The Role of Protein Kinase C in Cerebral Ischemic
and Reperfusion Injury
Rachel Bright, PhD; Daria Mochly-Rosen, PhD
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Background and Purpose—Stroke is a leading cause of disability and death in the United States, yet limited therapeutic
options exist. The need for novel neuroprotective agents has spurred efforts to understand the intracellular signaling
pathways that mediate cellular response to stroke. Protein kinase C (PKC) plays a central role in mediating ischemic and
reperfusion damage in multiple tissues, including the brain. However, because of conflicting reports, it remains unclear
whether PKC is involved in cell survival signaling, or mediates detrimental processes.
Summary of Review—This review will examine the role of PKC activity in stroke. In particular, we will focus on more
recent insights into the PKC isozyme-specific responses in neuronal preconditioning and in ischemia and reperfusioninduced stress.
Conclusion—Examination of PKC isozyme activities during stroke demonstrates the clinical promise of PKC isozymespecific modulators for the treatment of cerebral ischemia. (Stroke. 2005;36:2781-2790.)
Key Words: cerebral infarction 䡲 cerebral ischemia 䡲 neuroprotection
S
troke is the third leading cause of death in the United
States, with ⬎700 000 new incidents occurring each
year.1 Yet, we can claim only a modest understanding of the
complex network of cellular processes that regulates cerebral
injury. Although multiple agents have demonstrated efficacy
in reducing stroke injury in preclinical studies, the only
currently approved drug for stroke patients is a thrombolytic,
tissue plasminogen activator. However, the narrow therapeutic window (3 hours after stroke onset) and associated risks,
including hemorrhage,2 translate to limited therapeutic use;
⬍2% of stroke patients in the United States receive tissue
plasminogen activator.3 The great need for a new generation
of agents that confer neuroprotection against ischemic/reperfusion injury and extend the therapeutic window is clear.
considered “at-risk” tissue, affected by multiple stresses
including regional glutamate and potassium diffusion and
peri-infarct depolarizations emanating from the ischemic
core.7,8 Importantly, the effects of reperfusion, the return of
blood flow into an ischemic area after arterial recanalization,
may contribute significantly to stroke damage.4,9
It is now recognized that ischemia and reperfusion initiate
different intracellular responses.5 During reperfusion, the
inability of impaired mitochondria to use oxygen in electron
transport results in reduction in cellular ATP levels, production of reactive oxygen species (ROS), expression and activation of proapoptotic signaling intermediates, and initiation
of inflammatory processes.4,10 Reversing or halting some of
these processes can reduce damage,11–13 suggesting that
delivering neuroprotectants, even at reperfusion, may lead to
improved neurological outcome.
Salvage of “at-risk” tissue depends on reversing or
arresting detrimental intracellular processes that control
propagation of the infarct beyond the necrotic core. In
tissue with residual energy levels, such as the ischemic
penumbra, rapid changes occur in the activity of many
different signaling paths, involving diverse protein kinase
families. Alterations in the expression or activity of
calcium/calmodulin-dependent protein kinase II, mitogenactivated protein kinase (MAPK) family members c-Jun
N-terminal kinase and extracellular signal-regulated kinase
(ERK), protein kinase B (Akt), and protein kinase C (PKC)
suggest that multiple kinases participate in the response of the
tissue to ischemia and reperfusion.14 –18 The role of PKCs in
Molecular Mechanisms of Cerebral Ischemic
and Reperfusion Injury
Multiple cellular processes are rapidly activated in response
to ischemic/reperfusion-induced stress. The ischemic core, a
region of tissue that is immediately distal to an occluded
artery, undergoes rapid, anoxic cell death within minutes of
ischemia onset. Irreversible processes including mitochondrial collapse, rapid energy depletion, and ion pump failure
result in large increases in intracellular calcium, extracellular
potassium, and edematous cell swelling, characteristic of
necrotic cell death.4,5 However, in the ischemic penumbra, or
“shadow” surrounding the core, metabolism and intracellular
signaling cascades are maintained partly by hypoperfusion
from a diminished collateral blood supply.6 The penumbra is
Received March 7, 2005; final revision received July 21, 2005; accepted August 18, 2005.
From the Stanford University School of Medicine, California.
Correspondence to Daria Mochly-Rosen, Stanford University School of Medicine, CCSR, Room 3145A269 Campus Dr, Stanford, CA 94305-5174.
E-mail [email protected]
© 2005 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000189996.71237.f7
2781
2782
Stroke
December 2005
mediating stroke injury has received particular attention. PKC
activity has been seen in ischemic injury in multiple tissues,
including heart,19 liver,20 and kidney,21 suggesting it is
involved in a conserved ischemic response pathway. However, whether PKC mediates or is simply activated during
ischemic injury is controversial because of mixed reports on
PKC expression and activity, and thus is the focus of this
review.
The PKC Family
The PKC family of serine/threonine kinases consists of 10
different isozymes. In the brain and spinal cord, PKC␣,
TABLE 1.
PKC␤1, PKC␤2, PKC␥, PKC⑀, PKC␦, PKC␩, PKC␪, and
PKA␨ mRNA and protein are present and demonstrate unique
tissue, cellular, and subcellular localizations.22,23 However,
the relative levels of PKC isozyme expression in different
anatomic brain regions have not yet been examined in
detail, and alterations in the expression and levels of these
isozymes under conditions of cerebral ischemic stress have
not been systematically examined. Importantly, individual
PKC isozymes mediate different and sometimes opposing
functions after activation by the same stimulus.24 However, the use of nonspecific pharmacological tools conceals the role of individual PKC isozymes, contributing to
PKC Pharmacological Agents
Agent
Cerebral Ischemia
References
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
PKC Isozyme Specificity
Nonspecific Targets
Activity (EC50/IC50)
Mechanism/Target Site
␣, ␤1, ␤2, ␥, ␦, ⑀, ␯,
␪ PKC
Multiple DAG-binding
proteins
1–100 nmol/L
Mimic DAG
Reshef et al, 1997,
Maiese et al, 1993
Wang et al, 2004
Activators
PMA (phorbol
12-myristate
13-acetate)
␺␤RACK
␣, ␤1, ␤2, ␥PKC
NA
Translocation†
␺␦RACK
␦PKC
NA
Translocation†
␺⑀RACK
⑀PKC
NA
Translocation†
Di-Capua et al, 2003,
Raval et al, 2003
Inhibitors
Rottlerin
Calphostin C
␦PKC, lesser-other PKC
isozymes
CaM kinase III*, lesser PKA*,
casein kinase II
3–6 nmol/L
Catalytic domain
Hamabe et al, 2005,
Hsieh et al, 2005,
Jung et al, 2005
all
PKA*, myosin light chain
kinase
50 nmol/L
DAG and phorbol ester
binding site
Durkin et al, 1997,
Tauskela et al, 1999,
Cheung et al, 2003
Bisindolylmaleimide I
all
JNK
10 nmol/L
Staurosporine
all
serine/threonine and tyrosine
kinases
2.7 nmol/L
Substrate binding site
Hara et al, 1990,
Maiese et al, 1993,
Durkin et al, 1997
␣, ␤1, ␤2, ␥PKC
MAPK family
8 nmol/L
Substrate binding site
Hamabe et al, 2005,
Cheung et al, 2003
all
JNK1
10 nmol/L
Substrate binding site
5 nmol/L
Substrate binding site
Gö 6976
Ro 31-8220
LY333531
␤PKC
Chelerythrine
all
cyclic nucleotide
phosphodiesterases
660 nmol/L
Catalytic domain: ATP
binding site, phosphate
acceptor site
Tauskela et al, 1999,
Raval et al, 2003
H7
all
PKA*, PKG*, myosin light
chain kinase
NA
ATP-binding site,
catalytic domain
inhibitor
Felipo et al, 1993,
Maiese et al, 1993
␣C2-4
␣PKC
NA
Translocation†
␤1V5-3
␤1PKC
NA
Translocation†
␤2V5-3
␤2PKC
NA
Translocation†
␤C2-4
␣,␤PKC
NA
Translocation†
␥V5-3
␥PKC
NA
Translocation†
Wang et al, 2004
␦V1-1
␦PKC
⬎5 nmol/L
Translocation†
Bright et al, 2004,
Raval et al, 2005
⑀V1-2
⑀PKC
NA
Translocation†
Di-Capua et al, 2003,
Raval et al, 2003
␨V1-1
␨PKC
NA
Translocation†
Koponen et al, 2003
Wang et al, 2004
Koponen et al, 2003
*CaM kinase III indicates calcium/calmodulin– dependent kinase III; PKA, cAMP-dependent kinase; PKG, cGMP-dependent kinase; †translocation, subcellular
translocation inhibitor or activator.
Bright and Mochly-Rosen
inconsistency in reports on PKC function. With the availability of isozyme-specific modulators, the unique roles of
each PKC isozyme are becoming increasingly clear (note
1; Table 1).
PKC Activity in Stroke Models
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
When exposed to sublethal ischemic episode, the brain enacts
endogenous neuroprotective mechanisms to induce tolerance,
rendering it protected against a subsequent lethal transient
ischemic attack. Two temporal waves of preconditioning
have been described: a rapid induction phase, mediated in
part through adenosine, ATP-sensitive potassium (K⫹ATP)
channels, and low-level glutamate-induced calcium flux,25,26
and a delayed induction window, likely to rely on altered
gene expression and protein synthesis.5,27 Multiple agents
induce rapid tolerance in the brain, including exposure to low
levels of N-methyl-D-aspartate (NMDA),28 NO,29 and adenosine.30 Ischemic tolerance induced by these endogenous
preconditioning agents is dependent on PKC activation,26,28,31
suggesting that PKCs are key regulators of ischemic preconditioning in the brain.
A largely conflicting body of reports has emerged concerning the PKC response to ischemia (Table 2). Some studies
demonstrate that total PKC levels and activity are increased at
very early time points after ischemia in vivo32–34 and after
oxygen and glucose deprivation (OGD) and excitotoxic
damage in vitro.35 Inhibition of PKC using high concentrations of phorbol 12-myristate 13-acetate, or nonspecific PKC
inhibitors such as H7, calphostin C, or staurosporine, protect
cells against NO-, anoxic- or glutamate-induced excitotoxic
cell death in vitro36,37 and against ischemic damage in vivo.38
These data suggest that PKC is activated and plays a
damaging role during stroke. However, a greater majority of
studies report a rapid loss of total PKC activity and expression after ischemia, suggesting PKC is degraded.39 – 42 This
loss of total PKC activity, also seen in in vitro culture models
of ischemic and excitotoxic cell death,43,44 correlates with
neurodegenerative processes,45 implying that maintaining
PKC activity may confer protection against excitotoxic damage. These apparently conflicting reports may stem from
examination of varying animal models, brain regions, duration and intensities of ischemic/reperfusion insult, and may be
compounded by the different, possibly opposing roles of
individual PKC isozymes.
The mechanisms by which PKC is activated during stroke
are likely to be multifactorial. Here, we focus on 3 specific
PKC isozymes and examine their roles in widely studied
processes: (1) adenosine-mediated upregulation of PKCs in
the context of ischemic tolerance (preconditioning), and, in
particular, the role of ⑀PKC; (2) PKC response to glutamateinduced excitotoxicity during ischemia, focusing on the
neuronal ␥PKC isoform; and (3) ␦PKC activation in delayed
apoptotic processes during reperfusion. However, changes in
PKC activity and expression in response to other ischemic/
reperfusion processes have been reported, including inflammatory processes (as discussed with reference to ␦PKC
below), cell necrosis, and alterations in microvascular tone
and reactivity.46,47
Molecular Basis of PKC Activity in Stroke Injury
Ischemic Tolerance: Role for ⑀PKC
2783
The role of ⑀PKC in ischemic injury has been subject to
debate.26,48 Some reports demonstrate sustained activation of
⑀PKC after OGD35,49 and, in response to kainic acid, a
glutamate analog.50 Other reports suggest that ⑀PKC is not
activated in response to ischemia51,52 in an in vivo model of
spreading depression by cortical KCl application or by
ischemic preconditioning.31,53 ⑀PKC mRNA increases
slightly at 1 hour after reperfusion; however, more significant
upregulations were observed in the transcription of other
PKC isozymes.54 Importantly, many of these reports focus on
the RNA, protein, and activity levels of ⑀PKC during the
reperfusion period. However, a potential role for ⑀PKC may
exist during ischemia or after less severe injuries.
Studies using PKC isozyme-selective modulators have
demonstrated that ⑀PKC is required for induction of ischemic
tolerance; delivery of an ⑀PKC inhibitor peptide abates
NMDA-induced preconditioning in cell culture and isolated
hippocampal slice models.28 Correspondingly, delivery of an
⑀PKC-specific activator peptide reduces damage, as measured using lactate dehydrogenase (LDH) release, when
delivered before OGD in pure neuronal and mixed neuronal/
astrocyte cultures.49 Importantly, the protective effect of the
⑀PKC activator was lost when delivered after OGD in these
models. These data suggest that changes occur in ⑀PKC
activity over the time course of ischemic injury and begin to
address the cell type–specific effects of ⑀PKC.
The molecular basis of ⑀PKC-induced protection is unclear. One mechanism (Figure 1) implicates adenosine and
the mitochondrial K⫹ATP (mK⫹ATP). Under metabolic stress
such as ischemia, increases in adenosine levels (in addition to
bradykinin and opioids) initiate a series of intracellular
signaling events via G-protein– coupled receptor signaling,
leading to activation of phospholipases, production of diacylglycerol (DAG), calcium influx, and PKC activation.55
Multiple studies have now demonstrated that adenosine
administration protects neuronal cells against ischemic-type
injury via PKC,30,56,57 and more recent work has identified a
role for ⑀PKC in particular.57 Treatment of primary neuronal
cultures with N6-(R)-phenylisopropyladenosine, an A1 adenosine receptor agonist, causes extended activation of ⑀PKC (6
hours after treatment), whereas delivery of an ⑀PKC-selective
inhibitor peptide blocks adenosine-induced neuroprotection
against this chemical ischemia.57 Adenosine-mediated ⑀PKC
signaling is mediated in part through ERK, a MAPK family
member that has been implicated in antiapoptotic signaling.58
Interestingly, studies in cardiac mitochondria demonstrate
that ⑀PKC forms functional modules with MAPK family
members to maintain mitochondrial function, including inhibiting deleterious Bcl-2 associated death domain protein
(BAD; a Bcl-2 family member) activity.59 ⑀PKC activity at
the mitochondria may also contribute to regulation of mK⫹ATP
channels, important for preserving mitochondrial membrane
potential, maintaining energy and reducing calcium influx
during metabolic challenge.60 – 62 PKC is thought to mediate
the activity of this channel through direct phosphorylation63
and regulation of its internalization.64 In particular, reports in
cardiac ischemia models suggest ⑀PKC mediates adenosineinduced preconditioning via K⫹ATP function.65– 67 These data
2784
Stroke
December 2005
TABLE 2.
Studies of PKC Activity and Function in In Vivo Cerebral Ischemia Models
Model
Animal
PKC Isozyme
Major Findings
Reference
pc-cortical sd
Rat
␣, ␤, ␥, ␦, ⑀, ␨
Increase in ␦, ␨ transcription
Koponen et al, 1999
pc-cortical sd
Rat
␦
Increase in ␦ mRNA, protein, and activity*
Kurkinen et al, 2001
pc-2VO; 2 minutes
Rat
␣, ␦, ⑀, ␨
Increase in ␣, ␦ activity
Kurkinen et al, 2001
pc-2VO; 2 minutes
Gerbil
␥
decrease in ␥ activity
Katsura et al, 2001
Rat
␦, ⑀, ␨
Increase in ␦ mRNA and protein
Kaasinen et al, 2002
MCAO; 6 hours
Rat
n.s.
Decrease in PKC activity
Crumrine et al, 1992
Photochemically induced thrombus
Rat
n.s.
Increase in PKC activity
Kharlamov et al, 1993
Miettinen et al, 1996
Spreading depression/preconditioning
Sd
Permanent focal ischemia
Transient focal ischemia
Rat
␣, ␤, ␥, ␦, ⑀, ␨
Increase in ␦PKC mRNA, protein elevated
MCAO; variable times
Mouse
n.s.
Decrease of PKC activity
Hara et al, 1997
MCAO; 120 minutes
Rat
␦
␦ Activity detrimental during reperfusion
Bright et al, 2004
MCAO; 60–120 mins
Rat
␥
Increase in ␥ activity
Matsumoto et al, 2004
MCAO, CCAO; 150 minutes
␥ KO
␥
␥ Beneficial role in ischemia
Aronowski et al, 2000
MCAO; 60 mins and MCAO; 20 hours
(permanent)
␦ KO
␦
Neutrophil ␦ mediates reperfusion injury,
but does not play a role in permanent
focal ischemia
Chou et al, 2004
4VO; 10–20 minutes
Rat
n.s.
Increase in PKC activity, in particular
␥PKC
Saluja et al, 1999
4VO; 15 minutes
Rat
n.s.
PKC inhibition reduces NMDAR
phosphorylation
Cheung et al, 2003
4VO; 20 minutes
Rat
␣, ␤, ␥, ␦, ⑀, ␨
␦mRNA and protein induced
Koponen et al, 2000
4VO, 2VO; variable times
Rat
n.s.
Decrease in PKC activity
Louis et al, 1991
2VO; 5 minutes
Gerbil
n.s.
(1) Rapid activation of PKC in CA1 cells,
followed by decrease later in reperfusion;
(2) increase in ␥ mRNA
(1) Olah et al, 1990,
(2) Zablocka et al, 1998
2VO; 6 minutes
Gerbil
n.s.
Rapid activation of PKC, followed by
decrease later in reperfusion
Domanska-Janik and Zablocka,
1993
2VO; 10 minutes
Gerbil
␦, ⑀, ␨
(1) ␦, ␨ mRNA upregulated,
(2) elevated ␦, ⑀, ␨ mRNA
(1) Savithiry and Kumar, 1994
(2) Kumar and Wu, 1994
2VO; 15 minutes
Rat
Classical
(1) ␣, ␥ Activated during early
reperfusion, (2) variable changes in ␤2, ␥
␦uring ischemia and reperfusion, loss of
total PKC activity
(1) Cardell et al, 1990
(2) Wieloch et al, 1991
2VO; variable times
Rat
Classical
Time-dependent alterations in ␣, ␤2, ␥
levels
Cardell and Wieloch, 1993
Increased intracranial pressure 20
minutes
Dog
n.s.
PKC activity increased, atypical PKC levels
increased
Sieber et al, 1998
2VO⫹systemic hypotension
Rat
␣, ␤, ␥, ␦, ⑀, ␨
␣, ␤, ␥ translocation, no changes in
novel and atypical PKC
Harada et al, 1999
Postdecapitative ischemia
Rat
n.s.
Decrease total PKC activity
Domanska-Janik and Zalewska,
1992
Cardiac arrest
Rat
n.s.
Decrease in PKC activity
Crumrine et al, 1990
MCAO; 30 minutes, 90 minutes
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Transient global ischemia
n.s. indicates nonisozyme specific; pc, preconditioning; sd, spreading depression; KO, knockout; VO, vessel occlusion; MCAO, middle cerebral artery occlusion;
CCAO, common carotid artery occlusion.
*PKC activity was usually determined by increased association of the said PKC isozyme with the cell particulate fraction or changes in catalytic activity.
suggest that ⑀PKC confers cerebral ischemic protection, in
part by maintaining mitochondrial function via ERK activity and potentially by mediating adenosine-induced
mK⫹ATP channel function.
Ischemia and the Role of Neuronal ␥PKC
One of the central events that contributes to injury during and
after ischemic stroke is the release of the excitatory amino
acid glutamate. Extrasynaptic glutamate causes waves of
Bright and Mochly-Rosen
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Figure 1. ⑀PKC in cerebral preconditioning. Progression of
molecular events leading to ⑀PKC-mediated preconditioning. a,
Preconditioning stimuli, including hypoxia, lead to a decrease in
cellular ATP stores and the subsequent generation of adenosine, which is released extracellularly. Adenosine stimulates
G-protein– coupled receptors, such as the A1/A3 adenosine
receptors (ARs), to activate phospholipases (phospholipase C
[PLC]) via G-proteins (Gi/o). Other preconditioning stimuli,
including extracellular glutamate, stimulate NMDARs, leading to
increased cytosolic calcium and PLC activation. b, PLC causes
membrane damage, increasing DAG production, which activates
PKC isozymes including ⑀PKC. c, ⑀PKC may induce opening of
K (ATP) channels in the mitochondria (KATP), maintain ATP production and reduce generation of ROS, which, in turn, mediates
PKC activation. ⑀PKC leads to activation of ERK, an MAPK family member, which is involved in antiapoptotic signaling and cell
survival.
Molecular Basis of PKC Activity in Stroke Injury
2785
peri-infarct depolarization in surrounding cells and spreading
neuronal death. In part, via NMDA receptors (NMDARs),
glutamate induces an influx of calcium and sodium into the
cell, leading to release of intracellular calcium stores, lipid
peroxidation, and generation of free radicals.4 These events
lead to rapid activation and increased expression of multiple
PKC isozymes, as seen after glutamate, KCl or kainic acid
application in vitro and in vivo.50,68 –72 Correspondingly, PKC
inhibition reduces NMDA-mediated neurotoxicity,37,73,74 suggesting that PKC isozymes may mediate excitatory amino
acid–induced signaling.
␥PKC is expressed exclusively in neurons of the brain and
spinal cord.75 Therefore, this isozyme may play a central
nervous system (CNS)–specific role in mediating response to
ischemic injury. ␥PKC is activated rapidly during ischemia in
various models,17,52,76 –78 consistent with increases in intracellular calcium and phospholipid metabolism during ischemia,
required for ␥PKC activation. The use of ␥PKC knockout
mice suggest ␥PKC may play a detrimental role during
cerebral ischemic injury; ␥PKC knockout mice have significantly smaller infarcts, as measured using histological techniques, compared with wild-type animals after permanent
ischemia.79 However, in an in vitro model of OGD, inhibition
of ␥PKC using a ␥PKC-selective peptide inhibitor had no
effect on cell survival, as assayed by LDH release.49
Multiple reports now suggest that PKC may be involved in
a positive feedback loop to potentiate NMDAR activity,44,80
worsening calcium loading, mitochondrial dysfunction, and
promoting cell death (Figure 2).81 Modulating NMDAR
activity may be attributable to direct phosphorylation of NR1
receptor subunits by PKC,82 although recent evidence suggests PKC indirectly mediates NMDAR via regulation of
associated scaffolding proteins83 or Src-family tyrosine kinase activity.84 In particular, ␥PKC may regulate this feedback; ␥PKC interacts with NMDAR subunits in vivo to
regulate postsynaptic excitotoxic signaling.85 However, indirect evidence also indicates that ␤1PKC and ␤2PKC subspecies may also modulate NMDAR function.73,86
After extended ischemic injury and reperfusion, changes in
␥PKC activity are less clear. Several reports demonstrate that
␥PKC becomes inactive and downregulated,44,87 as occurs after
prolonged PKC activity,88 whereas others report that ␥PKC is
activated specifically during reperfusion.17,78 In fact, a contrasting role for ␥PKC has been suggested during reperfusion; ␥PKC
knockout mice demonstrate worsened injury after a transient
ischemia, suggesting ␥PKC may mediate beneficial signaling
processes during reperfusion injury.13
Together, these data suggest that ␥PKC may play a
deleterious role during early ischemic injury or permanent
ischemic injury, potentially activated by DAG formation and
flux of intracellular calcium that occur during early ischemic
signaling. During ischemia, ␥PKC may be involved in potentiating NMDAR function, leading to calcium overloading and
cell death. However, after reperfusion, ␥PKC may play an
opposite role: mediating protective signaling and reducing
cell death. These data demonstrate that individual PKC
isozymes may play differing or opposing roles at different
stages of injury, underscoring the importance of studying
2786
Stroke
December 2005
PKC␦ in Reperfusion Injury: Apoptosis,
Necrosis, and Inflammation
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Figure 2. Role of ␥PKC in cerebral ischemia. Progression of
molecular events leading to ␥PKC-mediated injury in response
to ischemia. a, Ischemia-induced reduction in blood oxygen and
glucose delivery to the brain causes reduction in cellular ATP
levels, leading to deregulation of cellular ion pumps and loss of
intracellular ion homeostasis. b, Rising intracellular calcium, in
part attributable to activation of NMDARs, causes activation of
phospholipase C (PLC), which increases ␥PKC activation.
Decreases in intracellular ATP also lead to ROS production,
contributing to PKC activation. c, It is thought that PKCs, and
␥PKC in particular, may potentiate NMDAR function via direct
phosphorylation or indirectly through scaffolding proteins and
Src tyrosine kinases. This feedback mediates increases in intracellular calcium, leading to mitochondrial dysfunction, ROS formation, and cell death.
PKC isozyme function during different periods of cell death
by examining transient and permanent ischemia models.
Other PKC isozymes are also likely to be involved in
mediating the cellular response to ischemia in the brain. For
example, ␨PKC mediates NMDA-induced injury in primary
neuronal cultures; inhibition of ␨PKC reduces cellular damage, as monitored by LDH release.74 A more detailed understanding of the molecular mechanism by which ␨PKC elicits
death in response to NMDA toxicity awaits further study.
With increasing evidence that cerebral reperfusion generates
its own host of detrimental intracellular signaling cascades
and contributes to expansion of the ischemic infarct, the
concept that reperfusion injury is a potent mediator of cell
death is now widely accepted. Understanding reperfusioninduced processes is of particular interest for therapeutic
targeting in combination with established thrombolytic therapy or mechanical recanalization techniques.
Multiple lines of data suggest that PKC plays an important
role in mediating cerebral reperfusion injury. In particular,
␦PKC has been implicated in mediating oxidative stress,
apoptosis, and inflammation, hallmarks of reperfusion
injury.89,90 Studies demonstrate that ␦PKC is specifically
upregulated and rapidly activated in response to reperfusioninduced intracellular signaling; in transient focal and global
ischemia models, ␦PKC mRNA is upregulated within 24
hours after ischemia, as seen in perifocal cortex and in CA1
neurons, respectively.51,91,92 Concomittantly, ␦PKC protein
levels are increased in the perifocal region during reperfusion,92 suggesting a role for this enzyme in mediating
delayed-injury processes. Studies in an in vivo transient
ischemia model show ␦PKC is rapidly activated at the onset
of reperfusion, although this activity is not sustained at 24
hours after ischemia.93
How ␦PKC mediates reperfusion injury, whether increasing activity of this enzyme promotes or reduces reperfusion
injury, has only more recently been addressed. Studies using
2 very different approaches, pharmacological agents and
knockout animals, indicate that ␦PKC plays a detrimental role
after stroke in vivo. Delivery of the ␦PKC-specific inhibitor
peptide ␦V1-1 significantly reduced ischemic injury when
delivered over an extended time window of reperfusion after
a 2-hour middle cerebral artery occlusion in vivo, as assessed
by histological staining techniques, and in an in vitro hippocampal slice model subject to OGD, as assessed by
propidium iodide staining of CA1 neurons. However, this
protective effect was not seen when ␦V1-1 was delivered
before ischemia, suggesting that ␦PKC specifically mediates
reperfusion-induced cell death in parenchymal cells of the
brain.93 A complementary study using ␦PKC knockout mice
demonstrates that ␦PKC may contribute to damage by mediating inflammatory processes during reperfusion damage;
␦PKC knockout mice have smaller infarcts after transient
focal ischemia compared with wild-type animals and demonstrate reduced neutrophil invasion into infarcted tissue. Further, wild-type animals that received bone marrow transplants from ␦PKC knockout donor mice also demonstrate
reduced ischemic injury,47 suggesting that ␦PKC activity
in neutrophils, rather than neuronal or glial cells, is critical
in this response.
A rapidly growing body of reports suggests a specific role
for ␦PKC in mediating apoptotic processes in a variety of cell
types.89,94 Apoptosis occurs largely in a delayed fashion during
cerebral reperfusion, correlating with the reported increases in
␦PKC activation and expression, as described above. Reduced
energy levels, as seen in the ischemic penumbra as well as
spreading waves of depolarization, induce the generation of
Bright and Mochly-Rosen
Molecular Basis of PKC Activity in Stroke Injury
2787
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
ROS such as hydroxyl radicals, superoxide, and singlet
oxygen.95 ␦PKC is responsive to ROS52,90,96 and other apoptotic mediators including caspase activation.97 In the heart,
␦PKC translocates to the mitochondria in response to oxidative stress,90 where it mediates superoxide production, pyruvate dehydrogenase inhibition, reduced ATP generation, and
increased free radical formation.98,99 Inhibition of ␦PKC
translocation reduces mitochondrial dysfunction, including
Bcl-2 family protein dimerization and release of apoptogenic
factors, and increases the rate of ATP regeneration and
correction of cellular acidosis.94,100 Parallel findings have
been demonstrated in a cerebral ischemia models. After
transient focal ischemia, delivery of a ␦PKC inhibitor peptide
reduced apoptotic cell death, as seen by TUNEL staining,
increased antiapoptotic Akt (protein kinase B) activity, and
reduced BAD translocation out of the cell cytosolic fraction,
indicating reduction of BAD deleterious activity.93 In a rat
cardiac arrest model, ␦PKC is activated via caspase-3–
mediated cleavage in hippocampal CA1 neurons, contributing
to reperfusion-induced hippocampal injury (Figure 3).101 The
role of ␦PKC in promoting reperfusion-induced apoptotic cell
death suggests it is an important therapeutic target for
extended time windows after stroke injury in patients.
PKC in Stroke: Unanswered Questions and
Potential Clinical Implications
A promising body of work supports a role for ⱖ3 different
PKC isozymes in mediating stroke-induced damage, with
unique functions in preconditioning, in ischemia, and in
reperfusion-induced signaling. Parallel findings concerning
the positive or negative role of these isozymes in multiple
models of ischemic/reperfusion damage, including focal and
global ischemia models, suggest that PKCs may play an
important role in mediating damage in multiple anatomical
territories of the brain and brain injury paradigms. However,
this underscores the need for more detailed studies about the
distribution and activity of individual PKC isozymes in
response to different stages and intensities of injury, different
brain regions, different cell types, and different subcellular
localizations. Identifying the roles for PKC in mediating
endothelial cell dysfunction,102 maintenance of microvascular
permeability,103 and cerebral vasospasm46 will provide a
more comprehensive understanding of PKC function in cerebral ischemic/reperfusion injury. Finally, elaborating our
understanding of the specific signaling pathways in which
PKC participates, including different downstream effectors in
different phases of stroke injury, will be critical to developing
PKC-based therapeutic strategies.
The use of PKC-isozyme selective tools has demonstrated
the importance of PKC signaling in mediating cerebral
ischemic/reperfusion injury. However, further studies on the
role of PKC isozymes in CNS ischemia are needed. Work to
date has been performed largely in rodent models without
translation to other animal models including primates and
often does not include dose-response or long-term behavioral
outcome analysis. The Stroke Therapy Academic Industry
Roundtable criteria for evaluating preclinical drugs is therefore not complete.104 However, current research strongly
suggests that regulating individual PKC isozymes has thera-
Figure 3. Role of ␦PKC in reperfusion-induced cell death pathways. Progression of molecular events leading to ␦PKCmediated injury in response to reperfusion. a, Reperfusion injury
contributes to detrimental cell signaling, in part via release of
glutamate and free radicals from the ischemic core and the
recruitment of inflammatory mediators. Glutamate causes rises
in intracellular calcium via the NMDAR, increasing activation of
phospholipases C and D (PLC/PLD) and deregulation of ion
channels (Na⫹/K⫹). b, The effects of PLC, as well as rises in
ROS, activate ␦PKC. ␦PKC is involved in a series of cell death
pathways that involve its translocation to the mitochondria,
where it mediates Bcl-2 family member BAD activity, release of
cytochrome c (cyt c), and additional ROS release. c, Release of
cytochrome c promotes apoptosis through the activation of
caspases and subsequent DNA damage events in the nucleus.
In addition, ␦PKC inhibits the activity of Akt, involved in cell survival signaling. Finally, not shown in this scheme, ␦PKCmediated mitochondrial damage causes loss of ATP regeneration and ROS production. This leads to necrosis and contributes
to infarction in the ischemic penumbra.112
2788
Stroke
December 2005
peutic value in ⱖ3 scenarios of CNS ischemic damage: (1)
predictable CNS ischemia, (2) damage that occurs during
transient ischemia followed by reperfusion, and (3) damage
that occurs because of a permanent occlusion. Present data
suggest that different PKC regulation should be used in the
above 3 scenarios as follows. For predictable ischemia, such
as that occurring during bypass surgery, it appears that ⑀PKC
activation before ischemic event will provide optimal outcome. For transient, unpredictable ischemia, treatment with a
short-acting ␥PKC inhibitor early during ischemia, and with
a long-acting ␦PKC inhibitor or ␥PKC selective activator
during reperfusion, will provide optimal therapy. Finally, in
cases of permanent occlusion, treatment with a ␥PKC inhibitor and a ␦PKC inhibitor should be protective. Clearly, more
work in animal models is required before the current findings
are translated to human studies. If substantiated, selective
PKC regulators will open new avenues for the treatment of
CNS ischemia and stroke.
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Note 1: Experimental Approaches to Study
PKC Activity
The majority of commonly used pharmacological tools to
study PKC function are nonspecific, altering the function of
non-PKC kinases, or are nonselective for individual PKC
isozymes. Designing agents that target individual PKC
isozymes has been particularly difficult because of the high
degree of homology between individual PKC isozymes and
broad substrate specificity.105,106 Commonly used pharmacological agents include the H7 and staurosporine family
members calphostin C, rottlerin, and chelerythrine (Table 1).
Although exhibiting potent inhibition of PKC, these agents
also inhibit other protein kinases (as catalytic domain inhibitors) and usually show no discriminatory activity on individual PKC isozymes. More comprehensive reviews of PKCmodulating compounds are given previously.105,106
The technical limitations of kinase selectivity and specificity have been addressed, in part, by the development of
peptide activators and inhibitors of individual PKC isozymes
that regulate subcellular localization of each isozyme.107,108
These peptides were developed based on a rational design
strategy that capitalizes on the unique interaction between
each PKC isozyme with an isozyme-specific anchoring protein, receptor for activated C kinase (RACK), necessary for
PKC function; the RACKs anchor each activated isozyme
near their substrates. Peptide inhibitors competitively block
the translocation and interaction of PKC with its RACK,
blocking access to substrates and therefore downstream
physiological signaling. PKC-derived peptide agonists act as
allosteric agonists by preventing intramolecular autoinhibitory interactions within PKC, leading to selective activation
of individual PKCs by enabling their anchoring to the
corresponding RACKs.109 The effect and isozyme selectivity
of these peptides have been demonstrated in numerous
systems, including in vitro and in vivo CNS models.49,74,93
The peptides can be effectively delivered to a number of tissues,
including the brain after intra-arterial and intraperitoneal injections and subcutaneous pump delivery,93,110,111 to modulate the
activation of individual PKC isozymes in vivo. A list of PKC
peptide activators and inhibitors is given in Table 1.
A useful review on this subject was published while this
paper was under review: Chou WH, Messing RO. Protein
kinase C isozymes in stroke. Trends Cardiovasc Med.
2005;15:47–51.
References
1. Heart Disease and Stroke, Statistical Update. American Heart Association; 2005.
2. Kaur J, Zhao Z, Klein GM, Lo EH, Buchan AM. The neurotoxicity of
tissue plasminogen activator? J Cereb Blood Flow Metab. 2004;24:
945–963.
3. Reed SD, Cramer SC, Blough DK, Meyer K, Jarvik JG. Treatment with
tissue plasminogen activator and inpatient mortality rates for patients
with ischemic stroke treated in community hospitals. Stroke. 2001;32:
1832–1840.
4. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999;79:
1431–1568.
5. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischemic
stroke: an integrated view. Trends Neurosci. 1999;22:391–397.
6. Phan TG, Wright PM, Markus R, Howells DW, Davis SM, Donnan GA.
Salvaging the ischemic penumbra: more than just reperfusion? Clin Exp
Pharmacol Physiol. 2002;29:1–10.
7. Dijkhuizen RM, Beekwilder JP, van der Worp HB, Berkelbach van der
Sprenkel JW, Tulleken KA, Nicolay K. Correlation between tissue
depolarizations and damage in focal ischemic rat brain. Brain Res.
1999;840:194 –205.
8. Hartings JA, Rolli ML, Lu XC, Tortella FC. Delayed secondary phase of
peri-infarct depolarizations after focal cerebral ischemia: relation to
infarct growth and neuroprotection. J Neurosci. 2003;23:11602–11610.
9. Yang GY, Betz AL. Reperfusion-induced injury to the blood-brain
barrier after middle cerebral artery occlusion in rats. Stroke. 1994;25:
1658 –1664; discussion 1664 –5.
10. Iadecola C, Alexander M. Cerebral ischemia and inflammation. Curr
Opin Neurol. 2001;14:89 –94.
11. Endres M, Namura S, Shimizu-Sasamata M, Waeber C, Zhang L,
Gomez-Isla T, Hyman BT, Moskowitz MA. Attenuation of delayed
neuronal death after mild focal ischemia in mice by inhibition of the
caspase family. J Cereb Blood Flow Metab. 1998;18:238 –247.
12. Zhao Q, Pahlmark K, Smith ML, Siesjo BK. Delayed treatment with the
spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) reduces infarct size
following transient middle cerebral artery occlusion in rats. Acta Physiol
Scand. 1994;152:349 –350.
13. Aronowski J, Grotta JC, Strong R, Waxham MN. Interplay between the
gamma isoform of PKC and calcineurin in regulation of vulnerability to
focal cerebral ischemia. J Cereb Blood Flow Metab. 2000;20:343–349.
14. Noshita N, Lewen A, Sugawara T, Chan PH. Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 2001;21:1442–1450.
15. Kitagawa H, Warita H, Sasaki C, Zhang WR, Sakai K, Shiro Y,
Mitsumoto Y, Mori T, Abe K. Immunoreactive Akt, PI3-K and ERK
protein kinase expression in ischemic rat brain. Neurosci Lett. 1999;
274:45– 48.
16. Hayashi T, Sakai K, Sasaki C, Zhang WR, Warita H, Abe K. c-Jun
N-terminal kinase (JNK) and JNK interacting protein response in rat
brain after transient middle cerebral artery occlusion. Neurosci Lett.
2000;284:195–199.
17. 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.
18. Skaper SD, Facci L, Strijbos PJ. Neuronal protein kinase signaling
cascades and excitotoxic cell death. Ann N Y Acad Sci. 2001;939:11–22.
19. Speechly-Dick ME, Mocanu MM, Yellon DM. Protein kinase C. Its role
in ischemic preconditioning in the rat. Circ Res. 1994;75:586 –590.
20. Piccoletti R, Bendinelli P, Arienti D, Bernelli-Zazzera A. State and
activity of protein kinase C in postischemic reperfused liver. Exp Mol
Pathol. 1992;56:219 –228.
21. Padanilam BJ. Induction and subcellular localization of protein kinase C
isozymes following renal ischemia. Kidney Int. 2001;59:1789 –1797.
22. Tanaka C, Nishizuka Y. The protein kinase C family for neuronal
signaling. Annu Rev Neurosci. 1994;17:551–567.
Bright and Mochly-Rosen
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
23. Naik MU, Benedikz E, Hernandez I, Libien J, Hrabe J, Valsamis M,
Dow-Edwards D, Osman M, Sacktor TC. Distribution of protein kinase
Mzeta and the complete protein kinase C isoform family in rat brain.
J Comp Neurol. 2000;426:243–258.
24. Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro
G, Banci L, Guo Y, Bolli R, Dorn GW II, Mochly-Rosen D. Opposing
cardioprotective actions and parallel hypertrophic effects of delta PKC
and epsilon PKC. Proc Natl Acad Sci U S A. 2001;98:11114 –11119.
25. Perez-Pinzon MA, Born JG. Rapid preconditioning neuroprotection following anoxia in hippocampal slices: role of the K⫹ ATP channel and
protein kinase C. Neuroscience. 1999;89:453– 459.
26. Reshef A, Sperling O, Zoref-Shani E. The adenosine-induced
mechanism for the acquisition of ischemic tolerance in primary rat
neuronal cultures. Pharmacol Ther. 2000;87:151–159.
27. Zemke D, Smith JL, Reeves MJ, Majid A. Ischemia and ischemic
tolerance in the brain: an overview. Neurotoxicology. 2004;25:895–904.
28. Raval AP, Dave KR, Mochly-Rosen D, Sick TJ, Perez-Pinzon MA.
Epsilon PKC is required for the induction of tolerance by ischemic and
NMDA-mediated preconditioning in the organotypic hippocampal slice.
J Neurosci. 2003;23:384 –391.
29. Huang PL. Nitric oxide and cerebral ischemic preconditioning. Cell
Calcium. 2004;36:323–329.
30. Heurteaux C, Lauritzen I, Widmann C, Lazdunski M. Essential role of
adenosine, adenosine A1 receptors, and ATP-sensitive K⫹ channels in
cerebral ischemic preconditioning. Proc Natl Acad Sci U S A. 1995;92:
4666 – 4670.
31. Kurkinen K, Keinanen R, Li W, Koistinaho J. Preconditioning with
spreading depression activates specifically protein kinase Cdelta. NeuroReport. 2001;12:269 –273.
32. Domanska-Janik K, Zablocka B. Protein kinase C as an early and
sensitive marker of ischemia-induced progressive neuronal damage in
gerbil hippocampus. Mol Chem Neuropathol. 1993;20:111–123.
33. Gajkowska B, Domanska-Janik K, Viron A. Protein kinase C-like
immunoreactivity in gerbil hippocampus after a transient cerebral ischemia. Folia Histochem Cytobiol. 1994;32:71–77.
34. Kharlamov AGA, Costa E, Hayes R, Armstrong D. Semisynthetic sphingolipids prevent protein kinase C translocation and neuronal damage in
the perifocal area following a photochemically induced thrombotic brain
cortical lesion. J Neurosci. 1993;13:2483–2494.
35. Selvatici R, Marino S, Piubello C, Rodi D, Beani L, Gandini E,
Siniscalchi A. 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.
36. Maiese K, Boniece IR, Skurat K, Wagner JA. Protein kinases modulate
the sensitivity of hippocampal neurons to nitric oxide toxicity and
anoxia. J Neurosci Res. 1993;36:77– 87.
37. Felipo V, Minana MD, Grisolia S. Inhibitors of protein kinase C prevent
the toxicity of glutamate in primary neuronal cultures. Brain Res. 1993;
604:192–196.
38. Hara H, Onodera H, Yoshidomi M, Matsuda Y, Kogure K. Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal
damage in the gerbil and rat. J Cereb Blood Flow Metab. 1990;10:
646 – 653.
39. Domanska-Janik K, Zalewska T. Effect of brain ischemia on protein
kinase C. J Neurochem. 1992;58:1432–1439.
40. Crumrine RC, Dubyak G, LaManna JC. Decreased protein kinase C
activity during cerebral ischemia and after reperfusion in the adult rat.
J Neurochem. 1990;55:2001–2007.
41. Hara H, Ayata G, Huang PL, Moskowitz MA. Alteration of protein
kinase C activity after transient focal cerebral ischemia in mice using in
vitro [3H]phorbol-12,13-dibutyrate binding autoradiography. Brain Res.
1997;774:69 –76.
42. Louis JC, Magal E, Brixi A, Steinberg R, Yavin E, Vincendon G.
Reduction of protein kinase C activity in the adult rat brain following
transient forebrain ischemia. Brain Res. 1991;541:171–174.
43. Durkin JP, Tremblay R, Buchan A, Blosser J, Chakravarthy B, Mealing
G, Morley P, Song D. An early loss in membrane protein kinase C
activity precedes the excitatory amino acid-induced death of primary
cortical neurons. J Neurochem. 1996;66:951–962.
44. Chakravarthy BR, Wang J, Tremblay R, Atkinson TG, Wang F, Li H,
Buchan AM, Durkin JP. Comparison of the changes in protein kinase C
induced by glutamate in primary cortical neurons and by in vivo cerebral
ischaemia. Cell Signal. 1998;10:291–295.
45. Durkin JP, Tremblay R, Chakravarthy B, Mealing G, Morley P, Small
D, Song D. Evidence that the early loss of membrane protein kinase C
Molecular Basis of PKC Activity in Stroke Injury
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
2789
is a necessary step in the excitatory amino acid-induced death of primary
cortical neurons. J Neurochem. 1997;68:1400 –1412.
Laher I, Zhang JH. Protein kinase C and cerebral vasospasm. J Cereb
Blood Flow Metab. 2001;21:887–906.
Chou WH, Choi DS, Zhang H, Mu D, McMahon T, Kharazia VN,
Lowell CA, Ferriero DM, Messing RO. Neutrophil protein kinase Cdelta
as a mediator of stroke-reperfusion injury. J Clin Invest. 2004;114:
49 –56.
Tauskela JS, Chakravarthy BR, Murray CL, Wang Y, Comas T, Hogan
M, Hakim A, Morley P. Evidence from cultured rat cortical neurons of
differences in the mechanism of ischemic preconditioning of brain and
heart. Brain Res. 1999;827:143–151.
Wang J, Bright R, Mochly-Rosen D, Giffard RG. Cell-specific role for
epsilon- and betaI-protein kinase C isozymes in protecting cortical
neurons and astrocytes from ischemia-like injury. Neuropharmacology.
2004;47:136 –145.
McNamara RK, Wees EA, Lenox RH. Differential subcellular redistribution of protein kinase C isozymes in the rat hippocampus induced by
kainic acid. J Neurochem. 1999;72:1735–1743.
Savithiry S, Kumar K. mRNA levels of Ca(2⫹)-independent forms of
protein kinase C in postischemic gerbil brain by northern blot analysis.
Mol Chem Neuropathol. 1994;21:1–11.
Harada KMT, Abu Shama KM, Yamashima T, Yoshida K. Translocation and down-regulation of protein kinase C- alpha, -beta, and
-gamma isoforms during ischemia-reperfusion in rat brain. J Neurochem. 1999;72:2556 –2564.
Koponen S, Keinanen R, Roivainen R, Hirvonen T, Narhi M, Chan PH,
Koistinaho J. Spreading depression induces expression of calciumindependent protein kinase C subspecies in ischaemia-sensitive cortical
layers: regulation by N-methyl-D-aspartate receptors and glucocorticoids. Neuroscience. 1999;93:985–993.
Kumar K, Wu XL. Post-ischemic changes in protein kinase C RNA in
the gerbil brain following prolonged periods of recirculation: a phosphorimaging study. Metab Brain Dis. 1994;9:323–331.
Schulz R, Cohen MV, Behrends M, Downey JM, Heusch G. Signal
transduction of ischemic preconditioning. Cardiovasc Res. 2001;52:
181–198.
Goldberg MP, Monyer H, Weiss JH, Choi DW. Adenosine reduces
cortical neuronal injury induced by oxygen or glucose deprivation in
vitro. Neurosci Lett. 1988;89:323–327.
Di-Capua N, Sperling O, Zoref-Shani E. Protein kinase C-epsilon is
involved in the adenosine-activated signal transduction pathway conferring protection against ischemia-reperfusion injury in primary rat
neuronal cultures. J Neurochem. 2003;84:409 – 412.
Lange-Asschenfeldt C, Raval AP, Dave KR, Mochly-Rosen D, Sick TJ,
Perez-Pinzon MA. Epsilon protein kinase C mediated ischemic tolerance
requires activation of the extracellular regulated kinase pathway in the
organotypic hippocampal slice. J Cereb Blood Flow Metab. 2004;24:
636 – 645.
Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli R,
Ping P. Mitochondrial PKCepsilon and MAPK form signaling modules in
the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions
and differential MAPK activation in PKCepsilon-induced cardioprotection.
Circ Res. 2002;90:390–397.
Teshima Y, Akao M, Li RA, Chong TH, Baumgartner WA, Johnston
MV, Marban E. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by
oxidative stress. Stroke. 2003;34:1796 –1802.
Yamada K, Inagaki N. ATP-sensitive K(⫹) channels in the brain:
sensors of hypoxic conditions. News Physiol Sci. 2002;17:127–130.
Sato T, O’Rourke B, Marban E. Modulation of mitochondrial ATPdependent K⫹ channels by protein kinase C. Circ Res. 1998;83:
110 –114.
Light PE, Bladen C, Winkfein RJ, Walsh MP, French RJ. Molecular
basis of protein kinase C-induced activation of ATP-sensitive potassium
channels. Proc Natl Acad Sci U S A. 2000;97:9058 –9063.
Hu K, Huang CS, Jan YN, Jan LY. ATP-sensitive potassium channel
traffic regulation by adenosine and protein kinase C. Neuron. 2003;38:
417– 432.
Liu GS, Thornton J, Van Winkle DM, Stanley AW, Olsson RA, Downey
JM. Protection against infarction afforded by preconditioning is
mediated by A1 adenosine receptors in rabbit heart. Circulation. 1991;
84:350 –356.
Baxter GF. Role of adenosine in delayed preconditioning of myocardium. Cardiovasc Res. 2002;55:483– 494.
2790
Stroke
December 2005
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
67. Ohnuma Y, Miura T, Miki T, Tanno M, Kuno A, Tsuchida A,
Shimamoto K. Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning.
Am J Physiol Heart Circ Physiol. 2002;283:H440 –H447.
68. Favaron M, Manev H, Siman R, Bertolino M, Szekely AM,
DeErausquin G, Guidotti A, Costa E. Down-regulation of protein kinase
C protects cerebellar granule neurons in primary culture from glutamateinduced neuronal death. Proc Natl Acad Sci U S A. 1990;87:1983–1987.
69. Vaccarino FM, Liljequist S, Tallman JF. Modulation of protein kinase C
translocation by excitatory and inhibitory amino acids in primary
cultures of neurons. J Neurochem. 1991;57:391–396.
70. Fukunaga K, Soderling TR, Miyamoto E. Activation of Ca2⫹/calmodulin-dependent protein kinase II and protein kinase C by glutamate in
cultured rat hippocampal neurons. J Biol Chem. 1992;267:
22527–22533.
71. Kaasinen SK, Goldsteins G, Alhonen L, Janne J, Koistinaho J. Induction
and activation of protein kinase C delta in hippocampus and cortex after
kainic acid treatment. Exp Neurol. 2002;176:203–212.
72. Kurkinen K, Busto R, Goldsteins G, Koistinaho J, Perez-Pinzon MA.
Isoform-specific membrane translocation of protein kinase C after ischemic preconditioning. Neurochem Res. 2001;26:1139 –1144.
73. Wagey R, Hu J, Pelech SL, Raymond LA, Krieger C. Modulation of
NMDA-mediated excitotoxicity by protein kinase C. J Neurochem.
2001;78:715–726.
74. Koponen S, Kurkinen K, Akerman KE, Mochly-Rosen D, Chan PH,
Koistinaho J. Prevention of NMDA-induced death of cortical neurons by
inhibition of protein kinase Czeta. J Neurochem. 2003;86:442– 450.
75. Saito N, Shirai Y. Protein kinase C gamma (PKC gamma): function of
neuron specific isotype. J Biochem (Tokyo). 2002;132:683– 687.
76. Wieloch T, Cardell M, Bingren H, Zivin J, Saitoh T. Changes in the
activity of protein kinase C and the differential subcellular redistribution
of its isozymes in the rat striatum during and following transient
forebrain ischemia. J Neurochem. 1991;56:1227–1235.
77. Cardell M, Wieloch T. Time course of the translocation and inhibition of
protein kinase C during complete cerebral ischemia in the rat. J Neurochem. 1993;61:1308 –1314.
78. Saluja I, O’Regan MH, Song D, Phillis JW. Activation of cPLA2, PKC,
and ERKs in the rat cerebral cortex during ischemia/reperfusion. Neurochem Res. 1999;24:669 – 677.
79. Aronowski J, Labiche LA. Perspectives on reperfusion-induced damage
in rodent models of experimental focal ischemia and role of gammaprotein kinase C. ILAR J. 2003;44:105–109.
80. 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.
81. Arundine M, Tymianski M. Molecular mechanisms of glutamatedependent neurodegeneration in ischemia and traumatic brain injury.
Cell Mol Life Sci. 2004;61:657– 668.
82. Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT,
Huganir RL. Characterization of protein kinase A and protein kinase C
phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using
phosphorylation site-specific antibodies. J Biol Chem. 1997;272:
5157–5166.
83. Zheng X, Zhang L, Wang AP, Bennett MV, Zukin RS. Protein kinase C
potentiation of N-methyl-D-aspartate receptor activity is not mediated by
phosphorylation of N-methyl-D-aspartate receptor subunits. Proc Natl
Acad Sci U S A. 1999;96:15262–15267.
84. Cheung HH, Teves L, Wallace MC, Gurd JW. Inhibition of protein
kinase C reduces ischemia-induced tyrosine phosphorylation of the
N-methyl-d-aspartate receptor. J Neurochem. 2003;86:1441–1449.
85. Suen PC, Wu K, Xu JL, Lin SY, Levine ES, Black IB. NMDA receptor
subunits in the postsynaptic density of rat brain: expression and phosphorylation by endogenous protein kinases. Brain Res Mol Brain Res.
1998;59:215–228.
86. Yaka R, Thornton C, Vagts AJ, Phamluong K, Bonci A, Ron D. NMDA
receptor function is regulated by the inhibitory scaffolding protein,
RACK1. Proc Natl Acad Sci U S A. 2002;99:5710 –5715.
87. Sieber FE, Traystman RJ, Brown PR, Martin LJ Protein kinase C
expression and activity after global incomplete cerebral ischemia in
dogs. Stroke. 1998;29:1445–1452.
88. Nishizuka Y. Studies and perspectives of protein kinase C. Science.
1986;233:305–312.
89. Brodie C, Blumberg PM. Regulation of cell apoptosis by protein kinase
c delta. Apoptosis. 2003;8:19 –27.
90. Majumder PK, Mishra NC, Sun X, Bharti A, Kharbanda S, Saxena S,
Kufe D. Targeting of protein kinase C delta to mitochondria in the
oxidative stress response. Cell Growth Differ. 2001;12:465– 470.
91. Koponen S, Goldsteins G, Keinanen R, Koistinaho J. Induction of
protein kinase Cdelta subspecies in neurons and microglia after transient
global brain ischemia. J Cereb Blood Flow Metab. 2000;20:93–102.
92. Miettinen S, Roivainen R, Keinanen R, Hokfelt T, Koistinaho J. Specific
induction of protein kinase C delta subspecies after transient middle
cerebral artery occlusion in the rat brain: inhibition by MK-801.
J Neurosci. 1996;16:6236 – 6245.
93. Bright R, Raval AP, Dembner JM, Perez-Pinzon MA, Steinberg GK,
Yenari MA, Mochly-Rosen D. Protein kinase C delta mediates cerebral
reperfusion injury in vivo. J Neurosci. 2004;24:6880 – 6888.
94. Murriel CL, Churchill E, Inagaki K, Szweda LI, Mochly-Rosen D.
Protein kinase C delta activation induces apoptosis in response to
cardiac ischemia and reperfusion damage: a mechanism involving BAD
and the mitochondria. J Biol Chem. 2004;279:47985– 47991.
95. Chan PH. Reactive oxygen radicals in signaling and damage in the
ischemic brain. J Cereb Blood Flow Metab. 2001;21:2–14.
96. Yoshida K, Mizukami Y, Kitakaze M. Nitric oxide mediates protein
kinase C isoform translocation in rat heart during postischemic reperfusion. Biochim Biophys Acta. 1999;1453:230 –238.
97. Emoto Y, Manome Y, Meinhardt G, Kisaki H, Kharbanda S, Robertson
M, Ghayur T, Wong WW, Kamen R, Weichselbaum R, et al. Proteolytic
activation of protein kinase C delta by an ICE-like protease in apoptotic
cells. EMBO J. 1995;14:6148 – 6156.
98. Churchill EN, Murriel CL, Chen CH, Mochly-Rosen D, Szweda LI.
Reperfusion-induced translocation of deltaPKC to cardiac mitochondria
prevents pyruvate dehydrogenase reactivation. Circ Res. 2005;97:
78 – 85.
99. Churchill EN, Szweda LI. Translocation of deltaPKC to mitochondria
during cardiac reperfusion enhances superoxide anion production and
induces loss in mitochondrial function. Arch Biochem Biophys. 2005;
439:194 –199.
100. Inagaki K, Chen L, Ikeno F, Lee FH, Imahashi K, Bouley DM, Rezaee
M, Yock PG, Murphy E, Mochly-Rosen D. Inhibition of delta-protein
kinase C protects against reperfusion injury of the ischemic heart in
vivo. Circulation. 2003;108:2304 –2307.
101. Raval AP, Dave KR, Prado R, Katz LM, Busto R, Sick TJ, Ginsberg
MD, Mochly-Rosen D, Perez-Pinzon MA. Protein kinase C delta
cleavage initiates an aberrant signal transduction pathway after cardiac
arrest and oxygen glucose deprivation. J Cereb Blood Flow Metab.
2005;25:730 –741.
102. Maczewski M, Beresewicz A. The role of endothelin, protein kinase C
and free radicals in the mechanism of the post-ischemic endothelial
dysfunction in guinea-pig hearts. J Mol Cell Cardiol. 2000;32:297–310.
103. Yuan SY. Protein kinase signaling in the modulation of microvascular
permeability. Vascul Pharmacol. 2002;39:213–223.
104. Recommendations for standards regarding preclinical neuroprotective
and restorative drug development. Stroke. 1999;30:2752–2758.
105. Mochly-Rosen D, Kauvar LM. Modulating protein kinase C signal
transduction. Adv Pharmacol. 1998;44:91–145.
106. Way KJ, Chou E, King GL. Identification of PKC-isoform-specific
biological actions using pharmacological approaches. Trends Pharmacol
Sci. 2000;21:181–187.
107. Souroujon MC, Mochly-Rosen D. Peptide modulators of protein-protein
interactions in intracellular signaling. Nat Biotechnol. 1998;16:919 –
924.
108. Mochly-Rosen D. Localization of protein kinases by anchoring proteins:
a theme in signal transduction. Science. 1995;268:247–251.
109. Schechtman D, Mochly-Rosen D. Isozyme-specific inhibitors and activators of protein kinase C. Methods Enzymol. 2002;345:470 – 489.
110. Begley R, Liron T, Baryza J, Mochly-Rosen D. Biodistribution of
intracellularly acting peptides conjugated reversibly to Tat. Biochem
Biophys Res Commun. 2004;318:949 –954.
111. Inagaki K, Begley R, Ikeno F, Mochly-Rosen D. Cardioprotection by
epsilon-protein kinase C activation from ischemia: continuous delivery
and antiarrhythmic effect of an epsilon-protein kinase C-activating
peptide. Circulation. 2005;111:44 –50.
112. Murriel CL, Mochly-Rosen D. Opposing roles of delta and epsilonPKC
in cardiac ischemia and reperfusion: targeting the apoptotic machinery.
Arch Biochem Biophys. 2003;420:246 –254.
The Role of Protein Kinase C in Cerebral Ischemic and Reperfusion Injury
Rachel Bright and Daria Mochly-Rosen
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Stroke. 2005;36:2781-2790; originally published online October 27, 2005;
doi: 10.1161/01.STR.0000189996.71237.f7
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2005 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/36/12/2781
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office.
Once the online version of the published article for which permission is being requested is located, click
Request Permissions in the middle column of the Web page under Services. Further information about this
process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Stroke is online at:
http://stroke.ahajournals.org//subscriptions/