect on the dissociation rate of [3H]-MK801 indi-cating a competitive interaction (Table 3, Figure 3).In contrast, DMI, imipramine and chlorimipraminesignificantly reduced the ligand dissociation rate.These effects were similar to those of Zn2 + and AP5,which both slow the dissociation rate of [3H]-MK801, whereas Mg2 + greatly enhances it

(Reynolds & Miller, 1988). However, the effects ofAP5 are competitive with glutamate, and we haveshown above that this is not the case for tricyclicantidepressant drugs. Similarly, a saturating concen-tration of glycine which was also included in theassay did not reverse the effects of the tricyclic anti-depressant drugs. Thus, the effects of these agentsmost closely resemble those of Zn2 +.

NMDA-induced Ca2+ influx

In order to determine the functional relevance of theobserved effects of the tricyclic antidepressant agentsin the binding assay, we examined the effects ofDMI, imipramine and ketamine on the Ca2+ influxproduced by the addition of NMDA to primary cul-tures of rat cortical neurones in Mg2 +-free solutions.This Ca2+ influx is blocked by low concentrations ofMK801 and is due to the passage of Ca2 + throughthe NMDA-gated ionophore (Murphy et al., 1987).When added to cells before the application of agon-ists, DMI completely inhibited the response produ-ced by a maximally effective concentration ofNMDA and glycine (Figure 4a). In order to quanti-tate the potency of inhibitors in producing this effect,we examined the ability of DMI, imipramine andketamine to reverse Ca2 + changes produced byNMDA and glycine. The addition of NMDA(100pM) and glycine (30pM) increased [Ca2+]i frombasal levels of 52 + 27 nm to a peak of1089 + 381 nm (mean + s.e.mean, n = 20) Figure 4bshows that DMI and imipramine reversed the effectsof NMDA and glycine with potencies of approx-imately 1.9 and 4.8 pM, respectively, which weresimilar to the values found in the [3H]-MK801binding assay. As a comparison, the phencyclidine-like agent ketamine also reversed the effect of theagonist, with an IC50 of 3.7 pm.

Discussion

The major finding of this study is that the tricyclicantidepressant drug DMI is a non-competitiveantagonist at the NMDA receptor as assessed bothin binding assays and in a functional assay using cul-tured cortical neurones. The action of DMI andrelated compounds is clearly not competitive withrespect to glutamate, glycine or MK801. Further-more, the effects of DMI on the dissociation rate of[3H]-MK801 are distinct from those of Mg2 + whichgreatly accelerates ligand dissociation (Reynolds &Miller, 1988). Thus, the inhibitory effects of DMIclosely resemble those of Zn2+ (Reynolds & Miller,1988). DMI may therefore represent the first organicmodulator of this site. It should be noted, however,

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100 I.J. REYNOLDS & R.J. MILLER

1500

-iC1000

+NcoS: 500

a

b120-,

100 -

80 -

60 -

40

20 -

n-4

g

10 20Time (min)

30

-8 -7 -6 -5

log [Drug] (M)Figure 4 Effects of desmethylimipraminramine and ketamine on the increase irduced by the application of N-met](NMDA, 100/AM) and glycine (30/AM) t

tures of rat cortical neurones. (a) Cellswith Mg2"-free HEPES-Hanks bufferarrow. Addition of NMDA and glycirrapid increase in [Ca2]i that was reversThe addition of NMDA and glycine in IDMI produced no change. This effectreversed by washing with buffer. The figutypical experiment that was repeated thessentially similar results. (b) Dose-redemonstrating the ability of DMI (El), ijand ketamine (A) to reverse the increaproduced by the addition of NMDAglycine (30/AM) to cells in Mg2"-freeresults represent the mean of 4-7 determdard error bars were omitted for clarity,ed less than 15% of the values shown.

that the techniques used in this stud:sely differentiate between direct actioibinding site, and 'Zn2+-like' actionsseparate site with the same functional

It is unlikely that these actions of Iramine represent the major site at whproduce their antidepressant effects. Dtially more potent in inhibiting noradithan inhibiting [3H]-MK801 binding1981; Rehavi et al., 1982; Javitch et c

)MI 30 F±M larly, imipramine is more potent in blocking 5-JMDA 100 FM + hydroxytryptamine (5-HT) uptake (Rehavi et al.,lIycine 30 FM 1982). Furthermore, if one compares the ability of

gg2+ free medium the antidepressants used in this study to block theNMDA receptor with their rank order in blockingnoradrenaline and dopamine uptake, measured bythe ability to block [3H]-mazindol or [3H]-DMIbinding, there is clearly no relationship (Javitch etal., 1984). Thus, the observed effects on NMDAreceptors are unlikely to play a role in the anti-

-, depressant effects of these drugs.40 Tricyclic and non-tricyclic antidepressant drugs

also act on several other neurotransmitter systems.Thus, tricyclic antidepressant agents are potentblockers of muscarinic cholinoceptors, a-adrenoceptors, histamine, 5-HT and dopaminereceptors and voltage-sensitive Ca2+ channels (Hall& Ogren, 1981; Hall et al., 1984; Isenberg &Tamargo, 1985). However, the blockade of NMDAreceptors does not correlate with the rank order ofpotency or absolute potency of any of these actions.The action described in this study appears, therefore,to be a novel facet of the action of these drugs.

In clinical studies of antidepressant actions,'4 _3 cerebrospinal fluid (CSF) concentrations of 100-

150 ngml- have been measured following routinetherapy (Potter et al., 1982). This corresponds to a

ke (DMI), imip- concentration of approximately 100nm. As the drugsm [Ca2 ]i pro- found to be effective against NMDA receptors havehyl-D-aspartate potencies rather lower than the concentrations of the

were perfused drugs found during clinical use, it might seemstarting at the unlikely that blockade of NMDA receptors wouldse produced a contribute to the spectrum of activity produced by,ed by washing. these compounds clinically. If, however, blockade ofthe presence of NMDA receptors were to occur during therapy, onet was partially might predict an anticonvulsant effect, as blockadeire represents a of NMDA receptors has previously been demon-iree times with strated to have an anticonvulsant effect in seizure-msponse curves prone mice (Croucher et al., 1982). Thus, it is

iase in [Ca2(]1 interesting to note that anticonvulsant effects of, (100iM) and DMI, imipramine and amitriptyline have beenmedium. The described, using in vivo, in vitro and genetic modelsinations. Stan- of epilepsy (Clifford et al., 1985; Dailey & Jobe,and represent- 1985). As tricyclic antidepressant drugs are lipid

soluble, it is possible that the effective concentrationsof these drugs used clinically might be somewhatgreater than those measured in CSF, resulting in a

y cannot preci- contribution to their clinical action due to the block-ns at the Zn2+ ade of NMDA receptors. Furthermore, it is notproduced at a entirely clear what fraction of NMDA receptors needconsequences. to be blocked in order to produce protection againstDMI and imip- ischaemia or epileptic episodes. If the necessary frac-lich these drugs tional occupancy is low then such effects may result)MI is substan- from the doses of the tricyclic antidepressant drugsrenaline uptake used clinically.(Lee & Snyder, Although Zn2+ can block the NMDA receptoral., 1985). Simi- and block neurotoxicity in vitro (Peters et al., 1987;

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NON-COMPETITIVE NMDA ANTAGONISTS 101

Westbrook & Mayer, 1987), the physiological func-tion of the Zn2+ binding site in situ is entirelyunknown. Thus, it is not clear whether Zn2+ toni-cally occupies such binding sites, or whether it is rel-eased in response to some modulatory input atNMDA synapses. The hypothesis that tricyclic anti-depressant drugs and Zn2+ interact with the NMDAreceptor at the same site requires further testingusing more direct approaches. The compoundsdescribed in the present study may not be specificenough to act as definitive probes for the actions ofZn2 + in NMDA-receptor-mediated glutamate

neurotransmission. However, it should be possible torefine the tricyclic structure and produce a potentand specific ligand which would be extremely usefulfor such studies. Such a substance may clearly alsopossess clinical utility.

The authors wish to acknowledge the gifts of [3H]-MK801and unlabelled MK801 from Dr Geoffrey Woodruff,Merck, Sharp and Dohme, Terlings Park, U.K. This workwas supported by Public Health Service grants DA 02121,DA 02575, MH 40165, grants from Miles and MarionPharmaceuticals to R.J.M. and by a grant from the ScottishRite Schizophrenia Research Program NMJ (U.S.A.) toI.J.R. and RJ.M.

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(Received December 7,1987Revised April 11, 1988

Accepted April 15, 1988)


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