N-Methyl-D-aspartate Induces
Neurogranin/RC3 Oxidation in Rat Brain Slices*
Junfa
Li,
Jhang Ho
Pak,
Freesia L.
Huang, and
Kuo-Ping
Huang
From the Section on Metabolic Regulation, Endocrinology and
Reproduction Research Branch, NICHD, National Institutes of Health,
Bethesda, Maryland 20892-4510
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ABSTRACT |
Neurogranin/RC3 (Ng), a postsynaptic neuronal
protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level
of Ca2+. In vitro, rat brain Ng can be
oxidized by nitric oxide (NO) donors and by oxidants to form an
intramolecular disulfide bond with resulting downward mobility shift on
nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as
compared with the reduced form, is a poorer substrate of PKC but like
the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced
form. To investigate the physiological relevance of Ng oxidation, we
tested the effects of neurotransmitter,
N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on
the oxidation of this protein in rat brain slices. Western blot
analysis showed that the NMDA-induced oxidation of Ng was rapid and
transient, it reached maximum within 3-5 min and declined to base line
in 30 min. The response was dose-dependent
(EC50 ~100 µM) and could be blocked by
NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO
synthase inhibitor
NG-nitro-L-arginine methyl ester
and NG-monomethyl-L-arginine. Ng
was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and
S-nitrosoglutathione, and H2O2 at
concentrations less than 0.5 mM. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by
dithiothreitol, ascorbic acid, or reduced glutathione. Reversible
oxidation and reduction of Ng were also observed in rat brain extracts,
in which oxidation was enhanced by Ca2+ and the oxidized Ng
could be reduced by NADPH or reduced glutathione. These results suggest
that redox of Ng is involved in the NMDA-mediated signaling pathway and
that there are enzymes catalyzing the oxidation and reduction of Ng in
the brain. We speculate that the redox state of Ng, similar to the
state of phosphorylation of this protein, may regulate the level of
CaM, which in turn modulates the activities of
CaM-dependent enzymes in the neurons.
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INTRODUCTION |
Rat brain neurogranin (Ng),1 also known as RC3 and
BICKS, is a 78-amino acid
neuron-specific, postnatal-onset, postsynaptic protein kinase C (PKC)
substrate (for review, see Ref. 1). Ng resembles a presynaptic PKC
substrate neuromodulin (Nm, also known as GAP-43, F1, or B-50) in its
high affinity binding of calmodulin (CaM) at low levels of
Ca2+ (for review, see Refs. 1-3). Both Ng and Nm are
phosphorylated by PKC at a single site located within a conserved
19-amino acid region, which also contains the predicted CaM-binding
domain (4-8). Phosphorylation of these two proteins by PKC weakens
their binding affinities for CaM. In addition to the PKC-mediated
regulation, both rat brain Ng and Nm are potential targets of nitric
oxide (NO) and other oxidants (9, 10). Treatment of cultured neurons with NO donors interferes with thioester-linked long chain fatty acylation of Nm and causes inhibition of neurite outgrowth (9). In vitro, oxidation of purified rat brain Ng with NO donors
or oxidants results in intramolecular disulfide formation and causes an
attenuation of its binding affinity for CaM (10). These previous findings point to an interesting possibility that both rat brain Ng and
Nm are common targets of both PKC- and NO-mediated signaling pathways.
For rat brain Ng, the PKC-mediated phosphorylation of this protein in
brain slices and cultured hippocampal neurons in response to phorbol
ester (4, 11), electrical stimulation (11, 12), and excitatory amino
acids (13) has been demonstrated; however, the oxidation of this
protein in intact cell has not been shown. Although the physiological
function of Ng is not clear, cumulating evidences suggest that Ng may
be involved in the regulation of CaM targets (14), transduction of the
Ca2+ signals (15), long term potentiation (LTP) (11,
16-18), and neuronal differentiation and synaptogenesis during
telencephalic development (19).
The endogenously formed oxidants, such as reactive nitrogen (nitric
oxide, NO) and oxygen (superoxide, O
2, and hydrogen peroxide, H2O2), and redox stress are thought to play
important roles in both physiological and pathological responses
(20-25). Some of these responses are due to a direct effect of these
oxidants and others arise from the reaction of NO with superoxide
(O
2) (26) or O2 (27) to form peroxynitrite
(ONOO
) or N2O3, respectively.
Protein thiols are critical sites of interaction with free radicals and
their intermediates. Oxidation of thiol on a protein by
H2O2 forms sulfenic acid (RS-OH), which in turn
interacts with a free thiol group to form disulfide products (28), and
with NO to form S-nitrosothiol and subsequent disulfide formation (27, 29). S-Nitrosation and subsequent disulfide formation of proteins are important physiological processes involved in
the regulation of blood pressure, host defense, and neurotransmission (30).
In this study we are attempting to answer the question if rat brain Ng
in intact cells can be oxidized by the commonly recognized oxidants
such as NO donors, hydrogen peroxide, and oxidized glutathione, and
more importantly, if any physiological agent that is known to stimulate
the production of oxidant will enhance the oxidation of this protein.
Since oxidation of rat brain Ng results in a preferential formation of
intramolecular disulfide bond and leads to an increase in the
electrophoretic mobility upon nonreducing SDS-PAGE (10), this unique
feature serves as a convenient assay to monitor the oxidation of this
protein by immunoblot analysis. Here, we demonstrate that rat brain Ng
in the brain slices can be oxidized by several exogenously added
oxidants. Furthermore, addition of neurotransmitter,
N-methyl-D-aspartate (NMDA), to the brain slices
caused a transient oxidation of this protein, and these effects of NMDA
can be blocked by its antagonist, 2-amino-5-phosphonovaleric acid
(AP5), as well as by NO synthase inhibitor,
NG-nitro-L-arginine methyl ester
(L-NAME) and
NG-monomethyl-L-arginine (L-NMMA).
Oxidation/reduction of Ng can also be shown in rat brain extracts, in
which the oxidation of this protein is stimulated by Ca2+
and the oxidized form can be reduced by NADPH.
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EXPERIMENTAL PROCEDURES |
Materials--
The following materials were obtained from the
indicated sources: S-nitrosoglutathione (SNOG),
(±)-S-nitroso-N-acetylpenicillamine (SNAP), NMDA, NADPH,
L-NAME,
NG-nitro-D-arginine methyl ester
(D-NAME), and L-NMMA from Calbiochem; H2O2 from Fisher; oxidized glutathione (GSSG),
sodium nitroprusside (SNP), glutathione (GSH), AP5, and ascorbic acid
from Sigma; dithiothreitol (DTT) from ICN; and horseradish
peroxidase-conjugated goat anti-rabbit IgG from Bio-Rad.
Preparation of Rat Brain Slices and Treatment with NO Donors and
Oxidants--
Brain from adult Sprague-Dawley rat (200-250 g) was
removed immediately after decapitation and placed in ice-cold pH 7.4 artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 26 mM NaHCO3, 1.25 mM
NaH2PO4, 1 mM MgCl2, 25 mM glucose, bubbled with 95% O2, 5%
CO2) for 3-5 min. Cortical slices (400 µm thickness) were prepared using a Vibratome 1000 sectioning device with the brain
submerged in ice-cold ACSF bubbled with 95% O2, 5%
CO2. The slices were maintained in ACSF in a storage bath
for 1-3 h at 30 °C to allow recovery from possible damage during
slicing. Each slice was transferred to a 20 ml scintillation vial
containing 1 ml of ACSF and various concentrations of NO donors, and
other oxidants. Incubation was carried out at 30 °C in a shaking
water bath under 95% O2, 5% CO2 atmosphere.
To determine the reversal of Ng oxidation, brain slices were washed
three times with 1 ml of ACSF in 15 min and then further incubated with
1 ml of ACSF containing DTT, reduced glutathione, or ascorbic acid.
Each slice was homogenized in the cell lysis buffer (50 mM
Tris-Cl, pH 7.5, 20 mM iodoacetamide, and 0.5% Nonidet
P-40). Extracted protein (60-80 µg) was analyzed for Ng oxidation by
immunoblot analysis.
Treatment of Brain Slices with NMDA--
Slices were kept at
30 °C in a storage bath containing ACSF for ~3 h, and the solution
was changed three times during storage. Ng in these slices was mostly
in the reduced form (>90%). These cortical slices were incubated at
30 °C in 1 ml of ACSF for 3 min with increasing concentrations of
NMDA from 100 to 500 µM for dose response or with 250 µM NMDA at the indicated times for the time course
experiments. To examine the effects of NMDA antagonist, AP5, the slices
were preincubated with 0-500 µM concentration of this
compound for 10 min before the addition of 250 µM NMDA. To test the effects of NO synthase inhibitors, L-NAME and
L-NMMA, on the NMDA-induced Ng oxidation, brain slices were
incubated with 0-200 µM concentration of the inhibitors
for 15 min, then 250 µM NMDA was added for 3 min.
Preparation of Rat Brain Extracts--
Adult rat brain, after
removal of cerebellum and brain stem, was homogenized in 4 volumes
(v/w) of ice-cold 20 mM potassium phosphate buffer, pH 7.5. The homogenate was centrifuged at 27,000 × g for 20 min, and the supernatant was either used directly after keeping on ice
for 2 h or added EDTA and ascorbic acid immediately after
centrifugation to final concentrations of 1 and 10 mM,
respectively. In the absence of EDTA and ascorbic acid, Ng in the brain
extract is spontaneously oxidized. For testing the effects of
Ca2+ and NADPH on the oxidation and reduction of Ng, the
extract containing KPO4/EDTA/ascorbic acid was used and for
testing the effect of reduced glutathione on the oxidized Ng, the
extract containing KPO4 only was used.
Determination of Ng Oxidation--
Conversion of the reduced Ng
to the oxidized form was analyzed by applying the sample (containing
60-80 µg of protein/lane in SDS gel sample buffer without
mercaptoethanol and tracking dye) to a 10-20% gradient SDS (0.1%)
gel, which had been prerun at 40 mA/slab gel for 15 min. After
electrophoresis, the proteins were transferred onto nitrocellulose
membrane. Immunoblot analysis was done using rabbit anti-rat brain Ng
polyclonal antibody and horseradish peroxidase-conjugated goat
anti-rabbit IgG and ECL reagent (5). Quantitative analysis of the
conversion of the reduced Ng to the oxidized form was done by scanning
the x-ray film and the data analyzed using the Fotodyne Gel-Pro
Analyzer program. The extent of Ng oxidation was calculated by dividing the intensity of the oxidized form with the sum of the oxidized and
reduced forms. To ensure that equal amount of protein from each sample
was applied to the gel, the intensity of immunoreactive band
corresponding to neuromodulin, which cross-reacts with our Ng
polyclonal antibody (10), was served as internal standard in the same
blot. Data for multiple comparisons versus a control were
analyzed with Bonferroni's t test and significance was
accepted for p < 0.05.
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RESULTS |
NMDA-induced Transient Oxidation of Ng in Brain Slices--
Ng
expression in rat brain increases during development, and its level
reaches maximum 2-3 weeks after birth (31, 32). Adult rat brain slices
were used to test the effect of NMDA on the oxidation of Ng. NMDA
caused a dose-dependent oxidation of Ng in these slices
(Fig. 1, panel A). Maximal
response was seen at 250 µM and at a higher
concentration, 500 µM, no further increase was seen. The
EC50 was ~100 µM. A 3-6-fold increase in
oxidation of this protein was observed following exposure to 250 µM NMDA for 3 min. At this concentration, the
NMDA-induced oxidation of Ng was transient, it reached a maximal level
within 3-5 min and declined to base line after 30 min (Fig. 1,
panel B). Pretreatment of brain slices with NMDA receptor
antagonist, AP5, for 10 min blocked the NMDA-induced oxidation of Ng
(Fig. 2). AP5 at concentrations over 250 µM completely blocked the enhanced oxidation of Ng by the
same concentration of NMDA. Pretreatment of brain slices with NO
synthase inhibitor, L-NAME, resulted in a
dose-dependent inhibition of the NMDA-induced oxidation of
Ng (Fig. 3, panel
A). The oxidation induced by 250 µM of NMDA
was completely inhibited by 100 µM of L-NAME;
the same concentration of the inactive enantiomer, D-NAME, was ineffective. Furthermore, the inhibition of the NMDA-induced oxidation of Ng by 100 µM L-NAME could be
blocked by over 500 µM L-arginine. Since NO
synthase also generates O
2, we tested the effect of another
inhibitor L-NMMA, which inhibits NO but not O
2
production (33). L-NMMA inhibited the NMDA-induced Ng oxidation dose-dependently (Fig. 3, panel
B), suggesting that NO, rather than O
2, is the
primary active species for Ng oxidation. These results demonstrate that
the oxidation of Ng in the neurons is triggered by the transient
production of NO following NMDA binding to its receptor.

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Fig. 1.
NMDA-induced oxidation of Ng in rat brain
slices. Cortical slices from adult rat brain (400 µm thickness)
were incubated with ACSF at 30 °C under 5% CO2, 95%
O2 atmosphere with increasing concentrations of NMDA for 3 min (panel A) or with 250 µM NMDA for the
indicated times (panel B). Brain slices were kept frozen at
70 °C after incubation and homogenized with lysis buffer (50 mM Tris-Cl, pH 7.5, 20 mM iodoacetamide, and
0.5% Nonidet P-40). Proteins (80 µg) were separated by nonreducing
10-20% gradient SDS gel, transferred to nitrocellulose membrane, and
the oxidized and reduced Ng detected by immunoblot and the extent of
oxidation quantified by densitometric scanning of the film. Each set of
data represents the average ± S.E. of six separate
experiments.
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Fig. 2.
Effect of preincubation with AP5 on the
NMDA-mediated oxidation of Ng in the brain slices. Rat brain
slices were incubated with ACSF under 95% O2, 5%
CO2 atmosphere and increasing concentrations of AP5 for 10 min before the addition of 250 µM NMDA. After additional
3 min of incubation, the slices were kept frozen at 70 °C and
homogenized with lysis buffer. Proteins (80 µg) were separated by
nonreducing 10-20% gradient SDS gel, transferred to nitrocellulose
membrane, and the oxidized and reduced Ng detected by immunoblot. The
extent of oxidation was quantified by densitometric scanning of the
film. Each set of data represents the average ± S.E. of six
separate experiments.
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Fig. 3.
Effect of NO synthase inhibitor
L-NAME and L-NMMA on the NMDA-mediated
oxidation of Ng in brain slices. Rat brain slices were incubated
with ACSF under 95% O2, 5% CO2 atmosphere and
increasing concentrations of L-NAME (panel A) or
L-NMMA (panel B) for 15 min before the addition
of 250 µM NMDA. After an additional 3 min of incubation,
the slices were kept frozen at 70 °C and homogenized with lysis
buffer. Proteins (80 µg) were separated by nonreducing 10-20%
gradient SDS gel, transferred to nitrocellulose membrane, and the
oxidized and reduced Ng detected by immunoblot. The extent of
oxidation was quantified by densitometric scanning of the film. Each
set of data represents the average ± S.E. of eight separate
experiments.
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Oxidation of Ng in the Brain Slices by NO Donors and Other
Oxidants--
To determine whether the exogenously supplied oxidants
can also have similar effects as those induced by NMDA, we tested the actions of several oxidants. Incubation of brain slices with NO donors
(SNP, SNAP, and SNOG), GSSG, and H2O2 all
resulted in the oxidation of Ng (Fig. 4).
Among these compounds, SNP, which contains a NO group with strong
NO+ character (34), appeared to be the most potent;
approximately 20-30% of Ng was converted to the oxidized form
containing intramolecular disulfide bond in 30 min at 0.1 mM. The same concentration of the light-inactivated SNP was
less effective than the freshly prepared SNP in oxidizing Ng. For
example, at 0.1 mM, the freshly prepared SNP caused a 36%
oxidation of Ng versus 5% for the light-inactivated SNP.
The high potency of SNP in causing Ng oxidation was further demonstrated by an effective oxidation of this protein in the brain
slices (up to 10-20%) even at 20 µM (data not shown).
The S-nitrosothiols, SNAP and SNOG, which release NO in the
presence of reduced sulfhydryl compounds to form mixed disulfide (35), were less potent than SNP. Significant oxidation of Ng was seen at a
0.5 mM concentration of these compounds. Previously, we
showed that SNAP and SNOG, when tested in vitro at 5 mM, were ineffective in oxidizing Ng (10, 36). Thus, the
potencies of these NO donors in oxidizing Ng in the brain slices are
different from those seen in vitro using purified Ng. It
seems that the effects of these compounds in the brain slices may be
mediated by other unknown intermediate(s).

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Fig. 4.
Oxidation of rat brain Ng in the brain slices
by NO donors and other oxidants. Cortical slices from adult rat
brain were incubated in ACSF at 30 °C under 5% CO2,
95% O2 atmosphere and incubated without addition
(lane 1) or with 0.1 and 0.5 mM NO donors: SNP
(lanes 2 and 3), SNAP (lanes 4 and
5), and SNOG (lanes 10 and 11) and
other oxidants: H2O2 (lanes
6 and 7) and GSSG (lanes 8 and 9) for 30 min. Brain slices were kept frozen at
70 °C and homogenized with lysis buffer. Proteins (80 µg) were
separated by nonreducing 10-20% gradient SDS gel, transferred to
nitrocellulose membrane, and the oxidized and reduced Ng detected by
immunoblot. The extent of oxidation was quantified by densitometric
scanning of the film. Each set of data represents the average ± S.E. of eight separate experiments.
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At 0.1 mM, both H2O2 and the GSSG
were ineffective in oxidizing Ng in the brain slices (Fig. 4).
Oxidation of Ng by these two oxidants were seen at 0.5 mM.
The effects of GSSG and H2O2 in the brain
slices are apparently mediated by different mechanisms. H2O2 is freely diffusible through cell
membrane, and it can oxidize Ng directly or indirectly after conversion
to other reactive oxygen species such as the hydroxyl radical
OH· (37) and the superoxide anion, O
2, through a
secondary effect (38). GSSG, on the other hand, is not permeable to the
cell membrane and its oxidation potential is either transmitted through other intermediate(s) or by transport of the cystine moiety inside the
cells following extracellular breakdown of GSSG (39).
Reversal of the SNP-mediated Oxidation of Ng in the Brain
Slices--
The SNP-mediated oxidation of Ng in the brain slices could
be reversed by reductants. Incubation of the brain slices with 0.1 mM SNP for 30 min resulted in approximately 30-40%
oxidation of Ng. Following washing of the slices with ACSF for 15 min
and replacing the medium with fresh ACSF containing 1 mM
DTT, ascorbic acid, or GSH for additional 30 min, the oxidized Ng was
reduced (Fig. 5, lanes
4-6). Among these three reductants, DTT appears to be the most potent. This reductant is also very effective in reducing purified oxidized Ng in vitro. GSH may either
function as antioxidant in the medium to promote Ng reduction inside
the cell or by repletion of intracellular GSH through uptake of the breakdown products and intracellular resynthesis of GSH. Ascorbate cannot reduce the oxidized Ng in vitro, thus, it may
function as an antioxidant, for instance to reduce thiyl radicals (40), produced by oxidation of Ng with NO that leads to the formation of
disulfide. Replacement of the medium with ACSF without reductant did
not cause a significant reversal (Fig. 5, lane 3); the
extent of oxidation of Ng was comparable with that treated with 0.1 mM SNP during the entire 75 min of incubation (Fig. 5,
lane 2). The Ng in the control slice incubated with ACSF
alone showed only basal level of oxidation (Fig. 5, lane
1).

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Fig. 5.
Reduction of the SNP-oxidized Ng in the brain
slices by DTT, ascorbic acid, and reduced glutathione. Cortical
slices were incubated at 30 °C in 1 ml of ACSF alone (lane
1) or with 0.1 mM SNP (lane 2)
for 75 min. Four samples incubated previously with SNP for 30 min were
washed with 1 ml of ACSF three times in 15 min and then incubated for
an additional 30 min with 1 ml of ACSF alone (lane
3) or with 1 ml of ACSF plus 1 mM each of DTT
(lane 4), ascorbic acid (lane
5), or GSH (lane 6). The oxidized and
reduced Ng were detected by immunoblot. The extent of oxidation was
quantified by densitometric scanning of the film. Each set of data
represents the average ± S.E. of six separate experiments.
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Oxidation/Reduction of Rat Brain Ng in the Crude Extract--
Ng
present in the rat brain extract prepared by homogenization in a buffer
containing 20 mM potassium phosphate, pH 7.5, was spontaneously oxidized when kept on ice for over 2 h (Fig.
6, panel A, 0 min
sample). Incubation of the extract with 3 mM GSH resulted
in a time-dependent reduction of Ng. A near complete reduction of the oxidized Ng was seen after 30 min (Fig. 6,
panel A). Ng in the brain extract prepared by
homogenization in 20 mM phosphate buffer, pH 7.5, and
followed by the addition of 1 mM EDTA and 10 mM
ascorbic acid showed a partial oxidation. The partially oxidized Ng was
used to demonstrate the interconversion between the oxidized and
reduced forms by either the addition of Ca2+ or NADPH. In
the presence of KPO4/EDTA/ascorbic acid, the ratio of the
oxidized and reduced Ng remained nearly the same upon incubation at
30 °C for up to 30 min (Fig. 6, panel B).
Addition of 1.1 mM CaCl2 to the extract
containing 20 mM KPO4, 1 mM EDTA,
10 mM ascorbic acid resulted in a rapid oxidation of Ng
(Fig. 6, panel C). Previously, we have shown that Ng in the
brain extract is completely dissociated from CaM in the presence of
Ca2+ (5). Thus, the free Ng becomes susceptible to
oxidation by an unknown mechanism, even in the presence of 10 mM ascorbic acid. Incubation of the partially oxidized Ng
in 20 mM phosphate, 1 mM EDTA, 10 mM ascorbic acid with 2 mM NADPH resulted in a
reduction of this protein (Fig. 6, panel D),
suggesting the presence of a Ng reductase. NADPH alone is ineffective
in reducing the purified oxidized Ng (data not shown).

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Fig. 6.
Oxidation and reduction of Ng in the rat
brain extracts. Rat brain extract prepared in 20 mM
KPO4, pH 7.5, was kept on ice for 2 h to allow a
complete oxidation of Ng for testing the effect of GSH as a reductant
in reversing the oxidation (panel A). The
extracts prepared by homogenization in 20 mM
KPO4, pH 7.5, and followed by centrifugation and addition
of EDTA (1 mM) and ascorbic acid (10 mM)
contained the oxidized and reduced Ng in nearly equal ratio
(panels B-D). Brain extracts (2 mg/ml) in 20 mM KPO4 were incubated with 3 mM GSH (panel A), and those in 20 mM KPO4, 1 mM EDTA, 10 mM ascorbic acid were incubated without further addition
(panel B), with 1.1 mM
CaCl2 (panel C), or with 2 mM NADPH (panel D). Samples (50 µg
of protein) were taken at timed intervals, SDS gel sample buffer
lacking mercaptoethanol and bromphenol blue added, and samples analyzed
by immunoblot to detect the reduced and oxidized Ng.
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DISCUSSION |
This study provides the first demonstration that rat brain Ng in
intact cells is subjected to modification by oxidation and reduction.
The redox of Ng in the neurons is likely a physiological event, since
NMDA can induce a transient oxidation of this protein. Rat brain Ng has
a high propensity to form a intramolecular disulfide bond, resulting in
a downward mobility shift on nonreducing SDS-PAGE; oxidation of this
protein in rat brain may conveniently serve as an indicator for the
generation of oxidants in neurons. Based on our previous mutagenesis
study (36) we have predicted that Ng oxidation results in
intramolecular disulfide bond formation between Cys-3, Cys-4, or Cys-9
and Cys-51. Electrospray ionization mass spectrometry analysis showed
that there were two pairs of disulfide bond in the oxidized Ng (data
not presented). The resulting oxidized Ng, with its N- and C-terminal
ends brought together by the disulfide bond, becomes more compact than
the reduced Ng. The calculated molecular weight of rat brain Ng is
7500, but it migrates as a Mr 14,000-17,000
protein on SDS-PAGE depending on the conditions of electrophoresis. The
oxidized Ng has an apparent molecular weight of approximately 10,000 on
nonreducing SDS-PAGE. Oxidation of the purified Ng in vitro
(10), in rat brain slices as induced by neurotransmitter NMDA and by
the various oxidants, and that seen in the crude rat brain extracts,
all result in the formation of the apparent Mr = 10,000 intramolecular disulfide form. It seems that formation of the
intramolecular disulfide bonds of rat brain Ng is a kinetically
favorable reaction.
Free radicals and redox stress are thought to participate in cellular
signaling (41). Ng in the brain slices is oxidized by the addition of
NMDA, NO donors, and several oxidants. The intramolecularly
disulfide-bridged form of Ng has a lower affinity for CaM and becomes a
poorer substrate of PKC than the reduced form (10). Both the
phosphorylation and oxidation of Ng seem to have a similar consequence
in reducing the affinity of this protein for CaM and thus render CaM
available for many CaM-dependent enzymes, including
CaM-dependent protein kinases, cyclic nucleotide phosphodiesterase, adenylyl cyclases, NO synthase, inositol
1,4,5-trisphosphate kinase, calcium pumps, CaM-dependent
protein phosphatase, nicotinamide adenine dinucleotide kinase, and
cytoskeletal proteins. Thus, the various oxidants either generated
in situ, as in the case of NO from NMDA treatment, or
present extracellularly may transduce their signals by oxidation of Ng
and modulate the level of CaM. The phosphorylated Ng can be
dephosphorylated by protein phosphatases 1 and 2A and by calcineurin
(42), whereas the enzyme(s) responsible for the reduction of the
oxidized Ng has yet to be identified. Nonetheless, there seems to be an
enzyme system in the brain that can efficiently reduced the oxidized Ng
as demonstrated by the transient effect of NMDA and that seen in the
crude rat brain extracts.
In the rat brain slices, the oxidant-induced oxidation of Ng can also
be reduced by exogenously added reductants, such as DTT, ascorbate, or
GSH, suggesting that these reductants can influence the intracellular
redox state in favor of the reduction of the oxidized Ng. Both DTT and
GSH can reduce the oxidized Ng directly in the in vitro
experiments, however, ascorbate cannot. Similarly, in the crude rat
brain extracts, DTT (data not presented) and GSH are effective in
reducing the oxidized Ng but not the ascorbate. It seems that these
reductants may either reduce the intracellularly oxidized Ng directly
or serve as antioxidants and thus favor the reduction of Ng by other
enzyme(s). In this respect, it is interesting to note that addition of
NADPH to the crude rat brain extracts containing endogenously oxidized
Ng causes a reduction of Ng, although NADPH itself is not effective in
reducing the purified Ng. It appears that there is a Ng reductase in
the neurons that utilizes NADPH as electron donor to reduce the
oxidized Ng. The Ng thiol-disulfide redox resembles those of
thioredoxins and glutaredoxins, both are small molecular weight
proteins (8000-12,000) containing redox-active thiols (43). Ng does
not exhibit any sequence homology to these proteins, including the
short sequence regions containing redox active Cys residues. A major
function of thioredoxin and glutaredoxin is to serve as a cofactor for
ribonucleotide reductase, which catalyzes the conversion of
ribonucleotides to deoxyribonucleotides (44). In the case of
thioredoxin, the reduced cofactor is regenerated by a specific
NADPH-dependent thioredoxin reductase (45). The oxidized
glutaredoxin is reduced directly by cellular GSH, and the resulting
GSSG is, in turn, reduced by NADPH via the glutathione reductase
(46).
The physiological relevance of Ng oxidation is best illustrated by the
NMDA-induced transient oxidation of Ng in rat brain slices, in which
oxidation of Ng is inhibited by the receptor antagonist, AP5, as well
as by the NO synthase inhibitors, L-NAME and
L-NMMA. It has been shown that activation of NMDA receptors and the subsequent increase in [Ca2+]i stimulate
NO synthase (47-49), which generates both O
2 and NO (33).
Since both L-NAME, which inhibits O
2 and NO production, and L-NMMA, which inhibits NO but not
O
2 production, are effective in inhibiting the NMDA-induced
oxidation of Ng, we infer that NO is the primary active species for the
oxidation of Ng. Thus, Ng oxidation serves as a downstream target of
the NO signaling pathway. Our results on the reversible
oxidation/reduction of Ng further support the contention that the redox
states of a protein may involve in the regulatory processes of NMDA.
The transient nature of Ng oxidation following treatment with NMDA is
indicative for the short burst of NO production after the
administration of this neurotransmitter.
Ng is a neuron-specific protein; its phosphorylation by PKC has been
linked to the induction of LTP in rat hippocampal slices (11, 16, 17),
and intracellular injection of antibodies against Ng prevents the
maintenance phase of LTP (18). The tetanus-induced LTP requires
activation of NMDA receptors (50) and an elevation of
[Ca2+]i (51, 52), which result in the activation
of PKCs and presumably stimulate phosphorylation of Ng located at the postsynaptic dendritic spine and shaft (53). However, phosphorylation of Ng was reported to be only at 60 min, but not at other time points
tested (15, 30, and 120 min), after tetanus-induced LTP (11). In
contrast, phosphorylation of presynaptic PKC substrate, B-50/GAP-43/neuromodulin, reached maximum within 15-30 min (11). As
the activation of NMDA receptors also stimulates NO synthase (47-49)
and causes oxidation of Ng, it is likely that Ng is oxidized during the
early phase of tetanic stimulation that renders Ng a poor substrate of
PKC (10). However, the oxidized Ng functions similarly as the
phosphorylated Ng in elevating the free CaM levels at the postsynaptic
sites, where the Ca2+/CaM-dependent protein
kinase II is also located. The activation of
Ca2+/CaM-dependent protein kinase II has been
linked to the postsynaptic mechanism for the enhancement of synaptic
transmission during LTP; thus, the oxidation of Ng may potentiate the
stimulatory effect during the early phase of LTP.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Bldg. 49, Rm. 6A36,
NIH, 49 Convent Dr., MSC 4510, Bethesda, MD 20892-4510. Tel.: 301-496-7827; Fax: 301-496-7434; E-mail: kphuang{at}helix.nih.gov.
The abbreviations used are:
Ng, neurogranin (RC3 protein); PKC, protein kinase C; Nm, neuromodulin; CaM, calmodulin; NO, nitric oxide; PAGE, polyacrylamide gel electrophoresis; SNOG, S-nitrosoglutathione; SNAP, (±)-S-nitroso-N-acetylpenicillamine; AP5, 2-amino-5-phosphonovaleric acid; L-NAME, NG-nitro-L-arginine methyl ester; D-NAME, NG-nitro-D-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine; SNP, sodium nitroprusside; GSH, glutathione; GSSG, oxidized glutathione; ACSF, artificial cerebrospinal fluid; NMDA, N-methyl-D-aspartate; LTP, long term
potentiation; DTT, dithiothreitol.
 |
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