National Research Council, Institute for Biological Sciences, Montreal Road Campus, Ottawa, Ontario, Canada K1A 0R6
Submitted 20 May 2003 ; accepted in final form 16 June 2003
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ABSTRACT |
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N-methyl-D-aspartate; Ca2+; antioxidants; mitochondria
Cultured neurons exposed to tolerance-inducing oxygen-glucose deprivation (OGD) preconditioning can be used to investigate neuron-specific stimuli that might mediate cerebral ischemic preconditioning (6). An initial attempt to correlate cellular signaling between the heart and brain revealed diverse pathways (63). Hence, an additional framework was sought to study the mechanism of neuronal OGD preconditioning.
Ischemic preconditioning protocols that induce tolerance are also capable of producing injury if too intensive or persistent. As for cerebral ischemic preconditioning, the threshold for OGD preconditioning and for neuronal injury may not be that far apart. The preconditioning paradigm employed in this study requires subjecting cortical cultures to as great an OGD stress as possible without causing toxicity. Thus we now consider the possibility that preconditioning initiates an endogenous neuroprotective response against OGD toxicity, primarily through cellular signaling that has the potential to be neurotoxic if of sufficient intensity. In cortical neurons, OGD of sufficient duration results in neuronal N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx, which may induce the formation of toxic reactive oxygen species. The feasibility of such a concept has already been demonstrated at the receptor level: transient activation of the neuronal NMDA receptor alone may precondition or kill cortical neurons, depending on the intensity of the stimulus (20).
Cerebral ischemic or excitotoxic generation of reactive oxygen species has been the subject of intense investigation. Intermediate between physiological and pathological activity, reactive oxygen species may also serve as activators of redox-sensitive signal transduction pathways during ischemic preconditioning. Mitochondrial Ca2+ uptake upregulates energy metabolism, resulting in increased generation of reactive oxygen species, although the mechanism (70) and temporal pattern of oxidant production by excitotoxicity (69) remains uncertain. The antioxidant MnTBAP [Mn(III)tetra(4-carboxyphenyl)porphyrin] suppresses ischemic tolerance by ischemic (30) and nonischemic preconditioning (37, 74) in nonneuronal tissue, consistent with oxygen radicals functioning as general mediators of preconditioning (66). Reactive oxygen species-dependent hypoxic preconditioning in cardiomyocytes is suppressed by an anion channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating superoxide transfer to the cytosol via mitochondrial anion channels (68). Reactive oxygen species have been implicated in ischemic tolerance generated by "chemical" preconditioning of the brain (for example, see Ref. 73). Superoxide radicals and H2O2 alone can "precondition" neurons against glutamate toxicity (48, 49) or ischemia (40).
In the current study, Ca2+-dependent neuronal signaling activated by NMDA receptors during OGD preconditioning is evaluated. Specifically, signaling pathways implicated in other models of ischemic preconditioning, as well as those linked to neurotoxic signaling activated by lethal OGD in cortical neurons, are investigated.
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MATERIALS AND METHODS |
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Tissue culture plates were purchased from either DuPont-Life Technologies (Burlington, ON, Canada) or VWR Canlab (Mississauga, ON, Canada), and 12-mm glass coverslips were purchased from Bellco Glass (Vineland). Fetal bovine serum and modified Eagle's medium (MEM) were obtained from Wisent Canadian Laboratories (St-Bruno, QC, Canada). Horse serum was acquired from Hyclone Laboratories (Logan). Fluo 3-AM, fura red-AM, Fluo-4FF, and DIDS were bought from Molecular Probes (Eugene, OR). 4,5-Diamin-ofluorescein diacetate (DAF-2) was purchased from Calbiochem (San Diego, CA). Diethylamine (DEA)/NONOate was purchased from Cedarlane Laboratories (Hornby, ON, Canada). MnTBAP and KN-62 were purchased from Alexis Biochemicals (San Diego, CA). Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], Mn(III)tetra(4-sulfonatophenyl)porphyrin [Mn(III)TPPS], and Mn(III)tetra(N-ethyl-2-pyridyl) porphyrin chloride [Mn(III)TE-PyP(2)] were purchased from Mid-Century Chemicals (Posen, IL). PD-98059 was purchased from Tocris (Ellisville, MO). NMDA, propidium iodide (PI), 7-nitroindazole (7-NI), NG-nitro-L-arginine (L-NNA), 1,5-isoquinolinediol (IQL), 8-cyclopentyltheophylline (8-CPT), 8-(p-sulfonyl)theophylline (8-SPT), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), N6-R-phenylisopropyladenosine (R-PIA), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), U0126, 2,4-dinitrophenol (DNP), tetrodotoxin (TTX), and all other reagents were purchased from Sigma (St. Louis, MO).
Preparation of Rat Cortical Cultures
Cultures of E18 rat cortical neurons were prepared as described previously (64). Timed-pregnant Sprague-Dawley rats (Charles River Canada, St. Constant, QC, Canada) were anesthetized with halothane and killed by cervical dislocation. After dissection of the cortical region of the fetal brain, cortical neurons were dispersed by trituration, centrifuged at 250 g for 5 min at 4°C, and suspended in a medium containing MEM supplemented to 25 mM glucose, 10% fetal bovine serum, 10% horse serum, and 2 mM glutamine. Cells were plated at 1.0 x 106 cells/ml on poly-L-lysine-coated glass coverslips (for confocal measurements) or at 1.7 x 106 cells/ml on tissue culture plates. Cultures were treated with 15 µg/ml 5-fluoro-2'-deoxyuridine and 35 µg/ml uridine after 4 days in vitro to minimize glial growth. At 7 days in vitro, one-half of the medium was replaced with medium consisting of MEM and 10% horse serum. Experiments were performed on cultures from 14-18 days in vitro. All experiments were approved by the Institute's Animal Care Committee.
Neurotoxic Insults
OGD. OGD was performed as previously described (64) by placing cultures in a 37°C incubator housed in an anaerobic glovebox (Forma Scientific, Marjetta, OH). Cultures were washed twice in a glucose-free balanced salt solution (BSS) at 25°C with the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 20 HEPES, and 0.03 glycine and maintained at pH 7.4. Cultures were then subjected to an anaerobic environment of 95% N2-5% CO2 for 75-90 min, producing an O2 partial pressure equal to 10-15 Torr, as measured with an oxygen microelectrode (Microelectrodes, Londonberry, NH). OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37°C in 95% O2-5% CO2. Control cultures were exposed to BSS that contained 3 mM D-glucose and maintained in the standard incubator. Durations of OGD employed were 60-70 min for OGD preconditioning or 75-90 min for lethal OGD. As described previously (64), constant durations for each condition were not employed due to variability in the susceptibility of cultures to OGD between, but not within, platings.
NMDA exposure. After being washed in glucose-containing BSS, cultures were exposed to 15-25 µM NMDA or 30-40 µM NMDA to precondition or to kill neurons, respectively, in glucose-containing BSS for 15-25 min at room temperature. After washout, the stored medium was returned to cultures.
Drug application. Drugs were either coapplied with OGD or NMDA treatment or applied 0.5-2 h before, during, and 24 h post-OGD or -NMDA treatment. For each 12-well plate subjected to OGD, 3 to 6 wells were devoted to drug-free OGD to account for variability arising between plates or platings of cells. All experiments were performed on 2-6 different platings of cells, with 3-6 wells devoted to each condition.
Assessment of Neuronal Injury
Neuronal injury was assessed 24 h after treatments by exposing cells to PI measurement of fluorescence intensities with a platereader (64). Briefly, media in 12-well plates were replaced with 0.5 ml/well of BSS containing glucose and 4.5 µM PI and incubated at 22°C for 30 min. The fluorescence intensity (Ex = 530 ± 20 nm; Em = 645 ± 20 nm) from four locations within each well was then measured on a Cytofluor 2350 platereader (Millipore, Bedford, MA). The %PI uptake was determined by subtracting the fluorescence measured in untreated sister cultures containing PI and then normalizing values to the fluorescence representing 100% neuronal death, which was obtained by exposing sister cultures to 100 µM NMDA for 15 min. Thus the %PI uptake is equivalent to the percentage of dead cells above control levels (death in untreated control wells was <5%).
Measurement of Neuronal Mitochondrial Ca2+ Uptake
Mitochondrial Ca2+ loading was evaluated by using a modified published procedure (8). Briefly, the uptake of mitochondrial Ca2+ after transient NMDA exposure was defined as the Ca2+ detected by cytosolic fluorescence indicators after mitochondrial depolarization induced by the protonophore FCCP. After coloading of the cultures with the Ca2+-sensitive fluorescence dyes, 5 µM fluo 3-AM and 10 µM fura red-AM, coverslips were placed in a microperfusion system housed on the stage of an LSM-410 Zeiss (Carl Zeiss, Thornwood, NY) inverted laser scanning microscope equipped with an argon-krypton ion laser and with a Fluar x40 1.3 oil-immersion objective. After a region of interest was chosen, fluorophores were excited with a 488-nm laser line, selected by a band-pass filter 485/20 and attenuated by a neutral density filter (to <1%). Emitted fluorescence from both fluorophores was separated at a cut-off point of 560 nm by dichroic beam splitters (FT560). Fluo-3 and fura red fluorescence were simultaneously detected after filtration through a 515- to 540-nm band-pass and a long-pass LP590 filter, respectively. Images of 256 x 256 pixels were acquired at a rate of one image/10 s, and images were averaged over 8 scans. Images were acquired with 50% of the light emitted from an optical slice 5 µm thick, with a confocal aperture adjusted to a full-width half maximum of 5 µm for both emitted wavelengths. Ratios of Fluo-3 to fura red intensities were evaluated for each image.
Measurement of Cytosolic Intracellular Ca2+
Cytosolic Ca2+ concentrations [Ca2+]i were
determined by using temperature-controlled platereader measurements of
fluorescence intensity from 12-well plates containing cultures loaded with a
Ca2+-sensitive low-affinity fluorescent dye, Fluo-4FF-AM
(Kd 9.7 µM; Ex = 485 ± 20 nm; Em = 530
± 20 nm). Cultures were washed in glucose-containing BSS and then
loaded with 4.5 µM Fluo-4FF in BSS for 45 min at 37°C, washed twice,
and allowed to equilibrate for
60 min at 22°C. Before treatment,
cultures were washed once in BSS and fluorescence intensity was measured from
four locations within each well in the platereader at 25°C. Fluorescence
was again measured during a 15- to 20-min exposure to NMDA ± inhibitor
in BSS (the increase in fluorescence intensity for NMDA was typically 4 times
that of the initial signal). The %increase in [Ca2+]i
was calculated by subtracting the initial reading from the final reading,
divided by the initial reading. Values were normalized relative to 0% for
control wash and 100% for NMDA alone.
Measurement of Extracellular Glutamate Concentration
Immediately after exposure of cultures to OGD, control wash, or glutamate, buffer solutions were collected and stored at -80°C. Glutamate concentrations were determined by using a commercially available kit (Molecular Probes), in which glutamate is enzymatically converted to H2O2, which in turn reacts with Amplex Red reagent to produce the fluorescent product resorufin, and were detected using the fluorescent platereader.
Statistics
Three to four wells were investigated per condition from a minimum of three different platings. Data are represented as means ± SE. Statistical comparisons were made by analysis of variance (ANOVA). When significant differences were observed, Tukey's test was employed for multiple comparisons. Statistical significance was inferred at P < 0.05.
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RESULTS |
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Role of Reactive Oxygen Species in OGD and NMDA Preconditioning
MnTBAP. Preconditioning by exposing cultures to OGD for 60-70 min rendered neurons significantly resistant to 75-90 min of otherwise lethal OGD 24 h later, as evaluated using %PI uptake 24 h later. Coapplication of 200 µM MnTBAP during OGD preconditioning significantly suppressed tolerance to otherwise lethal OGD (Fig. 1A). Preconditioning by exposing cultures to 15-25 µM NMDA for 15-25 min rendered neurons significantly resistant to 75-90 min of otherwise lethal OGD 24 h later (Fig. 1B). Coapplication of 200 µM MnTBAP during NMDA preconditioning significantly suppressed OGD tolerance (Fig. 1B). Preconditioning by exposing cultures to 15-25 µM NMDA applied for 15-25 min rendered neurons significantly resistant to otherwise lethal 30-40 µM NMDA applied for 15-25 min 24 h later (Fig. 1C). Application of 200 µM MnTBAP during NMDA preconditioning suppressed tolerance to otherwise lethal NMDA (Fig. 1C). None of the drugs investigated in this study was neurotoxic on their own (data not shown).
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Other antioxidants. Having identified reactive oxygen species as important to preconditioning, the effect of other antioxidants on NMDA preconditioning was investigated. Application of 100 µM Zn(II)TBAP, but not a less active analog, 160 µM Mn(III)TPPS, during NMDA preconditioning resulted in significant suppression of OGD tolerance (Fig. 2). Scavenging abilities by MnTBAP and Zn(II)TBAP include superoxide, H2O, peroxides, peroxynitrite, lipid peroxidation products, and carbonic radicals (13, 42, 62). Application of a much more potent superoxide dismutase and catalase mimetic, 50 µM Mn(III)TE-PyP(2), for 1 h before, during, and 24 h post-NMDA preconditioning did not significantly suppress OGD tolerance (Fig. 2).
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Role of Mitochondria Ca2+ in Preconditioning
Mitochondrial transport of superoxide. Tolerance to otherwise lethal OGD was significantly suppressed by inclusion of 0.5 mM DIDS during OGD or NMDA preconditioning (Fig. 3). Application of 0.5 mM DIDS during NMDA preconditioning also suppressed tolerance to otherwise lethal NMDA; however, in the nonpreconditioning control experiment, transient exposure to DIDS alone rendered cultures significantly more susceptible to NMDA applied 24 h later (Fig. 3).
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Mitochondrial Ca2+ loading. We considered whether a source of the MnTBAP/Zn(II)TBAP- and DIDS-sensitive component to OGD and NMDA preconditioning was nonlethal reactive oxygen species generated downstream of mitochondrial Ca2+ loading. Reactive oxygen species may be generated within cortical neuronal mitochondria as a result of mitochondrial Ca2+ loading consequent to an NMDA-initiated increase in [Ca2+]i (14, 50). MnTBAP can access neuronal cortical mitochondria (33).
We determined whether NMDA preconditioning caused mitochondrial Ca2+ loading by using a microperfusion protocol. Uptake of mitochondrial Ca2+ after transient NMDA exposure was measured as the Ca2+ detected by cytosolic fluorescence indicators after mitochondrial depolarization induced by the protonophore FCCP (8). Single-neuron fluorescence was monitored on cultures loaded with the Ca2+-sensitive dyes, Fluo-3 and fura red, and microperfused with glucose-containing buffer for 5 min, 25 µM NMDA for 5 min, buffer for 5 min, Ca2+-free buffer for 30 s, and finally 2 µM FCCP in Ca2+-free buffer to release Ca2+ present within mitochondria into the cytosol. NMDA typically induced a reversible rise in Fluo-3/fura red fluorescence ratio, followed by a substantial increase in the neuronal fluorescence ratio with FCCP treatment (Fig. 4). In this protocol, FCCP did not increase the fluorescence ratio in cultures that were not preexposed to NMDA (data not shown).
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Mitochondrial uncouplers. Mitochondrial generation of reactive oxygen species can be indirectly prevented by using the mitochondrial protonophore FCCP or the mitochondrial uncoupler DNP to depolarize the mitochondrial membrane and prevent mitochondrial Ca2+ loading (7, 50, 69). Coapplication of 0.75 µM FCCP, but not 200 µM DNP, during 25 µM NMDA preconditioning significantly suppressed tolerance to OGD 24 h later (Fig. 5). The loss of protection with FCCP was incomplete, though, because cultures were still significantly protected from otherwise lethal OGD.
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The effect of each of these inhibitors on cytosolic [Ca2+]i was also examined. Application of 0.75 µM FCCP or 200 µM DNP during 15 min of NMDA preconditioning resulted in increases in [Ca2+]i that were 115 ± 0 and 109 ± 3% that of a 25 µM NMDA induced increase, respectively. This confirmed that an NMDA preconditioning-induced increase in cytosolic [Ca2+]i was not significantly suppressed by FCCP or DNP (60). A 15-min application of 0.75 µM FCCP or 200 µM DNP alone increased [Ca2+]i by 68 ± 15 and 3 ± 3%, respectively, measured relative to a 25 µM NMDA preconditioning-induced increase. In control experiments, subjecting cultures to 15 min buffer alone caused no significant increase in [Ca2+]i.
Nitric oxide-linked pathways in OGD preconditioning. Inclusion of nitric oxide synthase (NOS) inhibitors, 100 µM 7-NI or 100 µM L-NNA, for 30 min before, during, and 24 h post-OGD preconditioning did not eliminate OGD tolerance (Fig. 6A). We addressed in further detail the basis for the negligible role for nitric oxide in our cultures, given a report of Gonzalez-Zulueta et al. (18) of the central role for this neurotransmitter in a similar model of OGD preconditioning. An intracellular dye, DAF-2, allows detection of NMDA receptor-mediated production of nitric oxide (31) and has been used to confirm nitric oxide production during hypoxic preconditioning in cardiomyocytes (32). As a positive control, cortical cultures preloaded with 10 µM DAF-2 were exposed to a nitric oxide donor, DEA/NONOate, which rapidly produces nitric oxide (halflife of 16 min at 25°C) (16). DEA/NONOate caused an immediate and sustained increase in neuronal fluorescence (Fig. 7, A and B). Plotting neuronal fluorescence intensity vs. [DEA/NONate] resulted in a slope of 1 (correlation coefficient = 0.99) (Fig. 7C). In contrast, a 10-min application of 100 µM NMDA did not significantly increase neuronal fluorescence (data not shown) and correlated with negligible generation of nitric oxide (Fig. 7C). NADPH-diaphorase staining, a marker of neuronal NOS-containing neurons (11), was also negligible (data not shown). Hence, the current cultures likely do not generate enough nitric oxide to participate in a lethal or preconditioning stress due to an insufficient number of neurons containing the neuronal isoform of NOS.
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Mitogen-activated protein (MAP) kinases are reported to be activated downstream of OGD preconditioning-induced nitric oxide production (18). However, inclusion of a MAP kinase inhibitor employed in that study, 50 µM PD-98059, or another inhibitor, 5 µM U0126, for 30 min before, during, and 24 h post-OGD preconditioning did not eliminate OGD tolerance (Fig. 6A).
Nitric oxide produced by NOS can activate poly-(ADP-ribose) polymerase (PARP) to neurotoxic levels (61), but moderate stimulation of PARP may suppress cerebral ischemia (39) or cardiac ischemic preconditioning (34). However, inclusion of the PARP inhibitor, 10 µM IQL, for 30 min before, during, and 24 h post-OGD preconditioning did not eliminate OGD tolerance (Fig. 6A).
Reactive oxygen species induce release of adenosine in the hippocampus (2), which may in turn stimulate nitric oxide production (4), possibly during ischemic or hypoxic preconditioning (9, 44). However, inclusion of the adenosine A1 receptor antagonists, 10 µM DPCPX, 10 µM 8-CPT, or 150 µM 8-SPT, for 30 min before, during, and 24 h post-OGD preconditioning did not significantly suppress tolerance to otherwise lethal OGD (Fig. 6B).
A1 receptor agonists can be beneficial in various models of cerebral ischemia, possibly through suppression of glutamate release through hyperpolarization of presynaptic neuronal terminals (53). Because glutamate is a key mediator in OGD preconditioning (20), the alternative possibility of activating the adenosine A1 receptor during OGD preconditioning was considered. However, application of 1 mM adenosine or the A1 agonist, 100 µM R-PIA, for 30 min before, during, and 24 h post-OGD preconditioning did not eliminate OGD tolerance (Fig. 6B).
Role of CaMKII in OGD and NMDA preconditioning. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a kinase the function of which is integrally coupled with NMDA receptor activation (15). After 75-90 min of otherwise lethal OGD, the %PI uptake was significantly higher between cultures preconditioned 24 h earlier in the absence and presence of a CaMKII inhibitor, 5 µM KN-62, applied for 180 min before and during 60-70 min of OGD preconditioning (Fig. 8A). This loss of protection was not complete, though, because cultures preconditioned in the presence of KN-62 were still significantly protected from otherwise lethal OGD. Similar results were obtained when examining NMDA preconditioning-induced tolerance to otherwise lethal NMDA (Fig. 8B). In contrast, inclusion of KN-62 during NMDA preconditioning did not significantly increase the %PI uptake in cultures subsequently exposed to otherwise lethal NMDA 24 h later (Fig. 8C). However, in the control experiment, treatment of cultures with KN-62 24 h before exposure to otherwise lethal NMDA also significantly decreased the %PI uptake, so a role for CaMKII cannot be ruled out in this pathway. KN-62 has been reported to inhibit voltage-dependent Ca2+ channels (58); however, acute application of KN-62 did not significantly inhibit an NMDA-induced rise in [Ca2+]i (data not shown).
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Comparison of cellular signaling activated by OGD/NMDA preconditioning or lethal OGD/NMDA. To compare cellular signaling activated by lethal vs. preconditioning stress, for each agent or class of agents the %suppression of OGD toxicity was plotted against the %suppression of OGD preconditioning-induced tolerance to OGD (preconditioning data were normalized relative to 0% for the drug-free condition) (Fig. 9A). A slope of unity (dashed lines) represents a perfect correlation between the degree of suppression of lethal and preconditioning stress for that particular agent or class of agents. The NOS inhibitors, MAP kinase inhibitors, PARP inhibitor, and adenosine A1 antagonists and agonists did not protect neurons against 75-90 min of lethal OGD, whereas MnTBAP, DIDS, and KN-62 were significantly protective (data not shown). These latter three compounds suppress OGD preconditioning (Figs. 1, 3, and 8, respectively) and, therefore, plot near the (100, 100) coordinate. In contrast, compounds that did not suppress OGD preconditioning or toxicity [inhibitors of MAP kinases, NOS, PARP, and adenosine A1 receptors (Fig. 6)] plot near the drug-free (0,0) coordinate. Hence, the degree of suppression of OGD preconditioning correlates with the degree of suppression of OGD toxicity for all compounds investigated.
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Similarly, the %suppression of NMDA-toxicity was plotted against the %suppression of NMDA preconditioning-induced tolerance to OGD (Fig. 9B). Inhibitors that suppress NMDA preconditioning fall into two categories. First, as for OGD, agents that suppress NMDA preconditioning [MnTBAP, Zn(II)TBAP, and KN-62 in Figs. 1, 2, and 8, respectively] also significantly protect against lethal NMDA (data not shown), whereas those compounds that do not suppress NMDA preconditioning [Mn(III)TPPS and Mn(III)TE-PyP(2) in Fig. 2] do not protect either (data not shown). Second, the mitochondrial uncouplers FCCP and DNP suppress lethal NMDA toxicity (data not shown) more effectively than NMDA preconditioning (Fig. 5), so the values for FCCP and DNP plot well below the unity slope (Fig. 9B).
Effect of preconditioning on extracellular glutamate levels during otherwise lethal OGD. Lethal OGD results in excessive levels of extracellular glutamate ([glutamate]ex). A reduction in [glutamate]ex observed during otherwise lethal ischemia may represent a common end effector of divergent models and signaling pathways initiated by preconditioning (20, 28, 56). Cultures were subjected to NMDA preconditioning or nonpreconditioning wash, and 24 h later buffer was collected at the termination of 75-90 min of lethal OGD. Preconditioning resulted in a significant reduction in [glutamate]ex (Fig. 10). PI analysis performed 24 h later confirmed significant protection (data not shown).
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DISCUSSION |
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MnTBAP- and Zn(II)TBAP-Dependent Preconditioning
The suppression of tolerance by including MnTBAP and the structurally related Zn(II)TBAP during OGD or NMDA preconditioning of cortical neurons implicates reactive oxygen species participation (Figs. 1 and 2). The fact that all three tolerance pathways investigated are suppressed by MnTBAP (Fig. 1, A-C) is consistent with our working hypothesis that NMDA preconditioning-induced tolerance to NMDA is a crucial subpathway of OGD preconditioning-induced tolerance to OGD and that reactive oxygen species are involved in this process (64). MnTBAP has been extensively investigated in neurons. Neurotoxic NMDA decreases aconitase activity in cultured cortical neurons, indicative of overproduction of intracellular reactive oxygen species, whereas MnTBAP protects by preserving aconitase activity (33). MnTBAP protects against the classic apoptotic agent staurosporine (41). MnTBAP does not block NMDA receptor-mediated whole cell currents, acute or delayed rises in [Ca2+]i, or mitochondrial depolarization (43, 69). Collectively, this evidence suggests specificity of MnTBAP against reactive oxygen species generated downstream of an NMDA receptor-mediated increase in [Ca2+]i (42). It is difficult to identify which reactive oxygen species are involved: possibilities include superoxide, H2O2, lipid peroxyl radicals, peroxynitrite, and carbonate anion radicals (12, 13, 47, 62, 75). Several possibilities could account for the different effects of MnTBAP and Mn(III)TE-PyP(2) in preconditioning. MnTBAP achieves higher cytosolic levels than Mn(III)TE-PyP(2) in cortical neurons (33). Intracellular localization or other properties may be affected by differences in overall net charge between MnTBAP (-3 charge) and Mn(III)TE-PyP(2) (+5 charge). Structure/activity relationships determined in cell-free assays may not directly translate to cells. The scavenging properties of each antioxidant can be differentially altered by endogenous reductants, reaction rates, intermediate species generated during reactions, intracellular compartmentalization/binding, or degradation of the antioxidant. N-alkylpyridiniumyl-based porphyrins may be inactivated through binding to intracellular proteins or degraded by reaction with H2O2 (51). Whatever the basis for these differences, the ineffectiveness of Mn(III)TE-PyP(2) is internally consistent, failing to suppress a lethal NMDA stress as well (Fig. 8B and Ref. 33).
Mitochondria-Independent Preconditioning
Despite mitochondrial Ca2+ loading during NMDA preconditioning (Fig. 4), the uncoupler-based data do not support a major role for generation of reactive oxygen species within the mitochondria. The inability of DNP to mimic FCCP by at least partially suppressing NMDA preconditioning-induced tolerance against OGD (Fig. 5) cannot be attributed to ineffective uncoupling of the mitochondria because both uncouplers completely protect neurons against lethal NMDA (60) (Fig. 8B). Hence, FCCP may partially suppress preconditioning through an alternative mechanism, perhaps related to the ability of FCCP to increase [Ca2+]i on its own, possibly through plasma membrane depolarization (72). Consequently, MnTBAP likely suppresses preconditioning by mitochondrial-independent scavenging of oxygen radicals. In studies reporting inhibition of preconditioning by MnTBAP, a potential link with mitochondrial production of oxidants has not been investigated (30, 37, 74).
The minimal involvement of mitochondrial reactive oxygen species in cortical neurons (Fig. 5) suggests that DIDS suppresses preconditioning (Fig. 3) by a mitochondria-independent mechanism. In contrast, suppression of hypoxic preconditioning by DIDS in myocytes has been attributed elsewhere to blocking the transport of superoxide through a mitochondrial anion channel, recently identified as voltage-dependent anion channels (22), to the cytosol (68). Complex III in the mitochondrial respiratory chain is believed to be the source of superoxide (68); however, a key distinction with the present study is the 10-min interval between preconditioning and the lethal insult. Alternative possibilities include a weak NMDA receptor block (65) or targeting downstream signaling common to excitotoxic and apoptotic stimuli (59).
Nitric Oxide-Independent Preconditioning
No evidence supporting production of nitric oxide or associated cellular signaling was found (Figs. 6 and 7), in contrast to a report that nitric oxide production is not only required during OGD preconditioning but can induce OGD tolerance on its own in cortical neurons (18). A wide range of concentrations of nitric oxide appear effective because identical concentrations of the two compounds used in that study to precondition would produce very different amounts of nitric oxide, i.e., NOR-3 and diethylenetriamine (DETA) NONOate have half-lives of 40 and 3,400 min, respectively (16). In vivo models of cerebral ischemic preconditioning also do not support NMDA receptor-mediated production of nitric oxide (17, 46). Possible reasons cited for negligible neuronal NOS expression in cortical culture (54), such as plating neurons on an established glial bed, cannot account for this discrepancy in the present cultures.
Agents that prevent preconditioning or toxicity in the current study can act independently of nitric oxide. For instance, hypoxic preconditioning-induced generation of reactive oxygen species, as well as subsequent hypoxic tolerance, is suppressed by DIDS but not by NOS inhibitors in cardiac myocytes (68). In neuron cultures for which NOS inhibitors are ineffective, FCCP, DNP, and MnTBAP suppress NMDA-mediated generation of reactive oxygen species and toxicity (14, 60, 67). Taken together, these findings show that OGD preconditioning can induce dramatically different cellular signaling to achieve OGD-resistant phenotypes in cortical neurons. Specifically, NMDA receptor-mediated Ca2+ influx and subsequent generation of MnTBAP/Zn(II)TBAP-sensitive reactive oxygen species represents a tolerance-inducing pathway that parallels a nitric oxide-based one in another laboratory (18).
The lack of effect of 50 µM PD-98059, which inhibits both MAPK/extracellular signal-regulated kinase 1 (MEK1) and MEK2 (1), as well as a more potent MEK1 and MEK2 inhibitor U0126, extends earlier work of ours, demonstrating a lack of effect of 20 µM PD-98059 (which inhibits MEK1) on OGD preconditioning-induced tolerance to otherwise lethal OGD (63). In contrast, 50 µM PD-98059 inhibited OGD preconditioning (18) in the same laboratory that demonstrated a nitric oxide-dependent component (11). These results add a further level of complexity to an already diverse role for MAP kinases in in vivo models of cerebral ischemic preconditioning, which generally report a necessary role for this kinase (26). PARP was investigated because this enzyme appears to be activated downstream of nitric oxide in cortical cultures during OGD or NMDA receptor activation (18), and pharmacological blockade of PARP is neuroprotective (61). As well, PARP may be activated during nonlethal transient cerebral global ischemia (39) and myocardial ischemic preconditioning (34). The inability of MAPK and PARP inhibitors to inhibit OGD preconditioning in the current study (Fig. 6A) might reflect a negligible role for nitric oxide.
The ineffectiveness of adenosine A1 receptor antagonists (Fig. 6B) contrasts with findings reported for in vivo models of delayed ischemic preconditioning in brain (23, 25) and in heart (45). A1 receptor activation can result in production of nitric oxide (44) or protein kinase C (5), events linked with ischemic preconditioning models (5, 44) other than ours (63). Similarly, the failure of agonists to abolish OGD preconditioning further highlights differences with intact brain because we have confirmed that adenosine A1 receptor agonists or antagonists may not protect against OGD in cortical neuron culture (35), despite benefits in various models of cerebral ischemia (53).
CaMKII-Dependent Preconditioning
A neurotoxic role for CaMKII has previously been identified in cerebral
ischemia. KN-62 protects cortical neurons against lethal OGD or NMDA
(21) and degradation of CaMKII
is enhanced in preconditioned CA1 hippocampal neurons after otherwise cerebral
ischemia, suggesting that downregulation of CaMKII contributes to the
neuroprotection (57).
Nevertheless, CamKII may also be neuroprotective because CaMKII-
knockout mice are more susceptible to cerebral ischemia
(71). A causal effect for
CaMKII in neuronal preconditioning has not been previously demonstrated. The
subcellular distribution of CaMKII-
in hippocampal CA1 neurons in rat
(57) or gerbil
(29) is not altered 1-2 days
after ischemic preconditioning (i.e., when neurons would be protected). Within
this interval, though, the ability of KN-62 to suppress OGD tolerance
(Fig. 8) suggests that
activation of CaMKII during OGD/NMDA preconditioning is necessary. An NMDA
receptor-mediated increase in postsynaptic Ca2+ results in
activation of the CaMKII-
-subunit, autophosphorylation, and
translocation from the cytosol to the membrane of postsynaptic densities.
Autophosphorylation renders CaMKII-
Ca2+ independent, thus
prolonging biological effects such as synaptic plasticity and trophism beyond
a transient Ca2+ elevation
(15). KN-62 has no effect on
the translocation of CaMKII in rat hippocampus shortly after cerebral ischemia
but does inhibit the autophosphorylation of CaMKII-
, as well as
phosphorylation of the NR2B subunit of the NMDA receptor and the binding of
CaMKII
to NR2B (36). A
reported inhibition of NMDA receptor-mediated nitric oxide production in
neurons by KN-62 (52) likely
does not apply in the nitric oxide-independent setting in the present study.
Thus the suppression of preconditioning by KN-62
(Fig. 8) may involve disrupted
CaMKII function and protein targeting of CaMKII as opposed to restricting
transit to subcellular locales. Taken together, these findings imply a dual
role for CaMKII in cerebral ischemic preconditioning: first, CaMKII activation
by preconditioning is required to induce tolerance
(Fig. 8), although suppression
of CaMKII activity may be required after the otherwise lethal event.
The mechanism by which CaMKII contributes to OGD tolerance may be pre- and postsynaptic in origin. CaMKII is prominent is glutamatergic synapses and phosphorylates multiple synaptic proteins and is involved in neurotransmitter synthesis and release (15). The lower [glutamate]ex observed during OGD in preconditioned cultures (Fig. 10) may result from CaMKII being involved in minimizing neuronal presynaptic glutamate release. CaMKII phosphorylates a number a proteins in the postsynaptic density, with NR2B functioning as a major acceptor site (15). Possibly, this could contribute to the tolerance observed against otherwise lethal NMDA. It is not known whether production of reactive oxygen species or CaMKII activation by OGD/NMDA preconditioning is coincident or represents separate paths toward inducing tolerance. It is noted, though, that kinases have been identified as targets for oxidants produced during cardiac preconditioning (10).
Preconditioning Results in Reduced Extracellular Glutamate Levels During Otherwise Lethal OGD
We chose to measure [glutamate]ex to determine whether the response or end effector of preconditioned cortical neuronal cultures to OGD can also differ between laboratories. The marked reduction in [glutamate]ex (Fig. 10) accounts for our previous finding that a lethal OGD-induced rise in [Ca2+]i is suppressed in NMDA-preconditioned cultures (64). In view of our finding of postsynaptic NMDA tolerance (64), the reduction in [glutamate]ex may consequently represent suppressed presynaptic glutamate release from neurons. A different laboratory reports that reduced glutamate release may be an important mediator in similar models of preconditioning in cortical neurons (20). Reduced [glutamate]ex during otherwise lethal hypoxia is also noted in acute models of hypoxic preconditioning (28, 56). Taken together, these findings indicate that reduced [glutamate]ex may represent a common end effector between different models of preconditioning.
Tolerance Induction Through Abbreviated Neurotoxic Cellular Signaling
Only cellular signaling that has the potential to be neurotoxic may be involved in preconditioning. Conversely, signaling that does not have the potential to be neurotoxic is not activated by preconditioning (Fig. 9). Investigation into the relationship between preconditioning and lethal stress was prompted by the realization that the laboratory reporting nitric oxide-dependent OGD preconditioning (18) previously reported profound nitric oxide-dependent OGD or NMDA toxicity (11). Hence, nitric oxide and other signalers may be involved in preconditioning only if the potential exists to be involved in a lethal stress. Reduction of a preconditioning stress may account for a recent finding that ischemic preconditioning is eliminated in neuronal NOS-knockout mice (3). Numerous laboratories have also reported NMDA induces nitric oxide-independent, but nonetheless reactive oxygen species-dependent, toxicity in cortical neuron cultures (for instance, see Ref. 24). OGD or NMDA preconditioning may also induce nitric oxide-independent signaling in those systems.
If tolerance- and toxicity-inducing stimuli are also strongly linked in other ischemia models, then the variability in preconditioning-induced signaling observed between models and tissues may largely reflect variability in toxic signaling. For instance, blockade of the NMDA receptor, which can be neuroprotective in ischemia models, almost universally inhibits ischemic/hypoxic/OGD preconditioning (for example see Ref. 19). Examples of this dual role in tolerance induction are becoming increasingly appreciated in ischemic preconditioning (38), as well as in "chemical" preconditioning, i.e., preconditioning could be triggered by molecules that in larger doses would be toxic.
The tolerance- and toxicity-inducing pathways diverge downstream of cytosolic Ca2+ at the mitochondrial Ca2+ level. Indeed, complete depolarization of the mitochondrial membrane by the uncouplers during NMDA preconditioning largely leaves the tolerance-induction machinery intact (Fig. 5). Hence, the beneficial effects provided by preconditioning may not preclude potential concomitant therapy based on neuronal mitochondrial uncoupling.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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---|
2. Almeida CG, de Mendonca A, Cunha RA, and Ribeiro JA. Adenosine promotes neuronal recovery from reactive oxygen species induced lesion in rat hippocampal slices. Neurosci Lett 339: 127-130, 2003.[ISI][Medline]
3. Atochin DN,
Clark J, Demchenko IT, Moskowitz MA, and Huang PL. Rapid cerebral ischemic
preconditioning in mice deficient in endothelial and neuronal nitric oxide
synthases. Stroke 34:
1299-1303, 2003.
4. Barth A, Newell DW, Nguyen LB, Winn HR, Wender R, Meno JR, and Janigro D. Neurotoxicity in organotypic hippocampal slices mediated by adenosine analogues and nitric oxide. Brain Res 762: 79-88, 1997.[ISI][Medline]
5. Baxter GF, Marber MS, Patel VC, and Yellon DM. Adenosine receptor involvement in a delayed phase of myocardial protection 24 hours after ischemic preconditioning. Circulation 90: 2993-3000, 1994.[Abstract]
6. Bruer U, Weih MK, Isaev NK, Meisel A, Ruscher K, Bergk A, Trendelenburg G, Wiegand F, Victorov IV, and Dirnagl U. Induction of tolerance in rat cortical neurons: hypoxic preconditioning. FEBS Lett 414: 117-121, 1997.[ISI][Medline]
7. Budd SL, Castilho RF, and Nicholls DG. Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. FEBS Lett 415: 21-24, 1997.[ISI][Medline]
8. Budd SL and Nicholls DG. A reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis. J Neurochem 66: 403-411, 1996.[ISI][Medline]
9. Centeno JM, Orti M, Salom JB, Sick TJ, and Perez-Pinzon MA. Nitric oxide is involved in anoxic preconditioning neuroprotection in rat hippocampal slices. Brain Res 836: 62-69, 1999.[ISI][Medline]
10. Das DK, Maulik N, Sato M, and Ray PS. Reactive oxygen species function as second messenger during ischemic preconditioning of heart. Mol Cell Biochem 196: 59-67, 1999.[ISI][Medline]
11. Dawson VL, Kizushi VM, Huang PL, Snyder SH, and Dawson TM. Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice. J Neurosci 16: 2479-2487, 1996.[Abstract]
12. Day BJ, Fridovich I, and Crapo JD. Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury. Arch Biochem Biophys 347: 256-262, 1997.[ISI][Medline]
13. Day BJ, Shawen S, Liochev SI, and Crapo JD. A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro. J Pharmacol Exp Ther 275: 1227-1232, 1995.[Abstract]
14. Dugan LL, Sensi SL, Canzoniero LM, Handran SD, Rothman SM, Lin TS, Goldberg MP, and Choi DW. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J Neurosci 15: 6377-6388, 1995.[ISI][Medline]
15. Fink CC and Meyer T. Molecular mechanisms of CaMKII activation in neuronal plasticity. Curr Opin Neurobiol 12: 293-299, 2002.[ISI][Medline]
16. Garthwaite G, Goodwin DA, Batchelor AM, Leeming K, and Garthwaite J. Nitric oxide toxicity in CNS white matter: an in vitro study using rat optic nerve. Neuroscience 109: 145-155, 2002.[ISI][Medline]
17. Gidday JM, Shah AR, Maceren RG, Wang Q, Pelligrino DA, Holtzman DM, and Park TS. Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning. J Cereb Blood Flow Metab 19: 331-340, 1999.[ISI][Medline]
18. Gonzalez-Zulueta M, Feldman AB, Klesse LJ, Kalb RG, Dillman JF, Parada
LF, Dawson TM, and Dawson VL. Requirement for nitric oxide activation of
p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning.
Proc Natl Acad Sci USA 97:
436-441, 2000.
19. Grabb MC and
Choi DW. Ischemic tolerance in murine cortical cell culture: critical role
for NMDA receptors. J Neurosci
19: 1657-1662,
1999.
20. Grabb MC, Lobner D, Turetsky DM, and Choi DW. Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release. Neuroscience 115: 173-183, 2002.[ISI][Medline]
21. Hajimohammadreza I, Probert AW, Coughenour LL, Borosky SA, Marcoux FW, Boxer PA, and Wang KK. A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death. J Neurosci 15: 4093-4101, 1995.[Abstract]
22. Han D, Antunes
F, Canali R, Rettori D, and Cadenas E. Voltage-dependent anion channels
control the release of the superoxide anion from mitochondria to cytosol.
J Biol Chem 278:
5557-5563, 2003.
23. Heurteaux C, Lauritzen I, Widmann C, and Lazdunski M. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. Proc Natl Acad Sci USA 92: 4666-4670, 1995.[Abstract]
24. Hewett SJ, Corbett JA, McDaniel ML, and Choi DW. Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-D-aspartate neurotoxicity. Brain Res 625: 337-341, 1993.[ISI][Medline]
25. Hiraide T, Katsura K, Muramatsu H, Asano G, and Katayama Y. Adenosine receptor antagonists cancelled the ischemic tolerance phenomenon in gerbil. Brain Res 910: 94-98, 2001.[ISI][Medline]
26. Irving EA and Bamford M. Role of mitogen- and stress-activated kinases in ischemic injury. J Cereb Blood Flow Metab 22: 631-647, 2002.[ISI][Medline]
27. Ishida T, Yarimizu K, Gute DC, and Korthuis RJ. Mechanisms of ischemic preconditioning. Shock 8: 86-94, 1997.[ISI][Medline]
28. Johns L, Sinclair AJ, and Davies JA. Hypoxia/hypoglycemia-induced amino acid release is decreased in vitro by preconditioning. Biochem Biophys Res Commun 276: 134-136, 2000.[ISI][Medline]
29. Katsura KI, Kurihara J, Kato H, and Katayama Y. Ischemic preconditioning affects the subcellular distribution of protein kinase C and calcium/calmodulin-dependent protein kinase II in the gerbil hippocampal CA1 neurons. Neurol Res 23: 751-754, 2001.[ISI][Medline]
30. Kevin LG,
Camara AK, Riess ML, Novalija E, and Stowe DF. Ischemic preconditioning
alters real-time measure of O2 radicals in intact hearts with
ischemia and reperfusion. Am J Physiol Heart Circ
Physiol 284:
H566-H574, 2003.
31. Kojima H, Nakatsubo N, Kikuchi K, Urano Y, Higuchi T, Tanaka J, Kudo Y, and Nagano T. Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF-2 DA. Neuroreport 9: 3345-3348, 1998.[ISI][Medline]
32. Lebuffe G,
Schumacker PT, Shao ZH, Anderson T, Iwase H, and Vanden Hoek TL. ROS and
NO trigger early preconditioning: relationship to mitochondrial
KATP channel. Am J Physiol Heart Circ
Physiol 284:
H299-H308, 2003.
33. Li QY, Pedersen C, Day BJ, and Patel M. Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation. J Neurochem 78: 746-755, 2001.[ISI][Medline]
34. Liaudet L, Yang Z, Al Affar EB, and Szabo C. Myocardial ischemic preconditioning in rodents is dependent on poly (ADP-ribose) synthetase. Mol Med 7: 406-417, 2001.[ISI][Medline]
35. Lobner D. Saturation of neuroprotective effects of adenosine in cortical culture. Neuroreport 13: 2075-2078, 2002.[ISI][Medline]
36. Meng F, Guo J,
Zhang Q, Song B, and Zhang G. Autophosphorylated
calcium/calmodulin-dependent protein kinase IIalpha (CaMKII) reversibly
targets to and phosphorylates N-methyl-D-aspartate
receptor subunit 2B (NR2B) in cerebral ischemia and reperfusion in hippocampus
of rats. Brain Res 967:
161-169, 2003.[ISI][Medline]
37. Mullenheim J, Ebel D, Frassdorf J, Preckel B, Thamer V, and Schlack W. Isoflurane preconditions myocardium against infarction via release of free radicals. Anesthesiology 96: 934-940, 2002.[ISI][Medline]
38. Murphy E,
Glasgow W, Fralix T, and Steenbergen C. Role of lipoxygenase metabolites
in ischemic preconditioning. Circ Res
76: 457-467,
1995.
39. Nagayama T, Simon RP, Chen D, Henshall DC, Pei W, Stetler RA, and Chen J. Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia. J Neurochem 74: 1636-1645, 2000.[ISI][Medline]
40. Ohtsuki T, Matsumoto M, Kuwabara K, Kitagawa K, Suzuki K, Taniguchi N, and Kamada T. Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons. Brain Res 599: 246-252, 1992.[ISI][Medline]
41. Patel M. Inhibition of neuronal apoptosis by a metalloporphyrin superoxide dismutase mimic. J Neurochem 71: 1068-1074, 1998.[ISI][Medline]
42. Patel M and Day BJ. Metalloporphyrin class of therapeutic catalytic antioxidants. Trends Pharmacol Sci 20: 359-364, 1999.[ISI][Medline]
43. Patel M, Day BJ, Crapo JD, Fridovich I, and McNamara JO. Requirement for superoxide in excitotoxic cell death. Neuron 16: 345-355, 1996.[ISI][Medline]
44. Peralta C, Hotter G, Closa D, Gelpi E, Bulbena O, and Rosello-Catafau J. Protective effect of preconditioning on the injury associated to hepatic ischemia-reperfusion in the rat: role of nitric oxide and adenosine. Hepatology 25: 934-937, 1997.[ISI][Medline]
45. Post H and Heusch G. Ischemic preconditioning. Experimental facts and clinical perspective. Minerva Cardioangiol 50: 569-605, 2002.[Medline]
46. Puisieux F, Deplanque D, Pu Q, Souil E, Bastide M, and Bordet R. Differential role of nitric oxide pathway and heat shock protein in preconditioning and lipopolysaccharide-induced brain ischemic tolerance. Eur J Pharmacol 389: 71-78, 2000.[ISI][Medline]
47. Quijano C,
Hernandez-Saavedra D, Castro L, McCord JM, Freeman BA, and Radi R.
Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal
center in decomposition kinetics and nitration. J Biol
Chem 276:
11631-11638, 2001.
48. Ravati A,
Ahlemeyer B, Becker A, Klumpp S, and Krieglstein J.
Preconditioning-induced neuroprotection is mediated by reactive oxygen species
and activation of the transcription factor nuclear factor-B.
J Neurochem 78:
909-919, 2001.[ISI][Medline]
49. Ravati A, Ahlemeyer B, Becker A, and Krieglstein J. Preconditioning-induced neuroprotection is mediated by reactive oxygen species. Brain Res 866: 23-32, 2000.[ISI][Medline]
50. Reynolds IJ and Hastings TG. Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J Neurosci 15: 3318-3327, 1995.[Abstract]
51. Riley DP. Functional mimics of superoxide dismutase enzymes as therapeutic agents. Chem Rev 99: 2573-2588, 1999.[ISI][Medline]
52. Rodriguez-Alvarez J, Lafon-Cazal M, and Bockaert J. The CaM-kinase II inhibitor KN-62 blocks NMDA but not kainate stimulation of NO synthesis. Neuroreport 7: 2525-2528, 1996.[ISI][Medline]
53. Rudolphi KA, Schubert P, Parkinson FE, and Fredholm BB. Neuroprotective role of adenosine in cerebral ischaemia. Trends Pharmacol Sci 13: 439-445, 1992.[ISI][Medline]
54. Samdani AF,
Newcamp C, Resink A, Facchinetti F, Hoffman BE, Dawson VL, and Dawson TM.
Differential susceptibility to neurotoxicity mediated by neurotrophins and
neuronal nitric oxide synthase. J Neurosci
17: 4633-4641,
1997.
55. Schaller B and Graf R. Cerebral ischemic preconditioning. An experimental phenomenon or a clinical important entity of stroke prevention? J Neurol 249: 1503-1511, 2002.[ISI][Medline]
56. Semenov DG, Samoilov MO, and Lazarewicz JW. Calcium transients in the model of rapidly induced anoxic tolerance in rat cortical slices: involvement of NMDA receptors. Neurosignals 11: 329-335, 2002.[ISI][Medline]
57. Shamloo M, Kamme F, and Wieloch T. Subcellular distribution and autophosphorylation of calcium/calmodulin-dependent protein kinase II-alpha in rat hippocampus in a model of ischemic tolerance. Neuroscience 96: 665-674, 2000.[ISI][Medline]
58. Sihra TS and Pearson HA. Ca/calmodulin-dependent kinase II inhibitor KN62 attenuates glutamate release by inhibiting voltage-dependent Ca2+-channels. Neuropharmacology 34: 731-741, 1995.[ISI][Medline]
59. Small DL, Tauskela J, and Xia Z. Role for chloride but not potassium channels in apoptosis in primary rat cortical cultures. Neurosci Lett 334: 95-98, 2002.[ISI][Medline]
60. Stout AK, Raphael HM, Kanterewicz BI, Klann E, and Reynolds IJ. Glutamate-induced neuron death requires mitochondrial calcium uptake. Nat Neurosci 1: 366-373, 1998.[ISI][Medline]
61. Szabo C and Dawson VL. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol Sci 19: 287-298, 1998.[ISI][Medline]
62. Szabo C, Day BJ, and Salzman AL. Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondrial respiration in immunostimulated macrophages using a manganese mesoporphyrin superoxide dismutase mimetic and peroxynitrite scavenger. FEBS Lett 381: 82-86, 1996.[ISI][Medline]
63. Tauskela JS, Chakravarthy BR, Murray CL, Wang Y, Comas T, Hogan M, Hakim A, and Morley P. Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart. Brain Res 827: 143-151, 1999.[ISI][Medline]
64. Tauskela JS, Comas T, Hewitt K, Monette R, Paris J, Hogan M, and Morley P. Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures. Neuroscience 107: 571-584, 2001.[ISI][Medline]
65. Tauskela JS, Mealing G, Comas T, Brunette E, Monette R, Small DL, and Morley P. Protection of cortical neurons against oxygen-glucose deprivation and N-methyl-D-aspartate by DIDS and SITS. Eur J Pharmacol 464: 17-25, 2003.[ISI][Medline]
66. Tritto I and Ambrosio G. Role of oxidants in the signaling pathway of preconditioning. Antioxid Redox Signal 3: 3-10, 2001.[ISI][Medline]
67. Urushitani M, Nakamizo T, Inoue R, Sawada H, Kihara T, Honda K, Akaike A, and Shimohama S. N-methyl-D-aspartate receptor-mediated mitochondrial Ca2+ overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca2+ influx. J Neurosci Res 63: 377-387, 2001.[ISI][Medline]
68. Vanden Hoek TL,
Becker LB, Shao Z, Li C, and Schumacker PT. Reactive oxygen species
released from mitochondria during brief hypoxia induce preconditioning in
cardiomyocytes. J Biol Chem
273: 18092-18098,
1998.
69. Vergun O,
Sobolevsky AI, Yelshansky MV, Keelan J, Khodorov BI, and Duchen MR.
Exploration of the role of reactive oxygen species in glutamate neurotoxicity
in rat hippocampal neurones in culture. J Physiol
531: 147-163,
2001.
70. Votyakova TV and Reynolds IJ. m-dependent and -independent production
of reactive oxygen species by rat brain mitochondria. J
Neurochem 79:
266-277, 2001.[ISI][Medline]
71. Waxham MN, Grotta JC, Silva AJ, Strong R, and Aronowski J. Ischemia-induced neuronal damage: a role for calcium/calmodulin-dependent protein kinase II. J Cereb Blood Flow Metab 16: 1-6, 1996.[ISI][Medline]
72. White RJ and Reynolds IJ. Mitochondria and Na+/Ca2+ exchange buffer glutamate-induced calcium loads in cultured cortical neurons. J Neurosci 15: 1318-1328, 1995.[Abstract]
73. Wiegand F, Liao W, Busch C, Castell S, Knapp F, Lindauer U, Megow D, Meisel A, Redetzky A, Ruscher K, Trendelenburg G, Victorov I, Riepe M, Diener HC, and Dirnagl U. Respiratory chain inhibition induces tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab 19: 1229-1237, 1999.[ISI][Medline]
74. Yamaguchi T, Dayton CB, Ross CR, Yoshikawa T, Gute DC, and Korthuis RJ. Late preconditioning by ethanol is initiated via an oxidant-dependent signaling pathway. Free Radic Biol Med 34: 365-376, 2003.[ISI][Medline]
75. Zhang H, Joseph
J, Gurney M, Becker D, and Kalyanaraman B. Bicarbonate enhances peroxidase
activity of Cu,Znsuperoxide dismutase. Role of carbonate anion radical and
scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme
mimetics. J Biol Chem 277:
1013-1020, 2002.
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