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Introduction
Neuronal injury, which is induced by cerebral ischemia/reperfusion, is a very complex process associated with excitotoxicity,
oxidative stress, apoptosis, and variations in gene expression or the activation of
kinase[1]. Scutellarin (Scu), a known flavone
glycoside, is a major active ingredient in breviscapine, which is a mixture extracted from the Chinese herb,
Erigeron breviscapus. Scu is used in clinics
to treat ischemic cerebrovascular diseases in China (Figure 1).
It is reported that Scu has protective effects against neuronal damage induced by chemical hypoxia or hydrogen peroxide
through interaction with a wide variety of targets,
including antioxidative action and attenuating neuron
damage[2,3]. There is no sufficient evidence proving the effects of Scu against ischemia/reperfusion injury, especially at molecular and cellular level.
Therefore, we used the oxygen and
glucose deprivation followed by reperfusion (OGD-Rep)
model, an in vitro model of cerebral ischemia/reperfusion, to
investigate the effects of Scu on protein kinase C
(PKC)g and PC12 cell injury.
PKCg, a serine/threonine kinase expressed exclusively in
neurons of the brain and spinal cord, is activated by
Ca2+, diacylglycerides (DAG) and free fatty acids
(FFA)[4]. Therefore, this isozyme plays a central nervous
system-specific role in mediating the response to ischemic/reperfusion
injury. Ischemia causes extensive membrane phospholipids
hydrolysis with accumulation of FFA, DAG, and other
breakdown products[5]. The changes in membrane phospholipids
may modulate the function of membrane proteins and also
shift the distribution of PKCg. Furthermore, during ischemia,
irreversible processes including mitochondrial collapse, rapid
energy depletion, and ion pump failure result in large
increases in intracellular calcium, leading to the activation and
increased expression of PKCg[6,7]. Multiple reports now
suggest that PKC may be involved in a positive feedback loop
to potentiate NMDAR activity, worsening calcium loading,
mitochondrial dysfunction, and promoting cell
death[8,9]. So as reported earlier,
PKCg is most effectively translocated to membranes and the nucleus during cerebral ischemia, and
plays an important role in neuronal
damage[10]. Previous studies have suggested that breviscapine inhibits the
activity of PKC. So we estimated that the protective effects of
Scu on neuron apoptosis might be related to this inhibitive
effect on PKC, especially on PKCg
[11,12].
PC12 cells have been extensively used as an in
vitro model system to study the mechanisms of neuronal cell death
after ischemic insult, and to develop potential
neuropro-tective agents. Ischemic stroke causes a significant amount
of cell damage resulted from an insufficient supply of
glucose and oxygen to brain
tissues[13,14]. PC12 cells are subjected to an initial short phase of OGD-Rep. The initial
phase of OGD mimics the lack of oxygen and glucose
supply, while the prolonged phase of OGD-Rep reflects the
reperfusion of oxygen and glucose supply to the injured
brain[15,16].
In the present study, we investigated whether Scu, the
major ingredient of Erigeron breviscapus, had neuronal
protective effects against ischemia/reperfusion injury by
influencing PKCg. So we used undifferentiated rat
pheochromocytoma PC12 cells and the OGD-Rep model to study the
effects of Scu on cell survival, apoptosis,
[Ca2+]i, and the expression and activation of
PKCg.
Materials and methods
Chemicals and drugs Scu (purity >92%) was supplied
by Kunming Institute of Botany, Chinese Academy of
Science (Kunming, Yunnan, China). Fura-2 acetoxymethyl
ester (Fura-2AM) and propidium iodide (PI) staining
solution were purchased from BD PharMingen (San Diego, CA,
USA). TRIzol Reagent was purchased from Invitrogen
(Carlsbad, CA, USA). M-MLV reverse transcriptase was
purchased from Promega (Madison, WI, USA).
Diphenyl-tetrazolium bromide (MTT) and monoclonal anti-protein
kinase Cg (PKCg) was purchased from Sigma (St Louis, MO,
USA). The SuperSignal West Pico Trial Kit was purchased
from Pierce (Rockford, MA, USA). Dulbecco's modified
Eagle's medium (DMEM) was purchased from Gibco (Carlsbad, CA USA). LDH activity kits were purchased from
Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
All other chemicals and solvents in experiments were of
analytical grade.
Cell culture The PC12 cell line was obtained from
American Type Culture Collection (ATCC, Manassas,
VA,USA) and maintained at 37 °C in a humidified atmosphere
containing 5% CO2 in high glucose DMEM supplemented
with 10% heat-inactivated fetal calf serum, 5%
heat-inactivated horse serum, 100 kU/L penicillin, and 100 mg/L
streptomycin.
PC12 ischemic model The PC12 cells were grown as
monolayers in tissue culture flasks under normal conditions.
To initiate OGD, the cell culture medium was removed and
the cells were washed twice with glucose-free DMEM, and
incubated in the same volume glucose-free DMEM
containing Scu (10, 30, and 100 μmol/L). The cell culture was
incubated at 37 °C in an oxygen-free incubator (95%
N2 and 5% CO2) for 4 h (OGD). Then the OGD glucose was
added to attain a final concentration of 4.5 mg/L, followed
by incubation for 20 h (OGD-Rep) in normal
conditions[17]. Scu was freshly prepared as stock solution with dimethyl
sulfoxide (DMSO) and diluted with glucose-free DMEM. Less
than 0.1% DMSO has no protective or toxic effects when
used alone.
Assay of cell survival and cell damage Cell survival was
evaluated by MTT assay. After reperfusion, MTT solution
in phosphate-buffered saline was added to attain a final
concentration of 0.5 g/L, then incubation continued for 4 h.
Finally, the media was removed and an equal volume of
dimethylfuramide was added. After the mixtures were kept
for 10 min, the amount of MTT was quantified by
determining its absorbance at 570 nm using a Spectra MAX 190
micro-plate reader (Molecular Devices Inc, USA). The
amount of LDH released by the cells was determined using
an LDH activity assay kit according to manufacturer's
instructions. LDH activity is proportional to the rate of
pyruvate loss, which was measured at 440 nm. LDH activity in
the cells was determined after incubation in a hypotonic
solution containing 1% Triton X-100, at pH 7.4.
Flow cytometric detection of apoptosis cells
The cells were collected and resuspended in ice-cold ethanol (70%),
then fixed at 4 °C for 24 h and resuspended in 1 mL DNA
staining reagent (RNase 50 mg/mL, 0.1% Triton X-100, 0.1
mol/L EDTA, and 50 mg/mL PI). Red fluorescence was
detected by using a FACS 440 flow cytometer (Becton Inc,
USA). Ten thousand cells in each sample were analyzed and
the percentage of apoptotic cell accumulation in the
sub-G1 peak was calculated.
Evaluation of DNA laddering The total DNA was
extracted according to a previous
study[18]. The DNA pellet was resuspended in loading buffer (10 mmol/L Tris, 1
mmol/L EDTA, 5% glycerol, and 0.25% bromophenyl blue, pH
7.5), and the entire sample was then subjected to
electrophoresis on a 2% agarose gel using a TBE running buffer
(89 mmol/L Tris, 89 mmol/L boric acid, and 2 mmol/L
EDTA, pH 8.3) containing 0.5 g/L ethidium bromide. The
fluorescence intensity of the DNA bands was measured
using the Bio-Rad Molecular Imaging System (Bio-Rad,
USA).
Measurement of [Ca2+]i
After reperfusion, the PC12 cells were loaded with 5
mmol/L (final concentration) Fura-2AM, 0.1% DMSO and 1% BSA for 30 min at room
temperature in dark conditions, then in a humidified
incubator for 30 min at 37 °C. Fluorescence measurement was
carried out with an F-4500 fluorescence
spectrophotometer (Hitachi, Japan). Fura-2AM-loaded PC12 cells were
exposed sequentially to an excitation wavelength of 340
nm and 380 nm (bandwidth 10 nm), and the emission signal
was monitored at a wavelength of 510 nm (bandwidth 10
nm). Fluorescence ratios were converted into calcium
concen-trations by using the following equation:
[Ca2+]i=Kd
(R-Rmin)/(Rmax
-R)B.
Preparation of total RNA and semiquantitative
RT-PCR The cells were collected and the total RNA was extracted
using TRIzol reagent according to the manufacturer's
instructions. RNA concentration was estimated by
absorbance at 260 nm using a UV spectrophotometer. The total
RNA of each sample was reverse-transcribed into cDNA
using the reverse transcription system. The cDNA was
amplified with the following specific primers:
PKCg: 5'-TTG ATG GGG AAG ATG GGG AGG-3' (upstream) and 5'-GAA
ATC AGC TTG GTC GAT GCT G-3' (downstream).
Amplifications were performed as follows: 30 cycles, at 94 °C for 30
s, 58 °C for 1 min, and 72 °C for 1 min. The PCR products
were normalized in relation to standards of GAPDH mRNA.
Preparation of total PKCg protein and
subcellular fractionation The cells were collected, and the total
PKCg protein was extracted using TRIzol reagent according to the
manufacturer's instructions. The subcellular fraction of
PKCg was extracted according to a previous
study[19]. Twenty µL of each sample was solubilized with NaOH (1 mol/L) for
protein concentration determination.
Polyacrylamide gel electrophoresis and Western blot
analysis Twenty µg protein per lane was subjected to 10%
SDS-PAGE electrophoresis for 2 h. Resolved proteins were
electroblotted onto NC membranes for 2 h at 250 mA. The
blots were incubated with TBST (0.1 mol/L Tris buffer, pH 7.4,
0.9% NaCl, and 0.1% Tween 20) containing 5% dry skim milk,
followed by incubation with TBS (0.1 mol/L Tris buffer, pH
7.4, 0.9% NaCl) containing anti-PKCg monoclonal antibodies and
3% BSA for 2 h, and incubation with secondary antibody
streptavidin-horseradish peroxidase-conjugated affinity
mouse anti-rat IgG (1:10000 dilution) for 2 h. The bound
immunoproteins were detected by enhancer
chemiluminescent assay, and band intensity was quantified with a
densitometric scanner.
Statistical analysis Data were expressed as mean±SD.
Statistical analysis was performed by ANOVA, with
P<0.05 as the significant level.
Results
Effect of Scu on PC12 cell viability and cell damage
The effect of Scu alone on cell viability was studied using MTT
assay. The results showed Scu (5_200 µmol/L) had no
effects on PC12 cells. After OGD-Rep, PC12 cell viability
decreased significantly (<20%) and LDH release increased from
6.77% to 12.30%. The addition of Scu at the concentration
of 10_100 µmol/L significantly attenuated the decrease of
cell viability and the increase of LDH release. LDH leakage
was expressed as the percentage of the total LDH activity
(LDH in the medium+LDH in the cells), according to the
following equation: %LDH=(LDH activity in the medium/total
LDH activity)×100 (Table 1).
Effect of Scu on cell apoptosis The
sub-G1 peak in the flow cytometry detection was considered an indicator of cell
apoptosis. The data was presented as the percentage of
apoptotic cells. OGD-Rep significantly induced the
sub-G1 peak, indicating an apoptotic cell accumulation of 33.34%.
The addition of Scu at the concentration of 10_100 µmol/L
significantly attenuated OGD-Rep-induced apoptosis
(25.88%, 21.67%, and 13.85%; Figure 2). The data is in
agreement with the results of the DNA ladder analysis (Figure 3).
Effect of Scu on [Ca2+]i
OGD-Rep induced a significant increase in the resting levels of
[Ca2+]i from 646±92 to 2112±
376 nmol/L. This increase was significantly inhibited by Scu
at 10, 30, and 100 µmol/L (Figure 4).
Effect of Scu on PKC mRNA expression in PC12 cells
After OGD-Rep, there was a significant increase in
PKCg mRNA expression. At the concentration of 10_100 µmol/L,
Scu was less potent in antagonizing the OGD-Rep-induced
increase of PKCg mRNA expression (P>0.05; Figure 5).
Effects of Scu on protein expression and subcellular
distribution of PKCg in PC12 cells The translocation of
PKCg from cytosol to the membrane and the nucleus is a hallmark
of PKCg activation. Western blotting experiments were
performed to detect the protein level of total
PKCg proteins and PKCg in cytosol, the membrane, and the nucleus of PC12
cells. Our results showed that neither reperfusion nor
treatment of Scu changed the expression of PKCg at protein level
(P>0.05), but in OGD-Rep injury, the protein level of
PKCg decreased in cytosol and increased in the membrane and
nucleus significantly. Scu at the concentration of 100
μmol/L significantly attenuated the decrease of the protein
level of PKC in cytosol and the increase of the protein level of
PKCg in the membrane and nucleus, whereas Scu at the
concentration of 30 μmol/L only attenuated the increase of the protein
level of PKCg in the membrane and nucleus (Figure 6;
Table 2).
Discussion
Stroke occurs when local thrombosis, embolic particles,
or the rupture of blood vessels interrupts the blood flow to
the brain. Excitotoxicity, oxidative stress, and apoptosis
propagates through a distinctive and mutually-exclusive
signal transduction pathway and contributes to neuronal
injury following
ischemia/reperfusion[20]. In the present study,
we demonstrated that Scu (10_100 µmol/L) significantly
increased cell survival (by MTT assay) and reduced LDH
release after OGD-Rep. Therefore, Scu has a protective
effect against PC12 cell injury induced by OGD-Rep.
Apoptosis that often occurs during the developmental
process is an additional route to pathological neuronal death
in the mature nervous system during ischemia/reperfusion,
and drugs inhibiting those changes attenuate neuronal
injury following
ischemia/reperfusion[21,22]. The alteration of
various apoptosis-associated genes and proteins, calcium
imbalance, and oxidative stress contribute to apoptosis
during ischemia/reperfusion. The results in our study indicate
that the OGD-Rep-induced PC12 cell death, in part due to
apoptosis and Scu (10_100 µmol/L), decreased the rate of
apoptosis. Therefore, it is tempting to suggest that the
neuroprotective effect of Scu is related to a reduction in
apoptosis following ischemia/reperfusion.
Ca2+ is a ubiquitous intracellular messenger, the
intracellular concentration of which is tightly regulated in order to
efficiently control signaling
mechanisms[23,24]. The selective accumulation of intracellular
Ca2+ following OGD-Rep injury can cause neuronal apoptosis through a mechanism that
involves the excessive release of glutamate and the activation
of several calcium-dependent/activated enzymes such as
proteases, protein kinases, phospholipases, NO syn-theses,
and endonucleases[25,26]. Our study shows that OGD-Rep
can significantly elevate the level of intracellular
Ca2+. This elevation could be attenuated by Scu at concentrations of
10_100 µmol/L. Previous studies in our laboratory on the
direct effects of Scu on calcium using the intracellular cation
measurement system, with the help of the
Ca2+-indicator Fura-2AM, show that Scu had no inhibitory effect on the increase
of [Ca2+]i induced by ATP and high
K+ in PC12 cells. It was reported that Scu could scavenge radicals and inhibit lipid
peroxidation, so we thought the inhibitive effect of Scu on
[Ca2+]i might be related to its antioxidation
activity[11]. PKCg is activated by
Ca2+, and the activation of PKCg also
increases intracellular Ca2+. Therefore, Scu might influence
PKCg by decreasing intracellular Ca2+ following OGD-Rep.
PKC is activated by stroke and plays a damaging role
during stroke. The mechanisms of PKC activation are
multifactorial: ischemia/reperfusion induces an influx of
calcium and sodium into the cell, leading to the release of
intracellular calcium stores, lipid peroxidation, and the
generation of free radicals[27,28]. These events lead to increased
expression and activation of multiple PKC isozymes. It was
reported that the expression of PKCg increases over a period
of postischemic reperfusion[29]. However, other studies
showed that the expression of PKCg decreased during an
extended reperfusion[30]. It was estimated that the alteration
of the expression depended on the ischemic model and the
period of reperfusion used in the studies. A previous study
also showed that Scu could inhibit PKCg activity in the rat
MCAO model. PKCg activity is regulated by 2 distinct
mechanisms: by phosphorylation, which regulates the
active site, and subcellular localization of the enzyme; and by
second messengers (Ca2+ and DAG) which promote
PKCg membrane association. In our previous study, we found
that Scu inhibited the increase of
[Ca2+]i and oxidative stress,
both of which influence the translocation of PKCg after
OGD-Rep, so we guess that the Scu inhibited the activity of
PKCg through inhibiting the translocation of PKCg by influencing
the cofactors Ca2+ and DAG. The results showed that the
expression of PKCg increased significantly following
OGD-Rep injury. Scu had no significant effect on this elevation at
concentrations of 10_100 µmol/L (P>0.05), but the results of
Western blotting showed that the total PKCg protein did not
change after OGD-Rep. It is thought that the
membrane-bound PKCg, compared to the cytosolic form, is more
sensitive to proteolysis, thus the degradation of
PKCg increased during reperfusion because of the activation of proteinase
by the elevation of [Ca2+]i.
Although reperfusion increased the expression of
PKCg at the mRNA level, it did not alter the expression of
PKCg at the protein level following OGD-Rep. Selective changes in the redistribution of
PKCg occurred after ischemic injury and reperfusion, but those
changes in PKCg activity are still not clear. Several reports
have demonstrated that PKCg becomes inactive and
down-regulated, whereas others report that PKCg is activated
specifically during reperfusion[19,31]. Activation of the
PKCg leads not only to their translocation from cytosol to the plasma
membrane, but also to various intracellular membranes and
to the cell nucleus[32,33]. Nuclear translocation of
PKCg is of particular importance because it may enable
PKCg to exert a direct role in nuclear signaling by phosphorylating nuclear
substrates, including DNA-modifying enzymes or
transcription factors. In our study, there was a significant increase of
PKCg in both the membrane and nucleus, accompanied with
a significant decrease in cytosol following OGD-Rep-induced
injury. Scu significantly attenuated the translocation of
PKCg to the membrane and nucleus at the concentration of 30 and
100 µmol/L. Scu also significantly attenuated the decrease
in cytosol at the concentration of 100 µmol/L. The data
showed that Scu, which attenuated the increase of
[Ca2+]i and oxidative stress, could protect PC12 cells against
OGD-Rep-induced injury as a PKCg inhibitor through inhibiting
the activation of PKCg, rather than the expression of
PKCg. However, there is no evidence that the effect of Scu on
PKCg is related to phosphorylation, the key step of the
translocation of PKCg. Further investigation is needed to show the
relationship between Scu and phosphorylation.
In conclusion, these results demonstrate that due to its
antioxidation activity against ischemia/reperfusion-induced
oxidative stress, Scu attenuates the increase of
[Ca2+]i and then inhibits the translocation of
PKCg, leading to a decrease in the rate of apoptosis, and possessing a significant
protective effect against OGD-Rep-induced injury in PC12 cells.
References
1 Seok JW, Doo YK, Byoung JG. Cellular and molecular pathways
of ischemic neuronal death. J Biochem Mol Biol 2002; 35:
67_86.
2 Hong H, Liu GQ. Protective against hydrogen peroxide-induced
cytotoxicity in PC12 cells by scutellerin. Life Sci 2004; 74: 2959_73.
3 Hu XM, Zhou MMOL, Zeng FD. Neuroprotective effects of
scutellarin on rat neuronal damage induced by cerebral ischemia/
reperfusion. Acta Pharmacol Sin 2005; 26: 1454_9.
4 Saito N, Shirai Y. Protein kinase C gamma (PKC gamma):
function of neuron specific isotype. J Biochem (Tokyo). 2002; 132:
683_7.
5 Bazan NG, Free arachidonic acid and other lipids in the nervous
system during early ischemia and after electroshock. Adv Exp
Med Biol 1976; 72: 317_35.
6 Lipton P. Ischemic cell death in brain neurons. Physiol Rev 1999; 79:
1431_568.
7 Matsumoto S, Shamloo M, Matsumoto E, Isshiki A, Wieloch T.
Protein kinase C-gamma and calcium/calmodulin-dependent
protein kinase II-alpha are persistently translocated to cell
membranes of the rat brain during and after middle cerebral artery
occlusion. J Cereb Blood Flow Metab 2004; 24: 54_61.
8 Chakravarthy BR, Wang J, Tremblay R, Atkinson TG, Wang F,
Li H, et al. Comparison of the changes in protein kinase C
induced by glutamate in primary cortical neurons and by
in vivo cerebral ischaemia. Cell Signal 1998; 10: 291_5.
9 MacDonald JF, Kotecha SA, Lu WY, Jackson MF. Convergence
of PKC-dependent kinase signal cascades on NMDA receptors.
Curr Drug Targets 2001; 2: 299_312.
10 Cardell M, Bingren H, Wieloch T, Zivin J, Saitoh T, Saitoh X.
Protein kinase C is translocated to cell membranes during
cerebral ischemia. Neurosci Lett 1990; 119: 228_32.
11 Yu G, Lou Y, Peng GG, Dong WW. Effect and mechanism of
protein kinase C inhibitor on cerebral ischemia in rats. Stroke
Nervous Disease 2002; 9: 321_4.
12 Shuai J, Dong WW. Experimental research of PKC inhibitor,
erigeron breviscapus on the ischemic/reperfusion brain injury.
Chin Pharmacol Bull 1998; 14: 77_9.
13 Siesjo BK. Pathophysiology and treatment of focal cerebral
ischemia. Part I: pathophysiology. J Neurosurg 1992; 77:
169_84.
14 Siesjo BK. Pathophysiology and treatment of focal cerebral
ischemia. Part II: mechanisms of damage and treatment. J
Neurosurg 1992; 77: 337_54.
15 Abu RS, Trembovler V, Shohami E, Lazarovici P. A tissue culture
ischemic device to study eicosanoid release by
pheochromocytoma PC12 cultures. J Neurosci Methods 1993; 50: 197_203.
16 Tabakman RP, Lazarovici P, Kohen R. Neuroprotective effects
of carnosine and homocarnosine on pheochromocytoma PC12
cells exposed to ischemia. J Neurosci Res 2002; 68: 463_69.
17 Tabakman R, Jiang H, Shahar I, Arien-Zakay H, Levine RA,
Lazarovici P. Neuroprotection by NGF in the PC12
in vitro OGD model: involvement of mitogen-activated protein kinases
and gene expression. Ann N Y Acad Sci 2005; 1053: 84_96.
18 Oberdoerster J, Kamer AR, Rabin RA. Differential effect of
ethanol on PC12 cell death. J Pharmacol Exp Ther 1998; 287:
359_65.
19 Huang HM, Weng CH, Ou SC, Hwang T. Selective subcellular
redistributions of protein kinase C isoforms by chemical hypoxia.
J Neurosci Res 1999; 56: 668_78.
20 Lopez-Neblina F, Toledo AH, Toledo-Pereyra LH. Molecular
biology of apoptosis in ischemia and reperfusion. J Invest Surg
2005; 18: 335_50.
21 Fukunaga K, Ishigami T, Kawano T. Transcriptional regulation
of neuronal genes and its effect on neural functions: expression
and function of forkhead transcription factors in neurons. J
Pharmacol Sci 2005; 98: 205_11.
22 Fujimura M, Tominaga T, Chan PH. Neuroprotective effect of
an antioxidant in ischemic brain injury: involvement of neuronal
apoptosis. Neurocrit Care 2005; 2: 59_66.
23 Gill DL, Ghosh TK, Mullaney JM. Calcium signaling
mechanisms in endoplasmic reticulum activated by inositol
1,4,5-trisphosphate and GTP. Cell Calcium 1989; 10: 363_74.
24 Carafoli E. Calcium pump of the plasma membrane. Physiol
Rev 1991; 71: 129£53.
25 Gwag BJ, Canzoniero LM, Sensi SL, DeMaro JA, Koh JY, Goldberg
MP, et al. Calcium ionophores can induce either apoptosis or
necrosis in cultured cortical neurons. Neuroscience 1999; 90:
1339_48.
26 Verkhratsky A, Toescu EC. Endoplasmic reticulum
Ca2+ homeostasis and neuronal death. J Cell Mol Med 2003; 7: 351_61.
27 Katsura K, Kurihara J, Siesjo BK, Wieloch T. Acidosis enhances
translocation of protein kinase C but not
Ca2+/calmodulin-dependent protein kinase II to cell membranes during complete
cerebral ischemia. Brain Res 1999; 849: 119_27.
28 Newton AC. Protein kinase C: structure, function, and regulation.
J Biol Chem 1995; 270: 28 495_8.
29 Savithiry S, Kumar K. The mRNA levels of Ca-independent
forms of protein kinase C in postischemic gerbil brain by
Northern blot analysis. Mol Chem Neuropathol 1994; 21: 1_11.
30 Zablocka B, Maternicka K, Zalewska T, Domanska-Janik K.
Expression of Ca2+-dependent (classical) PKC mRNA isoforms
after transient cerebral ischemia in gerbil hippocampus. Brain
Res 1998; 779: 254_8.
31 Selvatici R, Marino S, Piubello C, Rodi D, Beani L, Gandini E,
et al. Protein kinase C activity, translocation, and selective isoform
subcellular redistribution in the rat cerebral cortex after
in vitro ischemia. J Neurosci Res 2003; 71: 64_71.
32 Asaoka Y, Nakamura S, Yoshida K, Nishizuka Y. Protein kinase
C, calcium and phospholipid degradation, Trends Biochem
Sci1992; 17: 414_7.
33 Buchner K. Protein kinase C in the transduction of signals
toward and within the cell nucleus, Eur J Biochem 1995; 228:
211_21.
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