[125I]Thienylphencyclidine, a novel ligand for the NMDA receptor

l£uropeatt Joumal of l~harmacology - Molectdar Phamzacolo, w Section, 226 (1992) 53-58 .t', 1992 Elsevier Science Publishers B.V All righls reserved 0922-41t16/92/$05.011

53

EJPMOL 9{}297

[ 12~"l]Thienylphe~cyclidine, a novel ligand for the NMDA receptor Ian J. R e y n o l d s ", Kristi R o t h e r m u n d ", S u n i t a R a j d e v ", At~dul H. F a u q b a n d A l a n P. Kozikowski b " Department ol'Pharmacolog~', University of Pittshurgh, Pittsh~,rgh, P.d 15261. US\d, and ;' Dittsion c!l Netm;chemistO', Mayo (Tim, Jacksomilb.', Jacksom'dk,, 1"1. 32224. USA Received 21 JanuaD" 1992. accepted 28 January 19g2

We have monitored the bindi,,.g of [l:51]thicnylphcncyclidine ([i>I]TCP), a novel high affinity radioiodinated ligand that specifically recognizes the NMDA (N-methyl-D-aspartate} receptor in rat brain membranes. [~eSl]TCP binds with an affinity of about 30 nM, and recognizes a similar number of binding silos to previously employed ligands for this receptor. [t=.5I]TCP binding is characterized by slow association and dissociation rates, and the latter can be modified by the addition of Mg: ' {~r Zn: ~. as previously described for [3H]dizocilpinc ([ ~H]MK8[}I}. Oth~.i- phcncyelidine-tike iigands displaced [I>I]TCP binding with the order of potency dizocitpine > thienylphencyclidinc > ITCP > phenwelidine > ketaminc. The binding of [PSl]TCP was also increased by NMDA and glycine.-site agonists and inhibited by antagonists of these sites. Surprisingly. however, the polyamines spermidinc and spcrmit~c did not increase [1251]TCP, even though the polyamine antagonist arcainc was an effective inhibitor of binding. These results show that [PSl]TCP is a useful ligand for the NMDA receptor comptcx that binds to ~.tle receptor in a manner that is qualitatively distinct from previously dcscri')cd ligands. NMDA receptor: [lzSLrhienylphencyclidine: Potyamines

I. Introduction

The N M D A (N-methyI-D-aspartate) receptor complex is comprised of a number of ligand binding sites that together control the activation of the rcceptor-associamd ionophorc. These sites recognize the primary agonists for the receptor, glutamate and glycine, as well as a range of modulatory agents including Mg 2+, Zn 2+, polyamines and suifhydryl redox reagents (see Collingridge and Lester, 1989; Reynolds, 199{1a for review). Additionally, an important class of drugs related to phencyclidine inhibit N M D A receptor responses by binding to a site located within the receptor-associated ion channel (Johnson and Jones. 1990: Wong and Kemp, 19911. The latter class of drugs have ~mwcd invaluable as tools to probe drug interactions with the N M D A receptor complex because drugs such as [3H]dizociipinc ([~H]MKS01) and [3H]thienylphencyclidine ([3H]TCP) are specific high affinity ligands for the N M D A receptor (Maragos etal., 1986; Wong et al., 1986; Fagg, 1987; Reynolds et a l , 19871, Moreover, a number of studies have demonstrated that these ligands can effectively be employed to monitor the

C~'~rresl'umdence to: Inn J. Reynolds, Dcpa*rtmcnt of Pharmacnk~gy. University c[ Pittsburgh. E1354 Biomedical Science Tower. Phlsburgh. PA t52M, USA. Tel. {4!21 ¢~48.2t34; Fax {4121 ~48.1t~=15.

state of activation of the N M D A receptor (Fagg, 1987: Foster and Wong, 1987, Reynolds e t a l , , 1987). Studies using [3H]dizocilpine and [3H]TCP have revealed two broad types of interaction with ligands that bind in the ion channel. Binding of these ligands is accelerated by agonis;s that activate the channel, such as glutamate and giycine, and is retarded by drugs that close the channel such as competitive antagonists of the N M D A and glycine sites, and non-competitive antagonists like zinc (Bonhaus and McNamara, 1988; K!oog e t a l . , 1988b: Reynolds and Miller, 1988). The other principal mechanism for modulating [3H]dizocilpine and [3H]TCP binding is by direct allosteric modulation. This is the principal mechanism by which potyamines positively and Mg 2- negatively modulate [3H]dizocilpine (Reynnld~ and Miller. I988; Reynolds, I99uI'H. tJlvaient catR}n.,, .-,ucil a.', C,l ~ ,.,~Jd ~'ds "~ ,,L,,, act as agonists at ~:he polyamine site l Reynolds, 1990b: Sacaan and Johnson. 1990a). Thus, as polyamines. Ca: " and Mg:* are all present in the brain at concentrations that are sufficient or in excess of those required to bind to the N M D A receptor, understanding the action of phencyclidine-like drugs is dependent on the nature of the interaction between these modulators and phencyclidine-like ligands. We have recently characterized the bindfi~g of [l>I]dizocilpine (Raider and Reynolds, 19921. This ligand is distinct front [3H]dizocilpinc and [3H]FCP in

54 that Mg 2+ is tess effective as a negative modulator of [I-"~l]dizocilpine binding. In the present study we report the ligand binding characteristics of [~-~51]TCP. This ligand is an effective probe for the NMDA receptor complex that displays qualitatively distinct binding characteristics compared to those previously described.

2. Materials and methods

2.1. Materials

non-specific binding. Filters were washed twice with 5 ml buffer and radioactivity determined at an efficiency of 75% using an LKB-Wallac Clinigamma counter. Non-specific binding was defined by 30 /.tM dizocilpine. In association assays time points between 1 and 120 min were measured, and non-specific binding was determined at each point. For dissociation assays, 1 nM [J2Sl]TCP was incubated with 15 m g / m l tissue for 2 h. Aliquots (25 Izl) of this mixture were then diluted into 3 ml HEPES-NaOH which contained 100 g M glutamate, 30 g M glycine and 1 /.LM dizocilpine and other drugs as indicated. The dissociation reaction was allowed to proceed for 1-120 min and then terminated as described above. Membrane protein was determined using the BCA reagent (Pierce Chemical Co., Rockford, IL, USA) using bovine serum albumin (BSA) as a standard. Kinetic and saturation data from binding assays were analyzed using EBDA, L1GAND and KINET!C (Elsevier Biosofl, New York, NY, USA) as appropriate. Inhibition constants from competition exp~.;iments were obtaincd by linear regression of Hill plots prepared from data pooled from all of the experiments performed.

ITCP (fig. 1) was prepared from bromoTCP by a halogen metal exchange reaction with t-butyllithium followed by trapping of the anion in tetrahydrofuran/ 12. BromoTCP was prepared as previously described (Kozikowski et al., 19911. Radioiodination and purification of [IzSI]TCP (220(I Ci/mmol) was provided by Dr Garth Brown, NEN Research Products, E.I. Du Pont. Dizocilpinc was a gift of Mcrck. Sharp and Dohme (Westpoint, PA, USA). Ketamine was provided by Parke-Davis (Ann Arbor, MI, USA). 5,7-Dich!orokynurenate was generously provided by Macion Merrcll Dew (Cincinnati, OH, USA). Other drugs and chemicals were obtained from commercial sources, usually Sigma Chemical Co. (St. Louis. M e , USA).

3. Results

2.2. Bindhlg assays

3.1. KineHc characterization

Binding assays employed well-washed rat brain membranes prepared from frozen rat brains (cerebellum and brainstem removed) as previously described (Reynolds and Palmer. 1991). Assays were pertbrmed in borosilicate glass tubes and typically comprised (I.20.5 mg protein, 50-10(I pM [1251]TCP, and drugs as appropriate in a final volume of 0.5 ml 10 mM FIEPES-NaOH, pH 7.4. Assays wcrc incubated for 2 h at r
Wc initially evaluated ITCP using [3H]dizocilpine binding to rat brain NMI)A receptors. ITCP inhibited [3H]dizocilpine binding with an IC~. of 23.6 + 5.3 nM (mean + S.EM., n = 4). [12Sl]TCP binding trader similar conditions usually exhibited 60-70% specific binding with a KI) of 29.9 + 9.5 nM and a Bm,,~ of 3.52 + 1.53 pmol/mg protein in = 5, fig. 2). [12Sl]TCP bound rather slowly, v, ith K,,~,~ values of 0.04-0.086 min-I obtained (fig. 3A). Maximal levels of binding were observed after about ,," h in the presence of saturating concentrations of glutamate and glycinc. [t2~Sl]TCP showed monophasic dissociation curvcs with a dissociation constant of 0.0175 _+ 0.0017 rain-; (fig. 3B). The addition of Zn =+ significantly slowed the dissociation

/ 1

~f~ [~'~I1TCP_. !o 0.0041) +_ 0.0005 rain - ; (P < 0.05, t-test), ,,,I.:l.~ t,..I,24 ;~,~re,~,:~,! th,-~ r ; , t o ~t~ t h e e~tcv'll w h e r e it was difficult to determine a constant with confidence (fig. 3B).

3.2. Pharmacological characterization

',

Fig. I, Structure o¢ ITCP.

We next examined the pharmacological characteristics of [~251]TCP bi~ding. Compared to binding in the presence of 0.1 /.tM glutamate and 0.03 p.M glycine the k:.~.l:.. 11"1~¢'-'D wa~ iqcreased by the addition of t, lnuHtg of It 12~"lj,,.., glutamate and glycine (tab~,e 11. However, only the addition of glutamate :and glycine together was suffi-

55 TABLE l Effects of glutamate (Glut and glycine (Gly) on [t2Sl]TCP binding to the NMDA receptor complex, Agonis t. concentration

[;251]TCP binding (% Control} ~'

0,1 ,uM Gin +(}.(}3 ,aM Gly 100 ,aM Glu +1}.(}3 # M Gly 0.1 "aM G l u + 3 0 g M Gly

100 142± 19 108± 6

100 ~t M Glu + 3(} # M Gly

192 + 29 * *

t,

i

° 0

"~

°

'"~ 1 2 3 Bound, pMoles/mg protein

0

'P

4

Fig. 2. Scatchard plot for [1251]TCP binding to NMDA receptors. The data represent a single typical experiment, performed in the presence of 100 # M glutamate and 30 p.M glycine, that was repeated four more times with similar results. [USl]TCP concentrations were modified by adding unlabeled drug. The units for the y-axis are p m o l / m g p r o t e i n / n M . Data ,,"~fints represent actual data while the fine was drawn flom the data fitted by LIGAND.

" Data are expressed as a percentage of binding in the presence of 0.1 v M Gly and 0.03 ~ M Gly. Values represent the mean ( ± S.E.M.) of four determinaOons. ** Significantly different from binding in the presence of 0.1 ~xM Glu +0,03 ,ttM Gly, P < 0.05. t-test.

site antagonist aminophosphonovaleric acid (AP5) and the glycine site antagonist 5,7-dichlorokynurenate inhibited [t2Sl]TCP binding (IC50 14.5 and 2.66 #M respectively), and the action of these antagonists was

cient to significantly increase binding. Other phencyclidine-like ligands displaced [uSI]TCP binding with the

120

order of potency dizocilpine > TCP > ITCP > PCP > ketamine (fig. 4). ICso values for these ligands were

6.28, 13.7, 19.2, 28.8 and 357 nM respectively. We also tested the effects of several other NMDA receptor modulators. Mg 2+ and Zn 2+ were effective inhibitors of [tZSl]TCP binding with 1Cs0 values of 71,7 and 7.01 /zM respectively (fig. 5A), as anticipated from the dissociation experiments (fig. 3B). Interestingly spermidine did not increase [tZSl]TCP binding, and only displayed inhibition at concentrations between 1 and 1000 similar effects (data not shown). The competitive polyamine antagonist arcaine also inhibited [t25I]TCP binding with an .. ~,,~ t l c ,

,~t ~ ,, ~,a'* q p o r n l ; . o

prndnced

ICsn of 2.81 p.M (fig. 5A). As expected, the NMDA

:~

In, . 3000

®

{A) •

100

r3

so

60

["

~: A -,.0

-11.0

Ketamine ITCP -1 0.0

-9,0

-8,0

o7,0

-5.0

Fig. 4. Plmrnlacoiogicai characterization of [t-'~I]TCP binding. Exper, imenls were performed in the presence of 100 ~ M glutamate and 30 # M glycine and represent the mean (±S,E.M.) of 3-7 experiments perfl)rmed in duplicates.

1.0

IB)

•

m ....

-1.0i

L)

m = -2.0

~' looo ~-~ •~-

-3.0

--., .___~ ~ "~,'~~--~ • n

• 0

-6.0

Log [Drug], M

Control + Zn2+, 50gM + Mg2÷, 300l.tM

-4.0 100 150 0 50 100 Time, rain Tim':, rain Fig. 3. (A) Association rate of [1251]TCP performed in the presence of 100 ,~M glutamate and 311 ~ M glycmc. ]'he data represent a single typicai experiment that was repeated two more times with similar results, The eoncentrati,m of radioligaad in this c×periment was 6,3 pM. (B) Dissociation of [t2Sl]T-CI:'. Curves were performed in the presence of I00 # M gkttamate and 3[I g M glycine (C mlrul) to which either 1()0 p M Zn 2+ or 300 v.M Mg 2' were added at ~:lle point of dilution. The data represent tbc mean ( ± S , E . M . ) o f 4-5 experiment,'; performed in Juplieates. 0

50

~6

12o:

"~

i I r ,k--*.. ¥~ /T" /~ r~ 80 [- x ~, ~,~/ T'\ i ~, t "Iv ",.!t. \'T'\ " 6 0 " ~ ' ~ ' )':,, ~ -~a0 , "

~-w ,~

• ! • •

-7.0

-6.0

-5.0

Mg~* zn~" Sperrnldine Arcaine =~

"

.,--=... .~0 j

~2° i f ~ool\ i- "t--'

(A)

loo| I

-4.0

(B) •

60

| N - '~

40

~

T\~ .

,I

+Glu

~:

._

* :'It. ~ -3.0

" \ ~

~• '

-2.0

Log [Drug], M

,~

-7.0

-6.0

-5.0 -4.0 Log [Drug], M

-3.0

-2.0

Fig. 5. Ettects ol NMI)A rcccplol modulators on [I:~I]TCP binding. (A~ ('urves v,crc conMrllctcd ill !h;? presel~ce of lO(l/.tM glutamate and 30 ,aM glycinc, and represent the mean of 3--,1 cxpcrinlefHs p,ort\~rmcd in duplicale. (B) ('urees using AP5 were perfl>rmcd in the presence of 3(1/aM glycinc and 0.1 ,aM glutamate (closed circles} or 10t} a M gluta~alc (open circles). ('urvcs using 5.7-dichlorokynurenatc were performed in the presence of I{R} `aM glutamate and either (I.II3 `aM giycine (ck~sed squares) or 31} `aM glycine (open squares). The dala reprcscnl the mean ( "r S,E.M.) of 3-fi experiments performed in duplicates.

substantially reduced by increasing the appropriate agonist concentration Ifig. 5B).

4. D i s c u s s i o n

in this study wc have dcscribcd thc properties of a novel, high affinity, radioiodinatcd ligand for the NMDA receptor complex. [~:51]TCP shares many of the propt, rties found in other ligands that bind to the phcncyclidine site. Its binding is characterized by relatively slow association and dissociation rates, as has becn reported for [~H]dizocilpine (Kloog et al., 1988b: Rcynolds and Miller. 1988: Javitt and Zukin. 19891, [3H]TCP (Bonhaus and McNamara, 1988: Lamdaniltkin et al., 19901 and [~Z~l]dizocilpinc (Rajdcv and Reynolds, 19921, Moreover, thc dissociation of [t251]TCP is slowed by Zn :~, which is consistent with thc action of Zn 2~ on [3tt]di=ocilpinc binding (Reynolds and Millcr. 19881, and presumably ariscs from the ability of Zn: + to close the NMDA rcccptor-associatcd ion channel (Pcters ct al., 1987: Westbrook and Mayer, 19871. Additionally, Mg 2" accclcratcs thc dissociation of [~:5I]TCP, an effect that is bclicvcd to result from an allostcric interaction between the binding sites of Mg 2~ and phcncyclidinc-likc drugs (Reynolds and Miller. !9881. The binding of [~-'51]TCP is sensitive to glutamatc and glycine as anticipated for ligands that bind to the NMDA receptor-associated cbanncl (Loo et al.. 1986; Fagg. 1987: Foster and Wong. 1987; Reynolds et al., 19871. The degree of enhancemcnt of binding by glutamate and glycinc appears to be somewhat less than observed with other ligands (l.z~o et al.. 1986; Fagg. 1987; Foster and Wong. 1987: Reynolds et al., 19871. However, previous studies have shown that the prinei° pal mechanism whereby these agonists increase binding of phencyclidine-like drugs is the acccleration of the

association process (Bonhaus and McNamara. 1988: Kloog el al., 1988a: Reynolds and Miller, 1988). By this mechanism, the level of enhancement seen for a given ligand will also depend on the extent to which equilibrium has been approached, Even with glutamate and glycine present [3H]dizocilpine, lot example, does not reach equilibrium within 2 h, find without agonists equilibrium requires almost 24 h (Kloo~ ctal., 1988b). In contrast, [I"51]TCP is almost at equilibrium by 2 h, suggesting tha~ the level of enhanccment should be morc modest. Ncvcrthclcss, although the effects of glutamatc and glycinc arc relatively small, the presence of the binding sites for these agonists is clearly demonstratcd by thc inhibitory actions of AP5 and 5,7-dichlorokynurcnatc wh'=h arc antagonists for the glutamate and glycinc binding sitcs respectively (Watkins and Ewms. 1981: Baron ct al,, 19911). The most surprising result from this study is the failure of spermidinc and sperminc to increase [~:~I]TCP binding. Following thc initial obser'vations on the actions of polyamines with the NMDA receptor cor, plcx (Ransom and Stee. 1988) several studies have sh,own that potyamincs increasc [3H]dizocilpinc, [3H]TCP and [12~l]dizocilpinc binding, largely by increasing thc affinity of these ligands, although alterations in thc rate of binding cannot bc excluded (Wilhams c t a L , i';~,9; P.eynoid~, i09iib: Saca,m aud Juhnson, 19911b). It is also clear that the action,s of polyamines are complex, in that they enhance binding at low (1-100 g M ) concentrations and then inhibit binding at higher concentrations (Reynolds and Miller, 19891. Polyamine antagonists, such as arcaine (Reynolds, 1990c) compete against the high affinity effects but do not alter the low affinity effects (Reynolds, 1990b). In this study we observed that both spermidine and arcaine inhibited [~=Sl]TCP binding. The actkms of arcaine within the appropriate concentration range confirm the presencc of the high affinity polyamine

57 site. However, s p e r m i d i n e a n d spernfine were u n a b l e to increase b i n d i n g at all. Two possible e x p l a n a t i o n s might a c c o u n t for these findings. It is possible that [I'-Sl]TCP occupies the p h c n c y c l i d i n e site in a m e c h a nistically d i f f e r e n t way c o m p a r e d to dizocilpine a n d TCP, such t h a t the allosteric i n t e r a c t i o n b e t w c c n [~"51]TCP a n d the polyamine site is distinct from that which has b e e n o b s e r v e d for o t h e r ligands. However, this o p t i o n s e e m s less likely b e c a u s e arcaine still effectively inhibits b i n d i n g with the same efficacy a n d potency as previously o b s e r v e d ( R e y n o l d s , 19911c), indicating t h a t the p o l y a m i n e site a n d the [I:51]TCP are still ,~,,6, I it is possible t h a t t h e effectively. c o u p l e d . Al~dm,,:~e.y, inhibitory c o m p o n e n t is relatively m o r e p o t e n t w h e n [tZSl]TCP occupies the p h e n c y c l i d i n e site. In this way, lhe inhibitory c o m p o n e n t would effectively mask the increase in b i n d i n g n o r m a l l y associated with low conc e n t r a t i o n s of polyamines, However, it is clcar t h a t this is not a g e n e r a l p h e n o m e n o n associated with i o d i n a t e d ligands as [teSl]dizocilpine is actually less sensitive to the inhibitory cffects associated with high c o n c e n t r a tions of p o l y a m i n e s ( R a j d e v a n d Reynolds, 1992). T h e o b s c r v a t i o n s ol' t h e i n t e r a c t i o n b e t w e e n polyarnines a n d [JeSi]TCP have several i m p o r t a n t implications, Firstly, t h e s e results clearly d e m o n s t r a t e t h a t ligands for the p h e n c y c l i d i n c site do not all bind in a qualitatively similar fashion. This implies that ligand b i n d i n g to this site is not simply a passive process, but t h a t the precise n a t u r e of the i n t e r a c t i o n c f the iigand with the site will d e t e r m i n e the overall effect of alIostcric m o d u l a t i o n by ligands t h a t occupy the p o l y a m i n e site. T h e second i m p o r t a n t implication relates to the possible functional effectiveness of phencyclidine-like drugs. In more physiological p r e p a r a t i o n s the ionic c o m p o s i t i o n of the m e d i a can m a k e a prof o u n d difference to the effectiveness of d r u g s that act at the p h c n c y c l i d i n e site. It is possible tha! polyamines normally occupy the p o l y a m i n e site on the N M D A r e c e p t o r complex. Howevcr. it has recently b e e n d e m o n s t r a t e d that at n u m b e r of divalent cations, including Mg e + a n d Ca 2 ~, can also b i n d to the polyamine site a n d mimic thc effects of s p e r m i d i n e (Reynolds, 1990b; S a c a a n a n d J o h n s o n , ltJ9l}a). As these ions are normally f o u n d in biological fluids at c o n c c n t r a t i o q s in .... ,-~.~,~, ,,¢ ,i ........... ,,,ir,,A ~-¢~~cC!!Ov the nolwmaine site it s e e m s likely t h a t the p o l y a m i n e site is normally fully o c c u p i e d by t h e s e cations. T h u s , thc extent to which t h e s e cations a n d / o r p o l y a m i n c s alter the b i n d i n g of a given ligand to the p h e n c y c l i d i n e site will have a m a j o r i m p a c t o n the functional effectiveness of ligands for blocking the N M D A r e c e p t o r - a s s o c i a t e d ion c h a n n e l . Specifically, t h e s e studies suggest that ligands like I T C P will b e Icss effective 1.) ~ ecause of the i n h i b i t o u effects associated with o c c u p a t i o n o f the polyamine site. whi = iododizocilpine will he m o r e eft'ective duc t,,i its relative inscnsitivity to the inhibitory effects of divalent cations

( R a j d c v a n d Reynolds, 1992). T h e s e factors should make a c o n s i d e r a b l e impact o n the overall effectivehess of drugs that a n t a g o n i z e N M D A r e c e p t o r activity.

References

Baron. B.M., B,L. ltarrison, F.P. Miller. I.A. McDonaM. F.G. Saliluro, C.J. Schmidt, S.M. Sorcnsen. H.S. White and M.G. PaJfrcyman, 1991LActivity of 5,7-dichhm~kynurenic acid. a potent amagonist at the N-melhyl-D-aspartate receptor-associaled glycine binding site, Mol. Pharmacol. 38, 554. tsonhat,s. D.W. and J.O. McNamara. 1%;,', N-Melhyl-D-aspartale receptor regulation of uncompetitivc antagonist binding in rat brain membranes: kinetic analysis, Mot. Pharmac~;l, 34. 25(1. ('ollingridge. G.L. and R.A.J. Lester. 1989, Excitato w amino acid receptors in the vertebrate central nervous system, Pharmacot. Rev. 41, 143. Fagg. G.E., Itt87, Phencyclidine and related drugs bind to the activated N-methyI-D-aspartate receptor-channel con3plex in rat brain membranes. Neumsei. Len. 76, 221. Foster. A.C. and E.It.F. Wong, 1987, The novel antiamvuisant MK,g01 binds to the activated slate of the N-melhyI-D-aspartale receptor in rat brain. Br. J. Pharmacnl. 91,403. Javin, D.C and S.R. Zukin, 1989, Biexponenliat kinelics of [~tllMKS01 binding: evidence for access It'* closed and open N-melhyI-D-aspartatc receptor channels, Mol, Pharmacol. 35, 387. Johnson. K.M. and S.M. Jones, lug0, Neuropharmacol,agy of pht:ncyeli line: bze,ic mechanisms and therapeutic potential. :\nqn, Rev. Pharmacol. T~Ixicol. 30. 7117. Kloog, Y., R. [|aring and M. Sokt~lmsky, 1988a. Kinetic charac|erizalion of the phencyclidinc N-methyl-l)-asrpartate receptor intvra~,lion: evidence for a SleliC blockade of the channel Biochemist~3' 27. 843. Kloog. Y., V. Nadter and M. Sokohwsky, 198Sb, Mode oA binding ol [~tl]dibenz,,~cycloalkenimine (MK-8{II) to the N-melhyI-D-asparlate tNMDAt receptoz and its therapeutic implicati~m, FEBS [.en. 231L 10,7. Koziko~ki. A.P.. A.tt. Fauq, W. Tuckmanlel. SX). ('asaloni and K.F,. Krueger. l%q, SynHlcsis o[ TCP-based irrevcr,,ible alkyktt',~l's~`*flhc PCP recogniti~m site. Synlen. 654. Lamdani-hkin, H.. Y. Kloog and M. Sokohwsky, 1990. Modulation of glnlanlatc induced uncompetitivc bl~cker billding to the NMDA receptor by temperature and hy glycine, Biochemistr.~ 29, 3987. Loo. P.. A. Braunwalder. J. Lehclann and M. Williams, t986, Radioligand binding I¢~ central phcncydidine recognition ~ile,, is depcnden! tm excitamr- :.tMill~.`* acid receptor agOIlib|S. |'[[I|'. J, Pharmaco!. 123. 467, Maragos. W.F., D.CM. Chu. J.T. Greenam.vrc, J.B. Pcnne~ and A.B. Young, lt~Sfi, lligh correlation bct~een the h*calizalion of ['II]TCP binding and NMI)/~ itz~tUt,,i.,. ]-2::v ! Pharmacol. 123. 173. Peters. S.. J. Koh and D,W. Choi. U~ST,Zinc selectively blocks the i|ClitHl of N°methyl-D-aspartale on c~rtica[ neurons. Science 236. 5~9. Raider, S. and LJ. Reynolds. 1992, Elfects of monm'alen! and divalent cations on 34-. )[te51]-dizocilpine binding to the Nnlelhyl-D-asparia!c rcce]qlor ol l',~ll t"qilin nlenlbl'anc~. J. Ncllrochem,, in press. Ransom. R.W. and N.L. Sloe. 1988. Cnoperative modv~ation of [~[tlMK-NH bh~ding t~ the N-meth_vl-D-aspartale receplor-ion ctlanncl complex b.~ k-glu:amale, glycine and polyan3incs, ,!. Neurochem. 5t, 830.

58 Reynolds. l.J., lOg0a. Modulation of NMDA receptor responsiveness by neurotransmitters, drugs antt chemical modification. Life Sci. 47. 1785. Reynolds, l.J., 1991)b, Arcaine uncovers dual interaction of polyamines with the N-methyI-D-aspartate receptor. J. Pharmacol. Exp. Ther. 255, I(X)I, Reynolds, l.J.. 19t~Ic, Arcaine is a competitive antagonist of the polyamine site on the NMDA receptor, Eur..I. Pharmacol. 177, 215. Reynolds. IJ. and R,J. "tiller, !988, Multiple sites for the regulation of the N-methyI-D-aspartatc receptor, Mol. Pharmacol. 33. 581. Reynolds, l.J. and R.J. Miller, 1989, Ifenprodil is a noxc] type of NMDA receptor antagonist: interaction with polyamincs, Mol. Pharmacol. 36. 758. Reynolds, l.J, and A.M. Palmci-, It~')l, Rcgi6nal varia~k~rk~ i; ['XH]MKS01 binding to rat brain NMDA receptors, J. Ncurochem. 56, 1731. Reynolds. I.J.. S.N. Murphy and R.J. Miiier, t987. ~H-labelle~; MK801 binding to the exci|atory amino acid receptor complex frc~m rat brain is enhanced by glycinc, Proe. Natl. Acad. Sci. USA 84, 7744. Sacaan. A.I. and K.M. Johnson, I9¢,~11a. Competitive inhibition of magnesium induced [~II]N-( I-[thienyt]cyclohexyl)piperidinc bind-

ing by arcaine: evidence for a shared sr~ermidine-magnesium binding site, Mol. Pharmacol, 38, 705. Sacaan, A.I. and K.M. Johnson, 1990b, Characterization of the stimulatory and inhibitory effects of polyamines on [3H]TCP binding to the NMDA receptor ionophore complex, Mol. Pharmacol, 37, 572. Watkins, J.C. and R.H. Evans, 1981, Excitatory amino acid transmitters, Annu. Rev. Pharmaeol. Toxicol. 21, 165. Westbrook, G,L and M.L Mayer, 1987. Micromolar concentrations of Zn -~~ antagonize NMDA and GABA responses of hippocampal neurons, Nature 328. 6411. Williams. K., C. Romano and P.B. Molinoff. 1989. Effects of polyamincs on the binding of [~H]MK801 to the N-methyi-Daspartatc receptor: pharmacological evidence for the existence of a polyamine recognition site. Mol. Pharmacol. 36, 575. Wong, E.It.F. and J.A. Kemp, 1991. Sites t~)r antagonism on the N-melhyI-D-aspartate receptor channel complex, Annu. Rcv. Pharmacol. Toxicol. 31,401. Wong. E.1t.F., J.A. Kemp. T. Priestley, A.R. Knight, G.N. Woodruff anti E L . lversen. 1986, The anticonvulsant MK 8111 is a potent N-methyI-D-aspartate antagonist, Proc. Natl. Acad. Sci. USA 83, 7104.