0022-3565/01/2972-474 –478$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 297:474–478, 2001
Vol. 297, No. 2
900024/985998
Printed in U.S.A.
Perspectives in Pharmacology
Critical Role for Nitric Oxide Signaling in Cardiac and Neuronal
Ischemic Preconditioning and Tolerance
KRISHNADAS NANDAGOPAL, TED M. DAWSON, and VALINA L. DAWSON
Departments of Neurology (K.N., T.M.D., V.L.D.), Neuroscience (T.M.D., V.L.D.), and Physiology (V.L.D.), Johns Hopkins University School of
Medicine, Baltimore, Maryland
This paper is available online at http://jpet.aspetjournals.org
ABSTRACT
Preconditioning to ischemic tolerance is a phenomenon in
which brief episodes of a subtoxic insult induce a robust protection against the deleterious effects of subsequent, prolonged, lethal ischemia. The subtoxic stimuli that constitute the
preconditioning event are quite diverse, ranging from brief ischemic episodes, spreading depression or potassium depolarization, chemical inhibition of oxidative phosphorylation, exposure
to excitotoxins and cytokines. The beneficial effects of preconditioning were first demonstrated in the heart; it is now clear
that preconditioning can induce ischemic tolerance in a variety
of organ systems including brain, heart, liver, small intestine,
skeletal muscle, kidney, and lung. There are two temporally and
mechanistically distinct types of protection afforded by preconditioning stimuli, acute and delayed preconditioning. The sig-
Loss of blood flow to the heart or brain results in injury due
to oxygen and nutrient deprivation as well as the initiation of
toxic processes that compromise normal physiological function. The restoration of blood supply and containment of
secondary cardiotoxic or neurotoxic cascades is the focus of
therapeutic intervention aimed at limiting ischemic damage.
Preconditioning to ischemic tolerance is a phenomenon in
which brief episodes of a subtoxic insult induce robust protection against the deleterious effects of subsequent, prolonged, lethal ischemia. The profound protection derived
from preconditioning has now been established in a variety of
organ systems, including brain, heart, liver, skeletal muscle,
small intestine, kidney, and lung (Kloner and Yellon, 1994;
This work was supported by the National Institutes of Health Grant NS37090
and the American Heart Association Established Investigator Award.
naling cascades that initiate the acute and delayed preconditioning responses may have similar biochemical components.
However, the protective effects of acute preconditioning are protein synthesis-independent, mediated by post-translational protein modifications, and are short-lived. The effects of delayed
preconditioning require new protein synthesis and are sustained
for days to weeks. Elucidation of the molecular mechanisms that
are involved in preconditioning and ischemic tolerance and identification of drugs that mimic this protective response have the
potential to improve the prognosis of patients at risk for ischemic
injury. This article focuses on recent findings on the effects of
ischemic preconditioning in the cardiac and nervous systems and
discusses potential targets for a successful therapeutic approach
to limit ischemia-reperfusion injury.
Baxter, 1997; Chen and Simon, 1997; Ishida et al., 1997; Tomai
et al., 1999). Although certain pathophysiologic issues may be
similar, the mechanisms of induction and maintenance of tolerance in the brain are distinct from those described in the
heart. We review recent findings that describe the molecular
basis for ischemic tolerance in these organ systems and highlight the importance of nitric oxide-mediated protection. Pharmacologic preconditioning with drugs that mimic the beneficial
effects of ischemic preconditioning could lead to novel therapeutic approaches for the treatment of ischemic disorders including
myocardial infarction and stroke.
Cardiac Tolerance
Tolerance has been investigated extensively in the myocardium, in part, because the human heart can be precondi-
ABBREVIATIONS: NO, nitric oxide; NOS, NO synthase; iNOS, immunologic NOS; eNOS, endothelial NOS; nNOS, neuronal NOS; PKC, protein
kinase C; NF-␬B, nuclear factor-␬B; SNAP, S-nitroso-N-acetyl-DL-penicillamine; KATP channel, ATP-sensitive potassium channel; MAPK, mitogenactivated protein kinase; Erk, extracellular signal-regulated kinase; 8Br-cGMP, 8-bromo-3⬘,5⬘-cyclic guanosine monophosphate; NMDA, Nmethyl-D-aspartate; OGD, oxygen-glucose deprivation; GEF, guanine nucleotide-exchange factor; PI3K, phosphoinositide 3-kinase; BDNF,
brain-derived neurotrophic factor; Mek, MAPK/Erk kinase; CREB, cAMP response element-binding protein.
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Received September 15; 2000; accepted December 15, 2000
Nandagopal et al.
Fig. 1. Mediators of cardioprotection. Cardiac preconditioning occurs in
two distinct temporal phases: acute and delayed. Stimulation of the A1
and A3 adenosine receptor subtypes results in activation of PKC, p38
MAPK, and the opening of KATP channels in the acute phase. NO from
eNOS activation may also contribute to the regulation of KATP channels.
The delayed phase of preconditioning may involve adenosine receptor
activation of PKC, eNOS-generated NO, and peroxynitrite (ONOO⫺) may
activate PKC. PKC engages a complex signaling cascade. Downstream
signaling may involve p38 MAPK, Src and Lck tyrosine kinases, the Erks,
NF-␬B, or hypoxia inducible factor-1 (HIF-1) leading to new gene transcription and new protein synthesis. Ultimately iNOS is expressed and is
necessary for the development of cardiac tolerance. The recent observation that NO can regulate mitochondrial KATP channels provides a putative link between these preconditioning pathways.
couplers, nitric oxide (NO), as well as brief periods of sublethal ischemia (Rubino and Yellon, 2000).
Role of Nitric Oxide in Cardiac Tolerance. Functional
evidence indicates that NO plays a prominent role in both
initiating and mediating cardioprotective responses (Fig. 1).
It appears that brief ischemic stress causes increased production of NO via endothelial NO synthase (eNOS) leading to
the activation of protein kinase C (PKC) (Lowenstein, 1999;
Rakhit et al., 1999). In turn, activated PKC engages a complex signaling cascade involving the Src and Lck tyrosine
kinases, the p42 and p44 extracellular signal-regulated kinases (Erks), and nuclear factor-␬B (NF-␬B)-mediated increase in the transcription of immunologic NOS (iNOS)
(Jones et al., 1999; Lowenstein, 1999; Xuan et al., 1999; Bolli,
2000). Pharmacologic inhibition of iNOS induction prevents
ischemic tolerance in a rabbit model of myocardial preconditioning. Induction of iNOS in mice also provides cardioprotection (Rakhit et al., 1999). When iNOS⫺/⫺ mice are preconditioned 24 h before coronary occlusion, infarct size is not
reduced, but wild-type mice experience a profound protection
against ischemic injury. Disruption of the iNOS gene has no
effect on early preconditioning or on infarct size in the absence of preconditioning (Bolli, 2000). The measurement of redox potentials in rabbit ventricular myocytes shows that the
NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) potentiates oxidative effects of the mitochondrial ATP-sensitive potassium (KATP) channel opener diazoxide (Sasaki et al., 2000). The
observed effects of NO do not appear to involve cGMP-dependent mechanisms, since 8Br-cGMP failed to mimic the effects of
SNAP in this model system (Sasaki et al., 2000). However,
L-arginine preconditions isolated rabbit hearts through NO generation, and this response is mediated through a cGMP-dependent mechanism but is independent of the KATP channels (Rubino and Yellon, 2000). In another study, NO induced cardiac
tolerance, in part, through modulation of ATP sensitivity of the
mitochondrial KATP channel (Bolli, 2000; Rubino and Yellon,
2000). While there is clear evidence for NO mediation of both
acute and delayed preconditioning, there is controversy over the
biochemical pathways involved as well as the relative importance of NO versus other mediators of cardiac preconditioning.
As the field advances it is likely these apparent controversies
will be resolved.
Other Mediators of Cardioprotection. Cardiac preconditioning can also involve stimulation of the A1 and A3 adenosine receptor subtypes (Fig. 1), PKC activation, and the
opening of KATP channels (Rubino and Yellon, 2000). However, the functional significance of interactions between the
receptor subtypes in vivo remains unknown. Although the
precise mechanism of PKC activation by adenosine during
preconditioning has yet to be elucidated, signaling downstream of PKC may involve p38 MAPK, c-Jun NH2-terminal
kinase (JNK), or the Erks. The nuclear activation of MAPK
and induction of immediate early genes (c-fos, c-myc, c-jun)
possibly aid recovery of cardiac tissue from brief periods of
ischemia (Bolli, 2000). PKC activation facilitates opening of
the KATP channels via the p38 mitogen-activated protein
kinase. Potassium channels that are inhibited by internal
ATP (KATP channels) provide a critical link between metabolism and cellular excitability. PKC activation results in a
decreased Hill coefficient for ATP binding to cardiac KATP
channels, thereby increasing their open probability at physiological ATP concentrations. PKC activation may facilitate
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tioned ex vivo and in situ during elective procedures such as
angioplasty and coronary artery bypass grafting. Preconditioning may also occur naturally in some ischemic cardiac
syndromes, such as warm-up angina and preinfarction angina (Carroll and Yellon, 1999). The first evidence for myocardial ischemic preconditioning came from observations
that multiple episodes of brief ischemia do not lead to a
cumulative depletion of high-energy phosphate compounds
or impairment of cardiac function (Edwards et al., 2000).
Rather, preconditioning renders the myocardium tolerant to
subsequent lethal ischemia with reduction in infarct volume,
delay in onset of ultrastructural changes, and improved recovery of cardiac function during reperfusion (Carroll and
Yellon, 1999; Edwards et al., 2000). Cardiac preconditioning
occurs in two distinct temporal phases: acute and delayed
(Fig. 1). The acute phase is associated with post-translational
modifications of proteins and is observed within minutes and
dissipates after 2 to 3 h. The second delayed phase develops
hours after the preconditioning event, requires new protein
synthesis, and is sustained for several days (Carroll and
Yellon, 1999; Edwards et al., 2000; Rubino and Yellon, 2000).
However, the causal relationship(s) between the two phases
remains mechanistically undefined. Ischemic preconditioning may involve myocardial, vascular, and neural components that integrate multiple intracellular processes to ultimately curtail energy expenditure and mitigate reperfusion
injury. The protective effects of cardiac preconditioning can
be stimulated by diverse agents, including adenosine, norepinephrine, calcium, bradykinin, heat shock, mitochondrial un-
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Nitric Oxide in Ischemic Preconditioning and Tolerance
opening of the KATP channels either by direct phosphorylation of the channel or by modification of channel-associated
proteins. While sarcolemmal KATP channels were studied
initially with regard to the development of tolerance, recent
reports implicate the mitochondrial KATP channels in mediating preconditioning-induced cardioprotection (Rubino and
Yellon, 2000). The recent observation that NO can regulate
mitochondrial KATP channels provides a putative link between these preconditioning pathways.
Neuronal Ischemic Tolerance
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Kitagawa and coworkers first reported that gerbils subjected to sublethal transient global ischemia exhibited reduced hippocampal CA1 neuronal death after a severe ischemic insult 24 to 48 h later (Kitagawa et al., 1990). In the
brain, ischemic preconditioning is mediated largely through
calcium influx through the NMDA receptor, and neuronal
preconditioning requires new protein synthesis (Kato et al.,
1992; Kasischke et al., 1996; Roth et al., 1998; Grabb and
Choi, 1999; Gonzalez-Zulueta et al., 2000). Preconditioning is
triggered by diverse stimuli ranging from transient ischemic
episodes, spreading depression (Kawahara et al., 1997), hypoxia (Gidday et al., 1999), anoxia (Centeno et al., 1999),
adenosine (Heurteaux et al., 1995), chemical inhibition of
oxidative phosphorylation (Riepe et al., 1997), exposure to
excitotoxins (Grabb and Choi, 1999), and cytokines (Nawashiro et al., 1997). As in the heart, there is both acute and
delayed preconditioning. Cardiologists have exploited acute
tolerance clinically before invasive procedures. However, the
clinical utility of transient acute tolerance in the brain is not
apparent. Most investigators have focused on the acquisition
of delayed tolerance in the brain that occurs over a relatively
long period of time and persists for days to weeks. Requirements for the induction of tolerance depend, in part, on the
experimental model, whether global or focal ischemia, and
the animal species studied. There is a rich descriptive literature developing, illustrating changes in protein expression
such as induction of heat shock proteins (Sharp et al., 1999)
and bcl2 (Shimazaki et al., 1994) and decreased expression of
NMDA receptor NR2A and NR2B subunits (Shamloo and
Wieloch, 1999). Post-translational modifications of proteins
are also described including phosphorylation of protein tyrosines and the extracellular signal-regulated protein kinase
cascade (Shamloo et al., 1999; Shamloo and Wieloch, 1999).
However, exploring the functional relevance of these observed changes has proved difficult.
Investigators have modeled ischemic tolerance in culture
systems to gain a better understanding of the underlying
mechanisms. Ischemia can be mimicked in vitro by combined
oxygen-glucose deprivation (OGD) (Monyer et al., 1992). Preconditioning can be induced in vitro by brief exposure of neurons to OGD (Grabb and Choi, 1999; Gonzalez-Zulueta et al.,
2000). The salient features of ischemic tolerance observed in
vivo (Bond et al., 1999) can be replicated in this culture model
system: tolerance is dependent on NMDA receptor activation,
but not ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) or kainate receptor activation, requires calcium influx
and new protein synthesis (Grabb and Choi, 1999; GonzalezZulueta et al., 2000). The demonstration of OGD-induced tolerance in vitro has provided a powerful means of determining
which signaling pathways are activated during the precondi-
tioning exposure to OGD and assessing the functional significance of these signaling events.
Nitric Oxide in Neuronal Tolerance. In a newborn rat
model of hypoxic preconditioning, exposure to sublethal hypoxia for 3 h renders postnatal day 6 animals resistant to
cerebral hypoxic-ischemic insult imposed 24 h later. In this
model, preconditioning does not involve iNOS or neuronal
NO synthase (nNOS) but is dependent on NO produced by
eNOS to mediate protection (Gidday et al., 1999). In the rat
hippocampal slice model, nNOS-derived NO is involved in
neuroprotection mediated by anoxic preconditioning (Centeno et al., 1999). Preconditioning improves electrical recovery after anoxia in hippocampal slices with no significant
changes in NADH hyperoxidation (Centeno et al., 1999). In
culture models, a significant loss of neuroprotection occurs
when NMDA receptor antagonists are present during the
OGD preconditioning stimulus (Grabb and Choi, 1999;
Gonzalez-Zulueta et al., 2000). Application of the NOS inhibitor nitro-L-arginine during the preconditioning episode
blocks the protective actions of preconditioning by ⬃70%, and
coadministration of an excess of the NOS substrate L-arginine restores protection. NO donors induce tolerance in a
dose-dependent manner, indicating that NO is a key mediator in processes leading to tolerance against lethal ischemia.
The potent and selective inhibitor of guanylyl cyclase, 1H(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), has no effect on ischemic preconditioning nor does the cell permeable
cGMP analog 8Br-cGMP elicit tolerance, thus ruling out a
role for guanylyl cyclase in NO-mediated tolerance to OGD
(Gonzalez-Zulueta et al., 2000).
Functional analysis of OGD tolerance in cortical cultures
has been extended to signaling downstream of nNOS activation (Fig. 2). Lander and colleagues had previously shown
that p21Ras is a target of nitric oxide and is activated by
redox-sensitive mechanisms (Lander et al., 1995). Recently,
we demonstrated NO-induced activation of p21Ras following
NMDA receptor stimulation in primary cortical cultures
(Yun et al., 1998). How NO activates Ras is unknown. Direct
activation of Ras may occur via NO-mediated nitrosylation of
a critical cysteine residue (Lander et al., 1995), or NO nitrosylation of Ras could promote an interaction with a critical
GEF that leads to Ras activation. Ras-dependent signaling
pathways are involved in gene transcription, regulation of
synaptic plasticity, and neuronal growth and survival (Curtis
and Finkbeiner, 1999). Robust activation of Ras is observed
during a 5-min preconditioning exposure to OGD in a NMDA
receptor- and NO-dependent, but cGMP-independent, manner. Inhibition of Ras during the preconditioning event both
pharmacologically or with dominant negative mutants to Ras
completely abolishes the development of tolerance. Expression of a constitutively active form of Ras is sufficient to
induce protection against lethal OGD. These data provide
functional evidence for activation of Ras in the development
of tolerance (Gonzalez-Zulueta et al., 2000).
Ras signaling mediates cell survival through activation of
the PI3K/Akt or Raf/Erk effector cascades (Kolch, 2000). The
PI3K/Akt pathway is involved in anti-apoptotic signaling in
cerebellar granule cells and in peripheral sympathetic and
sensory neurons (Kolch, 2000). Neither pharmacologic inhibition nor dominant negative mutants to PI3K have any
effect on the development of tolerance to OGD indicating that
PI3K activity is not essential for ischemic preconditioning of
Nandagopal et al.
central neurons (Gonzalez-Zulueta et al., 2000). However,
PI3K/Akt-dependent signaling may be important for protection
of neurons located in the periphery, spinal cord, or cerebellum.
Glutamate receptor activation can elicit the production
and release of brain-derived neurotrophic factor (BDNF),
which can signal through activation of the Ras/Erk pathway
(Friedman and Greene, 1999). However, BDNF does not induce OGD tolerance in primary cortical cultures and specific
anti-BDNF antibodies or tyrosine kinase B (TrkB) receptor
bodies failed to block tolerance to OGD, thus ruling out a significant role for BDNF in OGD tolerance (Gonzalez-Zulueta et
al., 2000). However, BDNF may play a role in spreading depression-induced tolerance (Kawahara et al., 1997), and recent studies have ascribed a role for nNOS in mediating the neuroprotection induced by cortical spreading depression (Caggiano and
Kraig, 1998; Shen and Gundlach, 1999). The signaling intermediates that promote the development of tolerance in this model
merit further investigation. Additionally, BDNF is markedly
neuroprotective against neonatal hypoxic-ischemic brain injury
in vivo (Han and Holtzman, 2000). In this model, intracerebroventricular administration of BDNF to postnatal day 7 rats
resulted in phosphorylation of Erk1/2. Pharmacological inhibition of Erk reversed the neuroprotective effects of BDNF on
hypoxic-ischemic brain injury (Han and Holtzman, 2000).
Role for Erks in Neuronal Tolerance. The Ras/Erk
pathway is a hierarchical cascade that typically originates with
the recruitment of the p21Ras GTPase. Ras engages the serine/
threonine kinase Raf, which activates Mek (MAPK/Erk kinase).
Mek, in turn, phosphorylates and activates p42 and p44 Erks.
The sequential interactions in the Ras/Erk signaling pathway
permit regulation, integration, and enzymatic amplification of
the initial signals to promote a graded temporal and spatial
response. MAPK/Erk signaling cascades are linked to diverse
neuronal processes including long-term potentiation, synaptic
plasticity, consolidation of memory, development, cell survival,
and cell death (Impey et al., 1999).
Recent reports indicate that Erk signaling may be important for the development of ischemic preconditioning. Hypoxia stimulates rapid Erk phosphorylation in the cortex of
rats in a NMDA receptor-dependent manner (Gozal et al.,
1999). Increased phosphorylation of Mek and Erk has been
observed in the CA1 region of the hippocampus in a rat model
of global cerebral ischemic preconditioning (Shamloo et al.,
1999). We have recently demonstrated a functional requirement for the Ras/Erk signaling pathway in the acquisition of
tolerance in vitro (Fig. 2). Dominant negative mutants to Raf,
Mek, and Erk2 all blocked the development of tolerance to
OGD and the OGD-induced activation of Erk. Conversely,
constitutively active Raf and Mek mutants induced neuroprotection against 60 min of OGD and were capable of activating similar levels of Erk phosphorylation. Our results
indicate that all of these signaling mediators, Ras, Raf, Mek
and Erk, are required for the development of tolerance to
ischemia (Fig. 2) (Gonzalez-Zulueta et al., 2000).
Is Neuronal Preconditioning via Erk Activation Coupled to Transcription of Neuroprotective Genes? Since
induction of neuronal ischemic tolerance is dependent on new
protein synthesis, and the development of tolerance is blocked
by cycloheximide (Gonzalez-Zulueta et al., 2000), the profound
protection derived from preconditioning may result from transcriptional activation of neuroprotective proteins by the NMDA/
NO/p21Ras/Erk pathway. What are the neuronal targets of Erk
activity that stimulate preconditioning? Substrates for Erk include cytoskeletal proteins, cell adhesion molecules, ion channels, and pp90 ribosomal S6 kinases (Rsks) (Impey et al., 1999).
Since protein phosphorylation is a reversible modification, it
cannot be responsible for long-term plastic changes that result
in neuroprotection elicited by preconditioning. However, phosphorylation of transcriptional elements may regulate the expression of neuroprotective genes associated with the long-term
changes necessary for the acquisition of tolerance. Indeed, Erk
activation can stimulate nuclear transcription factors such as
Elk-1 and the cAMP response element-binding protein (CREB)
(Impey et al., 1999).
Activation of CREB is an attractive putative target as
CREB stimulates the transcription of immediate early response genes, which, in turn, induce the delayed response
genes that influence neuronal activity including growth factors, enzymes that synthesize neurotransmitters, synaptic
vesicle proteins, ion channels, and structural proteins (De
Cesare et al., 1999). Cortical plasticity involves activity-dependent changes in synaptic strength mediated by CREB
and is dependent on long-lasting biochemical changes in
postsynaptic neurons (Shieh and Ghosh, 1999). Calcium influx during ischemic preconditioning may thus activate
CREB in a manner that results in increased production of
neuroprotective molecules. Indeed, recent studies suggest
that CREB is important for cellular survival induced by
neurotrophins (Walton and Dragunow, 2000).
The expression of Elk-1 in various brain structures of the
adult rat is exclusively neuronal (Sgambato et al., 1998).
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Fig. 2. Mediators of neuroprotection. Neuronal ischemic preconditioning
is mediated largely through the activation of the NMDA receptors, involves increased intracellular calcium, and requires new protein synthesis. The NMDA receptor is coupled to nNOS via the scaffolding protein
PSD-95 effectively coupling calcium influx with nNOS activation and NO
production. In primary cortical cultures, NMDA receptor stimulation
leads to NO-induced activation of p21Ras, perhaps through a direct redoxsensitive activation of p21Ras or through activation of a NO-dependent
GEF. Subsequently, Raf, Mek, and Erk are activated. Inhibition of any of
these steps during the preconditioning event is sufficient to prevent
preconditioning from developing. It is yet unknown which transcriptional
elements are activated or which protein(s) mediate tolerance; however,
Elk-1 and CREB are attractive potential transcriptional candidates.
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Nitric Oxide in Ischemic Preconditioning and Tolerance
Stimulation of corticostriatal pathways or treatment of cultures with glutamate activates Erk, Elk-1 phosphorylation,
and immediate early gene (c-fos, zif268, and MAPK phosphatase-1) induction. These data implicate Erk and Elk-1 activation in the regulation of genes controlled by neuronal activity. In this model, CREB is also activated, and the
inhibition of Erk blocked activation of both Elk-1 and CREB
phosphorylation, providing evidence that Erk activity can
regulate both Elk-1 and CREB in neurons.
It is not yet known if CREB, Elk-1, or some other transcriptional pathway is activated by the induction of ischemic
tolerance. However, understanding the transcriptional elements responsible for preconditioning and tolerance will
point the direction toward the proteins and cellular changes
that mediate tolerance.
Summary
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