Pharmacology of ACEA-1 021 and ACEA-1 031: Systemically ...

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MOLECULAR

PHARMACOLOGY,

47:568-581,

Copyright

All rights

for Pharmacology

reserved.

1995.

and

Experimental

Therapeutics

In Vitro Pharmacology

of ACEA-1 021 and ACEA-1 031:

Systemically

Active Quinoxalinediones

with High Affinity and

Selectivity

for N-Methyl-D-aspartate

Receptor

Glycine Sites

R. M. WOODWARD,

J. E. HUETINER,

J. GUASTELLA,

J. F. W.

KEANA,

and

E. WEBER

Received

August

22, 1994; Accepted

December

1 5, 1994

SUMMARY

N-Methyl-D-aspartate

(NMDA)

receptor

antagonists

show

ther-

apeutic potential

as neuroprotectants,

analgesics,

and anticonvulsants.

In this context,

we used electrical

recording

techniques

to study

the in vitro

pharmacology

of two novel

quinoxalinediones,

i.e., ACEA-1 021 and ACEA-1 031 (5-nitro6,7-dichloroand

5-nitro-6,7-dibromo-1

,4-dihydro-2,3-quinoxalinedione,

respectively).

Assays

pressed

by rat brain poly(A)

NMDA receptors

in cultured

ACEA-1021

and

ACEA-1031

with

NMDA

receptors

ex-

RNA in Xenopus

oocytes

and with

rat cortical

neurons

indicated

that

are

potent

competitive

antago-

fists at NMDA

receptor

glycine

sites. Apparent

dissociation

constants

(Kb values) for ACEA-1021

and ACEA-1031

ranged

between

6 and 8 nM for oocyte assays and between

5 and 7 nM

for neuronal

assays.

Cloned

NMDA

receptors

expressed

in

oocytes

showed

up to 50-fold

variation

in sensitivity,

depend-

ing upon subunit composition.

For example,

using fixed agonist

concentrations

(1 0 M

glycine

and 1 00 M

glutamate)

IC50

values for ACEA-1021

with four binary combinations

were as

Channel

gands,

gating

glutamate

at NMDA

and

glycine,

receptors

acting

is effected

in conjunction

by two

at

lidis-

binding

sites (1). Experiments

in Xenopus

oocytes

and

cultured

neurons

suggest

that NMDA

by itself

causes only

low levels

of channel

activation,

indicating

that

glycine

should be considered

a ¡°coagonist¡±

at NMDA

receptors

(2-4).

Although

details

remain

uncertain,

evidence

from electrophysiological

and biochemical

studies suggests that glycine

and glutamate

binding

sites

are allosterically

coupled

(e.g.,

Refs. S and 6) and that glycine

reduces

one form

of receptor

desensitization

by increasing

the rate of recovery

from detinct

sensitized

states

(4,

7).

J.E.H.

was supported

by Grant

NS30888

Health.

J.F.W.K.

and E.W. were supported

from the National

Institute

on Drug Abuse.

ABBREVIATIONS:

soxazole-4-propionic

noxaline-2,3-dione;

from the National

in part by Grant

Institutes

RO1-DA06726

of

follows:

NMDA

receptor

(NR)1 A/2A,

29 nM; NR1 A/2B, 300 nM;

NR1A/2C,

120 nM; NR1A/2D,

1500 nM. Measurement

of EC50

for glycine

and calculation

of Kb for the inhibitors

indicated

that

differences

in IC50 values

are due to subunit-dependent

varia-

tions

in glycine

combined

selves

NMDA

affinity (EC50 ranged between

-0.1 and 1 M)

variations

in affinity

of the antagonists

them(Kb of --2-1 3 nM). In addition

to the strong

antagonism

of

receptors,

ACEA-1 021 and ACEA-1 031 were also mod-

erately

potent

activated

propionic

competitive

inhibitors

of

non-NMDA

receptors

either

by a-amino-3-hydroxy-5-methylisoxazole-4acid or by kainate.

Antagonist

affinities

were

whether

measured

with

receptors

(Kb of 1-2

expressed

by

similar

rat

brain

poly(A)

RNA in oocytes

j.tM) or with cultured

neurons

(Kb of 1 .5-3.3

p.M). Our results

suggest

that the in vivo neuroprotective

actions of ACEA-1 021 and ACEA-1 031 are predominantly

due

to

though

additional

an ancillary

role.

Studies

NMDA

at the

receptor

inhibition

at

inhibition

molecular

subunits,

NMDA

receptor

at non-NMDA

level

have

implying

glycine

receptors

revealed

that

sites,

may

al-

play

a diversity

a number

of

of differ-

ent receptor

subtypes

are present

in mammalian

nervous

systems (8). This

is supported

by binding

studies

and physiological

evidence

indicating

that, depending

on brain region

and stage of development,

NMDA

receptors

can show different pharmacologies

or electrical

properties

(eg., Refs. 5 and

9). At present,

two classes of subunit

have been identified,

(i)

NR1

subunits,

generated

which

by alternative

are

found

RNA

in

splicing

eight

different

(10,

11),

isoforms

and

(ii)

NR2

which

are found

in four distinct

subtypes,

each

encoded

by a separate

gene (12-14).

NR1A

(adopting

the

terminology

used in Ref. 11) appears

to be the predominant

isoform

in adult

rat

brain.

Expression

studies

in oocytes

suggest that

NR1

subunits

are sufficient

to produce

glutasubunits,

NMDA, N-methyl-D-aspartic

acid; ACPD, 1-aminocyclopentane-1

acid; BAPTA, 1 ,2-bis(2-aminophenoxy)ethane-N,N,N¡¯,N¡¯-tetraacetic

5,7-diCIKA,

5,7-dichlorokynurenic

acid; EGTA, ethylene

glycol

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid; NR1 and NR2, N-methyl-D-aspartic

568

with

,3-dicarboxylic

acid; AMPA,a-amino-3-hydroxy-5-methyliacid; DMSO, dimethylsulfoxide;

DNQX, 6,7-dinitroquibis(j3-aminoethyl

ether)-N,N,N¡¯,N¡¯-tetraacetic

acid; HEPES,

acid receptor

types 1 and 2.

Downloaded from molpharm. at Washington Univ Sch Med Libr on August 7, 2008

Acea Pharmaceuticals

Inc., Irvine, California

92715 (R.M. W., J.G.), Department

of Cell Biology and Physiology,

Washington

University

School

of Medicine,

St. Louis, Missouri

631 10 (J.E.H.), Department

of Chemist,y,

University

of Oregon, Eugene, Oregon 97403 (J.F.W.K.),

and

Department

of Pharmacology,

University

of California,

Irvine, Irvine, California

9271 7 (E. W.)

Pharmacology

mate

with NMDA-like

pharmacology

and electrical

(10, 11). However,

levels

of functional

receptor

in oocytes

are > 10-fold higher

using combinations

and NR2 subunits

(13), and functional

receptor

exin mammalian

systems

is detectable

only with het-

receptors

properties

expression

of NR1

pression

ero-oligomeric

expression

NMDA

hetero-oligomeric

sent

NR2

ingly

different

brain

(12-14).

are

has

receptors

led

more

of localization

patterns

that

Taken

subtype-selective

should

during

the

these

of brain

results

to regulate

effects

(21,

course

raise

22).

these

Glycine

site

antagonists

can

related

compounds

be divided

into

(25-28),

bioavailability

(19,

Quinoxaline-2,3-diones

29,

were

and

(vi)

ligands

for the non-NMDA

(AMPA/kainate)

mate

receptors

(3, 25, 27, 32). Molecules

usually

considered

to be moderate

or low

receptor

glycine

sites,

with

as selective

described

modest

of gluta-

family

of this

potency

levels

class

are

ligands

at

of selectiv-

ity between

NMDA

and non-NMDA

receptors

and little ability to penetrate

the blood/brain

barrier

(3, 25-28,

32, 33). We

report

on two quinoxalinediones

that

contradict

many

of

these

conceptions,

i.e., ACEA-1021

(5-nitro-6,7-dichloro-1,4quinoxalinedione)

ACEA-1031

quinoxalinedione)

of ACEA-1021

ACEA-1021

does

the

structurally

related

(Fig. 1). Pharmacological

characterization

and ACEA-1031

has been prompted

by conthat indicate

that both compounds

have sys-

not

in drug discrimination

In the present study

acterize

the

(5-nitro-6,7-dibromo-1,4-dihydro-2,3-

studies

temic bioavailability

in rat focal ischemia

current

and

basic

in

and

are

models

appear

efficacious

to substitute

studies

(35).

two assay systems

vitro

pharmacology

ACEA-1031

at mammalian

glutamate

oocytes,

where drug potencies

were

(34).

for

poly(A)

types

, ACEA-1

021

phencyclidine

were used to charand

receptors,

(i) Xenopus

assayed

against

NMDA,

metabotropic

and four

, 5,7-diCIKA,

031

and DNQX.

by mixtures

cultured

assayed

receptors

expressed

by rat

putative

NMDA

receptor

sub-

RNA

expressed

(ii)

rat

of subunit-encoding

cortical

against

neurons,

NMDA

and

cRNA,

where

non-NMDA

and

ACEA-1021

receptors.

was

pur-

For

of comparison,

the oocyte studies

included

5,7-diC1KA,

a selective

glycine

site antagonist,

and DNQX,

a quinoxaline2,3-dione

showing

selectivity

for

non-NMDA

receptors

(Fig. 1).

poses

Materials

and

Methods

Preparation

ofRNA.

Total RNA from whole rat brain (including

cerebellum

and a portion

of the brainstem)

was prepared

using the

acid guanidinium/phenol

method.

Poly(A)

mRNA

was isolated

from

total cellular

RNA by oligo(dT)-cellulose

chromatography.

All RNA

samples

were

cDNA

rat

stored

clones

NMDA

in sterile

encoding

receptor

Seeburg

and

homologs,

12-14).

Clones

were

have

been

enzyme

digestion

merase.

The

cRNA

and

reported

aliquots

until

injection.

was

Xenopus

oocyte

expression

cedures

(36),

mature

female

mm)

using

system.

laevis

3-aminobenzoic

acid

surgically

surrounded

ian

ovary.

were

dissected

from

the

bacteria

and

Following

Xenopus

se-

and

DNA purificaby restriction

T3 RNA

polystored

in 1-.il

with

ng/pi

and two to four ovarian

lobes were

developmental

stages V-VT, and still

tissues,

0.15%

host

conventional

was linearized

to 400

The

clones,

and their

(e.g. , Refs.

10, and

appropriate

synthesized

diluted

NR2D

by Dr. P. H.

Germany).

of these

with

clone

and

provided

previously

into

cRNA

was

NR2C,

Heidelberg,

properties

transformed

needed.

NR2B,

generously

University,

functional

were

- 80#{176}

until

NR2A,

plasmid

preparations

were made

tion techniques.

A sample

of each

(20-40

at

NR1A,

subunits

some

mouse

water

the

(Heidelberg

quences

established

were

ethyl

pro-

anesthetized

ester

(MS-222),

removed.

Oocytes

at

by enveloping

ovar-

Follicle-enclosed

oocytes

were microinjected

(pipette

mately

50 ng of whole-brain

cRNA

(NR1A

plus NR2A,

each

alone

Furthermore,

of ACEA-1021

5,7-diCIKA

of ACEA-1

and

non-NMDA,

brain

encoding

as neuroprotectants

of stroke

1. Structures

a

31).

initially

DNQX

Fig.

pro-

of specificity.

and

dthydro-2,3-

ACEA-1031

the

3-acyl-4-hydroxy-,

3-nitro-3,4-dihydro-,

and 3-phenyl-4-hydroxyquinolin-2(1H)-ones

(29-31).

The different

classes of

antagonists

show a wide range ofpotencies,

selectivities,

and

molecule

ACEA-1021

02N

antagonists

noxalinediones

NMDA

::xrx:

of

variety

of classes

(see Refs. 18, 19, and 23 for reviews),

(i)

partial

agonists

and related

compounds,

(ii) kynurenic

acids,

thiokynurenic

acids,

and the structurally

related

2-carboxytetrahydroquinolines,

(iii) indole-2-carboxylic

acids,

(iv)

dihydro-2,5-dioxo-3-hydroxy-1H-benzazepines

(24), (v) qui-

in vivo

:xzx:

in

Dis-

show therapeutic

potential

as

neuroprotectants

( 15), anticonvulsants

(16), and analgesics

(17).

From

a pharmacological

perspective

the glycine

site

provides

a target

for controlling

NMDA

receptor

activity,

which,

depending

on the circumstances,

could have advantages

over the use of ligands

acting

via other

sites

(18, 19).

For example,

glycine site antagonists

do not appear to induce

neuronal

vacuolation

(vacuolization),

a phenomenon

that has

been a cause for concern

with other

types

of antagonists

(20),

and have relatively

encouraging

profiles

of behavioral

side

receptor

NO2H

02N

subtypes

are inand, hence,

that

function

be able

NR2C

brainstem.

receptor

NMDA

drugs

a degree

structures,

together,

possibility

that

different

volved in discrete

aspects

H

repre-

and

569

Quinoxaline-2,3-diones

at

such that

between

susceptible

Oocytes

20

receptor

ng.

In

general,

maximum

we

currents,

aimed

to restrict

measured

at the

levels

second

of expression

phase,

ranged

100 and 500 nA. Larger

responses

(e.g., > 1 pA) were more

to contamination

by secondary

Ca2-gated

C1 currents.

were stored in Barth¡¯s

medium

[88 mM NaCl, 1 mM KCI, 0.41

mM

CaCl2,

mM

HEPES,

oocytes

tip diameter,

20-30

jim) with approxipoly(A)

RNA or with 1:1 mixtures

of

-2B, -2C, or -2D; -2, 5, or 20 ng of RNA

subunit).

NR1A-encoding

cRNA

was injected

0.33

were

znii

pH

still

Ca(N03)2,

7.4,

with

surrounded

0.82

0.1

nme

mg/ml

by

MgSO4,

2.4

gentamycin

enveloping

mM

NaHCO3,

sulfatel.

ovarian

tissues,

5

While

the

Downloaded from molpharm. at Washington Univ Sch Med Libr on August 7, 2008

(14).

development

with

idea

in adult

mammalian

NR2A

and NR2B

sub-

speaking,

Generally

expressed

in forebrain

and NR2D

in diencephalon

of NR2 subunits

also

varies

NMDA

to the

accurately

strongly

cerebellum,

tribution

cesses

This

NO

composition

of neuronal

receptors.

For the

in situ

hybridization

studies

indicate

strik-

the subunit

subunits,

units

(12).

of Novel

570

Woodward

et aL

Barth¡¯s

medium

was supplemented

with 0.1% bovine serum.

Oocytes

were defolliculated

1 day after injection

by treatment

with collagenase (0.5 mg/ml Sigma

type I, for 0.5-1 hr), vortex-mixed

to dislodge

epithelia,

and subsequently

stored

in serum-free

medium.

Electrical

recordings

were

made,

using

a conventional

in

for

rates

for

drug

at

and

among

7.4.

longer.

were

oocyte

surface

the

the

tangles

2 mi

injections

were

2-3

1.8

made

sec.

(i.e.,

Ringer

KC1,

mM

BaC12,

by

pneumatic

absence

using

1

Concentration-inhibition

was

com-

5 mii

HEPES,

pH

curves

were

fit with

I

in

which

i + ([antagonist]/10_P¡¯

=

is the current

evoked

is the concentration

¡®control

IC50

(IC50

inhibition),

antagonists

were

eq. 3,

1

i;;-

Dougall

solution

10-pKb

[agonist]

half-maximal

to

(2)

¡°

1+

-log

the

2.

+ [antagonist]

I

¡®\

appeared

eq.

A-pEC,oI

1

Exchange

beneath

of inhibitor,

I

two-elec-

of microvilli)

Zero-Ca2fBa2

of 115 mM NaC1,

Intraoocyte

changes

and

and

from

form

of the

the

agonist

of antagonist

n is the

determined

generalized

by

slope

factor.

inhibition

alone,

plC50

that

produces

The

Kb values

curves

Cheng-Prusoff

is

using

equation

for

a Leff-

(38),

eq. 4.

IC50

Kb

pressure-pulse

from micropipettes

(37). Injection

solutions

of EGTA (40-400

mM) and BAPTA

(50-500

mM) were

made

up in H20,

the pH was

adjusted

to 7.4 with KOH or HC1, and the solutions

were filtered

to

minimize

plugging

(Acrodisc-13

filters,

0.2-.tm

pore size). Pressure

was set between

200 and 400 kPa. The volume

of injections

was

regulated

by adjusting

the time of the pulses

(0.1-1

sec) and was

{2 + ([agonist]j/EC5o¡¯}

1

(44)

ejection

estimated

by

measuring

cortical

the

recordings.

Neuronal

neurons

were

diameters

Primary

prepared

of ejected

droplets.

dissociated

from

newborn

cultures

rats

of cerebral

as

described

from

Boralex

glass

capillaries

filled with an internal

solution

EGTA,

10 mM HEPES,

and either

(pH adjusted

to 7.4 with CsOH).

Excitatory

amino

acid

agonists

(Rochester

that

Scientific

contained

140

mM

and

Co.),

5 mM CsCl,

CsCH3SO3

antagonists

were

the

applied

in an

external

solution

composed

of 160 nmi NaCl,

2 mri CaC12, 500 nM

and 10 mrsi HEPES,

pH 7.4. The amplitude

of agonistevoked currents

were determined

relative

to the holding

current

in

control

external

solution,

which

lacked

agonist.

For experiments

with

kainate

and AMPA,

1 p.M dizocilpine

(MK-801)

was added

to the

tetrodothxin,

external

Drug

solution

to ensure

solutions

were applied

microcapillary

The time

100 msec.

tubes

constant

(2-pi

blockade

to the

Drummond

for external

lyzed

(Axon

as described

currents

Microcaps,

solution

Instruments).

were

55-mm

exchange

recorded

channels.

array

of

ranged

from 30

Currents

previously

were

to

(24). The logistic

with

equation

(eq.

1) was

by

=

a

i;;

=

i + (10_PEC50/[agonistj)n

(1)

determined

dissociation

constants

from a simultaneous

for the antagonists

(Kb values)

fit of concentration-response

were

data

and

are

times

tables,

scale.

as

curve

experiments

the

used

to

appropriate

confidence

Unless

given

necessary

to investigate

K,,

were

values

mean

construct

value

the

from

intervals

have

stated,

all data

otherwise

¡À standard

been

the

trans-

quoted

in

assays

of

error.

were

the

useful

mechanism

calculated

Statistical

by

using

conformity

calculating

to the

ratios

for

side-by-side

of inhibition.

both

approaches.

simple

competitive

of residual

variance

where

SS

is the

the sum

sum

=

ofsquared

of squared

df1 is the degree

of freedom

(number

fitted parameters)

for individual

fits,

for the simultaneous

fit.

Drugs.

ACEA-1021

were

3540)

(m.p.

synthesized

noxaline-2,3-dione

one,

elsewhere.¡¯

diC1KA,

(Natick,

was

from

MA).

mM

solutions

made

i

of

of freedom

ACEA-1031

(m.p.

352-

Details

will be provided

elemental

analyses.

5,7from Research

Biochemitetrapotassium

OR).

solution.

other

All

volume)

and

the

then

range

salt)

drugs

were

from

made

Tween-80

E. Weber,

DMSO

responses.

up

as

and

,.tM-30

unpublished

alone

had

solution

polyethylene

observations.

of

a series

mM.

of stock

Working

solutions

no measur-

As a vehicle

a stock

10%

concentrations

to generate

dilution

dilutions

current

at

made

of 0.3

1000-3000-fold

At these

ACEA-1021

dissolved

were

over

by

on membrane

J. F. W. Keana

initially

Dilutions

stock

(by

number

of 6,7-dichloro-1,4-dihydroqui-

(Eugene,

were

in DMSO.

were

0.5%

fit (eq. 2),

minus

Co.

solutions

Ringer

fits (eq. 1),

and df2 is the degree

(cell-impermeant

Probes

of DMSO

able effects

also assayed

points

KNO3

BAPTA

Molecular

Sigma Chemical

Quinoxalinediones

10-30

of data

and H2504.

drugs

gave satisfactory

and AMPA were obtained

DNQX,

cals

(5)

6,7-dibromo-1,4-thhydroquinoxaline-2,3-di-

using

Both

df1)

simultaneous

and

tested

5.

for individual

342-344#{176})

cases,

was

to eq.

for the

by nitration

and

respectively,

model

-

deviations

deviations

In most

according

S51)/(df2

SS1/df1

(552

Fdf,_df,df

with

Apparent

of agonist

the effects of different

antagonists

on a common

response

and, in the

case of cloned

NMDA

receptors,

for assessing

the relative

activity

(1C50 values)

ofinhibitors

at receptor

subtypes

with different

agonist

affinities.

Concentration-response

(Schild-type)

experiments

were

into

1

I

parameter

a linear

to

S

filtered

fit to the data

for individual

concentration-response

relations

adjusting

the slope factor,

n, and

the parameter

pEC50

(pEC50

-log EC50, where

EC50 is the agonist

concentration

that produces

half-maximal

response)

(Sigmaplot;

Jandel

Scientific).

each

In text

text

length).

an Axopatch

200

at 1-5 kHz (-3

dB, four-pole

Bessel

filter),

digitized

at 5 kHz, and stored

on computer.

Steady

state currents

were measured

by averaging

1-5 sec of

data during

the final third

of each agonist

application.

Membrane

potentials

have been corrected

for a junction

potential

of - 10 mV

between

the internal

solution

and the Tyrode¡¯s

solution

used to

perfuse

the bath.

Data analysis.

Agonist

concentration-response

curves

were anaamplifier

Whole-cell

of NMDA

receptor

neurons

from a linear

for

Inhibition

mM CsF

dose

EC50 is the half-maximal

agonist

concentration,

and b is the slope factor of the agonist

concentration-response

relation. In practice,

the parameter

10pIC50

was replaced

in eq. 3 by

(10¡¯b)[([2

+ ([agothst]1fEC5o)¡¯11¡±i

- 1], where n and pKb (-log

Kb)

were

the two free parameters.

The 95% confidence

intervals

for

pEC50, plC50, and pKb were obtained

as the product

of the standard

formed

were

is the fixed

curve,

distribution.

10 mM

or 140

[agonistl1

inhibition

deviation

(24).

Cultures

were

used

for electrophysiological

recordings

after

1-3

weeks in vitro.

The culture

dish was perfused

at a rate of 1-5 mI/mm

with Tyrode¡¯s

solution

(150 mM NaCl, 4 mM KCI, 2 mi CaC12,

2 mM

MgCl2, 10 ITIM glucose,

10 mM HEPES,

pH 7.4). Whole-cell

pipettes,

pulled

where

control

in

0.2

glycol.

M

we

Tris

This

Downloaded from molpharm. at Washington Univ Sch Med Libr on August 7, 2008

considerably

posed

solution

solutions

envelope

vitelline

be

mid-chamber

presence

:t-

trode voltage

clamp (Dagan

TEV-200),

over periods

ranging

between

3 and 14 days after injection.

Oocytes

were placed in a 0. 1-ml recording chamber

that was continuously

perfused

(5-15 mI/mm)

with frog

Ringer

solution

(115 mit NaC1,

2 mM KC1, 1.8 mri CaCl2,

5 m

HEPES,

pH 7.4). Drugs were applied

by bath perfusion.

The pH of all

solutions

(particularly

AMPA

and kainate)

was readjusted

to 7.4

where

necessary.

When

the more rapid flow rates

were used, halftimes

the

Pharmacology

of Novel Quinoxaline-2,3-diones

571

5,7-dicikA

showed

no difference

in potency

compared

with the

DMSO

stock solutions.

5,7-diC1KA

(10 mM) was made

up in 10-20

mM NaOH,

serial

dilutions

of stock solutions

were in water,

and

formulation

dilutions

into

DMSO

weeks

Ringer

solution

had

no

measurable

effect

stock solutions

of quinoxalinediones

were stored

in the dark at 4#{176}

without

apparent

reductions

solutions

of drugs were made up fresh each day

Ringer

on

ACEA-1 021

300 nM

10

pH.

100

Recordings

from

100

100

for up to 4

in

300nM

10iM

i2__

12_.

i.

[GLYfliM

i

100 (NMDA]

tM

potency.

of use.

I

Results

Electrical

10

10

Xenopus

f

Oocytes

receptors

expressed

by

rat

whole-brain

RNA. As reported

previously

for oocytes

expressing rat brain

poly(AY

RNA (2), NMDA

(1-100

p.vi) or glycine

(0.1-10

M)

elicited

minimal

membrane

current

responses

when applied alone but activated

large currents

when coapplied. Membrane

currents

activated

by NMDA/glycine

were

inward

at a holding

potential

of - 70 mV and

followed

a

relatively

complex time

course

involving

an initial

spike

of

NMDA

poly(A)¡¯

saturating

within

peak

concentrations

5-10 sec, followed

(Fig. 2). Repeated

ofagonists

2 mm

E

by a second,

exposures

to

resulted

in some degree

often followed

by a

intracellular

regula-

of ¡°run-down¡±

of the response

(10-80%),

gradual

increase

(data

not shown).

The

tory

mechanisms

current

responsible

were

amplitudes

for

not

In agreement

with

previous

of current

was

abolished

spike

Ba2

Ringer

solution

due

by

term

changes

that

(39,

to

the

in

with

oocyte

initial

through

0.01

0.1

1

10

[antagonist]

EGTA

or

x

Co

_E

-0.2-1

spike

NMDA

0.001

zero-Ca2/

concentration,

the response

influx

the

40),

switching

intraoocyte

to Ca2

long

studies

or by injecting

BAPTA

(100-500

pmol;

mM) (37). This confirmed

ary current

nels and

these

investigated.

10 0

tM

1.0

is a second-

receptor

chan-

endogenous

Ca2-gated

Cl

channels.

Interestingly,

the slowly developing

current

peak was largely

unaffected

by EGTA

or BAPTA

injections

and was still a prominent

feature

in oocytes

clamped

precisely at the chloride

equilibrium

potential

(data not shown);

these

results

argue

strongly

that this component

is not simply due to activation

ofCa2-gated

C1 channels.2

In barium

Ringer

concomitant

activation

solution

responses

became

injections

the second

Ba2

the

second

peak

an additional,

apparent.

This

(see also Ref.

current

component

could

through

of

was

slowly

could

40). The

receptor

Ca2-dependent

slow,

through

resultant

Ba2-stimulated

otherwise,

electrical

indirect

or slow

permeation

channels.

cological

measured

2, upper).

to current

under

steady

Preliminary

100

.tM

glutamate

at the

peak

of the

We considered

this a reasonable

flowing

directly

through

NMDA

state

desensitizing

dose-response

NMDA

and

and

10

glycine

these

concentrations,

(mean

¡À standard

conditions.

experiments

glycine

,.tM

recognition

current

error,

levels

258

the

Unless

made in normal

frog

current

was ignored,

(Fig.

tion

in

by

addi-

probably

arises

of intracellular

Ca2

and

of Cl

slow phase

approxima-

receptors

saturation

sites

(Figs.

ranged

¡À 58 nA;

stated

study

were

spike of C1

for pharma-

established

provided

R. M.

Woodward,

unpublished

observations.

1

10

100

[glycine]

jiM

2 and

that

of the

3). At

from 54 to 820 nA

n = 16). Antagonist

Fig. 2. Upper, sample

records

from a single oocyte, comparing

potencies

of ACEA-1 021 and 5,7-diCIKA against NMDA responses at

receptors

expressed by rat whole-brain

poly(A)

RNA. Unless stated

otherwise,

the holding potential

in this and the following

figures is -70

mV. Downward

deflections,

inward current; bars, solution changes

and

drug applications. The dead time of the perfusion system was 5-10 sec.

Responses

were separated

by 5-1 0-mm intervals of wash (not shown).

For pharmacological

assays the initial spike of Cl

current was ignored,

and response

amplitudes

were measured

at the peak of the slow phase

(arrow). GLY, glycine. Middle, concentration-inhibition

curves comparing potencies

of ACEA-1 021 , ACEA-1

031 , 5,7-diCIKA,

and DNQX at

NMDA receptors expressed by rat whole-brain

poly(A)

RNA. NMDA

was used at 100 tM and glycine at 10 tM. In this and all following

figures data are plotted as the mean ¡À standard error, expressed as a

fraction of either control responses (concentration-inhibition

curves) or

maximum

responses

(concentration-response

curves).

The number of

separate experiments

(cells) is given in parentheses.

Smooth

curves,

best fits of eq. 3 to the data for each drug; see Materials

and Methods

for details.

Curve

parameters

(IC50 and slope values) for these fits are

given in Table 1 Lower, effects

of 1 00 nM ACEA-1 021 and ACEA-1 031

on glycine concentration-response

curves for NMDA receptors

ex.

pressed

by rat whole-brain

was 1 00 jLM. Smooth curves,

Curve

2

0.1

0.01

by BAPTA

recordings

were

larger

current

whereas

in the present

Ringer

solution,

the initial

and response

amplitudes

assays

in

for the reduction

channels,

current

release

activation

but

inward

be abolished

reason

be block

NMDA

tional,

reduced,

developing,

parameters

values for paired

poly(A)

RNA.

The

NMDA

concentration

best fits of eq. 2 to the data for each drug.

(EC50 values for control curves and optimal slope

curves for control and with drug) are given in Table 1.

Downloaded from molpharm. at Washington Univ Sch Med Libr on August 7, 2008

current,

which

decayed

more

slowly developing,

EA

572

Woodward

et aL

x

receptor

Cs

_E 1.0

activation.

onists

mm.

variability,

and glycine

showed

0.8

NMDA

As shown

0.6

Even

0.4

at rat brain

compounds

0.2

represents

0.0

diC1KA

DNQX

1

100

10

1

1

1

tM

The

and

ing

[ACEA-1031]

jiM

ACEA-1031

for

[NMDA]jiM

glycine.

Both

(28,

using

2 mm

[ACEA-1031]jiM

[ACEA-1021]

jiM

10 [iS, 3R-ACPD]

jiM

the

given in Table 1 . Middle

and lower, sample

records

from

designed

to test whether

ACEA-1

021

or ACEA-1 031

showed measurable

inhibitory

effects

either

at NMDA receptor

glutamate binding sites or at metabotropic

receptor glutamate

binding sites.

Middle,

application

of 1 mM glycine (GLY) elicited a membrane current

response

predominantly

due to activation

of strychnine-sensitive

glycine receptors

that are coexpressed

by the rat brain poly(A)

RNA. This

response

was allowed to desensitize

to steady state levels and used as

a base-line for measuring

currents

mediated

by NMDA receptors.

Repeated

coapplications

of 1 M NMDA elicited

threshold

NMDA responses

(downward

notches

in the record) that were used to assay

inhibition

by antagonists.

Lower, an oocyte was repeatedly exposed to

10 .tM (1S,3R)-ACPD,

eliciting

a reproducible,

threshold,

oscillatory,

values

are

experiments

potency

was

initially

assessed

using

fixed

were assayed

agonist

for

concentra-

and 10 jiM glycine)

and varying

concentrations

of inhibitor.

In all cases, oocytes

were pretreated

with

glycine

for approximately

30 sec and receptors

were

then activated

by coapplication

of NMDA.

Antagonists

were

tions

applied

(100

and ACEA-1031

p.M NMDA

together

with

glycine

to promote

equilibration

competitive

F1116

parallel

rightward

2.6;

competitive

30,

the

32),

at the

inhibition

=

shifts

EC50

glycine

F144

ACEA-1031,

antagonism

Gaddum-Schild

between

Preliminary

tion

by ACEA-1021

for

for

glycine

before

relationship

the

two

all

binding

3.3).

=

four

antag-

measured

under

methods

(24).

There

of analysis

was

(Table

good

1).

experiments

in oocytes suggested

that inhibiand ACEA-1031

showed little or no voltover the range of -20 to - 100 mV. However,

voltage

range to positive

potentials

was com-

age dependence

extending

the

plicated

by activation

of the

study

performed

mm

Fig. 3. Upper, concentration-response

curves

for NMDA

at NMDA

receptors

and for (1S,3R)-ACPD

at metabotropic

glutamate

receptors,

in oocytes

expressing

rat whole-brain

poly(A)

RNA. Smooth

curves,

best fits of eq. 1 to the data for each agonist. EC50 values and slope

upon which ACEA-1021

caused

control

conditions

was used in conjunction

with

IC50 values

to estimate

Kb values

using

a Leff-Dougall

approach

(38). For

comparison,

shifts in EC50 induced

by fixed concentrations

of

ACEA-1021

and ACEA-1031

were used to calculate

Kb values,

thorough

Cl current,

inhibition.

receptors

expressed

in oocytes.

For both

was between

150 and 200 nii. This value

compounds

simple

agreement

2

antagonism

concentration-response

relation,

with approximately

20-fold

increases

in the EC50 and no clear reductions

in the maximum

response

(Fig. 2). Effects ofboth

drugs were

20

!2.

curves

potent

in the glycine

onists

.2_

indicated

-5

which

with

of inhibition

was investigated

by measurof fixed concentrations

of ACEA-1021

and

(100 nivi) on the concentration-response

relation

Assuming

!2___

ACEA-1031

NMDA

the IC50

consistent

with

site

(ACEA-1021,

:i_2____

12_.

of control

responses,

were

regularly

tested

effects

jiM

I

within

mechanism

the

[ACEA-1021]

;L.

of antagfully

of receptors.

[GLY]jiM

-

oocytes

out

using

A more

of large

endogenous

currents.

voltage

dependence

of inhibition

neuronal

NMDA

responses

to completely

was

(see below).

overcome

The ability

of 100 jiM glycine

inhibition produced

by 100 nM ACEA-1021

(Fig. 2) was consistent

with preliminary

binding

studies

that suggested

that ACEA1021 was relatively

inactive

at glutamate

recognition

sites on

NMDA

fore

receptors.3

designed

Electrophysiological

simply

to test

assays

whether

any

were

there-

inhibitory

actions

be detected

at the glutamate

binding

site. The protocol

used a high concentration

of glycine

( 1 mM), to promote

saturation

at glycine

sites, whereas

NMDA

was used at concould

centrations

thereby

glutamate

(1-3

maximizing

sites.

ACEA-1031

inhibition

sufficient

to elicit

threshold

responses,

the chances

of detecting

inhibition

at

Under

these

conditions,

ACEA-1021

and

jiM)

at concentrations

of NMDA

responses

of up to 1

(e.g.,

Fig.

p.M

showed

3). Using

NMDA

3

E. Weber,

of -27

unpublished

p.M (Fig.

3; Table

observations.

1, lower)

assuming

EC50

a

competitive

interaction,

we estimated

the K,, for both antagonists at glutamate

binding

sites to be >2 tM, i.e., 150-200fold lower than affinities

at glycine

sites. These experiments

were complicated,

a little,

by activation

of classical

strychnine-sensitive

glycine

receptors

that are coexpressed

in oocytes

injected

with

rat brain

poly(AY

RNA.

The glycine

refor

and

................
................

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