Q . Jiang et al. Brain Research 887 2000 285 –292
287
polyclonal, 1:10 000 or anti-active diphosphorylated ERK1 ERK2 antibody Sigma, monoclonal, 1:5000 at
48C overnight. Detection was carried out by alkaline phosphatase-conjugated
goat anti-rabbit
IgG Sigma,
1:20 000 or goat anti-mouse IgG Sigma, 1: 40 000 and developed with NBT BCIP color substrate Sigma. After
immunoblot, bands on filter were scanned, quantitative analyzed and printed by an image analyzer affiliated with
digital graphic printer LabWorks Software, UVP, Upland, CA, USA. Protein level and activation diphosphorylation
level of ERK1 ERK2, based on immunoreactivities of ERK1 ERK2 and active ERK1 ERK2, respectively, were
expressed as fold versus sham control of optical density of certain band from Western immunoblot.
2.4. Assessment of apoptotic-like cell death
5
Live cells grown on each coverslip 2310 cells were incubated with 10 mg ml fluorescent DNA binding dye
DAPI 49,6-diamidino-2-phenylindole, Sigma at 378C for 30 min, washed with PBS and excited with vertical
fluorescent at 400 nm. With fluorescence collected at 455 nm, apoptotic-like cells were characterized by the presence
of condensed and fragmented nuclei, as opposed to the
Fig. 1. Effects of antagonists of glutamate receptors,
L
-VGCC and
21
extracellular Ca elimination on ERK1 ERK2 activation in glutamate-
diffuse staining observed in nonapoptotic cells. Each
induced apoptotic-like death in cultured rat cortical neurons. Thirteen
sample was pooled from three coverslips. The proportion
days in vitro cortical neurons were exposed to 50 mM glutamate for 15
of apoptotic-like cells was calculated as a percentage of
min. MK-801 20 mM,
L
-AP-3 1 mM, DNQX 20 mM, nifedipine
total cells counted in 10 microscopic fields 3400.
ND, 20 mM and EGTA 5 mM were added, respectively, 20 min before and during the glutamate exposure. A Western immunoblot at 15
min of glutamate exposure with anti-ERK1 ERK2 antibody top or
2.5. Statistics
anti-diphosphorylated ERK1 ERK2 p-ERK1 ERK2 antibody bottom. B Quantitative representations expressed as fold versus sham control
Values were expressed as mean6S.D. from five in-
Sham, treated with no drug of optical density O.D. ERK2 or of
dependent cultures. One-way ANOVA was used. Com-
p-ERK2 band
from Western immunoblot.
Each point
represents
a b
parisons of each group to control were by LSD least
mean6S.D. of five independent cultures. P,0.05 versus sham, P,0.05 versus vehicle control Vehi, treated only with glutamate and vehicle.
significant difference test. Others were by q test New- man–Keuls test. A P value of ,0.05 was considered
significant. 1 mM [41], or KA AMPA-R with DNQX 20 mM [23],
or
L
-VGCC with nifedipine 20 mM [44]. Since the
3. Results alterations of p-ERK1 and p-ERK2 were much the same,
we directed attention to p-ERK2, which displayed a 3.1. The role of glutamate receptors,
L
-VGCC and relatively stronger immunoreactivity.
21
extracellular Ca in ERK
1 ERK2 activation in glutamate-induced apoptotic-like death in cultured rat
3.2. The role of protein kinases and NOS in ERK1 cortical neurons
ERK 2 activation in glutamate-induced apoptotic-like
death in cultured rat cortical neurons Protein level of ERK1 ERK2, as indicated by ERK1
ERK2 immunoreactivities, was unaffected Fig. 1A, top, As shown in Fig. 2, ERK1 ERK2 activation was largely
and B. While activation level of ERK1 ERK2, as indi- prevented by inhibition of either PTK with genistein 100
cated by diphosphorylated ERK1 ERK2 p-ERK1 ERK2, mM, also as an inhibitor of topoisomerase II kinase [1] or
was increased to 4.9-fold versus sham control at 15 min of PKC with sphingosine 10 mM [12]. It was weakly but
glutamate exposure Fig. 1A, bottom, and B. Such still significantly prevented by inhibition of CaM kinase II
activation was almost totally prevented by either blockage with KN-62 40 mM [45], but not significantly affected by
of NMDA-R with MK-801 20 mM or elimination of inhibition of PKA with H-89 40 mM, also as an inhibitor
21
extracellular Ca with EGTA 5 mM. It was not
of PKG [8], or NOS with
L
-NNA 100 mM [11]. The significantly affected by blockage of mGlu-R with
L
-AP-3 inhibitory effect of KN-62 was significantly weaker than
288 Q
21
Fig. 3. Effects of NMDA-R antagonist, extracellular Ca eliminator,
inhibitors of NOS and protein kinases on glutamate-induced apoptotic- like death in cultured rat cortical neurons. Thirteen days in vitro cortical
neurons were exposed to 50 mM glutamate for 15 min. MK-801 20 mM, EGTA 5 mM,
L
-NNA 20 mM, genistein Gen, 100 mM, KN-62 40 mM, sphingosine SS, 10 mM and PD98059 PD, 50 mM were added
into the medium 20 min before and during the glutamate exposure, respectively. DAPI staining at 18 h after exposure were quantitative
represented as percentage of total cells counted in 10 microscopic fields 3400. Each point represents mean6S.D. of five independent cultures.
a b
P,0.05 versus sham control Sham, with no drug treatment, P,0.05 Fig. 2. Effects of inhibitors of protein kinase and NOS on ERK1 ERK2
versus vehicle control Vehi, treated only with glutamate and vehicle, activation in glutamate-induced apoptotic-like death in cultured rat
c
P,0.05 versus single drug treatment. cortical neurons. Thirteen days in vitro cortical neurons were exposed to
50 mM glutamate for 15 min. Genistein Gen, 100 mM, H-89 40 mM, KN-62 40 mM, sphingosine SS, 10 mM and
L
-NNA 20 mM were added, respectively, 20 min before and during the glutamate exposure. A
PD98059 50 mM [26], or NOS with
L
-NNA 100 mM.
Western immunoblot at 15 min of glutamate exposure with anti-ERK1
A significant additive effect was observed in combined
ERK2 antibody top or anti-diphosphorylated ERK1 ERK2 p-ERK1
inhibition of CaM kinase II but not PKC or PTK and
ERK2 antibody bottom. B Quantitative representations expressed as
ERK1 ERK2. The number of total cells counted in 10
fold versus sham control Sham, treated with no drug of optical density
microscopic fields 3400 is about 34006360 cells.
O.D. of ERK2 or p-ERK2 band from Western immunoblot. Each point
a
represents mean6S.D. of five independent cultures. P,0.05 versus sham,
b
P,0.05 versus vehicle control Vehi, treated only with glutamate and
c
vehicle, P,0.05 versus single drug treatment.
3.4. The role of protein phosphatases in the reversion of ERK
1 ERK2 activation in glutamate-induced apoptotic- that of either sphingosine or genistein. Combined inhibi-
like death in cultured rat cortical neurons tion of CaM kinase II and PKC had a significantly additive
and completely preventive effect. Combined inhibition of As shown in Fig. 4, at 3 h after glutamate exposure,
PTK and PKC had no significant additive effect. ERK1 ERK2 activation reverted to sham control level.
Such reversion was largely prevented by inhibition of both
21
3.3. The role of NMDA-R, extracellular Ca , PKC,
protein phosphatase PP 1 and PP2A with 300 nM PTK
, CaM kinase II and NOS in ERK1 ERK2-mediated okadaic acid [4], or protein tyrosine phosphatase PTP
apoptotic-like death in glutamate-induced excitotoxicity with sodium orthovanadate 200 mM, also as an inhibitor
in cultured rat cortical neurons of ATPase and alkaline phosphatase [15], but not sig-
nificantly affected by PP2A with 5 nM okadaic acid [4], or As determined by DAPI staining Fig. 3, at 18 h after
PP 2B with cyclosporin A 2 mM, also as an immuno- glutamate exposure, the number of apoptotic-like cells was
suppressant [17]. Combined use of okadaic acid 300 nM significantly increased to 82 versus sham control 11.
and sodium orthovanadate 200 mM had no significantly Such increase was significantly prevented by elimination
additive effect. The concentration of each drug shown in
21
of extracellular Ca with EGTA 5 mM, or inhibition of
Figs. 1–5, except for okadaic acid 5 nM, was responsible NMDA-R with MK-801 20 mM, PTK with genistein
for the maximal effect of the drug data not shown. 100 mM, or PKC with sphingosine 10 mM, or CaM
Modified EBSS and vehicles had little effect on ERK1 kinase II with KN-62 40 mM, or MEK1 MEK2 with
ERK2 activation data not shown.
Q . Jiang et al. Brain Research 887 2000 285 –292
289
Fig. 5. Effects of inhibitors of protein phosphatases on glutamate-induced apoptotic-like death in cultured rat cortical neurons. Thirteen days in vitro
cortical neurons were exposed to 50 mM glutamate for 15 min. Okadaic acid OA, 300 nM and sodium orthovanadate Van, 200 mM were added
from 20 min before until 3 h after glutamate exposure, respectively. DAPI staining at 18 h after exposure were quantitative represented as per-
centage of total cells counted in 10 microscopic fields 3400. Each point
a
represents mean6S.D. of five independent cultures. P,0.05 versus sham
b
control Sham, with no drug treatment, P,0.05 versus vehicle control
c
Vehi, treated only with glutamate and vehicle, P,0.05 versus single drug treatment.
Fig. 4. Effects of inhibitors of protein phosphatases on the reversion of
death in cultured rat cortical neurons, and such activation
ERK1 ERK2 activation in glutamate-induced apoptotic-like death in
was almost completely prevented by either blockage of
cultured rat cortical neurons. Thirteen days in vitro cortical neurons were
21
NMDA-R or elimination of extracellular Ca [26].
L
-
exposed to 50 mM glutamate for 15 min. Sodium orthovanadate Van,
VGCC and glutamate receptors, including NMDA-R, a-
200 mM, okadaic acid OA, 5 nM, okadaic acid OA, 300 nM and cyclosporin A Cys A, 2 mM were added, respectively, from 20 min
amino-3-hydroxy-5-methyl-4-isoxazolepropionate kainate
before until 3 h after the glutamate exposure. A Western immunoblot at
receptor AMPA KA-R and metabotropic glutamate re-
3 h after glutamate exposure with anti-diphosphorylated anti-ERK1
ceptor mGlu-R, have all been shown involved in ERK1
ERK2 antibody top or ERK1 ERK2 p-ERK1 ERK2 antibody bot-
ERK2 activation in some other cases [18,20]. However, in
tom. B Quantitative representations expressed as fold versus sham
this study, blockage of none of them, except for NMDA-R,
control Sham, treated with no drug of optical density O.D. of ERK2 or p-ERK2
band from Western
immunoblot. Each
point represents
significantly affected ERK1 ERK2 activation. Therefore,
a b
mean6S.D. of five independent cultures. P,0.05 versus sham, P,0.05
in glutamate-induced cortical neurotoxicity ERK1 ERK2
versus vehicle control Vehi, treated only with glutamate and vehicle.
activation might be mainly mediated by NMDA-R-induced
21
influx of extracellular Ca .
3.5. The role of PP1 and PTP in glutamate-induced Stimulation of NMDA-R has been shown to mediate
excitotoxicity in cultured rat cortical neurons ERK1 ERK2 activation, which might involve PKC and
CaM kinase II in hippocampal neurons [18] and PTK in As shown in Fig. 5, inhibition of PP1 with 300 nM
striatal neurons [47]. However, the upstream cascades is okadaic acid and PTP with sodium orthovanadate 200
unknown in excitotoxicity. In this study, in glutamate- mM can promote glutamate-induced apoptotic-like death
induced cortical neurotoxicity ERK1 ERK2 activation was from 82 to 93 and 96, respectively. Modified EBSS and
largely prevented by inhibition of PKC, relatively mildly vehicles had little effect on apoptotic-like death data not
prevented by that of CaM kinase II, and combined shown.
inhibition of these two had an additive and complete inhibitory effect. These results strongly indicate that in
ERK1 ERK2 activation a PKC-dependent pathway was mainly involved, a CaM kinase II-dependent pathway was
4. Discussion relatively mildly involved, and these two pathways were
