286 Q
protein kinase MAPK superfamily. Both require specific glutamine, Gibco-BRL. Cultures were used after 13 days
diphosphorylation of both threonine and tyrosine residues in vitro when the cells were vulnerable to glutamate insult
at the regulatory sites by MEK1 MEK2 ERK1 ERK2 and verified .95 neurofilaments positive by immuno-
kinase for activation [33,42]. ERK1 ERK2 cascades play staining.
important roles in signal transduction from cell surface to nucleus. The well-documented neurotropic growth factor
2.2. Drug treatment receptor-mediated
activation cascade
Ras Raf MEK ERK has been thought to play important roles in cell
Excitotoxicity was induced by 50 mM glutamate expo- growth, proliferation and survival [10,18,20,22,32].
sure for 15 min, during which the medium was changed
21
Recently, ERK1 ERK2 have been found activated after into modified EBSS Mg
-free Earle’s balanced salt relatively mild stimulation of glutamate receptors and
solution EBSS Gibco-BRL supplemented with 5 mM involved in some activity-dependent functions [18,20].
glycine. For restoration, at the end of glutamate exposure, Furthermore, ERK1 ERK2 have also been found activated
cultures were rinsed twice with EBSS, and the original in some excitotoxicity-associated events, such as stroke,
feeding medium was restored. For drug treatments, seizure and Alzheimer’s disease [3,19,24]. We previously
PD98059 Calbiochem, San Diego, CA, USA, MK-801 observed that ERK1 ERK2 were transiently activated in
dizocilpine maleate, RBI, Natick, MA, USA., L-AP-3 glutamate-induced apoptotic-like death in cultured rat
L
-1-2-amino-3-phosphono-propionic acid,
RBI, cortical neurons, and PD98059, a specific inhibitor for
DNQX 6,7,-dinitroquinoxaline-2,3
1H,4H-dione, MEK1 MEK2, completely inhibited such activation and
Sigma, nifedipine Sigma, EGTA Sigma, genistein partially prevented the glutamate-induced apoptotic-like
Sigma, H-89 Calbiochem, KN-62 RBI, sphingosine
v
death [26]. Therefore, ERK1 ERK2 might be excessively RBI,
L
-NNA N -nitro-
L
-argine, Sigma were added form activated transiently and involved in the glutamate-induced
20 min before till the end of glutamate exposure, respec- cortical neurotoxicity.
tively, or in combination. In some other cases, sodium However, little is known about the upstream cascade of
orthovanadate Sigma, okadaic acid RBI and cyclos- the variation of ERK1 ERK2 in excitotoxicity. In this
porin A Sigma were added from 20 min before until 3 h study, we searched for upstream molecules responsible for
after glutamate exposure, respectively. PD98059, DNQX, the variation of ERK1 ERK2 and the ERK1 ERK2-me-
nifedipine, genistein, sphingosine, okadaic acid and cyclos- diated apoptotic-like death in the glutamate-induced neuro-
porin A were made as 5003 stocks in dimethyl sulfoxide toxicity in cultured rat cortical neurons. Several molecules
DMSO, respectively. KN-62 was made as 3003 stocks were investigated, including three subtypes of glutamate
in methanol. Other drugs were made as 2003 stocks in
21
receptors, L-type voltage-gated Ca channel L-VGCC,
water. Vehicle controls were treated only with 50 mM NOS, some protein kinases and protein phosphatases, each
glutamate and vehicle 0.5 water or 0.2 DMSO or of which has been implicated involved either in excitotox-
0.3 methanol in modified EBSS. Sham controls were icity or in the regulation of ERK1 ERK2 activation in
treated only with modified EBSS. other cases.
2.3. Cell extracts preparation and Western immunoblot
2. Materials and methods Cultured cells were rinsed with PBS, scraped off the
wells. Each sample was pooled from two wells approxi-
7
2.1. Neuronal cultures mately 1.2310 cells and homogenized in 160 ml ice-cold
buffer 50 mM 3-N-morpholino propane-sulfonic acid, Cortical neuronal cultures were prepared from 17-day-
MOPS, pH 7.4, 0.5 mM dithiothreitol, 2 mM sodium old Sprague–Dawley rat embryos as previously described
orthovanadate, 0.5 mM EDTA, 1 mM EGTA, 0.5 mM [6]. Briefly, neocortex was meticulously isolated in ice-
ouabain, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM cold high glucose Dulbecco’s modified Eagle medium
leupeptin and 0.5 mM pepstatin A, and centrifuged at h-DMEM, Gibco-BRL, Grand Island, NY, USA. Cortical
15 0003g for 15 min at 48C. Ten ml were removed for cells were dissociated by trypsinization 0.25 w v in
protein concentration determination by Lowry method
21 21
Ca - and Mg
-free Hank’s balanced salt solution Gib- [31], the remaining supernatant was incubated in sample
co-BRL, at 378C for 15 min, followed by gentle triturat- buffer 2 sodium dodecyl sulfate, 20 glycerol, 5
ing in plating medium h-DMEM supplemented with 10 b-mercaptoethanol, 62.5 mM Tris–HCl, pH 6.8, and
fetal bovine serum and 10 horse serum, Gibco-BRL. 0.01 bromphenol blue at 968C for 5 min. Equal amount
Cells were seeded onto poly-
L
-lysine Sigma, St. Louis, of proteins 40 mg were separated by 10 SDS–PAGE by
MO, USA-coated wells or coverslips at a density of the method of Laemmli [27] and electrotransferred onto
5 2
2310 cells per cm , incubated at 378C in 5 carbon
nitrocellulose filter pore size, 0.45 mm, Amersham, Buc- dioxide atmosphere, and fed by feeding medium Neuro-
kingham, UK by the method of Towbin [46]. The filter basal Medium supplemented with 2 B-27 and 0.5 mM
was probed with anti-ERK1 ERK2 antibody Sigma,
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,
