Introduction

Top Abstract Introduction Cardiac Tolerance Neuronal Ischemic Tolerance Summary References

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; 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

Top Abstract Introduction Cardiac Tolerance Neuronal Ischemic Tolerance Summary References

Tolerance has been investigated extensively in the myocardium, in part, because the human heart can be preconditioned 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 uncouplers, nitric oxide (NO), as well as brief periods of sublethal ischemia (Rubino and Yellon, 2000).

View larger version (26K): [in this window] [in a new window]   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.

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 (K_ATP) 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 K_ATP channels (Rubino and Yellon, 2000). In another study, NO induced cardiac tolerance, in part, through modulation of ATP sensitivity of the mitochondrial K_ATP 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 K_ATP 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 K_ATP channels via the p38 mitogen-activated protein kinase. Potassium channels that are inhibited by internal ATP (K_ATP channels) provide a critical link between metabolism and cellular excitability. PKC activation results in a decreased Hill coefficient for ATP binding to cardiac K_ATP channels, thereby increasing their open probability at physiological ATP concentrations. PKC activation may facilitate opening of the K_ATP channels either by direct phosphorylation of the channel or by modification of channel-associated proteins. While sarcolemmal K_ATP channels were studied initially with regard to the development of tolerance, recent reports implicate the mitochondrial K_ATP channels in mediating preconditioning-induced cardioprotection (Rubino and Yellon, 2000). The recent observation that NO can regulate mitochondrial K_ATP channels provides a putative link between these preconditioning pathways.

	Neuronal Ischemic Tolerance

Top Abstract Introduction Cardiac Tolerance Neuronal Ischemic Tolerance Summary References

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; Gonzalez-Zulueta 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 preconditioning 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).

View larger version (18K): [in this window] [in a new window]   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 redox-sensitive 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.

Role for Erks in Neuronal Tolerance. The Ras/Erk pathway is a hierarchical cascade that typically originates with the recruitment of the p21^Ras 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).

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/p21^Ras/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).

	Summary

Top Abstract Introduction Cardiac Tolerance Neuronal Ischemic Tolerance Summary References

Preconditioning sets in motion a series of signaling cascades that ultimately result in profound neuroprotection due to expression of newly synthesized proteins. Preconditioning leading to tolerance is a novel form of plasticity that may share common signaling pathways including the Ras/Erk signaling cascade. Understanding the transcriptional elements responsible for preconditioning and tolerance will point the direction toward the proteins and cellular changes that mediate tolerance. There may be other Ca²⁺-dependent pathways that participate in the development of tolerance in vivo. Identification and characterization of these pathways will undoubtedly enhance our understanding of the phenomena of tolerance and may lead to novel treatments for ischemia reperfusion injury in many different organ systems.