0145-h00X/9X/2204-08I0$03.00/0 Vol. 22, No.

4
CI INlCAL AND EXI’I‘RIMFNI;ZL RESEARCH
AI.C‘OHOI.ISM: June 1998

Mechanism of Action of Acamprosate. Part 11. Ethanol

Dependence Modifies Effects of Acamprosate on
NMDA Receptor Binding in Membranes from Rat
Cerebral Cortex
Mona al Qatari, Ouahiba Bouchenafa, and John Littleton

Acamprosate is a putative anticraving drug used to maintain absti- Ref. 1). At the molecular level, the mechanism of action is
nence in alcohol-dependent patients. Its mechanism of action is un-
obscure, but the drug is reported to reduce excitation in-
certain, but the drug is thought to interact with neuronal NMDA re-
ceptors and calcium channels, and these proteins are implicated in duced by excitatory amino acids in cortical neuron^.^ This is
the induction of alcohol dependence. In these experiments, the ef- consistent with inhibition of some putative mechanisms of
fects of acamprosate were studied on the binding of the NMDA re- negative r e i n f ~ r c e m e n t ~
and
’ ~ is thus a potential mecha-
ceptor ligand pH]dirocilpine to rat brain membranesunder nonequi- nism for the therapeutic effect. The characteristics of inhi-
librium conditions; 10 pM glutamate and 1 p M glycine were present
in the binding assays to partially activate the receptor. At clinically
bition in cortical neurons suggest an inhibitory effect of
relevant concentrations(in the micromolar range), acamprosatesig- acamprosate either on NMDA receptors, or on voltage-
nificantly enhanced [3H]dizocilpine binding to cortical membranes operated calcium channels, or both.4 Calcium flux studies
from control animals (suggesting that acamprosate may increase in similar cortical neuronal preparations show inhibition by
the rate of association of the radioligand),whereas at higher concen- acamprosate of calcium entry induced by glutamate or by
trations binding was inhibited. This effect is consistent with a partial
agonist effect of acamprosate on the NMDA receptor protein. How-
K+-depolarization6 and so do not resolve the issue. Radio-
ever, when rats were made dependent on ethanol (exposure to the ligand binding studies suggest that [3H]acamprosate inter-
drug for 10 days by inhalation) and cortical membranes were pre- acts with NMDA receptors, although not at the glutamate
pared from these animals, acamprosate in vitro no longer produced binding site,’ whereas studies with the calcium channel
any enhancement of rH1dizocilpinebinding. Similar results were ob-
ligand [3H]isradipine show that acamprosate potently dis-
tained when membranes were used from rats that had received 400
mg/kg/day of acamprosate in their drinking water with or without places this ligand from a low affinity site on cortical mem-
concurrent ethanol inhalation for 10 days. Thus, in brain membranes branes.8
from all these treatment groups, acamprosate in vitro caused inhibi- Thus, all of these studies suggest that acamprosate re-
tion of [3H]dizocilpine binding only. The results suggest that acam- duces neuronal excitation by inhibitory allosteric interac-
prosate may have excitatory or inhibitory effects on NMDA recep-
tions with NMDA receptors and/or calcium channel pro-
tors, depending on the experimental conditions. The effects of the
drug on this system appear to be shifted toward inhibition in alcohol teins. However, there are results that appear incompatible
dependence, and this finding may be important to its clinical mech- with this conclusion. Thus, electrophysiological investiga-
anism. tion of acamprosate in the rat hippocampus showed that
Key Words: Acamprosate, NMDA Receptor, Alcohol, Dependence, acamprosate enhanced NMDA receptor-mediated respon-
Binding.
ses’ at concentrations similar to those found to be inhibi-
tory in the earlier experiment^.^ There are several potential
CAMPROSATE IS the calcium salt of N-acetylhomo- explanations for this discrepancy, for example, acamprosate
A taurine and is now approved in Europe as a treatment may affect the receptor differently depending on the pres-
to reduce relapse in abstinent alcohol-dependent patients ence or absence of endogenous co-agonists; alternatively,
(e.g., see Ref. 1). It has been suggested that acamprosate receptors of differing subunit composition in different
acts as an anticraving drug, perhaps by suppression of brain areas may be affected differently by the drug. To help
negative reinforcement for drinking,2,3 and this is sup- resolve this situation, we chose to investigate effects of
ported by significantly lower self-reports of craving early acamprosate on the binding of the NMDA receptor ligand
during abstinence in the acamprosate treatment group (see [3H]dizocilpine to brain membranes and, for consistency
with our previous experiments, we used membranes from
From the Pharmacology Group, Kings College, London SW3 6Lx, United rat cerebral cortex. [3H]Dizocilpine binding can provide
Kingdom. information on the functional state of the NMDA receptor
Received for publication May 28, 1997; accepted January 7, 1998 because this ligand binds within the channel of the NMDA
Reprint requests: John Littleton, B.Sr., M.B., B.S., Ph. D., Tobacco and
Health Research Institute. University of Kentucky, Cooper and University
receptor, and so before equilibrium is reached its binding is
Drives, Lexington, KY 40545-0236. accelerated by receptor activation. Thus, effects on pre-
Copyright 0 1998 by The Research Society on Alcoholism. equilibrium binding of [3H]dizocilpinehave recently been
810 Alcohol Clin Exp Res, Vol22, No 4, 1998: pp 810-806
MECHANISM OF ACTION OF ACAMPROSATE. PART II 811

used to probe stimulatory and inhibitory polyamine sites on performed under nonequilibrium conditions, B,, and Kd values obtained
the NMDA receptor protein,” to search for novel poly- are “apparent” values only.
Data Analysis. Results were converted to dpm and specific binding
amine ligands,’ and to investigate differences between expressed as means (2SEM). For competition studies, values were con-
WSR and WSP mouse lines.’* The experiments described verted to percentages based on directly comparable binding data obtained
herein should thus enable us to assess whether direct ef- at the same time in the absence of added acamprosate. Statistical signif-
fects of acamprosate on the NMDA receptor protein are icance was determined in all cases by ANOVA and two-tailed Student’s
inhibitory or excitatory. ?test.
In addition to studying effects of acamprosate in brain
membranes from control animals, we chose to investigate Animal Treatments
membranes from rats that had been made physically de- To induce ethanol dependence, rats receiving food and water ad libi-
pendent on ethanol, and from rats that had received acam- tum were housed singly in cages in an inhalation chamber, and exposed to
increasing concentrations of ethanol vapor (10 to 12 muiter on day 1 to
prosate chronically in vivo. There are three main reasons 22 to 24 mditer on day 10). This exposure has repeatedly been shown to
for this: (1) we had observed differences in the effects of induce physical dependence, as evidenced by a withdrawal syndrome when
acamprosate in vitro on glutamate-induced calcium entry in ethanol administration is suspended (see Ref. 5). In these experiments,
brain slices from animals treated chronically with ethanol rats were sacrificed on day 10 of the ethanol exposure while still intoxi-
and/or acamprosate6; (2) it has been reported that, in vivo, cated (blood ethanol concentrations >50 mM). In some experiments,
animals received acamprosate in their drinking water while they were also
both drugs alter the expression of NMDA receptor NR1 receiving ethanol by inhalation. The concentration of acamprosate was
splice variants, a mechanism that might also affect the manipulated, based on daily water intake, to produce a daily dose as close
; we felt that as possible to 400 mglkg acamprosate. The presence of acamprosate in the
pharmacology of the NMDA r e ~ e p t o r ’ ~(3)
studying membranes from ethanol-dependent animals may drinking water did not itself appear to alter water intake, Another group
be of particular relevance to the mechanism for the clinical of animals received similar concentrations of acamprosate without etha-
nol, and a further group (untreated controls) received neither ethanol nor
action of the drug in alcohol-dependent patients. acamprosate. These two groups were not housed in the inhalation cham-
ber, but were in the same room, and so were exposed to many of the same
environmental factors as the other treatment groups.
MATERIALS AND METHODS
Preparation of Brain Membranes Materials
Male Sprague-Dawley rats (250 to 300 g) were sacrificed by cervical [3H](+)-5-Methyl-lO,ll-dihydro-5H-dibenzo~~]cyclohepten-5,10-
dislocation, and the brain was rapidly removed and placed on an ice-cold imine (dizocilpine/MK 801) was purchased from NENDupont (UK).
Petri dish. Cerebral cortices were dissected and homogenized at 0°C in a Acamprosate was provided by Groupe Lipha (Lyons, France). Ecoscint
Potter-Elvehjem homogenizer with a Teflon pestle in 0.32 M sucrose scintillation fluid (emuIsifier-safe LSC cocktail) was from NationaI Diag-
(10% w/v homogenate). The homogenate was then centrifuged at 4°C at nostics (UK). Ethanol was 99.7% pure Analar reagent grade from BDH
1,000 X g for 10 min. The pellet was discarded and the supernatant (UK). All other chenmicals used were of the highest purity commercially
recentrifuged at 4°C at 20,000 X g for 20 min. The pellet (P2 fraction) was available.
then resuspended in 15 ml of deionized water and centrifuged at 4°C for
20 min at 50,000 X g. This pellet was then washed three times in deionized
water and finally resuspended in 10 ml of 5 mM Tris HCl buffer (pH 7.4) RESULTS
and frozen rapidly. Membranes were stored at -80°C until the day of use
[3H]Dizocilpineshowed specific binding to cortical mem-
when they were thawed by washing three times in 15 ml of the Tris buffer.
branes from control rats with an affinity within previously
reported B m, values were below those previ-
[-’H]DizocilpineBinding ously reported,’l.l2 but this is partly explained by the non-
Competition Studies. An aliquot (150 to 300 pg) of membrane suspen- equilibrium binding conditions used. In control mem-
sion was incubated with 2 nM [3H]dizocilpine in 0.5 ml of 5 mM Tris HCl branes, an apparent Kd of 2.88 nM (22.2 nM) was observed
in the presence of glutamate (10 pM) and glycine (1 pM), and increasing with a B,, of 0.24 pmol/mg of protein (k0.012): all values
concentrations of acamprosate (500 nM to 10 mM) at room temperature
are from 4 to 7 separate experiments. Induction of ethanol
for 45 min. This time point was chosen on the basis of preliminary
experiments, which showed that [3H]dizocilpine binding reached equilib- dependence, with or without concomittant administration
rium at between 90 and 120 min. Based on the data in Ref. 10, binding at of acamprosate, was associated with an increase in appar-
this point in the time course should be capable of reflecting, enhancing, or ent binding affinity (Kd: 0.61 5 0.24 nM and 0.52 2 0.16
inhibiting effects of ligands for the polyamine site. Following the incuba- nM, respectively); but, in contrast, acamprosate treatment
tion of the samples, tubes were rapidly filtered using a 12-place Skatron
alone reduced apparent affinity for dizocilpine binding (Kd:
cell harvester. Ecoscint scintillation fluid (4 ml/sample) was then added to
each vial and radioactivity counted on a Beckman LS 6800 scintillation 7.0 2 4.2 nM). These results are undeniably variable, but
counter. Nonspecific binding was defined by the addition of 100 pM the range does not differ greatly from those previously
dizocilpine. Protein content of each sample was determined by the Lowry reported under similar conditions (e.g., see Ref. 10). The
method. variation is partly explained by the nonequilibrium binding
Saturation Studies. The membrane suspension was incubated for 45 min
conditions, which also makes the comparison between
with increasing concentrations of [3H]dizocilpine (2 to 48 nM) in the
presence of glutamate and glycine as described. Nonspecific binding was treatment groups of doubtful significance. Similarly, in all
defined by the addition of 500 p M dizocilpine. All other steps were as for the treatment groups, the apparent B,, for dizocilpine
competition studies. It should be noted that, because all these studies were binding was below that in control membranes; thus, for
812
zoo -
I 120
1
0
150-
Q m
.i
f loo--
.I
? ?
E
50-
0 1 . .
1;-7
...... ........ ........ ........ ........ ,....
1;s Id5 l b l b Id2
Concentration of Acamprosate
Fig. 1. Effect of acamprosate in vitro on binding of [3H]dizocilpine to mem-
branes from rat cerebral cortex; effect of induction of ethanol dependence.
Aliquots of membrane suspension were incubated with 2 nM rH1dizocilpinein the
presence of 10 p M glutamate and 1 FM glycine for 45 min. Effects of acampro-
sate were determined by including 500 nM to 10 mM acamprosate in the incu-
bation buffer. Open squares show the data obtained from cortical membranes
obtained from untreated control rats. Filled triangles show data from membranes
obtained from animals in which physical dependence on ethanol was induced
(see "Materials and Methods"). Values are shown as the means 2 SEM of
percentages calculated from mean values for specific binding obtained in the
absence of acarnprosate. Each value is obtained from 4 to 7 separate experi-
ments. The induction of ethanol dependence prevented the enhancement of
[3H]dizocilpine binding caused by acarnprosate in membranes from control ani-
mals.
acamprosate treatment, B,, was 0.136 0.031; for acam- *
prosate plus ethanol, B,, was 0.145 2 0.028 and for eth-
*
anol alone B,,, was 0.098 0.017 (all values are pmol/mg
of protein; mean ?SEM, n = 4 to 7). The nonequilibrium
nature of the binding conditions and the experimental de-
sign (which emphasized effects of acamprosate in vitro
above direct comparison between treatment groups) also
makes these differences of doubtful significance.
In the competition studies, acamprosate enhanced bind-
ing of ['H]dizocilpine to cortical membranes from control
rats at all concentrations between 500 nM and 100 pM (Fig.
1). The effect is moderate under the conditions of these
experiments, and the maximum effect is to increase binding
by -50% above the control level. (However, under the
binding conditions used in which the apparent B,, was
-60% of the B,, at equilibrium, this may be close to the
maximal enhancement achievable.) At concentrations
above 1 mM, acamprosate progressively inhibited ['Hldi-
zocilpine binding, with an IC,, of 2.1 5 0.21 mM. The
results obtained in membranes from ethanol-dependent
rats are also shown in Fig. 1. In these, no concentration of
acamprosate enhanced [3H]dizocilpine binding, and con-
centrations above 100 pM reduced the specific binding of
['H]dizocilpine. The IC,, for this effect (1.0 2 0.12 mM)
was lower than that observed in control membranes, but
this is probably an artefact caused by the initial enhance-
AL QATAR1 ET AL.
140 1
1
0
-3
a
e
.&
i
3
-20 '
1;-7
. .. ....a
1dB
. . ...... . ....... . . . .." . . . .....
d5 1 b 1 b lib
. . ...
3
Concentration of Acamprosate
Fig. 2. Effect of acamprosate in vitro on binding of rtlldizocilpine to mem-
branes from rat cerebral cortex; effect of acamprosate administration in vivo.
Binding conditions and data handling were as described in Fig. 1. Membranes
were obtained from rats that had received acampmsate at a dose of 400 mg/kg/
day via the drinking water. Open squares indicate the data from animals in which
this was the only treatment. Filled circles indicate data from rats that also received
ethanol by inhalation during the 10-day period of acamprosate administration.
The addition of acamprosate treatment did not change the effect of ethanol
dependence in abolishing the enhancing effect of acamprosate on rHJdizocilpine
binding, and acamprosate treatment in vivo also caused this effect to be lost.
ment of binding at lower concentrations of acamprosate in
controls so that 50% inhibition, as related to binding in the
absence of acamprosate, appears to occur at a higher con-
centration of the drug in these membranes (Fig. 1).
The results obtained in membranes from ethanol-depen-
dent rats were not affected by concomitant treatment with
acamprosate (Fig. 2). Once again, no enhancement of bind-
ing was seen, and only inhibition of [3H]dizocilpine binding
was seen with an IC,, in the 500 p M to 1 mM range.
Results from animals treated chronically with acamprosate
without any other treatment also showed no enhancement
of [3H]dizocilpine binding to brain membranes by acam-
prosate in vitro (Fig. 2), and the inhibition of binding at
high concentrations of acamprosate was similar to that seen
in all other groups.
DISCUSSION
These experiments were undertaken to establish whether
effects of acamprosate on [3H]dizocilpine binding are com-
patible with activation or inhibition of NMDA receptors on
neurons of rat cerebral cortex. All our previous data sug-
gested that acamprosate inhibits NMDA receptor function;
but, in the experiments reported herein, acamprosate, at
low concentrations, enhanced pre-equilibrium [3H]dizo-
cilpine binding to membranes from cortex of control rats.
This suggests that acamprosate may increase the rate of
association of the radioligand with its receptor site (as
polyamines do)," but this must now be confirmed by a
MECHANISM OF ACTION OF ACAMPROSATE. PART II
detailed investigation of the time course of binding under
different conditions of receptor activation and in the pres-
ence of different concentrations of acamprosate. The re-
sults are consistent with an excitatory effect of acamprosate
on NMDA receptors, and thus support the electrophysio-
logical results obtained in hippocampal neurons.' The re-
sult is similar to findings obtained independently by
Naassila et al. (see companion paper) in which the mech-
anism for enhancement of [3H]dizocilpinebinding is sug-
gested to involve modulation of a polyamine receptor site
on the NMDA receptor protein. Our experiments are com-
patible with this interpretation and suggest that, like the
polyamines," acamprosate may act as a co-agonist for the
NMDA receptor at micromolar concentrations, but inhibit
NMDA receptor function at higher concentrations.
If this interpretation is correct, then interactions between
acamprosate and endogenous polyamines may help explain
the conflicting electrophysiological finding^.^,^ Thus, the
enhancement of dizocilpine binding observed herein (and
in the companion paper) were observed under specific
conditions of submaximal activation of the receptor by
glutamate and glycine, in the absence of any endogenous
polyamines. However, if the receptor were maximally acti-
vated in the presence of polyamines, acamprosate might
reduce the effects of these and thus reduce NMDA recep-
tor activation. Alternatively, acamprosate may be capable
of activating or inhibiting NMDA receptors, depending on
their subunit composition, as polyamines are known to
do.14 These possibilities are important subjects for future
experiments, and should also focus attention on the poten-
tial roles of polyamine synthesis and release in ethanol
dependence (see Ref. 15 for review).
We also investigated [3H]dizocilpine binding in mem-
branes from ethanol-dependent, and chronically acampro-
sate-treated, rats and found no enhancement of [3H]dizo-
cilpine binding to cortical membranes from these treatment
groups at any concentration of acamprosate. The inhibition
of binding at high concentrations of acamprosate was, how-
ever, similar to that observed in control membranes. Be-
cause these studies are in isolated, washed membranes, it is
unlikely that the loss of the enhancing effect of acampro-
sate is due to the presence of competing endogenous li-
gands. Some treatment-induced change in the NMDA re-
ceptor itself seems likely, and alterations in subunit
composition are a potential explanation for the binding
differences. Thus, the ability of polyamines to enhance
NMDA receptor binding is NR2 subunit dependentI6 and
alterations in NR2 subunit expression are reported after
chronic ethanol exposure." Alternatively, ethanol and
acamprosate both change expression of the NRl splice
variants (see Refs. 3 and 13), and these subunits also alter
the spermidine sensitivity of the Whether any
of these alterations have implications for NMDA receptor
function remains to be investigated, but is an important
possibility.
The data suggests that chronic exposure to ethanol
813
and/or acamprosate produces changes that favor inhibitory
effects of acamprosate on NMDA receptor function. How-
ever, it must be emphasized once more that this is based on
receptor binding studies under highly specific conditions of
submaximal receptor activation, and cannot be interpreted
to mean that acamprosate produces only inhibitory effects
on NMDA receptor function in vivo. Indeed, the charac-
teristics described herein (and in the companion paper by
Naassila et al.) suggest that acamprosate is capable of
modulating the NMDA receptor as a partial agonist, tend-
ing to activate the receptor at low levels of physiological
stimulation and inhibit it at high, pathophysiological levels
of stimulation. However, much more detailed studies of the
functional interactions between acamprosate and endoge-
nous co-agonists of the NMDA receptor are necessary to
test this hypothesis. Whether such a mechanism could ex-
plain the action of acamprosate in relapse prevention is
also uncertain, but it is of interest that a very similar
mechanism has recently been proposed to explain the an-
ticraving effects of ibogaine."
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