@Pergamon

N 1C @ 1 E S r r

P G B0 $ +

PII:S0028-3908(96)00156-6

Activity of 2,3-benzodiazepines at Native Rat andRecombinant Human Glutamate Receptors In Vitro:

Stereospecificityand Selectivity Profiles

DAVID BLEAKMAN,l* BARBARA A. BALLYK,2DARRYLE D. SCHOEPP,3ANDREW J. PALMER,l CATHERINEP. BATH,l ERICA F. SHARPE,l MARIE L. WOOLLEY,lHYWEL R. BUFTON,4RAJENDER K. KAMBOJ,2ISTVAN TARNAWA5

IEli Lilly and Co., Lilly Research Centre, Erl Wood Manor, Sunninghill Rd, Windlesham, Surrey, GU20 6PH,U.K.; 2Allelti Biopharmaceuticals Inc., 6850 Goreway Drive, Mississauga, Ontario, Canada L4V1V7; 3Eli

Lilly and Co., Lilly Research Laboratories, Corporate Center, Indianapolis, U.S.A.; 4DepartmentPharmacology, Universi~ Bristol, Bristol, U.K. and ‘Institute for Drug Research, H-1325, P.O. Box 82,

Budapest, Hungaq

(Accepted 3 October 1996)

Summary-Theb

2 ,Gp

a I 6r I 5r n

ra c

kG

2 ,si k

s 2p a n

d dd

I 1h p s

2a @

Keywords-GYK152466,GYK153655,GYK153405,LY300168,LY293606,Purkinjeneurons,GIuR1,G1uR4,G1uR5,G1uR6,G1uR6+KA2,dorsalrootganglion,corticalwedge,NMDA,spinalcord.

Glutamate is the major excitatory amino acid in the activate receptors which form ligand gated ion channelscentral nervous system and is responsiblefor mediating (monotropicglutamate receptors) and those linked tofast excitatory transmission between neurons (Collin- second messenger systems (metabotropic glutamategridge and Lester, 1989). Glutamate has been shown to receptors) (Nakanishi, 1994). For the monotropicgluta-

mate receptors, functional studies using selective ago-*Towhomcorrespondenceshouldbe addressed.Fax:01236- nists and, more recently,

853525; b l1

al.

tropic glutamate receptors into either IV-methyl-D-aspartate (NMDA) or non-NMDAreceptors. In addition,non-NMDA glutamate receptors are further separatedinto a-amino-3-hydroxy-5-methyl-4-isoxazole propio-nate (AMPA receptors, GIuR 14) or kainate (GIuR 5–7,KAl and KA2) receptors (Hollman and Heinemann,1994).

The development of selective agents that are able todifferentiate between different glutamate receptor sub-types is a strategyfor improvingour understandingof therole of glutamate receptors in the central nervous systemand the potential for therapeuticinterventionin processesinvolving disturbed glutamatergic neurotransmission.Several compoundshave been developed as antagonistsof non-NMDA receptors (Danysz 1995). For thenon-NMDA receptor subtypes of monotropicglutamatereceptors the systemicallyactive quinoxalinedioneseriesof competitive anatagonists,which include CNQX andNBQX, have been useful for separatingNMDA and non-NMDA receptor-mediated responses (Sheardown et al.,1990). However, these compounds are unable todistinguishpharmacologicallybetween responseswithinthe non-NMDA receptor class, having activity at bothAMPA and kainate receptor subtypes (Watkins1990). The decahydroisoquinoline LY293558 showsgreater selectivity within the non-NMDA receptor classwith competitive antagonist activity at AMPA receptorsand G1uR5 kainate receptor but no activity at G1uR6(Bleakman et al., 1996).

Recent studies have described a class of orally active2,3-benzodiazepines that have been shown to be non-competitive antagonists at non-NMDA receptors (forreviews see Rogawski (1993); Vizi et al. (1996)). Themostwidely studiedof these compoundsare GYK152466and LY300168 (GYK153655).These compounds havebeen shown to antagonize AMPA receptor-mediatedresponses in hippocampal neurons and show selectivityfor AMPA receptorsover kainate receptors(Lerma1993, 1995; Wilding and Huettner, 1995). In order toevaluate the relative inb e

r 4r e

pe

in HEK293 cells. Wehave examined the relative selectivity of these com-pounds for kainate receptors existing in native form andalso human cloned and expressed GIuR5 and G1uR6expressed in HEK293 cells. Our studies have elucidatedthe rank order of potency of the 2,3-benzodiazepines,thestereospecificityof their action and indicate that use ofthe appropriate concentrations of these compounds canenable the pharmacological separation of AMPA andkainate-mediated responses in neurons.

MATERIALSANDMETHODS

All experimentswere performed at room temperature(20-22”C).

Acutely isolated cerebella Purkinje neurons

Cerebella Purkinje neurons were isolated via modi-fication of the method of Mintz (1992). Thecerebella vermis of 6-11 day old rat pups (Sprague-Dawley, Harlan) were removed and transferred to abuffer containing82 mM Na2SOq,30 mM K2S04,5 mMMgC12,1 mM HEPES, 1 mM glucose and 0.1% phenolred, pH 7. Tissuewas cleaned,choppedand digestedwithbuffer containing 1 mg/ml protease XXIII (SigmaChemical Co.) at 37°C for 6 min. After being digestedand washed, the tissue was transferred to buffersupplemented wth 1 mg/ml BSA (Sigma) and 1 mg/mltrypsininhibitor.Cellswere dissociatedby triturationandplated onto P coated glass coverslips (50 fig/ml). Purkinje cells were identified morphologically bytheir large cell bodies (15–30mm).

Acutely isolated dorsal root ganglion neurons

Dorsal root ganglion (DRG) neuronswere isolated viamodification of the method of Moises (1994)(Bleakman 1996). DRGs were dissected from thelumbar and thoracic regions of 4-7 day old rats andtreated with collagenase (type II, 3 mg/ml) for 50 min at37°C in a solutionconsistingof 82 mM Na2SOq,30 mMK2S04,5 mM MgC12,1 mM HEPES and 1 mM glucose,pH 7.4. After centrifugationand removal of the enzymesolution,a solutionof MEM (GIBCO Laboratories)withBSA (20 mg/ml) was added, and the tissue resuspendedand centrifuged. This procedure was repeated and theMEM/BSA solution replaced with an MEM solutionsupplementedwith 1.5 mg/ml NaHC03, 300 rig/mlnervegrowth factor, 10 pg/ml penicillin/streptomycin and10 mM glucose. Cells were mechanically dispersed withthe use of a fine-tippedplastic Pasteur pipette and platedontopoly-L-lysinecoated (50 pg/ml) glasscoverslips.Allrecordingswere performed within 36 hr of plating.

Transected cell lines

Stable cell lines of HEK293 cells transected withcDNA coding for the human GluR4(flip),G1uR5(Q)andG1uR6(Q)receptors were established as reported pre-viously (Hoo 1994;Korczak 1995;Fletcher

1995).The heteromericcell line G1uR6(Q)+ KA2was establishedby co-transfectionof HEK293 cells withG1uR6(Q)and KA2 cDNAs (Hoo 1994;Kamboj

1992), each incorporated into the mammalianexpression vector pRc/CMV (Invitrogen). Transectedcells were selected on the basis of G418 resistance, andexpression of both genes confirmed by RT–PCR. Thehuman GIuR1 receptor (humGluRl) was isolated byhybridization of human fetal brain cDNA/ZAPIIbacteriophagelibrary (Stratagene)with radiolabelled ratGIuR oligonucleotide probes. Stable cell lines wereestablished by transection of HEK293 cells with the

2

mammalian expression vector pRc/CMV (Invitrogen)incorporating the cDNA for human GIuR1. Transectedcells were selected on the basis of G418 resistance, andexpressionof the humGluRl gene was confirmedby RT–PCR. For electrophysiological recordings, cells weredissociated by trituration and plated onto P

coated (10 pg/ml) glass coverslips.

E l

Whole-cell voltage clamp recordingswere made fromsingle cells with use of the tight seal whole cellconfiguration of the patch-clamp technique (Hamill

1981). Glass fragments of coverslips with adherentcells were placed in a perfusionchamber and rinsedwithbuffer of composition 138 mM NaCl, 5 mM CaC12,5 mM KC1, 1 mM MgC12,10 mM HEPES and 10 mMglucose, pH to 7.5 with NaOH (osmolality 315 mosm/kg). Pipette solutions contained 140 mM CSC1,1 mMMgC12, 14 mM diTris creatine phosphate, 50 U/mlcreatinephosphokinase,14 mM MgATP, 10 mM HEPESand 15 mM BAPTA, pH to 7.2 with CSOH(osmolality295 mosm/kg). Experiments were performed at roomtemperature (20-22”C) and recorded on either a ListEPC-7 or an Axopatch ID amplifier.Pipette resistanceswere typically 1.5–2.5MQ. Drug application was via aseries of perfusion lines to a multi-barrelled applicator(Biologic Inc.) and exchange of solutions under thepresent recording conditions was approximately100 msec. Experiments using G1uR6, GluR6/KA2 andG1uR5 transected cells were performed followingremoval of agonist-induced desensitization by pre-incubation of coverslips with 250 pg/ml concanavalinA. Agonist-induced desensitization at GIuR1 and 4transected cells was removed by performing experi-ments in the presence of 100 ,uMcyclothiazide(Partin etal., 1993). For the acutely isolated cerebella Purkinjeneurons, drug application was by bath perfusion andoccurred within approximately 15 sec. We have demon-strated previously that under such recording conditionsand with the slow application of agonist, kainateproducesnon-desensitizingcurrentswhich are unaffectedby concanavalin A (Bleakman 1996) and thuslikely to be mediated by an action of kainate at AMPAreceptors in these cells. Curve fitting to data points wasbased upon the equationy = IOO(Dn/(Dn+ IC50”)),wherethe slope of the line n was fixed to a value of 1 and D isthe antagonistconcentration.Statistical significancewasdetermined by one-way ANOVA followed by Student–Newman–Keuls test.

p

Rats, 150-200 g, were killed by cervical dislocation,decapitated and the brains rapidly removed to ice-coldartificial cerebrospinal fluid (aCSF). Transverse 450 pmsections were cut from a block containing the rostralforebrain. Wedge shaped slices were taken from thecingulate cortex and placed in a two-compartmentchamber so that the mev matter was in one chamber

o +flcH3N‘4’o \,/ ,f/

o/\ INH2

G

( N-co-NII-cl13

NH2

L ( (L (L (

C*3

0(7- *N- cO-CH3

o \ ,N’

/

~T

NH2

L (L (L (

2

al.

Q

u L■ LA LO G

0.01 0.10 1 10 100 1000

IGYKI] (IIM)

IOUM LY300168— 30vM GYKI 52466

T f l— rwIOVM AMPA

IOpM AMPA

IOOpA IL5s

Fig. 2. W =c

2 ,(A), f a

c

and the white matter in the other, separated by a greaseseal barrier. D.C. potentials were recorded between thetwo chambers and recorded continuously via Ag/AgClelectrodes to a chart recorder. The grey matter wassuperfused with aCSF, composition in mM: NaCl 124,NaHC02 25.5, KC1 3.3, KH2P04 1.2, CaC12,2.5, D-glucose 10, equilibratedwith 5% COZin oxygen, to a pHof 7.4-7.5 at room temperature. The compartment withwhite matter was left in a static pool of aCSF containing1 mM MgS04. Compounds were tested as agonists byapplicationto the grey matter side in 4 ml aliquotsof thesuperfusing aCSF at not less than 20 min intervals.Compounds were tested as antagonists by continuoussuperfusion and 4 ml aliquots of standard agonistwere added to this solution. Data are expressed as a

percentage of the response evoked in the absence ofantagonist.

5 day old rats were killed by decapitation.InMg2+containing aCSF, the spinal cord was removedfrom the vertebral column taking care to preserve theventral and dorsal roots of the segment L4-L6. The cordwas hemisected and placed on a Perspex base so that thedorsal root (DR) and the ventral roots (VR) rested on Ag/AgCl stimulatingand recording electrodes, respectively.Grease seals were used to isolate these from the spinalcord itself. The cord was superfused continuously(1.5 ml/min) with an aCSF of composition (mM) NaCl126,KC13,NaH2P04 1.25,CaC122.4, MgS04 1.4 and D-

2

glucose 10,equilibratedwith 5% C02 in 02 to maintainapH of 7.4-7.5 at room temperature (20-22”C) (Patemainet 1995). To study the activity of the 2,3-benzodiazepineson depolarizationsproduced by AMPAand NMDA, the VR D.C. potential was recorded on achart recorder in response to 3 ml aliquots of Mg2+-freeaCSF containing AMPA (3 pM) or NMDA (30 PM)administeredat not less than 20 min intervals.Effects ofantagonists were assessed by superfusion for at least20 min prior to agonist addition. Cumulative antagonistdose–responsecurves were normalized by expression ofamplitudeas a percentageof the control agonistresponse(Palmer and Lodge, 1993).

G (and LY303071 (+) were synthesized at

the Institite of Drug Research, Budapest, Hungary.LY293606 (t), LY300164 (–) and LY300165 (+) andLY293558 were synthesized by Lilly Research Labora-tories, Indianapolis.NBQX, AMPA, NMDA and kainatewere obtained from Tocris Cookson (Bristol, U.K.). Allother reagents were obtained from Sigma ChemicalCompany (St. Louis, MO).

RESULTS

The 2,3-benzodiazepinesused in the present study areshown in Fig. 1.

2 ,

Figure 2 illustrates the activity of the three 2,3-benzodiazepinesshown in Fig. 1 and LY303070the (–)isomer of LY300168 on inward currents activated byAMPA (10 PM) in homomeric GIuR1 receptors ex-pressed in HEK293 cells. Inward currents were evokedunder voltage clamp conditions in transected cells at aholding potential of –70 mV by application of AMPA(10 PM) to cells. Applicationof the 2,3-benzodiazepinesto cells alone had no effect on the holding current (notshown). The three 2,3-benzodiazepines GYIU52466,LY293606 and LY300168 inhibited AMPA (10 pM)-induced responses with IC50values of 17.9 ~ 0.5 #M,23.9 + 0.8 VM and 5.9 t 0.1 pM, respectively. Inhi-bitions were readily reversible upon washout of com-pound (inset to Fig. 2). The (–) isomer of LY300168,LY303070 also inhibited responsesin GIuR1 transectedcells (Fig. 2) with an IC50of 2.9 f 0.5 PM. Similareffectswere observed for activity of these compoundsat G1uR4expressed in HEK293 cells. Figure 3 shows the dose–response curves for the same compounds evaluated atG1uR4. GYK152466, LY293606, LY300168 andLY303070 inhibited AMPA (10 PM) responses inrecombinant G1uR4expressing HEK293 cells with ICSovalues of 22.5 t 0.4 #M, 28.1 f 0.9 PM, 4.6 f--0.4PMand 2.9 f 0.4 pM, respectively.

Previous studies have suggested that the allosteric

100

1 U❑

90 ❑ LY303070■ LY300168

80 4 LY293606

70

60

50

40

30

20

10

0

0 GYKI 5

❑

1

0.01 0.10 1 10 100 1000

[Antagonist] (pM)

3. =

2

fa

modulator,cyclothiazide,is able to reduce the potency ofthe 2,3-benzodiazepine-dependent inhibition AMPAreceptor responses (Zorumski et al., 1993; Palmer andLodge, 1993;Johansen et al., 1995).We have examinedthe extent to which LY300168 (10 PM) was able toinhibit AMPA (30 vM) responses in GIuR1 transectedcells at variouscyclothiazideconcentrations.Using 3 PMcyclothiazidethe inhibitionproduced by LY300168 was79 ~ 2% = 4 cells). Increasing the cyclothiazideconcentration to 30 and 100 ,uM resulted in inhibi-tions of 63 ~ 6% = 4) and 59 f 4% = respec-tively.

2

In order to compare the activitiesof these compoundsat AMPA and kainate receptors we have also examinedthe effects of 2,3-benzodiazepines at homomeric andheteromeric human kainate receptors. Specifically, wehave investigated the actions of 2,3-benzodiazepinesathomomeric G1uR5,G1uR6and heteromeric GluR6/KA2glutamate receptors. The effects of LY300168 andLY303070 at G1uR5receptors are shown in Fig. 4. Forcomparison, the inhibitory effects of two competitiveantagonists,NBQX and LY293558, are also illustrated.NBQX and LY293558 have previously been shown toantagonizekainate currents in homomeric G1uR5recep-tors expressed in HEK293 cells and kainate activated

al.

100

901

(5)

T

10PM 10PM 30PM

(7)

(:)

100PM 30pM 100IJM

30uM Kalnate

!‘0’”’r20pA

LY293558 NBQX LY303070 LY300168

4.=

ip ( ~

currents in DRG neurons (Bleakman et al., 1996). In thepresent studieson G1uR5receptors, 10 VMLY293558orNBQX inhibited kainate (30 pM) -induced currents by77 f 4% (n= 6) and 56 f 7% (n= 6), respectively.LY300168 and its active isomer LY303070 producedonly small inhibitions of kainate-induced currents at30PM and inhibited kainate-induced currents at aconcentration of 100PM by 12 f 2 (n = 4) and 18 f 4(n= 4), respectively.The inset to Fig. 4 shows individualcurrent traces for inhibitionsof kainate-inducedcurrentsby LY293558 (10 ~M) and LY 300168 (100 PM).Further studies on cloned human kainate receptors areshown in Fig. 5(a, b). In these experiments we haveexamined the effects of several 2,3-benzodiazepinesonG1uR6receptors expressed in HEK293 cells. Figure 5(a)illustrates the inhibitory effects of GYIU52466,LY293606 and LY300168 on kainate (10 pM) -inducedcurrents. As with G1uR5 receptors, small inhibitoryeffects of these agents were seen at concentrations of

100PM.2

aa ~

a

f n =f 25Z0,n = 6). The

extentof inhibitionseen with these three compoundswasfound to be significantly different from each other(p< 0.05, one-way ANOVA followed by Student-New-man–Keuls test). Similarly, inhibition of 10 pM KA-evoked responses by 100PM of the racemic compoundLY293606was 12 f 4% (n = 5), while greater inhibitionwas seen with the (–) isomer, LY300164 (19 i 290,

5.=

i cp ~

%IO

pM

KA

-evo

ked

resp

on

se%

10P

MK

A-e

wkd

resp

on

sa

tI

I1

I1

I1

II

5)-

(9)%(6) 10uMKA

M -1 L

2s

G L L LY303070

m

Fig. 6.h + =

ci f

n = 3) and little inhibitionwas seen with its (+) isomer,LY300165 (3 ~ 1%, n = 6). Again, the extent of inhi-bition seen with these three compoundswas found to besignificantlydifferentfrom each other (p c 0.05, one-wayANOVA followed by Student–Newman–Keulstest). Wehave also examined the effects of GYK152466,LY293606, LY300168 and LY303070 on the hetero-meric G1uR6co-expressedwith the KA binding proteinKA2. As with the homomericG1uR6kainate receptors, ahigh concentration (100 pM) of each AMPA antagonistswas able to produce small (approximately 20%) inhi-bitions of 10 VM kainate-evoked currents in these cells(Fig. 6).

2 ,

activity and stereoselectivityof the 2,3-benzodia-zepines have also been examined on native AMPAreceptorsin cerebella Purkinjecells,on kainatereceptor-mediated responses in DRG neurons and on AMPA andNMDA receptor-mediated responses in the hemisectedspinal cord and cortical wedge preparations.

We have recently described experiments in which wehave used cerebella Purkinje neurons to examine theeffects of AMPA antagonists on AMPA receptor-mediated responses (Bleakman et al., 1996). In the

present study, the three 2,3-benzodiazepines,LY300168,LY293606 and GYK152466all inhibited AMPA recep-tor-mediated responses in cerebella Purkinje neurons(Fig. 7). IC50values for GYKI 52466, LY293606 andLY300168 of 9.9 f 0.6, 7.8 f 0.5 and 1.5 ~ 0.1 PM,respectively, were established. In addition, the stereo-selectivityof the compoundswas shown to reside in the(–) isomerof the compounds.Figure 8 shows the effectsof the ( —) isomer (LY303070) and the (+) isomer(LY303071) of LY300168 and also the (–) isomer(LY300164) and the (+) isomer (LY300165) ofLY293606. IC~~ values of the (–) isomers were2.5 + 1.7 pM and 0.7 ~ 0.1 flM for LY300164 andLY303070,respectively,while agonist-evokedresponseswere unaffected by the (+) isomers at concentrationstested up to 30 pM.

In order to relate the experiments performed usinghomomericG1uR5kainate receptorsto the effects of 2,3-benzodiazepineson native G1uR5receptors in neurons,we have investigated the effects of these compounds onacutely isolated DRG neurons.The currents activated bykainate in DRG neurons are likely to be mediated byeither homomeric or heteromeric G1uR5kainate recep-tors (Huettner, 1990;Partin et al., 1993;Bleakman1996). Studies by Wilding and Huettner (1995) haveshown that GYK152466 and LY300168 were weak

2

■ LA L

T O GYK152466

m

0.01 0.1 1 10 100 1000

[Antagonist](vM)

I

(A)and GYK152466(0) are shown.Each datapoint representsmean inhibitions~ SEM calculatedfrom

betweenthreeandsixseparatecells.

antagonistsat rat DRG kainate receptorswith IC50valuesof >200 PM. In our experimentswe have examined theeffects of LY300168 and the active isomer (–)LY303070 on currents activated by 30 .uM kainate inDRG neurons. Although both compounds produced aconcentration-dependent reduction in kainate currents,the inhibition was only approximately 45% at 100PM(Fig. 9).

H p

We have also performedexperimentsin which we haveexamined the activity of 2,3-benzodiazepineson nativeAMPA- and NMDA-mediated responses in the hemi-sected neonatal spinal cord preparation. In order toinvestigatenative AMPA responses,AMPA (3 PM) wasapplied to induce depolarizationsof motoneuronsin thespinal cord. Depolarizations were antagonized in aconcentration-dependent manner by LY303070 ((–)isomer of LY300168) (lC.XIvalue of approximately1 pM). LY303071 ((+) isomer of LY300168) had onlyminor effects (c1O%)on AMPA-induced depolarizationat concentrations up to 20 PM (Fig. 10 ). The stereo-selective effects of 2,3-benzodiazepines were alsoexamined on the short and long latency components ofthe dorsal root–ventral root reflex in the hemisectedspinal cord preparation as described in Materials andMethods. The (–) isomer LY303070 was effectiveagainstearly and late componentsof the reflex,producingestimated IC50 values of 2 VM and 10 PM, respectively,whereas the (+) isomer LY303071 was inactive on both

100

90

80

70

60

50

40

30

20

10

0

1 ●

/!/LY303070

● LY300164v LY300165A LY303071

0.01 0.1 1“ 10 100 1000

[Antagonist](#M)

-

uLJf’”2t

13

t

1

0.3

C

250pALImin

(f

componentsof the reflex at concentrationsup to 100 UM(Fig. 11).

Studies were also conducted on the ability of 2,3-benzodiazepinesto antagonizedepolarizationselicited inthe cortical wedge preparation by either AMPA, kainateor NMDA. Figure 12 summarizes the data obtained.Figure 12 (a) shows the effects of increasing concentra-tions of LY303070 (1–10 pM) against the AMPAdose–response relationship. LY303070 is an effectiveantagonist of AMPA-dependent depolarizations and at

1001 30PM I

901

10

0 I_

. .

1 (3). .

L

w J20PA

25s

I OL Y

k

( )p

a k a

high concentrations of antagonist the inhibition of the kainate dose–response relationship, the degree of shiftAMPA-induced depolarizations could not be overcome was markedly less pronouncedat 10 UMLY303070 thanby increasing agonist concentrations. The effects of the shift observed in Fig. 12(a) for the antagonism ofLY303070 on kainate-induced depolarizationsis shown AMPA-dependent depolarizations. The effect of

Fig. 12(b).Although LY303070-produceda shift in the LY303070 was also ~xamined

~oo

1 h

120

90110

1 e,zs

80 J

I \T

20

10 1I u

0 ~

o L

100< d

20

10 1

on depolarizations in-

L T S l + LY303071A I + L

H. s l + L❑ I + L

\

u I I 1 1 I 0.01 0.1 10.01

100.10 1 10 100

100

[[ (

Fig.D s 2

2 ,d3

A pd 3 70

a a fc

2

~ Control

d

~ lpMLY303070

~ 3.16KMLY303070

~ 10pM303070

P

J

# 1 11 10 100 1000

A Mc o n c e(

(b) 120

‘$!

60

40

20

0.1

1201

~ Control~ 10PM LY303070

k-1

1 10 100K c o(

$100-co~ 80-a)L

60-

20

t

LY303070

!

.1 1 1 1NMDA concentration (pM)

12.Effectsof LY303070at theAMPAreceptoron rat cerebro-corticalslices.Concentration–responsecurvesshowingtheA d a

1 D ka %

N d (A)

duced by NMDA in the same preparation.As can be seenin Fig. 12(c), 10 PM LY303070 has no effect on theNMDA-dependentdepolarizationsin the cortical wedgepreparation.

DISCUSSION

The present studies have investigated the potency,selectivity and stereoselectivityof three 2,3-benzodiaze-pines which are being used more frequently as selectivenon-competitive AMPA antagonists. We have used avariety of preparations including cloned andexpressed channels in HEK293 cells and also AMPA,kainate or NMDA receptors in either isolated cells orintact tissue preparations.

Recent studies in rat cloned and expressed AMPAreceptors have established that GYK152466 andLY300168 (GYIU53655) are effective antagonists atthese sites (Johansen 1995). In these studies theabilityof GYK152466was also examined on flip and flopvariants of either homomeric rat GIuR1(A) or G1uR4(D)or heteromeric constructsof these channels either in theabsence or presence of 50PM cyclothiazide. The studyshowedthat althoughthe potency of GYK152466was notdifferent for either flip or flop heteromeric constructscontaining AMPA receptors (IC50values approximately30PM in the absenceof cyclothiazide),GYK152466wasless potent at homomeric GIuR1 and 4 (IC50valuesapproximately 66 and 117 PM, respectively, in theabsence of cyclothiazide).In addition, it was shown that

1700 al.

cyclothiazide (50 PM) reduced the IC50 values forGYK152466 at heteromeric receptors with little effecton the degree of inhibitionby GYK152466at homomericGIuR1 and 4. In our experiments where we haveexamined the effects of 2,3-benzodiazepineson humanGIuR1 and 4 (flip versions of both) the potency ofGYK152466was found to be somewhathigher,with IC50values of 18 and 22 vM, respectively,for GYK152466inthe presence of 100 pM cyclothiazide.Partin and Mayer(1996) have recently demonstratedthat a point mutationof cloned rat homomeric AMPA receptor flip splicevariant, GluRli[S750Q], which lacks modulation bycyclothiazide,did not alter antagonismby 2,3-benzodia-zepines. Such results suggest that the moleculardetermi-nants for sensitivity to cyclothiazide are likely to bedifferent to those which interact with the 2,3-benzodia-zepines. Our observations,and those of others (Zorumski

1993; Palmer and Lodge, 1993; Johansen1995), suggest that cyclothiazide does reduce theinhibitory effects of 2,3-benzodiazepines (but also seeDesai (1995)), although the effect we saw wasmodest.Otherworkers have demonstratedthat cyclothia-zide and 2,3-benzodiazepinesappear to bind to differentsites on the AMPA receptor but can exert allostericinteractions with one another (Rammes 1996;Yamada and Turetsky, 1996).

Previous studies have compared the activities ofGYK152466 and LY300168 both in vivo and(Tamawa 1993;Donevan 1994).The presentresults have provided a rank order of potency for thethree racemic 2,3-benzodiazepines we have comparedof LY300168 (GYK153655)> GYK152466> LY293606(GYKY53405).This rank order of potency is conservedbetween the GIuR1 and G1uR4human AMPA receptors,with GYK152466being marginally more potent at thesereceptors than LY293606. However, in cerebellaPurkinje neurons, the rank order of potency isLY300168 > LY293606> GYK152466. Whether thissmall change in relative agonist potency betweenLY293606 and GYK152466reflects species differencesor the experimental conditions under which the com-pounds were examined (i.e in the absence of cyclothia-zide and using kainate as an agonist) is unclear. It hasbeen reported that GIuR1,2 and 3 subunitsare present incerebella Purkinje neurons (Pelligrini-Giampietro1991; Petralia and Wenthold, 1992).Although the exactstoichiometryof the subunitswhich make up the AMPAreceptors in these cells is unknown,it is possible that theheteromeric structure of the AMPA receptor proteinsmay underlie the small observed change in antagonistrank order of potency. A greater sensitivity to the 2,3-benzodiazepine, LY300168, for native AMPA receptorsgenerated from hippocampal and forebrain poly(A)-mRNA than for recombinant AMPA receptors, hasrecently been described (Partin and Mayer, 1996).

S

Previous studies demonstrate the potential for 2,3-

benzodiazepines as selective AMPA versus kainatereceptor antagonists when responses to kainate in ratabducens motoneurons were shown to beunaffected by GYK152466 at concentrations whichcompletely blocked responses to glutamate and AMPA(Ourdouz and Durand, 1991, 1993). Further differentia-tion of 2,3-benzodiazepineactivity at kainate receptorsrather than at AMPA receptorswas obtained in culturedchick neurons (Osipenko 1994) and cultured rathippocampal neurons (Patemain et al., 1995). Recentevidence that the 2,3-benzodiazepines block neuronalAMPA receptors more potently than currents mediatedby kainate receptors has been obtained by comparingresponses at AMPA receptors to those in either DRGneurons or rat cloned G1uR6 receptors (Wilding andHuettner, 1995; Johansen 1995; Yamada andTuretsky, 1996).Wilding and Huettner (1995) found thatLY300168 and GYK152466inhibited AMPA responsesin cultured rat cerebral cortex neurons (IC501 pM and18 pM, respectively), whereas activity at dorsal rootganglion neurons was markedly less (IC50>200 PM). Inaddition,Johansen (1995)showed that GYK152466was a weak antagonist of rat G1uR6receptors with anaverage IC50value of approximately 112 ,uM. Theseresultsare consistentwith thosepresentedin this study.Inisolated cells we have extended the investigation andcomparison of selectivity to include GIuR1 and 4receptors expressed in HEK 293 cells and native AMPAreceptors in cerebella Purkinje neurons with G1uR5,G1uR6 and G1uR6+ KA2 receptors expressed inHEK293 cells and also native kainate receptors in ratDRG neurons.The IC50values estimated for experimentsconducted in the presence (for the cloned receptors) orabsence (for the cerebella Purkinje neurons) of theallosteric modulator, cyclothiazide, ranged from 28 #Mfor LY293606 at G1uR4 receptors to 0.7 pM, for theactive isomer of the most potent 2,3-benzodiazepineonPurkinje cells (LY303070). For the range of kainatereceptors examined in the present study, significantinhibitoryactivitywas seen only at concentrationsof 2,3-benzodiazepines >30 pM. The effect of the 2,3-benzo-diazepines at kainate receptors appeared stereospecificand thus the activity of these compounds at kainatereceptorsis consistentwith a specific,albeit low, potencyaction at the channel. Use of 10-30 PM LY303070should allow inhibitionof AMPA-mediatedresponses inneuronswith little or no effect at kainate receptors.

Activity

Depolarizationsresulting from exogenous applicationof AMPA in the hemisectedspinal cord preparationwereshown to be stereoselectively inhibited by LY303070.The estimatedIC50value obtainedof approximately1PMis similar to the IC50value for the inhibitionof the shortlatency componentof the synapticallymediated DR–VRreflex. Previous studies have suggested that the both theshort and long latency components of this reflex aremediated by AMPA receptors, primarily based upon

2

compounds which are unable to discriminate betweenAMPA and kainate receptors. Given the much lowerpotency of LY303070 on the long latency componentofthe reflex,it is likely that the receptor subtypesmediatingthis response differ from those involved in the shortlatency component. Although the functional role ofkainate receptors in the spinal cord has yet to beestablished, there are suggestions of their involvementin primary afferent neurotransmission (Agrawal andEvans, 1986). Depolarizations of cortical neurons byAMPA but not by kainate have been shown to beselectively antagonized by both GYK152466 andLY300168 (Lodge et 1992; Palmer and Lodge,1993).Consistentwith the studieswe have performed onthe cloned AMPA receptors and Purkinje neurons, wehave been able to demonstrate that the (–) isomerLY303070 was a non-competitiveantagonist of AMPAdepolarizations of cerebro-cortical slices where it pro-duced a non-parallel shift in the AMPA dose–responsecurve. Similar results were also obtained in this studyusing cerebella Purkinje neurons (data not shown). Aswith previous studies, the effects on kainate-induceddepolarizations are much less pronounced. Althoughkainate can activateboth AMPA and kainate receptors inthis preparation, it is apparent from this and previousstudies (Palmer and Lodge, 1993), that the effects ofkainate in this study are mediated largely by 2,3-benzodiazepine-insensitivekainate receptors.

The present studies have enabled us to study in detailthe activity of the three 2,3-benzodiazepinesat AMPAand kainate receptors. They provide data which accom-pany studies on these compounds which have beenperformed (Lodge et 1996). Given theselectivity profiles of the compounds and the relativepotencies at AMPA receptors, it should be possible tochoose appropriate concentrations of these agents inorder to dissect pharmacologically AMPA receptor-mediated events in more complex systems. In addition,recent studies have shown that the decahydroisoquino-line, LY293558, is able to discriminate pharmacologi-cally between G1uR5 and G1uR6 kainate receptors(Bleakman et 1996).Combineduse of 2,3-benzodia-zepines and LY293558 should allow pharmacologicaldissection of non-NMDA receptor-mediated trans-mission.

REFERENCES

AgrawalS. G. and EvansR. H. (1986)The primaryafferentdepolarizingactionsof kainatein the rat.Br. J. Pharmacol.87,345-355.

P hd8

H -d eMolec. Pharmacol. 49, 581–585.

C

Pharmacol.Rev. 40, 143–210.

Drugs News Perspect. 8, 261–277.

a5

J. Pharmacol. Exp. Ther. 272, 3843.

N2

J. Pharmacol. Exp. Ther. 271, 25–29.

p aRecept. Channels 3, 21–31.

P@gersArch. 391, 85–100.

Ann. Rev. Neurosci. 17, 31–108.

aRecept. Channels 2, 327–337.

d Neuron 5, 255–266.

Molec.Pharmacol. 48, 946-955.

a Molec.Pharmacol. 42, 10-15.

c Recept. Channels 3, 41-49.

Proc. Natn. Acad. Sci. U.S.A.90, 11688-11692.

S 2 invivo: e

LodgeE Drug ResearchRelated to Neuroactive Amino Acids, A.

Nature 355, 827–829.

a 1Neurosci. 14, 3842–3851.

Mt Neuron 13,

1031–1037.

ak

Brain. Res. Devel. Brain Res. ’77,257-263.

m Neurosci. Lett. 125, 5-8.

t pm in vivo. Eur. J. Neurosci. 6, 1662–1668.

Cb

G

dc

c Neuron 11,1069–1082.Partin K. M. and Mayer M. A. (1996)Negativeallosteric

modulationof wild-typeand mutantAMPAreceptors53655.Molec. Pharmacol. 49, 142–148.

a kh Neuron 14,

185–189.

i mlJ. Comp. Neurol. 318, 329–354.

P e

in situProc. Acad. Sci. U.S.A. 88, 41474161.

Br. J. Pharmacol. 117, 1209–1221.

b Trends Pharmacol. Sci. 14, 325–331.

da n Science 247, 571–

574.

bBioorg. Med. Chem. Lett. 3, 99–104.

2

Drug Rev. 2, 91–126.K

Trends Pharmacol. Sci. 11,25–33.Wilding

-

b Molec. Pharmacol. 47, 582–587.

Br. J. Pharmacol. 117, 1663–1672.

A bNeuron 10,