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Excitotoxic Injury to Mitochondria Isolated from Cultured Neurons

2005; Elsevier BV; Volume: 280; Issue: 32 Linguagem: Inglês

10.1074/jbc.m503090200

1083-351X

Yulia E. Kushnareva, Sandra E. Wiley, Manus W. Ward, Alexander Y. Andreyev, Anne N. Murphy,

Metabolism and Genetic Disorders

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by ∼10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 ± 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 ± 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 ± 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca2+-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death. Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by ∼10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 ± 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 ± 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 ± 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca2+-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death. Glutamate excitotoxicity underlies neuronal loss in ischemic and traumatic brain injury (1Choi D.W. Ann. N. Y. Acad. Sci. 1994; 747: 162-171Crossref PubMed Scopus (305) Google Scholar) and also likely contributes to dysfunction in chronic forms of neurodegeneration. In particular, the evidence for involvement of excitotoxicity in Huntington disease has been significantly strengthened by recent studies (for a review see Ref. 2Bezprozvanny I. Hayden M.R. Biochem. Biophys. Res. Commun. 2004; 322: 1310-1317Crossref PubMed Scopus (208) Google Scholar). It is well established that glutamate-induced neuronal death depends on entry of extracellular Ca2+ as a result of activation of the NMDA 1The abbreviations used are: NMDA, N-methyl-d-aspartic acid; LDH, lactate dehydrogenase; HBSS, HEPES-buffered salt solution; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. subtype of glutamate receptors (3Choi D.W. Neuron. 1988; 1: 623-634Abstract Full Text PDF PubMed Scopus (4207) Google Scholar, 4Tymianski M. Charlton M.P. Carlen P.L. Tator C.H. J. Neurosci. 1993; 13: 2085-2104Crossref PubMed Google Scholar). The description of ensuing events in various types of cultured neurons is extensive (5Nicholls D.G. Curr. Mol. Med. 2004; 4: 149-177Crossref PubMed Scopus (230) Google Scholar) and includes an initial transient increase in cytoplasmic Ca2+, followed by a loss in ionic homeostasis (also termed delayed Ca2+ deregulation) (4Tymianski M. Charlton M.P. Carlen P.L. Tator C.H. J. Neurosci. 1993; 13: 2085-2104Crossref PubMed Google Scholar). The initial increase in cytoplasmic Ca2+ is associated with mitochondrial Ca2+ loading and slight mitochondrial depolarization followed by profound depolarization concurrent with the loss of ionic homeostasis. Other early events may include the release of apoptogenic cytochrome c and generation of reactive oxygen species and nitric oxide (5Nicholls D.G. Curr. Mol. Med. 2004; 4: 149-177Crossref PubMed Scopus (230) Google Scholar, 6Khodorov B.I. Membr. Cell Biol. 2000; 14: 149-162PubMed Google Scholar, 7Krieger C. Duchen M. Eur. J. Pharmacol. 2002; 447: 177-188Crossref PubMed Scopus (116) Google Scholar). As well, excitotoxic death in cultured neurons is preceded by a significant decline in cellular ATP (8Tsuji K. Nakamura Y. Ogata T. Shibata T. Kataoka K. Brain Res. 1994; 662: 289-292Crossref PubMed Scopus (28) Google Scholar, 9Budd S.L. Nicholls D.G. J. Neurochem. 1996; 67: 2282-2291Crossref PubMed Scopus (394) Google Scholar). Recent imaging studies of cultured neurons have demonstrated that mitochondrial Ca2+ loading is in large part responsible for induction of cell death following exposure to glutamate (9Budd S.L. Nicholls D.G. J. Neurochem. 1996; 67: 2282-2291Crossref PubMed Scopus (394) Google Scholar, 10Stout A.K. Raphael H.M. Kanterewicz B.I. Klann E. Reynolds I.J. Nat. Neurosci. 1998; 1: 366-373Crossref PubMed Scopus (529) Google Scholar, 11Castilho R.F. Hansson O. Ward M.W. Budd S.L. Nicholls D.G. J. Neurosci. 1998; 18: 10277-10286Crossref PubMed Google Scholar, 12Urushitani M. Nakamizo T. Inoue R. Sawada H. Kihara T. Honda K. Akaike A. Shimohama S. J. Neurosci. Res. 2001; 63: 377-387Crossref PubMed Scopus (100) Google Scholar), yet the nature of the Ca2+-induced mitochondrial injury remains controversial. An alternative approach to the elucidation of these mechanisms has been the study of mitochondria isolated from the brains of rodents exposed to excitotoxic stimuli. An inherent problem in such studies with brain mitochondria lies in the heterogeneity of these preparations that contain the organelles from both neuronal and glial cells (13Lai J.C. Clark J.B. Methods Enzymol. 1979; 55: 51-60Crossref PubMed Scopus (303) Google Scholar, 14Nicholls D.G. Proteins, Transmitters, and Synapses. Blackwell Scientific, Oxford1994: 3-14Google Scholar). Furthermore, frequently used isolation methods employing gradient centrifugation enrich the preparations in mitochondria from cell bodies, yet the excitotoxic events occur mostly within nerve terminals that are lost in the preparation as synaptosomal fractions. The use of digitonin to avoid the loss of synaptosomal mitochondria (15Rosenthal R.E. Hamud F. Fiskum G. Varghese P.J. Sharpe S. J. Cereb. Blood Flow Metab. 1987; 7: 752-758Crossref PubMed Scopus (232) Google Scholar, 16Andreyev A.Y. Fahy B. Fiskum G. FEBS Lett. 1998; 439: 373-376Crossref PubMed Scopus (129) Google Scholar) has been questioned recently (17Brustovetsky N. Jemmerson R. Dubinsky J.M. Neurosci. Lett. 2002; 332: 91-94Crossref PubMed Scopus (20) Google Scholar). Therefore, we have taken the approach of isolating mitochondria from relatively pure neuronal cultures exposed to glutamate to allow these problems to be overcome. We investigated the early bioenergetic consequences of exposure to glutamate by isolating mitochondria from primary neuronal cultures exposed to glutamate for 10 min, prior to a commitment to neuronal death. By doing so, we avoid studying potential secondary effects of the cell death process that may affect mitochondrial function. Estimation of the extent of glutamate-induced Ca2+ loading enabled us to test the effects of Ca2+ at concentrations that mimic the glutamate stimulus. Additionally, Ca2+ uptake capacities and susceptibility of mitochondria to the permeability transition were assessed. Our novel finding, which has not been revealed by other current approaches, is that mitochondria of neurons exposed to high levels of glutamate sustain significant Ca2+-induced injury to oxidative phosphorylation, and this injury occurs prior to any commitment to cell death. Preparation of Cortical Neurons—Primary cultures of cortical neurons were prepared from embryonic day 18 Sprague-Dawley rats. The cerebral cortices were collected and triturated gently (3–4 times) in ice-cold Hibernate E medium (Brain Bits, Southern Illinois University, School of Medicine) plus 1× B27 supplement (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin. After the tissue settled, the Hibernate E medium was aspirated, and the tissue was triturated for 1 min in 0.1% trypsin in a Ca2+/Mg2+-free phosphate-buffered saline solution supplemented with glucose (1.5 mm), after which trypsin was inactivated by addition of soybean trypsin inhibitor (0.1 mg/ml). The mixture was transferred into Hibernate E medium containing 20 units/ml DNase (Promega) in 0.2× reaction buffer (Promega), and the cells were centrifuged at 200 × g for 1.5 min. The supernatant was quickly aspirated, and the cells were resuspended in 10 ml of Neurobasal (E) medium (Invitrogen) plus glutamate (0.4 μg/ml), 0.5 mm l-glutamine, penicillin, and streptomycin (100 units/ml and 100 μg/ml, respectively), 1× B27 supplement, and 5 mm sodium pyruvate. Once in suspension, the cells were diluted into 30 ml of the same medium without pyruvate (initial plating medium), and the number of viable cells was determined by trypan blue exclusion. Cells were plated on poly-d-lysine-coated 10-cm plates at a concentration of 750,000 cells per ml in a volume of 10 ml per plate and kept at 37 °C in a 5% CO2 incubator. For measurements of cellular ATP and cytoplasmic Ca2+, cells were plated on Biocoat poly-d-lysine-coated black 96-well plates (BD Biosciences) at 50,000 cells per well. After 4–6 days in vitro, the initial plating medium was diluted with an equal volume of maintenance medium of the same composition lacking glutamate and l-glutamine and supplemented with 1% GlutaMAX-1 (Invitrogen). Cultures were fed every 3–4 days with fresh medium. All experiments were performed with cultures that were 13–15 days in vitro. These cultures were 91–95% neuronal, as estimated by immunocytochemical staining according to the manufacturer's protocols with anti-neuronal nuclei (Chemicon, mAB377) or anti-neurofilament 200 kDa (Calbiochem, IF06L). Measurement of Viability of Cortical Neurons Treated with Glutamate—Cell viability was measured in neurons cultured in 96-well plates using a cytotoxicity detection kit (lactate dehydrogenase, LDH) according to the manufacturer's recommendations (Roche Applied Science). Data are expressed as percent cell death based upon measurement of LDH activity that was cell-associated versus that which was released to the medium. Cells were treated in HEPES-buffered salt solution (HBSS, containing 137 mm NaCl, 5 mm KCl, 10 mm NaHCO3, 20 mm HEPES, 5.5 mm glucose, 0.6 mm KH2PO4, 1.4 mm CaCl2, 0.9 mm MgSO4) with 100 μm glutamate plus 10 μm glycine with or without pretreatment with 10 μm of the NMDA receptor antagonist MK-801. Control cultures were exposed to HBSS without glutamate or glycine. Following a 10-min incubation at 37 °C, an equal volume of maintenance medium was added (supplemented with glutamate plus glycine and/or MK-801 to maintain constant concentration of these effectors). The cells were then placed back in the incubator for 24 h prior to measurement of viability. Alternatively, at the addition of the maintenance medium, 2× (20 μm) MK-801 was included to block NMDA receptor activity. Measurement of Neuronal Ca2+—Cytoplasmic Ca2+ was measured in neurons cultured in the 96-well plates using magfluo-4, a cell permeant low affinity Ca2+ dye (Molecular Probes). A stock solution of magfluo-4 (1 mg/ml) was prepared on the day of the experiment in Me2SO and then diluted in HBSS to a final concentration of 4 μm. The culture medium was carefully aspirated, and cells were loaded with the dye (100 μl of a 4 μm solution per well) for 25 min in the incubator. The loading buffer was then replaced with dye-free HBSS, and the plates were assayed in a fluorescent plate reader (FLIPR, Molecular Devices) at 488 (excitation) and 525 nm (emission). Measurement of Neuronal ATP—The level of neuronal ATP was determined by using a CellTiter-Glo™ luminescent assay kit (Promega). Cells cultured in 96-well plates were treated with glutamate for varying lengths of time (from 10 to 40 min). Control cells were exposed to HBSS without glutamate and glycine for 10 min. At the end of the treatment, CellTiter-Glo™ reagent was added, and the plates were placed on a shaker for 5 min. In some experiments, the glutamate-containing medium was aspirated, and cells were incubated in Ca2+-free HBSS (containing 100 μm EGTA) for 3–6 min prior to the addition of CellTiter-Glo™ reagent. Luminescence was measured in a PolarStar plate reader (BMG). ATP levels are expressed as fmol/cell based upon an ATP standard curve, and data are corrected for background luminescent signal. Each set of data was collected from multiple replicate wells (n = 12) and plotted as mean ± S.E. Preparation of Cerebellar Granule Cells—Granule cells were prepared as described previously (18Courtney M.J. Lambert J.J. Nicholls D.G. J. Neurosci. 1990; 10: 3873-3879Crossref PubMed Google Scholar) from 7-day-old postnatal Wistar rats. Cells were plated on poly-d-lysine-coated 10-cm plates at a concentration of 1 × 106 cells per ml in a volume of ∼9 ml per plate. Cells were cultured in minimal essential medium containing Earle's salts (Invitrogen) plus 10% (v/v) fetal calf serum (HyClone), 25 mm KCl, 30 mm glucose, 2 mm glutamine, penicillin, and streptomycin (100 units/ml and 100 μg/ml, respectively). After 24 h, 10 mm cytosine arabinose was added to inhibit non-neuronal cell proliferation. Cells were maintained at 37 °C in a 5% CO2 incubator and were used after 7–8 days in vitro. Isolation of Mitochondria from Control and Glutamate-treated Neuronal Cultures—A method for isolation of functional mitochondria from primary neuronal cultures has been described previously (19Almeida A. Medina J.M. Brain Res. Brain Res. Protocols. 1998; 2: 209-214Crossref PubMed Scopus (70) Google Scholar). We used a modified protocol for rapid preparation of neuronal mitochondria suitable for assessment of bioenergetic parameters and the Ca2+ content. The culture medium was aspirated, and cells (cortical neurons or cerebellar granule cells) were exposed to 100 μm glutamate and 10 μm glycine in HBSS. Control cells were exposed to HBSS without glutamate and glycine. After glutamate addition, cells were returned to the incubator for 10 min. Next, the plates were placed on ice and washed twice with ice-cold Ca2+-free and Mg2+-free phosphate-buffered saline solution (Invitrogen) supplemented with 2 mm EGTA. Cells were rapidly scraped in 1 ml (per plate) of ice-cold mitochondrial isolation buffer containing 210 mm mannitol, 70 mm sucrose, 10 mm HEPES-KOH, pH 7.4, 2 mm EGTA, and 0.1% fatty acid-free bovine serum albumin and were then homogenized using a Dounce homogenizer (10 passes with a loose pestle and 10 passes with a tight pestle). Cell homogenates were centrifuged for 10 min at 1060 × g at 4 °C, and the supernatant was collected. The pellet was resuspended in the isolation buffer and centrifuged at 1060 × g for 5 min. The first and second supernatants were pooled together and centrifuged at 14,600 × g for 10 min at 4 °C. The mitochondrial pellet was resuspended in the isolation buffer lacking EGTA and albumin and centrifuged at 14,600 × g for 10 min. All isolation steps were performed on ice. The protein concentration was measured using the BCA protein assay kit (Pierce). Typically, 10–15 plates of cells were used for one mitochondrial preparation. Note that mitochondria were isolated in the absence of digitonin that is commonly used for disruption of the cholesterol-rich synaptosomal membranes to release mitochondria entrapped in synaptosomes (15Rosenthal R.E. Hamud F. Fiskum G. Varghese P.J. Sharpe S. J. Cereb. Blood Flow Metab. 1987; 7: 752-758Crossref PubMed Scopus (232) Google Scholar). Our mitochondrial preparations from cultured neurons appear to be relatively free of synaptosomes as evidenced by the lack of an effect of digitonin on the maximal rate of respiration in the presence of succinate and rotenone (data not shown). Succinate is a mitochondrial substrate that is poorly permeable to the plasma membrane. Measurements of Mitochondrial Respiration, Membrane Potential, and Ca2+ in Isolated Neuronal Mitochondria—Simultaneous measurements of mitochondrial respiration (O2 consumption), Ca2+ fluxes, membrane potential, and optical density of the mitochondrial suspension were conducted in a custom-constructed thermostatically controlled chamber (B. Krasnikov, Burke Medical Research Institute, NY). Membrane potential (Δψ) was monitored by distribution of tetraphenylphosphonium (2 μm) with a tetraphenylphosphonium-selective electrode (20Kamo N. Muratsugu M. Hongoh R. Kobatake Y. J. Membr. Biol. 1979; 49: 105-121Crossref PubMed Scopus (890) Google Scholar). Extramitochondrial Ca2+ was monitored with a Selectophore I membrane (Fluka)-based Ca2+-selective electrode. A Clark-type electrode (Diamond General) was used to measure oxygen consumption. In addition, measurements of optical density at 660 nm by means of a light-emitting diode and photodetector permitted changes in mitochondrial volume to be followed. This parameter was also used to confirm consistency of protein concentrations between experimental runs. Digital data acquisition using a DynaRes 16 board and WorkBench software (Strawberry Tree) permitted simultaneous recording of all four parameters as well as plots of their first derivatives in real time. The first derivatives were used for quantification of respiration and Ca2+ uptake rates. For respiration measurements, mitochondria were incubated at 25 °C in a basal saline medium (125 mm KCl, 5 mm HEPES/KOH, pH 7.4, and 2 mm phosphate) supplemented with 2 mm MgCl2 and either complex I-linked substrates (a mixture of 5 mm glutamate and 5 mm malate) or a complex II substrate (5 mm succinate in the presence of 2 μm rotenone). Protein concentrations were 1–1.5 mg/ml for cortical mitochondria and 0.7–1.3 mg/ml for cerebellar mitochondria. State 3 (phosphorylating) respiration was initiated by addition of 200 μm ADP, after which state 4 (resting) respiration was induced by addition of 100 μm atractyloside, an inhibitor of the ADP/ATP antiporter. Finally, to measure uncoupler-stimulated respiration (state 3u), sequential additions of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20–50 nm) were made until the respiration rate reached maximum. The acceptor control ratio was determined as the ratio of state 3 to state 4 respiration. The experiments were completed within 2–3 h after the isolation procedure. Mitochondrial Ca2+ content was determined in the same basal medium (above) supplemented with glutamate and malate. Protein concentrations were in the same range as for the respiration measurements. After 2–3 min of incubation to allow for uptake of contaminating Ca2+ from the medium, mitochondrial Ca2+ was released into the medium by addition of FCCP (0.5 μm) followed 5 min later by the addition of the pore-forming peptide alamethicin (40 μg/ml) to ensure complete Ca2+ release. In some experiments, A23187 (1.2 μm), a Ca2+ ionophore, was also used to release sequestered Ca2+. The amount of Ca2+ released from mitochondria was calculated based upon calibration of the Ca2+ electrode performed at the end of each experimental day by additions of known concentrations of CaCl2 to the medium. EGTA (1 mm) was then added to chelate free Ca2+ in the medium. According to the Nernst equation, the voltage changes on the electrode are linearly related to the logarithm of Ca2+ concentration values in the medium. The concentration of contaminating Ca2+ in the medium was also experimentally determined. It was defined as an independent variable in the regression analysis shown in Equation 1, ΔV=a+b×log([Ca2+]+[Ca2+]0)(Eq. 1) where a and b are regression parameters expressed in relative units, and [Ca2+]0 is the concentration of contaminating Ca2+. R2 values for all calibration curves were no less than 0.995. The best fit parameters were used to calculate Ca2+ concentrations from the voltage changes in the experimental runs. The mitochondrial Ca2+ load was determined as the difference in Ca2+ concentration after addition of the Ca2+-releasing agent (FCCP or alamethicin) minus the contaminating Ca2+ in the medium and was normalized to mitochondrial protein concentration. Mitochondrial Ca2+ uptake capacity was measured in the same medium as that used for mitochondrial Ca2+ content determination. Energized mitochondria were pulsed with successive additions of Ca2+ (30–50 μm) every 2–3 min. The increasing Ca2+ load caused a successive decline in the membrane potential and Ca2+ uptake rates. Maximal Ca2+ uptake capacity was defined as an amount of Ca2+ (per mg of protein) required to decrease the Ca2+ uptake rate by >90%. Note that because of the duration of the experimental run (25–40 min), most of these measurements were performed in an open chamber to avoid anoxia. Data are expressed as mean ± S.E., and n indicates the number of independent experiments. Each mitochondrial preparation was obtained from a separate neuronal harvest (or neuronal preparation). Statistical Analysis—One-way analysis of variance followed by post hoc Tukey test was performed using SigmaStat. Where differences are noted, analysis of variance detected significant variance at p < 0.001, and pairwise comparisons are indicated in the figure legends. Materials—Unless indicated otherwise, all reagents were obtained from Sigma. Measurements of Cytoplasmic Ca2+ in Cortical Cultures— Mitochondrial Ca2+ accumulation has been well established in different excitotoxicity models (21Peng T.I. Greenamyre J.T. Mol. Pharmacol. 1998; 53: 974-980PubMed Google Scholar, 22Nicholls D. Budd S. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1050) Google Scholar, 23Wang G.J. Thayer S.A. J. Neurophysiol. 2002; 87: 740-749Crossref PubMed Scopus (51) Google Scholar, 24Alano C.C. Beutner G. Dirksen R.T. Gross R.A. Sheu S.S. J. Neurochem. 2002; 80: 531-538Crossref PubMed Scopus (63) Google Scholar). An intact cell study utilizing a dye with low affinity to Ca2+ has demonstrated that a significant fraction of accumulated Ca2+ is retained within the mitochondria during brief glutamate exposures until FCCP is added to induce Ca2+ efflux (25Brocard J.B. Tassetto M. Reynolds I.J. J. Physiol. (Lond.). 2001; 531: 793-805Crossref Scopus (71) Google Scholar). Based on this approach, we performed measurements of cytoplasmic Ca2+ in situ with our cortical cultures to demonstrate that the conditions of glutamate treatment used in our model produces results consistent with literature data. Fig. 1 depicts results of a representative experiment in which cortical neurons were challenged with various concentrations of glutamate (in the presence of 10 μm glycine) for 10 min followed by chelation of extracellular Ca2+ with EGTA and subsequent addition of FCCP. The responses to glutamate at concentrations between 30 and 300 μm were similar, and the pattern is typified by an initial peak in cytoplasmic Ca2+ that is followed relatively rapidly in these cultures by a more sustained and profound rise in cytoplasmic Ca2+. The response to 100 μm glutamate appeared to be maximal with regard to cytoplasmic Ca2+ and FCCP-induced release of Ca2+, as noted previously by others (25Brocard J.B. Tassetto M. Reynolds I.J. J. Physiol. (Lond.). 2001; 531: 793-805Crossref Scopus (71) Google Scholar); therefore, we chose to probe in greater depth the bioenergetic changes occurring at this 10-min time point of glutamate exposure. Effect of Glutamate Treatment on Cellular ATP—As shown in Fig. 2, 10 min of glutamate exposure results in a significant drop in the ATP level (Fig. 2A). Longer exposures to glutamate (up to 40 min) did not further decrease the content of ATP. Partial ATP depletion could be a direct consequence of acute activation of ATP-requiring efforts by the cell to re-establish ionic homeostasis during glutamate stimulation, and/or the ATP loss could result from impairment of cellular ATP production. To test whether ATP could be readily recovered if the glutamate and Ca2+ challenge were halted, cells were exposed to glutamate for 10 min followed by incubation of the cells in a glutamate- and Ca2+-free buffer. Under these conditions, cytoplasmic Ca2+ fairly rapidly returns to a steady state level (see Fig. 1 described above); however, ATP did not recover within this time frame. This result suggests that the compromise in cellular ATP levels reflects a compromise in the ability of the cell to produce ATP at a normal rate. Effect of Glutamate Treatment on Neuronal Viability—We next tested the effects of glutamate exposure on the viability of the cortical cultures. By using LDH release as a method of assessing plasma membrane permeability, we found that 24 h of continuous exposure of these cultures to 100 μm glutamate resulted in a significant loss in viability that was inhibited by 10 μm of the NMDA receptor antagonist, MK-801 (Fig. 3). Even though ionic homeostasis appears to be lost and mitochondrial Ca2+ loading is maximal after 10 min of glutamate exposure (Fig. 1), if this treatment is halted by addition of MK-801 for the ensuing 24 h, the injury is no longer sufficient to induce LDH release substantially above control levels (Fig. 3), at least at this time point. Therefore, cellular events leading up to the 10 min time point may be critical to initiation of the cell death pathway, but these events have not resulted in commitment of the cells to acute cell death. Quantitative Estimation of Ca2+ in Mitochondria from Control and Glutamate-treated Neurons—The ability of mitochondria in glutamate-treated cells to release accumulated Ca2+ in response to FCCP argues against pervasive induction of a permeability transition in the mitochondrial population, as this would prevent retention of mitochondrial Ca2+. As shown on Fig. 4A, trace b, addition of FCCP to mitochondria from glutamate-treated cells caused a rapid release of a significant amount of Ca2+ presumably via reversal of the Ca2+ uniporter as a result of depolarization of the membrane (26Jurkowitz M.S. Geisbuhler T. Jung D.W. Brierley G.P. Arch. Biochem. Biophys. 1983; 223: 120-128Crossref PubMed Scopus (41) Google Scholar). This FCCP-induced release is relatively slow and was still progressing at a significant rate 5 min after the uncoupler addition (Fig. 4A, trace c; note that the y scale is logarithmic). Therefore, we used a pore-forming peptide alamethicin to quickly attain complete release (Fig. 4A, trace c). Quantitative estimation of mitochondrial Ca2+ (performed as described under "Experimental Procedures") revealed that the total Ca2+ released in response to FCCP and alamethicin reached 167 ± 19 nmol/mg protein (n = 8). In the 5-min time frame, FCCP-released about half of the total Ca2+ (86 ± 14 nmol/mg protein, n = 8). More importantly, the amount of Ca2+ released from mitochondria isolated from untreated cells was very low (1.9 ± 0.8 nmol/mg protein, n = 5; Fig. 4A, trace b). Note that the amount of contaminating Ca2+ taken up into mitochondria from the incubation medium (2.5 ± 1.6 nmol/mg protein, n = 13) was subtracted from the amount of Ca2+ released into the medium. We do not exclude the possibility that a fraction of damaged mitochondria could be lost during preparation, but there was no apparent difference in the yields of mitochondria isolated from control and glutamate-treated cells. As well, we cannot exclude the possibility that a subfraction of mitochondria may have undergone the permeability transition and released their accumulated Ca2+ prior to isolation. Both of these possibilities would result in an underestimation of the quantities of retained Ca2+. Although it is possible that mitochondrial Ca2+ could be lost during isolation, the standard conditions for preparation of mitochondria, such as low temperature, the presence of EGTA, and the absence of Na+ in the isolation buffers, minimize changes in mitochondrial Ca2+ during the isolation procedure. This statement is supported by direct Ca2+ measurements in Ca2+-loaded brain mitochondria after prolonged incubation under conditions that mimic our isolation procedure (27Zaidan E. Sims N.R. J. Neurochem. 1994; 63: 1812-1819Crossref PubMed Scopus (113) Google Scholar). Because alamethicin is nonselective, and endoplasmic reticulum is a persistent contaminant of mitochondrial preparations, we performed additional experiments to confirm that the released Ca2+ was primarily of mitochondrial origin. Pretreatment of intact cortical neurons for 15 min prior to glutamate addition with 10 μm cyclopiazonic acid, an endoplasmic reticulum Ca2+-ATPase inhibitor, did not affect Ca2+ release in response to FCCP and alamethicin (Fig. 4B, traces a versus b). This observation suggests that the Ca2+ released by alamethicin is primarily mitochondrial. Determination of Ca2+ Uptake Capacity in Mitochondria from Cortical Neurons—As discussed above, the results of Fig. 1 imply that the mitochondrial Ca2+ load is saturated under our standard conditions (100 μm glutamate and 10 min of treatment). At a low level of glutamate, the mitochondrial Ca2+ load appears to be limited by Ca2+ availability as indicated by a near basal steady state level of cytosolic Ca2+ (Fig. 1, trace e). In contrast, one might presume that unde