Protection of methylmercury effects on the in vivo dopamine release by NMDA receptor antagonists and nitric oxide synthase inhibitors.

Abstract

The possible protective effects of NMDA receptor antagonists dizocilpine (MK-801) and D(-)-2-amino-5-phosphonopentanoic acid (AP5), and nitric oxide synthase (NOS) inhibitors L-nitro-arginine methyl ester (L-NAME) and 7-nitro-indazol (7-NI) on the methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis. Intrastriatal infusion of 400 microM or 4 mM MeHg increased the extracellular DA levels to 1941+/-199 and 7971+/-534% with respect to basal levels. Infusion of 400 microM or 4 mM MeHg in 400 microM MK-801 pretreated animals, increased striatal DA levels to 677+/-126 and 2926+/-254%, with respect to basal levels, these increases being 65 and 63% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 microM or 4 mM MeHg in 400 microM AP5 pretreated animals, increased striatal DA levels to 950+/-234 and 2251+/-254% with respect to basal levels, these increases being 51 and 72% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 microM MeHg in 100 microM L-NAME or 7-NI pretreated animals, increased the extracellular DA levels to 1159+/-90 and 981+/-292%, with respect to basal levels, these increases being 40 and 50% smaller than those induced by MeHg in non-pretreated animals. In summary, MeHg acts, at last in part, through an overstimulation of NMDA receptors with possible NO production to induce DA release, and administration of NMDA receptor antagonists and NOS inhibitors protects against MeHg-induced DA release from rat striatum.

Full text

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Neuropharmacology 42 (2002) 612–618
www.elsevier.com/locate/neuropharm
Protection of methylmercury effects on the in vivo dopamine
release by NMDA receptor antagonists and nitric oxide synthase
inhibitors
L.R.F. Faroa, J.L.M. do Nascimentoa, M. Alfonsob, R. Dura ´nb,∗
aDepto de Fisiologia, Centro de Cie ˆncias Biolo ´gicas, UFPA, Bele ´m, PA, Brazil
bDepartamento de Biologı ´a Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, 36200-Vigo, Spain
Received 11 April 2001; received in revised form 27 November 2001; accepted 10 January 2002
Abstract
The possible protective effects of NMDA receptor antagonists dizocilpine (MK-801) and d(?)-2-amino-5-phosphonopentanoic
acid (AP5), and nitric oxide synthase (NOS) inhibitors l-nitro-arginine methyl ester (l-NAME) and 7-nitro-indazol (7-NI) on the
methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis. Intrastriatal
infusion of 400 µM or 4 mM MeHg increased the extracellular DA levels to 1941±199 and 7971±534% with respect to basal
levels. Infusion of 400 µM or 4 mM MeHg in 400 µM MK-801 pretreated animals, increased striatal DA levels to 677±126 and
2926±254%, with respect to basal levels, these increases being 65 and 63% smaller than those induced by MeHg in non-pretreated
animals. Infusion of 400 µM or 4 mM MeHg in 400 µM AP5 pretreated animals, increased striatal DA levels to 950±234 and
2251±254% with respect to basal levels, these increases being 51 and 72% smaller than those induced by MeHg in non-pretreated
animals. Infusion of 400 µM MeHg in 100 µM l-NAME or 7-NI pretreated animals, increased the extracellular DA levels to
1159±90 and 981±292%, with respect to basal levels, these increases being 40 and 50% smaller than those induced by MeHg in
non-pretreated animals. In summary, MeHg acts, at last in part, through an overstimulation of NMDA receptors with possible NO
production to induce DA release, and administration of NMDA receptor antagonists and NOS inhibitors protects against MeHg-
induced DA release from rat striatum.  2002 Elsevier Science Ltd. All rights reserved.
Keywords: Methylmercury; Dopamine; NMDA antagonists; Nitric Oxide Synthase inhibitors; Microdialysis
1. Introduction
The overall global increase of mercury, in various
forms, in the environment due to its excessive use in
industrial, agricultural, and mining practices has gener-
ated a serious toxicological problem and for this reason
the screening of detoxicant agent is becoming extremely
necessary (Week and Leicester, 1997).
Methylmercury (MeHg) is a very dangerous toxicant
which affects mainly the nervous system, creating rapid
changes and disturbing both the structural and biochemi-
cal machinery in the cell (Chang, 1977). However,
despite the broad reactivity of MeHg, selective impair-
∗Corresponding author. Tel.: +34-986-812-392; fax: +34-986-
812-556.
E-mail address: rduran@uvigo.es (R. Dura ´n).
0028-3908/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved.
PII: S0028-3908(02)00009-6
ment of cell functions is possible at low levels of
exposure (Brookes, 1992).
One of the biochemical effects of MeHg is an increase
in neurotransmitter release. For example, it increases the
in vivo dopamine (DA) release from rat striatum (Faro
et al., 1997, 1998, 2000). In previous studies, we have
observed that MeHg increased striatal DA levels, poss-
ibly through the membrane DA transporter (Faro et
al., 2002).
MeHg also increases glutamate release and inhibits its
transport into cultured mouse spinal cord and rat cerebral
cortical astrocyes thus increasing the extracellular con-
centration which leads to cell damage (Albrecht et al.,
1993; Aschner et al., 1993, 2000; Brookes and Kristt,
1989).
It has been reported that certain substances such as
vitamins C and E and the monothiol glutathione prevent
the effects of MeHg through its elimination from the

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brain (Sood et al., 1993; Usuki et al., 2001). It has also
been shown that these substances promote repair of the
various components and enzymes in cells affected by
MeHg (Vijayalakshmi et al., 1992).
Recently, it has been found that the ionotropic gluta-
mate receptor N-methyl-d-aspartate (NMDA) antagonist
dizocilpine (MK-801) protects against cortical neuronal
damage induced by MeHg (Miyamoto et al., 1999).
These data suggest that glutamate receptors could be also
involved in the effects produced by MeHg.
The striatum is a brain area highly enriched in NMDA
receptors (Greenamyre et al., 1995) and has been shown
to have high nitric oxide synthase (NOS) activity (Bredt
et al., 1991). The activation of NMDA receptors by
endogenous glutamate increases Ca++conductance lead-
ing to a rise in the intracellular Ca++concentration. This
Ca++binds to calmodulin and the Ca++/calmodulin com-
plex activates NOS (Garthwaite et al., 1988).
Stimulation of NMDA receptors by MeHg has been
proposed as one possible neurotoxic mechanism under-
lying the effects of MeHg (Yamashita et al., 1997).
Moreover, an overstimulation of NMDA receptors
induced by MeHg could produce an activation of NOS
and excessive nitric oxide (NO) production which could
lead to neuronal death (Himi et al., 1996; Snyder and
Bredt, 1992).
Therefore, it was considered of interest to investigate
the effects of the NMDA antagonists MK-801 (non-com-
petitive antagonist) and d(?)-2-amino-5-phosphonopen-
tanoic acid (AP5) (competitive antagonist) on the MeHg-
induced in vivo DA release from rat striatum. In
addition, to provide evidence for the proposed mech-
anism of the protective action of NMDA antagonists, the
inhibitors of NOS, l-nitro-arginine methyl ester (l-
NAME) and 7-nitro-indazol (7-NI) were also examined.
2. Methods
2.1. Animals, drug treatments and experimental
groups
Female adult Sprague–Dawley rats (weighing between
240 and 260 g) were used in all the experiments. Ani-
mals were housed under controlled conditions of tem-
perature (22±2 °C) and light (light:dark 14:10 h), with
free access to food and water. The experiments were per-
formed according with the Guidelines of the European
Union Council (86/609/EU) for the use of laboratory ani-
mals.
The drugs were dissolved in the perfusion fluid and
applied locally in the striatum through the dialysis probe.
MeHg, l-NAME, and 7-NI were purchased from Sigma,
St Louis (USA), MK-801 and d-AP5 were purchased
from Tocris (USA). All other chemicals and reagents
were of analytical grade.
The experimental groups were as follows: (1) 400 µM
MeHg; (2) 4 mM MeHg; (3) 400 µM MK-801; (4) 400
µM MeHg in 400 µM MK-801 pretreated animals; (5)
4 mM MeHg in 400 µM MK-801 pretreated animals;
(6) 400 µM AP5; (7) 400 µM MeHg in 400 µM AP5
pretreated animals; (8) 4 mM MeHg in 400 µM AP5
pretreated animals; (9) 100 µM l-NAME; (10) 400 µM
MeHg in 100 µM l-NAME pretreated animals; (11) 100
µM 7-NI; and (12) 400 µM MeHg in 100 µM 7-NI pre-
treated animals.
2.2. Microdialysis procedure
For microdialysis sampling, animals were anesthet-
ized (i.p.) with chloral hydrate (400 mg/kg) and placed
in a stereotaxic apparatus (Narishige SR-6) for the
implantation of a guide-cannula. A microdialysis probe
(CMA/12, 3 mm membrane length, CMA/Microdialysis,
Sweden) was implanted through the guide-cannula into
the left striatum at the following coordinates from
Bregma: A/P +2.0 mm; L +3.0 mm; V +6.0 mm. After
experiments, rats were given an overdose of chloral
hydrate, and the brains were fixed with 10% formalin
via intracardiac perfusion. Coronal sections (30 µm)
were made, stained with cresyl violet, and examined to
determine the precise location of the dialysis probe.
The experiments were carried out 24 h after implan-
tation of the guide cannula. Continuous perfusion was
performed with a Ringer’s solution (147 mM NaCl, 4
mM KCl, 3.4 mM CaCl2; pH 7.4) using a CMA/102
infusion pump (CMA/Microdialysis, Sweden) at a flow
rate of 2 µl/min.
At the beginning of our experiments with microdialy-
sis, we have made controls with different compositions
of Ringer medium in order to select the one most appro-
priate for our conditions. We also peformed periodical
control experiments to confirm the basal values and that
our conditions of microdialysis were correct.
All experiments were made with awake, conscious,
and freely-moving animals. The experiments were car-
ried out over 4 h periods, sampling striatal dialysates
every 15 min (30 µl). After collection of four basal
samples (60 min), MeHg was infused during 60 min;
after this, the medium was then switched back to the
unmodified Ringer’s solution and sampling was con-
tinued for an additional period of 120 min. In groups
pretreated with antagonists or inhibitors, the drugs were
infused from the beginning of experiment and then
together with MeHg.
2.3. HPLC–EC analysis
The samples obtained from the microdialysis pro-
cedure (30 µl) were collected by means of a CMA/142
microsampler (CMA/Microdialysis, Sweden) and DA
levels were quantified by High-Performance Liquid

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Chromatography (HPLC) with electrochemical detec-
tion. The dialysates were injected (20 µl) into a Hewlett-
Packard Series 1050 Liquid Chromatograph, using a
Rheodyne 7125 injection valve. The isocratic separation
of DA was achieved using Spherisorb ODS-1 reversed-
phase columns (10 µm particle size) according to Dura ´n
et al. (1998). The eluent (pH 4.0) was prepared as fol-
lows: 70 mM KH2PO4, 1 mM octanesulfonic acid, 1 mM
EDTA, and 7% methanol. Elution was carried out at a
flow rate of 2 ml/min. The DA detection was achieved
using an ESA Coulochem 5100A electrochemical detec-
tor (USA) at a potential of +400 mV.
2.4. Expression of results and statistics
Values of extracellular DA were corrected using the
percentage of relative recovery (the ratio between the
concentration of a particular susbstance in the perfusate
compared to its concentration in the medium outside the
microdalysis probe) estimated by an in vitro method
(Khan and Shuaib, 2001). The DA recovery was similar
for the different probes used (approximately 15%). The
averages of concentrations of DA in the three samples
before drug administration were considered as basal lev-
els. These basal levels were taken as 100% in order to
compare the changes in DA release following drug
administration. The results are shown as mean±S.E.M.
of five–six experiments, expressed as a percentage
respect to basal levels.
The rate of diffusion of MeHg through the microdialy-
sis probe was also estimated in vitro. MeHg was dis-
solved in Ringer solution and pumped through a 1-ml
glass syringe connected to the inlet of the microdialysis
probe placed in a conical tube containing 1 ml of Ringer
solution. Probes were perfused at a flow rate of 2 µl/min,
as used during the experiments with freely moving rats.
Under these conditions approximately 17% of MeHg dif-
fused out the microdialysis probe in 1 h. Sample mercury
levels were measured by Cold Vapor Atomic Absortion
(USA E.P.A., 1979). Thus, with 400 µM MeHg in the
perfusate, only 0.136 nmoles/min of MeHg are available
for diffusion through the dialysis membrane.
Statistical evaluation of the results was performed by
means of ANOVA and Student–Newman–Keuls mul-
tiple range test, considering the following significant dif-
ferences:∗P?0.05,∗∗P?0.01, and∗∗∗P?0.001, with
respect to basal.
3. Results
3.1. Effect of 400 mM and 4 mM MeHg on the basal
DA release
Intrastriatal infusion of 400 µM and 4 mM MeHg
increased the extracellular DA levels to 1941±199 and
7971±534%, respectively, with respect to basal values
(0.22±0.04 ng/15 min, n=15).
The data for the effects of MeHg on striatal dopamine
levels are plotted in Figs. 1–4 in order to compare with
the data obtained under other experimental conditions
(treatments with antagonists or inhibitors together with
MeHg).
3.2. Effect of NMDA receptor antagonists on MeHg-
induced DA release
To investigate the possible protective action of
NMDA antagonists, the effect of MeHg on DA release
was studied in the presence of MK-801 or AP5. One
hour infusion of 400 µM MK-801 had no significant
effects on striatal DA levels (Fig. 1). Under 400 µM
MK-801 pretreatment, infusion of 400 µM MeHg
Fig. 1.
400 µM and (B) 4 mM MeHg-induced DA release. MeHg infusion
started at the time indicated by the arrows. In the pretreated group,
MK-801 infusion started at the beginning of the experiment. The
results are shown as mean±S.E.M. of five–six experiments, expressed
as a percentage of basal levels (100%). Basal levels were considered
as the mean of substance concentrations in the three samples collected
before drug administration. Significant differences:
P?0.01 and∗∗∗P?0.001 with respect to basal levels andaP?0.05,
with respect to MeHg administration.
Protective effects of 400 µM MK-801 pretreatment on (A)
∗P?0.05,
∗∗

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L.R.F. Faro et al. / Neuropharmacology 42 (2002) 612–618
Fig. 2.
µM and (B) 4 mM MeHg-induced DA release. MeHg infusion started
at the time indicated by the arrows. In the pretreated group, AP5
infusion started at the beginning of the experiment The results are
shown as mean±S.E.M. of five–six experiments, expressed as a per-
centage of basal levels (100%). Basal levels were considered as the
mean of substance concentrations in the three samples collected before
drug administration. Significant differences:∗P?0.05,∗∗P?0.01 and
∗∗∗P?0.001 with respect to basal levels andaP?0.05, with respect
to MeHg administration.
Protective effects of 400 µM AP5 pretreatment on (A) 400
increased striatal DA levels to 677±126%, with respect
to basal (Fig. 1A). The increase in extracellular DA lev-
els induced by MeHg in MK-801 pretreated animals was
65% smaller than the increase produced in animals not
pretreated with MK-801. In the same way, infusion of
4 mM MeHg in 400 µM MK-801 pretreated animals
increased striatal DA levels to 2926±254% of basal (Fig.
1B), being this increase 63% smaller than that observed
with 4 mM MeHg in non-pretreated animals.
Intrastriatal infusion of 400 µM AP5 had no signifi-
cant effects on striatal DA levels (Fig. 2). Infusion of
400 µM MeHg in 400 µM AP5 pretreated animals
increased striatal DA levels to 950±234% with respect
to basal (Fig. 2A), while infusion of 4 mM MeHg under
400 µM AP5 pretreatment increased the extracellular
DA levels to 2251±254% with respect to basal (Fig. 2B).
These increases produced by MeHg following AP5 pre-
treatment were 51 and 72% smaller than those produced
by 400 µM and 4 mM MeHg, respectively, in animals
not pretreated with AP5.
Fig. 3.
µM MeHg-induced DA release. MeHg infusion started at the time indi-
cated by the arrows. In the pretreated group, l-NAME infusion started
at the beginning of the experiment The results are shown as
mean±S.E.M. of five–six experiments, expressed as a percentage of
basal levels (100%). Basal levels were considered as the mean of subst-
ance concentrations in the three samples collected before drug adminis-
tration. Significant differences:
P?0.001, with respect to basal levels andaP?0.05, with respect to
400 µM MeHg.
Protective effects of 100 µM l-NAME pretreatment on 400
∗P?0.05,
∗∗P?0.01 and
∗∗∗
Fig. 4.
MeHg-induced DA release. MeHg infusion started at the time indicated
by the arrows. In the pretreated group, 7-NI infusion started at the
beginning of the experiment The results are shown as mean±S.E.M.
of five–six experiments, expressed as a percentage of basal levels
(100%). Basal levels were considered as the mean of substance concen-
trations in the three samples collected before drug administration. Sig-
nificant differences:
respect to basal levels andaP?0.05, with respect to 400 µM MeHg.
Protective effects of 100 µM 7-NI pretreatment on 400 µM
∗P?0.05,
∗∗P?0.01 and
∗∗∗P?0.001, with
3.3. Effect of NOS inhibitors on MeHg-induced DA
release
To investigate if NO production could be implicated
in the effects produced by MeHg on striatal DA levels,
as well as the possible protective action of NOS inhibi-

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tors on MeHg-induced DA release, we infused l-NAME
or 7-NI through the microdialysis probe. Intrastriatal
infusion of 100 µM l-NAME for 1 h decreased the
extracellular DA levels 10±1% with respect to basal
(Fig. 3). This value was considered as basal for the
measurement of MeHg effects on DA release in l-
NAME pretreated rats. Thus, with 100 µM l-NAME
pretreatment, infusion of 400 µM MeHg increased stria-
tal DA levels to 1159±90%, with respect to basal (Fig.
3), this increase being 40% smaller than that produced
by 400 µM MeHg in animals not pretreated with l-
NAME.
Infusion of 100 µM 7-NI decreased the extracellular
DA levels 21±1% with respect to basal levels (Fig. 4)
and, in the same way, this value was considered as basal
for the measurement of MeHg effects on DA release in
7-NI pretreated rats. Infusion of 400 µM MeHg in 100
µM 7-NI pretreated animals increased the extracellular
levels to 981±292% with respect to basal (Fig. 4). This
increase was 50% smaller than that produced by 400 µM
MeHg in non-pretreated animals.
Fig. 5 shows the comparative effects of the different
treatments on MeHg-induced DA release. AP-5 and MK-
801 decreased the effect of 400 µM MeHg on striatal
dopamine levels by 51 and 65% respectively. l-NAME
and 7-NI decreased the effect of 400 µM MeHg on stria-
tal dopamine levels by 40 and 50%, respectively. More-
over, the competitive and non-competitive glutamate
receptor antagonists also decreased the effect of 4 mM
MeHg on striatal dopamine levels by 72 and 63%,
respectively.
Fig. 5.
obtained with the different drugs on MeHg-induced DA release were between 40–72%.∗P?0.05, comparing the protective effects of l-NAME
and 7-NI, respectively, with the protective effect of MK-801 on 400 µM MeHg-induced DA release.
Comparative effects of MK-801, AP5, l-NAME, and 7-NI on 400 µM and 4 mM MeHg-induced DA release. The protective effects
4. Discussion
The 1 h administration of MeHg increased striatal
dopamine levels. Under our experimental conditions, the
dopamine levels first increased and then recovered to the
initial levels. This dopamine behaviour is consistent with
the majority of microdialysis experiments, in which
drugs are administered though a microdialysis probe.
The purpose of the present study was to investigate
the ability of certain substances to impair the MeHg-
induced DA release from rat striatum and their possible
mechanisms of action.
It has previously been shown that MK-801 has a pro-
tective effect in MeHg-induced neuronal injury in cer-
ebral cortex in vitro and in systemic administration in
vivo (Miyamoto et al., 1999). Recently, Miyamoto et al.,
(2001) reported that enhanced sensitivity of NMDA
receptors is involved in the vulnerability of developing
cortical neurons to MeHg. The authors concluded that
the excytotoxic effects of MeHg in the cerebral cortex
could be glutamate receptor-mediated.
The effect of MeHg on DA release in the presence of
the NMDA receptor antagonists MK-801 and AP5, was
studied. Our findings demonstrate that pretreatment with
400 µM MK-801 decreases the DA release induced by
4 mM and 400 µM doses of MeHg (63 and 65%,
respectively). These results indicate that DA release
induced by MeHg could be partially mediated by the
activation of NMDA glutamate receptors.
The results obtained with AP5, a competitive NMDA
receptor antagonist, confirm the data observed with MK-