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Dopamine D2-type agonists protect mesencephalic neurons from glutamate neurotoxicity Mechanisms of neuroprotective treatment against oxidative stress.

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Dopamine D2-Type Agonists Protect
MesenceDhalic Neurons from Glutamate
Nebotoxicitv: Mechanisms of
Neuroprotective Treatment Against
Oxidative Stress
Hideyuki Sawada, MD, PhD,* Masakazu Ibi, MS,? Takeshi Kihara, MD,* Makoto Urushitani, MD,*
Akinori Akaike, PhD,? Jun Kimura, MD,* and Shun Shimohama, MD, PhD*
Oxidative stress, a process in which neurotoxic oxygen free radicals cause dopaminergic neuronal degeneration, has been
implicated in the degenerative process in Parkinson's disease. Glutamate-induced neurotoxicity is a model of oxidative
stress. We demonstrated that preincubation with D2-type dopamine agonists bromocriptine and quinpirole provides
neuroprotection against glutamate-induced neurotoxicity in cultured rat mesencephalic neurons. Simultaneous administration of D2 agonists, however, did not provide neuroprotection. The protective effects were dependent on the duration
of preincubation and were blocked by a D2 antagonist and a protein synthesis inhibitor. Furthermore, preincubation
with D2 agonists provided neuroprotection against toxicity induced by calcium overload and exposure to superoxide
anions. Confocal microscopic analysis, using 2,7-dichlorofluorescin diacetate, revealed that bromocriptine preincubation
suppressed the action of radicals on neurons. These findings indicate that dopamine D2 agonists provide protection
mediated not only by the inhibition of dopamine turnover but also via D2-type dopamine receptor stimulation and the
subsequent synthesis of proteins that scavenge free radicals.
Sawada H, Ibi M, Kihara T, Urushitani M, Akaike A, Kimura J, Shimohama S. Dopamine D2-type agonists
protect mesencephalic neurons from glutamate neurotoxicity: mechanisms of neuroprotective
treatment against oxidative stress. Ann Neurol 1998;44:110-119
Mechanisms of the lengthy process of dopaminergic
neuronal degeneration in Parkinson's disease remain
unsolved. Several possible causes, including chronic intoxication with neurotoxinslf2 and genetic
have been proposed to play a part in the neurodegenerative process. Oxidative stress is proposed as the
major possible mechanism of dopaminergic neuronal
degeneration in Parkinson's disease.'-13 One of the
neurotransmitters, glutamate, is an excitatory amino
acid14 and its excessive release can cause intracellular
calcium influx,' 5,16 activation of calcium-dependent
enzymes such as nitric oxide ~ y n t h a s e , 'and
~ production of toxic oxygen radicals. Excessive release of glutamate, therefore, can be used as a model of experimental oxidative
Neurotransmitters, in addition to classic synaptic
roles, have been revealed to have protective effects on
and dopamine was reported to affect
the morphology of developing cortical neurons.24 Dopamine agonists, such as bromocriptine, pergolide, and
pramipexole, which are widely used in the treatment of
Parkinson's disease to compensate for depleted dopamine in the nigrostriatal neurons, have been proposed
to have possible neuroprotective effect^.^^-^' Although
previous clinical trials concerning long-term effects of
dopamine agonists revealed beneficial effect^,^^-^' the
neuroprotective mechanisms were not revealed.
In this study, we investigated the neuroprotective effects of dopamine agonists against neurotoxicity induced by oxidative stress in dopaminergic neurons, to
clarify the neuroprotective mechanisms. First, we determined whether dopamine agonists provide neuroprotection against glutamate-induced neurotoxicity. Second, to clarify the neuroprotective mechanisms, we
investigated the effects of dopamine agonists on neuro-
From the *Department of Neurology, Graduate School of Medicine, and ?Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Address correspondence to Dr Shimohama, Department of Neurology, Graduate School of Medicine, Kyoto University, 54 ShogoinKawaharacho, Sakyoku, Kyoto 606, Japan.
Received Nov 11, 1997, and in revised form Feb 2, 1998. Accepted
for publication Feb 5 , 1998.
Copyright 0 1998 by the American Neurological Association
toxicity induced by calcium overload or oxygen free
radicals. Finally, using confocal microscopic analysis,
we investigated the effects of dopamine agonists on
radical scavenging.
Materials and Methods
Eagle's minimum essential medium (EMEM) was purchased
from Nissui Pharmaceutical (Tokyo, Japan). Glutamate,
quinpirole, domperidone, and hydrogen peroxide were obtained from Nacalai Tesque (Kyoto, Japan). Bromocriptine
was provided by the Sandoz Pharmaceutical (Tokyo, Japan).
Hypoxanthine and xanthine oxidase (from buttermilk), and
superoxide dismutase (from bovine erythrocytes), were purchased from Sigma Chemical (St Louis, MO). Polyclonal
anti-tyrosine hydroxylase (anti-TH) antibody was obtained
from Eugene Tech (Ridgefield Park, NJ), and monoclonal
anti-microtubule-associated protein 2 (anti-MAP2) antibody
for immunocytochemical studies was obtained from Sigma.
Cell Culture
Cultures of the rat mesencephalon were performed according
to methods described previo~sly.~'
The ventral two-thirds of
the mesencephalon was dissected from rat embryos on day
16 of gestation. The dissected regions included dopaminergic
neurons in the substantia nigra and the ventral tegmental
area but not noradrenergic neurons in the locus ceruleus.
Neurons were dissociated mechanically and plated on 0.1 Yo
polyethyleneimine-coated plastic coverslips at a density of
1.1 X lo5 cells/cm2. The culture medium consisted of
EMEM and 10% fetal calf serum for the first 1 to 4 days in
culture, and horse serum from day 5 onward. The animals
were treated in accordance with guidelines published in the
N I H Guide for the Care and Use of Laboratory Animals.
Treatment of the Cultures
We used SKF 38393 as a D 1 agonist, and bromocriptine
and quinpirole as D 2 agonists, because dopamine itself is oxidized and neurotoxic in the culture medium. In a pilot
study, concentrations of bromocriptine greater than 100
p M , quinpirole greater than 100 p M , and SKF 38393
greater than 30 p M were toxic against cultured neurons
(data not shown). The drug concentrations used were 1 to
30 p M for bromocriptine, 10 to 30 K M for quinpirole, and
3 and 10 p M for SKF 38393.
To investigate glutamate-induced neurotoxicity, cultured
neurons were exposed to 1 mM glutamate for 10 minutes on
day 9 of culture and then incubated in EMEM containing
10% horse serum for an additional 72 hours and fixed on
day 12. First, to determine the effects of preincubation of
D1 or D2 agonists on glutamate-induced neurotoxicity, the
cultures were preincubated with 3 or 10 p M SKF 38393, 1
or 10 p M bromocriptine, or 10 p M quinpirole, on day 8 in
culture, for 24 hours before glutamate exposure. O n day 9 in
culture, cells were exposed for 10 minutes to EMEM containing 1 mM glutamate but without dopamine agonists.
Cells were incubated in medium without drugs for an additional 72 hours and fixed on day 12. T o study the effects
afforded by simultaneous administration of dopamine ago-
nists on day 9, cells were exposed to EMEM containing 1
m M glutamate and dopamine agonists. Then, to determine
whether the effects of dopamine agonists are mediated by the
dopamine receptor, we investigated the effects of a dopamine
antagonist (domperidone). Control experiments were similar
to treatment, using EMEM containing no drugs. Furthermore, we researched the effects of a protein synthesis inhibitor, cycloheximide, on dopamine-induced neuroprotection.
The neuroprotective effects of dopamine against neurotoxicity induced by intracellular calcium overload or oxygen radicals were studied by using a calcium ionophore
(A-23187) and superoxide anions yielded by a reaction between hypoxanthine and xanthine oxidase.
Incubation with less than 0.1% dimethyl sulfoxide for 24
hours was found to have no effect on neuronal survival rates,
and so bromocriptine and A-23187 were dissolved in dimethyl sulfoxide. Glutamate, quinpirole, SKF 38393, and
domperidone were dissolved in water.
Evaluation of Neurotoxicity
The surviving numbers of neurons were determined by using
immunostaining as described in our previous study.32 In
brief, after fixation, cultured cells were incubated with
anti-TH (diluted at 1:1,000) or anti-MAP2 (diluted at
1:400) antibodies for 24 hours, with the secondary biotinylated antibody for 1 hour, and with avidin-biotin complex
solution (Vectastain) for 1 hour. Finally, the cultures were
reacted with diaminobenzidine solution for 6 minutes. The
number of cells stained with anti-TH antibody in the 10
randomly selected fields (X 200) was counted as the surviving
numbers of dopaminergic neurons, and those stained with
anti-MAP2 antibody in the 15 randomly selected fields
(X400) as those of total neurons, by investigators who were
blind to the experimental treatments. Neurotoxicity was evaluated by the reduction in neuronal survival rate in each experiment. Statistical analysis was performed by one-way analysis of variance and post hoc multiple comparison by the
Newman-Keuls method. Statistical significance was defined
a s p < 0.05.
Evaluation of Reactive Oxygen Species in
Cultured Neurons
Peroxide levels were measured by using the dye 2,7dichlorofluorescin diacetate (DCF).33 Using a confocal microscope (ACAS 570; Meridian, MI), fluorescence in the cultured cells was evaluated. Cultured cells were incubated in
EMEM containing 10 p M DCF for 20 minutes and washed
with EMEM without any drugs three times. Formation of
radicals in the cells was evaluated by DCF fluorescence intensity, which was measured before and after exposure to 10
p M hydrogen peroxide (H20,) for 10 minutes. In each experiment, 18 or 19 regions of interest were located on the
cell bodies of randomly selected cells. We compared H20,induced DCF fluorescent changes with and without bromocriptine preincubation.
Neuroprotection Against Glutamate Neurotoxicity
Immunostaining of neurons revealed that the neuronal
density i n control cultures was 1.4 t o 2.6 X lo4 cells/
Sawada et al: Neuroprotection by Dopamine Agonist
cm2 for MAP2-positive neurons and 500 to 700 cells/
cm2 for TH-positive dopaminergic neurons. Exposure
to 1 mM glutamate for 10 minutes significantly reduced the surviving numbers of both the dopaminergic
and the nondopaminergic neurons ( p < 0.001). After
preincubation with quinpirole (10 pM) or bromocriptine (1 and 10 pM), the numbers of surviving dopaminergic and nondopaminergic neurons after glutamate exposure were significantly greater than in
cultures incubated with glutamate only ( p < 0.001).
The protective effects of bromocriptine against glutamate neurotoxicity were significantly blocked by coadministration with a D2 antagonist, domperidone. In
contrast to preincubation, simultaneous coadministra-
tion of either bromocriptine or quinpirole did not improve the survival rate of either the dopaminergic or
the nondopaminergic neurons after exposure to glutamate (Fig 1). Bromocriptine, quinpirole, or domperidone alone did not have a significant effect on the
survival rate of dopaminergic or nondopaminergic neurons (data not shown). Preincubation with 3 to 10 pM
SKF 38392, a D1 agonist, for 24 hours did not significantly affect the survival rate of dopaminergic or nondopaminergic neurons after exposure to glutamate
(data not shown).
Immunocytochemical studies showed that exposure
to glutamate reduced the number and the size of the
surviving neurons. Glutamate-induced neurotoxicity
Fig 1. Surviving numbers of cultured mesencephalic dopaminergic (A) and nondopaminergic (B) neurons ajier glutamate (Glu)
exposure with or without preincubation with 0 2 agonists. Exposure to 1 mM glutamate significantly reduced the surviving numbers
of both dopaminergic and nondopaminergic neurons. Coadministration of either quinpirole (Qp) or bromocriptine (Br) did not
affect survival. However, preincubation with bromocriptine and quinpirole significantly increased the number of surviving neurons
( 7 . The protective effects provided by preincubation with bromocriptine were dose-dependent and significantly blocked by coadministration with domperidone (Dom) (#I. n = 4 coverslips/experirnent. Control = Cells exposed to Eagle? medium without any drugs;
Glu = cells exposed to 1 mM glutamate for 10 minutes; Glu +- Qp 10 p M and Glu +- Br 10 p M = cells exposed to 1 mM
glutamate and simultaneously administered 10 p M quinpirole or 10 F M bromocriptine; Qp 10 p M 24 hr/Glu, Br 1 pM 24
hr/Glu, and Br 10 p M 24 hr/Glu = cells preincubated with 10 pM quinpirole, or 1 or 10 p M bromocriptine, for 24 hours and
then exposed to 1 mM glutamate; Br 10 pM +- Dom 24 hr/Glu = cells preincubated with 10 p M bromocriptine and 5 p M
domperidone for 24 hours and then exposed to 1 mM glutamate. 9 < 0.001, compared with cells exposed to glutamate alone, by
one-way analysis of variance (ANOVA) and Newman-Keuls post hoc multiple comparison test. #p < 0.001, compared with cells
exposed to glutamate ajier bromocriptine preincubation by ANOVA. N S = not signz3cant. Symbols indicate mean +- SEM values.
Glu + Pp 1011M
Qp 10 II
M 24hr /Glu
Br 1IIM 24hr /Glu
Br 1011M 24hr /Glu
Br 1011M + Dcin 24hr /Glu
Qp 10 II M
24hr /Glu
Br 1 p M 24hr /Glu
E?f 1011M 24hr /Glu
Br 10 II M
112 Annals of Neurology
Dorn 24hr /Glu
Vol 44 No 1 July 1998
was ameliorated by 24-hour pretreatment with quinpirole or bromocriptine in that more dopaminergic neurons survived, and the neurites were longer (Fig 2).
The protective effect of bromocriptine against
glutamate-induced neurotoxicity was dependent on the
duration of preincubation. Preincubation with 10 p M
bromocriptine for 4 hours or more showed significant
protection against glutamate-induced dopaminergic
neuronal death, and 8 hours or more was required for
protection of nondopaminergic neurons (Fig 3). The
neuroprotective effects induced by bromocriptine were
blocked dose-dependently by coadministration with cycloheximide (0.1-1.0 pM) in both dopaminergic and
nondopaminergic neurons (Fig 4). Cycloheximide (1.O
pM) alone did not exert significant effects on neuronal
mocriptine for 24 hours before exposure to A-23 187
significantly blocked dopaminergic and nondopaminergic neuronal death induced by A-23187 (Fig 5).
Neuroprotection Against Neurotoxicity Induced by
Superoxide Anions
Exposure to a combination of hypoxanthine and xanthine oxidase significantly reduced the numbers of dopaminergic and nondopaminergic neurons; this neurotoxicity was ameliorated by preincubation with 10 p M
bromocriptine or 30 pM quinpirole. The toxic effects
induced by hypoxanthine and xanthine oxidase were
blocked by superoxide dismutase and catalase (Fig 6).
Neuroprotection Against Neurotoxicity Induced by
a Calcium Ionophore
Exposure to the calcium ionophore A-23187 (0.03-0.3
pM) for 10 minutes caused a significant reduction in
Ability to Scavenge Radicals Induced by
Hydrogen Peroxide
Confocal microscopic study using DCF revealed that
exposure to 10 p M hydrogen peroxide increased fluorescence intensity. Preincubation with 30 p M bromocriptine for 24 hours prevented the increase in flu-
dopaminergic and nondopaminergic neuronal survival
in a dose-dependent manner. Preincubation with bro-
orescence intensity by exposure to hydrogen peroxide
(Figs 7 and 8).
Fig 2. Immunostaining with anti-tyrosine hydroylase (TH) antibody. (A) Control. (B) Exposure to I mM glutamate for I 0 minutes reduced the length of neurites and the survival of dopaminergic neurons. (C and 0) Exposure to glutamate afer 24 hours of
preincubation with 10 p M bromocriptine (C) or 10 p M quinpirole (0).Ajier preincubation with either bromocriptine or quinpirole, the survival rate of the dopaminergic neurons was improved Bar = I00 pm.
Sawada et al: Neuroprotection by Dopamine Agonist
Br 4hr /Glu
Br 8hr /Glu
Br 16hr /Glu
Br 8hr /Glu
Br 16hr /Glu
Br /Glu
Br + Cyclo 0.1 P M /Clu
Br + Cyclo 0.3 P M /Glu
Cyclo 1 . 0 ~M
Fig 4. Bloc,king effects of cycloheximide on the neuroprotective effects
o f bromocriptine in dopaminergic
(A) and nondopaminergic (B)
neurons. Preincubation of 10 p M
bromocriptine (Br) coadministered
with 0.1 to 1.0 p M cycloheximide (Cyclo) blocked the neuroprotection aguinst glutamate
(Glu)-induced neurotoxiciiy s i p i f icantly and dose-dependently. Incubation of cells with up t o 1.0
p M cycloheximide alone f i r 24
hours did not have any significant
< 0.01, compared with
preincubation with bromocriptine
alone $lhwed by exposure to glutamate, by one-way analysis of
variance and post hoc multiple
comparison. n = 4 coverslips per
experiment. Symbols indicate
mean 2 X.44 values.
Br /Glu
Br + Cyclo 0.1 11 M /Glu
Br + Cyclo 0.3 P M /Glu
Br + Cyclo 1.011 M /Glu
Cyclo 1.011 M
Fig 3. Time dependency of the neuroprotective effects of bromocriptine
pretreatment in dopaminergic (A)
and nondopaminergic (0) neurons.
Preincubation with broinocr$tine
(Br)for 4, 8, and 1G hours before
exposure t o I mM gluturnate (Glu)
demonstrated a time dependency f i r
its protective effects on dopuminergic
and nondopaminergic neurons.
9 < 0.05, compared with treatment b y glutamate alone, by oneway analysis of variance and post
hoc multiple comparison. n = 4
coverslips per experiment. Symbols
indicate mean f SEM values.
Annals of Neurology
Vol 44
No 1
July 1998
Ca-I 0.03 @ M
Br /Ca-
Ca-1 0.03 p M
Ca-I 0.1 p M
Ca-I 0.3 j i M
Br /Ca-I 0.3 p M
0 2 Recepto r-Media teed Neurop ro tection
Consistent with previous s t ~ d i e s , " ~ ' brief
exposure to
glutamate significantly reduced the survival of dopaminergic and nondopaminergic neurons. The present
study demonstrates that preincubation with bromocriptine provides neuroprotection against glutamateinduced dopaminergic and nondopanii nergic neuronal
In addition to its actions as a doparnine agonisr,
there are sevcral possible mechanisms by which bromocriptine provides neuroprotection. First, bromocriptine can scavenge free radicals in ~ i t r o - ~and
~ , pro'~
vides neuroprotection against toxic Free radicals, such
as methamphetamine-induced hydroxyl radicals, in
v ~ v o Because
. ~ ~ glutamate-induced neurotoxicity can be
blocked by coadministration with a radical ~cavenger,'~
brornocriptine may scavenge toxic free radicals and prevent neuronal death induced by glutamate. However,
the simultaneous administration of bromocriptine did
not provide neuroprotection in this study, indicating
that bromocriptine did not act directly as a free radical
scavenger. Second, bromocriptine may reduce the extracellular glutamate concentration by activation of the
glutamate transporter.'* T o determine whether bromocriptine protects neurons by activation of the glutamate transporter, the glutamate conccntration in the
culture medium was measured and it was not changed
Fig 5. Neuroprotective effects of bromorriptine against neurotoxicity induced by calcium ionophore in dopaminergic (A) and nondoparninergic (B)
neurons. Exposure to A-23/87 (0.030.3 pM) fir 10 minutes reduced neuronal survival in dopaminergic (A) and
nondopaminergic (B) neuron., sign$cant4 and dose-dependently. AJ&r preincubation with bromocriptine, the
number of surviving dopaminergic and
nondopaminergic neurons was significant4 greater than those treated on!y
with A-23187. Ca-I 0.0.3-0.3 F M =
Cellr exposed to A-23187 (0.03-0.3
pM) fir 10 minutes; BrKa-I 0.3
p M = cells preincubated with I 0 p M
brornocriptine and then exposed to 0.3
p M A-2318Z p < 0.05, compared
with control, by one-way undysis of
variance (ANOVA) and NewmanKeuls post hoc multiple comparison test.
#p < 0.01, compared with treatment
w i d 0.3 p M A-23187 akone, by
ANOVA. n = 4 couersIips per experiment, Symbols indicate mean t SEM
by bromocriptine preincubation (data not shown).
Therefore, it appears unlikely that the neuroprotective
mechanism of bromocriptine is related to glutamate
transporter activation.
In addition to bromocriprine, preincubation with
another D 2 agonist, quinpirole, also showed significant
protective effects. However, preincubation with SKF
38393, a DI agonist, had no significant effect on
glutamate-induced neurotoxicity, indicating that D1
agonists do not provide neuroprotection. 'l'he neuroprotection was suppressed by coadministration with a
D 2 receptor antagonist. These findings indicate that
the protection is mediated by the dopamine D2-type
Neuroprotection Mechanisms of 0 2
Receptor StimuLation
D2-type dopamine receptors are localized mainly on
the cell body and the presynapric neurites of the mesencephalic dopaminergic neurons as dopamine autorec e p t ~ r s . ~ ~A
, * "small population of nondopaminergic
neurons also have D2-type receptors, but they are not
present on most nondopaminergic
Preincubation with D2 agonists provided neuroprotection not
only for dopaminergic neurons but also for nondopaminergic neurons, most of which have no D 2 receptors. Therefore, the neuroprotection mediated by D2
Sawada et al: Ncuroprotection by Dopamine Agonist
cells /cm2
200 250 300
HX 30pM
Br /HX 30pM
QP /HX 30 p M
HX 30 p M + SOD + Catalase
cells /cm2
15,000 20,000
/HX 3 0 p M
HX 30 p M + SOD + Catalase
Fig 6 Neuroprotective effects provided by bromocriptine or quinpirole against neurotoxicity induced by superoxide in dopaminergic
(A) and nondoparninergic (B) neurons. Hypoxanthine and xanthine oxidase (HX) signijcantly reduced the survival of dopaminergic
and nondopaminergic neurons. Preincubation with brornocriptine (Br) or quinpirole (Qp) signijcantly blocked the neurotoxicity
induced by the combination of bypoxanthine and xanthine oxidase. The latter combination was also inhibited by coadministration
with superoxide dismutase (SOD) and catalase. n = 4 coverslips per experiment "p < 0.001, compared with control by analysis of
variance (ANOVA). #p < 0.001, compared with treatment with 30 p M bypoxanthine and 0.024 U/ml xanthine oxidase by
ANOVA and Newman-Keuls post hoc multiple comparison test. HX 30 p M = Cells exposed to 30 p M bypoxanthine and 0.024
U/ml xanthine oxidase for I0 minutes; BdHX 30 p M and QPIHX 30 p M = after preincubation with 10 p M bromocriptine or
30 p M quinpirole, cells were exposed to 30 pM bypoxanthine and 0.024 U/ml xanthine oxidase; HX 30 p M -t SOD f Catalase = cells exposed to 30 p M bypoxanthine and 0.024 U/ml xanthine oxidase with coadministration of 10 U/ml superoxide dismutase and 100 U/ml catalase. Symbols indicate mean 2 SEM values.
agonists is indirect, via D 2 intracellular signaling
Recently, five different dopamine receptors, D 1, D2,
D3, D4, and D5, have been i d e n t i f ~ e d . ~Among
them, D 1 and D5 are included in a group of D1-type
receptors that couple to the Gs protein and activate
adenylate cyclase; and D2, D3, and D4 are categorized
in a group of D2-type receptors that couple to the Gi
protein, suppress adenylate cyclase activity, and inhibit
calcium channel a~tivity.~"" Inactivation of the calcium channel may play a role in neuroprotection
against glutamate-induced neurotoxicity because it is
calcium-dependent. However, the neuroprotection provided by D 2 agonist cannot be explained by only calcium channel inactivation, because preincubation with
D 2 agonists provided neuroprotection against neurotoxicity induced by calcium ionophore and superoxide
Our experiments demonstrated that the protective
116 Annals of Neurology
Vol 44 No 1 July 1998
effects of D2 agonists were also dependent on the duration of preincubation and were blocked by cycloheximide, a protein synthesis inhibitor. These results suggest that D 2 agonists stimulate dopaminergic neurons
to synthesize neuroprotective proteins that can be released to act on dopaminergic and nondopaminergic
neurons. Furthermore, preincubation with D 2 agonists
provided neuroprotection against not only glutamateinduced neurotoxicity but also against toxicity caused
by calcium overload or toxic oxygen radicals. In fact,
DCF study revealed that preincubation with bromocriptine suppressed intracellular peroxide levels of
the cultured neurons. These results suggest that D 2
agonists provide neuroprotection against glutamateinduced neurotoxicity by synthesis of proteins that
scavenge free radicals that are produced by glutamate
It is unclear which neuroprotective proteins were
synthesized by D 2 receptor stimulation. However,
Fig 7.Photograph of digital subtraction images observed by confocal microscopy after exposure to hydrogen peroxide (H202). The
2,7-dichlorojluorescein diacetate (DCF) values at baseline were subtractedjom those after exposure t o 10 p M H20, in each pixel.
Without preincubation, DCF jluorescence increased after H202 exposure (left, control). In contrast, the DCF increment was suppressed by preincubation with bromocriptine (right, bromocriptine).
Fig 8. Time course o f 2,7-dichlorojluorescin diacetate (DCF)
jluorescence intensity after exposure to hydrogen peroxide.
Compared with baseline, the jluorescence intensity in the cells
increased more than twofold 3 minutes after exposure to 10
p M H202 In contrast, there was no signijkant increase in
jluorescence in cells preincubated with bromocriptine. Symbols
indicate mean 2 SEM values of DCFjluorescence intensity in
regions of interest located on randomly selected cells. n = 18
(treated with Hz02 alone) and n = 19 (preincubated with
Time after exposure to H202
there may be several possible mechanisms in the neuroprotection provided by D2 agonists, including induction of both neurotrophic factors such as glial
cell line-derived neurotrophic factor (GDNF) and radical scavengers such as superoxide dismutase (SOD).
GDNF, one of a superfamily of the transforming
growth factor+, is a neurotrophic factor whose receptor is localized mainly on mesencephalic dopaminergic
neurons and which can protect neurons from radicalinduced n e u r o t o x i ~ i t y However,
. ~ ~ ~ ~ ~ GDNF may not
be related to the neuroprotection against glutamateinduced neurotoxicity because preincubation or simultaneous coadministration with GDNF did not show
neuroprotection against glutamate-induced neurotoxicity in mesencephalic culture in our study (data not
shown). Brain-derived neurotrophic factor can also be
a candidate for the neuroprotective agent induced by
D2 receptor stimulation. A previous study using ischemic neurons of the gerbil reported that bromocriptine
can preserve the activity of SOD in ischemic hippocampal neurons.54 Preservation of SOD could be
one of the mechanisms of neuroprotection provided by
D2 agonists. It has not been determined whether the
preservation of SOD by bromocriptine is mediated by
the D2 receptor; but these effects may be related to the
neuroprotection provided by D2 agonists observed in
the present investigation. It appears reasonable that
stimulation of the dopamine autoreceptor may provide
neuroprotection by scavenging radicals that are produced in the process of dopamine metabolism.
We conclude that preincubation with a dopamine
Sawada et al: Neuroprotection by Dopamine Agonisr
D2 agonist provides neuroprotection against oxidative
stress by protecting mesencephalic neurons from glutamate neurotoxicity. The neuroprotection may involve
synthesis of radical-scavenging proteins in dopaminergic neurons. Several types of neuroprotective proteins
are candidates for further study as contributing to this
neuroprotective phenomenon. Neuroprotection against
oxidative stress is an important factor in the clinical
treatment of Parkinson’s disease and the present findings provide a basis for neuroprotective therapy for this
This study was supported in part by Grants-in-Aid for Scientific
Research on Priority Areas from the Ministry of Education, Science
and Culture, and by grants from the Ministry of Welfare of Japan.
We thank Novartis Pharma (Tokyo, Japan) for providing us with
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Sawada et al: Neuroprotection by Dopamine Agonist
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treatment, neurotoxicity, dopamine, mechanism, typed, glutamate, neuroprotection, protect, mesencephalic, oxidative, agonists, neurons, stress
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