ORI GI NAL PAPER

Mechanism of Post-Translational Modication by Tyrosine

Phosphorylation of Apoptotic Proteins During Hypoxia
in the Cerebral Cortex of Newborn Piglets
Maria Delivoria-Papadopoulos Om Prakash Mishra
Accepted: 27 June 2009 / Published online: 12 July 2009
Springer Science+Business Media, LLC 2009
Abstract The present study aims to investigate the
mechanism of phosphorylation of apoptotic proteins and
tests the hypothesis that the hypoxia-induced increased
tyrosine phosphorylation of apoptotic proteins Bcl-2 and
Bcl-xl is Ca
2?
-inux-dependent. Piglets were divided in
normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-
pretreated with clonidine (Clo ? Hx, n = 4) groups. Hyp-
oxic animals were exposed to an FiO
2
of 0.06 for 1 h.
Clonidine (12.5 lg/kg, IV) was administered to piglets
30 min prior to hypoxia. Hypoxia was conrmed by ATP
and phosphocreatinine (PCr) levels. Cytosol was isolated
and separated by 12% SDSPAGE and probed with tyrosine
phosphorylated (p) -Bax, Bad, Bcl-2 and Bcl-xl antibodies
and bands were detected. The ATP levels (lmol/g brain) in
the Nx, Hx, Clo ? Hx were 4.3 1.0 (P\0.05 vs. Hx,
Clo-Hx), 0.9 0.8 and 1.5 0.3, respectively. The PCr
levels in the Nx, Hx, Clo ? Hx were 2.7 0.7 (P\0.05
vs. Hx, Clo-Hx), 0.9 0.2 and 0.9 0.9, respectively.
Ca
2?
-inux (pmoles/mg protein) was 4.96 0.94 in Nx,
11.11 2.38 in Hx, and 6.23 2.07 in Clo ? Hx
(P\0.05 Nx vs. Hx and Hx vs. Clo ? Hx). p-Bcl-2 density
was 21.1 1.1 Nx, 58.9 9.6 Hx and 29.5 6.4
Clo ? Hx (P\0.05 vs. Hx). p-Bcl-xl density was
29.6 1.5 Nx, 50.6 7.4 Hx and 32.1 0.1 Clo ? Hx
(P\0.05 vs. Hx). p-Bax density was 38.6 16.2 Nx,
46.1 5.5 Hx and 41.6 1.9 Clo ? Hx groups (P = NS).
p-Bad was 66.7 12.8 Nx, 71.2 6.8 Hx and 78.7 22.5
Clo ? Hx groups (P = NS). Results showed that clonidine
administration prior to hypoxia prevents the hypoxia-
induced increased nuclear Ca
2?
-inux and increased
phosphorylation of Bcl-2 and Bcl-xl while phosphorylation
of Bad and Bax was not altered. We conclude that post-
translational modication of anti-apoptotic proteins Bcl-2
and Bcl-xl during hypoxia is nuclear Ca
2?
-inux-depen-
dent. We propose that blockade of nuclear Ca
2?
-inux that
prevents phosphorylation of antiapoptotic proteins may
become a neuroprotective strategy.
Keywords Apoptotic proteins Bcl-2 Bcl-xl
Tyrosine phosphorylation Hypoxia EGFR kinase
Introduction
The Bcl-2 multigene family of proteins is an important
determinant of apoptotic cell death. It consists of pro-
apoptotic (Bax, Bcl-X
s
, Bak and Bad) and anti-apoptotic
(Bcl-2, Bcl-X
L
and Bcl-w) proteins. Bcl-2 and Bax proteins
play a crucial role in regulating cell survival and cell death.
Bcl-2 family members determine cell death and survival by
controlling mitochondrial membrane ion permeability,
cytochrome c release, and the subsequent activation of
caspase mediated executor functions [1, 2]. Bax is a
21 kDa protein that shares homology with Bcl-2, and
heterodimerizes with Bcl-2 or homodimerizes with itself.
When Bax is over-expressed in cells, apoptotic death in
response to a death signal is accelerated. When Bcl-2 is
over-expressed, it heterodimerizes with Bax and cell death
is repressed. Therefore, the ratio of Bcl-2 to Bax appears to
be important in determining susceptibility to apoptosis [3,
4]. Previously in our laboratory we have shown that during
hypoxia the expression of Bax protein was increased and
Bcl-2 protein expression was unchanged [5].
M. Delivoria-Papadopoulos (&) O. P. Mishra
Department of Pediatrics, Drexel University College
of Medicine and St. Christophers Hospital for Children,
245N 15th Street, New College Building, Room 7410,
Mail Stop 1029, Philadelphia, PA 19102, USA
e-mail: delivoria@drexelmed.edu
1 3
Neurochem Res (2010) 35:7684
DOI 10.1007/s11064-009-0032-7
Phosphorylation may affect the function of Bcl-2 or Bax
by altering the ability of these proteins to form heterodi-
mers or act independently of dimerization. Post-transla-
tional modication of Bcl-2 and Bax proteins through
phosphorylation changes the function of these proteins by
altering cell cycle events, by regulating cell proliferation as
well as programmed cell death [4, 6]. Studies have shown
that treatment of cells with a number of agents results in
phosphorylation of Bcl-2 and is associated with inhibition
of the anti-apoptotic function of Bcl-2 [3, 7, 8]. The present
study tests the hypothesis that cerebral hypoxia results in
increased tyrosine phosphorylation of anti-apoptotic pro-
teins Bcl-2 and Bcl-xl, in the cytosolic fraction of the
cerebral cortex of newborn piglets and the hypoxia-induced
increased tyrosine phosphorylation of apoptotic proteins is
prevented by administration of clonidine, an inhibitor of
high afnity calcium ATPase.
In our previous studies, we have also shown that brain
tissue hypoxia results in modication of the N-methyl-
D-aspartate (NMDA) receptor ion-channel and its recogni-
tion and modulatory sites [911] and increased NMDA
receptor-mediated Ca
2?
entry into synaptosomes. An
increase in NMDAreceptor-mediated Ca
2?
concentration in
hypoxic neurons [12] may activate several pathways of
oxygen free radical generation including the NOS pathway.
We demonstrated that cerebral hypoxia results in increased
generation of NO free radicals [13]. Peroxynitrite mediated
nitration of the NMDA receptor resulted in modication of
the NMDA receptor-ion-channel and the glutamate recog-
nition site similar to hypoxia [14]. Furthermore, hypoxia
resulted in nitration of the NMDA receptor subunits NR1,
NR2A, and NR2Bindicating that nitration is a mechanismof
modication of the NMDAreceptor function during hypoxia
[15]. We have also observed that cerebral hypoxia results in
increased expression of cell death promoter protein Bax,
whereas the expression of cell death repressor protein, Bcl-2,
was not increased [5]. Since the ratio of Bax/Bcl-2 deter-
mines neuronal survival or death, a selective increase in the
expression of Bax results in an increased ratio of Bax/Bcl-2
that favors cell death. We have shown that hypoxia results in
increased nuclear Ca
2?
-inux, increase in Ca
2?
/calmodulin-
dependent protein kinase (CaM kinase IV) activity in neu-
ronal nuclei, increase in cyclic AMP response element
binding (CREB) protein phosphorylation, increased
expression of cell death promoter protein Bax, activation of
poly(ADP-ribose) polymerase, caspase-3 activation and
damage to nuclear DNA [1623].
Previously we have shown that hypoxia results in
increased high afnity Ca
2?
-ATPase activity in neuronal
nuclei of newborn piglets [24]. We have shown that Clo-
nidine inhibits high afnity Ca
2?
-ATPase activity, and
decreases hypoxia-induced increased CaM kinase IV
activity and CREB protein phosphorylation in the neuronal
nuclei of the cerebral cortex of newborn piglets [25].
Clonidine, an imidazoline, is a lipid soluble drug, which
acts as a a
2
-adrenoreceptor agonist [17]. Studies have
shown that Clonidine reduces intracellular Ca
2?
levels by
modication of intracellular cAMP [26, 27]. Clonidine has
also been shown to be a noncompetitive inhibitor of high
afnity Ca
2?
-ATPase [28]. Thus the pharmacologic agent,
Clonidine was selected to elucidate the importance of
calcium inux in the apoptotic cascade and for its ability to
decrease high afnity Ca
2?
-ATPase activity and nuclear
Ca
2?
inux [28].
The present study specically focuses on investigating
the effect of hypoxia on tyrosine phosphorylation of apop-
totic proteins Bcl-2, Bclxl, Bax and Bad and the mechanism
of hypoxia-induced tyrosine phosphorylation of apoptotic
proteins in the cytosolic fraction of the cerebral cortex of
newborn piglets. In the present study we have tested the
hypothesis that hypoxia results in increased tyrosine phos-
phorylation of apoptotic proteins in the cerebral cortex of
newborn piglets and that the increased tyrosine phosphor-
ylation is mediated by Ca
2?
-inux. Therefore, administra-
tion of a high afnity Ca
2?
-ATPase inhibitor, clonidine,
prior to hypoxia, will prevent the increased tyrosine phos-
phorylation of apoptotic proteins in the cytosolic fraction of
the cerebral cortex of newborn piglets.
Experimental Procedures
Animal experimentation and induction of hypoxia Studies
were performed on 35 day old Yorkshire piglets obtained
from the Willow Glenn Farm, Strasburg, PA. The experi-
mental animal protocol was approved by the Institutional
Animal Care and Use Committee of Drexel University.
Newborn piglets were randomly divided into three groups:
normoxic (n = 5), hypoxic (n = 5), and hypoxic with clo-
nidine (hypoxic-clonidine n = 4). Clonidine (12.5 lg/kg)
was administered i.v. 30 min prior to hypoxia. The animals
were ventilated for 1 h under either normoxic condition
(FiO
2
= 0.21) or hypoxic condition; hypoxia was induced
by lowering the FiO
2
to 0.06 for 60 min. At the end of the
experimental period, the animal was sacriced; the cortical
tissue was removed and placed either in homogenization
buffer for isolation of cortical cell membranes or in liquid
nitrogen, and then stored at -80C for biochemical studies.
Preparation of Cytosolic Fraction from the Cerebral
Cortical Tissue
Cerebral cortical tissue was homogenized in 10 vol of
buffer 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS,
158 mM NaCl, 10 mM TrisHCl buffer, pH 7.0, 1 mM
Neurochem Res (2010) 35:7684 77
1 3
EGTA, 1 lg/ml each of aprotinin, leupeptin and pepstatin,
1 mM sodium orthovanadate (SOV), 0.1 mM phenylme-
thylsulphonyluoride (PMSF) and 0.1% IGEPAL. The
homogenate was centrifuged at 1,000g for 10 min. The
supernatant was centrifuged at 100,000g for 60 min and
the supernatant was collected. Protein was determined by
the method of Lowry et al. [29].
Western Blot Analysis of Tyrosine Phosphorylated
Apoptotic Proteins
The protein was brought to a nal concentration of 1 lg/ll
in a modied RIPA buffer (50 mM TrisHCl, pH 7.4,
1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.25% sodium
deoxycholate, 1 mM PMSF, 1 mM Na
3
VO
4
, 1 mM NaF,
and 1 lg/ml each of aprotinin, leupeptin and pepstatin).
Then 5 ll of Laemmli buffer (100 mM TrisHCl pH 6.8,
200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue,
20% glycerol) was added to each 20 lg of mitochondrial
membrane protein mixture. The samples were heated for
5 min at 95C. Equal protein amounts of each sample was
separated by using 10% sodium dodecyl sulfatepoly-
acrylamide gel electrophoresis (SDSPAGE). The proteins
were electrically transferred to nitrocellulose membranes.
The nitrocellulose membranes were blocked with 10%
non-fat dry milk in PBS buffer for 46 h at 4C. The
proteins on the nitrocellulose membranes were then probed
with primary antibodies directed against phosphotyrosine
(p)-Bcl-2, phosphotyrosine (p)-Bclxl, phosphotyrosine
(p)-Bax and phosphotyrosine (p)-bad antibodies (Santa
Cruz Biotech, Santa Cruz, CA) overnight at 4C on a
rocking platform. Immunoreactivity was then detected by
incubation with horseradish peroxidase conjugated sec-
ondary antibody (Rockland, Gilbertsville, PA). Specic
complexes were detected by enhanced chemiluminescence
using the ECL detection system (Amersham Pharmacia
Biotech, Buckinghamshire, UK) and analyzed by imaging
densitometry (GS 700 Imaging Densitometer, Bio-Rad)
using Quantity One Software (Bio-Rad). The data are
expressed as optical density (OD) 9 mm
2
.
Preparation of neuronal nuclei and determination of
nuclear Ca
2?
-inux Neuronal nuclei were isolated and
puried as described by us before [23, 25]. The nuclear
Ca
2?
-inux using Ca
45
was determined as previously
described [23].
Determination of ATP and phosphocreatine ATP and
phosphocreatine concentrations were determined according
to the method of Lamprecht et al. [30].
Statistical analysis Data was analyzed using one way
analysis of variance ANOVA to compare normoxic, hyp-
oxic, and hypoxic-nNOSi groups. A P value of less than
0.05 was considered statistically signicant. All values are
presented as mean standard deviation (SD).
Results
Cerebral cortical tissue hypoxia in newborn piglets was
documented by determining the levels of ATP and PCr in
the cerebral cortical tissue (Fig. 1a). The ATP levels
(lmoles/g brain) in the Nx, Hx, Clo ? Hx groups were
4.3 1.0 (P\0.05 vs. Hx, Clo ? Hx), 0.9 0.8 and
1.5 0.3, respectively. The PCr levels in the Nx, Hx,
Clo ? Hx were 2.7 0.7 (P\0.05 vs. Hx, Clo ? Hx),
0.9 0.2 and 0.9 0.9, respectively. The results dem-
onstrate that brain tissue hypoxia was achieved in the
hypoxic groups as indicated by decrease in cerebral tissue
high energy phosphates ATP and phosphocreatine. The
results also show that the tissue hypoxia within the two
hypoxic groups was comparable.
Ca
2?
-inux (pmoles/mg protein) was 4.96 0.94 in
Nx, 11.11 2.38 in Hx, and 6.23 2.07 in Clo ? Hx
(P\0.05 Nx vs. Hx and Hx vs. Clo ? Hx). The data
(Fig. 1b) demonstrate that clonidine prevents the hypoxia-
*p<0.05 vs. Nx
Nx Clo+Hx
*
*
Nx Hx Clo+Hx
5
4
3
2
1
0
6
5
4
3
2
1
0
6
A
T
P

(

m
o
l
e
s
/
g

b
r
a
i
n
)
P
C
r

(

m
o
l
e
s
/
g

b
r
a
i
n
)
*p<0.05 vs. Nx
Mean
I
n
t
r
a
n
u
c
l
e
a
r

C
a
+
+
-
i
n
f
l
u
x
(
p
m
o
l
e
s
/
m
g

p
r
o
t
e
i
n
)
Normoxia Hypoxia Clonidine+Hypoxia
*
*p<0.05 vs. Normoxia
15
5
0
10
Mean S.D.
S.D.
Hx
*
*
Fig. 1 a Levels of ATP and Phosphocreatine (PCr) in the cerebral
cortex of normoxic, hypoxic and hypoxic-clonidine piglets. ATP and
PCr values are expressed as lmoles/g brain tissue. The data are
expressed as mean SD. b Ca
2?
-inux in neuronal nuclei of the
cerebral cortex of normoxic, hypoxic and hypoxic-clonidine piglets.
Nuclear Ca
2?
-inux is expressed as pmoles/mg protein/min. The data
are expressed as mean SD
78 Neurochem Res (2010) 35:7684
1 3
induced increase in Ca
2?
-inux. The results conrm our
previously published observation.
Representative Western blots of phospho (pTyr)-Bcl-2,
phosphor (p-Tyr)-Bcl-xl, phospho (pTyr)-Bax and phos-
phor (p-Tyr)-Bad for normoxic, hypoxic and hypoxic-clo-
nidine groups are shown in Fig. 2 The results show an
increased expression of total tyrosine phosphorylated Bcl-2
and Bcl-xl in the Hx group indicating increased level of
tyrosine phosphorylated Bcl-2 and Bcl-xl in the cerebral
cortex of newborn piglets during hypoxia. The expression
of Bcl-2 and Bcl-xl did not increase during hypoxia as
previously reported. The results also indicate that clonidine
administration prevents the hypoxia-induced increased
tyrosine phosphorylation of Bcl-2 and Bcl-xl. The results
also show that expression of phosphorylated (p-Tyr) Bax
and Bad proteins was not increased during hypoxia.
The data (Fig. 3a) show that the density (expressed as
optical density 9 mm
2
) of tyrosine phosphorylated p-Bcl-2
was 21.1 1.1 in Nx, 58.9 9.6 in Hx and 29.5 6.4
in Clo ? Hx (P\0.05 vs. Hx). Tyrosine phosphorylated
p-Bcl-xl density was 29.6 1.5 in Nx, 50.6 7.4 in Hx
and 32.1 0.1 in Clo ? Hx (P\0.05 vs. Hx). The data
show that hypoxia results in increased tyrosine phosphor-
ylation of anti-apoptotic proteins Bcl-2 and Bcl-xl in the
cytosolic fraction of the cerebral cortex of newborn piglets.
The results also show that administration of clonidine
prevents the hypoxia-induced increased tyrosine phos-
phorylation of anti-apoptotic proteins.
The data (Fig. 3b) show that the density of tyrosine
phosphorylated p-Bax was 38.6 16.2 in Nx, 46.1 5.5 in
Hx and 41.6 1.9 in Clo ? Hx groups (P = NS). Tyrosine
phosphorylated p-Bad was 66.7 12.8 in Nx, 71.2 6.8 in
Hx and 78.7 22.5 in Clo ? Hx groups (P = NS). The
data show that hypoxia did not increase the tyrosine phos-
phorylation of pro-apoptotic proteins Bax and Bad. Cloni-
dine administration prior to hypoxia had no effect on the
tyrosine phosphorylation of pro-apoptotic proteins. Hypoxia
did not increase the expression of anti-apoptotic proteins
under the same experimental conditions whereas the
expressions of pro-apoptotic proteins increased.
pY- Bax
23 kDa
Bax
pY- Bad
Bad
21 kDa
pY- Bcl-2
Bcl-2
26 kDa
28 kDa
pY- Bcl-xl
Bcl-xl
Normoxia Hx+Clo Hypoxia
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7
1 2 3 4 5 6 7 8
a b c d e f
a b c d e f
a b c d e f
Fig. 2 Representative Western blots of phospho-(pTyr)-Bcl-2
(p-Bcl-2), p-Bcl-xl, p-Bax and p-Bad in the cytosolic fraction of the
cerebral cortex of normoxic, hypoxic and hypoxic-clonidine piglets.
Western blot analysis was performed using anti-phospho tyrosine
(p-Tyr) Bcl-2, anti-phospho tyrosine (p-Tyr) Bcl-xl, anti-phospho
tyrosine (p-Tyr) Bax and anti-phospho tyrosine (p-Tyr) Bad (Santa
Cruz biotechnology, CA) and anti-actin antibody (Chemicon). Protein
Bands were detected using enhanced chemiluminescence detection
system and analyzed by imaging densitometry. Lanes 1, 2 and 3
represent normoxic; lanes 4 and 5 represent Clo ? Hx; lanes 6, 7 and
8 represent hypoxic piglets. Lanes a and b represent normoxic, lanes c
and d represent Clo ?Hx, lanes e and f represent hypoxic piglets
T
y
r
o
s
i
n
e

p
h
o
s
p
h
o
r
y
l
a
t
e
d

B
c
l
-
2

(
O
D
m
m
2
)
*p<0.05 vs. Nx
Mean S.D.
Hx Hx Nx
T
y
r
o
s
i
n
e

p
h
o
s
p
h
o
r
y
l
a
t
e
d

B
c
l
-
x
l

(
O
D
m
m
2
)
100
80
60
40
20
0
100
80
60
40
20
0
*p<0.05 vs. Nx
T
y
r
o
s
i
n
e

p
h
o
s
p
h
o
r
y
l
a
t
e
d

B
a
x

(
O
D
m
m
2
)
T
y
r
o
s
i
n
e

p
h
o
s
p
h
o
r
y
l
a
t
e
d

B
a
d

(
O
D
m
m
2
)
Nx Hx Hx Nx
p=NS
100
80
60
40
20
0
100
80
60
40
20
0
p=NS
Mean S.D.
Clo+Hx Nx Clo+Hx
Clo+Hx Clo+Hx
Fig. 3 a Effect of hypoxia on tyrosine phosphorylation of anti-
apoptotic proteins Bcl-2 and Bcl-xl in cytosolic fraction of the
cerebral cortex of normoxic, hypoxic and hypoxic-clonidine piglets.
The protein density (OD 9 mm
2
) is presented on Y-axis. The data are
expressed as mean SD. b Effect of hypoxia on tyrosine phosphor-
ylation of pro-apoptotic proteins Bax and Bad in cytosolic fraction of
the cerebral cortex of normoxic, hypoxic and hypoxic-clonidine
piglets. The protein density (OD 9 mm
2
) is presented on Y-axis. The
data are expressed as mean SD
Neurochem Res (2010) 35:7684 79
1 3
Discussion
Perinatal hypoxicischemic brain injury continues to be a
signicant cause of long-term neurodevelopmental
impairment in prematurely born infants, for which there is
no specic treatment [31]. Although the etiology of brain
injury in premature infants is complex and multifactorial, a
subset of larger premature infants from 31 to 36 weeks of
gestation with metabolic acidosis on umbilical cord blood
have a high rate of acute clinical encephalopathy [32] and
subcortical gray matter damage [33], and thus may be
candidates for experimental neuroprotective treatments
[32].
Studies at older ages suggest that the early or latent
phase of recovery from hypoxicischemic brain injury is a
critical period when the fate of injured cells is determined
[34]. Consistent with this, it has been reported that exposure
to severe hypoxia induced by reversible occlusion of the
umbilical cord in preterm fetal sheep is associated with
subsequent secondary loss of mitochondrial activity and
seizures, and subcortical neuronal loss after 3 days
recovery [35]. Intriguingly, in the same model blockade of
the neuroinhibitory a
2
-adrenergic receptor for 4 h in the
immediate recovery period, before the known time of sec-
ondary deterioration, signicantly increased hippocampal
damage [36]. Others have reported similar outcomes after
hypoxiaischemia in the newborn rat [37]. Conversely, in
neonatal rodents pretreatment with a
2
-adrenergic receptor
agonists reduced excitotoxic [38, 39] and hypoxicischemic
brain damage [40], while post-treatment improved brain
weight decit after hypoxiaischemia [41], suggesting that
this may be a clinically useful therapy. Partial neuropro-
tection with low dose infusion of clonidine was observed in
the preterm fetal sheep [42].
Previously we have shown that hypoxia results in
increased nuclear Ca
2?
inux and increased activity of
CaM kinase IV which is predominantly located in the
nucleus [16, 17, 23]. Hypoxia resulted in increased phos-
phorylation of cyclic AMP response element binding
(CREB) protein and increased expression of proapoptotic
protein Bax [18, 19]. The present study specically focuses
on investigating the effect of hypoxia on tyrosine phos-
phorylation (post-translational modication) of apoptotic
proteins Bcl-2, Bclxl, Bax and Bad and determining the
mechanism of hypoxia-induced tyrosine phosphorylation
of apoptotic proteins in the cytosolic fraction of the cere-
bral cortex of newborn piglets. In the present study we have
tested the hypothesis that hypoxia results in increased
tyrosine phosphorylation of apoptotic proteins in the
cerebral cortex of newborn piglets and that the increased
tyrosine phosphorylation is mediated by Ca
2?
-inux.
Therefore, administration of a high afnity Ca
2?
-ATPase
inhibitor, clonidine, prior to hypoxia, will prevent the
increased tyrosine phosphorylation of apoptotic proteins in
the cytosolic fraction of the cerebral cortex of newborn
piglets.
The results of the present study show that cerebral
hypoxia results in increased tyrosine phosphorylation of
antiapoptotic proteins Bcl-2 and Bclxl in the cytosolic
fraction of the cerebral cortex of newborn piglets. The data
show that administration of clonidine prior to hypoxia
prevented the hypoxia-induced increased tyrosine phos-
phorylation of Bcl-2 and Bcl-xl. Hypoxia did not increase
the tyrosine phosphorylation of proapoptotic proteins
Bax and Bad. The results also conrmed that clonidine
administration blocked the hypoxia-induced Ca
2?
-inux in
neuronal nuclei indicating the effect of clonidine on Ca
2?
-
inux mechanisms. Furthermore, clonidine administration
did not affect the cerebral levels of high energy phosphates.
These results demonstrate that hypoxia-induced increased
tyrosine phosphorylation of apoptotic proteins is Ca
2?
-
inux-dependent. It addition, the data indicate that hypoxia
results in differential tyrosine phosphorylation of pro-
apoptotic and anti-apoptotic proteins. This may be due to
the structure of proteins itself and exposure of tyrosine
residues for phosphorylation.
The increased tyrosine phosphorylation of anti-apoptotic
proteins Bcl-2 and Bcl-xl will alter their ability to form
heterodimers with pro-apoptotic proteins such as Bax and
Bad or their homodimerization with themselves. We have
previously observed that hypoxia results in increased phos-
phorylation of Bcl-2 in neuronal nuclei of newborn piglets.
The lack of heterodimerization results in increased free Bax
while tyrosine phosphorylation of Bcl-2 or Bcl-xl results in
decreased Bcl-2 and Bax thus leading to an increased ratio of
proapoptotic/anti-apoptotic proteins. The increase proa-
poptotic proteins in the cytosol will activate procaspase-9 to
active caspase-9, the initiator of programmed cell death. The
activated caspase-9 then by proteolytic cleavage activates
procaspase-3 into active caspase-3. Activated caspase-3
leads to break down of a number of cytoskeletal proteins.
Activates caspase-3 by proteolytic cleavage separates cas-
pase-activated DNase (CAD) from its endogenous inhibitor
ICAD. The free CAD then moves into the nucleus to frag-
ment nuclear DNA and subsequent neuronal death. We
propose that during hypoxia increased phosphorylation of
anti-apoptotic proteins that results in loss of their antiapo-
ptotic potential is the potential mechanism of hypoxic neu-
ronal death which is Ca
2?
-inux-dependent. The results
with administration of clonidine prior to hypoxia provide a
proof of principle and mechanism for the use of clonidine
and similar compounds for screening as a neuroprotective
strategy in the hypoxic newborn brain.
Clonidine functions presynaptically as well as postsyn-
aptically. Presynaptically it is associated with inhibition of
glutamate release [43, 44]. Post-synaptically, clonidine
80 Neurochem Res (2010) 35:7684
1 3
decreases N-methyl-D- aspartate (NMDA) receptor-medi-
ated intracellular Ca
2?
entry [45]. It is suggested that
neuroprotection effect of clonidine is most probably related
to its anti-excitotoxic effect [38]. Since the NMDA
receptor channel mediated entry of Ca
2?
activates neuronal
nitric oxide synthase which is postsynaptically bound to
PSD-95 that is associated with the NMDA receptor subunit
NR2B. Therefore, administration of clonidine might pre-
vent the hypoxia-induced activation of nNOS by blocking
NMDA receptor ion channel-mediated Ca
2?
entry.
During hypoxia, the increased activation of nNOS
leading to increased generation of nitric oxide free radicals
may result in activation of EGFR kinase and Src kinase by
inactivating protein tyrosine phosphatases, SH-PTP-1 and
SH-PTP-2. Since all protein tyrosine phosphatases contain
a cysteine residue at their active site, enzyme activity can
be affected by redox mechanisms [4648]. NO free radicals
generated during hypoxia can combine with superoxide
radicals to produce peroxynitrite. The reaction between NO
free radical and superoxide to form peroxynitrite is favored
over the reaction between superoxide and superoxide dis-
mutase [49, 50]. Hypoxia-induced nitration of NMDA
receptor subunits indicates formation of peroxynitrite dur-
ing hypoxia [15]. Therefore, during hypoxia the perox-
ynitrite anion (ONOO
-
), which is structurally similar to a
phosphate group in terms of molecular diameter and ionic
charges, can be easily attracted to the active site loop of
PTP. This loop contains a negatively charged cysteine
residue surrounded by positively charged arginine residues
to accommodate the negatively charged phosphate moiety.
Once attracted to the active site, peroxynitrite will result in
oxidation of the essential cysteine residue and inactivation
of the enzyme. We have also observed that inactivation of
PTP in the presence of sodium nitroprusside (a NO-donor)
is only 1015% of the total activity as compared to
8590% inactivation of the enzyme in presence of perox-
ynitrite. Therefore, we propose that during hypoxia
NO-mediated peroxynitrite-dependent inactivation of SH-
PTP-1 and SH-PTP-2 leads to increased activation of
EGFR and Src tyrosine. The increased activation of EGFR
kinase and Src kinase may lead to increased tyrosine
phosphorylation of antiapoptotic protein during hypoxia,
Thus NO-mediated activation of EGFR and Src kinases is
the potential mechanism of tyrosine phosphorylation of
anti-apoptotic proteins Bcl-2 and Bcl-xl. Blockade of
intracellular Ca
2?
-inux by clonidine thus preventing
nNOS activation leading to tyrosine phosphorylation of
anti-apoptotic proteins is a new mechanism of clonidine
action.
On the other hand, NO-mediated activation of EGFR
kinase may lead to cell proliferation. EGFR has been shown
to translocate to the nucleus upon ligand binding, where it
acts as a transcription activator. Over expression of EGFR
has been detected in many epithelial malignancies including
cancers of the bladder, breast, lung, brain, stomach, prostate
ovary and pancreas. It is overexpressed in high grade gli-
omas and plays an important role in the development of
astrocytes. A number of excellent reviews are available
[5155].
Since clonidine prevents the hypoxia-induced increase
in intranuclear Ca
2?
. It may prevent or attenuate the fol-
lowing cascade of nuclear mechanisms that lead to cell
death. The increased intranuclear Ca
2?
leads to increased
activation of Ca
2?
/calmodulin-dependent protein kinase IV
and results in activation of Ca
2?
-dependent nuclear
mechanisms and activates cascades of post-hypoxic pro-
grammed cell death. We have observed that hypoxia results
in increased activation of CaM kinase IV in neuronal nuclei
of newborn piglets. We have also demonstrated that
administration of nitric oxide synthase (NOS) inhibitor,
N-nitro-L-arginine (NNLA) prevented the hypoxia-induced
increase in CaM kinase IV activity, increase in CREB
phosphorylation, increase in the expression of pro-apop-
totic protein Bax and increased fragmentation of nuclear
DNA [1719]. The increased expression of pro-apoptotic
proteins leading to increased levels in mitochondrial
membranes. The expression of Bcl-2 did not increase in
nuclei, mitochondria or cytosol. The increased ratio of Bax/
Bcl-2 will increase the permeability of the mitochondrial
membrane. In addition, the increased Bax may also
increase the activation of caspase-9 within the mitochon-
dria and may lead to proteolysis of mitochondrial proteins.
Increased permeability and increased activation of caspase-
9 may result in activation of mitochondrial endonuclease
leading to fragmentation of nuclear DNA. Therefore, the
results of our previous studies indicated that NO-mediated
alterations in nuclear events leading to increased expres-
sion of Bax and alteration in the ratio of Bax/Bcl-2 protein
in mitochondria results in mitochondrial membrane per-
meability and activation of caspase-9 in mitochondria. The
NO-mediated increased expression of proapoptotic protein
Bax that subsequently alters the mitochondrial Bax mem-
brane concentration thus altering mitochondrial membrane
permeability and activation of caspase-9 within mito-
chondria are our new proposed mechanisms of hypoxic
neuronal injury. Thus blockade of intranuclear Ca
2?
by
clonidine can have consequences through blocking CaM
kinase IV activation as well as preventing proapoptotic
protein expression.
Bax overexpression or an increase in the ratio of Bax to
Bcl-2, leads to programmed cell death [4]. Up-regulation of
Bax and/or down-regulation of Bcl-2 mRNA or protein
levels have been observed in several experimental models
including transient global ischemia [5658]; the expression
of Bax and Bcl-2 genes may be regulated by p53 [59].
Using a permanent middle cerebral artery occlusion model,
Neurochem Res (2010) 35:7684 81
1 3
DNA breaks occurred within 6 h and levels of Bax mRNA
signicantly increased within the infarcted hemisphere,
indicating a shift in the gene expression ratio of Bcl-2 to
Bax [53]. Using immunohistochemistry before and after
10 min of global ischemia, there were high levels of Bax,
low levels of Bcl-2, and DNA-strand breaks in the same
population of neurons found to be degenerating morpho-
logically [58]. Neurons with elevated Bax levels almost
uniformly had morphologic evidence of ischemic degen-
eration with apoptotic features including nuclear DNA
fragmentation [60].
Bax activated caspase-9 that activates caspase-3 may
result in caspase-activated DNAse dependent degradation
of nuclear DNA. In addition, NO may also lead to free
radical-induced mitochondrial DNA damage. Programmed
cell death is also assessed by cleavage of genomic DNA and
has been shown to occur in the brain following ischemia and
oxygen deprivation [57, 61, 62]. In the newborn piglet,
fragmentation of neuronal genomic DNA occurs following
1 h of hypoxia, and the degree of fragmentation correlates
with the severity of cerebral hypoxia [63]. Two distinct
patterns of DNA degradation during hypoxicischemic
brain cell death have been observed [64]. First, there is a
random cleavage of the DNA, including nick formation in a
single strand that appears as a smear after agarose gel
electrophoresis. The second pattern is characterized by
double-stranded DNA degradation into oligonucleosomal
fragments that appears as a ladder after agarose gel elec-
trophoresis. The ladder pattern of genomic DNA fragmen-
tation has been associated with programmed cell death [65,
66], although morphologic characteristics of programmed
cell death are not always associated with ladder-type DNA
fragmentation [67] and similarly ladder-type fragments
have been observed in some necrotic cells [68]. Mecha-
nistically, the cleavage of DNA at its internucleosomal
linker regions is produced by a specic endonuclease that is
Ca
2?
dependent [65, 69].
In view of these observations, clonidine can prevent cell
death by blocking intracellular Ca
2?
-dependent events as
well as intranuclear Ca
2?
-dependent cascades. First, the
NMDA receptor mediated intracellular Ca
2?
potentially
initiates a number of reactions leading to increased free
radical generation via a number of enzymatic pathways
such as Ca
2?
activation of phospholipase A
2
, causing
release of arachidonic acid which then can be metabolized
by cyclooxygenase and lipoxygenase, the conversion of
xanthine dehydrogenase to xanthine oxidase by Ca
2?
-
dependent activation of proteases and activation of nitric
oxide synthase by Ca
2?
to further generate NO leading to
formation of peroxynitrite and oxygen free radical species.
Therefore, clonidine can prevent the free radicals-induced
increase in peroxidation of cellular and sub-cellular
membranes leading to necrotic cell death. Second, by
blocking the hypoxia-induced increase in intranuclear
Ca
2?
, clonidine can prevent the nuclear cascade of pro-
grammed neuronal death. Increased intranuclear Ca
2?
may
activate Ca
2?
-dependent endonucleases leading to DNA
fragmentation. In addition, increased intranuclear Ca
2?
can
activate CaM kinase IV in the nucleus leading to increased
phosphorylation of cyclic AMP response element binding
protein (CREB) resulting in increased transcription of
apoptotic genes such as Bax and initiating the early events
of DNA fragmentation and programmed cell death.
In summary: These results show that cerebral tissue
hypoxia results in increased tyrosine phosphorylation of
antiapoptotic proteins Bcl-2 and Bcl-xl in the cytosolic
fraction of the cerebral cortex of newborn piglets. Admin-
istration of clonidine prior to hypoxia prevented the hypoxia-
induced increased tyrosine phosphorylation of Bcl-2 and
Bcl-xl. Tyrosine phosphorylation of proapoptotic proteins
Bax and Bad did not change. Clonidine also prevented the
hypoxia-induced increase in nuclear Ca
2?
-inux. We con-
clude that the mechanism of increased tyrosine phosphory-
lation of antiapoptotic proteins Bcl-2 and Bcl-xl during
hypoxia is Ca
2?
-inux dependent. We propose that post-
synaptic generation of nNOS-derived NO, by inhibiting
protein tyrosine phosphatases (SH-PTP-1 and SH-PTP-2)
mediates the increased activation of EGFR and Src kinases
that leads to increased tyrosine phosphorylation of anti-
apoptotic proteins Bcl-2 and Bcl-xl resulting in loss of their
antiapoptotic potential and hypoxic neuronal death.
Acknowledgments This study was supported by the National
Institute of Health grants HD-20337 and R56 HD038079. The authors
thank Mrs. Anli Zhu for her expert technical assistance.
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