Vol. 91, No. 3, 1979 December
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14, 1979
Pages
1013-1024
COMPETITION FOR DIAZEPAM RECEPTOR BINDING BY DIPHENnHYDANTOIN AND ITS ENHANCEMENT BY y-AMINOBUTYRIC ACID Godfrey
Tunnicliff
& Julie
A. Smith
Evansville Center for Medical Education Indiana University School of Medicine 8600 University Boulevard, Evansville, IN 47712 and That T. Ngo Faculty Research Facility University of California Irvine, CA 92717 Received October 25, 1979
SUMMARY The anticonvulsant diphenylhydantoin competitively inhibited the binding of [3aldiazepam to synaptic membranes of rat cerebral cortex. The Ki was calculated as 0.9 PM. In the presence of y-aminobutyric acid the Ki was reduced to 0.3 PM. A similar change in Ki was also observed in the presence of muscimol, a potent agonist of y-aminobutyric acid. y-Aminobutyric acid also reduced the dissociation constant (Kd) of diazepam binding from 16.7 nM to 3.9 nM. Thus it appears that diphenylhydantoin and diazepam are binding at the same site and the presence of y-aminobutyric acid increases the affinity of the receptor for each of the drugs. These observations indicate that the anticonvulsant effects of diphenylhydantoin and diazepam may be related to an interaction with a common binding site and to the modification of this binding site by y-aminobutyric acid. INTRODUCTION several
For
most
popular
In addition, and cardiac
membranes. is 0006-291 Copyright All rights
that X/79/231
used
in the
this
drug
has been
of action that
of
diphenylhydantoin
drug
arrhythmias
mechanism however,
decades
it
control
121.
(DPH) has been of grand
prescribed
Despite
its
For
instance,
sodium
into
with
calcium stimulated
01 3-07$01.00/O
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
1018
long There
of DPH is unknown. can interfere
for
the
ma1 epilepsy. myokymia history
[l] the
are
indications,
ion
flux
across
neural
influx
is
inhibited
[3],
nerve
cells
[4].
Recently
as
Vol. 91, No. 3, 1979
Perry
et al
resting
[5]
sodium Diazepam
reported
that
channels
in
is a drug
of anxiety.
However,
anticonvulsant still
BIOCHEMICAL
this
drug
itory
There
interacts
ition
have
binding
of
presence
butyric
acid
nature
y-aminobutyric
acid,
that
diazepam acid
acid
treatment
important
of its
action
however, the
is that
major
inhib-
potentiates [6,7].
is
the
inhib-
In addition,
stimulated
the
in the
[8,9].
that
to synaptic enhances
the
Electrophysiological
to receptors
evidence
markedly
in
possesses
brain.
passive
axon.
evidence,
in the
of diazepam
blocks
considerable
of y-aminobutyric
binding
RESEARCH COMMUNICATIONS
used
also
by y-aminobutyric
We now present the
with
13Hldiazepam
squid
is widely DPH it
demonstrated
produced
giant
The exact is
neurotransmitter
studies
the
that like
BIOPHYSICAL
diphenylhydantoin
properties.
unclear.
AND
this
diphenylhydantoin membranes
can inhibit and that
y-amino-
inhibition.
MATERIALS AND METHODS 5,5-Diphenylhydantoin was purchased from Sigma Chemical Company, St. Louis, MO. Muscimol was supplied by Research Organics Inc., Cleveland, OH. Diazepam was kindly donated by Hoffmann La Roche, Nutley, NJ. [JH]Diazepam (36 curies/mmol) was obtained from Amersham, Arlington Heights, IL. Preparation of membranes. The cerebral cortex from male Sprague Dawley rats was homogenised in 9 vol of 0.32 M sucrose containing 1 mM HEPES buffer (pH 7.4) and centrifuged at 4O.C for 10 min at 1000 g. The supernatant was diluted 4-fold with 50 mM TRIS citrate buffer (pH 7.4) and centrifuged at 30,000 g for 20 min at 4°C. The precipitate was suspended in 20 vol (based on the original weight of tissue) of the TRIS buffer and recentrifuged as previously. This procedure was repeated 3 times to ensure thorough washing of the membranes. The pelleted membranes were stored overnight at -20°C. Measurement
of [3H]diazepam binding. The frozen membranes were suspended in 50 mM TRIS-citrate buffer (2.5 ml per 1 g of tissue). Twenty five microlitres of membranes (about 0.3 mg protein) was transferred to 0.975 ml of TRIS citrate-buffer containing 1.4 nM [jH]diazepam (70,000 dpm). The mixture was incubated for 30 min at 23°C prior to centrifugation for 10 min in an Eppendorf 3200 centrifuge. The pellet was washed twice with 1.5 ml of the TRIS citrate buffer. The membranes were dissolved in 0.3 ml of tissue
1019
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
solubiliser (NCS, Amersham) and, after the addition of 10 ml of toluene containing 0.1% PPO and 0.03% POPOP, were counted in a Nuclear Chicago Unilux IIA. Binding of diazepam was taken as that radioactivity able to be displaced by 10 PM non-radioactive diazepam. Protein
serum
The method of Lowry was used as a standard.
assay.
albumin
[lOI
followed.
was
Bovine
RESULTS Effects
of DPH and anulogues
Synaptic ence
of
membranes
were
are
presented
Table
1.
the
absence
related
compounds. gave
Acetyl-2-thiohydantoin
inhibited
binding
of
compounds
The remainder
or pres-
Diphenylhydantoin
of binding.
and phenyl-5-ureido-oxadiazole 29% respectively.
in
structurally
in
to an 83% inhibition
binding.
incubated
10 uM DPH and several
The results rise
on diazepam
the
by 31% and produced
no
inhibition. inhibition
Competitive
Membranes concentration experiments were
of diazepam
binding
incubated
with
were range
1.25
plotted
of
1.4
~J.Mor 2.5
by the method
TABLE 1.
Effects
binding
21.4
of Lineweaver
of DPH and related of diazepam
to rat
over
X lo-9M.
UM DPH was present.
a ligand In some
The results
and Burk
[ll]
52.;
; 6.9 - 2.7
29:2 f 3.2
1.3 f 0.2 0.8 l 0.2
acid
5.1 f 2.6
-3.3
l 0.5
Membranes (about 0.3 mg protein) were incubated in 1.4 nM [3H]diazepam at 23"~ for 30 min in the absence or presence of each compound. The values represent the mean (*S.E.M.) of 3 experiments.
1020
and
compounds on the synaptic membranes. % Inhibition
Diphenylhydantoin Acetyl-2-thiohydantoin Phenyl-5-ureido-oxadiazole
Pentobarbital Uric acid Allantoin
[3B]diazepam
X 10Wg to
Compound (10 PM)
Barbituric
by DPH.
BIOCHEMICAL
Vol. 91, No. 3, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2
0I _ I25
T P P m
2.5
02
DIAZE PAM-‘hM)-’
Fig
1.
Fig
2.
shown
in
Double reciprocal plot of the effects of DPH on diazepam binding to synaptic membranes over a ligand concentration range. Membranes were incubated at 23"~ for 30 min in the absence (closed circles) or presence of DPH (triangles = 1.25 PM; stars = 2.5 PM). The inset is the data shown as a Dixon plot. Each point is the mean of 3 experiments. Binding = pm01 per mg protein. Double reciprocal plot of the effects of DPH on diazepam binding to synaptic membranes over a ligand concentration range in the presence of 10 uM y-aminobutyric acid. Membranes were incubated at 23"~ for 30 min in the absence (closed circles) or presence of = 1.25 PM). DPH (squares = 0.625 uM; triangles The inset is the data shown as a Dixon plot. Each point is the mean of 3 experiments. Binding = pm01 per mg protein. Fig.
itively
inhibits
plotted
by the
1.
It
diazepam
binding
evident
binding
method
a Ki =0.9
of
J.IM.
reduced
a competitive
of
to
diazepam.
time
1.25
PM and 0.625
inhibition
of
1021
Fig.
re-
1) and (Kd)
for
the
acid and muscimol.
was repeated This
were
nM.
by y-wninobutyric
(10 PM).
in
constant
as 16.7
compet-
The data
(inset
The dissociation
experiment acid
diphenylhydantoin
[12]
was calculated
The previous y-aminobutyric
that
Dixon
of DPH inhibition
Enhancement
uced
is
the
revealed
DPH were
DIAZE PAM-’ (“MY-’
in the
JAM.
[xH]diazepam
the
presence
concentrations Once again binding
of of
DPH prod(Fig.
2).
Vol. 91, No. 3, 1979
However,
BIOCHEMICAL
the
Ki was reduced
Furthermore, similar
the
degree If
place
Kd for (from
muscimol,
BIOPHYSICAL
by a factor
diazepam
16.7
nM to 3.9
acid
PM and a Kd of
6.6
to 0.31
was reduced
uM.
by a
nM). acid
in the
nM were
RESEARCH COMMUNICATIONS
of three
binding
a y-aminobutyric
of y-aminobutyric
0.35
AND
agonist,
above
was used
experiment,
in
a Ki
of
obtained.
DISCUSSION It
is
action
clear
between
from
our
experiments
DPH, diazepam and an amino
sions
Diphenylhydantoin
competing
for
diazepam
is
the about
That
900 nM). same binding blance
in
drugs
also
fact
site their
for
share
in the
a reduction the
onset
of
brain
y-aminobutyric
On the
other
that
tend
to protect
y-aminobutyric
that
y-aminobutyric
acid
ated
the
of diazepam
ort
-for
inhibition the
idea
that
in
is
and its
binding
DPH and diazepam
1022
is
related
to
blockage [15].
acid It
[18].
by DPH is to the
binding The fact
study.
muscimol
concen-
is known
receptor
present
bind
that
convulsions
diazepam
agonist
the
available
y-aminobutyric
the
resem-
significant.
Further,
seizures
the
neurotrans-
acid
induces
can enhance
with
thus
inhibitory
in brain
confirmed
for
with
is highly
[13,141.
against
acid
an observation
[8,91,
actions
amino
receptor
increases
hand,
this
seizures acid
affinity
Each of these
Evidence
1171.
of
experimental
of the
trations
levels
to be
of a close
[lb].
the major
brain
appear the
light
a common receptor
mammalian in
in
antiepileptic
is
to convul-
interact
conformations
acid
two
DPH (16 nM compared
unexpected
important
seem to
although
and diphenylhydantoin
molecular
y-Aminobutyric mitter
that
-
linked
and diazepam
60 times
is not
is an interacid
intimately
site
possess
they
acid
same receptor
diazepam
there
and y-aminobutyric
anticonvulsants 113-151.
that
also strong
stimulsupp-
same site.
Vol. 91, No. 3, 1979
BIOCHEMICAL
Moreover
the
increase
is
the
same (3 to 4-fold)
about
to speculate increase
that
in
the
changes
and that the
shape
of the
fit
occurs
[o],
of rats
with
binding
[ 191.
These
since
the
that
occupancy to affinity
changes
differ
its
that
conciderably. site
is
diazepam
so that
necessarily
of
acid
binding
diazepam
binding
the
to each other
altered
stating
do not
at the
with
the
adjacent
occupies is
inviting
The mechanism
that
enhanced
diazepam
is
acid
acid-elicited
is associated
are
receptor
results
It
by y-aminobutyric
has appeared
two studies of the
drugs.
compounds.
about
diphenylhydantoin
ours
leads
these
neurotransmitter
a report
both
y-aminobutyric
receptors
diazepam
RESEARCH COMMUNlCATlONS
by y-aminobutyric
has been postulated
acid
when the
Of late
of
it
and y-aminobutyric
for
binding
brought
However,
induced
observed
actions
affinity
unknown.
affinity
DPH and diazepam
anticonvulsant the
in
AND BIOPHYSICAL
site a better
pretreatment receptor disagree It
is
by DPH in
with feasible vivo
receptor.
REFERENCES 1.
Isaacs, H. and Frere, G.S. (1974) S. Afr. med. J. 48, 1601-1607. 2. Bigger, J.T. (1972) Adv. intern. Med. 18, 251-281. 3. Sohn, P.S. and Ferrendelli, J.A. (1973) J. Pharmacol. exp. Ther. 185, 272-275. 4. Lipicky, R.J., Gilbert, D.L. and Stillman, I.M. (1972) Proc. natl. Acad. Sci., USA 69, 1758-1760. 5. Perry, J.G., McKinney, L. and De Weer, P. (1978) Nature, Lond. 272, 271-273. 6. Pole, P. and Haefely, W. (1976) Naunyn-Schmiedeberg's Arch. expl. Path. Pharmak. 294, 121-131. Raabe, W. and Gumnit, R.3.(1977) Epilepsia 18, 117-120. 87: Tallman, J.F., Thomas, J.W. and Gallager, D.W. (1978) Nature, Lond. 274, 383-385. 9. Briley, M.S. annanger, S.Z. (1978) Eur. 3. Pharmacol. 52, 129-132. 10. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. J. biol. Chem. 193, 265-275. 11. Lineweaver, H. aniBurk, D. (1934) J. Amer. Chem. Sot. 56, 12.
658-666.
Dixon,
M. (1953)
Biochem.
J..g,
1023
170-197.
Vol. 91, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
13. Maynert, E.W., Marczynski, T.J. and Browning, R.A. (1975) Adv. Neurol. 13, 79-147. 14. Wood, J.D. (19E) Progr. Neurobiol. 5, 77-95. 15. Curtis, D.R., Duggan, A.W., Felix, D. and Johnston, G.A.R. Nature, Lond. 226, 1222-1224. 16. Camerman, A. anmamerman, N. (1970) Science 168, 1457-1458. 17. Roberts, E. (1974) Biochem. Pharmacol. 23, 26r2649. 18. Anlezark, G., Horton, R.W., Meldrum, B.S. and Sawaya, M.C. Biochem. Pharmacol. 25, 413-417. 19. Tallman, J.F. and GalEger, D.W. (1979) Pharmacol. Biochem. Behav. 10, 809-813.
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